Bovine Antibodies Contributes to AIDS Vaccines, and benefits for AIDS Treatment

Cows are entering the field of HIV vaccine research. As described in a research report published in the journal Nature, Devin Sok, director of antibody discovery and development at the International AIDS Vaccine Initiative (IAVI), reported a strong HIV-blocking antibody within a few weeks - one that usually takes years The process that can be completed. At the moment when new energy is injected into HIV vaccine research, unexpected animal models provide clues to important issues.

One of the study's first authors, Sok, said: "One approach to preventive HIV vaccines involves trying to trigger a wide range of neutralizing antibodies in healthy people, but so far these experiments have not been successful in human and animal studies." This experiment shows that it is possible not only to produce these antibodies in animals, but also to do this reliably and quickly, and to use relatively simple immunization strategies in the right environment. ”

Scientists have known for some time that some people with chronic HIV infection produce a wide range of neutralizing antibodies (bnAb), which can overcome the high level of HIV diversity. A type of bnAb, first reported by IAVI, the Scripps Research Institute (TSRI) and Theraclone in 2009, used a long arm-shaped ring to reach hidden areas on the surface of the virus to stop infection. Experiments led by Vaughn Smider, a bovine antibody specialist at the Scripps Research Institute (TSRI), have shown that bovine antibodies also have ultra-long cycles that may be exposed to difficult epitopes that human antibodies cannot. This feature is related to HIV researchers because the virus has a sugar that is used to stop most antibodies from reaching their vulnerable sites.

Sok is a subsidiary of the IAVI Neutralizing Antibody Center (NAC), part of TSRI where several scientists work together to study antibody-based HIV vaccines. NAC was led by Dennis Burton, lead author of the study, TSRI Professor of Immunology and Microbiology. Like a bull in a science porcelain store, the Alliance of HIV, Antibody and Veterinary Scientists from IAVI, TSRI and Texas A&M University raises a colorful question: What happens if we immunize cattle with HIV immunogens ?

“This is a very simple and profound idea,” Sok said, working closely with TSRI's Dennis Burton. “Because we know that some human bnAbs have longer loops than the average length, will immunization of animals with similar antibody structures lead to bnAbs against HIV?”

The answer starts with a single protein on the surface of HIV as a target for bnAb - developing an antibody that recognizes variants of this protein on different HIV viruses and you may be protected from all of these viruses. One of the many techniques that HIV uses to stop humans from developing correct antibodies is to show an unrelated form of this protein to disperse the immune system. Scientists believe they have overcome this challenge by developing an immunogen called BG505 SOSIP, which mimics protein targets. Immunization with rhesus monkeys, guinea pigs, and rabbits with this immunogen is both encouraging and frustrating - so far, it has triggered very good antibodies against a strain of virus, but failed to lead to overcome HIV. Globally diverse antibodies.

All four cows immunized with BG505 SOSIP induced bnAbs to infect HIV within 35-52 days. In contrast, HIV-positive people take years to develop appropriate responses, and only 5-15% can even develop these responses.

Of course, cows cannot be infected with HIV. But these findings have set new targets for HIV vaccine researchers: By increasing the number of long-circulating human antibodies, we may be more likely to obtain protective bnAbs by vaccination.

There is no doubt that the ability of cows to produce bNAbs against complex pathogens (such as HIV) in a few weeks highlights a broader sense, especially for emerging pathogens.

IAVI CEO Mark Feinberg said: "Scientific innovations like this have driven this area ahead. "This surprising result deserves further exploration and has potential for HIV prevention and treatment, but also Rapid development of antibodies and vaccines against other infectious diseases. ”

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Significant Progress in The Field of AIDS---Antibodies Successfully Cleared SHIV in Baby Monkeys

Scientists at the Oregon National Primate Research Center revealed today that infants exposed to SHIV (chimeric monkey virus with HIV envelope protein) are treated with antibodies within 24 hours to completely eliminate the virus. The study, published in the journal Nature Medicine, showed that antibody administration to infants exposed to SHIV can successfully eliminate the virus, a major advance in the AIDS scientific community.

SHIV-infected non-human primates can transmit SHIV to their offspring through breastfeeding, just as HIV can be transmitted mother-to-child through breastfeeding and childbirth. People often use antiretroviral therapy (ART), caesarean section delivery and formula feeding to block mother-to-child transmission of HIV-1, which has reduced the mother-to-child transmission rate of HIV from 25% in 1994 to 2%. . Although it is much reduced, there are still about 200,000 children infected with AIDS every year, and it mainly occurs in developing countries where ART treatment is immature.

"We understand that HIV can quickly infect babies in mother-to-child transmission, so we need to treat baby monkeys as soon as possible, but we were not sure that antibody treatment could completely eliminate the virus." Haigwood and colleagues first, after the monkeys were exposed to the virus, The mice were neutralized by subcutaneous injection at 7 and 10 days. Infant monkeys without antibody treatment were observed to be found in various tissues on the first day of exposure. Instead, the effect is immediate after the first needle is injected. Injection of a potent antibody in the early stages of infection completely cleared the virus on day 14. That is, no virus was detected in any tissue in the baby monkey using a highly sensitive method.

Often, HIV is rapidly infected, spreads in local lymph nodes, and spreads throughout the body within a week. In the experimental model of this study, viral replication was detected in lymphoid tissues 24 hours after SHIV oral exposure of newborn rhesus monkeys, and was detected in the blood after 5 to 7 days.

Studies have shown that: antibodies injected subcutaneously can be rapidly distributed in blood and tissues, maintaining neutralizing activity at different locations, and antibodies can effectively eliminate viruses. Unlike the mechanism of ART treatment, ART is a variety of anti-retroviral Drugs can slow the rate of HIV replication in the body.

Experiments with other non-human primates have shown that antiretroviral treatment is too late after three days of infection, preventing the establishment of HIV reservoirs. The researchers recommend ART in the last month of pregnancy, a few days after delivery, and breastfeeding. But the risks remain, including the toxicity of long-term ART treatment, the emergence of virus-resistant strains and the lack of care before delivery. New research registration, using antibody therapy and other methods may be beneficial for neonatal infection suppression.

The authors of the study acknowledge that there are still many questions that are unanswerable about HIV-infected babies and mothers, including practical and cultural aspects of breastfeeding, whether antibodies can be used to expose humans to HIV, and optimized antibodies Composition and other issues.

Clinical trials of therapeutic antibodies for HIV-exposed neonates have been conducted in the United States and South Africa. Previous clinical trials have shown antibody safety and good tolerance in HIV-1 negative adults.

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Function and Applications of Squalene in Clinical Treatment

Squalene has a red blood cell-like oxygen-carrying function, which combines with oxygen to form activated oxygenated squalene in the body. It is transported to the terminal cells of the body through the blood circulation to release oxygen, which promotes the biooxidation-reduction reaction in the body's metabolism. Thereby increasing the utilization ability of tissue cells for oxygen, improving the body's tolerance to hypoxia, and preventing various diseases caused by hypoxia. Because squalene has a red blood cell-like oxygen-carrying function, it makes sharks have strong hypoxia tolerance in the deep sea anoxic environment.

Qiu Chunmei et al studied the effect of squalene soft capsule on hypoxia tolerance in mice. The results showed that squalene was the recommended dose of 20 times and 30 times the dose of normal hypoxia and prolonged survival time of sodium nitrite poisoning. . Under the conditions of polar cerebral ischemic hypoxia, the 10, 20, and 30-fold doses of squalene recommended doses significantly prolonged the gasping time in mice compared with the control group. Growth has no effect.

It can be seen that squalene has oxygen carrying capacity and can improve hypoxia tolerance in mice. The higher content of squalene in the yak in the plateau area may be closely related to the yak's adaptation to the environment of hypoxia, cold and low pressure.

Regulate the metabolism of cholesterol in animals

In the 1950s, researchers discovered a key intermediate metabolite in the study of the biochemical metabolic mechanism of human cholesterol, which was identified as squalene by structure, which confirmed for the first time that squalene was present in humans. Squalene can be converted to lanosterol by lanosterol synthase, converted to cholesterol, and further metabolized to produce bile acids and steroid hormones. Given that squalene can be converted to cholesterol, there has been a view in the academic world that exogenous squalene increases cholesterol synthesis and increases the risk of atherosclerotic disease in humans.

However, with the deepening of research, it has been found that the intake of exogenous squalene does not increase the level of cholesterol in the serum, and even lowers the serum cholesterol. The mechanism of action of squalene to lower serum cholesterol levels may be that exogenous squalene can reduce the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to inhibit cholesterol synthesis. It depends on the amount of exogenous squalene absorbed. At the same time, through the feedback regulation of cholesterol, the cholesterol is converted into fecal bile acid, which is excreted with the stool.

Anti-cancer and anti-cancer effects

Squalene can effectively prevent and inhibit the occurrence of various cancers such as breast cancer, colon cancer, pancreatic cancer, lung cancer and skin cancer in chemically induced rodents. People in the Mediterranean region consume large amounts of olive oil in their daily lives, and the probability of breast and pancreatic cancer in the population is extremely low. This is closely related to the daily diet of local residents. This is due to the high content of squalene in olive oil. The average daily intake of squalene in the region is 200-400 mg, which is much higher than other parts of the world. The average daily intake of the population.

Radiation resistance

Storm et al studied the anti-radiation effect of squalene on male mice, and found that the mouse squalene intake group can improve the resistance of the mouse body to radiation damage compared with the control group. The mechanism of action may be that squalene can eliminate the free radical or singlet oxygen generated by radiation to stimulate the body's immune response, protect the organelles and improve the cell repair ability.

Applications

Squalene is widely used as an emollient because it has anti-oxidant, anti-UV damage and moisturizing effect. Squalene can block the pores of the skin and is easily absorbed by the skin, showing a good moisturizing effect. Squalene is an important component of sebum and provides nutrition to the skin. Squalene is also a moisturizing substance in the stratum corneum. It is reported that fetal sebum is a highly effective moisturizer that has a water retention effect on the stratum corneum. When squalene is used in combination with triacylglycerol, cholesterol, ceramide and fatty acid, it can produce a water retention effect similar to fetal sebum.

In order to enhance the therapeutic antibodies effect of drugs, drug sustained release agents have received widespread attention. Squalene is widely used as a drug sustained release agent. Contains squalene emulsion drugs to prolong the half-life of the drug. Wang et al. reported that squalene emulsions delay the release of morphine prodrugs by stabilizing phosphatidylethanolamine or copolymer. The squalene emulsion can also be used for the sustained release of lipophilic prodrugs or psoralen capsules of cyclobutalol. In the early years of Japan, squalene was used to treat tuberculosis. In recent years, since squalene has been infiltrated, diffused, and sterilized, it can absorb a large amount of oxygen in daily service or on the skin, strengthen cell metabolism and eliminate fatigue, and thus has become a functionally defined active ingredient in functionality. Widely used in food. In recent years, many countries have included it in the ranks of drugs, such as the Chinese Pharmacopoeia, to use squalene as an oral nutrient, at a dose of one gram per day. Japan has extended it to be used as an internal medicine for the treatment of hypotension, anemia, diabetes, cirrhosis, cancer, constipation, tooth decay, and as a treatment for gallbladder and bladder stones, tonsillitis, rheumatism, neuralgia, bronchitis, colds, External application of rhinitis, asthma, gout, stomach and duodenal ulcer.

Squalene is a free radical scavenger, which is used as a functional food additive in foods because it promotes human metabolism and enhances the body's immunity. At present, the squalene health foods on the domestic and international markets include squalene capsules, squalene capsules and squalene soft capsules, and the content of squalene in each capsule/capsule is 500-1000 mg. In addition, squalene is added to edible vegetable oils such as soybean oil and peanut oil because of its good antioxidant activity, thereby inhibiting or delaying the oxidation of oils and fats, thereby improving the stability of edible vegetable oils and prolonging the shelf life of the products.

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Squalene---Extracted From Sharks?

Squalene, also known as squalene, shark, squalene, is an important chemical active substance in the liver of sharks. Squalene is an all-trans isomer with six double bonds and is a highly linear, unsaturated triterpenoid. Squalene is a colorless or slightly yellow transparent oily liquid that absorbs oxygen and becomes linseed. It is easily soluble in petroleum ether, ether and acetone, slightly soluble in glacial acetic acid and ethanol, and incompatible with water.

Discovery 

Squalene is a colorless or slightly yellow transparent oily liquid that absorbs oxygen and becomes linseed. It is easily soluble in petroleum ether, ether and acetone, slightly soluble in glacial acetic acid and ethanol, and incompatible with water.

Distribution of squalene

Squalene is not only found in animals, plants, but also in microorganisms. Deep sea sharks are one of the animals with high levels of squalene found in nature to date. Tsujimoto found that squalene is the main component of shark liver oil unsaponifiable matter, which is 89.62 g/100 g and 71.64 g/100 g of Ai-zamé and Heratsuno-zamé shark liver oil, respectively. The content of squalene in deep sea shark liver oil is above 40 g/100 g, which is considered to be the main source of early squalene.

Because of the different geographical conditions of shark species, gender, age and growth, there is a big difference in the content of squalene in liver oil. The content of squalene in the liver oil of deep sea and toothed shark is above 90 g/100 g, while the river The content of squalene in shark liver oil is lower. Following the deep sea shark, sebum is an animal tissue found to have a high squalene content. The content of squalene in sebum is approximately 13 g/100 g.

Downing and other studies have found that the content of squalene in the sebum of the mole is 70 g / 100 g, which is significantly higher than the content of squalene in human and other animal sebum, which may be closely related to the adaptation of the mole to the humid living environment. . Generally, human skin is exposed to sunlight for a long time, and ultraviolet rays easily cause oxidative stress damage to the skin. Squalene in sebum can effectively block the reaction and inhibit the peroxidation of sebum, thereby protecting the skin from harm.

Recent studies have found higher levels of squalene in yak meat. In 2012, Luo Zhang detected squalene in freeze-dried yak meat, which can reach 328.28μg/kg. It can be seen that yak meat can be used as a new resource for the development and utilization of squalene.

Kopicov et al found that the content of squalene in the muscles of 20 freshwater fish such as red-eye fish was 9.80~153.68 mg/100 g, and the content of squalene in visceral fat was 7.01~180.38 mg/100 g. The content of squalene in freshwater fish is significantly lower than that in deep sea sharks, which may be closely related to the environmental conditions in which they live.

Dewitt et al found that trace amounts of squalene were present in the blood of humans and rats, containing 30 to 35 μg of squalene per 100 mL of blood.

In addition, shark mab has been found that squalene is detected in vegetable oils and by-products such as olive oil, amaranth oil, rice bran oil and palm oil, crop seed oil and vegetable oil deodorized distillate, especially in olive oil and amaranth oil. The high content of squalene can be used as an important industrial raw material for the preparation of squalene. Crude olive oil is a vegetable oil obtained by crushing olive kernels. It is considered to be one of the main sources of squalene in the current plant, and its squalene content is 100 mg/100g~800 mg/100 g.

Squalene is also present in microbial cells. Bhattacharjee et al. produced squalene by anaerobic fermentation of Saccharomyces cerevisiae isolated from Saccharomyces cerevisiae and molasses, with dry yields of 4.12 mg/100 g and 23.72 mg/100 g, respectively.

In addition, Brid et al. found that trace squalene was also detected in microorganisms such as Staphylococcus, Hansen Debaryomy, Rhodospirillum and Aspergillus nidulans (filamentous fungi). By screening high-yield strains of squalene and then producing squalene by bio-fermentation, it opens up a new way to solve the squalene resources.

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Analysis of Recombinant Antibodies and Separation and Purification Methods of Its Fragments

Antibodies are proteins secreted by a plasma cell in the human body. At the production stage, antibodies are usually present in complex cells, and most of their applications require purification. The isolation and purification of recombinant antibodies and their fragments are relatively simple compared to other recombinant proteins. The ideal purification method can obtain antibody products of sufficient purity in one step from the crude extract of recombinant E. coli protein. Therefore, the rational selection of purification methods is particularly important. A series of processes for protein isolation and purification are designed to isolate a single type of protein from a complex mixture. Protein Separation offers a wide range of purification systems for simple protein separation and purification.

Antibodies can identify some foreign substances in the human immune system, such as viruses, bacteria, etc. All antibodies belong to immunoglobulins, so antibodies can be said to belong to proteins, but not all proteins are antibodies. Antibodies are constantly being used in a variety of scientific research, including but not limited to basic research, chromatographic analysis, targeted analysis, imaging, diagnosis and treatment. However, most of these applications require high purity homogeneous antibodies. Thus, for the purification of antibodies from complex mixtures such as plasma, serum, ascites, cell culture media, egg yolks, plant extracts and bacterial or yeast cultures, there is an increasing demand for simple, efficient and economical purification methods. Protein separation companies often use affinity chromatography to separate and purify proteins. Medicilon is a preclinical CRO company with extensive experience in recombinant protein expression and purification services, with a variety of protein expression systems, including prokaryotic protein expression systems, yeast protein expression systems, and insect cell protein expression systems (baculovirus). The mammalian cell protein expression system, with a variety of fusion technologies, provides customers with a wide range of options for protein expression and purification.

Purification of recombinant antibody and fragments thereof can generally be accomplished in several ways:

1. Bacterial affinity chromatography.

Affinity chromatography is a common method for purifying recombinant proteins. It utilizes the specificity of the protein structure, can bind to the corresponding specific molecule, and can be dissociated under specific conditions, and can obtain high-purity protein in one step. For example, the specific enzymes, receptors, antibodies, and the like can be purified by utilizing specific binding of an enzyme to a substrate, binding of a receptor to a ligand, and binding of an antibody to an antigen. It is also possible to add a tag such as His, Flag, GST, HA, c-Myc, GFP, SUMO, MBP, Avi, etc., when the recombinant protein is expressed.

Some bacteria can synthesize proteins that specifically recognize and bind to higher mammalian immunoglobulins, such as proteins A, B, G, and L. The binding sites are mainly located in the constant region of the antibody molecule, and only a few are present in the variable region. Therefore, this method is more suitable for isolating recombinant antibody intact molecules as well as monovalent and bivalent FAB fragments, but is ineffective for the isolation of FV or scFV fragments.

2. Antibody affinity chromatography

Antibody fragments expressed as fusion proteins can often be separated by selection of a suitable antibody affinity chromatography column depending on the characteristics of the target protein. Currently used target proteins such as alkaline phosphomonoesterase, peroxidase and some toxin proteins have corresponding commercial antibody affinity maturation services media, but if E. coli itself can also synthesize this target protein or target protein The homologous protein, the specificity of this method will be affected. To overcome this difficulty, the Flag and Myc TAG tag sequences in the pIG vector can also serve as target sequences for anti-Flag antibodies and anti-Myc TAG antibodies, and fusion proteins containing these sequences can be separated by corresponding antibody affinity chromatography columns. However, the above antibodies are expensive and generally only used on a laboratory scale.

3. Antigen affinity chromatography

If a specific antigen corresponding to a recombinant antibody or antibody fragment is readily available, separation of the expression product using this antigen affinity chromatography column is the best choice because it is not only highly selective but also can be obtained from any non- Rapid separation of the target antibody or antibody fragment in a properly folded protein mixture. During hapten affinity chromatography, the isolated product typically requires elution with a soluble hapten under very mild conditions. However, for some harmful antigens (such as tumor antigens, etc.), it is generally inappropriate to use this method to separate antibody fragments for use in vivo.

4, ligand affinity layer column method

Phosphocholine affinity chromatography columns, which were used to isolate intact antibody molecules, can also be used directly to purify recombinant FAB, FV, scFV fragments and various bivalent minibodies from crude E. coli protein extracts, all with the proviso that all The recombinant antibody fragment must have a good folded structure. In addition, the His tag tag sequence mounted on some pIG vectors was designed specifically for the ligand affinity chromatography purification process for expression products. The histidine residue in the polypeptide chain can be combined with a plurality of divalent heavy metal ions, and the protein in which the histidine residue is concentrated in the primary sequence can theoretically be separated by a divalent heavy metal ion affinity chromatography column. The good separation effect depends to a large extent on the combination of metal ions and eluent. For example, the Zn2+ column is usually eluted with a diacetic acid imide solution, while the Ni2+ column is eluted with an imidazole or triacetonitrile solution. Because of the low cost of heavy metal ion affinity chromatography media, this method is more suitable for large-scale production of recombinant antibody fragments.

Affinity chromatography as a method for purification of recombinant protein, according to its uniqueness, easy operation, relatively high yield and throughput, make it the most efficient and widely used chromatographic technology. A general technique for antibody purification.

Differentiation of Four IgG Antibody Subtypes & Application in Recombinant Antibody Drugs

There are four subtypes of IgG antibodies: IgG1, IgG2, IgG3, IgG4. Although the positions and numbers of disulfide bonds are different, the spatial structures of the four subtype antibodies are very similar. This paper combs the differences in the structure and physiological activities of four IgG antibody subtypes, and then discusses its application in the development of recombinant antibody drugs.

IgG1 is the most abundant in plasma and is the most subtype of recombinant antibody. The FcRn affinity of IgG3 is weak and the half-life is only 9 days. Considering that pharmacokinetics requires more frequent administration, it is rarely used to develop antibody drugs. In addition, various subtypes of antibodies will change during development, and children's IgG1 levels will reach adulthood at 5 years of age, while other subtypes of antibodies will slowly increase.

In recent years, with the expansion of new indications and the discovery of new therapeutic mechanisms, the application of IgG2 and IgG4 subtypes has gradually increased.

Taking an immunological checkpoint inhibitor as an example, the PD-1/PD-L1 pathway theoretically blocks the pathway with a PD-1 antibody or a PD-L1 antibody, thereby suppressing inhibition of T cells and the like, thereby killing the cells. This mechanism of action is different from previous mechanisms such as anti-cancer antibodies relying on ADCC activity to kill cells. Therefore, PD-1 antibodies Opdivo and Keytruda are designed to use the IgG4 subtype with weak ADCC activity, and the PD-L1 antibody Tecentriq uses IgG1 subclass. Type but using antibody engineering to remove glycosylation and no ADCC activity.

Of course, the various mechanisms of action of antibody drugs interact and are very complex. In theory, ADCC activity can also synergize with the PD-1/PD-L1 pathway to kill cancer cells. More is the balance between safety and effectiveness, which is waiting to accumulate more clinical experience and further understand its network of mechanisms of action. For example, the IgG1 subtype of strong ADCC activity used by the Pfizer PD-L1 antibody Avelumab, and the PD-1 antibody of Baekje Shenzhou also undergo antibody engineering of Fc (the modification of Fc is mostly the modification of glycosylation modification). The comparison of these attempts with accumulated clinical data has helped us to gain a deeper understanding of the more detailed mechanism of action of PD-1/PD-L1 antibody drugs.

As mentioned above, antibody subtype selection and antibody engineering are all specific attempts in the development of antibody drugs. Taking antibody engineering as an example: Roche's third-generation CD20 antibody is transformed into a cell line with no salt-to-alkali carbonylation by cell line (glycosylation-related enzyme), thereby expressing antibodies with high ADCC activity, which is the transformation of cell line level.

A more mature technique is protein-level modification. For example, the Roche PD-L1 antibody Tecentriq achieves deglycosylation by mutating the Asn-298 glycosylation site, and the Korean-American LAPS long-acting protein technology is modified by E. coli expression without aglycosylation. Fc, chemically coupled to protein drugs in vitro (using only its long-acting mechanism); Genexine's HyFc long-acting fusion protein technology, through hybrid Fc, combines the flexibility of the IgD hinge region with the low ADCC/CDC activity of IgG4 , get long-acting, safe protein drugs.

With the development of new antibody drugs such as bispecific antibodies and ADCs, the application range of antibody engineering has been greatly expanded. If the subtypes of bispecific antibodies have been found to be different, the ADCC activity can be significantly affected.

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MHC Tetramer Application---Development of TCR-like Antibodies

The tetramer technology enables specific, efficient and direct quantification of antigen-specific CTI-activity assays, and can be applied to immunological research and detection, specific immunotherapy, and vaccine efficacy monitoring.

MHC class I molecules serve as antigen presentation and recognition in the CD8+ cellular immune system. Due to their specificity, MHC-peptide complexes play an important role in immunotherapy. Although MHC-polypeptide complexes are specifically recognized by TCR, most natural TCR receptors are not only less stable but also have lower affinity for MHC-polypeptide complexes.

In order to solve the above two deficiencies, targeting tumor epitopes, the preparation of TCR-like antibodies with high affinity and solubility is the best alternative to wild-type TCR receptors. As a new immune molecule, this type of TCR-like antibody plays a role in recognizing and killing tumor cells.

Immunotherapy targeting T cell epitopes

Cells and cancer cells in the autoimmune system disorder express specific peptides that bind to cell surface MHC molecules. Tumor cells expressing specific short peptides in cancer patients can be recognized by CTLs. Despite this recognition mechanism, tumor cell apoptosis escape is common in tumor tissues.

In recent years, with the gradual deepening of research on tumor-associated antigens, people are also designing and validating relevant immunotherapy programs for tumor-specific antigens, and have achieved certain results in the specific treatment of cancer patients. There are many researches on tumor-associated antigens expressed by tumor cells, which can be roughly divided into three categories:

1) Tumor-specific antigens caused by protein mutations, such as B-catenin mutations in colon cancer.

2) Antigen expressed by certain defined cell lines

3) derived from high expression or amplification of genes, such as HER-2/neu

As for immunotherapy programs, most researchers have focused on how to enhance acquired immunity to tumor cells. These include acquired immune T cells, cancer cell short peptide vaccines, autologous cancer vaccines, and DC vaccines. These different methods have made great progress in the clinic.

Cell-based research efforts face challenges such as the mechanism of apoptosis, although these challenges limit the anti-tumor activity of CTL. However, the use of antibodies, through the simulation method can better solve this problem. Specific tumor-associated MHC I-polypeptide complexes play a specific immunomodulatory role. Using this property, an MHC I-polypeptide complex containing a specific short peptide can be used as a specific antigen on the surface of a tumor cell.

Such an antigen not only serves as a targeting molecule for cell therapy, but also serves to distinguish between cancer cells and normal cells. Moreover, a series of short peptides that are well-recognized by TCR are selected. These short peptides and MHC form an MHC-short peptide complex and can recognize most common tumors.

The mechanism by which TCR-like antibodies specifically recognize tumor cells is identical to that of MHC-short peptide complexes recognized by T cells. Based on the cell therapy program, it is easily restricted by many factors such as the state of the cell itself. Compared with cell therapy, soluble antibodies have higher yield; tumor infiltration ability is stronger than T cells, and it is superior to T cells as a treatment.

TCR-like antibodies have two major advantages in the immune system: from a biological point of view, TCR-like antibodies have better antigen specificity. As a drug, its stability is stronger than that of T cells, and T cells are more affected by the outside world. At the same time, tumor cells are affected by the tumor microenvironment, and the expression of MHC molecules on the surface of most tumor cells is down-regulated. Based on this understanding, high-affinity TCR-like antibodies are particularly suitable for low-density MHC-expressing antigens.

T cell receptor-like antibody

MHC-restricted antigen-specific T cell antibodies are few and difficult to prepare. TCR recognizes linear short peptides that bind to MHC molecules. This mechanism differs from the B cell recognition mechanism in that B cells recognize the three-dimensional structure of soluble or cell membrane antibodies and are not restricted by any MHC. At this point, T and B cells exhibit recognition mechanisms for two different functions in the immune system.

T cells recognize antigenic peptides presented by MHC class I or class II molecules on the cell membrane surface to recognize cells. B cells do not have a selective recognition mechanism. In recent years, research and development work on TCR-like antibodies has made great progress. Many research teams have the ability to make this antibody, and many TCR-like antibodies against tumor and viral T cell epitopes have been developed.

It is possible to obtain a THC-like antibody preparation that is recognized by T cells and naturally folds the MHC-short peptide complex after purification. The MHC-short peptide complex can be obtained by E. coli expression. Escherichia coli expresses MHC heavy chain and β-globulin, respectively, and after folding in vitro, the MHC-short peptide complex is obtained by affinity with a short peptide. The folded MHC-short peptide complex is required to be high, and the prepared complex is required to be very pure and folded correctly.

The prepared MHC-short peptide complex is biotinylated to screen for specific TCR-like antibodies in vitro. Many laboratories try to obtain epitopes that can be recognized by T cells, but many laboratories have difficulty preparing them. In 1996, Andersen et al. demonstrated the use of phage display technology as a means to obtain specific antibodies.

After they fuse the antigenic protein on the surface of the phage particle, they display the corresponding antibody by display screening. There are different genes in the phagemid that express different sequence proteins on the surface of the phage. After several rounds of screening and bacterial culture, the desired phage is eluted. Phage display exhibits a stronger positive selectivity than hybridoma technology.

Many research teams have built phage peptide libraries to screen for appropriate antibodies. The phage display technology was first able to isolate mouse MHC class I antibodies with short peptide specificity and MHC restriction. Thereafter, human HLA-related disease antibodies were also prepared.

TCR-like antibody screening

The specificity and affinity of a TCR-like antibody determines whether the antibody is good or bad. Specific T cell antibodies with MHC restriction are more difficult to prepare. However, in recent years, through the efforts of many teams, the use of tumor or viral T cell epitopes to prepare TCR sample antibodies has been greatly developed.

A number of TCR-like antibodies are obtained by combining phage display in vitro screening methods and developing appropriate immunization procedures. Phage display screening, high flux, screening pressure. Before screening, a large number of gene banks can be constructed, and the gene bank can be introduced into the phage, and the optimal antibody can be screened as much as possible.

3.1 phage display library

Phage display technology is mainly to fuse foreign genes to the surface of phage particles. Each phage particle displays an antibody. A display library is constructed and the library sequence is constructed into phage by genetic means. Each phage is shown to display a unique antibody gene. The larger the library, the more information is covered, and the more useful antibodies are available through display technology.

3.1.1 original library

Phage display screens for affinity TCR antibodies and is often not effective for therapeutic purposes. Selection of affinity TCR antibody genes, both random mutations and target mutations can increase TCR-like antibody affinity. Chames et al (2000) reported that after screening for TCR samples with affinity for tumor soluble T cell receptor (scTCR) epitopes, random mutations were made in Fab, and their affinity was increased by 18-fold by in vitro screening.

Renner et al. (2003) analyzed the TCR-like Fab 3M4E5 protein structure of HLA-A2/NY-ESO-1, identified some key amino acids that determine the affinity, and made site-directed mutagenesis at key amino acid positions without changing the helical structure of the MHC recognition site. In this case, the TCR-like Fab affinity is increased by a factor of 20.

TCR-like antibodies derived from phage display are presented as monovalent antibodies. At the same time, such antibodies can be purified in the field with higher stability in bacteria. Monovalent fragments can be used to conduct studies such as antigen screening and structural analysis. In addition, monovalent fragments can be used as targets for transposable proteins.

However, the antibody that appears in the form of monovalent has a weak affinity, especially in the research of low-density epitopes of tumors. To compensate for this deficiency, Fab or scFV is prepared in a tetrameric form, and the labeling of fluorescent molecules to the tetramer can be used to analyze the affinity for the MHC-short peptide complex.

3.1.2 immune library

Phage display immunoblot construction, this step complements phage display screening. The recombinant MHC-short peptide complex was used to immunize mice, and then mouse spleen cells were used to construct a phage display system. The mice used should use mice with their own MHC deletion and stably expressing the human HLA gene. This type of mouse is immunized with an MHC-polypeptide complex. This type of mouse can increase the rate of TCR-like antibody acquisition.

Phage screening yields a series of affinity antibody Fab fragments. The affinity Fab fragment gene information is thus obtained. Such gene sequences are cloned into a vector to prepare an antibody. What is the affinity of such antibodies? Further evaluation is needed. The ELISA method for evaluating antibody affinity is generally accepted by researchers.

TCR-like antibodies were evaluated using the MHC-peptide complex and coated by ELISA. Bhavna Verma et al (2010) used breast cancer as a model to select hCGB Ag antigen. The HLA-short peptide form was prepared by the short peptide GVLPALPQV on the Ag antigen and HLA-A2. The mice were immunized to obtain antibodies.

In evaluating the antibody, the HLA-short peptide complex was prepared as an MHC tetramer, and the tetramer was coated on the enzyme-linked plate. Different candidate TCR-like antibodies were prepared, and ELISA values were determined to evaluate different candidate TCR-like antibodies.

The MHC tetramers are cross-linking of four MHC-short peptide complexes on streptavidin. The MHC tetramer is coated on the enzyme-linked plate, and the affinity of the antibody is determined by ELISA to achieve high-throughput detection.

Anna Sergeeva et al. (2011) used mouse HLA-A2-PR1 peptide complex to immunize mice and isolate mouse spleen cells in the study of TCR-like antibodies in human acute myeloid leukemia. Monoclonal cells are cultured to prepare antibodies. The MHC-PR1 polypeptide complex was coated on an enzyme-linked plate and subjected to the same quantitative ELISA screening. Of the 2850 clones, one (8F4) affinity antibody was screened.