Recombinant monoclonal anti csf1r antibody to CSF-1R, CXIIG6, is a mouse monoclonal antibody intended for the prophylaxis and treatment of related disease.
Tuesday, September 3, 2019
Sunday, August 4, 2019
anti mouse cd3
The anti-CD3 monoclonal antibody specifically target CD3 and can induce downmodulationg of the TCR/CD3 complex in mouse CD4+ T cells without activating T cells. Thus, the specific antbody can be used for suppressing immune responses and inducing immunosupression.
Specifications
- Host
- Rat
- Specificity
- Mouse
- Clone
- Dow2
Target
- Alternative Names
- CD3; CD3 T-Cell Co-receptor;CD3D; CD3E; CD3G
- For lab research use only, not for diagnostic, therapeutic or any in vivo human use.
Thursday, July 11, 2019
cd14 mouse
This antibody is a mouse monoclonal antibody that binds specifically to CD14, and it can neutralize the bioactivity of CD14.
For lab research use only, not for diagnostic, therapeutic or any in vivo human use.
Friday, June 21, 2019
Principle of Humanization of Antibodies and Classification of Humanized Antibodies
The entry of non-human antibodies into the human body can cause serious body rejection, which in turn affects the safety and therapeutic effect of the antibody in clinical application. Therefore, it is necessary to humanize the antibody to minimize the heterogeneity of the antibody. And keep its specificity and affinity unchanged.
Each variable region of the antibody contains three amino acid sequence hypervariable regions, which are binding sites of the antigen and are complementary to the structure of the antigenic determinant, and are referred to as antibody complementarity determining regions (CDRs), amino acid sequences and spaces of the CDRs. Polymorphism in the structure is a factor determining the heterogeneity and affinity of antibodies. Other amino acids in the variable region serve as a backbone support moiety, called Framework Residue (FR), which is not in direct contact with the antigen. The amino acid sequence and spatial structure are relatively conservative, providing a skeleton for maintaining the typical three-dimensional structure of the antibody, indirectly affecting the antibody. The specificity and affinity of the antibody constant region and the antibody variable region FR are relatively conservative during the evolution process, and the species are specific to the species, which is the main factor causing the body to reject the reaction. Therefore, the basic principle of humanization antibody is to preserve the conserved sequence of the antibody as a human sequence, reduce the body rejection reaction, replace the antigen-binding region with the sequence of the antibody produced by the animal immunization, and maintain the specificity and affinity of the antibody.
Humanization of antibodies requires the use of genetic engineering methods. Therefore, humanized antibodies belong to genetically engineered antibodies, and humanised antibodies can be classified into chimeric antibodies, modified antibodies, and fully humanized antibodies depending on the degree of antibody sequence modification.
Modified antibody
The modified antibody is also called CDR grafting antibody, and the CDR grafting method is used for preliminary humanization of the antibody, that is, the 6 CDR regions of the non-human antibody are moved to the human skeleton, due to the human FR skeleton. The region will indirectly affect the specificity and affinity of the antibody. Based on the CDR grafting, the individual amino acid residues in the human FR framework region are further adjusted to further improve the specificity and affinity of the CDR-grafted antibody.
Full human antibody
The entire sequence of the fully human antibody antibody is a human sequence, which is realized by the antibody library technology. The main antibody library technology is a phage display and ribosome display technology.
Antibody Library Technology Basic Approach to Obtaining Fully Humanized Antibodies: Selecting a Variable Region Light Chain (or Heavy Chain) Gene of a Parent Mouse Monobody to Pair with a Variable Region Heavy Chain (or Light Chain) Gene Library of a Human Antibody The mouse-to-human hybrid antibody library is constructed, and the corresponding antigen is used to select a clone capable of producing a specific binding antibody, thereby obtaining an Fv fragment capable of binding to the parent mouse monoclonal antibody and having specific binding ability to the composition. Human heavy chain (or light chain) variable region gene, combined with another human antibody variable region light chain (or heavy chain), construct a human antibody library, and again use antigen screening to obtain specificity and mouse The human antibody with the same source parent antibody is identical.
Sunday, May 19, 2019
Common antibody drug development strategy
Monoclonal antibodies are the most shining and mature technology in the field of modern biomedicine. Whether it is tumor targeting drugs, immunological checkpoint blocking drugs, or the hot market CAR-T technology, it is based on monoclonal antibodies and their downstream basis. Above. In general, a humanized mouse monoclonal antibody represented by a murine monoclonal antibody, after demonstrating its pharmaceutical value, requires a series of engineering modifications to improve its pharmacokinetics in vivo. Common antibody engineering strategies include antibody humanization, antibody affinity maturation and FC modification, or reduced antibody aggregation.
Humanization of antibodies
Mouse monoclonal antibody is the first monoclonal antibody to enter the clinic, but early clinical application found that because mouse monoclonal antibody is a heterologous protein, it will gradually cause HAMA response in clinical use, and the infused mouse monoclonal antibody will gradually be Neutralizing antibodies were neutralized, so that the murine monoclonal antibody was ineffective after infusion. Grafting the CDR regions of the murine monoclonal antibody humanization into the FRAME WORK region of the human antibody by antibody engineering techniques can effectively reduce the HAMA response induced by the antibody in clinical use. This approach is more advantageous than human-chimeric chimeric antibodies in terms of humanization. In fact, the most widely used antibody drugs in clinical practice are humanized mab or human and mouse chimeric antibodies.
With the development of bioinformatics, antibody humanization technology has been rapidly updated, and it is usually possible to recognize that some of the CDRs of the antibody antigen-binding region are grafted into the framework region of human antibodies, and some also have mouse anti-CDR regions and framework regions. The surface residues are replaced to be closer in sequence to human antibodies; in addition, the key amino acid sequence of the antibody antigen recognition region is replaced by the antibody antigen recognition region of the human antibody. In addition, different strategies have been proposed, such as CDR compensation, positioning reservation, and template replacement.
At the same time, full-human antibodies obtained by phage display library technology, or fully human antibodies expressed by humanized mice have gradually become an important direction in the field of antibody research and development, but different strategies have certain advantages and disadvantages in evaluating patients. The risk-return rate should always be considered when considering this issue.
Different companies have roughly similar technical pathways in the field of antibody humanization, but how to obtain the most humanized monoclonal antibodies while maintaining their high affinity for target proteins, for each company's technicians It is a big test. The technical team of Elken Bio has accumulated considerable experience in the field of antibody humanization. Based on years of experience in antibody drug research and development, it has provided antibody humanization services for many pharmaceutical companies. Some antibodies have entered the clinical trial stage. .
In vitro affinity maturation
Usually, the murine monoclonal antibody has a good affinity for the target antigen, but the affinity of the antibody obtained by humanization or by the phage display library technology to the target antigen may be unsatisfactory, which is not conducive to reducing the clinical use of the therapeutic antibody. The amount and toxic side effects, at this time the affinity maturation of the antibody, will contribute to future clinical use. The theoretical basis for antibody affinity maturation in vitro is the process of mimicking the affinity of antibodies in vivo. High-affinity antibodies can be screened by constructing a random mutagenesis library that mimics high-frequency mutations in B cells in vivo. In fact, in the course of monoclonal screening, affinity-maturation of any one of the antibodies results in an antibody with improved affinity.
Fc modification enhances effector function and half-life
FC receptors play an important role in the process of cellular immunity, and the modification of the FC region can produce profound effects on monoclonal antibodies with specific pharmacological effects. Monoclonal antibodies can mediate immune responses via Fcγ receptor binding (FcγR). The binding of different types of FcγR to IgG enhances/inhibits the immune response, and therefore preferentially enhances binding to FcγRIII while reducing binding to FcγRIIB to enhance clinical efficacy. At the same time, the half-life of the antibody can be prolonged by modification of the Fc. Since the binding of the antibody to FcRn is a pH-sensitive form, the antibody enters the cell by pinocytosis, in the endosome of acidic pH, the antibody binds to FcRn, and the FcRn mediates the antibody back to the extracellular, in extracellular neutral pH conditions. Next, dissociate from FcRn. In this way, FcRn avoids the fate of other proteins in the blood to be degraded by lysosomes into the cells through the pinocytosis, thereby achieving long-term effects, and the half-life is as long as several weeks (IgG1, IgG2, IgG4 half-life three weeks, IgG3 is 9 days). A number of pharmaceutical companies have attempted to modify antibodies to specifically increase the affinity for FcRn under low pH conditions for further long-acting effects. Although Fc modifications to enhanced cycle time may affect the effector function of Fc, studies have shown that mutations in different AAs of bevacizumab and cetuximab lead to more potent antitumor activity in mouse cancer models, Thus a strategy to increase the half-life of therapeutic antibodies was verified. Since then, a large number of favorable mutations have been reported. Unfortunately, the results of animal models are not always related to humans. However, an increase in affinity with FcRn by a factor of 5-10 usually results in a 2-4 fold increase in half-life.
The binding of the antibody to FcRn is a pH-sensitive form, and the antibody enters the cell by pinocytosis. In the acidic pH endosome, the antibody binds to FcRn, and the FcRn mediates the antibody back to the extracellular, under extracellular neutral pH conditions. , dissociation from FcRn
Improve stability and reduce polymerizability
Since the production of therapeutic antibodies is free from the physiological environment of the body, its thermal stability and stability of the colloids are limited, which in turn causes certain problems in the production and preservation of the drug. By technical means, improving the physical and chemical properties of the antibody helps to improve the overall efficacy and productivity of the antibody. By altering the antibody framework structure, the antigen binding domain of the antibody helps to reduce the polymerization of the antibody. It is usually possible to reduce the polymerization of the antibody by: 1. by changing the antibody dosage form; 2 by changing the framework and CDR regions, 3; adding additional disulfide bonds to the CH2-CH3 region; 4, changing the Fab The disulfide bond structure of the region; 5 increases the fusion tag with dots. In fact, with the development of computer simulation technology, the polymerization of antibodies can be offset by different designs and in vitro screening methods, and the chemical degradation rate and serum clearance rate of antibodies can be predicted.
An Overview of Major Histocompatibility Complex(MHC)
The major histocompatibility complex is a collective term for a group of genes encoding the major histocompatibility antigens of animals. The human MHC is located on the short arm of human chromosome 6, and the mouse MHC is located on chromosome 17 of the mouse. The length of the MHC is approximately 4 x 10^6 bp. The human MHC is also called the HLA complex. The MHC of mice is called the H-2 gene. Due to the polygenic nature of MHC, it can be divided into MHC class I, MHC class II, and MHC class III genes, which encode MHC class I molecules, MHC class II molecules, and MHC III, depending on the structure, tissue distribution, and functional differences of the coding molecules. Class of molecules. The human MHC product is commonly referred to as HLA, the human leukocyte antigen.
MHC molecule
1. Types of MHC molecules
Different MHC-encoded products have different functions.
MHC class I (MHC I): Located on the surface of a general cell, it can provide some conditions in general cells. For example, if the cell is infected with a virus, the amino acid peptide of the outer membrane fragment of the virus is prompted to pass through the MHC to the outside of the cell. Can be identified by killer CD8+ T cells for culling.
MHC class II: only located on antigen-presenting cells (APC), such as macrophages. This kind of supply is external to the cell. If there is bacterial invasion in the tissue, the macrophage will be swallowed, and the bacterial fragments will be prompted by the MHC to help the T cells to initiate the immune response.
MHC class III: mainly encodes complement components, tumor necrosis factor, heat shock protein 70 and 21 hydroxylase genes.
2. Physiological significance of MHC molecules
MHC antigens were originally discovered as transplant antigens and are the major antigenic systems responsible for transplant rejection. This antigen is incompatible, which can cause the immune response of the receptor and reject the transplanted donor tissue. After the 1970s, MHC molecules also proved to have important immunophysiological functions.
MHC molecules are involved in antigen recognition during the immune response. In the 1970s, RM Zinkner Zeer and other mice found that killer T cells can kill the target cells infected with the same cells when killing the target cells infected with the virus, but have no killing effect on the infected target cells of different lines. The phenomenon is genetically restricted. It was subsequently confirmed that the killer T cells must be consistent with the MHC of the target cells to have a killing effect, so this phenomenon is also called MHC restriction.
This reveals the role of MHC in T cell recognition of heterologous antigens. Further studies have shown that T4 T cells are restricted by MHC class II molecules when recognizing heterologous antigens, while T8 T cells are restricted by MHC class I molecules when recognizing xenoantigens. This restrictive mechanism is that T cells, through their antigen recognition receptors, can simultaneously recognize new complex antigenic determinants formed by heterologous antigenic determinants and their own MHC molecules.
It has also been found that non-T cells such as peripheral blood B cells and monocytes can induce proliferative responses in certain autoreactive T cells in vitro, which is called self-mixed lymphocyte reaction (AMLR) and proves that this is a non- Caused by MHC class II antigens on T cells. Such autoreactive T cells may have an effect of enhancing or inhibiting immune function in vivo, thereby maintaining the immune stability of the body, and thus MHC molecules are also involved in immunomodulatory effects.
Studies have shown that MHC molecules also play an important role in the differentiation and maturation process of T cells in the thymus. In vitro studies have found that the removal of MHC class II antigen-positive stromal cells in the thymus inhibits the development of T4T cells, and the addition of monoclonal antibodies against MHC class II antigens in thymic culture cells can also prevent the development of T4 T cells. It is currently believed that MHC molecules play an important role in the formation of T cell self-tolerance and the production of T cell pools.
MHC gene
The understanding of the discovery, gene composition and function of MHC is based on mouse experiments. Therefore, it has been determined since the 1930s that the mouse MHC is located on chromosome 17, called the H2 complex. The H2 complex is composed of K region, I region, S region and D region, wherein the I region is further divided into two sub-regions, IA and IE, and the gene-encoded product is called the I region-associated antigen.
In 1958, Dausset et al found that there were different specific leukocyte antibodies in the serum of patients who received blood transfusions, multi-partum women and volunteers immunized with the same kind of leukocytes. These antibodies were used to identify many different specific white blood cell antigens. Human leukocyte antigen. Through genetic analysis of families and populations, it was found that human MHC is located on chromosome 6, called HLA complex.
All vertebrates have their own MHC. In addition to human HLA and mouse H2, the MHC of rhesus monkeys, chimpanzees, dogs, rabbits, guinea pigs, rats and chickens are called RhLA, ChLA, DLA, RLA, GpLA, AgBI (H-1I) and B.
In 1980, the Nobel Prize in Physiology or Medicine was awarded to Baruj Benacerraf, George D.Snell and Jean Dausset (three people). The study laid the foundation for the establishment of transplant immunology. Benaselav was an American medical scientist and immunologist who discovered immune response genes in MHC Tetramer when studying organ transplant rejection. Ir), pointing out that the immune phenomenon is controlled by this gene, and the immunology is pushed to the climax on the basis of genetics.
Snell is an American immunologist who proposed through tissue transplantation experiments in mice: The transplantability of tissues between different individuals is determined by specific antigens on the cell surface, ie histocompatibility antigen (also known as H antigen). , controlled by the H gene. This gene is present in a limited area of a chromosome called the major histocompatibility complex (MHC). Dosser, a French immunologist, discovered the human leukocyte antigen (HLA) and the HLA gene that determines these antigens, the H gene equivalent to mice; it also confirmed that humans and many other animals have MHC.
(1) Polygenicity: A gene complex consists of a plurality of closely adjacent gene loci whose encoded products have the same or similar functions.
For example: mouse H-2: chromosome 17: short arm of chromosome 6 (6P21.31), full length 3600-4000 kb, 224 gene loci (128 functional genes, 96 pseudogenes).
(2) Polymorphism: There are two or more alleles in the same HLA gene locus in the population.
The significance of MHC polymorphism:
1. Expanding the population's presentation of antigenic peptides is conducive to maintaining the survival and continuation of the population.
(The polymorphism of HLA products is mainly manifested in the difference in composition and sequence of amino acid residues in the antigen-binding groove)
2. It is not conducive to the choice of donors in organ transplantation.
It has been shown that MHC not only controls allograft rejection, but more importantly, it is closely related to the immune response, immune regulation and the production of certain pathological states. Thus, the complete concept of MHC refers to a group of closely linked genes that encode major histocompatibility antigens on a chromosome of a vertebrate, control mutual recognition between cells, and regulate immune responses.
Combination of TLR7 Agonists and Neutralizing Antibodies Can Kill Latent HIV Virus Libraries
Given that more than 35 million people worldwide are infected with HIV and nearly 2 million new cases of HIV infection each year, the virus remains a major global epidemic. Existing antiretroviral drugs (ART) do not cure HIV infection because the virus can enter a dormant state and persist in the presence of immune cells. These infected immune cells (called latent virus banks) -- despite the use of ART drugs, remain in a latent state -- can be active again at any time.
Dr. Dan H. Barouch, director of the Virology and Vaccine Research Center at the Beth Israel Deaconess Medical Center, said, "This latent virus library is a key barrier to the development of a cure for HIV-1 infection. There is a hypothesis that activation of these latent viral pool cells may Make them more vulnerable to damage."
In a new study, Barouch and colleagues demonstrated that the combination of a broadly neutralizing agonistic antibody (bNAb) targeting HIV and a Toll-like receptor 7 (TLR7) agonist that stimulates the innate immune system can delay HIV cessation. Monkeys taking ART drugs rebounded. These findings suggest that this two-pronged approach represents a potential strategy for targeting this virus pool. The results of the study were published online October 3, 2018 in the journal Nature, entitled "Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys".
Barouch and colleagues studied 44 rhesus monkeys infected with HIV-like virus (SHIV) and started treatment with ART for two and a half years after infection. After 96 weeks, these rhesus monkeys were divided into four groups. One group, the control group, did not receive any further study treatment. The other two groups were given only TLR7 antibody agonists or only bNAb antibodies. The fourth group was given both a TLR7 agonist and a bNAb antibody. All rhesus monkeys continued to receive ART medication until the 130th week of stopping the treatment, at which time the researchers began monitoring whether the rhesus monkeys showed signs of a rebound in SHIV.
As expected, in the control group, all rhesus monkeys rapidly rebounded with SHIV and they had a relatively high viral load peak, while in rhesus monkeys given only this TLR7 agonist, almost all The same is true for rhesus monkeys. However, among the rhesus monkeys receiving this combination therapy, 5 of the 11 rhesus monkeys did not rebound from the SHIV virus within 6 months. In addition, another 6 rhesus monkeys with SHIV virus rebound showed lower viral load peaks than rhesus monkeys in the control group. Rhesus monkeys given only bNAb antibodies showed detectable SHIV virus rebound, but this virus rebound was delayed.
"The combination of bNAb antibody and TLR7 agonist has led to the best killing of immune cells infected with SHIV," Barouch said. In summary, our data show that this combination therapy stimulates the innate immune system and allows infected cells to become more A mechanism that is easy to remove. T
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