Transformation Made to Therapeutic Antibodies

Today, more than 40 therapeutic antibodies in the United States and Europe have been approved for clinical use, demonstrating its value in cancer, autoimmunity, transplantation and other indications. Most of them are monospecific full-length IgG1 antibodies. However, at least 100 bispecific antibodies are in preclinical and clinical development. When developing therapeutic antibodies, scientists must not only ensure that it has the highest affinity with the target, other factors are also critical, including antibodies that can elicit an immune response and avoid off-target effects.

What is an antibody?

It is clear to everyone that a typical IgG antibody is a Y-type structure consisting of two arms (Fab region) and a bottom region (Fc region). An IgG consists of four antibody sequencing amino acid chains, a pair of identical heavy chains extending from the bottom to two arms; a pair of identical light chains, one on each arm. The complementarity determining regions (CDRs) are at the front end of the two arms; they are responsible for recognizing and binding to the antigen. Both CDRs recognize the same antigen, which makes the antibody bivalent, monospecific.

The Fc region binds to natural killer (NK) cells, macrophages, neutrophils, and Fc receptors on other cells in the immune system. This, in turn, is responsible for inducing phagocytosis, cytokine release, antibody-dependent cell-mediated cytotoxicity (ADCC), and other downstream effects. Similarly, binding of the Fc region to a particular Fc receptor (FcRn) protects the IgG from degradation, thereby extending the half-life of the antibody.

Humanized transformation

Typically, monoclonal antibodies for research are made by injecting mice with antigen. The antibody-producing cells are collected, fused with myeloma cells to form a hybridoma, and the extent to which the antibody recognizes the antigen is screened. However, such antibodies, if used for treatment, will be considered by the human immune system to be foreign proteins.

"We produce our own antibodies to drug antibodies, so the concentration of the drug must be reduced," explains David Bramhill, founder of Bramhill Bio Consulting. "Only three of the current market are pure mouse antibodies." Most therapeutic antibodies are "chimeric" or "human", meaning that the variable region (or CDR) of the mouse Fab is cloned into The backbone of a human antibody, or sequence, is altered so that it is not rejected by the human immune system.

However, the disadvantage of this approach is that most antibodies undergo a process of central tolerance, ie, the animal recognizes and removes autoreactive antibodies. "If you compare human and mouse proteins, you will find that important regions are often homologous. So you may lose a lot of therapeutically effective epitopes," Paul Kang, Chief Scientific Officer, Innovative Targeting Solutions (ITS) Point out.

ITS uses a system to produce antibodies in which all genetic elements are engineered by human embryonic kidney cells (HEK). "We amplify cells and induce recombination, so each cell undergoes a unique V(D)J recombination reaction, displaying antibodies on the surface, just as it happens in the body," Kang said. This produces billions of cells, each expressing a unique antibody without being centrally tolerated.

Today, there is another concept in antibody engineering: developability, meaning that they not only have the right combination of biological properties, but are also manufacturable. Residues that may oxidize or glycosylate antibodies during manufacture should be removed. These antibodies are designed to provide better stability, better folding, and higher yields. Using fed-batch culture, the yield is generally 2-5 g/L.

The part that can be engineered in the antibody is the Fc region. All four IgG subtypes have different interactions with different Fc gamma receptors, and have the ability to activate and inhibit the function of these receptors. Researchers are trying to exchange or mutate a part of these areas. Similarly, antibody re-use can be adjusted by engineering regions that interact with FcRn.

Some tumors and pathogens produce proteases that cleave the hinge structure of the antibody, making it less able to induce ADCC or complement cascades. Therefore, it is necessary to modify this region of anti-tumor or anti-bacterial antibodies so that it is not susceptible to proteolysis, Bramhill said.

Of course, the therapeutic affinity reagent does not have to be a standard IgG. Currently, at least three Fab segments are licensed. They block receptors but do not cause Fc-related effector functions. In one approach, a Fab2 fragment (two Fab regions are chemically linked, but no Fc) can be used to crosslink the target. The two Fab regions do not need to recognize the same epitope. This bispecific antibody recognizes both tumor-associated antigens and T cell receptors. In addition, an antigen recognition domain can be added to make it a trivalent antibody.

There are many goals that people need to target. “The key is to find the wonderful 'best combination,'” Kang said. The combination of drugs, mono- and bispecific antibodies, and effector functions is really a specific analysis of the specific problem, and the situation is different.

Other related product:

humanized antibody

Q & A about De Novo Sequencing II

Q: What factors can affect the quality of sequencing results?

A: (1) Individual heterozygosity: The higher the individual heterozygosity, the more difficult the splicing is, and may even lead to the splicing of the sequence.

    (2) Polymorphism of the species genome: Since the individuals of some species are too small, the amount of genomic DNA extracted by a single individual may be difficult to meet the sequencing requirements (such as some parasites), so it is necessary to mix multiple individuals for genomic DNA pumping. Used for sequencing. For such cases, the polymorphism of the genome of the species needs to be assessed, and if the polymorphism of the genome is too high, it will affect the splicing of subsequent genomes.

    (3) Quality of DNA samples: For bacteria and fungi, the source of the sample must be single colony free of contamination, and the animal and plant samples should be as homozygous as possible, and there is no pollution, otherwise the quality of the sequencing results will be seriously affected. In addition, the prepared genome can not be less than 23Kb. If the fragment is too small, small fragments are easily lost during the process of genomic fragmentation, resulting in the complete sequencing of the constructed sequencing library, which has a significant impact on the sequencing results.

    (4) In addition, if the GC content of some regions of the genome is too high (GC% ≥ 65%), the bias will occur during the sequencing process, resulting in too low coverage in some areas, thus affecting subsequent splicing and annotation.

(5) For species with too many repeats, the presence of a large number of repeats creates many erroneous overlaps, causing the contigs produced by splicing to be too short, resulting in severe deviations in the results.

Q: How is the genome assembled?

A: In general, the assembly strategy based on Roche 454 FLX+ sequencing results is as follows:

     (1) First use the short sequence assembly software to de novo sequencing splicing the paired-end data and assembling it into contigs. This stage generally needs to provide paired-end sequencing data with high coverage, which requires a lot of computer memory, which is also the most genome assembly. a difficult step;

     (2) Gradually add the mate-pair data of the long insert to build the scaffold. In general, the sequencing depth of the mate-pair is not too high. The contigs are connected to a larger scaffold by the mate-pair double-end distance information.

     (3) Review the paired-end and mate-paired insert length information to fill the gap;

     (4) Sometimes adding Sanger data will greatly help fill gaps and extend contigs.

Q: What are the common contents of comparative genomic analysis?

A: Comparative genomics refers to the comparison of known genes and gene structures based on genomic maps and sequence analysis to understand the functions of genes, the mechanisms of expression regulation and the evolution of species. Generally include the following aspects:

    (1) Pairwise genome alignment with closely related species. The sequence and structural homology between the two genomes can be utilized to map the genes in the other genome by mapping information of the known genome, thereby revealing the potential function of the gene and the changes in the internal structure of the genome.

(2) Multi-genomic alignment with closely related species. When sequence comparisons are made between two or more genomes, the evolutionary relationship of the sequences in the phylogenetic tree is essentially obtained. The increase in genomic information makes it possible to study molecular evolution and gene function at the genomic level. By studying a variety of biological genomic data and its vertical evolution and horizontal evolution process, we can understand the structure and regulation of genes.

De Novo sequence is also called de novo sequencing, and a species can be sequenced without any genetic sequence information. The sequence is spliced and assembled by bioinformatics analysis to obtain the genomic sequence map of the species. It is currently widely used to denovo sequencing analyze the genomic sequence, gene composition, and evolutionary characteristics of unknown species.

Q & A about De Novo Sequencing I

Q: What are the advantages of next-generation sequencing technology over traditional Sanger sequencing?

A: Sanger sequencing took 13 years to complete the first human genome map, and today the next-generation sequencing technology platform can be completed in just a few months. The next-generation sequencing technology platform uses a large number of parallel processing capabilities to read multiple short DNA fragments and then stitch them into longer contig and scaffold. The technology is more advanced, with advantages of high accuracy, high throughput, high sensitivity and low operating cost.

Q: What do contig, scaffold, N50, etc. represent from scratch?

A: Read: the length of the nucleotide read by the instrument in one sequencing

Contig: A unit formed by assembling adjacent reads by overlapping portions is called contig.

Scaffold: Using information from other methods such as double-end sequencing, locates the linear or relative positional relationship of contigs on chromosomes and joins them to form longer scaffold sequences.

N50: Sorts contig or scaffold from large to small and accumulates their length. When the accumulated length reaches half the length of the genome sequence, the last contig or scaffold length.

Q: What is the frame picture, the fine picture, and the completed picture in the de novo antibody sequencing?

A: The frame diagram refers to the genome coverage greater than 95%, the coverage of the gene region is over 98%, the contig N50 reaches 5Kb, the scaffold N50 reaches 20Kb, and the single base error rate is 100,000. One or less.

     The fine map refers to the genome coverage greater than 98%, the coverage of the gene region is over 99%, the contig N50 reaches 20Kb, the scaffold N50 reaches 300Kb, and the single base error rate is less than 100,000 points after bioinformatics analysis. One of them, the number of gaps does not exceed 100.

     The completion map refers to the complete genomic sequence obtained by bioinformatics analysis, and the single base error rate is less than one in 100,000.

Q: What are the requirements for whole-genome sequencing for DNA samples?

A: The OD value of DNA should be between 1.8 and 2.0; the higher the concentration of the sample, the better, the minimum should not be less than 200 ng / ul; the total amount of sample DNA is not less than 10 ug.

Q: What are the requirements for sampling the sample by animal genome sequencing?

A: Samples should be extracted from areas with low fat content such as muscle and blood. Try to use the same individual to sample. If the species size is small, the amount of DNA extracted by a single body is less than one sequencing reaction. In the case of guaranteed amount, the number of species should be minimized to reduce the influence of individual differences on subsequent splicing.

Q: Should be 454 or solexa suitable to De Novo igg sequencing?

A: (1) Both sequencing methods can complete the De Novo protein sequencing splicing of the genome. It is recommended to use two sequencing methods, and the results can be combined and spliced to reuse the advantages of the two technologies.

     (2) According to the situation of the genome, if the heterozygosity is high and the repeat sequence is too high, it is recommended to increase the proportion of 454 sequencing.

     (3) If the genome is relatively simple, it is recommended to increase the Solexa sequencing ratio from the cost considerations.

     (4) 454 is mainly a cost and flux limitation. Solexa is mainly unable to overcome short tandem repeats above 150 bp.

     (5) 454 can't test short tandem repeats above 700bp. If this level of repeats causes a splicing bottleneck, Sanger data needs to be added.

...

Application of Phage Display Technology in Antibody Drug Development

The 2018 Nobel Prize in Chemistry was settled and awarded to Frances Arnold, George Smith and Gregory Winter. Half of the prize for the Chemistry Prize was awarded to American scientist Frances Arnold, who rewarded her work for the directed evolution of the enzyme; the other half was awarded to British scientist Gregory Winter and American scientist George Smith to reward their phage display technology in peptides and antibodies. Great achievement.

The development of science and technology is the driving force for drug research and development. Smith et al. have developed phage display technology, which has made breakthroughs in the research and development of antibody drugs. Phage display technology has become one of the most important drug screening platforms. It can be used not only for the screening of peptides, monoclonal antibodies, but also for the screening of other proteins. The diversity of screening is also one of the highlights of this phage display library construction protocol.

So, what is phage display technology? The technology was developed by Smith in 1985, and Winter continued to develop the technology. The tools they used were genetically engineered phage, a virus that infects bacteria, as a host. The specific process is shown in the following figure:

Phage display technology schematic

A. Genetic engineering inserts the DNA sequence encoding the polypeptide or protein library into the coat protein gene of the phage, so that the polypeptide or protein can be expressed on the surface of the phage; B. The phage is transformed into the host cell, and the mature phage is released from the host come out. 1. Capturing a phage capable of specifically binding to a target protein with a plate immobilized with a target protein; 2. Washing out the phage not bound to the target; 3 eluting the phage bound to the target for the next round of transformation; Carry out the next round of infection and transformation; 5. Further enlarge the number of target phage. Thus, by repeating the cycle, the desired high affinity antibody can be obtained.

So, what are the benefits of doing this? First, not all protein epitopes expressed in the library are useful, so that the epitopes of interest can be screened to ensure optimal activity of the drug candidate. Second, in vitro, human and non-human sources can be screened. Target, this saves time in preclinical testing.

At present, a variety of antibody drugs have been successfully marketed using this technology. Adalimumab monoclonal antibody is one of the best examples. The first fully human recombinant antibody IgG1: κ monoclonal antibody successfully developed has been the best-selling drug in recent years. At the top of the list, in 2017, its sales were $18.4 billion. In addition, a number of drugs using this technology have been successfully marketed, and a number of drugs to be marketed are in clinical trials. Let us know about these successful drugs.

Adalimumab

Adalimumab is a fully human recombinant IgG1-κ monoclonal antibody, the first approved anti-tumor necrosis factor TNFα drug for the treatment of rheumatoid arthritis in the United States in 2002. Adalimumab was screened using the "Guided Selection" of phage display technology. This step was divided into two steps. First, the researchers developed the murine anti-human TNF antibody MAK195, but the murine antibody could not be used as an autoimmune disease drug, so they used this mouse antibody to guide the isolation of people with the same epitope as MAK195. Source antibody. The paired human DNA sequences were found in the protein pool using the heavy and light chains of MAK195, and then further screened using phage display technology to obtain high affinity anti-TNF antibodies. This formed the Adalimumab mAb.

Belimumab

Belimumab is a human IgG1 lambda monoclonal antibody. Developed by Cambridge Antibody Technology and Human Genome Sciences (GlaxoSmithKline), it was approved for marketing in 2011 for the treatment of systemic lupus erythematosus and was the first targeted drug for the treatment of systemic lupus in more than 50 years. Belimumab binds to B lymphocyte stimulating factor (BLyS), prevents BLyS from binding to B lymphocytes, and promotes apoptosis of B lymphocytes. The researchers screened 1,200 antibodies using single-chain variable fragment (scFv) phage display technology to obtain higher affinity Belimumab. Preclinical studies have shown that this drug can inhibit the growth of B lymphocytes in cynomolgus macaques. Clinical trials have shown that Belimumab can effectively improve the patient's condition.

Ranibizumab (Lucentis®)

Ranibizumab, developed by Genentech, is an antigen-binding fragment (Fab) that binds to VEGF-A and inhibits its activity. The murine antibody A4.6.1 has a good application in the mouse tumor model, and the antitumor drug Bevacizumab (Avastin®), which is also a candidate compound for the mutation site of A4.6.1, was obtained. Clone Y0317 (also known as Ranibizumab) has an affinity for VEGF-A of 0.1 nM, contains only 6 mutations different from the parent, and increases the affinity for VEGF by more than 100-fold, greatly reducing the dose. The drug was approved for marketing in 2006 for the treatment of age-related macular degeneration and was approved for the treatment of diabetic macular edema in 2010 and was approved for the treatment of diabetic retinopathy in 2015.

Ecallantide (Kalbitor®)

Ecanllantide is composed of 60 amino acids and is a recombinant kallikrein protein inhibitor. Ecallantide was discovered using a phage display library constructed from the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1) as a scaffold. The drug was approved for marketing in 2009 for the treatment of hereditary angioedema (HAE), a rare hereditary disease.

Romiplostim (Nplate®)

Romiplostim contains two identical subunits, each consisting of an IgG1 Fc structural region and a c-Mpl binding region covalently bound, combined with a platelet-producing receptor (TPOR), approved by the US FDA in 2008. The drug is the first FDA-approved peptide drug and the first and only platelet-generating drug.

Raxibacumab (Abthrax®)

Raxibacumab is a human monoclonal IgG1 lambda antibody used to treat and prevent diseases caused by Bacillus anthracis infection. Raxibacumab binds to the protective antigen of Bacillus anthracis and prevents the release of bacterial toxins. The drug was developed by Human Genome Sciences and tested to show that Raxibacumab significantly increased the survival of rabbits and monkeys.

Necitumumab (Portrazza®)

Necitumumab was developed in collaboration with ImClone Systems, Eli Lilly and Bristol-Myers Squibb using the "de Haard" Dyax Fab phage display technology library, which targets the epidermal growth factor receptor (EGFR). During the development of the drug, epidermal-like cancer cells were used as targets for antibody screening, and the affinity of Necitumumab for the cells was 3.3 ± 0.5 nM. In 2015, Necitumumab was approved for the treatment of squamous non-small cell lung cancer in combination with gemcitabine and cisplatin.

Ramucirumab (Cyramza®)

Ramucirumab is also screened from the deHaard Fab phage display technology library, VEGFR2 drug, Ramucirumab is a human heavy and light chain amplified by PCR using a library of non-immunized phage display technology of human Fabs. In preclinical studies, the drug showed excellent antitumor activity, and it was shown in clinical III trials that the drug has a significant inhibitory effect on a variety of tumors. It was approved by the US FDA in 2014 for the treatment of advanced gastric cancer or adenocarcinoma of the gastroesophageal junction.

Conclusion

In addition to the above listed drugs, there are more drugs in clinical trials. In the screening of antibody drugs and protein drugs, phage display technology has shown great advantages, improved the success rate of drug development, and also saved the time and cost of drug screening.

Shotgun Protein Sequencing Method

Introduction

The most common method for obtaining a gene of interest directly from the genome of a biological cell is the "shotgun method", also known as the "shotgun method." The "shotgun method" is to separate the genomes by chromosomes, scramble them all, cut them into pieces, perform random sequencing, and then splicing them together after sequencing. It has the advantage of being fast and in a short time to get the genome of a biological cell (for example, 95% of the human genome). The idea of the law is unique. It seems that a large group of birds have been parked in the woods. Many people shoot and shoot, and in a short period of time, most of the birds in the forest can be hit. The "shotgun" is a bit like a jigsaw puzzle played by people. The jigsaw puzzle divides a complete picture into disorganized pieces and then reassembles them. The "shotgun method" first scrambles the entire genome, cuts it into random fragments, then measures each small fragment sequence, and finally uses a computer to sort and assemble the slices and determine their correct position in the genome.

The shotgun protein sequencing method is a sequencing method in which the target DNA is randomly processed into fragments of different sizes, and the sequences of these fragments are connected together, belonging to the first generation sequencing technology.

Application

The "shotgun method" was originally used primarily to determine the microbial genome sequence. In recent years, the biological genius Craig Venter and its company, Celera, have used the improved genome-wide “shotgun method” to complete the sequencing of the fruit fly and human genome, demonstrating its ability to determine large genomes. Feasibility and effectiveness

Features

Advantages

The advantage of shotgun method is that it is fast, simple and easy to operate, and the cost is low. But it is not easy to use for sequencing service, and the final assembly result is not easy. Chinese scientists have designed a sequence assembly software that can effectively overcome the "shotgun method". Difficulties in the whole genome sequencing monoclonal antibodies assembly process.

In the study, they first generated a number of DNA (deoxyribonucleic acid) slices of known length across the entire rice genome and then arranged them in overlapping regions of the DNA sequence. The number of these slices is sufficient to cover the rice genome 4 times. The scientists then determined the base pair sequence for each slice and assembled it into longer pieces using a computer program, then sorted and assembled the pieces into more than 100,000 larger components called stents.

The software they designed focused on assembly at the level of the scaffold, and adopted a unique repetitive sequence processing algorithm that identified and temporarily blocked approximately 40% of the repeats of the rice genome. This has the advantage of reducing the amount of computation and minimizing the possibility of false stitching.

Bennett Zeen, a plant geneticist at Purdue University in the United States, commented that the Chinese scientist's rice genome project "provides an excellent example of the speed and efficiency of shotgun sequencing."

Limitation

When eukaryotes express genes, introns in structural genes cannot guide protein synthesis. Therefore, the target gene of eukaryotes cannot be obtained by the "shotgun method".

The "shotgun method" is a method of extracting a gene of interest from a biological genome. First, cleavage of biological cell chromosomal DNA into many fragments at the gene level using physical methods (such as shear, ultrasound, etc.) or enzymatic chemistry (such as restriction endonucleases), and then combining these fragments with appropriate vectors, The recombinant DNA is transferred into a recipient strain to obtain a gene library of asexual reproduction, and a strain containing a certain gene is selected from a plurality of transformant strains, and the recombinant DNA is separated and recovered therefrom.

This method is to use genetic engineering technology to isolate the target gene, which is characterized by bypassing the difficulty of directly isolating the gene and screening the target gene in the genomic DNA library. It can be said that this is to use the "slack shot" principle to "hit" a certain gene. Because the target gene is too small and too small in the whole genome, and to a certain extent depends on "take a chance", people call this method "shotgun" or "shotgun" experimental method.

Conclusion

Most of the eukaryotic genes consist of a sequence with coding function, an exon and a sequence without coding function, and the ratio of the two is 1:4. The target gene selected by the shotgun cloning method in the genomic DNA library is the same as the natural gene in the chromosome, and contains an intron. The junction region at both ends of the structural gene also has a transcriptional regulatory sequence fragment, that is, a regulatory gene. Such genes are available for regulatory analysis of gene expression. Because of the presence of introns, the structural genes thus obtained are not suitable for expression in prokaryotic host tissues. Therefore, in order to obtain the target gene of eukaryotes, it is not appropriate to use the shotgun method to obtain the coding sequence of the gene for the eukaryote.

Types of Monoclonal Antibody Technology

A monoclonal antibody is an antibody that is highly uniform and unique to a particular epitope produced by a single B cell clone, and is referred to as a monoclonal antibody. Hybridoma technology is usually used to prepare hybridoma antibody technology. Based on cell fusion technology, sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproduction ability are fused to B cell hybridization tumor.

Types

Murine antibody

The basic principle of hybridoma monoclonal antibody preparation technology is to use polyethylene glycol as a cell fusion agent to fuse the spleen cells of the immunized mouse with the mouse myeloma cells with the ability to reproduce in vitro, in HAT selective medium. Under the action of the fusion, only the hybridoma cells with successful fusion are grown, and repeated immunological detection and single cell culture (cloning) are finally obtained to obtain a hybridoma cell line which can produce the desired monoclonal antibody and can continuously multiply. The cells are expanded and cultured, and inoculated into the peritoneal cavity of the mouse, and a high-priced monoclonal antibody can be obtained in the ascites produced. Since the application of monoclonal antibodies is in vitro development from in vitro diagnostics to in vivo tumor localization and treatment, murine monoclonal antibodies have been difficult to overcome, especially in vivo, when there are major histocompatibility antigens (MHC) and super Sensitive reaction problem. With the increasing use of murine monoclonal antibodies in clinical treatment, the need to reduce the immunogenicity of antibodies has become more and more urgent. To overcome this problem, scientists have used genetic engineering methods to humanize antibodies in mice. By constructing a human-mouse chimeric antibody, human anti-mouse antibodies were attenuated to some extent.

Human and mouse chimeric antibody

To overcome the problems of murine monoclonal antibodies, scientists have used genetic engineering methods to humanize antibodies in mice. By constructing a human-mouse chimeric antibody, human anti-mouse antibodies were attenuated to some extent. In 1984, Morrison et al. successfully constructed the first human murine monoclonal antibody, a chimeric antibody. A chimeric antibody refers to a monoclonal antibody produced by the constant region gene of a murine monoclonal antibody which is encoded by a genetic region recombination technique and which is encoded by a human antibody constant region gene and expressed in a suitable host cell.

Rationale: The specific recognition of antibody molecules, antigen binding is determined by the light chain and heavy chain variable regions, and the human anti-mouse antibody produced by the heterologous protein is mainly the antibody constant region. The mouse mAb constant region was replaced with a humanized constant region and spliced ​​into a chimeric antibody, the variable regions of the heavy and light chains were derived from the mouse, and the constant region was derived from human. Briefly, chimeric antibodies have both antigen binding specificity and greatly reduce the heterogeneity of murine mAb. Chimeric antibody is an antibody originally developed by genetically engineered antibodies and has been widely used in tumor therapy and diagnostic methods. Although chimeric antibodies attenuate human anti-mouse antibody responses to some extent, there is still a small fraction of murine components. This directly leads to the rapid elimination of antibodies, thereby reducing the therapeutic effect.

Humanized antibody

Reshaped antibody

In 1986, Jones et al. successfully constructed the first modified antibody, also known as CDR-grafted antibody and humanized antibody, which refers to the replacement of the corresponding CDR sequence of the human antibody by the complementarity determining region (CDR) sequence in the murine monoclonal antibody variable region. Recombinant constitutes a CDR-grafted antibody that has both murine monoclonal antibody specificity and human antibody affinity. To date, more than 100 murine monoclonal antibodies have been humanized by CDR grafting. Rationale: The variable regions of the antibody heavy and light chains are composed primarily of CDRs and framework regions (FR). The six CDRs of the variable region are the regions responsible for the recognition and binding of antigens, and they are directly in contact with the antigen, which determines the specificity of the antibody. The framework region is other than the variable region, mainly plays a role in supporting CDRs, and their amino acid composition and arrangement are relatively difficult to change. Therefore, the CDR of the murine monoclonal antibody can be transplanted into the framework region of the human monoclonal antibody. It is possible to obtain the same antigen specificity as the murine mAb, and to minimize the heterogeneity of the murine mAb, thus obtaining a modified antibody. Compared with chimeric antibodies, the modified antibody further reduces the proportion of the murine fraction in the antibody and reduces the human anti-mouse antibody, but there are still antibodies that may lead to the production of anti-idiotypic antibodies and have some limitations, such as the relative construction method. Complex, time-consuming and labor-intensive; the crystal structure of the antibody and the micro-structure of the computer-simulated antibody have great problems; there are still many problems in reducing immunogenicity and maintaining antigen binding activity. Of course, it is imperative to find a simple and easy way to achieve both humanization and high immunological activity.

Surface amino acid residues humanized one-faced antibody

A method for reducing the immunogenicity of murine antibodies, which was completely different from CDR grafting, proposed by Padlan in 1991. [9] The theoretical basis is to analyze the surface exposure of a large number of murine monoclonal antibody variable regions and human monoclonal antibody variable region amino acid residues, and found that the position and number of these exposed amino acid residues are very conservative, not because of species Change with type. On the surface of the study, these exposed amino acid residues are the main source of immunogenicity in the murine variable region. By changing the amino acid residue corresponding to the human variable region in the amino acid residue of the framework region exposed by the variable region of the murine mAb to human, the surface of the variable region can be humanized, and the heterogeneity is eliminated without affecting The overall spatial conformation of the variable region.

Episode imprint selection

Epitope selection refers to a method of humanized antibody that binds to the highly efficient screening of phage antibody library technology, and can be obtained by digital screening to obtain fully human antibodies. Rationale: A heavy or light chain variable region gene of a murine mAb is paired with a heavy chain or light chain variable region gene library of a human antibody to obtain a heterozygous human mouse antibody library. With the efficient screening method of phage antibody library, the desired antibody can be obtained quickly, which is simpler than CDR transplantation and can obtain real human antibodies. Disadvantages: The screening workload is particularly large.

Small molecule antibody

Small molecule antibodies, as the name implies, are antibody fragments of smaller molecular weight, and the antigen binding site of the antibody molecule is limited to the variable regions of the heavy and light chains. Although the molecule is small, it retains the same specificity as the parental monoclonal antibody with the affinity of the parental monoclonal antibody. The categories mainly include: antigen binding sheet (Fab) antibody, Fv antibody, single chain antibody, single domain antibody and minimal recognition unit.

Fab antibody

The Fab antibody is a Fab-only molecule, and the Fab segment is formed by a disulfide bond between the intact light chain (constant region CL and variable region VLCL) and the heavy chain Fd segment (first constant region CH1 and variable region VH). The dimer, which is about one-third the size of the total antibody, contains only one antigen-binding site. The coding genes of the complete light and heavy chain Fd are ligated and expressed in E. coli to form a complete disulfide bond and a three-dimensional fold, which can preserve the function of Fab break. Often used in laboratory research tools.

Fv antibody

The Fv antibody consists only of the variable regions of the light and heavy chains by non-covalent linkages, and is the smallest functional fragment of the antibody that retains the intact antigen binding site. Since the fragment is connected by a non-covalent bond, the stability is not good and it is very easy to dissociate. An appropriate method is used to solve the problem of Fv fragment stability.

Single-chain antibody

The advent of single-chain antibodies solves the problem of stability of Fv antibodies. It is formed by linking the light chain and heavy chain variable regions by a suitable oligonucleotide sequence to form a single-stranded molecule, so it is called a single-chain antibody. The structure of a single chain greatly increases the stability of the Fv fragment. Single-chain antibodies have many advantages in clinical use as therapeutic agents relative to fully antibodies. But it also has shortcomings such as decreased affinity.

Single domain antibody

The single domain antibody contains only the heavy chain variable region and has a smaller structure than the subunit of Fv, which is a molecule having antigen binding activity. Single domain antibodies still have the same ability and stability to bind to antibodies as compared to intact antibodies.

Minimum identification unit

The smallest recognition unit smaller than the single domain antibody contains only a single CDR structure in the variable region, the molecular weight is very small, only about 1% of the complete antibody, and the affinity is also relatively low, so it is named as the smallest recognition unit. Although the minimal recognition unit has a small molecular weight and low affinity, it has the ability to bind to an antigen.

Human antibody

From the initial murine monoclonal antibody technology to humanized technology, monoclonal antibodies have made rapid progress in decades. The advent of humanization has made monoclonal antibodies basically solve the problem of human anti-rat origin. However, useful humanized antibody genes are derived from hybridoma cells. This process of hybridomas is complex and time consuming, and it is difficult to prepare autoantigen antibodies and fully human antibodies using hybridoma technology. These two major disadvantages have become a stumbling block in the application of genetically engineered antibodies. With the continuous advancement of science and technology, monoclonal antibodies have entered a new stage of development - human-derived monoclonal antibodies. It mainly includes phage antibody library technology, preparation of human-derived antibodies in transgenic mice, and ribosome display technology.

The development of monoclonal antibody drugs originated in 1975, and the advent of hybridoma technology has made it possible to prepare a large number of uniform murine monoclonal antibodies. In 1986, the first murine monoclonal antibody mumomonab-CD3 (OKT3) against post-transplant immune rejection was approved by the US Food and Drug Administration (FDA), but derived from murine lymphocyte hybridization. Tumor antibodies are recognized by the human immune system and cause severe human anti-mouse antibody (HAMA), which not only shortens the half-life of therapeutic monoclonal antibodies, but also reduces the efficacy and sometimes causes serious adverse reactions.