What can be done if cancer turns resistance to KRAS inhibitors?

Recently, KRAS and KRAS-G12C mutant inhibitors, which were previously considered to be “non-drugable” targets, have ignited a development boom. However, KRAS inhibitors are not a “magic bullet” for once and for all. Cancer resistance is a major problem in cancer treatment. It is usual in clinical practice that tumor tissue becomes significantly smaller after targeted therapy, but soon deteriorates again. Can we develop a multi-mechanical combination therapy to eradicate the disease in order to solve the potential drug resistance problem before the tumor develops resistance to the research drug?

After screening 16,019 genes by shRNA screening, researchers at the Francis Crick Institute and the British Cancer Institute found that the KRAS-G12C inhibitor ARS-1620, and the insulin growth factor-1 receptor inhibitor linsitinib, were studied. The combination therapy with mTOR inhibitors can significantly reduce the tumor size of lung adenocarcinoma, and the effect is more durable. The results of this trial were recently published in the journal Science Translational Medicine.

RAS family proteins are mainly divided into three categories: KRAS, HRAS, NRAS, and the KRAS gene mutation rate is more than 80%, which is one of the most common oncogenes. RAS protein regulation includes the MAPK signaling pathway, as well as multiple downstream pathways such as PI3K/AKT/mTOR, which control several key cellular activities, including proliferation, differentiation, survival, and angiogenesis. KRAS mutations occur in more than 90% of pancreatic cancers, 40% of colorectal cancers, and 16% of lung adenocarcinoma cases.

IGF1R is a membrane protein receptor with tyrosine kinase activity, usually overexpressed in lung cancer. It is a key factor for malignant transformation upstream of the MARK and PI3K signaling pathways and is one of the leading causes of acquired resistance to EGFR inhibitors.

Membrane receptor protein IGF-1R regulates downstream MAPK and PI3K/AKT/mTOR signaling pathways

The researchers found that knocking out the MTOR gene made the cells significantly sensitive to KRAS and IGF1R inhibitors. While blocking the three signaling pathways IGF1R, MAPK and PI3K/AKT/mTOR, cancer cells carrying KRAS mutations could not survive. The KRAS-G12C inhibitor ARS-1620 develop resistance and re-grow in a few weeks. The combination of KRAS-G12C inhibitor ARS-1620, IGF1R inhibitor linsitinib, and mTOR inhibitors can significantly reduce the size of mouse and human tumors, and the effect is significantly longer. This combination therapy has the potential to prevent or delay the resistance of KRAS-G12C inhibitors.

Combination therapy under the study, KRAS-G12C inhibitor ARS-1620, IGF1R inhibitor linsitinib, and mTOR inhibitors has significant effect for lung cancer carrying KRAS-G12C mutation. Dr. Julian Downward, one of the authors, believes that the study provides a new approach to improve the efficacy of targeted mutant KRAS proteins. KRAS mutations often lead to greater invasiveness. “This provides a clear direction on how to better use KRAS-G12C inhibitors in the clinic and how to circumvent the possible evolution of cancer when used as a single drug.”

References

  1. 1. Romero-Clavijo. et al. Development of combination therapies to maximize the impact of KRAS-G12C inhibitors in lung cancer. Science Translational Medicine. https://doi.org/10.1126/scitranslmed.aaw7999
  2. 2. Researchers Develop New Class of Lung Cancer Drugs. Retrieved Sep. 19, 2019, from https://www.biospace.com/article/new-class-of-drug-in-combination-shows-promise-for-lung-cancer/
  3. 3. Could a combo treatment boost KRAS inhibitors in lung cancer?. Retrieved Sep. 19, 2019, from https://www.fiercebiotech.com/research/improving-kras-inhibitors-lung-cancers-a-combo
  4. 4. Two Is Better Than One: Combining IGF1R and MEK Blockade as a Promising Novel Treatment Strategy Against KRAS-Mutant Lung Cancer. Retrieved Sep. 19, 2019, from https://cancerdiscovery.aacrjournals.org/content/3/5/491.figures-only

 

A detailed introduction to recombinant protein drugs (Part Three)

  1. 2.Technical methods for expressing host cell construction

(1). Target gene acquisition

1). Primer synthesis technology

Primer refers to a single-stranded DNA molecule that can be covalently bound to a nucleic acid template at the beginning of nucleotide polymerization and function as a replication extension origin. The synthesis of primers uses solid phase DNA synthesis technology. The specific technical process is as follows:

The nucleotide attached to the controlled pore glass is reacted with trichloroacetic acid to remove the protecting group DMT (dimethoxytrityl) to obtain a free 5′-hydroxyl end. Adding a mixture of a phosphite amide monomer and a tetrazolium activator to form a phosphite amide tetrazole active intermediate, which is condensed with a nucleotide free 5′-hydroxyl group in the reaction system, and the nucleotide chain is extended by one base; The subsequent reaction is terminated by the addition of acetic anhydride and methyl imidazole, and the 5′-hydroxyl group which is not involved in the condensation reaction is removed in the final purification stage; the phosphite amide formed in the condensation reaction is converted into a stable phosphate triester by adding a solution of iodine in tetrahydrofuran. The above four steps complete the attachment of a deoxynucleotide to the original nucleotide chain. This process can be repeated to synthesize the desired primer DNA.

2). Gene amplification technology

Generally speaking, it refers to polymerase chain reaction (PCR). When DNA is denatured to a single strand at a high temperature of 95 °C, the primers at 60 °C and the template DNA single strand are refolded by base-pair pairing principle, about 72 °C. The DNA polymerase extends along the direction of phosphoric acid to the five-carbon sugar (5′-3′) to form a complementary strand, which alternates between denaturation, renaturation, and extension temperature, and the DNA strand is continuously replicated and amplified. Now PCR has developed nested PCR, reverse transcription PCR (RT-PCR), in situ PCR (in situ POR), real-time quantitative PCR (RT-PCR), derivative upgrade technology such as digital PCR. There are also new DNA amplification technologies such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA).

3). Reverse transcription PCR to obtain the target protein gene

Earlier, the acquisition of target gene is usually obtained by reverse transcription PCR, which is, tissue samples are obtained from human or animal body, and the specific mRNA extraction kit is generally used to extract messenger RNA (mRNA) in the tissue sample, so that the gene sequence is not an intron containing a gene of a eukaryotic cell. Then, using mRNA as a template, in the presence of appropriate primers, the corresponding DNA single strand is synthesized by reverse transcriptase, which is called cDNA (complementary DNA). After the corresponding RNA is removed by alkali treatment, the DNA is polymerized by using single-stranded cDNA as a template. The enzyme acts to synthesize double-stranded cDNA. At the time of primer design, an appropriate cleavage site is set for subsequent recombination with the vector. Before constructing the expression vector, the cDNA is ligated into a cloning vector, amplified, and then subjected to DNA sequencing, and the codon substitution which is more favorable for expression is selected according to the preference of the degenerate codon of the host cell for sequence optimization.

4). Whole gene chemical synthesis

Currently, as the sequencing of the human genome and other biological genes is completed, more and more functional genes or structural gene sequences are clear. The level of gene synthesis technology is continuously improved, the synthesis time is shortened, and the cost is reduced. On this basis, the full-length gene of the target protein can be obtained by chemical synthesis according to the optimal gene sequence.

The chemical synthesis method is designed into a short gene fragment (<100bp), and the appropriate restriction sites are designed on both sides of the fragment; the full-length primers are synthesized by solid phase DNA synthesis, and each method is obtained by PCR amplification. Fragmented double-stranded DNA; these fragments are then stepwise ligated by DNA ligase, and if necessary, subcloning is used to obtain the complete gene sequence. Each subclone can be identified separately, thus reducing sequence errors. Full-gene chemical synthesis is currently the most accurate and fastest method for obtaining recombinant protein genes.

5). Gene library technology

In molecular biology and genetics, a genome refers to the entire genetic material of an organism, generally referred to as DNA, including genes, non-coding DNA, mitochondria, and DNA in chloroplasts.

A gene library is a collection of all genes in a certain organism. Generally, it is divided into a genomic library and a partial gene library. A genomic library is a collection of clones formed by recombining all genomic DNA of a certain organism to a certain vector and transforming the recipient cells. A partial gene library refers to a collection of clones, such as a cDNA library, in which a part of the gene DNA of a certain organism is ligated to a vector and transformed into a recipient cell.

According to the recombinant vector function of the library, it can also be divided into a clone library and an expression library. The commonly used recombinant vectors include plasmids, phages, cosmids, and bacterial artificial chromosomes (BAC).

The general method for genomic library construction is to first extract and fragment genomic DNA, select a suitable vector and prepare in large quantities, connect the DNA fragment to the vector to form a recombinant vector, and transfect the recombinant vector into the host cell. The cDNA library usually first obtains the total RNA of the cells, and isolates and extracts the mRNA, obtains the cDNA by reverse transcription PCR, and then constructs according to the vector ligation and transformation of the host cells. DNA recombination and its transfected cell technology are described later.

The gene library is usually screened by nucleic acid hybridization method, that is, a probe is formed by labeling a probe with a known sequence, and under certain conditions, the probe can be transferred to the nitrocellulose after being denatured by nucleotide pairing hybridization. The library DNA sequences on the membrane are combined to screen for the gene sequence of interest. For expression libraries, antibody molecules and immunoblotting methods can also be used to bind the protein of interest expressed by the library to determine the gene of interest. The phage vector library can also be diluted in a 96-well plate, and the appropriate PCR buffer system is added to amplify the library gene by PCR, and then the labeled DNA probe is used to bind the target gene, and the dilution is repeated several times to finally select the target gene.

To be continued in Part Four…

 

What are tumor markers and how important are them?

First, what is the tumor marker?

Tumor cells release certain substances, such as hormones, enzymes and antigens, which may be metabolites of tumor cells. These substances may be present in tumor cells or may be present in the body fluids of patients through the immunological properties of these substances to judge and identify the tumor. The metabolism of tumor cells is different from that of normal cells, so there will be some changes in the humoral environment of tumor patients. This change can be judged by detecting tumor markers. In clinical practice, the role of tumor markers is mainly to discover primary tumors. At the same time, it is possible to screen the high-risk population of tumor, and also to judge the therapeutic effect of the tumor and the prediction of the later treatment stage.

Second, does the tumor marker elevated mean cancer present?

It is not necessarily. Although the tumor marker is a metabolite produced by tumor cells, it does not mean that normal cells won’t. Some studies have found that healthy tissues and cells may also produce tumor markers, so it is actually not 100 percent for sure to check tumor by tumor markers.

Moreover, not every tumor patient has obvious tumor markers, so this method can only be used as a reference of the results. If one wants to confirm the diagnosis, you need to go through various inspections before you can determine it.

Third, the significance of tumor markers

  1. Auxiliary support

Tumor markers can be used to assist in the examination of tumors, and for some special cancers, the reference value is relatively high. If there is a problem with the tumor markers, further confirmation by biopsy is needed.

  1. 2.Develop a treatment plan

Cancer patients often need to perform a tumor marker test before making a production plan. This test can reflect the symptoms of the disease and help the doctor to develop a treatment plan.

  1. 3.Monitor the treatment effect

Tumor markers can be used to judge the symptoms during treatment and the therapeutic effect over a period of time. If the tumor markers do not change, in most cases, it means the therapeutic effect is not significant.

  1. 4. Check the recurrence

Tumor markers can also be used to examine tumor recurrence, which can be used as a basis test.

The maximum value of tumor markers should be in their fluctuations. If they fluctuate frequently within a short period of time, attentions should be paid to.

Creative Biolabs provides a comprehensive range of recombinant antibodies of the key tumor for research processes and diagnostic assay developments. The ranges include but not limited to:

 

The knowledge of metastasis, proliferative signaling, metabolism, inflammation, apoptosis is the focus of research. Hence, the identification of tumor markers using antibodies is widely used in cancer diagnostics and research. Tumor markers are proteins or antigens which are used to detect elevated in the body due to the presence of cancer. Elevated quantities of tumor markers in the blood, urine, stool, tumor tissue for cancer diagnosis and prognostic evaluation can be detected by the tumor marker-specific antibodies.

Discovering and detecting cancer cell signaling pathways and the cellular process will help us to find a link between its genotype and phenotype.

The Introduction of Disialic Ganglioside (GD)2

3F8 antibody

Recombinant Mouse Antibody (3F8 antibody) is capable of binding to GD2 ganglioside, expressed in Chinese Hamster Ovary cells (CHO). It is a murine IgG3 with a moderate affinity for GD2.

What is GD2?

In 1935, researchers first discovered a new substance, ganglioside, in the Ganglionzellen cells of the gray matter of the brain. There are many kinds of gangliosides. So far, more than 70 kinds of gangliosides have been isolated and identified. Gangliosides are a group of sphingolipids containing sialic acid. They are composed of hydrophobic ceramide and hydrophilic oligosaccharide chains containing sialic acid. They are widely distributed in the cell membranes of vertebrate tissues, and the nervous system is the most abundant one. According to the number of sialic acids, it can be divided into monosialic ganglioside (GM), disialic ganglioside (GD) and trisialic ganglioside (GT). Each ganglioside can also be divided into several subgroups according to the number of glycosyl groups, such as GD1, GD2, GD3, etc. Ganglioside GD2 is an important component of the cell membrane of nervous system. It plays an important role in regulating the proteins in the cell membrane. In addition, ganglioside GD2 mediates melanoma cell adhesion and can be used as a target for clinical immunotherapy of neuroblastoma.

Biosynthesis and structure of GD2

Sugar chains are not only structural substances in vivo, but also play an important role in signal transduction between cells and cells. Abnormal expression of glycosylation is usually a marker of the transformation of cancer cells. Glycosyl antigens of some tumors are considered to be closely related to proliferation, invasion, angiogenesis and metastasis of cancer cells. Ganglioside is a glycolipid containing sugar chain. It is mainly synthesized in the endoplasmic reticulum and Golgi body. First, in the endoplasmic reticulum, serine and fatty acid coenzyme A (CoA) synthesize mother nucleus ceramide. Then, ganglioside is processed by a series of glycosyltransferases step by step in the Golgi body to form various forms of gangliosides. Ganglioside GD2, a B-series ganglioside, contains two sialic acid units and plays an important role in cell adhesion and recognition related signal transduction. Gangliosides are the main components of glycolipid-rich microstructural domains, which can bind to sphingomyelin, cholesterol, glycosylphosphatidylinositol anchoring protein and tyrosine receptor kinase.

Anti-GD2 antibody therapy

 

Figure1. Action mechanism of anti-GD2 antibody

Based on the in-depth study of the structure and function of ganglioside GD2, it is recognized that anti-GD2 antibody therapy may have a therapeutic effect on neuroblastoma. However, in order to play an anti-tumor role, anti-GD2 antibodies are confronted with two great challenges: 1) Usually high-affinity antibodies rely on Fc receptor-mediated action to kill cancer cells, but the immune response to sugar chains is usually lack of T cell intervention, so the antibodies produced by anti-GD2 antigens are mostly low-affinity IgM antibodies, while the high molecular weight of IgM is well known. It is often more difficult to invade tumor cells, so the anti-tumor effect is not ideal. 2) The presence of blood-brain barrier prevents intravenous injection of anti-GD2 antibodies into the central nervous system, and may cause adverse reactions to peripheral nerve cells and melanogenesis cells with low GD2 expression. Over the past 30 years, anti-GD2 antibody therapy represented by the Dinutuximab monoclonal antibody has been developing steadily, which has brought the treatment of high-risk neuroblastoma into a new stage.

  1. 3F8 is the first anti-GD2 antibody to be used for the treatment of neuroblastoma in clinical trials. It is a mouse-derived IgG3 antibody with a maximum affinity of 5 nmol/L to GD2. Pre-clinical studies have shown that 3F8 can kill neuroblastoma in a dose-dependent manner by effectively binding with lymphocytes, granulocytes and Fc gamma receptors, further through complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).
  2. ME36.1 is another mouse-derived IgG3 antibody which is still in the early stage of research. Its affinity to GD2 is lower than 3F8 and 77 nmol/L, but it not only binds to GD2, but also has partial affinity to GD3. Pre-clinical studies have shown that the IgG2a subtype variant of ME36.1 not only has no decrease in antigen affinity, but also has certain antitumor activity.
  3. The most striking mouse-derived IgG3 antibody is14.18, and its affinity to GD2 is between 3F8 and ME36.1, which is 19 nmol/L. Subsequently, according to the amino acid skeleton of 14.18, mouse-derived IgG2a antibody, human-mouse chimeric antibody (ch14.18 antibody) and humanized antibody (hu14.18) are further developed, among which ch14.18 is Dinutuximab monoclonal antibody.

Strategies to improve the effectiveness of anti-GD2 antibody

Although anti-GD2 antibody represented by ch14.18 has achieved some success, there are still some challenges in its efficacy and safety. Like other anti-GD2 drugs, there are three strategies to improve the efficacy of anti-GD2 antibodies: 1) coupling with radioactive reagents, toxins and cytokines to enhance the lethality of anti-GD2 antibodies; 2) forming bispecific antibodies with anti-immune cell antibodies, or specific modification of T cells to enhance the tumor immunity of T cells; 3) using single-chain antibodies as nuclei. Through a series of functional improvements, the heart can improve the activity of antibodies and enhance their invasiveness to tumors at the same time.

  1. Coupled antibodies

Previous studies have shown that anti-GD2 antibody combined with cytokine IL-2 and GM-CSF has a certain therapeutic effect on cancer. Therefore, scientists couple cytokines with anti-GD2 antibodies for cancer treatment. In addition to coupling cytokines, some bacterial or plant-derived protein immunotoxins have also been attempted to couple with anti-GD2 antibodies to enhance the anti-GD2 antibody’s tumor killing activity.

  1. Nanoparticle coupled antibody

Nanoparticles are usually 3-200 nm-sized particles composed of polymers, liposomes and viruses. They have the ability to carry large payloads for the treatment of cancer diseases. Liposomes are the most widely used nanoparticles in anti-GD2 antibody drugs. The greatest advantage of drug liposome nanoparticles is that they can through the capillary space of tumor tissue, the infiltration of drugs is effectively improved, while the normal tissue is more closely linked, which limits the entry of drugs, thus further improving the targeting of drugs.

  1. T-cell enhanced antibodies

Activating T cells in human body is the most effective treatment for cancer. Therefore, in recent years, CTLA-4, PD-1 and other immunological checkpoint inhibitors have achieved remarkable results in the treatment of melanoma and other diseases. However, the immunogenicity of most tumors in human body is not strong, so the therapeutic effect of these immuno-checkpoint inhibitors is not obvious. Fortunately, neuroblastoma is more sensitive to antibody-mediated killing effects. Therefore, T cell-related immunotherapy can be widely used in the treatment of neuroblastoma.

Generally speaking, the introduction of Dinutuximab monoclonal antibody is only the beginning of clinical application of anti-GD2 therapy, and there are still many challenges in the treatment of high-risk neuroblastoma. On the one hand, the relationship between GD2 and neuroblastoma remains to be further studied, and the regulatory mechanism of the interaction between GD2 and glycosyltransferase is still unknown. On the other hand, the current course of Dinutuximab monoclonal antibody immunotherapy is relatively complex, and its side effects and adverse reactions cannot be ignored. Therefore, the structure and function of anti-GD2 antibody should be optimized to further improve the safety and efficacy of anti-GD2 therapy. It is believed that with more advanced treatment technologies such as CAR-T coming into clinical practice, anti-GD2 therapy will make greater progress in the treatment of high-risk neuroblastoma.

 

The history of autoimmune disease and animal models (part three)

  1. Rheumatoid arthritis 

Rheumatoid arthritis is considered to be a clinical syndrome spanning several disease subsets. These different subsets entail several inflammatory cascades, which all lead to a final common pathway in which persistent synovial inflammation and associated damage to articular cartilage and underlying bone are present. One key inflammatory cascade involves overproduction and overexpression of TNF. This pathway drives both synovial inflammation and joint destruction. TNF overproduction has several causes, including interactions between T and B lymphocytes, synovial-like fibroblasts, and macrophages. This process leads to overproduction of many cytokines such as interleukin, which also drives persistent inflammation and joint destruction. Overproduction of other pro-inflammatory cytokines (eg, interleukin 1) differs from the interleukin production in that the production is either less pronounced or is specific to one or more disease subsets. This is best illustrated by the blockage of interleukin 1 in sub-forms of juvenile idiopathic arthritis or adult-onset Still’s disease. The dominant local cell populations in joints affected by rheumatoid arthritis are synovial and cartilage cells. Synovial cells can be divided into fibroblast-like and macrophage-like synoviocytes. Overproduction of pro-inflammatory cytokines is believed to be caused predominantly by macrophage-like synoviocytes. Fibroblast-like synoviocytes show abnormal behaviour in rheumatoid arthritis. In experimental models, co-implantation of fibroblast-like synoviocytes with cartilage causes fibroblasts to invade cartilage, and this behaviour is associated with joint destruction. Considerable information has been accumulated about the joint destruction and the role of osteoclast activation as a key process leading to bone erosion. It has been proven that specific inhibition of osteoclast activation can reduce joint destruction but does not affect joint inflammation. It is unclear whether arthritis starts primarily in the bones and subsequently moves to the joints, or the other way around. One argument for rheumatoid arthritis starting in the joint is the observation that fibroblast-like synoviocytes showing altered behaviour can spread between joints, suggesting how polyarthritis might develop. Regulation of immune inflammation depends on the balance between the number and strength of different cells. Control of arthritogenic immune-responses has been studied in mice known to have specific antigen. Infusion of a small number of T cells with specific characteristics can ameliorate arthritis in a rodent model of the disease, showing that T cells have protective effects. Ongoing research should translate these experimental findings into clinical practice.

Rheumatoid arthritis antibodies are classic autoantibodies in rheumatoid arthritis. IgM and IgA rheumatoid factors are key pathogenic markers for the Fc fragment of IgG. Additional (and increasingly important) types of antibodies are those against citrullinated peptides (ACPA). Although most, but not all, ACPA-positive patients are also positive for rheumatoid factor, ACPA appear to be more specific and sensitive to diagnosis and seem to be better predictor of poor prognostic features such as progressive joint destruction. Ongoing research aims to identify antibody specificities associated with different patients’ subsets and disease stages. Composition of the antibody response varies over time. In early rheumatoid arthritis, there is limited specificity, and in late disease, there is a mature response, in which more epitopes are recognised and more isotypes used. Evidence from animal models and in-vivo data suggests that ACPA are pathogenic on the basis of induction of arthritis in rodent models because immunological responses are present in ACPA-positive patients in a citrulline specific manner. Findings of clinical studies have shown that patients with rheumatoid arthritis, rheumatoid factor and ACPA (autoantibody-positive disease) differ from individuals with so-called autoantibody-negative disease. For example, histologically, people with ACPA-positive disease have more lymphocytes in synovial tissue, whereas those with ACPA-negative rheumatoid arthritis have more fibrosis and ynovial lining layers with increased thickness. ACPA-positive disease is associated with increased joint damage and low rates of remission.

50% of risk of developing rheumatoid arthritis can be attributed to genetic factors. Much progress has been made in identification of genetic regions tagged by structural variation (single nucleotide polymorphisms). Rheumatoid arthritis is associated with more than one genetic region. At present, apart from PTPN and HLA genes, no major pathogenic insights have come from these genetic associations. However, progress has been demonstrated by the realisation that from a putative 2 mm of DNA harbouring candidate variants, these regions are all contained within 2 mm of DNA. Using current sequencing methodology, 2 mm of DNA can be sequenced in large cohorts. So, we can reasonably expect that new mechanisms can be identified in the next few years. The existence of many risk alleles discovered in recent years is fairly common in the population as a whole, and individually they have modest effects on the risk of rheumatoid arthritis. However, ongoing research suggests that several risk loci are linked to other autoimmune diseases, and some genes fall within discrete biological pathways that drive inflammation. Findings of genetic studies have found that differences in ACPA status of patients with rheumatoid arthritis are related to the number of specific HLA-DRB1 alleles. These HLA alleles share a common motivation, which is known as shared epitope. Currently, antigens are believed to be modified by a process called citrullination, which entails post-translational modification of the aminoacid arginine to citrulline. This modification is thought to allow antigens to fit in the HLA alleles that harbour this shared epitope. The end result is disruption of tolerance that allows antibody formation against these antigens. Genetic risk factors associated with rheumatoid arthritis are primarily thought to be specifically associated with either ACPA-positive or ACPA-negative disease. The most studied environmental factor for rheumatoid arthritis—smoking—seems to be a risk factor for ACPA-positive disease, especially when the shared epitope of HLA-DRB1 is positive. Genetic research supports the idea that rheumatoid arthritis is a heterogeneous group of overlapping syndromes.

Reference

[1] Vyse T J, Todd J A. Genetic analysis of autoimmune disease [J]. Cell, 1996, 85(3):311-8.

[2] Clark N M, Brown R W, Parker E, et al. Childhood asthma[J]. Environmental Health Perspectives, 1999, 107:421-429.

[3] Xu H H , Werth V P , Parisi E , et al. Mucous Membrane Pemphigoid[J]. Dental Clinics of North America, 2013, 57(4):611-630.

[4] Klareskog L, Catrina A I, Paget S. Rheumatoid arthritis. [J]. British Medical Journal, 1950, 373(9664):659-672.

Introduction to FcRn

Firstly, let us learn some antibodies:

Anti-Human FcRn Antibody

The FcRn-specific human antibody targets FcRn which is a receptor with a high affinity for IgG. The binding of the antibody to FcRn can reduce serum pathogenic auto-antibody levels, and therefore the FcRn antibody can be used to treat autoimmune diseases such as immune neutropenia, Guillain-Barre syndrome, epilepsy, autoimmune encephalitis, Isaac’s syndrome, nevus syndrome, pemphigus vulgaris and so on.

HL161

Recombinant Human monoclonal antibody expressed in CHO binds to Human FcRn. HL161 is a novel anti-FcRn antibody that can be used as a new therapeutic option for pathogenic IgG-mediated autoimmune diseases.

What is FcRn?

 

Figure1. FcRn transmits maternal antibodies to the fetus

Immunoglobulin G (IgG) is the most abundant immunoglobulin component in colostrum. Both maternal IgG secretion to breast and uptake by newborn animals need to cross the epithelial cell barrier through cell transfer. This process requires the participation of a receptor with transport function, namely neonatal Fc receptor (FcRn). FcRn was first found in rodents transporting maternal IgGs to newborns in the intestine. With the deepening of research, more data show that FcRn not only transports IgGs through the placental barrier during pregnancy, but also maintains the serum IgG level, which plays an important role in many organs and tissues.

Molecular structure of FcRn

FcRn is a heterodimer consisting of two subunits, large subunit with molecular weight ranging from 45 kD to 53 kD called alpha chain. Small subunit is beta-2 microglobulin with molecular weight of 14 kD, called beta chain. The two chains are bound together in the form of non-covalent bonds. Beta-2 microglobulin plays an important role in the function of FcRn. Alpha chain must be assembled with beta-2 microglobulin before it can be put into operation. Like MHC-I, the alpha chain has three extracellular functional regions, one transmembrane region and one tail region. The homology of transmembrane region, extracellular functional region and MHC class I molecule is high, but the homology of cytoplasmic tail region is low. The tail region of cytoplasm consisting of 44 amino acid residues may contain signals that mediate intracellular pathways. The bovine cytoplasmic tail region is the shortest found so far among all kinds of animals. In addition, ruminant FcRn alpha chain amino acids are highly similar to human newborn Fc receptor (hFcRn).

Function of FcRn

  1. The role of FcRn in placenta

Ruminant maternal IgG transports through mammary gland epithelium and small intestinal epithelium. Colostrum and normal breast are the main sources of IgG, which are mainly transported to the fetus through maternal-fetal barrier or yolk sac in primates and rodents. In humans, FcRn is expressed in the vesicles in the syncytiotrophoblast. The syncytiotrophoblast contacts with maternal blood and sinks into the endosomes, then the IgG and FcRn bind tightly during the acidification of the endosomes. The vesicles move to the syncytiotrophoblast and the fetus surface fuses with the membrane. IgG is separated from FcRn at physiological pH. FcRn passes through trophoblast cells and returns to the mother, transporting more IgG to the fetus.

  1. The role of FcRn in blood circulation

FcRn can prolong half-life of IgG in serum, maintaining high level of antibody concentration in blood circulation and dynamic balance. FcRn was expressed in human, pig and bovine vascular endothelium. Many experiments have proved that vascular endothelium is an important part of FcRn to protect IgG from metabolism. FcRn not only absorbs IgG from extracellular acidic environment, but also participates in the steady-state regulation of circulating IgG level in endothelial cells.

  1. The role of FcRn in intestinal tract

The expression pattern of FcRn in human intestine is significantly different from that in rodents. FcRn is expressed in human fetal and adult intestinal epithelial cells. However, FcRn was less expressed in the intestine of adult rodents, and the highest expression was found in proximal intestinal epithelial cells in neonatal period, which declined rapidly after weaning. In addition to regulating serum IgG levels and transporting maternal IgGs, FcRn transport may have other significance: oral IgG may be transported through FcRn for passive immunization; by analogy with dairy cows, FcRn transports IgG to the intestine may play a role in eliminating IgGs.

  1. The role of FcRn in breast

FcRn is expressed in mammary glands of many animals such as pigs, rodents, ruminants and humans. FcRn of breast directly combines with Fc fragment of IgG to transport IgG, which is involved in maintaining the dynamic balance of IgG.

Influencing factors of FcRn expression

  1. The effect of hormones on FcRn

Hormones affect the transport of IgG and the expression of IgG in FcRn. In mammary gland, FcRn participates in the secretion of IgG1 and is affected by hormone regulation. Prolactin regulates the activity of IgGl and IgG2 transport receptors. Prolactin can inhibit the transport of IgG to breast and reduce the expression of IgG binding protein in mammary epithelial cells. Exogenous corticosteroids and thyroid hormones affect immunoglobulin transport and FcRn expression in a dose-and time-dependent manner.

  1. The effect of different physiological stages on FcRn

The expression of FcRn varies in different physiological stages. In rodents and human placentas, FcRn transports IgG to the fetus through the epithelial cell barrier. The expression of FcRn was almost not detected in intestinal epithelial cells of rodents after weaning, and IgG was not absorbed by adult animals after oral administration. Adult intestinal epithelial cells still express FcRn. FcRn was detected in cultured epithelial cells in vitro, and it can transport IgG bi-directionally across monolayer cells.

  1. The effect of gene polymorphism on FcRn

Gene polymorphism affects the expression of FcRn, even in different individuals and corresponding parts of the same species.

Expectation

FcRn is expressed in many tissues and organs such as human, cattle, sheep, pigs, rats and mice. The function is also influenced by hormones, physiological stages and gene polymorphisms. In view of the basic role of FcRn in IgG metabolism, the application value of FcRn can be realized through the following ways: (1) FcRn binds to immunoglobulin, saturates FcRn by intravenous injection of immunoglobulin or specific antibody in order to shorten the half-life of pathogenic IgGs in autoimmunity and reduce the pathological response; The IgGFc region of n-interaction prolongs the half-life of therapeutic antibodies and enhances the immune function of the body. (2) Studying the metabolism of IgG in animals can serve the production of polyclonal antibodies. At present, human polyclonal antibodies are widely used in treatment, but their sources are limited. If IgG is selectively transported from serum to milk, it can not only reduce the production cost of immunoglobulin, but also improve the safety of the product, making it possible to produce large-scale therapeutic antibodies. (3) The study of FcRn gene polymorphism has broad prospects. The haplotypes of dairy cows can affect the acquisition of maternal antibodies, thus affecting the morbidity and mortality of calves. Through genetic improvement, cattle screening can enhance the passive immunity of calves and improve the economic benefits of animal husbandry production. In addition, gene polymorphism can cause specific pharmacological and toxicological effects in different individuals after taking drugs, resulting in differences in drug treatment effect, providing a scientific basis for clinical drug treatment.

 

CAR-T Cells Are Expected to Treat Solid Tumors Based on Nanobody

Most CAR-T cell therapies require targeting cancer cell-specific antigens. Now, there is a new way to target the environment around the tumor, which comes from nanobodies naturally produced by free alpacas, camels and llamas. Using this method in mouse models, the researchers successfully inhibited melanoma and colon cancer that currently cannot be treated with CAR-T cell therapy.

In 1989, two undergraduates of Free University of Brussels stumbled upon an unknown antibody while testing camel’s frozen serum. It is a miniaturized version of human antibodies and consists of only two heavy chains, not two light chains and two heavy chains. Their final report says the presence of the antibody has been confirmed not only in camels, but also in alpacas and alpacas. Thirty years later, in the PNAS, researchers at Boston Children’s Hospital and the Massachusetts Institute of Technology showed that further reduction of these mini antibodies could create so-called nanobodies. It may help solve the problem in the field of cancer: making CAR-T cell therapy work in solid tumors.

Chimeric antigen receptor (CAR) T cell therapy through genetic engineering to modify the patient’s own T cells, so that it can better attack cancer cells, which shows a great development prospect for blood tumors. For example, Dana-Farber / Boston Children’s Cancer and Blood Disease Center is currently using CAR-T cell therapy to treat recurrent acute lymphoblastic leukemia (ALL). However, CAR-T cells still face great challenges in eliminating solid tumors, and it is difficult to find tumor-specific proteins that can be used as safe targets in solid tumors. At the same time, solid tumors are also protected by extracellular matrix, which is a supporting network composed of proteins and acts as a barrier. At the same time, immunosuppressive molecules can also weaken the attack of T cells.

Rethinking CAR-T cells

For two decades, antibody patents have been largely held by Belgium. But that changed after the patent expired in 2013. Dr. Hidde Ploegh, a senior researcher and immunologist at Boston Children and PNAS Research, said: “A lot of people are aware of this field and are beginning to recognize the unique nature of nanobodies. “A useful feature is that they enhance their positioning capabilities. Ploegh of Boston Children’s Hospital and his team, in collaboration with Dr. Noo Jalikhani and Dr. Richard Hynes of the Koch Comprehensive Cancer Institute at the Massachusetts Institute of Technology, have used nanoparticles to carry imaging agents to accurately visualize metastatic cancer.

The Hynes team maked nanobodies target the tumor’s extracellular matrix (ECM), the environment around the cancer, rather than the cancer cell itself. This marker is common in many tumors, but usually does not appear in normal cells. “our laboratory and Hynes laboratory are one of the few laboratories that actively study this method of targeting the tumor microenvironment, and most laboratories are looking for tumor-specific antigens,” Ploegh said.

Targeted tumor microenvironment

Ploegh and his team targeted factors that make solid tumors difficult to treat and applied the idea to CAR-T cell therapy. They created CAR-T cells filled with nanosomes that recognize specific proteins in the tumor environment and carry signals instructing them to kill any cells that bind to them. One of the proteins, EIIIB, is a variant of fibronectin, which exists only in newly formed blood vessels that provide nutrition for tumors. The other is PD-L1, an immunosuppressive protein that most cancers use to inhibit close T cells.

Doctor of the Dana-Farber Cancer Institute, Jessica Ingram, drove to Amherst, Massachusetts, to collect T cells from two alpaca Bryson and Sanchez, inject them with interested antigens and collect blood. Further processed in Boston to produce nanobodies.

Treatment of melanoma and colon cancer

It was tested in two independent melanoma mouse models and a colon adenocarcinoma mouse model. CAR-T cells based on nanobodies kill tumor cells, significantly slow down tumor growth, improve the survival rate of animals, and have no obvious side effects.

Ploegh believes that engineered T cells work together through a variety of factors. They cause damage to tumor tissue, which often stimulates the inflammatory immune response. Targeted EIIIB may damage blood vessels in a way that reduces tumor blood supply while making them more permeable to cancer drugs. “If you disrupt the local blood supply and cause vascular leakage, it may improve the delivery of other more inaccessible things, and I think we should see this as part of a combination therapy,” Ploegh said.

Future development direction

Ploegh thinks his team’s approach may be useful for many solid tumors. He was particularly interested in testing CAR-T cells based on nanobodies in pancreatic and cholangiocarcinoma models. “Nanobodies may carry a cytokine to enhance the immune response to tumors, such as toxic molecules that kill tumors and radioisotopes that irradiate tumors at close range,” Ploegh said. “CAR-T cells are the vanguard of breaking through the gates and the other elements then complete their mission. In theory, you can equip a T cell with multiple chimeric antigen receptors and achieve higher accuracy, which is what we want to pursue.”

An overview of mass spectrometry

  1. Overview

The development history of mass spectrometry (MS) dates back to the beginning of the 20th century. The original mass spectrometer was mainly used to determine the atomic mass of elements or isotopes. With the development of ion optics theory, mass spectrometers have been continuously improved, and their applications are constantly changing. It has been widely used in the determination of inorganic compounds and organic compounds in the late 1950s. After the 1980s, the development of new disciplines and technologies such as materials science, precision mechanics, electric vacuum and computers has promoted the advancement of high-performance mass spectrometer manufacturing. Today, mass spectrometry technology has been extensively applied to chemical analysis tests.

Basic theory of mass spectrometry

(1). Principles of mass spectrometry

Mass spectrometry is an analytical method for ionizing a substance to be measured by mass spectrometry, separating the mass-to-charge ratio of ions, and measuring the intensity of various ion peaks for analysis purposes. Quality is one of the intrinsic characteristics of matter. Different substances have different mass spectra, ie mass spectrometry. With this property, qualitative analysis (including molecular mass and related structural information) can be performed; and peak intensity and compound content represented by it can be quantified.

 (2). Composition of the mass spectrometer

The mass spectrometer is generally composed of a vacuum system, a sample introduction system, an ion source, a mass analyzer, a detector, and a data processing system.

  1. Performance indicators of mass spectrometer

There are many indicators for measuring the performance of a mass spectrometer. These indicators include resolution, mass range, sensitivity, quality stability, mass accuracy, and more. There are many types of mass spectrometers, and the representation methods of their performance indicators are not identical. The main indicators are as follows.

2.1. Resolution

The resolution of the mass spectrometer represents the ability of the mass spectrometer to separate adjacent two masses. Commonly represented by R. It is defined as if a mass spectrometer can just separate the two masses of ions M and M+∆M at mass M. The resolution of the mass spectrometer is R=M/ ∆ M.

2.2. Quality range

The mass range is the range of ionic mass-to-charge ratios that can be measured by the mass spectrometer. For most ion sources, the ions ionized are singly charged ions. Thus, the mass range is actually the range of molecular weights that can be determined; for electrospray sources, the ions formed are multi-charged, and although the mass range is only a few thousands, the molecular weight that can be determined can be more than 100,000. The size of the mass range depends on the mass analyzer. The upper limit of the mass range of the quadrupole analyzer is generally around 1,000, and some can reach 3,000, while the time-of-flight mass analyzer can reach hundreds of thousands. Due to the different principles of mass separation, different analyzers have different mass ranges. It doesn’t make sense to compare each other. The same type of analyzer reflects the performance of the mass spectrometer to some extent.

2.3. Sensitivity

Sensitivity represents the amount of sample required to produce a certain signal-to-noise ratio of molecular ion peaks at a given resolution for a given sample.

2.4. Quality stability

The quality stability mainly refers to the condition that the quality of the instrument is stable during operation, and is usually expressed by the mass unit of mass drift within a certain period of time. For example, the quality stability of an instrument is: 0.1 amu / 12 hr, which means that the instrument drifts within 12 hours without a mass drift of more than 0.1 amu.

2.5. Quality accuracy

Mass accuracy refers to the accuracy of mass determination. Commonly represented by relative percentage. Mass accuracy is an important indicator of high-resolution mass spectrometers and does not make much sense for low-resolution mass spectrometers.

  1. Tandem mass spectrometry and applications

3.1. Tandem mass spectrometry

Two or more mass spectra connected together, are called tandem mass spectrometry. The simplest tandem mass spectrometry (MS/MS) consists of two mass spectra connected in series, with the first mass analyzer (MS1) pre-separating or energizing the ions and analyzing the results by a second-stage mass analyzer (MS2). The most basic functions of MS/MS include the description of the relationship between the parent ion in MS1 ​​and the daughter ion in MS2.

MS/MS has many advantages in mixture analysis. When mass spectrometry is combined with gas chromatography (GC) or liquid chromatography (LC), it can be identified even if the chromatogram fails to completely separate the material. The MS/MS can select the parent ion from the sample for analysis without interference from other substances.

3.2. Combined technology

Chromatography can be used as a sample introduction device for mass spectrometry, and the sample is initially separated and purified. Therefore, chromatography/mass spectrometry can separate and analyze complex systems. Since chromatography can give retention times of compounds, mass spectrometry can give molecular weight and structural information of compounds, so it is very effective for the identification and determination of compounds in complex systems or mixtures.

To be continued in Applications of mass spectrometry in chemical analysis.

Study on function of plant growth regulator

Plant growth regulator is a kind of substance which has similar physiological and biological effects with plant hormones. Those that have been found with functions of regulating Plant Growth and development of Plant Growth Regulators mainly include auxin, gibberellin, ethylene, cytokinins, abscisic acid, br, salicylic acid, jasmonic acid and polyamine, and the first six categories have been applied in the agricultural production.

 

For the target plant, through the nutrient detection process, results can be found that plant growth regulator is exogenous of nonnutritive chemicals, usually in the plant body parts to effect, at very low concentration could promote or inhibit some link of the process of its life, to meet the needs of human development. Each plant growth regulator has a specific purpose, and the application of technical requirements are quite strict, only under specific application conditions to produce a specific effect on the target plant. Changes in concentration tend to have the opposite effect, such as a stimulant at low concentrations and an inhibitor at high concentrations.

 

Auxins, as the main plant growth regulator, mainly refers to indoleacetic acid, which is an important pathogenic factor. Indoleacetic acid can be synthesized by many pathogenic fungi and bacteria. Some pathogenic bacteria themselves do not produce indoleacetic acid, but because the indoleacetic acid oxidase in the body of plants is inhibited, blocked the degradation of indoleacetic acid, leading to the increase of indoleacetic acid level. Indoleacetic acid accumulation can be detected in tomato inoculated with solanella, and the content of indoleacetic acid increases continuously after inoculation for a period of time. After inoculation of tobacco with this pathogen, the indoleacetic acid content in the diseased plant increased nearly 100 times than that in the uninoculated plant.

 

In addition, Plant Hormone Detection found that growth Hormone may also enhance the pathogenicity of pathogens by inhibiting Plant defense response. When arabidopsis thaliana was applied to naphthalene acetic acid, the susceptibility was enhanced and the infection of pseudomonas clove increased. Suspension of auxin producing tumorigenic soil bacillus was injected into tobacco leaves, and then inoculated with pseudomonas Eugenia. As a result, the allergic reaction that should have occurred was restrained, and the degree of inhibition was related to the expression of auxin synthesis function gene of tumorigenic soil bacillus.

 

Cytokinins, another plant growth regulator, not only promote cell division, but also make cells larger. However, different from auxin, cytokinin increases cell volume through lateral enlargement and thickening rather than promoting longitudinal elongation of cells, which has a certain inhibitory effect on cell elongation. To eliminate apical advantage, auxin is the main cause of apical advantage, while cytokinin can eliminate apical advantage and promote the rapid growth of lateral buds. In this respect, auxin and cytokinin show obvious antagonistic effects, and the production site and operation mode of both determine the growth of roots and shoots. Cell division consists of two processes, nuclear division and cytoplasmic division. Auxin only promotes nuclear division, but has nothing to do with cytoplasmic division. Cytokinin, on the other hand, mainly ACTS on cytoplasmic division. Therefore, the effect of cytokinin promoting cell division can only be shown in the presence of auxin.

 

Cytokinins can also promote the biosynthesis of proteins. Because cytokinins are present on ribosomes, they encourage ribosomes to bind to mrnas to form multiple ribosomes, speeding up translation to form new proteins. It was proved that cytokinin could induce the protein synthesis of tobacco cells to form a new nitrate reductase.

 

Plant Growth Regulators functions and features:

 

(1) Plant growth regulator has a wide range of effects and many application fields. Plant growth regulator can be applied to almost includes all the higher and lower in the planting industry plants, such as field crops, vegetables, fruit trees, flowers, trees, etc., and through the regulation of plant photosynthesis, respiration, material, absorption, distribution and function, signal transduction, stomatal opening and closing, osmoregulation, transpiration processes such as regulation and control of plant growth and development, improve the relationship between plants and the environment interaction, enhance crop resilience, increase crop yield, improve quality of agricultural products, make crop agronomic traits expressed according to the demand by people in the direction of development.

 

(2) It can regulate both the external characters and the internal physiological processes of plants. Plant growth regulators are highly targeted and can solve some problems difficult to be solved by other means, such as the formation of seedless fruits, prevention and control of wind, control of plant shape, promotion of cuttings rooting, fruit ripening and coloring, inhibition of axillary bud growth, promotion of cotton leaf shedding.

 

(3) The use effect of plant growth regulator is affected by many factors and it is difficult to achieve the best. Climatic conditions, application time, dosage, application method, application site and the absorption, integration and metabolism of the crop itself will affect its effect.

A prediction of gene sequencing application areas and scale

The upstream of the gene sequencing industry is the supply of sequencing instruments, sequencing reagents and consumables. The midstream is mainly the processing and interpretation of sequencing services and sequencing data, and the downstream is the terminal application of sequencing services, including clinical testing and life science research. Benefiting from the decline in sequencing costs and the maturity of sequencing technologies, the application fields of gene sequencing are rapidly expanding. They can be divided into clinical applications and non-clinical applications. The application in clinical fields is currently developing rapidly, mainly including reproductive health screening, investigation, tumor gene detection and pathogen infection detection.

  1. Tumor gene detection

Prevention, diagnosis, treatment, monitoring, gene sequencing, comprehensive coverage.

The current application of gene sequencing in tumors mainly includes tumor susceptibility gene screening, early tumor diagnosis, tumor accompanying diagnosis and medication guidance, and tumor surveillance.

  1. Tumor susceptibility gene screening

The occurrence and development of tumors are the result of the combination of genetic factors and environmental factors. Gene changes are at the core of them, abnormal expression of oncogenes or inactivation of tumor suppressor genes, and unlimited tumor cells are the molecular basis of proliferation. Therefore, through genetic testing, it is helpful to assess the risk of tumors from the source, and implement effective monitoring, early warning and intervention before the individual suffers from the disease and reduce the risk of cancer.

  1. Early diagnosis of tumors

About 90% of malignant tumors have no obvious symptoms in the early stage. When they are found, they are usually in the middle and late stage. Therefore, early detection, prevention and treatment are important means to prevent cancer and slow down the development of cancer. At present, the clinically mature early tumor diagnosis technology is mainly realized by detecting circulating tumor cells (CTC), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA) and exosomes. When a tumor occurs, a small number of tumor cells and DNA and RNA fragments released from apoptotic tumor cells enter the bloodstream. By extracting peripheral blood and capturing this part of cells, DNA and RNA, they can be sequenced in the early stage of tumorigenesis. Thus to confirm it and improve the cure rate.

  1. Tumor-associated diagnosis and medication guidance

Studies have shown that tumors are often not the result of a single genetic mutation, usually caused by the interaction of multiple genes. Therefore, even with the same type of tumor, the difference at the genetic level may be huge, so it is necessary to group the tumors by gene sequencing, and then adopt different treatment plans. In addition, the treatment effect of the same drug on different patients often differs greatly. This individual difference depends largely on the genetic difference of the individual. Therefore, according to the patient’s genetic test results and the pharmacogenetic mechanism, a more targeted drug regimen can be developed for the patient, which can greatly improve the effectiveness of the treatment and maximize the survival of the patient.

  1. Surveillance of tumors

Most tumors have a greater risk of recurrence after treatment. By means of genetic testing, tumor recurrence can be monitored in a timely and accurate manner, and the goal of timely treatment is finally achieved.

  1. Detection of pathogen infection

The market potential should not be underestimated

The detection of pathogenic infection mainly uses the second-generation sequencing technology, combined with the analysis method of metagenomics, to quickly and accurately detect the pathogens infected by the patients, so as to timely solve the problem and improve the cure rate. The detection of pathogenic infections at this stage is also mainly used for difficult and critically ill patients.

We believe that in the future, improving the quality of gene sequencing detection, speeding up detection, and broadening the number of diseases covered by gene sequencing to reduce detection costs are the development direction of the industry. In addition, the gene bank accumulated in the previous clinical tests is expected to be gradually converted into products after 5-10 years, becoming the key factor of the genetic sequencing company to finally establish its market position.