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.
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