A Brief Look of Variable Domain

The variable domain is a region in which the immunoglobulin light chain and the heavy chain vary greatly near the N-terminal amino acid sequence. An immunoglobulin refers to an animal protein having antibody activity. It is mainly found in plasma and is also found in other body fluids, tissues and some secretions. Most of the immunoglobulins in human plasma are present in gamma globulin (gamma-globulin). Immunoglobulins can be classified into five classes: IgG, IgA, IgM, IgD, and IgE. It consists of two identical light chains and two identical heavy chains and is an important class of immune effector molecules.

The basic structure of the Ig molecule consists of a tetrapeptide chain consisting of two identical light chain chains of smaller molecular weight (L chain) and two identical heavy chain heavy chains (H chains). The L chain and the H chain are disulfide-bonded to form a tetrapeptide chain molecule, a monomer called an Ig molecule, which is a basic structure constituting an immunoglobulin molecule. It is known that the H chain of IgG, IgA and IgD in each of the five immunoglobulins has one variable region (VH) and three constant regions (CH1, CH2 and CH3) in total.

The main function of Ig is to combine with antigens (including foreign and self) to effectively remove foreign bodies such as microorganisms and parasites invading the body. Antibodies are antigen-specific and antigen-specific. Combined protein. Each antibody binds to a specific epitope. This combination can inactivate the antigen, but it may also be ineffective, but it may also cause pathological damage to the body, such as anti-nuclear antibodies, anti-double-stranded DNA antibodies, anti-thyroglobulin antibodies, and other autoantibodies. The characteristic of the Ig variable region is that the amino acid species, arrangement order and configuration variation of the region are large.

According to the target, it can be divided into anti-toxin, anti-bacterial antibody, anti-viral antibody and pro-cell antibody (immunoglobulin capable of binding to cells, such as lgE-reactive antibody in type 1 allergy, which can be adsorbed on the target cell membrane).

1. Heterogeneity of antibodies. The composition of antibodies is extremely complex and consists of thousands of different immunoglobulin (Ig) molecules. These Ig molecules are similar and different in shape, size, structure, and composition and arrangement of amino acids. Due to differences, their electrophoretic activity varies greatly.

2. The antibodies in the serum of any normal human are all a mixture of thousands of immunoglobulin molecules with various idiotype antigenicities. The κ chain and the λ chain can be combined with various types and subclasses. The heavy chain and the light chain have various isoforms, and the Ig produced by each clone has its own unique type.

Ig class switching: In the process of antibody response, after the antigen activates B cells, the Ig class expressed and secreted on the membrane will be converted from IgM to Ig of other classes or subclasses such as IgG, IgA, IgE, also known as the same species. Type conversion. At this time, the Ig variable region is unchanged, that is, the specificity of the binding antigen is the same, but the heavy chain type (constant region) is changed.

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Principle and Practice of Phage Display Antibody Technology

Abstract

In October 2018, half of the Nobel Prize in Chemistry was awarded to American scientist George P. Smith and British scientist Gregory P. Winter. The two winners were the phage display technology for peptides and antibodies. Among them, George P. Smith is the creator of phage display technology, which creatively pioneered the platform of this technology, and Gregory P. Winter used this technology to develop the first fully human antibody drug for rheumatoid treatment. Arthritis, psoriasis, etc., is the famous drug king adalimumab. In 2017, its global sales reached 18.5 billion US dollars, with accumulated sales of 115.9 billion US dollars. Phage display technology has a wide range of applications in biomedical research and development, and is the source technology for the development of new biopharmaceuticals.

Introduction

Phage display technology is called the power to control evolution. The most famous thing about biological evolution and evolution theory is Darwin's theory of evolution. "The natural selection of the game, the survival of the fittest", Darwin believes that there is a struggle between the creatures, the adaptors survive, and the discomfort is eliminated, this is the natural choice. It is through biology, variation and natural selection that biology evolves and develops from low to high, from simple to complex. The inheritance and variation of organisms have created the diversity of the population. Some key harsh conditions of the natural environment are used as screening pressures to make the diversity of the population partially lost. At the same time, the population can survive the adaptation to the screening pressure. The interaction between biology and the environment is a complex interaction between multiple trait diversity and multiple conditional screening pressures in complex systems. In contrast, phage display technology is a single trait diversity and selection of replicated organisms. A single screening pressure interacts to purposefully obtain a technique that is responsive to the trait of the screening pressure. Specific to phage antibody display technology, usually by genetic engineering of Escherichia coli filamentous bacteriophage M13 to replicate the genetic diversity of the antibody of the organism, and fusion of the gene-encoded antibody and phage membrane protein on the surface of the phage, select whether The ability to bind to a specific target protein as a screening pressure, a technique for obtaining antibodies and genetic information capable of binding to the specific target protein. The breakthrough contribution of phage antibody display technology lies in establishing a direct link between the in vitro antibody function and its corresponding genetic information, and screening the phage of the functional antibody to obtain the genetic information corresponding to the encoded antibody in the phage.

Diversity of protein and genetic information of antibodies

The antibody protein has a monoclonal antibody in a single Y-shaped monomer or a plurality of Y-shaped monomers, and each Y-shaped monomer consists of four polypeptide chains, comprising two identical heavy chains and two identical light chains. The top of the Y-shaped structure is the variable region, which is the antigen binding site, and the rest is a more conserved constant region. The single antibody variable region is called scFv (single-chain variable fragment). The heavy chain variable region and the light chain variable region are folded into a globular structure by covalent bond and non-covalent bond, and the light and heavy chain variable region is composed of 4 The frame region (FWR) with relatively constant frequency of change and the inclusion of three hypervariable regions (CDRs), the hypervariable region is also called the complementarity determining region, and the six complementary determining regions of the light and heavy chain are close to each other in the three-dimensional structure. Forming a protein cyclic structure is a key region that binds to the antigen.

The light and heavy chain variable regions are encoded by the light chain V gene and the heavy chain V gene, respectively, wherein the light chain V gene has two types, a kappa light chain and a lamda light chain, assembled by a VL fragment and a JL fragment or a VΚ fragment and a JΚ fragment. The heavy chain V gene is assembled from VH fragments, DH and JH fragments. According to the following figures, more than 30 light chain heavy chain V fragments, 23 heavy chain D fragments, and 4-6 light and heavy chain J fragments in the human body are not completely counted, and their arrangement constitutes the diversity of antibody genetic information. Generally, the CDR3 region of the heavy chain of the antibody protein is a key region for antigen binding, and its main body is determined by the DH fragment, but during the assembly of the heavy chain V gene V, D, J, the VH fragment and the JH fragment are also inserted in a small number. The nucleotides of the nucleus enter the CDR3 region, further increasing the diversity of the CDR3. In theory, the diversity of the human antibody genetic information can be as high as 5E13, and the 10 trillion-level antibody library accumulated by humans for a long time. It can be said that it can react with any known or unknown macromolecular small molecule on the earth, which shows the great power of life evolution, and the phage display technology will harness the power of this evolutionary diversity.

Marketed antibody using phage display technology

According to incomplete statistics, 10 listed antibodies using phage display technology in the antibody development stage are limited by the patent restriction of phage display technology. The use of this technology is only in the hands of a few companies for a period of time, which is also the first use. The phage display technology antibody adalim was listed in 2002 for the next decade and there are few root causes of the market for antibodies using this technology.

Phage display technology advantages and applications

Phage display technology, whether it is the source technology for antibody discovery or the screening tool for antibody characterization in antibody engineering, has the irreplaceable advantages of existing traditional technologies and the application of multi-directional fields.

1. Compared with the traditional murine immune hybridoma technology, the antibody screened by the human natural phage library is fully human and can effectively reduce the risk of immunogenicity. This chronic disease such as rheumatoid which requires long-term regular injection. It is a key factor in the treatment of diseases.

2. The discovery of fully human antibodies relative to transgenic mice requires in vivo immunization of mice, some humans with highly homologous proteins, and more toxic proteins such as the marketed antibody Raxibacumab against anthrax toxin, antigen-labile Proteins, allosteric proteins, and transmembrane proteins are not suitable for in vivo screening, and human phage libraries are in vitro screened for antibody discovery of more target molecules. Moreover, the phage display technology has not undergone in vivo immunization, and is capable of detecting antibodies against a single target multi-epitope (especially a conserved epitope with high homology of human mouse).

3. Relative to immune-based in vivo antibody discovery technology, animal immunization, late hybridoma fusion, monoclonal screening and other cumbersome cell culture processes, phage display for panning, screening process in E. coli, amplification process is very simple, antibody The time period found was short, the risk of unexpected accidents in the experiment was low, and reagent consumables and labor costs were lower.

4. Immunized animals can extract their B cells for the establishment of an immunophage library, combining the advantages of both in vivo immunization and phage in vitro screening. For example, the first listed Nanobody Caplacizumab is an immune alpaca and then screened using phage library technology. This method has a great role in the screening and discovery of tools for scientific research.

5. Random or site-directed mutagenesis of existing antibody gene sequences to construct a mutant library for humanization or affinity maturation, screening for more affinity antibodies or conditionally activating antibodies, such as Genetech's Ranibizumab and Bevacizuma antibodies. Derived from the murine clone monoclonal antibody A4.6.1, the humanization of the Bevacizuma antibody is obtained by transplanting the CDR regions into the human framework region (VLκ1, VHIII) and then site-directed mutagenesis, while the humanization of Ranibizumab is to transplant the CDR regions. The light and heavy chain framework regions of different humans construct high affinity antibodies 2 screened by phage library.

6. Using the existing antibody as a Guided selection, using a natural phage library to screen out a plurality of new sequence antibodies with the same epitope as the antibody acting on the target, and obtaining a new drug consistent with the in vivo efficacy of the guide antibody. Sequence antibodies, such as adalims, were screened by Guided selection using mouse monoclonal antibody MAK195 as a guide, using phage display method.

7. Selecting a relatively fixed combination of antibody light and heavy chain framework regions to construct a diverse array of synthetic phage libraries in the CDR regions. The relatively fixed combination of light and heavy chain framework regions removes potential post-translational modification sites (PTMs), allowing the selected antibodies to be screened. It has better physical and chemical properties and reduces the difficulty of CMC in the later stage. Ylanthia synthetic phage library 4 as MorphoSys.

8. The CAR of chimeric antigen receptor T cell (CAR-T) therapy is usually scFv, which has a similar display environment as the phage displayed scFv library, using phage display technology to find CAR (chimeric antigen receptor) in CART. It is the key to innovative CAR-T therapy.

9. The technology of phage display screening is also widely used in the field of drug research and development.

Phage display technology is the source technology of bio-innovative medicine research and development. It plays a central role in the discovery and optimization of new antibodies and peptides, and can effectively interface with traditional antibody discovery platforms. It can be widely used in antibodies and antibody couples. Combined drugs and CAR-T/TCR-T and other fields.

Four Days for Phage Amplification & Titer Method

Day one:

17:00: After inoculation with a single loop of bacteria inoculated in LB medium, shake at 37 ° C overnight (do not exceed 16 hours)

Day two:
7:30: Turn on the UV lamp and illuminate for half an hour (put the instrument into the fume hood); 8:00: Put 20mL of LB broth into a 250mL Erlenmeyer flask and dilute it at a ratio of 1:100. (ie put 200ul of bacterial liquid), put it into a 37 °C shaker, culture for about 3.5 hours; 11:00: turn on the UV lamp, irradiate for half an hour; 11:30: take 10μL of positive phage solution to 20mL In the bacterial culture (early logarithmic growth phase), put it into a shaker and shake vigorously (300 rpm) at 37 °C for 4.5 hours; 15:30: turn on the UV lamp and irradiate for half an hour. At the same time, the high-speed centrifuge was pre-warmed; 16:00: The culture in the Erlenmeyer flask was transferred to a centrifuge tube, centrifuged at 10,000 rpm for 10 min at 40 ° C, and the supernatant was transferred to another clean centrifuge tube and centrifuged again. Aspirate part of the supernatant and mix it with glycerin in a ratio of 1:1 and store at -200C. Pipette 80% supernatant into another centrifuge tube, add 1/6 volume PEG/NACL, overnight at 40 ° C; 17:00 inoculation with a monoclonal bacteria inoculated in LB medium, 37 ° C shaker Stay overnight (do not exceed 16 hours).

Day three:

7:30: Turn on the UV lamp for half an hour (put the instrument into the fume hood); 8:00: Dilute the overnight culture bacteria in a ratio of 1:100 (put 50 μl of the bacterial culture into 5 ml of LB) Put in a 370C shaker and incubate for about 3.5 hours; 8:30: turn on the UV lamp, illuminate for half an hour (put the instrument into the fume hood), and let the high speed centrifuge preheat; 9:00:40 °C 10,000 rpm Centrifuge for 15 min and discard the supernatant. Resuspend the pellet by adding 1ml TBS, transfer to a microcentrifuge tube, add 1/6 volume of PEG/NACL to precipitate again, incubate on ice for 15-60min, discard the supernatant, and add 200ul TBS to resuspend the pellet, which is positive phage expansion. Add liquid. After several rounds of amplification to the required titer; 10:30: Place the IPTG/Xgal plate in a 37 ° C incubator for pre-warming, prepare a water bath and other titer supplies; 11:00: use LB medium The phage were serially diluted at 100/1000 fold. Dissolve the top coat in a microwave oven, dispense 3 mL per 3 mL in a sterile culture tube, one for each dilution of the phage, and place the tubes in a 45 ° C water bath for use; 11:30: dispense every 200 ul of bacterial culture Into the microcentrifuge tube. Each phage dilution of 10 uL was separately added to the above bacterial culture, and incubated at room temperature for 1-5 minutes. Transfer to the above sterile culture tube containing the top coat, quickly suspend, immediately spread onto the pre-warmed LB/IPTG/Xgal plate, and tilt the plate to spread the top coat. The plate was chilled for 5 min at room temperature, inverted, and incubated overnight at 37 °C.

Day four:

Plaque count, the optimal dilution of about 100 per plate. Phage titer = number of plaques × dilution (pfu/10uL)

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Notices Before Using Raptiva

Raptiva is a kind of man-made form of a protein similar to human antibodies to treat plaque psoriasis in adults. And efalizumab is made to target and destroy only certain cells in the body, which is able to protect those healthy cells from damage. However, it is well-known that raptiva may increase the risk of serious infections, such as viral brain infection. So it is important for customers to use raptiva in our daily life.

How should we use raptiva?

Generally speaking, the raptiva is usually prescribed by doctors and is forbidden to be used in larger amounts or for longer than recommended.

What should we do if a dose is missed?

Do not be worried, we can immediately call our doctors for some instructions, which will dig us out of predicament.

What should we do if overdose?

Firstly, you should understand the symptom of overdosing. Overdose will cause severe vomiting, of course, it has different symptoms in different body conditions. If you do think so, you have to seek emergency medical attention.

What should be paid more attention or what should we avoid?

Firstly, we should keep distance from people who have got flu, colds or other contagious illnesses. And then, “live” vaccines, such as mumps, rubella, chichenpox, etc. are not allowed while we are using raptiva. What’s more, do not be near to those who have a “live” vaccine recently. Once these are violated, virus may have the chance to be pasted onto you.

Drugs list affecting the function of raptiva:

Ø drugs that weaken your immune system

Ø mycophenolate mofetil (CellCept)

Ø other psoriasis medications or phototherapy

Ø basiliximab (Simulect), muromonab-CD3 (Orthoclone)

Etc.

Of course, it is possible to list all drugs that affect raptive’s function. So before taking raptiva, we should tell doctors about all medicine or other treatment we are being through recently.

Some side effects of raptiva we should learn:

Ø skin infection, including redness, tenderness and swelling

Ø Infection, such as chills, fever, flu symptoms, bleeding and so on

Ø Problems with walking, speaking, decreased vision, swallowing, etc.

Ø Neck stiffness

Ø Numbness of your joints

Some other serious side effect of raptiva

Ø Back pain

Ø Joints pain

Ø Swelling

Ø Headache, muscle pain and nausea

Notices before using raptiva

Once you are allergic to efalizumab, you should not take it due to a serious infection, such as a viral infection of brain which may increase the risk of disability or death, which will be more higher in those who have a weak immune system or are receiving certain medicines.

Before using raptiva, make sure that your doctor know all about your condition, such as any active or chronic infection, arthritis or a weak immune system, which can be shown from your diseases or certain medicines.

The most important advice is for pregnant women. FDA now can not make sure if the raptiva may be harmful to an unborn baby, so you should tell your doctor if you are pregnant during taking raptiva.

Those suggestions above may not be complete. We also hope to give you a detailed instruction on the surface. And it is admired if you can find more detailed information about the medication, please tell us for the benefit of more people.

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How to Identify the Anti-ErbB2 Dual Variable Domain Immunoglobulin (DVD-Ig™) Proteins

Restraining ErbB2 motioning with monoclonal antibodies (mAbs) or little atoms is a set up helpful technique in oncology. We have created hostile to ErbB2 Dual Variable Domain Immunoglobulin (DVD-Ig) proteins that catch the capacity of a mix of two enemy of ErbB2 antibodies. What's more, a portion of the counter ErbB2 DVD-Ig proteins gain the new elements of improving ErbB2 flagging and cell multiplication in N87 cells. We further discovered that two DVD-Ig proteins, DVD687 and DVD688, have two unmistakable components of activities in Calu-3 and N87 cells. DVD687 improves cell cycle movement while DVD688 instigates apoptosis in N87 cells. Utilizing a half DVD687, we found that energy may assume a key job in the agonist movement of DVD687 in N87 cells.

Materials and Methods

Cell Lines and Cell Culture Conditions

Calu-3, N87 and MDA-MB175 cells were acquired from the American Tissue Culture Collection (ATCC, VA). All cells were kept up in DMEM medium enhanced with 10% fetal cow-like serum (FBS), 50 units/mL penicillin, and 50 µg/mL streptomycin.

293G cells steadily transfected with both YFP and luciferase-labeled ErbB2 (C-end erasure) were kept up in DMEM medium enhanced with 10% fetal ox-like serum (FBS), 50 units/mL penicillin, 50 µg/mL streptomycin, 500 µg/ml G418, and 250 µg/ml hygromycin.

Binding Analysis

For ELISA, plates were covered with 1 µg/ml of enemies of his neutralizer (Invitrogen, CA) in carbonate cradle at 4°C medium-term. Subsequent to obstructing with Superblock (Pierce, CA) at room temperature for 60 minutes, His-labeled ErbB2 proteins (R&D Systems, MN) were included 1% BSA in PBS at room temperature. In the wake of washing, different groupings of antibodies were included for 1 hour at room temperature and caught antibodies were identified by HRP-conjugated goat-hostile to human antibodies.

Immunoblot Analysis

For cell flagging examinations, cells were plated into 6-well plates and hatched medium-term. Cells were serum-starved for 24 hours and pre-brooded with 100 nM antibodies or DVD-Ig proteins each for 2 hours and invigorated with 10 nM HRG for 10 minutes and after that reaped. Cells were lysed with RIPA cushion enhanced with protease and phosphatase inhibitor mixed drinks. Cell lysate proteins were settled by SDS-PAGE and immunoblots were tested with antibodies against phosphorylated Tyrosine, trailed by brooding with IRDye 700 conjugated objective enemy of mouse and IRDye 800 conjugated goat hostile to rabbit. Add up to protein was recognized with hostile to PCNA pursued by IRDye680 CW conjugated goat against mouse. Smears were envisioned utilizing an Odyssey Imaging framework.

Co-immunoprecipitation Assay

Calu-3 or N87 cells were serum-starved for 24 hours and pre-brooded with 30 nM antibodies and DVD-Ig proteins each for 2 hours and animated with 10 nM HRG for 10 minutes and after that gathered. Cells were lysed with RIPA cushion enhanced with protease and phosphatase inhibitor mixed drinks. The cell lysate were hatched with hostile to ErbB2 conjugated Protein A/G globules vulnerable space for a hour and a half. The dabs were then washed multiple times with RIPA support and bubbled in 2X SDS stacking cushion for five minutes. Proteins were settled by SDS-PAGE and immunoblots were examined with antibodies against EGFR, ErbB2, ErbB3, and PCNA.

293G cells steadily transfected with both YFP and Luciferase labeled ErbB2 (C-end cancellation) were treated with 30 nM DVD-Ig protein or mAbs for two hours and afterward collected. Cells were lysed with RIPA cradle enhanced with protease and phosphatase inhibitor mixed drinks. The cell lysate were brooded with against YFP conjugated Protein A/G dots in the driving rain space for a hour and a half. The globules were then washed multiple times with RIPA cushion and bubbled in 2X SDS stacking cradle for five minutes. Proteins were settled by SDS-PAGE and immunoblots were tested with antibodies against YFP, Luciferase, and PCNA.

Cell Proliferation Assays

For 3H thymidine fuse measures, cells refined in 96-well tissue-culture plates (1000∼5000 cells per well) in medium were treated with sequential weakened antibodies or DVD-Ig proteins for 96 hours, at that point 3H thymidine (1 µCi per well) was included and hatched medium-term. The cells were collected and the measure of 3H thymidine take-up was estimated utilizing microplate sparkle and a glow counter.

For province development test, N87 cells (1,000/well) were seeded into 96-well plates and hatched medium-term. Antibodies were included at the dosages demonstrated and cells were treated for ten days. Following washing, cells were settled with 4% formaldehyde, and recolored with 0.1% precious stone violet. Recolored gem violet was separated with 10% acidic corrosive and quantitated at A540.

Apoptosis Assay

Calu-3 or N87 cells (10,000/well) were seeded into 6-well plates and brooded medium-term. Antibodies or DVD-Ig proteins were included at 100 nM and apoptotic cells were meant the ensuing three days by FITC-Annexin V and propidium iodide marking as per maker's convention.

BrdU-incorporation Assay

Calu-3 or N87 cells (100,000/well) were seeded into 6-well plates and brooded medium-term. Antibodies or DVD-Ig proteins were included at 100 nM and cells were treated for two days. Cells were beat named with 10 mM BrdU for 30 minutes at 37°C and afterward collected and recolored with BrdU-FITC as per maker's convention.

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Study of scfv Antibody Library Screening

Screening and Activity of Human ScFv Antibody Targeting the Extracellular Region of Fibroblast Growth Factor R3

Fibroblast growth factor (FGF) is the largest family of growth and differentiation polypeptide factors in mesodermal and epithelial cells. Fibroblast growth factor plays an important role in many biological processes, such as embryonic development, wound healing, hematopoiesis and angiogenesis. In addition, studies have shown that fibroblast growth factor can increase the infiltration of many kinds of tumor cells, such as prostate, bladder, kidney, breast, pancreas and so on.

At present, more than 20 kinds of fibroblast growth factors have been found, which have different effects on different types of cells. However, only five kinds of fibroblast growth factor receptors (fibroblast growth factor receptors) were found. At the protein level, these receptors have 55% to 72% homology. The structure of FGFR includes an extracellular ligand binding region, a transmembrane region and an intracellular kinase region. Ligand binding regions contain three distinct immunoglobulin-like domains (called immunoglobulin I, II and III). Different splicing effects of fibroblast growth factor R1-3 mRNA form two subtypes of alpha and beta. Fibroblast growth factor R3 has two different mutants, IIIb and IIIc. The two variants have different affinity activities: IIIC is more widely distributed and can bind to various kinds of fibroblast growth factors (FGF1, FGF2, FGF4, and FGF9); IIIb preferentially binds to fibroblast growth factor 1, which can bind to fibroblast growth factor 8 and fibroblast growth factor 9 at a lower level. In the presence of heparin, the binding of fibroblast growth factors and fibroblast growth factors (FGFRs) induces receptor dimerization, which results in the phosphorylation of intracellular kinase region and the activation of downstream signal cascades. After ligand receptor binding, fibroblast growth factor (FGFs) initiates various signal transduction pathways: elevated intracellular calcium level, induction of mitogen-activated protein kinase and protein kinase C pathway, activation of adenylate cyclase and induction of proto-oncogenes c-myc and c-fos.

It has been found that special mutations of fibroblast growth factor R3 lead to activation of tyrosine kinase activity, leading to some syndromes related to skeletal development, multiple myeloma, neck tumors and bladder tumors. Recent studies have found that the signal of fibroblast growth factor receptor (FGFR) is essential for the survival of prostate cancer cells in vitro. Recently, FGFR3 has been used as a potential therapeutic target for multiple myeloma. Although there is evidence that the mutation of activated fibroblast growth factor R3 exists in cancer tissues, little is known about the expression of fibroblast growth factor R3 in cancer tissues. Recently, after gene expression analysis using gene chip technology, it was found that fibroblast growth factor R3 was overexpressed in bladder cancer samples compared with normal tissues. The level of gene expression was further confirmed at protein level by Western blot and immunohistochemical analysis. In fact, the overproduction of this protein seems to be more likely to occur in transitional tumors than in gene mutations. All these data suggest that fibroblast growth factor R3 may be a very attractive therapeutic target for urological tumors. Bladder cancer is one of the second most common malignant tumors in the reproductive and urinary system. About 40% - 50% of bladder tumors show mutations in the FGFR3 gene; epidermal tumors are more likely to occur (80%) than invasive tumors.

With the increasing interest of fibroblast growth factor R3 as a therapeutic target for different tumors and the discovery of its overexpression in transitional cell tumors, we have begun to develop human antibodies for treatment using phage display technology. Phage antibody display is currently the best way to develop human antibodies for research, clinical and therapeutic purposes. However, for antibody development, the FGFR3 molecule is very difficult to understand, because the homology of mouse and human fibroblast growth factor R3 is very high (92%). Only recently, it has been reported that a Fab fragment targeting a subtype of FGFR3 has been developed by using a commercialized Fab library with a very large storage capacity (2.1*1010). In our experiment, we used two open scFv antibody libraries Tomlinson_I+J(MRC Geneservices,Cambridge,United_Kingdom). The storage capacity of both libraries is about 1.4*108. Compared with IgG and Fabs, scFvs have better invasiveness, faster clearance and better specificity. In this report, we have screened some specific human scFv antibodies against subtype III C of FGFR3a. These antibodies have been demonstrated by FACS to react with bladder cancer cell line RT112 and inhibit cell proliferation, which has potential for further treatment.

Materials and methods

Cell lines, proteins, antibodies:

RT112, HEK293; recombinant human fibroblast growth factor R3a (IIIc)/Fc, fibroblast growth factor R1a (IIIc)/Fc, fibroblast growth factor 9, fibroblast growth factor 1 and epidermal growth factor. Mouse anti-human FGFR3 monoclonal antibody, sheep anti-human IgG (Fc specificity), mouse anti-c-myc monoclonal antibody, tubulin inhibitor; HRP-anti-c-myc antibody, anti-6His antibody, anti-M13 antibody, HRP-anti-M13 monoclonal antibody, FITC-rabbit anti-mouse IgG, R-phycoerythrin sheep anti-mouse IgG, mouse IgG TrueBlot.

Cloning and Cell Transfection of Fibroblast Growth Factor R3_cDNA

The extracellular region of fibroblast growth factor R3 (FGFR3) and the Fc_C end of human IgG were fused and expressed. The expression vectors pcDNA3.1-fibroblast growth factor R3 (IIIc) WT-Fc and pcDNA3.1-fibroblast growth factor R3 (IIIc) S249C-Fc were constructed. Then, HEK293T cells were transfected. Western blotting and FACS were used to detect protein expression and activity.

Screening of anti-fibroblast growth factor R3 specific scFv antibodies from phage library

Human scFv phage library Tomlin-son_I+J, auxiliary phage KM13, E.coli_TG1 and HB2151. Two phage libraries were cultured separately, and then 1:1 mixed phages were used for screening. Phage library screening and scFv expression were performed as shown in Figure 1. In the first round of screening, microporous plates were coated with 1ug_FGFR3 or human IgG protein. The phages were incubated with human IgG for 1 hour to remove the phages that could react with Fc, and then incubated with the holes coated with fibroblast growth factor R3 for 2 hours. Finally, the microporous plate was washed 10 times with 0.1% PBST (20 times in the next round of screening) and treated with 100 UL trypsin to elute the bound bacteriophage. Phage obtained by elution was screened in another round according to the method described by Goletz et al.

Phage ELISA

Using 0.3ug of fibroblast growth factor R3, fibroblast growth factor R1 or human IgG protein to coat the microporous plate, wash, seal, and add different concentrations of screened and purified phage suspension. After incubation, HRP-anti-M13 antibody was added, substrate color was added, and the results were read at 450 nm.

Monoclonal Phage ELISA

After two or three rounds of screening, the harvested bacteriophages were cultured and cloned. Then the monoclones were picked up by QPix high throughput automated clone screening system (Molecular Devices) and cultured on 96 microporous plates containing 100ul_2TY medium at 37 degrees. The next day, the cultures were diluted by the same medium at 1:100 degrees, then cultured in 37 degrees shaking for 2 hours, and added 25ul containing 109 auxiliary bacteriophages. Body KM13 was cultured on 2TY medium for 1 hour. After centrifugation, the bacteria were suspended in 2TY medium and cultured overnight at 30 degrees. Finally, 50 UL supernatant was centrifuged and analyzed by monoclonal phage ELISA.

Expression, Purification and ELISA Detection of Soluble ScFv Antibody

The highly specific clones were induced by E. coli_HB2151, harvested by centrifugation, and purified by affinity chromatography. The purified components were analyzed by SDS-PAGE and Coomassie brilliant blue staining. The purified components were further separated from scFv protein monomers by molecular sieve chromatography (scFv is prone to dimer or polymer).

Surface plasmon resonance analysis

BiacoreX was used to analyze the binding kinetics of soluble scFv and FGFR3, and the Kd value of each purified scFv was calculated. The specific binding sites of each scFv were determined by competitive binding analysis. The same method was used to analyze whether scFvs and FGF9, FGF1 and EGF could compete to bind to FGFR3.

Flow cytometry and confocal microscopy analysis

The binding activity of soluble scFv to fibroblast growth factor R3 at cell level was analyzed by loss cytometry. The antibody binding activity of RT112 cells was further analyzed by confocal microscopy.

Cell Proliferation Analysis

RT-112 cells were treated with different concentrations of anti-fibroblast growth factor R3_scFvs antibodies (0.02-2umol/L). After 48 hours, the cells were stained with MTT, and then 570 nm reading was performed. Cell viability was calculated by the following formula: Abs-scFv treated cells/Abs-control cells.

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