WO2015133882A1 - Scfv antibody library, method for preparing same, and scfv antibody screening method using same - Google Patents

Abstract

The present invention relates to a ScFv antibody library, a method for preparing the same, and a ScFv antibody screening method using the same and, specifically, to a library presenting a ScFV antibody comprising a heavy chain variable region (V_H) and a light chain variable region (V_Lκ or V_Lλ) of an immune organ-derived antibody, to a method for preparing the same, and a ScFv antibody screening method using the same.

Description

ScFv antibody library, preparation method thereof, and ScFv antibody screening method using the same

The present invention relates to a ScFv antibody library, a method for preparing the same, and a method for screening ScFv antibodies using the same, specifically, a ScFv comprising a heavy chain variable region (V _H ) and a light chain variable region (V _Lκ or V _Lλ ) of an antibody derived from an immune organ. The present invention relates to a library for presenting an antibody on a phage surface, a method for preparing the same, and a method for screening ScFv antibodies using the same.

Antibody drugs are growing rapidly in the biopharmaceutical field due to their high therapeutic effect and target therapeutic properties. The global market for antibody drugs continues to grow at an average annual rate of 10-15%, forming a market of $ 44.7 billion in 2010. Most of the diseases that are the main targets of antibody drugs are concentrated on specific disease indications such as intractable cancer (49%) and immune diseases (35%), and competition between similar indication products is intense. Nevertheless, the development of anti-cancer antibody therapeutics will continue to grow in the future as there are therapeutic targets segmented within the diseases.

In addition, in recent years, the field of personalized medicine is expanding due to the development of various analytical technologies. This customized disease treatment strategy is expected to be optimal for combining existing therapeutics (antibodies, synthetic drugs) or applying antibody therapeutics to new targets. Therefore, the R & D field of antibody development is expected to grow accordingly. .

Currently, techniques used for identifying and securing antibody candidates include the development of chimeric or humanized antibodies using hybridoma cell lines, methods using transgenic mice, and methods using antibody display technology. It can be divided into

Early antibody drugs were developed through antibody engineering in the form of chimeric (human / mouse) or humanized monoclonal antibodies, along with mouse monoclonal antibody development using hybridoma cell lines. However, although CDR-Grafting technology has been developed to solve the human-anti-mouse antibody (HAMA) reaction, and thus immunogenicity has been reduced, there has been a problem for the reduction of antigen binding capacity of the improved antibody. CDR-Walking technology has improved the antigen binding capacity of antibodies degraded during humanization, but has shown a problem of reducing or eliminating immunogenicity.

Due to the reduced efficacy and technical difficulties, researches for the development of human antibodies have been actively conducted. Human antibodies have a 100% human-derived antibody sequence, which completely solves the problem of immunogenicity and is an ideal therapeutic antibody.

The technical field for producing human antibodies can largely exemplify transgenic mice and antibody displays (phage display, yeast display, ribosomal display, etc.). The development of antibodies using transgenic mice, which have been tried a lot recently, is a technique for producing human monoclonal antibodies by applying the existing hybridoma technology to transgenic mice transplanted with human antibody genes. This technology can produce in vivo affinity maturation, which can produce antibodies with high affinity, and can make human antibodies effectively. However, the conditions for use of transgenic mice are expensive and manufactured. Technological entry is difficult such as knowhow.

Antibody fragment display technologies, such as phage display, yeast display, and ribosomal display, have been combined with research into the development of humanized antibodies, and have been approached with various strategies to date to identify antibody candidates. to be. Human antibody phage library (Human Antibody Phage Library) technology has been developed since the late 1990s, and has been used in various antibody development studies.

Among these, phage display is a technique for screening antibodies by expressing antibody fragments on the surface of bacteriophage, and existing antibody development techniques (chimeric / humanized antibody development using hybridomas, antibody development using transgenic transgenic mice). Compared to the above, there is an advantage in that the antigen-specific antibody can be identified in a short time. Although there is a disadvantage in that an effective antibody can be identified only when a high diversity of libraries is secured, securing a library is largely solved due to the recent development of gene amplification and cloning techniques.

Compared to the natural library based on human genes, the synthetic antibody library is created by adding random synthetic sequences to the complementarity determining regions (CDRs) of antibodies. However, synthetic antibody libraries have a lower percentage of antibody fragments that can function normally due to mutations, frameshifts, and the like, compared to natural human libraries. In recent years, strategies for using antibody libraries to identify new target antigens have been diversified, and new antigen-specific antibodies have been identified through cell panning using Tumor-derived primary cells. (Zhu X. et al., Mol. Cancer Res. 2010).

As such, since a variety of approaches are possible, phage display is an efficient approach because it can secure candidate antibodies and produce antibodies through cloning. Candidate antibody drugs targeting various types of cancers identified by applying such phage display technology are entering clinical trials. In order to utilize phage display technology, the library of antibody variable region genes is required for the identification of a desired antibody, and various library constructions are essential.

Under this background, the inventors of the present application provide a scFv antibody comprising a heavy chain variable region (V _H ) and a light chain variable region (V _Lκ or V _Lλ ) of an antibody derived from an immune organ through a library and a method for preparing the same. By confirming that human antibodies that do not induce a HAMA response can be screened conveniently and efficiently, the present invention has been completed.

Summary of the Invention

It is an object of the present invention to provide a ScFv library for screening human antibodies that can be effectively used for the treatment or diagnosis of a disease, a method for preparing the same, and a method for screening antibodies using the same.

The present invention is a library comprising a phage to present a ScFv antibody on the surface,

The ScFv antibody comprises a heavy chain variable region (V _H ) and a light chain variable region (V _Lκ or V _Lλ ) of an antibody derived from an immune organ,

The ScFv antibody was encoded from a nucleic acid synthesized using a heavy chain variable region (V _H ) and a light chain variable region (V _Lκ or V _Lλ ) specific primer of an antibody derived from an immune organ, using a nucleic acid extracted from an immune organ-derived cell as a template. It is about a library characterized in that.

The ScFv antibody may be expressed in a transformant into which a vector having the following structure in which a nucleic acid has been cloned is introduced, and present on a phage surface included in the transformant:

A-X-B-Linker-C-Y-D-E

A to D are any one restriction enzyme recognition site selected from the group consisting of SfiI, NheI, BglII and NotI,

E is a His tag or HA (Hemagglutinin) tag insertion site,

X is a gene insertion region encoding a heavy chain variable region (V _H ), Y is a gene insertion region encoding a light chain variable region (V _Lκ or V _Lλ ).

The invention also comprises the steps of extracting a nucleic acid from an immune organ-derived cell; The nucleic acid _encoding the heavy chain variable region and the light chain variable region using the extracted nucleic acid as a template and using the heavy chain variable region (V _H ) and the light chain variable region (V _Lκ or V _Lλ ) specific primers of the antibody derived from an immune organ. Amplifying; Cloning a nucleic acid encoding the heavy and light chain variable regions into a vector and then introducing the transformant into a transformant; And expressing the ScFv antibody in the transformant to present the ScFv antibody expressed on the phage surface included in the transformant.

The present invention further relates to a ScFv antibody screening method comprising reacting the library with an antigen to screen for antigen specific ScFv antibodies.

1 is a map of a pSIA23 vector constructed in accordance with the present invention.

2 is a schematic diagram illustrating a process of constructing a pSIA23 vector according to the present invention.

Figure 3 is a photograph showing the results of confirming the V _H and V _Lλ / V _Lκ gene amplification of the cDNA obtained from the bone marrow / blood / tonsil samples using the primer mixture.

Figure 4 is a photograph showing the colony PCR results of the V _H and V _Lλ / V _Lκ gene inserted in the T-vector.

5 is a photograph showing the results of confirming the insertion efficiency of V _Lλ / V _Lκ through colony PCR.

6 is a schematic diagram showing the construction of a human antibody library.

7 is a photograph showing the results of confirming the insertion of V _H through colony PCR.

8 is a graph that plots subfamily.

9 is a graph showing the results of confirming the length distribution of the sequenced antibody fragment (scFv) CDR-H3 using KABAT DB.

10 is a graph showing the results of confirming the distribution of various amino acids for each KABAT position.

Figure 11 is a photograph showing the results confirmed whether the expression of the antibody fragment (scFv) through a dot-blot experiment.

12 is a table showing the library panning results for each antigen.

Figure 13 shows the results of the screening of each antigen-specific antibody fragment (scFv).

Figure 14 shows the result of confirming the binding pattern through ELISA after phage recovery of DLL4_C01 clone.

15 shows the results of confirming the expression and purification of pure antibody fragments after transformation with the antibody fragment expression cell line (TOP10F ′).

Figure 16 shows the results confirming the binding pattern of the antibody fragment to DLL4.

Fig. 17 is a schematic diagram of cell panning identifying EGFRvIII specific antibodies using 626T, glioblastoma (GBM) patient cell.

FIG. 18 identifies EGFRvIII mutations in 626T patient cells and shows that EGFRvIII specific antibody fragments are enriched through cell panning.

19 confirms antigen-specific binding of three EGFRvIII antibodies identified in the present antibody library, measures binding constants (SPR analysis) through surface plasmon resonance, and shows cell growth inhibition. will be.

Detailed Description of the Invention and Specific Embodiments

In one aspect, the present invention is a library comprising a phage to present a ScFv antibody on the surface, the ScFv antibody comprises a heavy chain variable region (V _H ) and light chain variable region (V _Lκ or V _Lλ ) of the antibody from the immune organs And a nucleic acid extracted from an immune organ-derived cell as a template and encoded from a nucleic acid synthesized using a heavy chain variable region (V _H ) and a light chain variable region (V _Lκ or V _Lλ ) specific primers of an immune organ-derived antibody. It is about a library.

As used herein, the term "antibody" is an immunoglobulin selected from the group consisting of IgA, IgE, IgM, IgD, IgY, and IgG, and can specifically bind to a target antigen. It consists of two light chains and a heavy chain, each of which consists of a variable domain in which the amino acid sequence is variable and a constant domain having a constant sequence. The antigen binding site is located at the end of the three-dimensional structure of the variable region, which is formed by the collection of complementarity determining regions, each of which is present in the light and heavy chains. Complementarity determining regions are particularly highly variable parts of the amino acid sequence among the variable region, the antibody specific for a variety of antigens can be found by this high variability.

"ScFv (single-chain Fv, single chain fragment antibody or antibody fragment)" is an antibody connecting the variable region of the light chain and heavy chain. In some cases, it may include a linker consisting of a peptide chain of about 15 amino acids linked, wherein the ScFv is a light chain variable region-linked region-heavy chain variable region, or heavy chain variable region-linked It may have a structure of a site-light chain variable region and has the same or similar antigenic specificity as the original antibody.

An "antibody (or ScFv) library" is a collection of various antibody genes with different sequences. Very high diversity is required to isolate antibodies specific for any antigen from an antibody library, and libraries of different antibody clones are constructed and used. The antibody gene constituting such an antibody library can be cloned into a phagemid vector, for example, and transformed into a transformant (E. coli).

The “nucleic acid” may be used interchangeably with genes or nucleotides, for example, but may be selected from the group consisting of natural / synthetic DNA, genomic DNA, natural / synthetic RNA, cDNA, and cRNA, but is not limited thereto.

A "phagemid" vector is used for phage display and is a plasmid DNA having a phage origin of replication, and typically has an antibiotic resistant gene as a selection marker. The phagemid vector used for phage display includes the gIII gene of M13 phage or a part thereof, and the ScFv gene is ligated at the 5 'end of the gIII gene and expressed through a transformant.

A "helper phage" is a phage that provides the genetic information needed for phagemids to be assembled into phage particles. Since phagemid contains only gIII or a part of phage gene, the host cell (transformer) transformed with phagemid is infected with a helper phage to supply the remaining phage gene. M13K07 or VCSM13 are available and most include antibiotic resistance genes such as kanamycin, which allows the selection of transformants infected with helper phage. In addition, since there is a defect in the packaging signal, the phagemid gene is selectively assembled into the phage particle rather than the helper phage gene.

A "signal sequence" is a nucleotide sequence or the corresponding amino acid sequence which is located at the 5 'end of a gene and functions as a signal required when a protein encoded from the gene is secreted to the outside.

In one embodiment, the ScFv antibody may be expressed in a transformant into which a vector having the following structure in which a nucleic acid is cloned is introduced, and present on a phage surface included in the transformant.

A-X-B-Linker-C-Y-D-E

A to D are any one restriction enzyme recognition site selected from the group consisting of Sfi I, Nhe I, BglII and Not I,

E is a His tag or HA (Hemagglutinin) tag insertion site,

X is a gene insertion region encoding a heavy chain variable region (V _H ), Y is a gene insertion region encoding a light chain variable region (V _Lκ or V _Lλ ).

In the AXB-linker-CYDE structure according to the present invention, the gene sequence for the restriction enzyme recognition site of A to D is a gene encoding V _H to be inserted into the X site and a gene encoding V _Lκ or V _Lλ to be inserted into the Y site. In consideration of the sequence of, each gene should be selected within a range that does not cleave.

The vector comprising the structure according to the present invention may be, for example, a pSIA23 vector, the cleavage map of which is as shown in FIG. 1. Referring to the structure of the pSIA23 vector shown in FIG. 1, the structure may include a recognition site of a restriction enzyme of SfiI-NheI-BglII-NotI in the A to E direction, that is, the 5 'to 3' direction. 1, A may be SfiI, B is NheI, C is BglII, and D may be a recognition site of NotI restriction enzyme.

The linker is a linking region connecting the heavy and light chain variable regions in the ScFv described above, and is a hydrophilic flexible peptide chain mainly composed of glycine and serine ("Gly-Gly-Gly-Gly-Ser"). Genes encoding 15 amino acid sequences of ₃ "or similar sequences can be used.

The process of producing a vector comprising the structure according to the invention is shown in FIG. 2. Referring to Figure 2, based on the pCANTAB 5E phagemid vector construct a restriction enzyme recognition site for insertion of the heavy chain variable region (V _H ) and light chain variable region (V _Lκ or V _Lλ ), and inducing expression into the surrounding cytoplasm The pelB sequence can be added as one of the signal sequences. In addition, the label tag can be replaced with His6-tag and HA-tag which are used more widely than the E-tag of the pCANTAB 5E vector. Subsequently, a library vector pSIA23 including restriction enzyme recognition sites excluding restriction enzymes capable of cleaving X and Y through restriction enzyme recognition site optimization may be prepared.

Antibodies expressed in the transformants into which the vector is introduced can be identified through labeling or ELISA through antigens tagged with fluorescent markers.

Once a transformant is expressed that expresses an antibody having the desired properties, the nucleic acid encoding the antibodies can be isolated and tested again to see if the nucleic acid encodes an antibody with the desired biological properties.

In one embodiment, the ScFv according to the present invention may be an unsensitized human antibody. The ScFv may be an unsensitized human antibody derived from an immune organ such as bone marrow, blood or tonsils. The heavy chain variable region (V _H ) and light chain variable region (V _Lκ or V _Lλ ) genes of the _ScFv may be obtained by extracting RNA from cells isolated from human immune organs. In particular, the inventors of the present application secured RNA (cDNA) for ScFv library construction using tonsil tissue with various B cell repertoires (Example 2).

For example, the sequence number for the primer mixture, for example, V _H gene amplification of the extracted synthesizing a cDNA of the RNA, then Table 1 (SEQ ID NO: 7 to 58) from the mononuclear cells isolated from the human immune system 7 and Amplifying a cDNA with a mixture comprising at least one primer selected from the group consisting of the sequences represented by 24 and at least one primer selected from the group consisting of the sequences represented by SEQ ID NOs: 25-58 for amplification of the V _L gene, V _H And genes encoding V _L can be obtained. Since the heavy chain variable region (V _H ) and the light chain variable region (V _Lκ or V _Lλ ) are 100% human-derived, the ScFv according to the present invention does not exhibit the HAMA response seen in existing techniques (eg hybridomas, etc.). .

In this case, the obtained heavy chain variable region (V _H ) and the light chain variable region (V _Lκ or V _Lλ ) may have the sequences of Tables 6, 7 and 8 (SEQ ID NOs 63 to 254), respectively. Specifically, the heavy chain variable region is SEQ ID NO: 63-104 (heavy chain variable region of V _H -V _Lλ having the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4), 147-200 (V _H -V _Heavy chain variable region of _Lκ may have any sequence selected from the group consisting of the sequence represented by FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the light chain variable region is SEQ ID NO: 105 -146 (V _Lλ , has the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4), 201-254 (V _Lκ , has the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) It may have any one sequence selected from the group consisting of the sequence represented by.

In consideration of the HAMA response, research / product development of human antibodies has been actively conducted, and since ScFv to be identified through the library according to the present invention has a size of about 1/6 of the antibody, penetration rate into tissues or tumors is increased. As it is excellent, it can be used for biological diagnosis.

In some cases, ScFv can be converted into immunoglobulin to be used as a therapeutic antibody. In addition, although the half-life is short in the human body, it is possible to develop a therapeutic antibody by increasing the half-life through PEGylation (PEGylation) technology, a drug delivery system. In addition, it is applicable to the development of new types of antibody platform, such as bispecific antibodies.

The diversity of the library according to the present invention can exhibit a diversity of about 10 ⁸ to 10 ¹⁴ at a high level, and the antibody library of about 10 ¹⁰ level can be constructed by the present invention. In some cases, the diversity may be further increased by any combination _resulting from the heavy (V _H ) and light (V _Lκ or V _Lλ ) gene combination process of the antibody during cloning.

In another aspect, the present invention comprises the steps of extracting the nucleic acid from immune-derived cells; The nucleic acid _encoding the heavy chain variable region and the light chain variable region using the extracted nucleic acid as a template and using the heavy chain variable region (V _H ) and the light chain variable region (V _Lκ or V _Lλ ) specific primers of the antibody derived from an immune organ. Amplifying; Cloning a nucleic acid encoding the heavy and light chain variable regions into a vector and then introducing the transformant into a transformant; And expressing the ScFv antibody in the transformant to present the ScFv antibody expressed on the phage surface included in the transformant.

The preparation method according to the present invention comprises the step of extracting nucleic acids from cells derived from immune organs. In one embodiment, RNA can be obtained by extracting RNA from cells isolated from an immune organ, such as bone marrow, blood or tonsils.

In the preparation method according to the present invention, the extracted nucleic acid is used as a template, and the heavy chain variable region and the light chain using heavy chain variable region (V _H ) and light chain variable region (V _Lκ or V _Lλ ) specific primers of the antibody derived from an immune organ. Amplifying the nucleic acid encoding the variable region. For example, after synthesizing cDNA from the extracted RNA, the primer mixture of Table 1 (SEQ ID NO: 7 to 58), for example, in the group consisting of the sequence represented by SEQ ID NO: 7 to 24 for V _H gene amplification as for the selection of one or more primers and V _L gene amplification SEQ ID NO: consisting of a sequence represented by 25-58 mixture comprising at least one primer selected from the group amplifies the cDNA to obtain a gene encoding the V _H and V _L can do.

Thereafter, the nucleic acid encoding the heavy chain variable region and light chain variable region is cloned into a vector and then introduced into a transformant. The ScFv antibody may be introduced into a transformant into which a vector of the following structure into which a nucleic acid has been cloned has been introduced:

A-X-B-Linker-C-Y-D-E

A to D are any one restriction enzyme recognition site selected from the group consisting of Sfi I, Nhe I, BglII and Not I,

E is a His tag or HA (Hemagglutinin) tag insertion site,

X is a gene insertion region encoding a heavy chain variable region (V _H ), Y is a gene insertion region encoding a light chain variable region (V _Lκ or V _Lλ ). The detailed description of each structure is as mentioned above.

The production method according to the present invention includes expressing the ScFv antibody in the transformant, and presenting the ScFv antibody expressed on the phage surface included in the transformant. The type of the transformant is preferably, for example, Escherichia coli. E. coli DH5a, E. coli JM101, E. coli K12 294, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, FMB101, NM522, NM538 and NM539 Including but not limited to.

The invention also relates to a ScFv antibody screening method comprising the step of screening an antigen specific ScFv antibody by reacting the library with an antigen.

The screening method may include culturing the transformant and phage of the library, binding the ScFv antibody expressed on the phage surface with the antigen, and screening and selecting a transformant expressing the desired antibody. Such screening and screening steps can be performed using various techniques known in the art.

For example, a ScFv antibody that binds to a specific antigen can be isolated and prepared from the library by a panning method. The panning is a process of binding a phage to a target antigen and removing unbound phage, amplifying the phage by recovering the bound phage and infecting the host cell, and repeating this process 2-4 times. It may include.

According to the screening method of the present invention, ScFv antibodies for treating autoimmune diseases or cancer can be screened. For example, ScFv antibodies can be screened specifically for binding to factors known to cause autoimmune diseases or cancers, and the screened ScFv antibodies act as antagonists, leading to expression of autoimmune diseases or cancer causing targets. By suppressing, the disease can be treated.

Autoimmune disease refers to a disorder caused by an autoimmune process in which immune system components, such as antibodies or lymphocytes, attack or harm molecules, cells or tissues of the organisms that produce them, for example multiple sclerosis, allograft rejection. , Autoimmune thyroid disease, inflammatory bowel disease (Crohn's disease, ulcerative colitis, local enteritis, etc.), psoriasis, rheumatoid arthritis or systemic lupus erythematosus.

Cancer may mean a disease caused by neoplastic cell growth and proliferation and unregulated cell growth / proliferation. Examples of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, Hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, gastric cancer, melanoma, and various types of head and neck cancer However, the present invention is not limited thereto.

In one embodiment, the ScFv antibody targeting the DLL4 (Delta like ligand 4) protein can be screened by the screening method according to the present invention, and the screened ScFv antibody was found to have a significant binding capacity to the DLL4 protein. (See Example 13-16). In another embodiment, EGFRvIII specific antibodies were identified through cell panning techniques, and the screened ScFv antibodies were found to have significant binding capacity to EGFRvIII, and showed cell growth inhibition (see Examples 17-19). ).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example

Example 1: phagemid vector construction for antibody library construction

Based on the pCANTAB 5E phagemid vector, a restriction enzyme recognition sequence insertion for V _H and V _L insertion and a pelB sequence for insertion of expression into the periplasm were inserted into the vector. In addition, pSIA23 vector, which is a vector for constructing a library of itself, was constructed by changing a label tag. In order to construct a library through sequential insertion of V _L → V _H , restriction enzymes Sfi I, Nhe I recognition sequences are inserted at the sites where V _H is to be inserted, and BamH I and Not I recognition sequences are inserted at the sites where V _L is to be inserted, and the pelB sequence is also inserted. Four oligonucleotides were designed for insertion. Oligonucleotides used are as follows.

SEQ ID NO: 5'-TAC GCC AAG CTT GGA GCC ATG AAA TAC CTG CTG CCG ACC GCT GCT GCT GGT-3 ',

SEQ ID NO: 5'-TCA GCG CAA CAC GCT AGC GGT GGA GGC GGT TCA GGC GGA GGT GGA TCC GGC GGT GGC GG-3 ',

SEQ ID NO: 3'-CTG GCG ACG ACG ACC AGA CGA CGA GGA GCG CCG GGT CGG CCG GAG TCG CGT TGT GCG AT-5 '

SEQ ID NO: 3'-TAG GCC GCC ACC GCC TAG CCC TAG GAC ACT TAC CCT GCG CCG GCG TTA AT-5 '.

The template to be inserted was completed by assembly PCR using four oligonucleotides, and insertion into the skeletal vector (pCANTAB 5E) cut with HindIII and NotI was completed. His6-tag and HA-tag, which are used more widely than the E-tag of the existing pCANTAB 5E vector, were inserted. To replace the label tag, a primer set in which restriction enzyme NotI and EcoRI recognition sequences were introduced at both ends was synthesized to amplify His6-HA-Amber codon-pIII sequence from pCOMB3X phagemid vector. Primers used were as follows (SEQ ID NO: 5 (F-primer (Not I)): 5'-TTT GCG GCC GCC ACC ATC ACC ATC ACC ATG G-3 ', SEQ ID NO: 6 (R-primer (EcoR I)): 5'-CGG AAT TCA GAC TCC TTA TTA CGC AGT ATG TTA GC-3 ']. It was inserted into pSIA23 vector digested with NotI / EcoRI. After confirming in literature that the restriction enzyme recognition sequence of BamHI used in the initial vector design was inside the light chain sequence, BamHI was converted into BglII, and the construction of the final library vector (pSIA23) was completed as shown in FIG. 1.

Example 2: Obtaining Each Organ Tissue and cDNA for ScFv Library Construction

The variable region gene extraction of the bone marrow (20) / blood (40) / tonsil (16) -derived antibodies was carried out as follows. The library of the present invention is technically distinctive and unique in that RNA for constructing ScFv libraries (cDNA) was obtained using tonsil tissue having various B cell repertoires.

Bone marrow from healthy donors was obtained through STEMCELL Technologies in the form of frozen primary cells (~ 1.5 × 10 ⁷ cells / vial, total 20 vial). After securing only mononuclear cells using a ficoll gradient, RNA extraction was performed as follows. After addition of 1 mL of Tri-reagent (Molecular Research Center) and repeated pipetting, the cells were allowed to stand at room temperature for 5 minutes and lysed. Then, 100 μL of biphasic calcium phosphate (BCP) was added and shaken several times for 15 seconds. After centrifugation at 4 ° C and 13,000 rpm for 15 minutes, only the supernatant was carefully separated, 500 μL of isopropanol was added, and then allowed to stand at room temperature for 10 minutes. RNA granules were obtained by centrifugation at 15 ° C. and 13,000 rpm for 10 minutes. In order to increase the purity of the RNA obtained, the mixture was stirred with 1 mL of 75% EtOH and stirred vigorously, followed by centrifugation at 15 ° C. and 13,000 rpm for 5 minutes. After discarding EtOH, the granules were dried at room temperature (about 10 minutes), suspended in 50 μL of RNase-free water (DEPC-treated), and RNA concentration was measured using a Bioanalyzer (Nanodrop). In the case of blood, 10 mL of healthy donor blood was mixed with 10 mL of PBS, and mononuclear cells were obtained by Ficoll gradient, and RNA was extracted in the above manner. In the case of tonsil, 2 mL of Tri-reagent (Molecular Research Center) was added to 200 mg of tonsil tissue, followed by tissue disruption. After 200 μL of BCP was added to the crushed tissue, the experimental method was performed as described above. Using 5 μg of each prepared RNA, cDNA was synthesized using a superscript III cDNA synthesis kit (Invitrogen), and used for amplifying the variable region of the antibody.

Example 3: V _H and V _{L (λ / κ)} Gene Amplification

A cDNA mixture synthesized in three bone marrow / blood / tonsil tissues was used as a template, and PCR was performed using primers of 14 heavy chains and 28 light chains (kappa 13 and lambda 15). Primer sets for amplification of V _H , V _Lκ , V _Lλ fragments were designed and used as shown in Table 1 below.

Table 1

Restriction sequences inserted for cloning variable region genes in the constructed pSIA23 phagemid were SfiI-NheI (for V _H insertion) and BglII-NotI (for V _L insertion). Accordingly, as shown in Table 1, SfiI-NheI was inserted at both ends of the V _H fragment to be amplified, and BglII-NotI was inserted at both ends of the V _L fragment. For heavy chain variable region gene amplification, 14 F-primers and 4 R-primers were used, and the synthesized primers were divided by the difference in gene expression of the variable region subgroups, and V _H is a total of 5 sets as shown in Table 2. V _H1 (SEQ ID NOs 7-10), V _H3 (SEQ ID NOs 13-15), V _H5 (SEQ ID NO 18), V _H6 (SEQ ID NO 19), V _{H2 + 4 + 7} (SEQ ID NOs 11-12, 16 -17, 20) divided into F-primers, and primers in which JH ₁₂ , JH ₄₅ , JH ₃ , JH ₆ (SEQ ID NOs: 21-24) R-primers were mixed in equal moles were used.

TABLE 2

Light chain variable region gene amplification was also divided into four sets of F-primers, each of 13 F-primers (SEQ ID NOs: 25-37) for kappa amplification and 15 F-primers (SEQ ID NOs: 38-52) for lambda amplification. , Amplification was performed by mixing the R-primer mixture (kappa: SEQ ID NO: 53-54, lambda: SEQ ID NO: 55-58) corresponding to each molar number. PCR conditions for V _H / V _L amplification are as follows. 95 ° C., 5 minutes; (95 ° C., 30 sec-56 ° C., 30 sec-72 ° C., 45 sec) 30 repetitions; 72 ° C., 7 minutes. Upon completion of the amplification, the V _H fragment was amplified to about 400 bp including Sfi I and Nhe I, and the V _L fragment was about 360 bp including BglII and Not I sites. The results are shown in FIG. 3. Referring to FIG. 3, it was confirmed that various V _H and V _Lλ / V _Lκ genes were successfully amplified. The amplified PCR result was purified and concentrated through EtOH precipitation after PCI extraction. Concentrated PCR results were subjected to electrophoresis on 2% agarose gel to confirm their size (V _H = ~ 400 bp, V _Lλ / V _LK = ~ 350 bp), and each recovered fragment was subjected to cloning after DNA concentration measurement. Store at 20 ° C.

Example 4: Subcloning with V _H and V _{L (λ / κ)} Gene T-Vector

Before proceeding with the antibody library construction, T-vector subcloning was performed to confirm that there was no problem with the sequence of the inserted variable region. The ligation was carried out at 16 ° C. overnight culture conditions using 100 ng and 40 ng of the T-vector: molar ratio of V _H & V _Lλ / V _Lκ fragment as 1: 3. After incubation for 10 minutes at 65 ℃, the enzyme was eluted with 10μL in dH ₂ O using a microcon centrifugal filter devices (Millipore) was purified and concentrated. T-vector, 5μL of antibody variable region gene, and 25μL of electro-competent cell (E. cloni 10G, Lucigen) were mixed and transferred to 0.1cm electro-cuvette and transformed at 1.8kV. The transformed host was suspended in 1 mL SOC medium and incubated for 1 hour at 37 ° C., and then titrated through 10 μL sequential dilution (10 ⁻² to 10 ⁻⁶ ), and the remaining cultured transformants were 15 cm plate (LB / ampicillin). /0.5% glucose) to obtain host cells grown by incubating overnight at 37 ℃, overnight, divided and stored at -80 ℃. As a result, the V _Lκ T-vector library (total ~ 1.29 × 10 ⁸ cfu) was obtained through 10 T-vector-V _Lκ transformations as shown in Table 3, and 8 T-vector-V _Lλ fragment transformations. Through the V _Lλ T-vector library (total 1.05 × 10 ⁸ cfu) was obtained. In addition, six T-vector-V _H transformations yielded a V _H T-vector library (total -3.03 × 10 ⁸ cfu).

TABLE 3

Example 5: Performance Evaluation of V _H and V _{L (λ / κ)} Genes

Using a T-vector in a transformed colony in which VH and V _{L (λ / κ)} fragments were subcloned as a template, PCR was performed using a primer that recognizes a specific site in the plasmid, thereby performing V _H and V _{L (λ / κ). κ) the} fragment was inserted correctly. After V _H and V _{L (λ / κ)} transformation, colonies were tipped and mixed into the PCR mixture. The primer sequences used for colony PCR are as follows.

SEQ ID NO: 59 (F-primer (M13-F)): 5'-ACA GGA AAC AGC TAT GAC-3 ',

SEQ ID NO: 60 (R-primer (M13-R)): 5'-GTT TTC CCA GTC ACG A-3 '.

PCR was carried out, and 2% agarose gel was used to confirm proper insertion, and the results are shown in FIG. 4. Referring to Figure 4, it was confirmed that most of them are properly inserted without problems. V _H and V _{L (λ / κ) were} transformed, plasmids of individual colonies were obtained, and then functionalized by analyzing the cloned VH and V _{L (λ / κ)} sequences using M13 F-primers. It was confirmed whether ScFv without expression can be expressed. After DNA sequence analysis, germ-line sequences (complementarity determining regions, skeletal regions) of antibodies were analyzed using VBASE2 DB (http://www.vbase2.org). Since a simple check is made before moving on to the next step, the sequences analyzed here are not shown extensively, only the number of clones and the percentage of clones that can function normally. A total of 77 clones of the heavy chain variable region and 140 clones of the light chain variable region were analyzed, and the 'VBASE2' antibody germ-line seq. As a result of sequence analysis through the DB, 36 problematic sequences were identified, including 14 'out of frame', 13 'stop codon insertion' and 9 'problematic chain'. As a result, 181 clones out of 217 clones were found to have the correct sequence, confirming that about 83.4% were effective clones. 83.4% of effective V _H and V _L are considered in the case of building libraries using human immune tissues considering the factors that affect the normal sequence maintenance of variable regions such as similar genes, invalid skeletal genes, and PCR error rate. _It is believed that the T-vector _{sublibrary with the (λ / κ)} gene was constructed appropriately.

Example 6: pSIA23-LC (light chain) construction

To identify antigen-specific antibody fragments using phage display technology, the V _H and V _{L (λ / κ)} genes were inserted into the library construction vector (pSIA23), and then transferred through an appropriate E. coli host (eg, TG1, ER2537). It should be expressed on the surface of the M13 bacteriophage in ScFv form. After inserting the V _{L (λ)} gene and the V _{L (κ)} gene into the pSIA23 vector, two sub-libraries are made. After confirming that each light chain gene is properly inserted into the pSIA23 vector, the V _H gene is inserted. Hosts with T-vector-V _{L (λ)} and T-vector-V _{L (κ)} were grown in 500 mL LB / ampicillin at 37 ° C. overnight, followed by Maxi-prep.

The pSIA23 vector, T-vector-V _{L (λ)} and T-vector-V _{L (κ)} were treated with 5U / μg of restriction enzymes BglII and NotI, respectively, and cleavage was performed at 37 ° C. overnight. Restriction digestion was repeatedly performed to obtain a large number of V _L genes and a pSIA23 vector that was cleaved and inserted into the V _L gene in the next step. T4 ligase in the middle of the night culture azepin 16 ℃ insert the V _L genes in pSIA23 vector and the resulting pSIA23-V _L using Microcon centrifugal filter devices (Millipore) and concentrated to pSIA23-V _L to a final volume of 10μL. 5 μL of concentrated pSIA23-V _L and 25 μL of electro-competent cells (E. cloni 10G, Lucigen) were mixed and transferred to 0.1 cm electro-cuvette and transformed at 1.8 kV. The transformed host was suspended in 1 mL SOC medium and incubated for 1 hour at 37 ° C., and then titrated through 10 μL serial dilution (10 ⁻³ to 10 ⁻⁷ ), and about 1 mL of the transformants were 15 cm plate (LB / Ampicillin). /0.5% glucose) and then cultured overnight at 37 ℃, to ensure the transformed host, aliquoted and stored at -80 ℃. Through nine transformations of pSIA23-VLλ, a pSIA23-VLλ library (total ˜6.44 × 10 ⁸ cfu) was obtained as shown in Table 4.

Table 4

Colony PCR was performed using the following primers to confirm that the V _{L (λ)} gene was correctly inserted into the library construction vector, and the results are shown in FIG. 5.

SEQ ID NO: 61 (F-primer (pelB-F)): 5'-ATG ATT ACG CCA AGC TTG GAG CC-3 ',

SEQ ID NO: 62 (R-primer (pelB-R)): 5'-GGA ACC AGA GCC ACC ACC GGA A-3 ']

Referring to FIG. 5, it was confirmed that 93.8% was properly inserted, and 90% of sequencing analysis confirmed that there is no problem in complementarity determining and skeletal sites. Nine times through transfection with _{pSIA23-V Lκ, pSIA23-V} Lκ library (total ~ 5.23 × 10 ⁸ cfu) were obtained (Table 4). Colony PCR was performed in the same manner as above, and 87.5% confirmed that the V _{L (κ)} gene was correctly inserted, and 90% confirmed that there was no problem in complementarity determining and skeletal regions.

Example 7: Completed library construction by constructing pSIA23-ScFv (V _H -V _L ) and inserting into host cell (TG1)

Inserting the V _H gene into the previously constructed pSIA23-V _L to prepare for constructing pSIA23-ScFv (pSIA23-V _H -V _L ), E. coli with T-vector-V _H and pSIA23 obtained in the previous step Escherichia coli transformed with -V _Lλ & pSIA23-V _Lκ were _grown in 500 mL LB / ampicillin at 37 ° C., overnight culture conditions. After securing a large-scale plasmid by Maxi-prep, restriction enzyme cleavage was repeatedly performed on pSIA23-V _Lλ & pSIA23-V _Lκ and T-vector-V _H under the following conditions. Since restriction enzyme cleavage conditions were different, NheI digestion (5U / ug, 37 ° C, midnight culture) was performed first, followed by SfiI (5U / ug, 50 ° C, midnight culture) digestion. A large amount of V _H gene and restriction enzyme truncated pSIA23-V _Lλ & pSIA23-V _Lκ were obtained and proceeded repeatedly. Inserting the VH gene into the pSIA23-V _L vector under the following conditions, and the resulting pSIA23-ScFv (pSIA23-V _H -V _Lλ , pSIA23-V _H -V _Lκ ) using Microcon centrifugal filter devices (Millipore), PSIA23-ScFv was concentrated to 10 μL of final volume dH ₂ O. Since the light and heavy chains are sequentially inserted, the light chain and the heavy chains can be utilized for light chain shuffling as compared to a library made through the conventional PCR method (SOE-PCR). In particular, when a library is prepared through overlap extension PCR, the combination of the light chain-linker-heavy chain is inserted into the phagemid vector at once, and thus, HC-shuffling or LC-shuffling techniques cannot be applied. However, in the library prepared according to the method of the present invention, the light and heavy chain sequences were inserted sequentially, and since there are restriction enzyme sequences at both ends of the light and heavy chain sequences, the light and heavy chain sequences may be separated if necessary. In this case, through affinity maturation by applying HC-shuffling or LC-shuffling, it is possible to finally obtain a better antibody fragment binding capacity. A schematic diagram of the pSIA23-ScFv library constructed according to the present invention is shown in FIG. 6.

5 μL of pSIA23-ScFv and 25 μL of electro-competent cells (TG1, Lucigen) were mixed and transferred to 0.1 cm electro-cuvette for transformation at 1.8 kV. The transformed host was suspended in 1 mL SOC medium and incubated at 37 ° C. for 1 hour, and then 10 μL was titrated through sequential dilution (10 ⁻³ to 10 ⁻⁷ ), and the remaining culture solution was 15 cm plate (LB / ampicillin / 0.5%). Glucose), and cultured overnight at 37 ° C. to ensure transformed E. coli, and then aliquoted and stored at −80 ° C. pSIA23-V _H -V _Lλ (~ 6.14 × 10 ⁹ cfu) and pSIA23-V _H -V _Lκ (~ 5.64 × 10 ⁹ cfu) were obtained as shown in Table 5 through 44 transformation of pSIA23-ScFv into TG1. .

Table 5

The final library's diversity, combining the two sub-library, is 1.18 × 10 ¹⁰ cfu. The size of a library is one of the most important factors in assessing its quality. The larger the library is, the more various antibodies it has, and the higher the probability of finally identifying an antibody specific for a desired antigen. Additionally, 92.2% (pSIA23-V _H -V _Lλ ) 93.8% (pSIA23-V _H -V _Lκ ) in colony PCR with the pelB primers used previously to confirm that the V _H -V _L gene was inserted correctly. It was confirmed that the insertion was properly, the results are shown in FIG. Even if properly inserted, if there is a mutation such as a frameshift in the sequence, the protein may not be properly expressed due to a stop codon, and even if expressed, a protein that cannot function as an antibody may be expressed. The following sequencing is intended to analyze the number of functionally benign antibody clones.

Example 8: Antibody Library Sequencing

For variable region sequencing of the human Naive ScFv Library with 1.18 × 10 ¹⁰ levels of transformants, sequencing was performed after each plasmid was obtained by culturing any transformant. The results are shown in Tables 6, 7 and 8. 42 (65.6%) of 64 analyzed pSIA23-V _H -V _Lλ (V _H : SEQ ID NO: 63-104, V _Lλ : SEQ ID NO: 105-146) have normal complementarity determining and skeletal regions Confirmed. In the combination of V _H -V _{L (λ)} , 02, 15 and 48 clones cannot be expressed in the form of an antibody such as scFv because they have a stop codon on the variable region of the heavy chain. In the case of 24 clones, the antibody heavy chain D and J gene fragments are analyzed to be absent from the sequence and thus cannot function as antibodies. In the case of 05, 46, 54, the antibody cannot perform the function of the antibody because it is not a native sequence of the antibody due to the insertion, deletion, or mutation of the nucleotide sequence in the antibody heavy chain variable region. The nucleotide sequence of the 32, 59 clone heavy chain was excluded because it is not the sequence of the antibody. For clones 06, 07 and 38, stop codons were identified in the antibody light chain variable region. Clones 09, 21, 58 do not have an antibody-specific sequence due to the insertion, deletion, mutation, etc. of the nucleotide sequence in the antibody light chain sequence. For clones 22 and 50, the sequence of the light chain is not the sequence of the antibody. In the case of the 27 clone, the antibody heavy chain D and J gene fragments were analyzed to be absent from the sequence, so that the antibody could not function as the antibody, and the light chain was also a clone that could not function as an antibody. For clones 33 and 59, neither the antibody heavy chain nor the light chain shows the antibody sequence. For 63 clones, stop codons were found in both the heavy and light chains of the antibody.

In addition, 54 out of 78 (69.2%) of the analyzed pSIA23-V _H -V _Lκ (V _H : SEQ ID NO: 147-200, V _Lκ : SEQ ID NO: 201-254) are normal complementarity determining regions and skeletal regions. It was confirmed to have. 30, 40, 45, 51, 59 clones in the combination of V _H -V _{L (κ)} cannot be expressed in the form of an antibody such as scFv because they have a stop codon on the variable region of the heavy chain. In the case of 21, 24, 56 clones, it is not possible to perform the function of the antibody because it is not the native sequence of the antibody due to the insertion, deletion, mutation, etc. of the nucleotide sequence in the antibody heavy chain variable region. The base sequence of the 43, 55, 69, 75 clone heavy chain was excluded because it is not the sequence of the antibody. In the case of 71 clones, a stop codon was identified in the antibody light chain variable region. 14, 21, 27, 44, 61, 65, 66, 77 clones do not have an antibody-specific sequence due to the insertion, deletion, mutation, etc. of the nucleotide sequence in the antibody light chain sequence. For clone 08, the sequence of the light chain is not the sequence of the antibody. For 16 clones, neither the antibody heavy chain nor the light chain shows the antibody sequence. For 64 clones, stop codons were found in both the heavy and light chains of the antibody.

Table 6

lambda	VH			VL			IGHV Subfamily	IGKV Subfamily
Clone	CDR1	CDR2	CDR3	CDR1	CDR2	CDR3
VH-VL (λ) 01	GYTFTNDY	INPSGGST	ARERSSTGDNNWFDP	SLRPYY	TNT	LSRDITANYVL	One	3
VH-VL (λ) 02	EDTFKRNA	IVPILGLA	ARSGVVAAKVDQ	SLRSCS	GKN	NSRDIS	One	3
VH-VL (λ) 03	GASVSRWS	VLHSGST	ARDGRFSGSGSYAFDV	SSNIGSNT	SNN	AAWDDSLNGVV	4	One
VH-VL (λ) 04	GGSFSGYY	INHSGRT	VQQKLGNDY	SDHVGSYNL	EGN	SSYTGVAIFV	4	2
VH-VL (λ) 05	GIIFSDN	ISSSSGYT	ASHKGFGSRLITGAREPWSP	SLRSYF	GAN	NSRDSSGNRWV	3	3
VH-VL (λ) 06	GGSFSEYY	INHAGRT	ALELDAARRGVDY	SSNIGVNS	RNT	AAWDDSLSACV	4	One
VH-VL (λ) 07	GGTFRRYA	IIPTLDTP	ARDMNSYYDGRGSPGASDL	TSNIGAGYD	GNT	ETYDSSLSAVIFGGGTS * PSx	One	One
VH-VL (λ) 08				SLRSYY	GKN	NSRDSSGNHLV	―	3
VH-VL (λ) 09	GGTFSSFA	IIPMFDTP	ARDARDYYDSEGYLGAFDI	SLRSYY	GKN	NSRDSSGNHVVFGGRTKVTVL	One	3
VH-VL (λ) 10	GFSLTTYW	INREGTVT	ARDNTHYGSSALEYYDAFDL	TGAVTNGHY	STA	LLFYGGAQLV	3	7
VH-VL (λ) 11	GFSLSAPSVG	IYWNDNK	AHGAGWLFDY	SLRSYF	GNN	NSRDSGAGHPYV	2	3
VH-VL (λ) 12	GFSLSTSGLT	IYWNNDK	GHRRRVNEGGKGGFDY	ALPKHF	KDT	QSADSSDSYVV	2	3
VH-VL (λ) 13	GFKFSDYY	IGNDDSVK	ARDGGRYYDYNGLDV	SLRSYY	GKN	NSRDSSGNHLV	3	3
VH-VL (λ) 14	GFTFDTYW	IKEDGTET	AGFAPF	SSNIGSNS	ADS	APWNSSLNGYV	3	One
VH-VL (λ) 15	GFSLITPGMC	IDWDDKK	ARKVYGNIDAFDI	SSYIGNNY	DNN	GTWDSSLSAGV	2	One
VH-VL (λ) 16	GFTFNDYY	ISGAGNTI	ARGRYFYYMDV	SLRSYY	LEN	NSRDSSGDRVL	3	3
VH-VL (λ) 17	GYSFSNYW	INPGDSDV	ARLHTGQHLVGDWYFDI	SSDVGAYDY	DVI	CSYVGSYTYV	5	2
VH-VL (λ) 18	GGFISSTTYY	IYHTGNT	ARQACYRGSCYSRATYFDQ	ALPKKY	EDD	FSTDSSGKHGV	4	3
VH-VL (λ) 19	GYTFSSYW	IYPGDSDA	ARQVQPVYHYHYLDV	SSNIGRNT	KND	AAWDDSLSVV	5	One
VH-VL (λ) 20	GFTFSSYS	ISSSSSTI	ARKETTVNYYMDV	SSNIGSSF	DNN	GTWDSSQSAVV	3	One
VH-VL (λ) 21	GFTFSNYW	IRRVGSDK	ARDLNPRTNSRDPLDAYDV	SLRSYY	GKN	NSRDSSGNHLVFGGSTQLTVL	3	3
VH-VL (λ) 22	GFTFTSSA	IVVASGNT	AADYDFSFYYASDV				One	―
VH-VL (λ) 23	GDSVGSGNYY	IYYDGRT	ATGVEAGSQGYFDV	SLRSYY	GKN	NSRDSSGNHLV	4	3
VH-VL (λ) 24	GDFFTSYG	ISAFNGNR		SLRSYY	GKN	NSRDSSGNHLV	One	3
VH-VL (λ) 25	GDTFSAYA	IVPIFDRT	ARDHRDGYKYYFDS	LLGDNY	ENT	QAWASSTVV	One	3
VH-VL (λ) 26	GYTFTVYY	INPIGGAT	ARGPSSGDFDY	NLGKRY	QGS	QAWDSTTDYV	One	3
VH-VL (λ) 27	GYTFTGYY	INPNSDGT		SNDVSGYNR	GVN	SSRSSTNSYVFGTRTKLTVL	One	2
VH-VL (λ) 28	GGSITGYY	IYPTGKT	ARLEFGVRFFDF	SLRSYY	GKN	NSRDSSGNHLV	4	3
VH-VL (λ) 29	GFSFTGYY	INPKSGDT	LRSGYPLDH	SLRSYY	GKN	NSRDSSGNHLV	One	3
VH-VL (λ) 30	GGSLSTYY	IYNTGTT	ARQYSSSRYTV	SGYSSHA	LSSDGSH	QTWGDGVHV	4	4
VH-VL (λ) 31	GASISSTNW	IYHGGHT	ARVVRGRHQFIDN	ALPKKY	EDS	YSTDSSGSQWV	4	3
VH-VL (λ) 32				SLRISY	GRN	GSRDISGDHPV	―	3
VH-VL (λ) 33	GYSFTTYW	IYPGHSDV	ARLPYCGTECYPHLDNWARAPWSPS				5	―
VH-VL (λ) 34	GFSVDTGAVG	IFGDGDK	AHTDLKYGDFSFDD	SLRSYY	GKN	NSRDSGGNHQV	2	3
VH-VL (λ) 35	GGSVSNYNYH	FTYSGNI	VNYLRGNGGRGP	SSDFGGYNY	DVS	CSYAGTYTSGV	4	2
VH-VL (λ) 36	GYAFSSYG	ISAVNGNT	ARDDFSSYCFSH	NIGSKS	DNR	QVWDSSSEHYV	One	3

TABLE 7

VH-VL (λ) 37	GYSFTNDW	IYPGDSDV	ARRTFCGGDCDAFDI	NIGSKS	SDS	QVWDSSSDHYV	5	3
VH-VL (λ) 38	GFKFSSYG	ISQDGSNK	AKTTTYSIQMGFDS	SLRSYY	GKN	NSRDSSGNHLV	3	3
VH-VL (λ) 39	GYTFTNYG	ISAYNGNT	ARDDGPVATIGTWVLFDY	SLRSYY	GKN	NSRDSSGNHLV	One	3
VH-VL (λ) 40	GASVDSGRNY	FSYSGST	ARLSPVAGNYYFDY	SLRSYY	GKN	NSRDSSGNHLV	4	3
VH-VL (λ) 41	GSTFSNFL	INQDGNEQ	AKPLLRLVSSSWAGH	SLRSYY	GKN	NSRDSSGNHLV	3	3
VH-VL (λ) 42	GFSFSIYG	IWCDGSHQ	ARRGSSGILGPDTFDL	SSNVGNNY	DNN	GTWDSRLIVGI	3	One
VH-VL (λ) 43	GGSVSSNY	VSNIGSP	ARGVLALSHGDFHFDS	GVGRKS	YDT	QVWHTTTNRVV	4	3
VH-VL (λ) 44	GFLLSEHGLG	IFSNDEK	VRIFSSHNGKLWFPYKSDY	SSNIETNY	KDD	ASWDDSLTDVV	2	One
VH-VL (λ) 45	GFTFTNYW	IYPADYDT	ARRGDDWNYVRY	TSNIGSNS	NNN	AAWDDSLNGWNVF * TGTQLTVL	5	One
VH-VL (λ) 46	GGSFTGYS	INPSGTT	ARLKTSRTYYSDSGSYLSVGYFDSWAQGTLVTVA	SLRSYY	GKN	NSRDSSGNHLV	4	3
VH-VL (λ) 47	GGSFSGYY	TIPRGRA	ARHQTGERAFDV	GSDVGGYNY	EVT	WSYAGSYTLV	4	2
VH-VL (λ) 48	GGSINSTSSY	IYYSGNT	ARSRDYGSFFDS	SSDVGSFDR	DVI	CSVADSGTLVLGGGTKLTVL	4	2
VH-VL (λ) 49	GYKFTNYW	IHPDDSES	AKTVTGDRPIAGDGFGL	SLRSYY	GKN	NSRDSSGNHLV	5	3
VH-VL (λ) 50	LHWVRQAP	IAEDESKA	TRDVGFADYYMDV				3	―
VH-VL (λ) 51	GYTLTNYG	FYPGDSAS	ARGSYCTAGVCTTDAFDI	SGSVSTSYY	STN	VLYVGNGIVL	5	8
VH-VL (λ) 52	GYTFTNNW	IDPSDSQT	ARGGFDYDDRGNPTPDYFDS	SLRSYY	GKN	NSRDSSGNHLV	5	3
VH-VL (λ) 53	GFSLKNYG	LWYDGTTE	TRISNRWGGDYFGY	NIGSKS	YDS	QVRDGLTDQVV	3	3
VH-VL (λ) 54	RFNFSSYA	ISGSDGST	RRREGLVVVPAAPHDGLLISGAKGQWSPSL	SSNIGSNT	DNS	AAWDDSPNGHCV	3	One
VH-VL (λ) 55	GFSLTTRGEG	IYWDDVE	VRKIIITNVSRKVLVPYFDH	SGSVSTSYS	GTN	ALHLSSGIWV	2	8
VH-VL (λ) 56	GYAFTSYG	ISAHNGNT	ARDGFSSYYFPL	SLRSYY	GKN	NSRDSRGVV	One	3
VH-VL (λ) 57	GFTFGDAW	IKSNSAGGTT	ATDRDYAFQI	GSNIGDNA	NGD	AAWDGSLNGWV	3	One
VH-VL (λ) 58	GSTFTGYF	TNPKSGDS	AGQQLVPANDVFDL	SSDVGAYYR	AVS	TSYTTTKTYVFGTGPRSPSx	One	2
VH-VL (λ) 59							―	―
VH-VL (λ) 60	GFSLNSPRLG	LFSDDEK	ARSTNPYSGSYFSAYFDL	LLAKKC	KDS	YSAADYKIV	2	3
VH-VL (λ) 61	GFTFSSYW	INKDGSEK	ARDGADYGDDFDY	SSNIGSND	TND	AAWDDSLSGVL	3	One
VH-VL (λ) 62	GDSVSDSY	IFPSAST	ARRYSTSLSDSFDI	SLRSYY	GKN	NSRDSSGNHLV	4	3
VH-VL (λ) 63	GSSIHYYY	IYSRGST	CARIRVDDRDFWGREYYV * LLGPVDAGHG	YLQNYY	GER	DSLDSSGNHMV	4	3
VH-VL (λ) 64	GFSLSTTGMS	IDWDNDH	ARATSDSWSGYRNYYFDS	SSDVGGYNY	DVS	CSYGGHHSVV	2	2

Table 8

kappa	VH			VL			IGHV Subfamily	IGKV Subfamily
Clone	CDR1	CDR2	CDR3	CDR1	CDR2	CDR3
VH-VL (K) 01	GYKFTGFW	IDPDDSHT	ATTGGAFA	QSISTW	KAS	QQYNGFPT	5	One
VH-VL (K) 02	GYTFTRYY	IDANGGGT	VRDADNVVVTALAF	EDVSRW	GAS	QQGYSLPIT	One	One
VH-VL (K) 03	GYTFTNYL	ISVYNGNT	ARDGSGGSCYSDDSCALDI	QTITTK	DVS	QQYGIWTS	One	3
VH-VL (K) 04	GYSFSTYW	IYPGDSDT	ATYSSGWY	RTINMY	GAS	LQHNYYIFS	5	One
VH-VL (K) 05	GYTFTAYY	FNPNSNDA	ARLPSTPCTGGSCHDY	RDITNY	VAS	QQSADLPTT	One	One
VH-VL (K) 06	GFSFSNYW	IKEDGAET	ARADFGVVRTFYYYYNALDV	QDIRND	AAS	QQYNTYPLT	3	One
VH-VL (K) 07	GANISGTSDY	LRYGGTT	ARQAVEGRWYGGRRVYCP * QLLL	QTVSDW	KAS	QQYHSYPWT	4	One
VH-VL (K) 08	GFTFSSYS	ISSSSSYI	ARDGIAARPIDP				3	―
VH-VL (K) 09	GGPISSTTFY	VHHTGSS	ARQRRGTSSWSSRRLQFDL	QSISNY	AAS	QQSYSTPNT	4	One
VH-VL (K) 10	ADTFSRFA	IIPLFGTG	ATSHYYGSDADPAWHFGL	QSVVRY	AAS	QQYNSFSWT	One	One
VH-VL (K) 11	GFSLASTGMC	IDWDEDT	GRIQPQRGIDF	QSIGKW	KAS	QQYSSYSPFT	2	One
VH-VL (K) 12	GFSLSTSGMC	IDWDDDK	ARTQGASGYSYGYVFGY	EDVSRW	AAS	QQVDKMPVT	2	One
VH-VL (K) 13	GGSIGSFS	IYSSGST	ARDFRDYNSGTYEVYNGSDP	QSILTSSNGNNF	WAS	QQSYNTPFT	4	4
VH-VL (K) 14	GFSLSNRRVG	IFSNDGK	ARTGYNYGGSFFFHGIDI	QSISDY	LAS	LQDYSYPRT	2	One
VH-VL (K) 15	GFSLNTSGMC	IDWDDDK	ARMLYRYYFQTSGLDS	QNVNR	YGS	QQYDDWPQT	2	3
VH-VL (K) 16	GGSFCGYN						4	―
VH-VL (K) 17	GGSVSAYY	IYHSGSS	ARDSGSRVDF	QSVLYSYTKKMY	WAS	QQYGTTPYT	4	4
VH-VL (K) 18	GYGFSNYG	IHVYDGKT	ARNLLGGGTTLYPRDESLDV	QSVGSN	AAS	QHYHDWPQT	One	3
VH-VL (K) 19	GFSLTSPGAG	IHWDDDK	AREGRFPDAFDI	QSISSY	AAS	QQSYSAPPVT	2	One
VH-VL (K) 20	VSNTRASWN	YRSKWYY	ARSRPYGVYNDYFGY	QGIDTY	AAS	LQDYNYPRT	6	One
VH-VL (K) 21	DSPSPDYG	TSPRDSNI	ARRALFREETAHMDFGGCGPRAMV				5	―
VH-VL (K) 22	GFSFNTGGVG	IFWDDEK	AYFGALGFGFKD	QNINNY	KAS	LQHNSYPRT	2	One
VH-VL (K) 23	GGIFTDFS	IIPMSGSS	ARHRSRSWFKMSMDV	QDIRHW	AAS	QQADSFPLT	One	One
VH-VL (K) 24	GYYFIDHY	INPNNGAT	LTDGGMGQGTMVTV	HNVFSSY	RAS	QQYGNSPRT	One	3
VH-VL (K) 25	GFSVNCDGVG	IFWDGDK	GHSLRGPGCRGGACYFFDY	QNIKNY	AIS	QQSYSTPYT	2	One
VH-VL (K) 26	GFSVNTGAVG	IFGDGDK	AHTDLNYGDFSFDD	QSVSGY	DAS	HQRANWPLT	2	3
VH-VL (K) 27	GFSSSTYW	IHSGGSRT	AVLPPGD	QSISTW	ETS	QQYSGYLYSFGQGPGGNQ	3	One
VH-VL (K) 28	GFTFGDAW	IKSYSAGGAT	ATDRDYAFQV	QSVSNL	AVS	QQSYSALYS	3	One
VH-VL (K) 29	GFSLTTSGMC	IDWDDEK	ARMCGYYDTSSGYDEASDM	QSVLYSSNNKNC	WAS	QQYYSTPFT	2	4
VH-VL (K) 30	SHSTLVQWV	IYWDDDK	AHSPAYKIGFYKIAI	QPIGRW	EAS	QQYDSFPHT	2	One
VH-VL (K) 31	GGSINTYY	IHSSGTT	ARDRVLVQGTHYYYYMDV	RDISDR	GAT	QQHDDVPYT	4	One
VH-VL (K) 32	GYSFNDFY	INPDSGDT	ARARGSRLPRYNDNDGSVDHYYYYTDV	QSISSY	AAS	QQSYSTPWT	One	One
VH-VL (K) 33	GFSLYTPRMG	IYWDNDE	AHSQYFGSNNDAFDV	ESISTY	AAS	QQSYSTPQWT	2	One
VH-VL (K) 34	GDSITSSTYN	LYSSGGT	ATDSNNVVPAARGLTFHH	RDISHY	GAS	QQYDNLPGIT	4	One
VH-VL (K) 35	GFSLDTFGVG	IYWDDDK	AYTYNTGSIDWFDP	QGVLNS	ASH	QQYNYMST	2	One
VH-VL (K) 36	GFTFSSYS	ISSSSSTI	ARKETTVNYYMDV	QHITDF	DAS	QQNDDLPPYT	3	One
VH-VL (K) 37	GYTFTKYG	ISTYNGNT	AREDGDSFGLMDYYYAMDV	QGISNY	AAS	LQDFGYPRT	One	One
VH-VL (K) 38	GFTVSSNY	IYSNGDT	ARDSPLDWYHEY	RDISNH	GAS	QQYDHVPPA	3	One
VH-VL (K) 39	GGSISGTSYF	LFNTDGI	AAYQSTSYYRPFED	QDIDSW	SAS	QQYHDVPLT	4	One
VH-VL (K) 40	GGTFSCYA	IIPIFGTT	ARGHTSSGWDYWGQSTLVTVA	QSLTTY	DAS	QQSYSAPLT	One	One
VH-VL (K) 41	GGTFSNYA	IIPIFATA	ARGWDYYGSAPYYLPFSY	QSIGVW	RAS	QQYNLYPLT	One	One
VH-VL (K) 42	DGSFASGGYY	VYFTGDT	ARGVHSSSSGGRAFDV	QSVRRN	GAS	QQYNNWPLWT	4	3
VH-VL (K) 43				EDVSRW	AAS	QQVDKMPAT	―	One
VH-VL (K) 44	GGSISTDY	ISYSGST	ARAGQVILGNYYYYMDV				4	―
VH-VL (K) 45	GYIFTNIW	MYPGDSDT	ASAVFLGGGNDAFDI	QSVFSSSNNKNY	WAS	QHYYTYSPM	5	4
VH-VL (K) 46	GGSINSADDY	IFYSGNT	ARGGDSSSWFSYYFQD	QSISTD	AAS	QQSYSTPYT	4	One
VH-VL (K) 47	GFSLNTDKVS	IYSNYDK	VFRSIRLRAFDI	ESVSSN	GAS	QQYTNWPRT	2	3
VH-VL (K) 48	GYSFTSYW	IYPGGSES	ASRYSSTWFL	QGISNS	AAA	QQHITSPLT	5	One
VH-VL (K) 49	GASISSTLYY	IYYSGNS	ARNEPNWNYVFDY	QGINSW	AAS	QQANNFPLT	4	One
VH-VL (K) 50	GYTFSDYY	INPNRGDT	ARDRVGYCRGGTCYSNAYDV	QGVGPW	STS	LQSYSFHRT	One	One
VH-VL (K) 51	ERSFIGYY	INRSVTT	ARGLRRAAPGPFRY	QDIDSW	GAS	QQTTNFPIT	4	One
VH-VL (K) 52	SDAICSGSDY	IYTSGST	ARERKSFVVRGGYYYYYMDV	QDISNF	DAS	QQYDNVPFT	4	One
VH-VL (K) 53	GFSLSTSGMC	IDWDDDN	ARLTYYYDSSAYLPGAFDT	QTISTY	AAS	QQSYSTPFD	2	One
VH-VL (K) 54	GYTFTGYY	INPKNDET	ARDPGSSSATNNWFDP	QSISSY	AAS	QQTYSNFFT	One	One
VH-VL (K) 55				QDISTY	DAA	QQYENLPVT	―	One
VH-VL (K) 56	GLIVSGNY	IYSGGNT	ARHVDTTMSYFFDSWGQGQWSPSL	QDITTF	DAV	QQFDSLPYT	3	One
VH-VL (K) 57	SITNSYYY	IYYLYNRGRT	ARQGQVVPSAHDPFYM	QSVNSF	DAS	HLYGSSPPWT	4	3
VH-VL (K) 58	VFSNAASWN	YRIKWFH	ARALLNGHFDY	QDISNY	DAS	QQYDNLPFT	6	One
VH-VL (K) 59	GGSISGYY	IHIVGALT	EDGG * LSRTTLTTGAREPWSPS	QDIKTH	AAS	QQTTTFPIT	4	One
VH-VL (K) 60	GGSITTSSSF	LYYNEIT	VRPRDHGFDL	QGISSA	DAS	QQFNNYLPLT	4	One
VH-VL (K) 61	GYFFTDHY	FDPKSGRT	ARVAVAGRLEFYQH	QSVGSY	DVS	QQSYSTRRTFGQRTKVEIK	One	One
VH-VL (K) 62	GFSLTNARMG	IFSDDDT	ARIRAMGMRDYYYYMDV	QSISSGY	GPS	QQYGSPPLT	2	3
VH-VL (K) 63	GGSISSSF	ISYSGSS	ARDRSGTYYTFDI	QSLFFSSTNKTF	WAP	QQSSSTPYT	4	4
VH-VL (K) 64	GYSFTHFE	VNPDSGNT	ARGMTTVANEDVYLYYMDV	QSISSY	GAS	QQNYITL * T	One	One
VH-VL (K) 65	GFSLSTSGAG	LFWDADR	AHNFHMTPVM	EDISGW	GAS	QQANTFPPAFGQGTKWISx	2	One
VH-VL (K) 66	GGSISHYY	IYGSGST	ARDGADDGAQHYHLYGMDV	ESISGR	SAS	LQDFSYPRTFGQGPRWRSx	4	One
VH-VL (K) 67	GFSLSTNGVG	IFWDDDK	AHMGYTSSWYDY	QNIESY	KAS	QQYHSYPWT	2	One
VH-VL (K) 68	GGSFSGYY	INHSGST	ARGNEQWAVGAFDI	QSISSS	TAS	QQSRT	4	One
VH-VL (K) 69				RGIGNY	AAS	QKYNSAPYT	―	One
VH-VL (K) 70	GYILIDYW	IWPSDSDT	ARPRDYSRSSSLDL	QGISSW	AAS	QQTYSFPLT	5	One
VH-VL (K) 71	GYPFIGYY	INPNSGGT	ARENQREITIFGMDPFDH	EDVSRW	AAS	QQVDKVPVT	One	One
VH-VL (K) 72	GYIFTSYY	INPSGGGA	ARDRAKGGDYEFAY	QGISNS	AAS	QQYYSYPLT	One	One
VH-VL (K) 73	GASFNGYY	ITPRGEV	ARDVNTYDRDIGHHPFDV	QGIRNK	DAS	LQNYNYPYT	4	One
VH-VL (K) 74	GFSFSNYW	IDPSDSYT	ARQGYCNGNSCSNFYSYMDV	QSAGKW	KTS	QQYNLYPLT	5	One
VH-VL (K) 75				QDISSY	AAS	QQSYSVPQTT	―	One
VH-VL (K) 76	GDTFNNYA	IIPIFSTT	ARDWDPVDAARPDAFDF	QGILYSSNNKNY	WAS	QQYYSTPIT	One	4
VH-VL (K) 77	GASISISSYY	IGYSGST	ATHYCDSVGLDWFDP	RNIRTS	AAT	QQYNSYSPLTFGGGDQAGDQ	4	One
VH-VL (K) 78	GYKFSNYW	IYPDDSDV	ARRSHNWNDLDF	QSISSY	AAS	QQSYSTLALT	5	One

The functional diversity of the library is determined by the percentage of clones that can actually express the antibody. Clones that do not function or express these normal functions are most likely due to errors in the PCR process during library construction. Due to the nature of library construction, a lot of PCR work was performed, and as these errors accumulated, the proportion of clones capable of functioning was about 69%.

Finally, a human unsensitized antibody library having a transformant of 1.18 × 10 ¹⁰ cfu was obtained, and it was confirmed that 8.10 × 10 ⁹ cfu could be expressed as a normal ScFv. In general, since it is recognized that the use is at least 10 ⁸ or more, the library according to the present invention can be determined to be properly constructed.

In addition, subfamily distribution and complementarity determining site sequence diversity of the variable region were confirmed, and the results are shown in FIG. 8. The subfamily distribution heavy chain clones total 135 that can determine the variable region, the light chain Total 134 (kappa 74 gae, lambda 60 pieces) clones were analyzed, 'VBASE2' sequence analysis through the DB, V _H region Although the distribution of V _H1 and V _H4 subfamily was rather high, relatively even distributions of V _H1 and V _H5 were shown. V _Lλ regions showed a tendency that _λ3 subfamily V high, V _Lκ region was observed to have the V _κ1 subfamily present in 78%.

9, amino acid length and distribution of the CDR-H3 region, which is an important site for maintaining the diversity of the antibody library, are also about 4 to 25 different lengths as in many libraries, and 98.4% is recommended in the KABAT DB. Has the correct length of CDR-H3. Referring to FIG. 10, it was confirmed that various distributions of amino acids in the KABAT position (H95-H102) of the CDR-H3 region were also present. CDR-H3 plays an important role in antigen recognition, and due to the diversity of amino acid composition and amino acid length by position of the CDR loops, as shown in the above results, it is possible to produce a significant change in the conformational structure in the process of binding antigen to several antigens. It seems to be suitable for recognition.

Example 9: Dot-blot Analysis

As shown in FIG. 11, Dot-blot analysis was performed to confirm whether the constructed library can be properly expressed in ScFv form. 96 clones are randomly selected from the prepared library and incubated at 37 ° C. in 200 μL SB / Ampicillin medium in 96 well plates. When the absorbance at O.D 600 was about 0.5 to 0.8, IPTG was added to a final concentration of 1 mM and incubated overnight at 30 ° C. Next day, the supernatant (culture solution) was obtained by centrifugation, and the precipitated Escherichia coli was mixed with 40 μL of 1X TES, and then lysed by adding 60 μL of 0.2X TES. The debris was precipitated by centrifugation and supernatant (peripheral cytoplasmic extract) was taken. Since the pSIA23 vector contains the pelB leader sequence, the expressed ScFv migrates to the periplasm. The nitrocellulose membrane was treated with 1 μL of the culture solution and the peripheral cytoplasmic extract, and then dried at room temperature for 1 hour. After blocking with 5% skim milk (TBS-0.1% tween 20), anti-HA-HRP was detected. Referring to FIG. 11, it was confirmed that 64 clones out of 96 analyzed HA-Tag were properly detected. Through this, it was confirmed that the antibody fragment is properly expressed.

Example 10 Library Amplification and Recovery of ScFv-Expressing Bacteriophage

To amplify the pSIA23 vector introduced into Escherichia coli host TG1, two sub-libraries of V _H -V _Lλ and V _H -V _Lκ were each added in 1 L of culture medium (SB / ampicillin / 2% glucose) to 37 ° C., Incubated at 220 rpm for about 5 hours. Host cells cultured at OD 600 with an absorbance of 0.5 to 0.8 were centrifuged at 5,000 g for 20 minutes to remove supernatant, and then the precipitated host cells were suspended in SB medium and completely mixed with 0.5 volume of 50% glycerol. Thereafter, 1 mL was dispensed and stored at -80 ° C. Two sub-libraries amplified before, V _H -V _Lλ , V _H -V _Lκ were incubated in 400 mL culture medium (SB / ampicillin / 2% glucose) until OD ₆₀₀ became 0.5. The supernatant was removed by centrifugation, suspended in 400 mL of glucose-free culture medium (SB / Ampicillin), and 10 ¹² pfu (plaque forming unit) of VCSM13 helper phage was added at 37 ° C. Cultured at 80 rpm for 1 hour. The kanamycin antibiotic (an antibiotic gene introduced into the helper phage) was then added at a final concentration of 70 μg / mL, followed by overnight culture at 30 ° C. to allow phage libraries to be made out of the host cell. The culture supernatant was then centrifuged to recover the phage library by precipitation of phage particles using PEG8000 (polyethylene glycol) solution. Each sample was diluted and counted in LB / ampicillin culture medium again after infection with host cells (TG1) to count phage recovered from each sublibrary. Since phage particles expressing Sc12F of 10 ¹² ~ 10 ¹³ pfu level was used for panning.

Example 11: Antigen Specific Antibody Identification Via Biopanning

Phage display screening was performed through repeated round panning. The counted _sublibrarys were assembled to a level of about 4.0 × 10 ¹³ pfu (V _H −V _Lλ : about 2.0 × 10 ¹³ pfu, V _H −V _Lκ : about 2.0 × 10 ¹³ pfu) and then 5 μg / mL in PBS. Nine antigens diluted at levels (c-Met, HGF, ANG2, DLL4, EGFR, TNF-a, NRP1, VEGF, VEGFR2) were treated with coated immunotubes. Prior to treatment, the immunotubes and phage particles were treated for 1 hour with a blocking solution containing 3% skim milk in PBS to prevent non-specific binding. The phage library was treated with an antigen-coated immunotube for 1 hour, followed by washing the immunotube with PBST (0.1% Tween 20) washing solution, and then adding 1 mL of 100 mM triethylamine to the antigen for 10 minutes. Bound phage particles were recovered and neutralized with 1M Tris-Cl (pH 7.4). In order to confirm the number of recovered phages (output), the recovered solution was diluted and infected with host cells, and counted in the culture medium. The remaining recovered solution was plated in a 15 cm culture medium and cultured, and 5 mL of SB culture medium (50% glycerol) was added to collect and store colonies (-80 ° C).

Phage particle amplification was performed by taking 50 μL of the previous round phage solution stored for subsequent panning cycles. Phage particles recovered by adding VCSM13 helper phage (10 ¹¹ -10 ¹² pfu) after incubation in host cells were prepared by PEG precipitation, and the next round panning was performed by the same method as the previous round panning. Panning was performed up to 3 to 4 times for the antigen, and the phage display panning results are shown in FIG. 12.

Example 12 ELISA Screening for Anti-Antigen ScFv Candidate Selection

After a total of three to four pannings for each antigen were completed, the phage particles recovered in the final round were identified as colonies in the culture medium through infection with host cells (TG1). These colonies were randomly taken and cultured after each inoculation in 96-well plates containing 200 μL SB / ampicillin culture medium (within 37 ° C., within 3 hours). Then, to induce the expression of the ScFv-pIII protein, each well was treated with IPTG at a final concentration of 1 mM and incubated overnight at 30 ° C. and 220 rpm. Afterwards, the culture plate was centrifuged and the supernatant was removed, and then 1XTES solution (20% w / v sucrose, 50 mM Tris, 1 mM EDTA) was maintained at 40 μL at 4 ° C. to recover periplasm sites of cultured cells in each well. , pH 8.0) to lyse cells by standing at 4 ° C for 30 minutes. Subsequently, 60 μL of a 0.2 × TES solution was again treated, and allowed to stand for 30 minutes. Finally, the plate was centrifuged and the supernatant was recovered to produce a small scale ScFv-pIII protein. Meanwhile, antigen-coated 96-well plates were prepared at the same time, and then 25 μL of the recovered periplasmic sites were taken and bound to each well for 1 hour. BSA protein was used as a control. After 3-4 times of washing using TBST, HRP conjugated anti-HA antibody was bound for 1 hour, followed by washing and color reaction (TMB substrate), and the value was measured at OD 450 nm. It was. The result of analyzing the number of clones having a binding capacity multiple of 2 or more is shown in FIG. 13. Referring to Figure 13, it was confirmed that the ScFv having a binding capacity for the various antigens are recovered.

Example 13: Phage ELISA

In FIG. 13, the C01 clone showing a strong toxic signal was selected from the panning result of the DLL4 antigen. Since DLL4_C01 ScFv was selected through phage display screening, it was confirmed that it showed concentration-dependent binding ability to DLL4 while being expressed in the structure of phage. Phage particles were individually collected from host cells expressing the DLL4_C01 clone and counted. Thereafter, each phage particle was diluted in a DLL4 coated 96 well plate and treated according to its concentration, and the binding ability thereof was confirmed by ELISA analysis using an anti-phage antibody, and the results are shown in FIG. 14.

As a result, it was confirmed that the binding capacity decreases as the number of phage particles decreased, and it was confirmed that the signal was saturated at a specific particle number or more. Through this, it was possible to test the specificity for DLL4 by the number of phage particles. Therefore, the ScFv screened on the basis of the binding capacity to DLL4 during the screening process was able to test the binding ability to DLL4 even in the form labeled on the phage structure.

Example 14 ScFv Production and Purification Using Protein Expression Strain TOP10F '

In order to confirm the binding capacity and function of ScFv alone, the protein expression strain (TOP10F ') was used for expression and purification. The basic structure of the phagemid vector can be seen in FIG. 1, where the host cell (TG1) carrying the screened phagemid inhibits the amber codon (UAG) present between ScFv and the phage pIII protein. As it is impossible to express ScFv alone, an expression strain (TOP10F ′), which is a non-suppressor strain, was used.

Phagemid encoding the ScFv was recovered from the host cell and then transduced into the expression strain. Subsequently, the DNA strains were used to confirm the expression strains to which each phagemid was successfully introduced. The expression strains to which each ScFv was introduced were taken in single colonies, inoculated in 3 mL of LB / ampicillin culture medium, and then cultured overnight at 37 ° C and 220 rpm. . After overnight culture, the culture medium was transferred to 400 mL of culture medium (SB / Ampicillin), and further cultured until the OD ₆₀₀ reached 0.5-0.8, and overnight culture was again carried out at 30 ° C. and 220 rpm by adding IPTG at a final concentration of 1 mM. It became. After centrifugation, the cells were suspended using 16 mL of 1X TES solution, an additional 24 mL of 0.2X TES was added to dissolve the expression host, and only the surrounding cytoplasm was recovered by centrifugation. The recovered culture solution was 0.45. It was filtered through a μm filter.

ScFv protein in the filtered lysate was bound with 1 mL of Ni-NTA beads (Qiagen) for 1 hour at 4 ° C for His-tag purification. Then, it was packed in a gravity column (gravity column, Bio-rad), washed with PBS (with 10 mM imidazole), and recovered in ScFv form using PBS (with 200 mM imidazole). Purified ScFv was confirmed by SDS-PAGE and coomassie blue staining, and the results are shown in FIG. 15. Referring to Figure 15, each ScFv was confirmed to have a size of about 28 kDa. Each purified ScFv protein was used for storage after concentration measurement through the Bradford protein measurement method and for later experiments.

Example 15: Confirmation of direct binding patterns of ScFv by DLL4 concentration

The binding capacity of the produced ScFv to DLL4 protein was confirmed by concentration-specific ELISA. ScFv was treated for each concentration produced in DLL4 coated at a concentration of 4 μg / mL in a 96-well plate, and the HF-coupled anti-HA antibody was treated for 1 hour to detect ScFv binding to DLL4, and the results are shown in FIG. 16. It was. As a result, it was confirmed that DLL4_C01 ScFv showed a binding pattern in a concentration-dependent manner, and it was confirmed that the DLL4_C01 ScFv had a significant binding capacity to the DLL4 protein compared to the control group (BSA).

Example 16: EGFRvIII Specific Antibody Screening with GBM Patient Derived Cells

EGFRvIII is a cancer cell-specific antigen that is a mutation resulting from overexpression and amplification of EGFR in cancer cells, resulting in the deletion of 6-273 amino acids. Most existing EGFR antibody therapeutics, such as Cetuximab and Panitumumab, bind between 310-480 amino acids of EGFR. Generic EGFR antibody therapies bind to both EGFR and EGFRvIII and are unable to specifically target EGFRvIII and are reported to be ineffective in EGFRvIII mutant cancer patients. In addition, EGFRvIII antigen is an antigen optimized for the development of antibody-drug conjugates (ADCs) because it is a cancer-specific antigen that is expressed only in cancer cells but not in normal cells.

Antibodies were identified by cell panning techniques in the libraries prepared through the examples in the manner shown in FIG. 17 using patient derived cells overexpressed with EGFRvIII for the development of antibodies that bind to EGFRvIII. Since purified recombinant proteins (antigens) do not reproduce the three-dimensional structure of proteins, conventional panning methods cannot efficiently identify antibodies. However, since cell panning uses cancer antigens overexpressed in patient cells, antibodies that recognize natural forms can be more efficiently developed. After developing cancer cells from glioblastoma (GBM) cancer patients to develop EGFRvIII specific antibodies using cell panning, the expression level of EGFRvIII was confirmed using RT-PCR and Western blot as shown in FIG. , 626T glioblastoma patient cells with the highest expression of EGFRvIII were selected as the cells for final antibody development. It was confirmed that EGFRvIII expression was inhibited using EGFRvIII shRNA, and the proportion of EGFRvIII specific antibodies increased through repeated panning of EGFRvIII specific antibodies after negative selection in patient-derived cells. After finally securing three candidate antibodies in FIG. 19, EGFRvIII specific binding ability was confirmed by ELISA analysis. Surface Plasmon Resonance was performed using Biacore T100 to measure the binding constant. Three scFvs were found to show a binding capacity of several tens of nM. As a result of confirming that three identified candidate antibodies can effectively inhibit cell growth of 626T glioblastoma patient cells, it was confirmed that 1B07N effectively inhibited growth as shown in FIG. 19.

Those skilled in the art to which the present invention pertains will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

Through the present invention, not only the construction of a library having a high level of diversity is possible, but also ScFv is a human antibody, so it is possible to screen for antibodies which do not exhibit HAMA response as compared to antibodies screened through the prior art, and for treating or diagnosing a disease. To enable the development of antibodies that can be used effectively.

Electronic file attached.

Claims (20)

1. A library containing phage that present ScFv antibodies to a surface,

   The ScFv antibody comprises a heavy chain variable region (V _H ) and a light chain variable region (V _Lκ or V _Lλ ) of an antibody derived from an immune organ,

   The ScFv antibody was encoded from a nucleic acid synthesized using a heavy chain variable region (V _H ) and a light chain variable region (V _Lκ or V _Lλ ) specific primer of an antibody derived from an immune organ, using a nucleic acid extracted from an immune organ-derived cell as a template. Library.
2. The library of claim 1, wherein the immune organ is at least one selected from the group consisting of bone marrow, blood, and tonsils.
3. The library of claim 1, wherein the ScFv antibody is expressed in a transformant into which a vector having the following structure in which nucleic acid has been cloned is introduced, and is present on a phage surface included in the transformant.

   A-X-B-Linker-C-Y-D-E

   A to D are any one restriction enzyme recognition site selected from the group consisting of Sfi I, Nhe I, BglII and Not I,

   E is a His tag or HA (Hemagglutinin) tag insertion site,

   X is a gene insertion region encoding a heavy chain variable region (V _H ), Y is a gene insertion region encoding a light chain variable region (V _Lκ or V _Lλ ).
4. The library according to claim 3, wherein A is SfiI, B is NheI, C is BglII and D is a restriction enzyme recognition site of NotI.
5. The library of claim 1, wherein the ScFv antibody is a human antibody.
6. The method of claim 1, wherein the primers include one or more primers selected from the group consisting of sequences represented by SEQ ID NOs: 7-24 and one or more primers selected from the group consisting of sequences represented by SEQ ID NOs: 25-58 Featuring a library.
7. The library of claim 1, wherein the heavy chain variable region has any one sequence selected from the group consisting of SEQ ID NOs: 63-104 and 147-200.
8. The library of claim 1, wherein the light chain variable region has any one sequence selected from the group consisting of SEQ ID NOs: 105-146 and 201-254.
9. The library of claim 1, wherein the diversity of the library is 108 to 1014.
10. Extracting a nucleic acid from an immune organ-derived cell;

    The nucleic acid _encoding the heavy chain variable region and the light chain variable region using the extracted nucleic acid as a template and using the heavy chain variable region (V _H ) and the light chain variable region (V _Lκ or V _Lλ ) specific primers of the antibody derived from an immune organ. Amplifying;

    Cloning a nucleic acid encoding the heavy and light chain variable regions into a vector and then introducing the transformant into a transformant; And

    Expressing a ScFv antibody in the transformant, the method of producing a library comprising the step of presenting the ScFv antibody expressed on the phage surface contained in the transformant.
11. The method of claim 10, wherein the immune organ is at least one selected from the group consisting of bone marrow, blood, and tonsils.
12. The method of claim 10, wherein the ScFv antibody is expressed in a transformant into which a vector having the following structure is introduced.

    A-X-B-Linker-C-Y-D-E

    A to D are any one restriction enzyme recognition site selected from the group consisting of Sfi I, Nhe I, BglII and Not I,

    E is a His tag or HA (Hemagglutinin) tag insertion site,

    X is a gene insertion site expressing the heavy chain variable region (V _H ), Y is a gene insertion site expressing the light chain variable region (V _Lκ or V _Lλ ).
13. The method of claim 12, wherein A is SfiI, B is NheI, C is BglII and D is a restriction enzyme recognition site of NotI.
14. The method of claim 10, wherein the ScFv antibody is a human antibody.
15. The method of claim 10, wherein the primers include one or more primers selected from the group consisting of sequences represented by SEQ ID NOs: 7-24 and one or more primers selected from the group consisting of sequences represented by SEQ ID NOs: 25-58 Characterized in the manufacturing method.
16. The method according to claim 10, wherein the heavy chain variable region has any one sequence selected from the group consisting of sequences represented by SEQ ID NOs: 63-104 and 147-200.
17. The method of claim 10, wherein the light chain variable region has any one sequence selected from the group consisting of sequences represented by SEQ ID NOs: 105-146 and 201-254.
18. A method for screening ScFv antibodies, comprising screening an antigen specific ScFv antibody by reacting a library according to any one of claims 1 to 9 with an antigen.
19. The method of claim 18, wherein the ScFv antibody is specifically screened for binding to an autoimmune disease or cancer causing factor.
20. The method of claim 18, wherein the ScFv antibody specifically binds to a DLL 4 (Delta like ligand 4) protein.

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