KR100963507B1 - A vector for expressing antibody fragments and a method for producing recombinant phage that displays antibody fragments by using the vector - Google Patents

Abstract

The present invention provides phage with plasmid vectors (pLA-1 or pLT-2) and heavy chain antibody fragments (VH + CH1) -ΔpIII fusion protein expression and genotype-phenotype linkage functions for producing water soluble light chain antibody fragments (VL + CL). The present invention relates to a medium vector (pHf1g3T-1 or pHf1g3A-2), a host transformed with the vector, and a method for producing and selecting recombinant phage that display water-soluble antibodies and antibodies to the host.

Water soluble antibody fragment, vector, plasmid, phagemid, phage display

Description

A vector for expressing antibody fragments and a method for producing recombinant phage that displays antibody fragments by using the vector}

The present invention provides phage with plasmid vectors (pLA-1 or pLT-2) and heavy chain antibody fragments (VH + CH1) -ΔpIII fusion protein expression and genotype-phenotype linkage functions for producing water soluble light chain antibody fragments (VL + CL). The present invention relates to a medium vector (pHf1g3T-1 or pHf1g3A-2), a host transformed with the vector, and a method for producing and selecting recombinant phage that display water-soluble antibodies and antibodies to the host.

Phage display technology was first developed in 1990 by the Medical Research Council in the United Kingdom, and produced human antibody libraries to express antibody clones against specific antigens by expressing them on the surface of bacteriophages in the form of antibody fragments (Fab, ScFv). It is a technique to do.

The importance of phage display technology in the production of human recombinant antibodies is well recognized (see Clackson, T., Hoogenboom, HR, Grifiths, AD, Winter, G .. 1991. Making antibody fragments using phage display libraries. Nature.352, 624 .; Hoogenboom, H., Charmes, P., 2000. Natural and designer binding sites made by phage display Technology.Imunmun.Today 21, 371 .; Hoet, RM, Cohen, EH, Kent, RB, Rookey, K., Schoonbroodt, S., Hogan, S., Rem, L., Frans, N., Daukandt, M., Pieters, H., van Hegelsom, R., Neer, NC, Nastri, HG, Rondon , IJ, Leeds, JA, Hufton, SE, Huang, L., Kashin, I., Devlin, M., Kuang, G., Steukers, M., Viswanathan, M., Nixon, AE., Sexton, DJ, Hoogenboom, HR, Ladner, RC 2005. Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity.Nat Biotechnol. 23 (3), 344.), almost reacting specifically with antigen Human recombinant monoclonal antibody of all kinds The possibility to pull screening antibody libraries from single pot systems have been proposed (reference:. Nissim, A., et al 1994. Antibody fragments from a 'single pot' phage display library as immunological reagents. EMBO J. 13, 692 .; Griffiths, AD, Williams, SC, Hartley, O., Tomlinson, IM, Waterhouse, P., Crosby, WL, Kontermann, RE, Jones, PT, Low, NM, Allison, TJ, Prospero, TD, Hoogenboom, HR, Nissim, A., Cox, JPL, Harrison, JL, Zaccolo, M., Gherardi, E., Winter, G. 1994. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13 (14), 3245.). This can be achieved by phage display antibody technology. It means that a variety of antibody fragments (scFv or Fab form) can be obtained for diagnosis or treatment (Ref .: McCafferty, J., Griffiths, AD, Winter, G., Chiswell, DJ 1990. Phage antibodies Filamentous phage displaying antibody variable domains.Nature 348, 552; Winter, G., Griffiths, AD, Hawkins, RE, Hoogenboom, HR 1994. Making antibodies by phage display technology.Annu. Rev. Immunol. 12, 433 .; Griffiths , AD, Duncan, AR, 1998. Strategies for selection of antibodies by phage display.Curr Opin Biotechnol. 9, 102). However, the above-described ideal antibody engineering technique is not fully realized because various technical problems exist in phage display antibody technology (Ref .: Knappik, A., Pluckthun, A., 1995. Engineered turns of a recombinant antibody improve its in vivo folding.Protein Eng . 8, 81; McCafferty, J. 1996. Phage display: factors affecting panning efficiency.In: Kay, BK, Winter, J., McCafferty, J. (Eds.) , Phage Display of Peptides and Proteins, a Laboratory Manual.Academic Press, San Diego, p. 261; Krebber, A., Burmester, J., Pluckthun, A. 1996.Inclusion of an upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p.Gen. 178, 71; Assazy, HME, Highsmith, WE, 2002. Phage display technology: clinical applications and recent innovations.Clin Biochem. 35, 425; Baek, H., Suk, KH , Kim, YH, Cha, S. 2002. An improved helper phage system for efficient isolation of specific antibody molecules in phage display.Nucleic Acids Res. 30 (5), e18; Corisdeo, S., Wang, B., 2004. Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions. Protein Expr Purif. 34, 270). In other words, this technique has the advantage of being able to isolate monoclonal antibodies in just a few weeks, but has the disadvantage that the affinity of the separated antibodies is not so high. In order to make up for this drawback, in vitro affinity maturation was performed by varying the residues of the CDRs and FRs of the isolated antibody clones and then re-selecting higher affinity human antibody clones using phage display. Considering.

One of the decisive factors affecting the quality of the antibody library is the number of clones with phagemids in which the antibody gene has been inserted (Ref. McCafferty, J. 1996. Phage display: factors affecting panning efficiency.In: Kay, BK, Winter, J., McCafferty, J. (Eds.), Phage Display of Peptides and Proteins, a Laboratory Manual.Academic Press, San Diego, p. 261). It can be estimated that the greater the number of clones present in an antibody library, the greater the diversity of the library. The minimum number of clones in the library required to ensure that a particular target molecule-specific recombinant antibody can always be successfully selected from the antibody library. It is almost impossible to define. On the other hand, assuming that there is about 5 x 10 ⁸ antibody diversity in mice, it has been suggested that the size of the library should be much larger than 5 x 10 ⁸ to obtain an antibody with the desired affinity and catalytic function from the antibody library ( Reference: Ostermeier, M., Benkovic, SJ 2000. A two-phagemid system for the creation of non-phage displayed antibody libraries approaching one trillion members.J Immunol Methods.237 (1-2), 175), in vitro Because there is no affinity maturation mechanism in the in vitro system, only low affinity antibodies (10 ^{-6-10 -7} M) can be selected from antibody libraries with a diversity of 5 x 10 ⁸ (see De Bruin, R., Spelt, K., Mol, J., Koes R., Quattrocchio, F. 1999. Selection of high-affinity phage antibodies from phage display libraries.Nat Biotechnol. 17 (4), 397). . In addition, it has been suggested that the diversity of antibody libraries should be higher than 10 ¹⁰ in order to obtain high affinity antibodies (10 ^-9 to 10 ^-10 M) due to other experimental problems (Ref .: Griffiths, AD, Williams) , SC, Hartley, O., Tomlinson, IM, Waterhouse, P., Crosby, WL, Kontermann, RE, Jones, PT, Low, NM, Allison, TJ, Prospero, TD, Hoogenboom, HR, Nissim, A., Cox, JPL, Harrison, JL, Zaccolo, M., Gherardi, E., Winter, G. 1994.Isolation of high affinity human antibodies directly from large synthetic repertoires.EMBO J. 13 (14), 3245; Sheets, MD, Amersdorfer, P., Finnern, R., Sargent, P., Lindquist, E., Schier, R., Hemingsen, G., Wong, C., Gerhart, JC, Marks, JD, Lindqvist, E. 1998. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens.Proc Natl Acad Sci US A. 95 (11), 6157; Vaughan, TJ, Williams, AJ, Pritchard, K. , Osbourn, JK, Pope, AR, Earnshaw, JC, McCafferty, J., Hodits, RA, Wilton, J., Johnson, KS 1996.Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol. 14 (3), 309). Unfortunately, when the genes ligated between vector DNA and antibody DNA are introduced into Escherichia coli host cells using electroporation methods, antibodies with a variety of strains of about 10 ¹⁰ due to the low transformation efficiency of Escherichia coli are introduced. Manufacturing a library is very difficult and time consuming.

To avoid these technical difficulties, lambda phage att recombination sites and Int recombinases (see Geoffroy, F., Sodoyer, R., Aujame, L., 1994. A new phage display system to construct multicombinatorial libraries of very large antibodies Gene 151, 109) or the loxP site and phage P1 Cre recombinase system (Ref .: Waterhouse, P., Griffiths, AD, Johnson, KS, Winter, G. 1993. Combinatorial infection and in vivo recombination: a strategy for making large phage antibody repertoires.Nucleic Acids Res. 21, 2265; Griffiths, AD, Williams, SC, Hartley, O., Tomlinson, IM, Waterhouse, P., Crosby, WL, Kontermann, RE, Jones, PT, Low, NM, Allison, TJ, Prospero, TD, Hoogenboom, HR, Nissim, A., Cox, JPL, Harrison, JL, Zaccolo, M., Gherardi, E., Winter, G. 1994.Isolation of high affinity human antibodies directly from large synthetic repertoires.EMBO J. 13 (14), 3245; Tsurushita, N., Fu, H., Warren, C., 1996. Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.Gen. 172, 59) In vivo combinations of the VL and VH genes encoded by plasmids and phagemids, respectively, have been attempted, but it is difficult to confirm the actual diversity of antibody libraries prepared by this method.

In addition, in order to avoid the low E. coli transformation efficiency of the DNA vector in the production of the antibody library, a method of introducing the DNA into the host cell through the phage infection method has been attempted to prepare a combination antibody library in addition to the two-vector system. For example, Hoogenboom et al. Used fd-tet-DOG1 phage vector and pHEN1 phagemid vector, which produce heavy and light chain antibody fragments, respectively, to ensure that the two vectors are properly maintained in the same host cell to express functional Fab antibody molecules. It has been shown that Fab antibody libraries can be prepared with a viable two-vector system (Reference: Hoogenboom, HR, Griffiths, AD, Johnson, KS, Chiswell, DJ, Hudson, P., Winter, G. 1991. Multi -subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.Nucleic Acids Res. 19 (15), 4133). However, this method, although capable of displaying functional Fab molecules on the surface of the phage, is impractical for use in the preparation of antibody libraries because of the presence of recombinant fd-tet-DOG1 phage as well as TG1 cells into which the pHEN1 phagemid vector has been inserted. This is because host cell infection rate of phage descendants obtained by infecting recombinant fd-tet-DOG1 phage is very limited. This loss of infection has already been described in the M13δg3 system (Ref .: Rakonjac, J., Jovanovic, G., Model, P. 1993. Filamentous phage infection-mediated gene expression: construction and propagation of the gIII deletion mutant helper phage R408d3. 198, 99; McCafferty, J. 1996. Phage display: factors affecting panning efficiency.In: Kay, BK, Winter, J., McCafferty, J. (Eds.), Phage Display of Peptides and Proteins, a Laboratory Manual. As pointed out in Academic Press, San Diego, p. 261), these recombinant phages do not have the native g3p that interacts with the sex pili of the host bacteria.

Another two-vector system proposed by Ostermeier and Benkovic (Reference: Ostermeier, M., Benkovic, SJ 2000. A two-phagemid system for the creation of non-phage displayed antibody libraries approaching one trillion members.J Immunol Methods 237 (1-2) and 175 have a more serious problem, and in detail, their invention is made by first constructing heavy and light chain gene libraries in two phagemid vectors, and then using the VCSM13 helper phage. The combinatorial Fab library is produced by producing recombinant phage with the mid genome and infecting the resulting phage simultaneously with bacterial host cells. However, these libraries are of little value when applied to phage display because they are expected to contaminate very serious helper phages, and the limitation that technical strategies for the selection of more targeted molecule-specific phages cannot be provided. Because it has.

The present invention aims to provide a combinatorial Fab antibody library easily without the helper phage contamination problem by continuously transforming two vectors encoding heavy and light chain fragments into host cells in the form of circular DNA.

The present invention also provides phagemid vectors with plasmid vectors (pLA-1 or pLT-2) and Fd (VH + CH1) -ΔpIII fusion protein expression and genotype-phenotype linkage functions for producing water soluble light and heavy chain antibody fragments. pHf1g3T-1 or pHf1g3A-2), a host transformed with the vector and a method for producing and selecting recombinant phage that display the antibody using the water-soluble antibody and phage display technology.

In order to achieve the above object, the present invention comprises the steps of (1) enzymatic digestion of pBR322 plasmid with Pst I and Eco RI to generate DNA fragments, (2) pCMTG-SP112 phagemid as SEQ ID NO: 1 and SEQ ID NO: 2 PCR reaction using a primer set consisting of the step of enzymatic digestion of the PCR product with Pst I and Mun I to generate a DNA fragment, (3) the DNA fragment of step (1) and the DNA fragment of step (2) Ligation, (4) transforming the cells for TG1 transformation using the DNA ligated in step (3), and (5) TG1 transformed in step (4). It provides a method for producing pHf1g3T-1 phagemid, comprising culturing cells and separating and purifying phagemid therefrom, and pHf1g3T-1 phagemid prepared according to the above method.

In another embodiment, the present invention provides a DNA fragment comprising (1) PCR reaction of a pBAD / gIII plasmid using a primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4, and enzymatic digestion of the PCR result with Cla I and Spe I (2) PCR reaction of the pCDFDuet-1 plasmid using a primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6, and enzymatic digestion of the PCR result with Cla I and Spe I to generate DNA fragments. Step, (3) ligating the DNA fragment of step (1) and the DNA fragment of step (2), (4) TG1 transformed cells using the DNA ligated in step (3) Transforming, (5) culturing the TG1 cells transformed in step (4) and separating and purifying the plasmid therefrom, (6) plasmid purified in step (5) with Nco I and Xho I Enzymatic digestion produces DNA fragments That step, (7) pCMTG-SP112 phagemid the SEQ ID NO: 7 and SEQ ID NO: step of a PCR reaction using a primer set consisting of 8 and, by enzymatic digestion the PCR products with Nco I and Xho I produced a DNA fragment (8) ligation of the DNA fragment of step (6) and the DNA fragment of step (7), (9) transducing cells for TG1 transformation using the DNA ligated in step (8) Converting, (10) culturing the TG1 cells transformed in step (9) and separating and purifying the plasmid therefrom, (11) enzymatically treating the plasmid purified in step (10) with Sac I and Sac II. Digesting to generate DNA fragments, (12) pCMTG-SP112 phagemid was subjected to PCR reaction using a primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10, and the PCR result was enzymatically digested with Sac I and Sac II. Generating a DNA fragment, (13) ligating the DNA fragment of step (11) and the DNA fragment of step (12), (14) transforming a cell for TG1 transformation using the DNA ligated in step (13) And (15) culturing the TG1 cells transformed in step (14) and separating and purifying the plasmid therefrom, and the method for preparing pLA-1 plasmid and pLA- prepared according to the above method. Provide 1 plasmid.

In another embodiment, the present invention provides a DNA fragment by (1) enzymatically digesting the pLA-1 plasmid of claim 2 with Xho I and Sal I, (2) converting pCMTG-SP112 phagemid to SEQ ID NO: 11 and sequence. No .: PCR reaction using a primer set consisting of 12 and enzymatic digestion of the PCR product with Xho I and Sal I to generate DNA fragments, (3) the DNA fragment of step (1) and the step (2) Ligation of the DNA fragment of (4) transforming the cells for TG1 transformation using the DNA ligated in step (3), and (5) the TG1 transformed in step (4) It provides a method for producing pHf1g3A-2 phagemid, comprising culturing cells and separating and purifying phagemid therefrom, and pHf1g3A-2 phagemid prepared according to the above method.

In another embodiment, the present invention provides a method for preparing a DNA fragment by (1) enzymatic digesting pBR322 plasmid with Pst I and Eco RI, and (2) pCMTG-SP112 phagemid consisting of SEQ ID NO: 13 and SEQ ID NO: 14 PCR reaction using a primer set, and the result of PCR enzymatically digested with Pst I and Mun I to generate a DNA fragment, (3) the DNA fragment of step (1) and the DNA fragment of step (2) Ligation, (4) transforming the cells for TG1 transformation using the DNA ligated in step (3), and (5) culturing the transformed TG1 cells in step (4) It provides a method for preparing a pLT-2 plasmid, and the pLT-2 plasmid prepared according to the above method comprising the step of separating and purifying the plasmid therefrom.

In another embodiment, the present invention provides a method of transforming TG1 cells using (1) pHf1g3T-1, and (2) transforming the cells transformed in step (1) using pLA-1. It provides a dual vector system-IA (DVS-IA) comprising the step of, and (3) culturing the cells transformed in the step (2).

In another embodiment, the present invention (1) transforming TG1 cells using the pLA-1, (2) transforming the cells transformed in the step (1) using the pHf1g3T-1 And (3) culturing the cells transformed in step (2), thereby providing a dual vector system-IB (DVS-IB).

In another embodiment, the present invention (1) transforming TG1 cells using the pLT-2, (2) transforming the cells transformed in the step (1) using the pHf1g3A-2 It provides a dual vector system-II-A (DVS-II-A), and (3) culturing the cells transformed in the step (2).

In another embodiment, the present invention (1) transforming TG1 cells using the pHf1g3A-2, (2) transforming the cells transformed in the step (1) using the pLT-2 It provides a dual vector system-II-B (DVS-II-B) comprising the step of, and (3) culturing the cells transformed in the step (2).

In another embodiment, the present invention provides a method of expressing a human Fab antibody gene using said DVS-I-A, DVS-I-B, DVS-II-A or DVS-II-B.

An important feature of the vectors according to the present invention is summarized in Table 1 below.

Table 1. Comparison of Features of Vectors

DVS-I DVS-II vector pLA-1 pHf1g3T-1 pLT-2 pHf1g3A-2 Plasmids and Phagemids Plasmid Phagemid Plasmid Phagemid Promoter P _BAD P _lac P _lac P _BAD Coded Antibody Fragments L chain Fd-ΔpIII L chain Fd-ΔpIII Signal sequence gIII ompA ompA gIII derivative Arabinos IPTG IPTG Arabinos Replication origin CDF ori pBR ori pBR ori CDF ori f1 origin none has exist none has exist Packaging with phage descendants none has exist none has exist Antibiotic resistance amp ^R tet ^R amp ^R tet ^R

In the present specification, a dual vector system refers to a system for preparing two vectors each containing two different foreign genes and transforming the same vector to a host simultaneously or continuously to obtain a desired product. In particular, a system using host cells transformed with pLA-1 plasmid vector and pHf1g3T-1 phagemid vector was transformed with dual vector system (DVS) -I, pLT-2 plasmid vector and pHf1g3A-2 phagemid vector. The system using the host cell is called Dual Vector System (DVS) -II.

In the present invention, using a pyruvate dehydrogenase complex (Pruvate dehydrogenase complex) -E2 (PDC-E2) specific SP112 Fab clone as a model, a phage display system consisting of a dual-vector system (DVS) Provided. In addition, the present invention can provide a combination phage display Fab library using the dual vector system.

Hereinafter, the present invention will be described in more detail with reference to Examples, but this does not limit the present invention.

Example 1 Preparation of DVS-I and DVS-II

1.1 bacterial cell lines

Escherichia coli Cloning cell line TG1 ( supE thi-1 Δ (lac-proAB) Δ (mcrB-hsdSM) 5 (rK-mK-) [F ′ traD36 proAB lacIq lacZ Δ M15 ]) (Amersham Pharmacia Biotech, Sweden) And as a bacterial host for recombinant phage production.

1.2. PCR Amplification and Synthesis of Oligonucleotides

Ex-Taq polymerase (available from Takara, Japan) was used for all PCR amplifications and all restriction enzymes were purchased from Takara. In addition, all PCR primers used in the present invention were synthesized by order from Bioneer (Bioneer, Korea).

1.3. Preparation of Recombinant Vectors

All DNA cloning experiments were performed using standard methods (J. Sambrook, EF Fritsch, T. Maniatis, Molecular Cloning. A laboratory Manual, 2 ^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.). Followed.

1.3.1. Dual Vector System-I (DVS-I) (combination of pHf1g3T-1 phagemid and pLA-1 plasmid )

1.3.1.1 Preparation of pHf1g3T-1

pBR322 plasmid (provided by Dr. M. Eric Gershwin, University of California) was digested with Pst I and EcoR I, and the digested 4 kb DNA fragment was subjected to electrophoresis using 1% agarose gel, followed by Wizard DNA purification. Acquired using the kit (Promega, USA). Next, 30 units of CIP (calf intestinal phosphatase; obtained from Roche) were added and reacted in a 37 ° C. water bath for 1 hour, and then 1 μl of 0.5 M EDTA was added thereto and inactivated at 65 ° C. for 1 hour. Consisting of P _lac + Fd (V _H + C _H1 ) + delta gIII + f1 ori from pCMTG-SP112 (IG Therapy Co.) with Fab gene of SP112, a PDC-E2-specific human monoclonal antibody PCR method for DNA fragments (sense primer: 5′-GGGCTGCAGACGCGGCCTTTTTACGGTGGTTCCT-3 ′ (SEQ ID NO: 1), and anti-sense primer: 5′-GGGCAATTGCCGCGCACATTTCCCCGAAAAG-3 ′ (SEQ ID NO: 2) ) Was amplified under conditions of 35 cycles at 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, and 10 minutes at 72 ° C. At this time, the PCR machine used Perkin Elmer 9700 (manufacturer: Perkin Elmer Inc.). The PCR result was electrophoresed on a 1% agarose gel to separate DNA fragments (about 2.1 kb), followed by restriction enzyme treatment with Pst I and Mun I. After quantitating the prepared pBR322 vector and PCR results, T4 DNA ligase (obtained from Takara) was added and reacted O / N (overnight) at 4 ° C. and the reaction solution was 2.5 kV in TG1 electrocompetent cells. Transformation was performed using Gene-pulser II (manufacturer: Bio-rad, USA) at 25 ㎌ and 200 조건 conditions. Thereafter, 1 ml of LB culture was added and incubated for 1 hour at 37 ° C., and then plated on LB agar plates (LB / T plates) containing 10 μg / ml tetracycline for antibiotic selection and incubated overnight at 37 ° C. .

Phagemid was isolated and purified from the cultured cells to obtain pHf1g3T-1 phagemid.

1.3.1.2 Preparation of the pLA-1 Plasmid Vector

PCR method for gene fragments containing AraC ORF + ara BAD promoter + Multicloning site-AmpR ORF from pBAD / gIII (Invitrogen, USA) (sense primer: 5′-GGGATCGATTCAATTGTCT GATTCGTTACCAA-3 ′ (SEQ ID NO: 3) And anti-sense primers: 94 ° C. 1 minute, 55 ° C. 1 minute, 72 ° C. 1 minute, 35 cycles, and 72 ° C. for 10 minutes using 5′-GGGACTAGTTCAGTGGAA CGAAAACTCACG-3 ′ (SEQ ID NO: 4)) Amplified by. The obtained 2.9 kb PCR product was isolated using a 1% agarose gel, and purified using the Wizard DNA purification kit. DNA fragments were subjected to restriction enzyme treatment with Cla I and Spe I followed by CIP treatment as described above. Gene fragments containing CDF ori (approximately 850 bp) were also obtained from pCDFDuet-1 (Novagen, USA) by PCR method (sense primer: 5'-GGGATCGATATAGCTAGCTCACTCGGTCG-3 '(SEQ ID NO: 5) and anti-sense primer). : 5′-GGGACTAGTGCACTGAAATCTAGAGCGGAA-3 ′ (SEQ ID NO: 6)) was amplified under conditions of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 1 cycle, 35 cycles, and 72 ° C. for 10 minutes. After restriction enzyme treatment with Cla I and Spe I, the prepared pBAD / gIII vector fragments were mixed with T4 DNA ligase (obtained from Takara), followed by O / N reaction at 4 ° C. The reaction solution was transformed into TG1 transformed cells using Gene-pulser II under conditions of 2.5 kV, 25 Hz and 200 Hz. Thereafter, 1 ml of LB medium was added thereto, followed by incubation for 1 hour at 37 ° C., and then plated on LB agar plate (LB / A plate) to which 50 μg / ml ampicillin was added for antibiotic selection, and O / N culture at 37 ° C. It was. E. coli strains were obtained from the LB / A plate, and a single colony was incubated in LB / A liquid medium, and pBAD / gIII / CDF ori recombinant plasmid was isolated and purified using the Wizard plasmid purification kit. This plasmid was subjected to restriction enzyme treatment with Nco I and Xho I followed by CIP treatment. On the other hand, human C _L kappa gene fragments with Sal I and Sac II cloning sites inserted at the 5 ′ terminal were PCR-produced from pCMTG-SP112 (sense primer: 5′-GGGCCATGGGATTTAGGTGACACTATAGGATCTCGATCCCGCGAAAT-3 ′ (SEQ ID NO: 7) and anti-sense). Primer: 5'-GGGCTCGAGTTATCAACACTCTCCCCTGTTGCTC-3 '(SEQ ID NO: 8)) was used to amplify at 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, 35 cycles, and 72 ° C for 10 minutes. The obtained PCR result was subjected to restriction enzyme treatment with Nco I and Xho I, and then mixed with the vector DNA at an appropriate concentration, T4 DNA ligase and O / N reaction at 4 ° C. The reaction solution was transformed into TG1 transformed cells using Gene-pulser II under conditions of 2.5 kV, 25 Hz and 200 Hz. Thereafter, 1 ml of LB medium was added thereto, followed by incubation at 37 ° C. for 1 hour, and then plated on LB / A agar plates for antibiotic selection and O / N culture in 37 ° C. incubator. The single colonies thus obtained were cultured in LB / A liquid medium, and then plasmids cloned with the human CL gene were obtained using the Wizard plasmid purification kit. The obtained plasmid was treated with Sac I and Sac II restriction enzymes, and 30 units of CIP were added and reacted in a water bath at 37 ° C. for 1 hour, and then 1 μl of 0.5 M EDTA was added thereto. I was. Human V _L gene fragment was PCR amplified from pCMTG-SP112 (V _L κaSal 5-GGGGTCGACA TGGACATCCAGATGACCCAGTCTCC-3 ′ (SEQ ID NO: 9) and JκSac 5′-GGGCGGCGGATAC GTTTGATHTCCASYTTGGTCCC-3 ′ (SEQ ID NO: 10)) (Degeneracy codons : H = A / C / T, S = G / C, Y = C / T)) at 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 cycle, 35 cycles, and 72 ° C for 10 minutes Amplified under conditions. The obtained PCR results were subjected to restriction enzyme treatment with Sac I and Sac II, and the vector DNA and PCR products were mixed, and then T4 DNA ligase was added and reacted at O / N at 4 ° C. The reaction solution was transformed into TG1 transformed cells using Gene-pulser II under conditions of 2.5 kV, 25 Hz, and 200 Hz. Thereafter, 1 ml of LB medium was added thereto, followed by incubation at 37 ° C. for 1 hour, and then plated on LB / A agar plates for antibiotic selection and O / N culture in 37 ° C. incubator.

The plasmid was isolated and purified from the cultured cells to obtain a pLA-1 plasmid.

1.3.2. Dual Vector System-II (DVS-II) (combination of pHf1g3A-2 phagemid and pLT-2 plasmid )

1.3.2.1 Preparation of pHf1g3A-2 Phagemid Vector

This vector was created in 1.3.1.2 Prepared from pLA-1 vector. The pLA-1 vector was digested with Xho I and Sal I to cut human light chain antibody gene portions, and 4.3 kb gene fragments were obtained. This gene was isolated using 1% agarose gel, purified using Wizard DNA purification kit, and then subjected to CIP treatment. DNA fragments comprising Fd (VH + CH1) + ΔgIII + f1 ori were PCR method from pCMTG-SP112 (sense primer: 5'-GGGCTGCAGACGCGGCCTTTTTACGGTGGTTCCT-3 '(SEQ ID NO: 11) and anti-sense primer: 5'-GGGCAATTGCCGCGCACATTTCCCCGAAAAG -3 '(SEQ ID NO: 12)) was amplified at 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, 35 cycles, and 72 ° C for 10 minutes. The obtained PCR result was treated with restriction enzymes by Xho I and Sal I, mixed with the vector DNA in a 1: 2 molar ratio, and then added with T4 DNA ligase (obtained at Takara) at 4 ° C. O / N reaction at. The reaction solution was transformed into TG1 transformed cells using Gene-pulser II under conditions of 2.5 kV, 25 Hz and 200 Hz. Thereafter, 1 ml of LB medium was added thereto, followed by incubation at 37 ° C. for 1 hour, followed by antibiotic selection using LB / A agar plates for antibiotic selection.

The antibiotic-selected cells were cultured and phagemids were isolated and purified to obtain pHf1g3A-2 phagemids.

Meanwhile, the Fd (VH + CH1) gene of PDC-E2 specific SP112 present in pHf1g3A-2 for use as a negative control for bio-panning experiments was converted to BCKD-E2 (branched-chain alpha-keto acid dehydrogenase). The pHf1g3A-2-BCKD vector was prepared separately by substituting the Fd (VH + CH1) gene (IG Therapy Co.) of the antibody specific for complex-E2).

1.3.2.2 Preparation of the pLT-2 plasmid vector

This vector was prepared using pBR322. The pBR322 plasmid vector (provided by Dr. M. Eric Gershwin, University of California) was subjected to restriction enzyme treatment with Pst I and EcoR I to isolate 3.6 kb of the gene fragment from a 1% agarose gel using a Wizard DNA purification kit. Purified and CIP treatment was performed. DNA fragments containing P _lac + SP112 light chain gene were PCR-prepared from pCMTG-SP112 (sense primer: 5'-GGGATCGATTCAATTGTCTGATTCGTT ACCAA-3 '(SEQ ID NO: 13) and anti-sense primer: 5'-GGGACTAGTTCAGTGGAACGAAAACTC ACG-3' (SEQ ID NO: 14)) for 35 cycles at 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute, and at 10 ° C for 72 minutes. The obtained PCR result was subjected to restriction enzyme treatment with Pst I and Mun I, and mixed with the prepared pBR322 vector in a 1: 2 molar ratio. Then, T4 DNA ligase was added and O / N reaction at 4 ° C. I was. The reaction solution was transformed into TG1 transformed cells using Gene-pulser II under conditions of 2.5 kV, 25 kV and 200 kV. Thereafter, 1 ml of LB medium was added thereto, followed by incubation at 37 ° C. for 1 hour, followed by antibiotic selection using LB / T agar plates for antibiotic selection.

The antibiotic-selected cells were cultured and the plasmids were isolated and purified to obtain a pLT-2 plasmid.

Example 2: E. coli Transformation of

2.1 Transformation Experiments of Dual Vector System-I (DVS-I)

Fresh TG1 E. coli was incubated in LB culture and then centrifuged at 4000 X g for 15 minutes using a J2-MC centrifuge (manufactured by Beckman). The supernatant was removed and TG1 cells were washed with sterile distilled water containing 10% glycerol (obtained from Duchefa). This process was repeated three times to prepare a transformed cell line, and then transformed TG1 cells with Gene-pulser II at 100 kng of pLA-1 or pHf1g3T-1 vector at 2.5 kV, 2 ㎌, and 200 조건. It was. TG1 cells were then plated onto LB / A and LB / T plates, respectively, and cultured at 37 ° C. Cells containing pLA-1 and cells containing pHf1g3T-1 were selected from the resulting E. coli colonies, followed by LB / A (LB / AG) or LB / T containing 2% glucose (Duchfa). LB / TG) was cultured to OD ₆₀₀ = 0.5 in the culture. Each cultured cell was centrifuged at 4000 X g for 15 minutes using a J2-MC centrifuge (manufactured by Beckman), then the supernatant was removed and washed with sterile distilled water containing 10% glycerol. This process was repeated three times to prepare transgenic TG1 cells containing pLA-1 or pHf1g3T-1. Subsequently, the transformed TG1 cell line containing pLA-1 was transformed by electroporation of 100 ng pHf1g3T-1, and the transformed TG1 cell line containing pHf1g3T-1 was 100 ng of pLA-1. Transformation was performed by electroporation. The transformed cells were incubated at 37 ° C O / N in LB / AT plates containing both ampicillin and tetracycline for antibiotic selection. After culturing, the number of generated colonies was measured to determine CFU (colony forming unit), which is shown in FIG. 3A.

From FIG. 3A, when transforming TG1 cells containing pHf1g3T-1 phagemid with pLA-1 plasmid (DVS-IA), about 9 × 10 ⁸ CFU / μg DNA was obtained, but the vector was introduced into TG1 host cells. When changing the order of (DVS-IB), the transformation efficiency of the host cell was reduced by about 45 times. Can be.

2.2. Transformation of Dual Vector System-II (DVS-II)

TG1 cell lines were transformed using Gene-pulser II under conditions of 2.5 kV, 25 Hz and 200 Hz using 100 ng of pLT-2 or pHf1g3A-2 vectors. After transformation, the cells containing pLT-2 or pHf1g3A-2 were selected from E. coli colonies generated by plating on LB / T and LB / A plates and incubating at 37 ° C, respectively, and then LB / containing 2% glucose. Incubated in TG or LB / AG culture to OD ₆₀₀ = 0.5. Each cultured cell was centrifuged at 4000 X g for 15 minutes using a J2-MC centrifuge. The supernatant was removed and washed with sterile distilled water containing 10% glycerol, and this process was repeated three times to prepare a transgenic TG1 cell line containing pLT-2 or pHf1g3A-2. Then, the transforming TG1 cell line containing pLT-2 was transformed using 100 ng of pHf1g3A-2, and the transforming TG1 cell line containing pHf1g3A-2 using 100 ng of pLT-2. Transformed. The transformed cells were incubated at 37 ° C. O / N in LB / AT plates for antibiotic selection. After incubation, the number of generated colonies was measured to determine the CFU and are shown in FIG. 3B.

From FIG. 3B, the number of TG1 cells exhibiting the phenotypes of amp ^R and tet ^R was nearly 7.8 × 10 ⁸ CFU / μg DNA and 6.7 × 10 ⁸ CFU / μg DNA in DVS-II-A and DVS-II-B, respectively. The results showed that the DVS-II had little effect on the transformation efficiency of the host cell due to the difference in the order of introduction of the pHf1g3A-2 and pLT-2 vectors. The stability of the vector was higher than that of I.

Example 3: ELISA Analysis for Water-Soluble Fab Molecules

TG1 cells into which pCMTG-SP112, DVS-I, or DVS-II were inserted were cultured under the following conditions to prepare water-soluble Fab molecules. pCMTG-SP112 used 10 ml of LB / AG culture and DVS-I and DVS-II were incubated to approximately OD ₆₀₀ = 0.5 using 10 ml of LB / ATG culture. Each culture was centrifuged at 3300 X g for 10 minutes and then the supernatant was removed. PCMTG-SP112 was then added to LB / A culture (LB / AI) with 0.1 mM IPTG (isopropyl-β-D-1-thiogalactopyranisid), while DVS-I and DVS-II were 0.1 mM IPTG and 0.02% arabinose. After resuspending with LB / A culture (LB / ATIA) containing (arabinose), and incubated for 15 hours at 27 ℃, the culture was centrifuged to obtain a supernatant containing a water-soluble Fab. Maxi-sorp immune plates (Nunc, Denmark) at 10 μg / ml of PDC-E2, glutathione-s-transporase (GST), human interleukin-15 (IL-15), and bovine serum albumin (BSA) ) Were diluted in coating buffer (0.1 M NaHCO 3, pH 9.6), added 50 μl per well, and then adsorbed at 4 ° C. O / N. After washing the plate three times with 0.1% Tween containing phosphate buffered saline (PBS-Tween), 200 μl of blocking buffer (containing 3% skimmed milk in PBS) was added and reacted at 37 ° C. for 1 hour. . After washing three times with PBS-Tween again, 50 μl of the obtained water-soluble Fab supernatant was added to each well and reacted at 37 ° C. for 1 hour. After washing three times with PBS-Tween, goat anti-human kappa L-chain antibody-HRPO conjugated pAb (Sigma) diluted in blocking buffer at 1: 5000 was added to the plate, and each water-soluble Fab antibody was labeled with PDC. It was confirmed to have a specific reactivity to -E2. The binding reaction was confirmed using a 3.3 ', 5.5'- tetramethyl benzidine (TMB) substrate and measured using an ELISA reader (manufactured by Boirad) at an absorbance of 450 nm. The results are shown in FIG.

From FIG. 4, DVS-I produced at least about five times less SP112 Fab molecules showing antigen binding activity as compared to pCMTG-SP112, whereas DVS-II produced SP112 Fab antibody production compared to pCMTG-SP112. It was found that the differences were almost similar and produced Fab antibodies with about four times more antigen binding activity than DVS-I. In addition, the Fab present in these three TG1 cell cultures did not bind to the negative control antigens (IL-15, GST and BSA).

On the other hand, phage ELISA was performed in the same manner as above, at which time 50 μl of phage supernatant (5 × 10 ⁷ PFU / well) was added and reacted at 37 ° C. for 1 hour. After washing the PBS-Tween, chlorine anti-M13 HRPO conjugated pAb (obtained from Sigma) diluted in blocking buffer at 1: 5000 was added and the phage was confirmed to have a specific reactivity with respect to PDC-E2. Is shown in FIG. 7.

As can be seen in FIG. 7, all recombinant phages produced by pCMTG-SP112, DVS-I or DVS-II showed specific binding activity for PDC-E2 and negative control antigens (IL-15, GST , BSA).

Example 4 Comparison of Expression of Fab Fragments by Western Blot Assay

TG1 cells containing recombinant vectors (pCMTG-SP112, DVS-I, and DVS-II) were cultured in a culture medium containing IPTG and arabinose as mentioned in Example 3, and then cell precipitates were centrifuged. Obtained. The obtained precipitate was resuspended 1: 1 with SDS-sample buffer, and then bathed in boiling water for 5 minutes and a 12% SDS-PAGE experiment was performed. Proteins in SDS-PAGE were then delivered to the nitrocellulose membrane (Amersham pharmacia biotech) for 90 minutes at 65 V using a Ready gel precast gel system (Biorad). The membrane to which the protein was transferred was reacted with blocking buffer for 1 hour at room temperature, washed three times with PBS-Tween for 5 minutes each, and then mouse anti-diluted 1: 3000 in blocking buffer for detection of fused Fd-ΔpIII. myc mAb (obtained from IG Therapy Co.) was reacted with the membrane for 1 hour at room temperature. After washing the membranes with PBS-Tween three times for 5 minutes each, goat anti-mouse IgG AP conjugated pAb (obtained by Sigma) diluted 1: 5000 in blocking buffer was reacted for 1 hour. On the other hand, goat anti-human kappa L chain AP conjugated pAb (Sigma) was used for the detection of human light chain fragments. Nitro Blue Tetrazolium Chloride (NBT) / 5-Bromo-4-chloro-3-indoliphosphate (BCIP) substrate (Sigma) was used as the substrate, and the signal appearing on the membrane was measured using a densitometer ( Manufacturer: Biorad) and the results are shown in FIG. 5.

As can be seen in Figure 5, in the Fd-ΔpIII molecule, DVS-I produced about 3-4 times more than pCMTG-SP112 and DVS-II in TG1 host cells, and conversely, expression of human light chain fragments. The amount was reduced by about 6 to 10 times compared to pCMTG-SP112 and DVS-II. Thus, Fabs with antigen-binding ability are optimally produced by the combination of light chain fragments with Fd of the same number of molecules, so the production of low antigen-binding active Fab molecules shown in DVS-I is dependent on the disproportionate expression of the antibody fragments that make up Fab molecules. It is assumed to be due.

Example 5: Amplification of Recombinant Phage

Amplification of recombinant phage is described in the prior art (Mcafferty, J. 1996. Phage display: factors affecting panning efficiency.In: Kay, BK, Winter, J., McCafferty, J. (Eds.), Phage Display of Peptides and Proteins, a Laboratory Manual.Academic Press, San Diego, p. 261; Baek, H., Suk, KH, Kim, YH, Cha, S. 2002.An improved helper phage system for efficient isolation of specific antibody molecules in phage display Nucleic Acids Res. 30 (5), e18), with reference to what was reported in some modified methods to obtain recombinant phage as follows. Briefly, first, each TG1 cell containing a recombinant vector (pCMTG-SP112, DVS-I or DVS-II) was cultured. pCMTG-SP112 was incubated in 10 ml of LB / AG culture, and DVS-I and DVS-II were incubated in 10 ml of LB / ATG culture to approximately OD ₆₀₀ = 0.5. Each culture was centrifuged at 3300 × g for 10 minutes and then resuspended with 10 ml of LB / G culture. M13K07 or Ex-12 helper phage was added to the suspension to be 20 MOI (multiplicity of infection), and then incubated at 37 ° C for 1 hour. The mixture of cell line and helper phage was again centrifuged at 3300 X g for 10 minutes, and then the supernatant was removed to harvest the cells. PCMTG-SP112 is then 100 ml LB / AK (containing 100 μg ampicillin and 50 μg kanamycin), while DVS-I and DVS-II are 100 mL LB / ATKA (100 μg ampicillin, 10 μg After resuspending with tetracycline, 50 μg kanamycin and 0.001% arabinose), the cells were incubated at 27 ° C. for 15 hours. The culture was centrifuged at 3300 X g for 20 minutes to obtain a supernatant with recombinant phage. Phage particles were precipitated using PEG / NaCl solution and then resuspended in 1 ml of sterile PBS to obtain concentrated phage.

Phage titers were measured by the following plaque forming unit (PFU) assay. TG1 cells were cultured in LB culture so as to have an OD ₆₀₀ = 0.8, and 1 μl of the phage concentrate obtained in 100 μl of the culture was mixed and mixed, and then 10 ^-2 , 10 ^-4 , 10 ^-6 using TG1 cell culture. , Diluted 100-fold with 10 ^-8 . After reaction at 37 ° C. for 30 minutes, the mixture was mixed with 4 ml of top agar and poured flat onto the LB plate. Plates left at room temperature for 10 minutes were incubated at 37 ° C. for 15 hours, and the titers of phage obtained by measuring the plaques generated on the plates were calculated, and the results are shown in FIG. 6.

As can be seen in FIG. 6, when ex12 helper phage was used for phage rescue, DVS-II showed the highest phage titer (approximately 7 × 10 ¹⁰ PFU / mL) and pCMTG-SP112 and DVS-I, respectively, Phages of 5 × 10 ¹⁰ PFU / ml and 2 × 10 ¹⁰ PFU / ml were produced, showing the best recombinant phage productivity in DVS-II. The M13K07 helper phage showed a similar phage titer of approximately 2 X 10 ¹¹ PFU / mL in pCMTG-SP112 and DVS-II, whereas DVS-I showed a phage titer of approximately 10 ¹⁰ PFU / mL. Compared to SP112 and DVS-II, Ex12 helper phage showed 2-3 times lower yield and M13K07 showed 20 times lower recombinant phage yield.

In addition, the production of recombinant phage for bio-panning was performed in the same manner as above, except that DVS-II-inserted TG1 cells contained DVS-II-BCKD (pLT-2 and pHf1g3A-2-BCKD). Strains diluted in the ratio of 1:10 ⁴ , 1:10 ⁶ or 1:10 ⁸ were used as TG1 cells, and Ex-12 helper phage was used as the helper phage.

Example 6: Bio-panning

The selection of recombinant phage binding to PDC-E2 was performed by the panning method as shown in the schematic diagram of FIG. 8 (Ref. Baek, H., Suk, KH, Kim, YH, Cha, S. 2002). An improved helper phage system for efficient isolation of specific antibody molecules in phage display.Nucleic Acids Res. 30 (5), e18). First, 10 μg / ml PDC-E2 antigen was reacted with Maxi-sorp immunoplate at 4 ° C. O / N using a coating buffer. Thereafter, the plate was washed three times with PBS-Tween, 200 μl of blocking buffer was added thereto, and reacted at 37 ° C. for 1 hour. Recombinant phage obtained from a mixed culture of TG1 cell lines containing a positive control DVS-II and a negative control DVS-II-BCKD, respectively, at a ratio of 1:10 ⁴ , 1:10 ^6, or 1:10 ⁸ , respectively. Was added to 24 microwells at a total concentration of 1.2 × 10 ⁹ (5 × 10 ⁷ / well) and then reacted at 37 ° C. for 2 hours. After washing 10 times with PBS-Tween, 50 μl of elution buffer (0.1 M glycine-HCl, pH 2.5) was added per microwell to react for 10 minutes to elute phage from the plate. The obtained phages were infected with fresh TG1 cells for 1 hour, plated on LB / T plates and incubated overnight at 27 ° C. Escherichia coli colonies grown on LB / T plates were obtained using sterile glass rods and pHf1g3A-2 phagemid DNA was isolated and purified from the E. coli using the Wizard plasmid purification kit. 100 ng of this phagemid DNA was electroporated into TG1 cells into which pLT-2 had already been inserted, plated on LB / AT plates, and screened. Recombinant phage was again amplified using Ex12 helper phage from selected cell lines. . This amplified recombinant phage was used again for the panning and the experiment was repeated four times in total. The binding reactivity of the recombinant phage and E. coli clones obtained in each step with PDC-E2 was measured by ELISA method and the results are shown in FIG. 9.

From FIG. 9, PDC-E2-specific selection is progressed from the first panning under conditions of 10 ⁴ more than that of the DVS-II-BCKD, which is a negative control, and PDC-E2 from the second panning under 10 ⁶ more conditions. It was confirmed that -specific selection was in progress. However, PDC-E2-specific selection of recombinant phage did not proceed even if the negative control group DVS-II-BCKD proceeded to the 4th panning under the condition of more than 10 ⁸ more than DVS-II. On the other hand, in order to confirm the PDC-E2-specific selection of the recombinant phage shown in the phage ELISA at the clone level, the phagemid genome was isolated from the recombinant phage obtained after each round of panning, and inserted into TG1 cells carrying pLT-2. 24 Escherichia coli colonies were randomly cultivated, and then subjected to ELISA with the culture to check whether each E. coli clone produces a PDC-E2-specific Fab antibody.

Table 2. Frequency of Escherichia coli clones secreting anti-PDC-E2 Fab molecules after each panning round

Input rate
(Positive / voice) Yield of anti-PDC-E2 clone ^a 1 ^st round 2 ^nd round 3 ^rd round 4 ^th round 1: 10 ⁴ 4/24 24/24 24/24 24/24 1: 10 ⁶ 0/24 4/24 21/24 24/24 1: 10 ⁸ 0/24 0/24 0/24 0/24 Negative control 0/24 0/24 0/24 0/24

24 clones were randomly extracted for antigen binding ELISA. This table shows the ratio of (number of positive clones) / 24 clones.

From Table 2, a negative control group of DVS-II-10 ⁴ BCKD produces a number of conditions under the 24 clones all PDC-E2- specific Fab antibody obtained after the second panning than DVS-II, 10, ⁶ under many conditions After about 4 th panning it was confirmed that all clones produced PDC-E2-specific Fab antibodies. This is consistent with the phage ELISA of FIG. 9 and demonstrates that screening of antigen-specific recombinant phage proceeds about 100-fold in one panning.

In summary, DVS-II was found to be stable compared to DVS-I regardless of the sequence of vector transduction efficiency of host cells into the host cells. The amount of expression of the molecule, the titer of the recombinant phage and the amount of Fab-ΔpIII displayed on the surface of the phage progeny were similar to the phage display system using a conventional single phagemid vector, and was also targeted using a panning method. Recombinant phage displaying specific Fab-ΔpIII molecules could be successfully selected, allowing antigen-specific Fd genes to be isolated from pHf1g3A-2 phagemids.

The DVS-II technique can be used as a very useful tool for the preparation of highly versatile combinatorial phage display Fab libraries. Furthermore, if the light chain's flexibility in the antigen-antibody binding reaction of antibodies is taken into account, the desired antibody clone can be obtained through a panning method. It may be practically used for the selection of humans, and at least a monoclonal antibody of a rodent origin may be manipulated by guided-selection or chain shuffling to prepare humanized antibodies.

DVS-II is a single phage in all areas, including vector stability, expression of water soluble Fab molecules, titer of recombinant phage produced, amount of antibody molecules displayed on the phage surface, and the selection of recombinant phage to display antigen-specific antibodies. It is comparable to conventional phage display systems using mid vectors.

The usefulness of an antibody library is directly related to the number of clones that make up the antibody library, so it can be assumed that the larger the number of clones present in the library, the greater the antigen binding specificity of that library is, The possibility of obtaining useful antibodies that bind to the fluorescence is increased. In this respect, DVS-II can be very useful for the preparation of combinatorial Fab antibody libraries.

In addition, in the conventional single vector system, considering that both heavy and light chain fragments must be expressed by a single vector, the dual vector system of the present invention is composed of two independent vectors. In manufacturing, there is an advantage in that the restriction enzyme used in the antibody cloning process can prevent the diversity of the antibody genes as much as possible.

In addition, DVS-II can screen for target molecule-specific heavy chain genes paired with specific monoclonal light chain genes and chain shuffling to convert monoclonal antibody genes of rodent origin into human genes of human origin. Immediately available for use in shuffling or guided-selection techniques, the biggest advantage of DVS-II is that once a good heavy chain gene library is prepared in pHf1g3A-2 phagemid, light chain genes of any desired rodent origin The library can be used to obtain human heavy chain genes that bind to and display specific antigen binding specificities.

In conclusion, DVS-II is a very practical technique for producing antibody libraries with higher diversity than existing original phagemid vectors without experimental difficulties due to phage contamination and reduction of antibody concentration displayed on the recombinant phage surface. It has the potential to develop a kit that can select humanized antibody genes from various mouse antibody genes can be very useful in the field of antibody drug development.

1 shows a schematic diagram of a vector produced in the present invention. In A, dual vector system-I (DVS-I) uses a combination of pLA-1 plasmid and pHf1g3T-1 plasmid, and in B, dual vector system-II (DVS-II) is pLT-2 plasmid and pHf1g3A- It shows that the 2 plasmid combination is used.

Figure 2 shows four different strategies for transforming host cells for TG1 transformation in DVS-I (DVS-IA and DVS-IB) or DVS-II (DVS-II-B or DVS-II-B). have.

3 shows a comparison of the number of E. coli colonies produced by different transformation strategies in a dual vector system. Colony forming units (CFUs) were measured from E. coli colony counts that exhibited amp ^R and tet ^R phenotypes after secondary transformation into cells according to FIG. 2. The sequence of vectors introduced into fresh TG1 cells is as follows: (A) DVS-I is in the order of DVS-IA (pHf1g3T-1 → pLA-1) and DVS-IB (pLA-1 → pHf1g3T-1), (B) DVS-II is in the order of DVS-II-A (pLT-2 → pHf1g3A-2) and DVS-II-B (pHf1g3A-2 → pLT-2). Data represent mean ± standard deviation of experiment 3.

4 shows the antigen binding specificity of soluble SP112 molecules produced by TG1 cells carrying pCMTG-SP112, DVS-I and DVS-II. PDC-E2 and other negative control antigens including GST, IL-15, BSA were coated on microtiter plates. Supernatants were received from cultures of TG1 cells with pCMTG-SP112, DVS-I and DVS-II and subjected to ELISA. Goat anti-human kappa light chain antibody conjugated with HRPO was used as the secondary antibody. Binding signals were visualized with TMB substrate and analyzed at OD _{450 nm} . Data represents the mean ± standard deviation of experiment 3.

5 shows Western blot analysis to determine expression of Fd-ΔpIII and kappa light chains in TG1 host cells with different vector configurations. SP112, DVS-I and DVS-II were incubated in the presence of 0.1 mM IPTG and 0.02% arabinose. Whole cell lysates were obtained from precultured cells to obtain the same concentration and loaded into each well of 12% SDS-PAGE. The Fd-ΔpIII fusions used goat anti-mouse IgG conjugated with mouse anti-Myc tail mAb and AP (A), and the kappa light chain fragment replaced the goat anti-human kappa light chain antibody (B) conjugated with AP. Used. Visualization using NBT / BCIP substrate. Lane 1 is TG1 cells with pCMTG-SP112, lane 2 is TG1 cells with DVS-I, and lane 3 is TG1 cells with DVS-II.

6 shows the measurement of PFU following phage obtained from TG1 cells with different vector sets.

FIG. 7 shows phage ELISA showing antigen specific reactivity of phage preparations obtained from TG1 cells with different vector sets.

8 shows a schematic diagram showing affinity screening of DVS-II.

9 shows a polyclonal phage ELISA that determines PDC-E2 specific abundance after rounds of continuous panning. TG1 cells with positive controls (pHf1g3A-2 and pLT-2) and negative controls ((pHf1g3A-2-BCKD and pLT-2) were harvested at a ratio of 1:10 ⁴ , 1:10 ⁶ , 1:10 ⁸ Or mixed with a negative control.

10 shows a schematic of the pBR322 vector.

11 shows a schematic of the pCMTG vector.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> A vector for expressing antibody fragments and a method for          producing recombinant phage that displays antibody fragments by          using the vector <130> IPM-34365-01 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Sense primer <400> 1 gggctgcaga cgcggccttt ttacggtggt tcct 34 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Anti-sense primer <400> 2 gggcaattgc cgcgcacatt tccccgaaaa g 31 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sense primer <400> 3 gggatcgatt caattgtctg attcgttacc aa 32 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Anti-sense primer <400> 4 gggactagtt cagtggaacg aaaactcacg 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Sense primer <400> 5 gggatcgata tagctagctc actcggtcg 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Anti-sense primer <400> 6 gggactagtg cactgaaatc tagagcggaa 30 <210> 7 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Sense primer <400> 7 gggccatggg atttaggtga cactatagga tctcgatccc gcgaaat 47 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Anti-sense primer <400> 8 gggctcgagt tatcaacact ctcccctgtt gctc 34 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> VLkappaaSal <400> 9 ggggtcgaca tggacatcca gatgacccag tctcc 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> JkappaSac <220> <221> misc_feature <222> (21) <223> H is A, C or T <220> <221> misc_feature <222> (26) <223> S is G or C <220> <221> misc_feature <222> (27) <223> Y is C or T <400> 10 gggcggcgga tacgtttgat htccasyttg gtccc 35 <210> 11 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Sense primer <400> 11 gggctgcaga cgcggccttt ttacggtggt tcct 34 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Anti-sense primer <400> 12 gggcaattgc cgcgcacatt tccccgaaaa g 31 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Sense primer <400> 13 gggatcgatt caattgtctg attcgttacc aa 32 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Anti-sense primer <400> 14 gggactagtt cagtggaacg aaaactcacg 30  

Claims (6)

(1) binding a light chain of a human antibody to the pBR322 plasmid to prepare a first vector; (2) preparing a first DNA fragment through a PCR reaction with a primer set consisting of pBAD / gIII plasmid consisting of SEQ ID NO: 3 and SEQ ID NO: 4, and the pCDFDuet-1 plasmid consisting of SEQ ID NO: 5 and SEQ ID NO: 6 PCR reaction was performed with a primer set to prepare a second DNA fragment; After joining the first DNA fragment and the second DNA fragment; Binding a heavy chain of a human antibody to prepare a second vector; (3) transforming the first vector of step (1) into a host cell; And (4) transforming the dual vector system-II-A (DVS-II-A), comprising transforming the second vector of step (2) further to the transformed host cell of step (3) How to produce a convertible. (1) binding a light chain of a human antibody to the pBR322 plasmid to prepare a first vector; (2) preparing a first DNA fragment through a PCR reaction with a primer set consisting of pBAD / gIII plasmid consisting of SEQ ID NO: 3 and SEQ ID NO: 4, and the pCDFDuet-1 plasmid consisting of SEQ ID NO: 5 and SEQ ID NO: 6 PCR reaction was performed with a primer set to prepare a second DNA fragment; After joining the first DNA fragment and the second DNA fragment; Binding a heavy chain of a human antibody to prepare a second vector; (3) transforming the second vector of step (2) into a host cell; And (4) transforming the dual vector system-II-B (DVS-II-B), comprising transforming the first vector of step (1) further to the transformed host cell of step (3) How to produce a convertible. A method for expressing a human Fab antibody gene using the DVS-II-A of claim 1. A method of expressing a human Fab antibody gene using the DVS-II-B of claim 2. A method of producing recombinant phage displaying human Fab antibodies by the method of claim 1 or 2. A method for screening recombinant phage having a target molecule-specific VH + CH1 antibody gene phagemid genome by the method of claim 1 or 2.

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