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WO2019078591A1 - Système d'affichage à protéine multimère utilisant la fluidité de la membrane cellulaire - Google Patents

Système d'affichage à protéine multimère utilisant la fluidité de la membrane cellulaire Download PDF

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WO2019078591A1
WO2019078591A1 PCT/KR2018/012212 KR2018012212W WO2019078591A1 WO 2019078591 A1 WO2019078591 A1 WO 2019078591A1 KR 2018012212 W KR2018012212 W KR 2018012212W WO 2019078591 A1 WO2019078591 A1 WO 2019078591A1
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monomer
microorganism
protein
target protein
vector
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정상택
조미경
윤현웅
황보라
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Kookmin University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)

Definitions

  • the present invention relates to a display system for multi-chain objective proteins using flow properties of cell membranes.
  • Protein display systems have physically linked genotypes and phenotypes. Bacterial display systems, bacteriophage display systems, and yeast display systems have been developed and are now widely used for high-speed search for improving the functionality of pharmaceutical / industrial proteins (Directed Evolution Library Creation: Methods and Protocols 2003 Arnold, Frances H., Georgiou, George, Directed Enzyme Evolution: Screening and Selection Methods 2003 Arnold, Frances H., Georgiou, George)
  • Therapeutic antibodies are considered to be one of the most effective cancer treatment methods because they exhibit very high specificity to the target, low bio-toxicity and low side effects, as well as excellent blood half-lives of about 3 weeks, compared with conventional low-molecular drugs.
  • Therapeutic antibodies are composed of Fab (Fragment antigen binding), which recognizes the target antigen, and Fc (fragment crystallizable), which collects immune cells and removes the target. Since the effector function on Fc is mediated by the binding of Fc ⁇ Rs (Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII), many global pharmaceutical companies are exploring Fc variants with increased binding capacity to Fc ⁇ Rs to increase the efficacy of antibody drugs .
  • Roche is a leading company in the development of therapeutic antibody medicines including Roche, Amgen, Johnson & Johnson, Abbott and BMS. Especially Roche has developed Herceptin, Avastin, Rituxan ) Is a representative product. With these three therapeutic antibodies, it generates a huge profit of around US $ 19.5 billion in the global market in 2012, and is leading the global antibody drug market. Johnson & Johnson, who developed Remicade, is also growing rapidly in the global antibody market due to increased sales, and pharmaceutical companies such as Abbott and BMS are also known to have a number of therapeutic antibodies at the developmental stage. As a result, biopharmaceuticals, including therapeutic antibodies specific to disease targets and low side effects, are rapidly replacing the global pharmaceutical market, where low-molecular drugs have taken the initiative.
  • Conventional antibody Fc variant detection systems using bacteria include full-length IgG display and Fc display using PelB. All of them are displayed on the inner membrane of the bacteria and then spheroplasted to peel off the outer membrane Analysis system.
  • the full-length IgG display system is a system in which bacteria are co-transformed with plasmids coding for heavy and light chains, and heavy and light chains expressed in the periplasmic region around the plasma membrane are assembled (Jung et al., 2013 ACS Chem Biol). Since this method requires two plasmids encoding the heavy chain and the light chain respectively, two promoters (Lac promoter and PBAD promoter) must be considered at the same time during the culture and overexpression, and the culture conditions and time (20 hours) The longer the screening process, the greater the rate of negative clone that grows fast. In addition, since the display level is not constant, the deviation (CV: coefficient of variation) is high when the flow cytometer is actually analyzed.
  • Fc display using PelB leader peptide is a system in which trap is increased due to viscosity of trehalose due to Fc protein secreted to the cytoplasmic space region by sec pathway (Jung et al., 2010 Proc Natl Acad Sci USA).
  • This method is inconvenient to separately prepare a medium to which 0.5 M of trehalose is added at a high concentration without using a general medium, and the amount of the Fc fragment to be washed by the washing step in the spheroplasting process It is very difficult to control the display level of the trapped Fc protein. Indicating that they are not suitable in terms of uniformity and reproducibility essential for a series of screening processes.
  • the present inventors have sought to develop a display system with increased convenience and efficiency over a full-length IgG display system, which is a conventional display system, or an Fc display system using PelB.
  • a display system can be manufactured simply and conveniently by forming a multiplexed body by self-assembly due to the fluidity of the cell membrane. And that the degree of display is uniform.
  • an object of the present invention is to provide a method for producing a multi-chain objective protein display system using the fluidity of a cell membrane.
  • It is another object of the present invention to provide a target protein display system comprising a monomer of a target protein to which an anchor polypeptide is fused.
  • Still another object of the present invention is to provide a composition for a target protein display comprising a monomer of a target protein to which an anchor polypeptide is fused, a nucleic acid molecule that encodes a monomer of a target protein to which the anchor polypeptide is fused or a vector comprising the nucleic acid molecule .
  • the present invention provides a method of producing a multimeric target protein display system comprising the steps of:
  • a vector comprising (i) a nucleotide sequence encoding a monomer and anchor polypeptide of the target protein to be displayed and (ii) a vector comprising a promoter operatively linked to the coding nucleotide sequence Constructing a second image;
  • the monomer of the objective protein to which the anchor polypeptide is fused is self-assemble by membrane fluidity to form a multimer.
  • the present inventors have sought to develop a display system with increased convenience and efficiency over a full-length IgG display system, which is a conventional display system, or an Fc display system using PelB.
  • a display system can be manufactured simply and conveniently by forming a multiplexed body by self-assembly due to the fluidity of the cell membrane. And the degree of display is uniform.
  • " target protein " is used herein to mean any protein or peptide as an object to be displayed on the display system.
  • VH or / and VL a single-chain variable fragment (scFV), an Fc (humanized antibody) or a fragment thereof, an antibody or a portion thereof, Binding protein or a binding domain thereof, a peptide, an antigen, an adhesion protein, a structural protein, a regulatory protein, a toxin protein, a cytokine, a transcription regulator, a blood coagulation factor, a plant bio- But are not limited to, CSF (Macrophagecolony stimulating factor), hemoglobin, G-protein, CD28, interleukin, interferon- ⁇ , TGF- ⁇ (Transforming growth factor- ⁇ )
  • a target protein of the present invention is an antibody, an antibody antigen binding region (VH or / and VL), an scFv, a Fab, an Fc protein, a G- protein, a hemoglobin, Interferon-gamma, TGF-beta, or a fragment thereof.
  • Antibodies are proteins that specifically bind to specific antigens. There are five major classes of natural antibodies: IgA, IgD, IgE, IgG and IgM, usually two identical light chains (L) and two identical heavy chains (H) , which is a heterodimeric glycoprotein of about 150,000 daltons.
  • antibody refers to polyclonal antibodies, monoclonal antibodies, human antibodies, and humanized antibodies and fragments thereof.
  • Papain degradation of the antibody forms two Fab fragments and one Fc fragment, and in the human IgG molecule, the Fc region is generated by papain digestion of the N-terminus of Cys 226 (Deisenhofer, Biochemistry 20: 2361-2370, 1981) .
  • the antibody Fc domain may be the Fc domain of an IgA, IgM, IgE, IgD, or IgG antibody, or a variant thereof.
  • the target protein of the present invention is in a multimer form including a dimer.
  • the target protein of the present invention is expressed in a periplasmic region in the form of a monomer, floated by the fluidity of the cell membrane, and self-assemble with other monomers to form a multimer.
  • the monomer of the target protein of the present invention is one or more monomers selected from the group consisting of heavy chain variable region (VH), light chain variable region (VL) and fragments thereof.
  • the monomer of the target protein of the present invention is an Fc protein monomer or a fragment thereof.
  • &quot refers to an anchor polypeptide that binds to the N-terminal or C-terminal of a monomer of the target protein and is expressed and anchored in the cell membrane of the microorganism and the monomer of the target protein is expressed in the periplasmic space (NlpA leader peptide), MalF (Jung et al., 2007 Biotechnol Bioeng), and Pf3 coat protein (hereinafter referred to as " (Kleiner et al., 2008 FEBS Lett), M13 procoat protein (Czwizinski and W Wickner, 1982 EMBO J), ProW Nt / TM1 / 3K (Linda Froderberg et al., 2003 Molecular Microbiology), KdpD protein (Sandra J. Facey, Andreas Kuhn 2004 Biochim Biophys Acta).
  • the anchor polypeptide may be linked to the monomer of the target protein by a linker.
  • a nucleotide sequence encoding a monomer and an anchor polypeptide of the target protein to be displayed is constructed as a vector together with a promoter operatively linked to the coding nucleotide sequence.
  • &quot operably linked " means a functional linkage between a promoter that is a nucleic acid expression control sequence and another nucleic acid sequence, whereby the promoter regulates transcription of the other nucleic acid sequence.
  • the nucleotide or nucleic acid molecule may be isolated or recombinant, and includes DNA and RNA in single and double stranded form as well as corresponding complementary sequences.
  • the isolated nucleic acid is a nucleic acid isolated from a peripheral genetic sequence present in the genome of the isolated nucleic acid, in the case of a nucleic acid isolated from a naturally occurring source.
  • a nucleic acid that is enzymatically or chemically synthesized from a template such as a PCR product, a cDNA molecule, or an oligonucleotide
  • the nucleic acid generated from such a procedure can be understood as an isolated nucleic acid molecule.
  • the isolated nucleic acid molecule represents a nucleic acid molecule as a separate fragment or as a component of a larger nucleic acid construct.
  • a nucleic acid is operably linked when it is placed in a functional relationship with another nucleic acid sequence.
  • the DNA of a full-length or secretory leader is operably linked to the DNA of the polypeptide when expressed as a preprotein in the form before secretion of the polypeptide, and the promoter or enhancer comprises a polypeptide sequence
  • the ribosome binding site is operably linked to a coding sequence when it is placed to facilitate translation.
  • operably linked means that the DNA sequences to be linked are located adjacent to each other, and in the case of the secretory leader, it means that the DNA sequences are adjacent to each other in the same reading frame.
  • the enhancer need not be located contiguously. Linking is accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional methods.
  • vector refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence.
  • the nucleic acid sequence may be exogenous or heterologous.
  • Vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophage). Those skilled in the art can establish a vector by standard recombinant techniques (Maniatis, et al, Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al, In:.. Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994).
  • the term expression vector refers to a vector comprising a nucleic acid sequence encoding at least a portion of the gene product to be transcribed. In some cases, the RNA molecules are then translated into proteins, polypeptides, or peptides.
  • the expression vector may contain various regulatory sequences. Vectors and expression vectors may also include nucleic acid sequences that provide another function, as well as regulatory sequences that regulate transcription and translation.
  • microorganisms are transformed using the constructed vector.
  • the microorganism can be transfected or transformed by the vector, which means that the exogenous nucleic acid molecule is transferred or introduced into the host cell.
  • the microorganism of the present invention is a bacterial cell.
  • the cells are suitable for the practice of the present invention in that they have a periplasmic region between the inner membrane and the outer membrane.
  • Examples of preferred bacterial cells of the present invention include E. coli , Pseudomonas aeruginosa , Vibrio cholera , Salmonella typhimurium , Shigella flexneri , Haemophilus influenza , Bordotella pertussi , Erwinia amylovora , Rhizobium sp. But are not limited thereto.
  • the step of expressing the monomer of the target protein fused with the anchor polypeptide is anchored to the inner membrane of the microorganism, and the monomer of the target protein is displayed in the periplasmic region of the microorganism .
  • the monomer of the target protein to which the anchor polypeptide is fused floats the cell membrane due to the membrane fluidity of the cell membrane, and the monomers self-assemble as a pair to form a multimer, .
  • the term " multimer " includes a dimer, wherein the dimer may be a homodimer with the same monomers attached, or a heterodimer with different monomers combined.
  • the display system of the present invention is self-assembled by expressing and self-assembling Fc monomers to form homodimers (Examples 1 to 3), or by expressing the heavy and light chains of the antibody, respectively It was confirmed that heterodimers (Examples 4 to 5) can be formed.
  • a target protein display system produced by the above method.
  • the objective protein display system manufactured according to the present invention is superior to conventional full-legged IgG display systems or PelB-based Fc display systems in convenience (simplicity of medium production, reduction of incubation time for screening, (%) CV value is smaller than that of the conventional full-field IgG display technique and the fluorescence intensity value (Mean fluorescence intensity, MFI) between the negative clone and the positive clone is larger than that of the conventional full IgG display technique.
  • MFI an fluorescence intensity
  • a method for producing an anchor polypeptide comprising the steps of: (a) preparing an anchor polypeptide comprising a monomer of a target protein to which an anchor polypeptide is fused, a nucleic acid molecule encoding a monomer of a target protein to which the anchor polypeptide is fused, Thereby providing a composition for a target protein display.
  • the monomer of the objective protein to which the anchor polypeptide of the present invention is fused comprises a sequence selected from the group consisting of SEQ ID NO: 35 to SEQ ID NO: 39.
  • the nucleic acid molecule of the present invention comprises a sequence selected from the group consisting of SEQ ID NOS: 30 to 34.
  • the present invention provides a method of screening Fc variants with improved affinity for Fc [gamma] R comprising the steps of:
  • a vector comprising (i) a nucleotide sequence encoding a monomer and anchor polypeptide of the Fc variant to be displayed and (ii) a vector comprising a promoter operably linked to the coding nucleotide sequence Constructing a second image;
  • said Fc variant comprises one or more amino acid substitutions selected from the group consisting of the following amino acid substitutions according to the Kabat numbering system: L235V, G236A, S239D, F243L , V264E, S267E, R292P, Q295R, S298G, T299A, Y300L, K326I, A327Y, L328F, L328G, L328W, A330L, P331A, I332E, I332Y, T350A, D357G, E382V, N390D, T394A, P396L, F405S, M428I and M428L .
  • amino acid residue number of the antibody Fc domain herein refers to the Kabat numbering system conventionally used in the art (Kabat et al., In Proteins of Immunological Interest 5th Ed., US Department of Health and Human Services , NIH Publication No. 91-3242, 1991).
  • the present invention provides a method for screening an antigen binding region (VH and / or VL) having a binding force to an antigen comprising the steps of:
  • a vector comprising (i) a nucleotide sequence encoding an heavy chain or light chain variable region of an antibody to be displayed and an anchor polypeptide, and (ii) a vector comprising a promoter operatively linked to the coding nucleotide sequence Constructing a second image;
  • the heavy or light chain variable region of the antibody fused with the anchor polypeptide is self-assemble with the light or heavy chain variable region of the antibody by membrane fluidity of the antibody to form a multimer ;
  • nucleotide sequence encoding the heavy chain variable region of the antibody and the nucleotide sequence encoding the light chain variable region can be constructed in the same vector or constructed in different vectors, preferably constructed in the same vector and expressed by a single promoter .
  • the present invention provides a multi-chain target protein display system using the fluidity of cell membranes.
  • the display system of the present invention has much higher convenience (such as simple medium production, reduction of incubation time for screening, use of a single promoter, etc.) compared with the conventional full-field IgG display system or PelB-based Fc display system, ,
  • The% CV value is smaller than that of the full-length IgG display technique, and the difference between the vocal clone and the positive clone fluorescence signal value is larger, which is more effective for screening using the target protein display.
  • Figure 1 shows the Fc that is self-assemble by the flowability of gIII protein and cell membrane to form a dimer.
  • Figure 2 shows Fc with NlpA leader peptides and dimers fused by self-assemble by the flowability of cell membranes.
  • FIG. 3 shows the comparison of Fc ⁇ RIIIa binding and signal difference between the dimeric Fc display using the gIII domain and the NlpA leader peptide and cell membrane fluidity and the conventional full-length IgG display technique.
  • FIG. 4 shows the results of assemble with bevacizumab VH / VL by antigen binding in A cell lining.
  • FIG. 5 shows a schematic diagram of the library constructed and an antibody Fc domain display schematic diagram using gIII.
  • FIG. 6 is a bar graph comparing the fluorescence intensities of the wild-type Herceptin Fc with the Fc ⁇ RIIIa affinity (6A) of each round of library after 5 rounds of screening (6B).
  • Figure 7 shows variants with high affinity for Fc [gamma] RIIIa (A: No. 25 (HW 25), B: No. 86 (HW 86)
  • Fig. 10 shows experimental results of T394A mutagenesis found in HW86.
  • FIG 11 shows the results of the ADCC efficacy test of HW86.
  • Fig. 13 shows a cleavage map of pAK200-Fc-gIII.
  • Example 1 Vector construction for dimeric Fc display using gIII domain and cell membrane fluidity (wild type WT Fc, MG48)
  • Plasmids were prepared to self-assemble the monomeric form of the Fc protein with the gIII protein into dimers by the flowability of the cell membrane (Fig. 1). Vent polymerase (New England Biolab) and primer MJ # 183, each amplifying the wild-type WT Fc and MG48 gene using a MJ # 184 Sfi I (New England Biolab) in a restriction enzyme-treated to create an insert Sfi I restriction enzyme treatment pAK200 vector.
  • Escherichia coli Jude1 (F '[Tn 10 (Tet r) proAB + lacI q ⁇ (lacZ) M15] mcrA ⁇ (mrr - hsdRMS - mcrBC) 80d lac Z ⁇ M15 ⁇ lacX74 deoR recA1 araD139 ⁇ (araleu) 7697 galUgalKrpsLendA1nupG) ( Kawarasaki et al., 2003) to obtain a single clone.
  • WT Fc and MG48 were successfully inserted into pAK200.
  • Example 2 Vector construction for dimeric Fc display using NlpA leader peptide and cell membrane fluidity (wild type WT Fc, MG48)
  • a plasmid was prepared to allow the NlpA leader peptide to be self-assemble with the dimer by the flowability of the membrane-type Fc protein fused to the monomer (FIG. 2).
  • Vent polymerase New England Biolab
  • primer MJ # 183 each amplifying the wild-type WT Fc and MG48 gene using a MJ # 184 Sfi I (New England Biolab) in a restriction enzyme-treated to create an insert Sfi I restriction enzyme treatment pMopac12 And then ligation was performed with -NlpA-FLAG vector. After that, a single clone was obtained by transformation into Escherichia coli Jude1, and it was confirmed by sequencing that WT Fc and MG48 were successfully inserted into the corresponding vector.
  • Example 3 Vector construction for antibody fragment display using cell membrane fluidity
  • VH and VL portions of Bevacizumab were amplified with a vent polymerase (New England Biolab) using primers MJ # 232 and MJ # 233, MJ # 228 and MJ # 229, respectively.
  • a gene amplification was conducted for each of the ligation pMopac12-NlpA-FLAG vector and pMopac12-NlpA-His-cMyc vector prepared with the same restriction enzymes and then treated with restriction enzyme Sfi I (New England Biolab).
  • This plasmid was transformed into Jude1 and the pMOPac12-NlpA-VH (bevacizumab) -FLAG and pMopac12-NlpA-VL (bevacizumab) -His-cMyc were confirmed by confirming the base sequence of single colony. Then, VH and VL were produced in bicistronic form to express in one vector. The previously prepared pMopac12-NlpA-VH (bevacizumab) -FLAG was used as a template and amplified with primers MJ # 1 and MJ # 236.
  • This DNA fragment was restriction enzyme treated with XbaI (New England Biolab) and then ligated into the same restriction enzyme-treated pMopac12-NlpA-VL (bevacizumab) -His-cMyc vector. Sequence analysis revealed that a bicistronic plasmid of pMopac12-NlpA-VH (bevacizumab) -FLAG-NlpA-VL (bevacizumab) -His-cMyc was produced.
  • Example 4 Escherichia coli culture for full-length IgG display
  • VL-CK-NlpA-VL (Cys-PelB-VL-CK-NlpA-VL) was introduced into Jude1 cell by pBAD30-Km-PelB-VH-CHl-CH2-CH3 (WT) -FLAG, pMopac12- -Ck-His-cMyc plasmid to prepare heavy chain and light chain to be expressed in the intercellular region, respectively.
  • 5 ml of glucose containing 2% of glucose was cultured at 37 ° C for 16 hours, and 5.5 ml of TB was inoculated into a 100 ml flask at a ratio of 1: 100.
  • Example 5 Cell membrane fluidity and coliform culture for dimeric Fc display using NlpA or gIII
  • Example 6 Escherichia coli culture for antibody fragment display based on cell membrane fluidity
  • the pMOPac12-NlpA-VH (bevacizumab) -FLAG-NlpA-VL (bevacizumab) -His-cMyc plasmid was transformed into Jude1 bacterial cells and cultured in 5 ml of TB + 2% glucose medium at 37 ° C for 16 hours The cells were then inoculated 1: 100 in 5 ml of TB medium and cultured to an OD 600 of 0.6. Immediately after cooling at 25 ° C and 250 rpm for 20 minutes, 1 mM IPTG was added and overexpressed at 25 ° C and 250 rpm for 5 hours. After overexpression, the OD 600 value was measured and the cells were recovered by centrifugation at 14000 rpm for 1 minute.
  • Example 7 Elimination of extracellular membrane and peptidoglycan layer for protein variant search and analysis
  • the cells were resuspended in 1 ml of 10 mM Tris-HCl (pH 8.0) and centrifuged for 1 min. The wash procedure was repeated twice.
  • the cells were resuspensioned with 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] and the extracellular membrane was removed by rotation at 37 ° C for 30 minutes. After centrifugation, the supernatant was removed and 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8] was added and centrifuged with resuspension.
  • Example 8 Verification of Fc variant display system based on cell membrane fluidity using flow cytometry
  • the binding capacity with Fc ⁇ RIIIa was compared and analyzed. After centrifugation, the supernatant was removed and resuspension in 1 ml of PBS. After taking 300 ⁇ l, 700 ⁇ l of PBS and 5 nM of tetrameric Fc ⁇ RIIIa-Alexa488 probe were added together and the fluorescent probe was labeled on spheroplast by rotation at room temperature. After labeling, the plate was washed once with 1 ml of PBS and analyzed using Guava (Merck Millipore) equipment (FIG. 3).
  • Example 9 Screening of cell membrane fluidity based antibody fragment display system using flow cytometry analyzer
  • VH and VL are expressed together, it is confirmed that VH and VL are assimilated due to the fluidity of the bacterial intracellular membrane, thereby successfully binding to the antigen.
  • Fc protein various proteins forming multimers were displayed and confirmed to be able to self-assemble.
  • Example 10 Establishment of a library for enhancing binding ability to Fc [gamma] RIIIa
  • primer # The nucleotide sequence (5 ⁇ 3) 1Fw (SEQ ID NO: 9) GACAAAACTCACACATGCC CACCGTGCCCAGCACCTGAA 2Rv (SEQ ID NO: 10) GAGGGTGTCCTTGGGTTTTGGG GGAARGAGGAAGACTGACGGTCCCCCTAMGAG TTCAGGTGCTGGGCACGGTG 2Rv S239D (SEQ ID NO: 11) GAGGGTGTCCTTGGGTTTTGGG GGAARGAGGAAGACATCCGGTCCCCCTAMGAG TTCAGGTGCTGGGCACGGTG 3Fw (SEQ ID NO: 12) CCCAAAACCCAAGGACACCCTC ATGATCTCCCGGA CCCCTGAGGTCACATGCGTG 4Rv (SEQ ID NO: 13) CAGTTGAACTTGACCTCAGGGTCTTC GTGGCTCACGTCTWCCAC CACGCATGTGACCTCAGGGG 4Rv S267E (SEQ ID NO: 14) CAGTTGAACTTGACCTC
  • the constructed library was cultured in 25 ml of TB medium (Becton, Dickinson and Company, New Jersey, USA) + 2% glucose (Sigma Aldrich, Missouri, USA) , The cells were inoculated at 1: 100 in terrific broth and cultured at 37 ° C until OD600 reached 0.5. After the incubation, induction was carried out for 5 hours at 25 ° C using 1 mM IPTG (Biosesang (Sungnam, South Korea)), and the cell solution was recovered at 8 / OD600 after induction. The recovered cells were washed with 1 ml of Tris-HCl (pH 8.0), centrifuged at 14000 rpm for 1 minute, and the same operation was performed once more.
  • Tris-HCl pH 8.0
  • the recovered cells were then resuspensioned with 1 ml of STE (0.5 M Sucrose-10 mM Tris-10 mM EDTA (pH 8.0)) and stirred at 37 ° C for 30 minutes. After stirring, the cells were centrifuged at 14,000 rpm for 1 minute and solubilized with solution A (0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8). After centrifugation at 14,000 rpm for 1 minute, the pellet was resuspended in solution A containing 1 mg / ml lysozyme (Sigma Aldrich, Missouri, USA) and stirred at 37 ° C for 15 minutes.
  • STE 0.5 M Sucrose-10 mM Tris-10 mM EDTA (pH 8.0)
  • the agitated cells were centrifuged at 14000 rpm for 1 minute and then dissolved with 1 ml of PBS.
  • the mixture was mixed with PBS containing fluorescently labeled Fc ⁇ RIIIa at a ratio of 7:15 to make 1 ml, and the mixture was stirred at room temperature for 1 hour.
  • the fluorescently labeled spheroplasts were centrifuged at 14,000 rpm for 1 minute, washed with PBS, and centrifuged again at 14,000 rpm for 1 minute.
  • Sprague-lavaged endothelial cells with Fc ⁇ RIIIa were diluted in 1 ⁇ PBS at a ratio of 1:15 and screened using a flow cell analyzer (S3 cell sorter, BioRad). Since the high fluorescence intensity of spiroflavast, which is indicated by fluorescence-labeled Fc ⁇ RIIIa, means that it has a mutant with high affinity for Fc ⁇ RIIIa, the top 3% of the fluorescence intensity is collected through screening . Screening of the recovered spooflast was performed by flow cytometry again to remove negative clones that were recovered in the screening process and to increase the proportion of mutants having high affinity for Fc ⁇ RIIIa.
  • the obtained spiroplast was amplified and recovered by PCR using positive mutant DNA, and cloned into a display vector to construct a library. After this screening, the library construction process was repeated five times in total, and as the screening progressed, the library was amplified with mutants having higher affinity for Fc ⁇ RIIIa by gradually decreasing the concentration of the fluorescent label Fc ⁇ RIIIa used.
  • the libraries constructed in all the screening steps and the starting library were cultured in 25 ml of TB + 2% glucose medium in 250 ml plats at 37 ° C for 4 hours, : 100 and the cultivation was continued at 37 ° C until OD600 reached 0.5. After the incubation, induction was carried out for 5 hours at 25 ° C using 1 mM IPTG. After induction, 8 ml / OD600 cells were recovered. In addition, 5 ml of TB + 2% glucose medium was inoculated into the untreated wild-type Fc test tube used as the comparative group, pre-cultured overnight at 37 ° C, and then cultured in 5 ml of TB medium Cell recovery was performed.
  • the individual mutants were selected from the libraries constructed after the last round screening to measure the binding capacity of the mutants to Fc ⁇ RIIIa.
  • the test tube was inoculated with 5 ml of TB + 2% glucose medium and incubated overnight at 37 ° C.
  • the cells were inoculated at a ratio of 1: 100 in 5 ml of TB medium and cultured at 37 ° C until OD600 reached 0.5.
  • Induction was carried out at 25 ° C for 5 hours using 1 mM IPTG. After the induction was completed, E.
  • HW 25 and HW 86 discovered unintended mutations introduced during the PCR process to amplify DNA during the screening process, rather than the locations of mutations envisioned for building libraries.
  • PCR was carried out using Quik change kit (Agilent) to return each mutation to the wild-type Fc sequence.
  • the cloned mutants were analyzed by flow cytometry To compare the affinity for Fc [gamma] RIIIa.
  • HW 25 and HW 86 both of the mutations were found to contribute to the enhancement of affinity to Fc ⁇ RIIIa (FIG. 9).
  • T394A this T394A mutation was introduced into Fc 1004 / IYG, a mutant possessed by the present inventors. After the cloning was completed, the affinity for Fc [gamma] RIIIa was analyzed through a flow cytometer and it was confirmed that the affinity for Fc [gamma] RIIIa was increased by introducing the T394A mutation (FIG. 10).
  • Example 14 IgG expression and purification in animal cells
  • a DNA coding for the antibody heavy chain is obtained by linking with DNA encoding VH-CH1 of trastzumab, HII / Xba I < / RTI > PMAZ-IgH (No. 25), pMAZ-IgH (No), and pMAZ-IgH (trp) were obtained by treating with restriction enzyme Bss HII / Xba I and then ligation, .25-DG), and pMAZ-IgH (No. 86).
  • the pMAZ plasmid encoding the light chain of trastzumab was transformed into HEK293F to express the antibody.
  • the cells were centrifuged at 3000 ⁇ g for 20 minutes, and the supernatant was collected.
  • the supernatant was collected, diluted with 25 ⁇ PBS to 1 ⁇ PBS, and filtered with a 0.22 uM filter.
  • the protein solution was equilibrated with 1 ⁇ PBS in the supernatant.
  • the column was filled with resin and 1 ⁇ PBS was flowed 50 times the resin volume.
  • the antibody bound to 100 mM glycine (pH 3.0) was taken and immediately neutralized with neutral pH by adding 1 M Tris (pH 8.0).
  • the purified antibody was centrifuged through a 3 kDa column tube at 7,500 x g for 25 minutes and then filled with 1 x PBS. After centrifugation at 7,500 x g for 25 minutes, 1 x PBS was repeated to repeat the procedure.
  • the ratio of 1 x PBS was set to 99% or more.
  • Example 15 Expression and purification of IgG in E. coli
  • a plasmid was constructed using the PelB signaling protein while encoding the heavy and light chains of trastzumab. This plasmid was introduced into MG1655 Escherichia coli as a plasmid encoding DsbC-chaperon, and was transformed into R / 2 medium (KH 2 PO 4 6.75 g / l, (NH 4 ) 2 HPO 4 2.0 g / l, citric acid monohydrate 0.93 g / l, MgSO 4 .7H 2 O 0.7 g / l, TMS 5.0 ml / l, 2% glucose) in the after subculturing, after creating the R / 2 medium in a 2 L jar fermantor 1: 100 dilution in a ratio of And the cultivation was continued.
  • the culture was performed so that the OD600 was 70, and the induction was performed using 1 mM IPTG.
  • Escherichia coli was resuspended in 1.2 L of 100 mM Tris, 10 mM of EDTA (pH 7.4), lysozyme was added in an amount of 4 mg / wet cell g and incubated at 30 ° C for 16 hours and the E. coli was disrupted by incubation.
  • the disrupted Escherichia coli was centrifuged at 14,000 x g for 30 minutes, and the supernatant was recovered and purified by the method described above.
  • SKBC-3 a target cell for measuring ADCC, was cultured in McCoy's medium + 10% FBS + 1 x anti-anti at 37 ° C and 5% CO 2 and then assay buffer (RPMI + 10% heat inactivated FBS + 30 ng / ml recombinant human IL-2) and inoculated in a 96-well plate (V-bottom) at 1 ⁇ 10 4 cells / 50 ⁇ l / well per well.
  • Serial dilutions of IgG expressed in animal cells in 1/5 of the highest concentration of 20 ⁇ g / ml to 0 ⁇ g / ml in the assay buffer were prepared at a total of 8 concentrations, and 10 ⁇ l of each of the SKBR-3 cells After the addition, the cells were cultured at 37 ° C and 5% CO 2 for 1 hour.
  • the PBMC were then rapidly dissolved in a 37 ° C water bath for 2-3 minutes, transferred to a 50 ml tube, treated with 10 ml of anti-aggregation buffer (1 ml of CTL Anti-aggregate Wash TM 20xSolution + Was mixed well, and the supernatant was carefully removed by centrifugation at 300 g for 10 minutes, 10 ml of an anti-clotting buffer was added, and the cells were suspended. After centrifugation at 300 ⁇ g for 10 minutes, the supernatant was carefully removed, and 1 ml of assay buffer was added to suspend the cells.
  • the PBMCs were diluted in assay buffer at a concentration of 2.5 ⁇ 10 6 cells / mL, added with 50 ⁇ l / well of each well of the target cells and the plate, and incubated at 37 ° C in a 5% CO 2 incubator Lt; / RTI > After 4 hours, centrifugation was carried out at 300 ⁇ g for 5 minutes. 50 ⁇ l of the supernatant was transferred to SpectraPlate 96-well plate, and 50 ⁇ l of CytoTox 96 Reagent was added to each well.

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Abstract

La présente invention concerne un système d'affichage d'une protéine cible multimère utilisant la fluidité de la membrane cellulaire. Un système d'affichage de la présente invention présente un niveau pratique remarquablement amélioré (préparation simple du milieu, temps d'incubation réduit pour le criblage, utilisation de promoteurs uniques, etc.) comparé à celui de systèmes d'affichage IgG de pleine longueur classique ou de systèmes d'affichage Fc utilisant PelB et faisant preuve d'une plus petite valeur de % CV et d'une plus grande différence dans l'intensité du signal de fluorescence entre les clones négatifs et positifs que les techniques d'affichage d'IgG de pleine longueur classique, devenant ainsi plus efficace pour le criblage en utilisant un affichage à protéine cible.
PCT/KR2018/012212 2017-10-20 2018-10-17 Système d'affichage à protéine multimère utilisant la fluidité de la membrane cellulaire Ceased WO2019078591A1 (fr)

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WO2012109133A1 (fr) * 2011-02-07 2012-08-16 Research Development Foundation Polypeptides fc modifiés d'immunoglobuline
EP2155789B1 (fr) * 2007-05-01 2013-07-24 Research Development Foundation Banques d'immunoglobulines fc
WO2017040380A2 (fr) * 2015-08-28 2017-03-09 Research Development Foundation Variants de fc d'anticorps modifiés

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EP2155789B1 (fr) * 2007-05-01 2013-07-24 Research Development Foundation Banques d'immunoglobulines fc
WO2012109133A1 (fr) * 2011-02-07 2012-08-16 Research Development Foundation Polypeptides fc modifiés d'immunoglobuline
WO2017040380A2 (fr) * 2015-08-28 2017-03-09 Research Development Foundation Variants de fc d'anticorps modifiés

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NG, TING WUN: "Improving protein therapeutics through quantitative molecular engineering approaches and a cell -based oral delivery platform", THESIS, 2013, University of Pensilvania, pages 1 - 275 *

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