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WO2019124461A1 - Molécule multimère de fragment d'anticorps, agent pharmaceutique, agent antitumoral, agent de traitement de maladie auto-immune, et procédé de production de molécule multimère de fragment d'anticorps - Google Patents

Molécule multimère de fragment d'anticorps, agent pharmaceutique, agent antitumoral, agent de traitement de maladie auto-immune, et procédé de production de molécule multimère de fragment d'anticorps Download PDF

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WO2019124461A1
WO2019124461A1 PCT/JP2018/046869 JP2018046869W WO2019124461A1 WO 2019124461 A1 WO2019124461 A1 WO 2019124461A1 JP 2018046869 W JP2018046869 W JP 2018046869W WO 2019124461 A1 WO2019124461 A1 WO 2019124461A1
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antibody fragment
scfv
chain
variable region
antibody
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Japanese (ja)
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勝実 前仲
高志 田所
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Hokkaido University NUC
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Hokkaido University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present invention relates to antibody fragment multimer molecules, drugs, antitumor agents, agents for treating autoimmune diseases, and methods for producing antibody fragment multimer molecules.
  • Antibody therapeutics for cancer treatment target antigens expressed specifically in cancer, and since high therapeutic effects may be obtained, they are expected as cancer therapeutic agents.
  • a full-length antibody of IgG has a large molecular weight of about 150 kDa, so that the tissue permeability and retention in blood are poor, and the production cost is high, and the drug price is high. Furthermore, it is difficult to be taken into cells because the binding to cancer antigens is too strong, and in cancer tissues, the tissue morphology and antigen expression are not uniform, and unwanted cancer antigens are expressed due to the high affinity of the antibody. In addition, it has been revealed that only a very small amount (0.003-0.01%) of IgG administered actually accumulates in cancer tissues (Non-patent Document 1). Thus, it has been viewed as problematic that the efficacy of antibody drugs may be insufficient.
  • Non-Patent Document 2 reports an example in which a certain therapeutic effect is recognized by the combined use of an antibody drug and a low molecular weight drug.
  • ADC antibody-drug conjugate
  • Non-patent Document 3 a method using an antibody-drug conjugate (ADC) in which a drug is linked to an antibody has also been proposed.
  • ADCs are designed to carry an antibody with high target selectivity as a drug carrier and to exert drug effects on the drug linked to the antibody.
  • Non-Patent Document 2 when used in combination with the low molecular weight drug described in Non-Patent Document 2, the low molecular weight drug itself often exhibits strong side effects, and there are still problems in terms of therapeutic effect. Further, in the ADC described in Non-Patent Document 3, the one having a full-length antibody as a basic skeleton is the mainstream, and there are problems such as side effects and high drug price.
  • the present invention has been made in view of the above circumstances, and has high target selectivity and affinity as well as low cost antibody fragment multimer molecule, drug, antitumor agent, autoimmune disease therapeutic agent, antibody fragment It is an object of the present invention to provide methods for producing multimeric molecules and single chain Fv antibody fragments.
  • an antibody fragment multimer molecule is Two or more multimers of single-chain Fv antibody fragments, It has an apparent dissociation constant K D of 10 -3 to 10 3 nM for the antigen,
  • the single-chain Fv antibody fragment has a dissociation rate constant k off of 10 -5 to 10 2 s -1 and a dissociation constant K D of 10 -1 to 10 5 nM with respect to the antigen.
  • the single-chain Fv antibody fragment has a mutation in at least one amino acid sequence of the variable region of heavy chain and the variable region of light chain.
  • one or several amino acids are substituted, deleted, added or inserted in the variable region of the heavy chain and the variable region of the light chain of the single-chain Fv antibody fragment.
  • one or several amino acids are substituted, deleted, added or inserted in at least one amino acid sequence of the variable region of the heavy chain, and at least at least the variable region of the light chain.
  • one or several amino acids are substituted, deleted, added or inserted.
  • the pharmaceutical preparation according to the second aspect of the present invention comprises the antibody fragment multimer molecule according to the first aspect of the present invention.
  • the antitumor agent according to the third aspect of the present invention comprises the antibody fragment multimer molecule according to the first aspect of the present invention.
  • the autoimmune disease therapeutic agent according to the fourth aspect of the present invention comprises the antibody fragment multimer molecule according to the first aspect of the present invention.
  • the antibody fragment multimer molecule according to the fifth aspect of the present invention is an antibody fragment multimer molecule for use in treating a tumor.
  • the antibody fragment multimer molecule according to the sixth aspect of the present invention is an antibody fragment multimer molecule for use in the treatment of an autoimmune disease.
  • a method for producing an antibody fragment multimer molecule according to the seventh aspect of the present invention A single-chain Fv antibody fragment having a dissociation rate constant k off of 10 -5 to 10 2 s -1 to the antigen and having a dissociation constant K D of 10 -1 to 10 5 nM is two or more by multimerized to multimers, with respect to said antigen, comprising the step of obtaining antibody fragments multimeric molecule having a dissociation constant, K D, the apparent 10 -3 ⁇ 10 3 nM.
  • mutations are generated in at least one amino acid sequence in the variable region of the heavy chain and the variable region of the light chain of the single-chain Fv antibody fragment.
  • one or several amino acids are substituted, deleted, added or inserted in the variable region of the heavy chain and the variable region of the light chain of the single chain Fv antibody fragment.
  • one or several amino acids are substituted, deleted, added or inserted in at least one amino acid sequence of the variable region of the heavy chain, and at least at least the variable region of the light chain.
  • one or several amino acids are substituted, deleted, added or inserted.
  • a method for producing an antibody fragment multimer molecule according to the eighth aspect of the present invention A single-chain Fv antibody fragment having a dissociation rate constant k off of 10 -5 to 10 2 s -1 to the antigen and having a dissociation constant K D of 10 -1 to 10 5 nM is used.
  • the single-chain Fv antibody fragment according to the ninth aspect of the present invention, has a dissociation rate constant k off of 10 -5 to 10 2 s -1 and a dissociation constant K D of 10 -1 to 10 5 nM.
  • antibody fragment multimer molecules having high target selectivity and affinity as well as low cost, pharmaceuticals, antitumor agents, agents for treating autoimmune diseases, methods for producing antibody fragment multimer molecules, and Chain Fv antibody fragments can be provided.
  • (A) is a figure which shows typically the full-length antibody of trastuzumab
  • (b) is a figure which shows scFv typically
  • (c) is a figure which shows the structure of scFv typically.
  • (A) is a conceptual diagram of molecular design
  • (b) is a diagram for explaining the expected effects of antibody fragment multimeric molecules. It is a figure explaining how to obtain kinetic parameters.
  • (A) is a figure which shows typically the structure of the scFv variant by one Embodiment
  • (b) is a figure which showed each CDR of trastuzumab typically.
  • FIG. 5 shows amino acid residues around light chain CDRs.
  • A) is a view schematically showing a GCN leucine zipper forming a dimer
  • (b) is a view schematically showing a cartilage matrix protein forming a trimer
  • (c) is a view FIG. 7 schematically shows a cartilage oligomeric matrix protein that forms a pentamer
  • (d) schematically shows an antigen fragment multimer molecule that is a dimer
  • (e) shows a ternary amount.
  • FIG. 5 is a view schematically showing an antigen fragment multimer molecule which is a body
  • FIG. 5 (f) is a view schematically showing an antigen fragment multimer molecule which is a pentamer.
  • FIG. 5 is a diagram illustrating pET-22b (+) vector.
  • FIG. 2 shows gene sequences of scFv-LH-dimer.
  • FIG. 2 shows gene sequences of scFv-LH-trimer.
  • FIG. 2 shows the gene sequence of scFv-LH-pentamer.
  • (A) is a figure which shows the preparation methods of pET22b-wt-pentamer
  • (b) is a figure which shows the preparation methods of pET22b-wt-dimer and pET22b-wt-trimer. It is a figure explaining the method of mutation introduction. It is a figure explaining the method of mutation introduction to a multimer.
  • (A) is a view schematically showing a scFv variant
  • (b) is a view schematically showing a wild-type multimeric molecule of a comparative example
  • (c) is an antibody fragment multimer of an example It is the figure which showed the molecule
  • (A) is a figure explaining SEC-MALS
  • (b) is a graph figure of multimerization progress confirmation by SEC-MALS.
  • (A) is a diagram schematically showing the state of interaction between the analyte and the immobilized ligand
  • (b) is the velocity of the scFv variant or antibody fragment multimer molecule of the example with the antigen HER2 Theoretical interaction analysis.
  • (A) schematically shows HER2-ECD and EGFR-ECD
  • (b) schematically shows the structures of EGFR and EGFR-ECD
  • (c) explains pFastBac vector
  • D) is a graph of the antigen binding specificity experiment. It is a graph of avidity effect verification by multimerization.
  • FIG. 2D is a drawing schematically showing the state of the interaction with the above, and FIG. 2D is a drawing of the kinetic interaction analysis of the scFv mutant with the antigen.
  • antibody fragment multimeric molecules according to the present embodiment will be described in detail.
  • the present inventors highly discriminate between normal cells and target cells (eg, cancer cells) for the purpose of improving the rate of accumulation in target tissues (eg, cancer tissues) and preventing binding to normal cells.
  • target tissues eg, cancer tissues
  • an antibody fragment multimer molecule according to the present embodiment was invented. Specifically, not a high molecular weight full-length antibody (FIG. 1 (a)), but an antibody fragment that has been denatured while having the ability to target to a target cell, specifically, a single-chain Fv antibody fragment (single chain fragment variable (scFv) (FIG. 1 (b)) is used. Since scFv has a lower molecular weight than a full-length antibody, it is known that tissue permeability and retention in blood are improved.
  • the affinity per monovalent is intentionally reduced and the antibody is multimerized.
  • Fragment multimeric molecules are produced ( Figure 2 (a), (b)).
  • the antibody fragment multimer molecule according to the present embodiment can obtain an avidity effect by the increase of the valence number, and has high target selectivity and high affinity.
  • retention of normal cells with minimal antigen expression can be reduced, and the antigen can be specifically accumulated in target cells (eg, cancer cells) in which the antigen is closely expressed.
  • target cells eg, cancer cells
  • they can be easily detached from normal cells, and excellent medicinal effects and reduction of side effects can be realized.
  • the antibody fragment multimer molecule according to the present embodiment is a multimer of two or more single chain Fv antibody fragments, and has an apparent dissociation constant K D of 10 -3 to 10 3 nM, preferably 10 -3. It has an apparent dissociation constant K D of ⁇ 10 1 nM.
  • scFv single chain fragment variable
  • the single-chain Fv antibody fragment according to the present embodiment has a dissociation rate constant k off of 10 -5 to 10 2 s -1 as a degree of antigen binding ability, and a dissociation constant K of 10 -1 to 10 5 nM. Have D.
  • the single-chain Fv antibody fragment according to this embodiment preferably has a dissociation constant K D of 1 to 10 5 nM.
  • the dissociation rate constant (disassociation rate constant, unit: s ⁇ 1 ) k off of the single-chain Fv antibody fragment (scFv) can be determined by any method, for example, surface plasmon resonance It can be determined from a sensorgram measured by (for example, interaction analysis using Biacore system (manufactured by GE Healthcare)). In the interaction analysis using Biacore, more specifically, according to the following (i) to (iv) (FIG.
  • the dissociation constant K D may be determined from any arbitrary method such as surface plasmon resonance, ELISA, isothermal titration calorimetry, etc., by performing analysis according to the Scatchard plot or the attached document of each device. Can.
  • the antibody fragment multimer molecule according to the present embodiment has an apparent dissociation constant K D of 10 -3 to 10 3 nM with respect to the antigen.
  • (Sensogram A)-(Sensogram B) is performed.
  • (Iii) Perform similar experiments for different analyte concentrations. Repeat (i) and (ii).
  • a sensorgram represents a sensorgram when all three of three polymers are bonded and a sensorgram when two or one is bonded. Is regarded as being 1: 1 coupled for convenience, and analysis is performed by approximation. Therefore, the analyzed result is expressed as "apparent" 1: 1 binding, and each parameter is interpreted as when 1: 1 binding occurs in the system. The approximate expression is shown below.
  • dissociation constant, K D the apparent dissociation constant, K D, and antibody fragments multimeric molecules of single-chain Fv antibody fragments
  • the dissociation constant, K D the apparent of the antibody fragments multimeric molecules, single chain Fv antibody fragments preferably lower than the dissociation constant, K D,, more preferably dissociation constant, K D, the apparent of the antibody fragments multimeric molecule is 1/10 or less of the dissociation constant, K D, single chain Fv antibody fragments, antibody fragments multimeric dissociation constant, K D, the apparent molecule more preferably 1/100 or less of the dissociation constant, K D, single chain Fv antibody fragments.
  • the “single-chain Fv antibody fragment (scFv)” has a reduced antigen-binding ability by having a mutation in at least one amino acid sequence of the variable region of the heavy chain and the variable region of the light chain. May be In the present specification, a single-chain Fv antibody fragment (scFv) having a mutation in at least one amino acid sequence of the heavy chain variable region and the light chain variable region is referred to as “single-chain Fv antibody fragment variant (scFv variant In some cases.
  • the single chain Fv antibody fragment variant may have a mutation only in the variable region of the heavy chain, and has a mutation only in the variable region of the light chain Or may have mutations in both the heavy chain variable region and the light chain variable region.
  • variable region of heavy chain and variable region of light chain means, for example, that single-chain Fv antibody fragment variants (scFv variants) have reduced antigen binding ability.
  • scFv variants single-chain Fv antibody fragment variants
  • one or several amino acids may be substituted, deleted, added or inserted in at least one amino acid sequence of the variable region of the heavy chain and the variable region of the light chain.
  • One or several refers to 1 to 20, preferably 1 to 15, and more preferably 1 to 10.
  • single-chain Fv antibody fragment variants have a dissociation rate constant k off of 10 -5 to 10 2 s -1 and a dissociation constant K D of 10 -1 to 10 5 nM
  • the number of "one or several" may be selected without particular limitation.
  • the means for causing a mutation in the amino acid sequence of a single chain Fv antibody fragment (scFv) in order to reduce the antigen binding ability For example, the variable region of heavy chain and the variable region of light chain By structurally altering at least one of (eg, binding a small molecule, etc.), the interaction with the antigen may be weakened to reduce the antigen binding ability.
  • amino acid residues involved in antigen binding can be substituted with other kinds of amino acid residues .
  • amino acid residues in the amino acid sequence of a single chain Fv antibody fragment scFv
  • the amino acid sequence of a single chain Fv antibody fragment The cationic residue or the anionic residue in may be replaced with a neutral amino acid residue.
  • amino acid residue may be replaced with another amino acid residue (eg, a neutral amino acid residue).
  • variable region (VH) of the heavy chain and the variable region (VL) of the light chain for example, H-CDR1 (complementarity determining region 1 of VH), H-CDR2 ( One or several amino acids are substituted in at least one amino acid sequence of VH complementarity determining region 2), L-CDR1 (complementarity determining region 1 of VL) and L-CDR2 (complementarity determining region 2 of VL) It may be In the heavy chain variable region (VH) and the light chain variable region (VL), amino acid residues that are deeply involved in antigen binding may be substituted with other amino acid residues in terms of conformation.
  • one or several amino acids are substituted, deleted, added or inserted in at least one amino acid sequence of the variable region of heavy chain, and at least at least the variable region of light chain
  • one or several amino acids are preferably substituted, deleted, added or inserted. That is, it is preferable to have a mutation (in which one or several amino acids are substituted, deleted, added or inserted) in both VL (variable region of light chain) and VH (variable region of heavy chain).
  • Interaction of a single-chain Fv antibody fragment (scFv) with an antigen more efficiently by substitution, deletion, addition or insertion of one or several amino acids in both the VL and VH amino acid sequences Can be weakened to reduce the antigen binding ability.
  • the antibody fragment multimer molecule according to the present embodiment is a multimer of two or more single chain Fv antibody fragments (scFv), but the number of multimerization is not particularly limited, and, for example, a dimer or a trimer It may be pentamer, decamer or the like, and any number of multimerization can be adopted as long as the effects of the present invention are exhibited.
  • the method of multimerization can use arbitrary methods, for example, as shown to Fig.4 (a), you may utilize a well-known multimer formation peptide (multimer).
  • single-chain Fv antibody fragments may be linked by SS bond or the like to be multimerized.
  • other methods for example, Philipp Holliger & Peter J Hudson, Engineered antibody fragments and the rise of single domains, NATURE BIOTECHNOLOGY, VOLUME 23 NUMBER 9 SEPTEMBER 2005, 1112-1136; Maneesh Jain et al, Engineering antibodies for clinical applications, TRENDS in Biotechnology Vol. 25 No. 7, 307-316; Nature Reviews Drug Discovery, VOLUME 17, AUGUST, 2 It may be multimerized by 18,531-533).
  • monoclonal antibody may be used as a full-length antibody from which a single chain Fv antibody fragment (scFv) is derived.
  • scFv single chain Fv antibody fragment
  • monoclonal antibodies that can be used as antibody drugs can be used.
  • monoclonal antibodies listed below may be used, but are not limited to the following.
  • the antibody fragment multimer molecule according to the present embodiment is a multimer of two or more single-chain Fv antibody fragments (scFv), but the type of the full-length antibody which is the origin of the scFv may be one (homo large amount Body molecules), may be two or more (heteromultimeric molecules).
  • the antibody fragment multimer molecule is a trimer using trastuzumab
  • all three scFvs may be trastuzumab scFvs (homomultimeric molecules);
  • two may be scFvs of trastuzumab and one may be scFv of an antibody different from trastuzumab (heteromultimeric molecule),
  • one of the three scFvs is a scFv of trastuzumab, the other 1
  • One may be a scFv of an antibody different from trastuzumab, and the other may be a scFv of an antibody different from trastuzumab or another (a heteromultimeric molecule).
  • the molecular weight of the antibody fragment multimeric molecule is preferably smaller than the molecular weight of the full-length antibody that is the source of the single chain Fv antibody fragment (scFv), in the case of a homomultimeric molecule, from the viewpoint of good permeability.
  • scFv single chain Fv antibody fragment
  • it is preferably smaller than the molecular weight (150 to 160 kDa) of the IgG antibody.
  • the "antigen” can be selected without limitation as long as it is a substance that causes an immune response in vivo.
  • trastuzumab is used as a full-length antibody on which a single-chain Fv antibody fragment (scFv) is derived
  • the antigen is, for example, HER2 (human epidermal growth factor receptor 2).
  • VH and VL are linked by an arbitrary linker (FIG. 4 (a)), and examples thereof include (G 4 S) 3 .
  • a multimer for multimerization can be linked to any region, for example, a linker (eg, amino acid sequence GSAGSAAGSGEF) at the C-terminus of VH
  • a linker eg, amino acid sequence GSAGSAAGSGEF
  • the multimer may be linked via.
  • a multimer may be conjugated with a conjugate for the purpose of retention in blood, cancer tissue specific drug delivery and the like.
  • the method for producing an antibody fragment multimer molecule has a dissociation rate constant k off of 10 -5 to 10 2 s -1 and a dissociation constant K of 10 -1 to 10 5 nM with respect to the antigen.
  • the details and methods of determining the dissociation rate constant k off and dissociation constant K D of the scFv, and the details and method of determining the apparent dissociation constant K D of the antibody fragment multimeric molecule are as described above.
  • the method for preparing antibody fragment multimeric molecules is, for example, first, using any genetic recombination technology, constructing a recombinant vector in which a gene encoding scFv and multimer-forming peptide (multimer) is incorporated into an expression vector or the like. Then, the constructed recombinant vector is introduced into a host by any of various transformation methods to obtain a transformant, which is cultured to express an antibody fragment multimeric molecule and recover it. be able to.
  • the details of the multimer-forming peptide (multimer) are the same as described above.
  • antigen binding ability can be achieved by causing mutations in at least one amino acid sequence of the variable region of heavy chain of scFv and the variable region of light chain May be lowered.
  • the antibody fragment multimer molecule is a multimer of “scFv variant” having a predetermined dissociation rate constant k off and dissociation constant K D.
  • a recombinant vector in which genes encoding scFv and multimer have been incorporated into an expression vector or the like, one of at least one amino acid sequence of the variable region of heavy chain and the variable region of light chain
  • mutations may be introduced into the gene encoding scFv by any method such as PCR so that several amino acids are substituted, deleted, added or inserted.
  • scFv is selected by any method such as PCR so as to substitute, delete, add, or insert one or several amino acids in at least one amino acid sequence of the variable region of heavy chain and the variable region of light chain.
  • the recombinant vector may be constructed after introducing a mutation into the gene encoding.
  • mutation may be introduced so as to have a mutation only in the variable region of heavy chain, or may be introduced so as to have a mutation only in the variable region of light chain, or It may be mutagenized to have mutations in both heavy chain variable region and light chain variable region.
  • the details of mutagenesis are the same as described above.
  • scFv including scFv variants
  • scFvs are linked by an S-S bond or the like according to any method. And may be multimerized.
  • the host used for producing the transformant is not particularly limited as long as it can express scFv (including scFv variants) from the introduced recombinant vector etc., for example, human, mouse, etc. Any cell that can serve as a host can be used, such as cells derived from various animals, cells derived from various insects, prokaryotic cells such as E. coli, eukaryotic cells such as yeast, and plant cells.
  • scFv is carried out by a method comprising the steps of culturing the above-mentioned transformants, and collecting scFv (including scFv variants) from the obtained culture. It can be carried out.
  • “culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. Culturing of the transformant can be carried out according to a conventional method used for culturing a host. The protein of interest is accumulated in the culture.
  • scFv including scFv variants
  • the culture solution is used as it is or cells are removed by centrifugation, filtration or the like. After that, scFv (including scFv variants) are collected from the culture by extraction with ammonium sulfate precipitation etc. if necessary, and if necessary, dialysis, various chromatography (gel filtration, ion exchange chromatography, affinity chromatography Can be isolated and purified using
  • scFv When scFv (including scFv variants) are produced intracellularly, scFv (including scFv variants) can be collected by disrupting cells. When the soluble fraction contains scFv (including scFv variants), after disruption, the cell debris (including the cell extract insoluble fraction) is optionally removed by centrifugation, filtration or the like. The supernatant after residue removal is a cell extract soluble fraction, and can be a crudely purified protein solution.
  • the scFv (including the scFv variant) is expressed as an inclusion body in the insoluble fraction
  • the insoluble fraction is isolated by centrifugation, and washing and centrifugation are repeated with a buffer containing a surfactant etc. By removing the debris from the cells.
  • the obtained inclusion body is solubilized in a buffer containing a denaturing agent such as guanidine or urea, and then the protein is rewound using a dilution method or a dialysis method.
  • Purification of the functionally refolded scFv (including scFv variants) can be isolated and purified using various chromatography (gel filtration, ion exchange chromatography, affinity chromatography, etc.).
  • production of scFv can be performed using a cell-free protein synthesis system that does not use living cells at all, and the produced scFv (
  • the scFv variants can be purified by appropriately selecting a means such as chromatography.
  • Method for producing an antibody fragment multimeric molecules in another aspect, to an antigen has a dissociation rate constant k off of 10 -5 ⁇ 10 2 s -1, and 10 -1 ⁇ 10 5 nM dissociation constants K It is characterized in that a single-chain Fv antibody fragment having D is used.
  • the details and methods of determining the dissociation constant k off and dissociation constant K D of the single-chain Fv antibody fragment and scFv are as described above.
  • the single-chain Fv antibody fragment (scFv) according to this embodiment has a dissociation rate constant k off of 10 -5 to 10 2 s -1 and a dissociation constant of 10 -1 to 10 5 nM with respect to the antigen. with a K D.
  • the details and methods of determining the single-chain Fv antibody fragment (scFv), the dissociation rate constant k off of the scFv and the dissociation constant K D are as described above.
  • the single-chain Fv antibody fragment (scFv) may have, for example, mutations in at least one of the amino acid sequences of the heavy chain variable region and the light chain variable region, for example, heavy chain variable
  • One or several amino acids may be substituted, deleted, added or inserted in at least one amino acid sequence of the region and the variable region of the light chain.
  • the scFv preferably has one or more amino acids substituted, deleted, added or inserted in at least one amino acid sequence of the variable region of the heavy chain, and at least one amino acid sequence of the variable region of the light chain, One or several amino acids have been substituted, deleted, added or inserted.
  • the medicament, the antitumor agent and the therapeutic agent for an autoimmune disease according to the present embodiment consist of the antibody fragment multimer molecule described above.
  • the antitumor agent according to the present embodiment comprises the antibody fragment multimer molecule described above, and includes, for example, breast cancer, liver cancer, non-small cell lung cancer, adrenocortical cancer, anal cancer, cholangiocarcinoma, bladder cancer, cervical cancer, colon cancer Endometrial cancer, esophageal cancer, Ewing's tumor, gallbladder cancer, Hodgkin's disease, hypopharyngeal cancer, laryngeal cancer, lip and mouth cancer, non-Hodgkin's lymphoma, melanoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic cancer It exerts a therapeutic effect on tumors such as prostate cancer, gastric cancer, testicular cancer, thyroid cancer, chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL).
  • CML chronic myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • the therapeutic agent for an autoimmune disease comprises the antibody fragment multimer molecule described above, and includes, for example, polymyositis, vasculitis syndrome, giant cell arteritis, Takayasu's arteritis, recurrent polychondritis, acquired blood. Hemophilia A, Still's disease, Adult-onset Still's disease, Amyloid A amyloidosis, polymyalgia rheumatica, spondyloarthritis (arthritis), pulmonary arterial hypertension, graft vs.
  • autoimmune myocarditis contact hypersensitivity (Contact dermatitis), gastroesophageal reflux disease, erythroderma, Behcet's disease, amyotrophic lateral sclerosis, transplantation, neuromyelitis optica, rheumatoid arthritis, juvenile rheumatoid arthritis, malignant rheumatoid arthritis, drug resistant rheumatoid arthritis Kawasaki disease, polyarticular or systemic juvenile idiopathic arthritis, psoriasis, chronic obstructive pulmonary disease (COPD), Castleman's disease, asthma, allergic asthma, allergic encephalomyelitis, cancer Inflammation, progressive chronic arthritis, reactive arthritis, psoriatic arthritis, enteritis arthritis, osteoarthritis, rheumatoid disease, spondyloarthritis, ankylosing spondylitis, Reiter's syndrome, hypersensitivity (airway hypersensitivity and skin hypersensitivity) Allergies, including systemic l
  • the pharmaceutical preparation according to the present embodiment is composed of the antibody fragment multimer molecule described above and is used for a desired pharmaceutical use, and can be used, for example, as the aforementioned antitumor agent, autoimmune disease therapeutic agent and the like.
  • the administration method of the medicine, the antitumor agent and the therapeutic agent for an autoimmune disease may be appropriately selected from oral administration, topical administration, intravenous administration, intraperitoneal administration, intradermal administration, sublingual administration and the like.
  • the dosage form may also be arbitrary, for example, oral solid preparations such as tablets, granules, powders, capsules etc., oral liquid preparations such as internal liquid solutions and syrups, parenteral liquid preparations such as injections etc. Can be prepared accordingly.
  • any suitable drug delivery system (DDS) may be used.
  • an antibody fragment multimer molecule for use in treating a tumor and an antibody fragment multimer molecule for use in treating an autoimmune disease according to the present embodiment will be described.
  • the antibody fragment multimer molecule for use in treating a tumor relates to the use of the antibody fragment multimer molecule described above as an antitumor agent, and the type of tumor is the same as described above.
  • the antibody fragment multimer molecule for use in the treatment of an autoimmune disease relates to the use of the above antibody fragment multimer molecule as a therapeutic agent for an autoimmune disease, and the type of autoimmune disease is the same as above. is there.
  • the antibody fragment multimer molecule according to the present embodiment is a multimer of single-chain Fv antibody fragments (scFv) having a predetermined dissociation rate constant k off and a dissociation constant K D, and has a predetermined apparent appearance. having a dissociation constant K D.
  • scFv single-chain Fv antibody fragments
  • an avidity effect can be obtained by the increase of the valence number, and it has high target selectivity and high affinity.
  • retention of normal cells with minimal antigen expression can be reduced, and the antigen can be specifically accumulated in target cells (eg, cancer cells) in which the antigen is closely expressed.
  • target cells eg, cancer cells
  • they can be easily detached from normal cells. Therefore, the medicine, the antitumor agent and the therapeutic agent for an autoimmune disease according to the present embodiment can realize excellent medicinal effects and reduction of side effects, and significantly reduce the dose, thereby reducing the side effects and medical expenses. We can expect reduction.
  • the antibody fragment multimer molecule according to the present embodiment is not a high molecular weight full-length antibody, but is a single-chain Fv antibody fragment (scFv) which is reduced while having the ability to target a target cell (for example, a cancer cell).
  • scFv single-chain Fv antibody fragment
  • the pharmaceutical agent, the antitumor agent and the therapeutic agent for an autoimmune disease according to the present embodiment have improved tissue permeability and retention in blood, and can be produced at low cost.
  • Example 1 Using trastuzumab, an antibody drug, as a model antibody, mutations were introduced into the variable region, and multimerization was attempted by the addition of multimerization peptides.
  • Trastuzumab is a molecular targeting drug whose antigen is HER2 (human epidermal growth factor receptor 2) of the epidermal growth factor receptor family, and is a humanized monoclonal antibody adapted for breast cancer and gastric cancer in which HER2 overexpression has been confirmed ( Fig. 1 (a).
  • Trastuzumab is an immunoglobulin G full-length antibody, binds to an antigen in the Fv domain, and exerts functions such as an antitumor effect by ADCC activity and an increase in blood half life by recycling in the Fc domain.
  • a full-length antibody has a high molecular weight of about 150 kDa, it has been a problem that the production cost is high in an animal cell culture system and the permeability to cancer tissue is low. Furthermore, it may be difficult to correctly deliver to target cancer cells, and therapeutic effects may be low, and side effects that may be caused by acting on normal cells that express target antigen HER2 may also be raised as issues. It was
  • a single-chain Fv antibody fragment (scFv) (Fig. 1 (b)) was prepared from trastuzumab, and scFv variants whose antigen binding ability per molecule was intentionally reduced were multimerized by a protein engineering approach I did.
  • the amino acid sequence of trastuzumab VL is shown in SEQ ID NO: 1
  • the amino acid sequence of VH is shown in SEQ ID NO: 2.
  • a fusion protein in which the multimerization peptide and the scFv were expressed as one polypeptide chain was prepared (FIG. 4 (a)).
  • a multimer-forming peptide (multimer) was used, and a construct expressed as a fusion protein was prepared by linking to the C-terminus of scFv with a linker.
  • GCN4 GCN4 leucine zipper
  • CMP cartilage matrix protein
  • CMP cartilage oligomer matrix protein
  • FIG. 6 (c) Each multimer forming peptide (multimer) forms a coiled coil in one direction from the N-terminus to the C-terminus.
  • a multimer was linked to the C-terminus of the scFv variants to form dimers, trimers and pentamers respectively (FIG. 6 (d)-(f)).
  • CMP forms an intermolecular disulfide bond at the N-terminus and COMP at the C-terminus.
  • Linker 1 links the C-terminal side of VL (SEQ ID NO: 1) with the N-terminal side of VH (SEQ ID NO: 2) (- GGGGS) 3-: SEQ ID NO: 3), “Linker 2” is for linking a multimer-forming peptide (multimer) to the C-terminal side of VH (SEQ ID NO: 2) (GSAGSAAGSGEF: SEQ ID NO: 4, Waldo GS. Et al., 1999, Nature Biotechnology. 17: 691-695).
  • PET22b-scFv-LH (monomer)
  • the gene encoding the scFv (SEQ ID NO: 5) is inserted into the NdeI-BamHI site of pET22b (+).
  • PET22b-wt-dimer (dimerized peptide)
  • a gene (SEQ ID NO: 6, FIG. 8) encoding a scFv, linker2 and GCN4 leucine zipper-derived peptide is inserted Amino acid sequence of GCN4 leucine zipper derived peptide: SEQ ID NO: 7).
  • PET22b-wt-trimer (trimerization peptide) As shown in FIG. 4 (a), a gene encoding a scFv, linker 2 and cartilage matrix protein (CMP; Cartilage Matrix Protein) -derived peptide in the NdeI-XhoI site of pET22b (+) (SEQ ID NO: 8, FIG. 9) ) Is inserted (amino acid sequence of cartilage matrix protein (CMP) derived peptide: SEQ ID NO: 9).
  • PET22b-wt-pentamer (pentameric peptide) In the NdeI-XhoI site of pET22b (+), as shown in FIG.
  • a gene encoding a scFv, linker 2 and a cartilage oligomeric matrix protein (COMP) -derived peptide (SEQ ID NO: 10, Figure 10) is inserted (amino acid sequence of cartilage oligomeric matrix protein (COMP) derived peptide: SEQ ID NO: 11).
  • This pET22-LH-linker is used as a template with the above pET22b-scFv-LH (monomer) as a template and LH-Fw (5'-GAAGACTTACATATATGGATATCCAGATGACCCAG-3 '; SEQ ID NO: 13) It was amplified by PCR using 3 ', SEQ ID NO: 14) as a primer, and inserted into the NdeI and HindIII sites of pET22b (+).
  • LH-Fw (5'-GAAGACTTACATATATGGATATCCAGATGACCCAG-3 ', SEQ ID NO: 15) using the above-mentioned pET22-LH-linker as a template and Linker 1-COMP-Rv (5'-GTGGGGCTAGGTCAAGCTTAACTACGCCCAAGAC-3' , PCR reaction using SEQ ID NO: 16), and pET22b-COMP as a template, Linker 1-COMP-Fw (5′-CTGGCGAGTTTAAGCTTGACC CCCCACACAGATG-3 ′, SEQ ID NO: 17) and COMP-Rv (5′-CCGCTCGAGTTATCGCAAGCGTCACATTCCATC-3C , PCR reaction using SEQ ID NO: 18) was performed respectively 1st PCR reaction).
  • PCR reaction is carried out using pET22b-GCN4 as a template and GCN4-Fw (5'-CGGAAGCTTATGAAACAGCTGGAAGACAAAG-3 ', SEQ ID NO: 21) and GCN4-Rv (5'-CCGCTCGAGTTATTCACCAACACGTTTCTTCAGAC-3' , PCR using SEQ ID NO: 22) as a primer, and pET22b-CMP as a template, CMP-Fw (5'-CGGAAGCTTGAAGAGATCCCGTGCGAATGC-3 ', SEQ ID NO: 23) and CMP-Rv (5'-CCGCTCGAGTTAGATTGTTTTTTTCAGCGCGC-3', SEQ ID NO: 24) ) was used as a primer.
  • heavy chain 50th and 59th arginine residues in CDR2 are 558th glutamic acid and 560th aspartic acid of HER2-ECD.
  • Strong formation of a salt bridge, and the tyrosine residue at heavy chain 33 in CDR1 (hereinafter referred to as HC_Y33) were assumed to interact with phenylalanine 573 at HER2-ECD (FIG. 5).
  • B phenylalanine residues in CDR2
  • the light chain 30 asparagine residue in CDR1 (hereinafter referred to as "LC_N30”) has a polar interaction with the glutamine residue 602 in HER2-ECD, and the light chain in the framework region near CDR2
  • the 66th arginine residue (hereinafter referred to as “LC_R66”) was assumed to form a strong salt bridge with the 598th glutamate (Fig. 5 (c)). Therefore, HC_R50, HC_R59, HC_Y33, LC_N30, and LC_R66, which are important residues for these bonds, were each substituted with alanine, which is a neutral amino acid.
  • FIG. 14 (b) which is a comparative example, no mutation was introduced.
  • HC_R50A / HC_R59A Uses scFv_HC_R50A as a template DNA and In the amplification of mer HC_R59A_F and HC_R59A_R, LC_N30A / HC_R50A / HC_R59A, a scFv_HC_R50A / HC_R59A is used as a template DNA, and the above-mentioned primers LC_N30A_F and LC_R30G / HC_R50A / HC_R are It was used.
  • the PCR amplification product was purified by ethanol precipitation and used for preparation of an E. coli expression plasmid.
  • the prepared plasmids are pET22b-HC_Y33A, pET22b-HC_R50A, pET22b-HC_R59A, pET22b-LC_N30A, pET22b-LC_R66G, pET22b-HC_R50A / HC_R59A, HC24H-HCH and Gh
  • the reaction solution is as follows. 1 ⁇ L of template DNA 10x PCR Buffer 5 ⁇ L 2 mM dNTP mix 5 ⁇ L 4 mM of 25 mM MgSO4 Primer 1 1.5 ⁇ L Primer 2 1.5 ⁇ L 1 ⁇ L of KOD Plus DNA polymerase Add sterile distilled water to make 50 ⁇ l
  • the thermal cycler was programmed as follows. (1) 94 ° C. 2 minutes (2) 98 ° C. 10 seconds (3) 55 ° C. 30 seconds (4) 68 ° C. 6 minutes 30 seconds (5) 68 ° C. 1 minute (2) to (4) were repeated 30 cycles.
  • Preparation of dimeric variants R59A-dimer and HC_R50A / HC_R59A-dimer involves linking the NdeI-NcoI treated fragment of pET22b-HC_R59A or pET22b-HC_R50A / HC_R59A with the NdeI-NcoI treated fragment of pET22b-wt-dimer respectively Made in The prepared plasmids were named pET22b-HC_R59A-dimer and pET22b-HC_R50A / HC_R59A-dimer.
  • the trimeric variant HC_R59A-trimer, pET22b-HC_R50A / HC_R59A-trimer and pET22b-LC_R66G / HC_R50A / HC_R59A-trimer are pET22b-HC_R59A, pET22b-HC_R50A / HC_R59A or GEThIh.
  • the fragment was prepared by ligating the -NcoI-treated fragment with an NdeI-NcoI-treated fragment of pET22b-wt-trimer to obtain pET22b-HC_R59A-trimer, pET22b-HC_R50A / HC_R59A-trimer and pET22b-LC_R66G / HC_R50A / HC_R59A-trimer.
  • the plasmid DNA was purified from the resulting E. coli pellet using alkali-SDS method, PCI extraction and alcohol precipitation.
  • E. coli BL21 DE3 pLysS strain (Novagen) was transformed with the plasmid DNA obtained as described above, seeded on Luria-Bertani (LB) agar medium containing 100 ⁇ g / mL ampicillin, and then cultured overnight at 37 ° C. The resulting colonies were inoculated into 2 ⁇ YT medium (10 mL) containing 50 ⁇ g / mL ampicillin and 20 ⁇ g / mL chloramphenicol, and shake cultured at 37 ° C. for 2 to 4 hours (pre-culture).
  • LB Luria-Bertani
  • the cells were suspended in suspension buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) and sonicated on ice.
  • suspension buffer 50 mM Tris-HCl pH 8.0, 150 mM NaCl
  • the disrupted solution is centrifuged (8,000 rpm, 4 ° C., 5 minutes), and the resulting precipitate is suspended in a washing buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% Triton X-100)
  • the washing was repeated three times by washing the suspension by centrifugation (8,000 rpm, 4 ° C., 5 minutes).
  • the suspension After washing, in order to remove the surfactant Triton X-100 from the obtained precipitate, the suspension is suspended using a suspension buffer, and then the suspension is centrifuged (8,000 rpm, 4 ° C., 5 The washing operation for 3 minutes was repeated 3 times to obtain an inclusion body.
  • a solubilization buffer 50 mM Tris-HCl pH 8.0, 6 M guanidine-HCl, 10 mM EDTA
  • each molecule was prepared from the inclusion body by unrolling by dilution. Dilute the final protein concentration to 1 to 2 ⁇ M with unwinding buffer (0.1 M Tris-HCl pH 8.0, 1 M L-arginine, 2 mM EDTA, 3.73 mM cystamine, 6.73 mM cysteamine) After, the film was unwound at 4 ° C. Subsequently, DTT is added to the solubilized inclusion body to a final concentration of 10 mM, and the same rewinding buffer is added dropwise to the protein solution incubated for 1 hour at room temperature until the guanidine concentration becomes 1.5 M.
  • unwinding buffer 0.1 M Tris-HCl pH 8.0, 1 M L-arginine, 2 mM EDTA, 3.73 mM cystamine, 6.73 mM cysteamine
  • the diluted solution was added dropwise to a rewinding buffer, diluted to a final protein concentration of 1 to 2 ⁇ M, and then stirred at 4 ° C. for 72 hours.
  • the protein solution after unwinding was subjected to ultrafiltration concentration with a VIVAFlow system (MWCO: 10,000, Sartorius), Amicon Ultra (MWCO: 10,000 Da, Millipore).
  • the concentrated protein solution was purified by liquid chromatography using AKTA purifier system (GE). Size exclusion chromatography was performed using a HiLoad 26/60 Superdex 75TM Prep grade column (GE). At this time, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl was used as a buffer. A HiLoad 26/60 Superdex 200TM Prep grade column (GE) was used for purification of each dimer, and a Superose 6TM GL column (GE) was used for purification of each trimer and each pentamer. The buffer used was 20 mM Tris-HCl pH 8.0, 400 mM NaCl when purifying the dimer or higher molecule.
  • the absorbance of the purified protein solution was measured using a spectrophotometer (ND-1,000 Spectrophotometer, NanoDrop Technologies).
  • concentration of each protein solution was calculated from the measured value of absorbance (Abs) using an ultraviolet absorption method.
  • the molecular weight of the protein used at the time of calculation of the concentration of each monomer was 26,000 Da, and the value of the molar absorption coefficient was 48,000 L / mol ⁇ cm.
  • HC_Y33A mutant a molecular weight of 26,000 Da and a molar absorption coefficient of 47,000 L / mol ⁇ cm were used.
  • the molecular weight of the protein used in calculating the concentration of each dimer was 62,000 Da, and the molar absorption coefficient was 49,000 L / mol ⁇ cm.
  • the molecular weight of the protein used at the time of concentration calculation of each trimer used 97,000 Da, and the value of the molar absorption coefficient 48,000 L / mol * cm was used.
  • the molecular weight of the protein used in calculating the concentration of each pentamer was 160,000 Da, and the molar absorption coefficient was 48,000 L / mol ⁇ cm.
  • HC_Y33A a scFv variant in which the 33rd tyrosine residue (HC_Y33) of heavy chain of CDR1 is substituted with alanine •
  • HC_R50A a scFv variant in which the 50th arginine residue (HC_R50) in heavy chain of CDR2 is substituted with alanine •
  • HC_R59A ScFv variant in which arginine residue (HC_R59) of heavy chain 59 of CDR2 is substituted with alanine ⁇ scFv variant with both HC_R50 and HC_R59 substituted with alanine in HC_R50A / HC_R59A ⁇ LC_N30A: light chain 30th of CDR1 ScFv variant in which asparagine
  • HC_R59A-dimer dimer of HC_R59A HC_R50A / HC_R59A-dimer: dimer of HC_R50A / HC_R59A HC_R59A-trimer: trimer of HC_R59A HC_R50A / HC_R59A-trimer: HC_R50A / HC_L50A / HC_R50A / HC_R59A-trimer: trimer of LC_N30A / HC_R50A / HC_R59A ⁇ LC_R66G / HC_R50A / HC_R59A-trimer: trimer of LC_R66G / HC_R50A / HC_R59A-trimer: trimer of LC_R66G / HC_R50A / HC_R59A
  • SEC-MALS Size exclusion chromatography
  • MALS multi angle light scattering
  • FIG. 14 (b) It analyzed by (FIG. 15 (a)).
  • SEC-MALS the external shape and absolute molecular weight of the molecule are revealed from static scattered light obtained by irradiating the molecule separated by SEC, and the size of the molecule can be analyzed with higher accuracy than SEC alone.
  • the measurement conditions are as follows. ⁇ Measurement equipment: DAWN 8 (Showa Scientific) Column: Superdex 200 increase 10/300 (GE Healthcare) Buffer: 20 mM Tris-HCl (pH 8.0), 400 mM NaCl
  • the absolute molecular weight was wt-dimer: 66 kDa, wt-trimer: 78 kDa, wt-pentamer: 150 kDa. From the analysis results and the comparison of molecular weights, it was confirmed that dimers, trimers and pentamers of wild type scFv were formed as intended.
  • Example 3 Boding analysis of scFv variants
  • scFv variants HC_Y33A, HC_R50A, HC_R50A, LC_N30A, HC_R50A / HC_R59A, and LC_R66G / HC_R50A / HC_R59A, FIG. 14 (a)
  • an antibody fragment multimeric molecule HC_R59A-
  • SPR method surface plasmon resonance method
  • HER2-ECD-Fc immobilized on the sensor chip, scFv mutants and antibody-terminated multimeric molecules of each concentration were added, these sensorgrams were obtained, and kinetic parameters were calculated.
  • the measurement conditions are as follows. Bovine serum albumin (BSA) was used as a negative control.
  • BSA Bovine serum albumin
  • HER2-ECD-Fc a protein purchased from R & D Systems (Recombinant Human ErbB2 / Her2 Fc Chimera Protein, CF) was used.
  • BIAcore 2000 and 3000 GE healthcare
  • HER2-ECD-Fc or BSA as a ligand is immobilized on the sensor chip CM5 for about 1200 RU by amine coupling method, and each scFv variant and antibody fragmented molecule molecule Dilution series (4, 8, 16, 32 nM) as an analyte were prepared, and the liquid was sequentially delivered from the low concentration side (FIG. 16 (a)).
  • the sensorgram (A) with each scFv mutant as a ligand and the sensorgram (B) with BSA as a ligand were subtracted (Sensogram A-Sensorgram B).
  • HC_R50A / HC_R59A is 5.0 ⁇ 10 -3 (1 / s)
  • LC_R66G / HC_R50A / HC_R59A is 3.6 ⁇ 10 -2 (1 / s) It became.
  • K D is, wt whereas was 0.2 (nM) with (wild type), 3.1 HC_Y33A (nM), 7.2 in HC_R50A (nM), 5.1 in HC_R59A ( nM), LC_N30A at 2.2 (nM), HC_R50A / HC_R59A at 70 (nM), LC_R66G / HC_R50A / HC_R59A at 1100 (nM), and it became clear that the mutant had reduced binding affinity.
  • HC_Y33A, HC_R50A, HC_R59A, and LC_N30A which are single mutants, are 10 times or more than wild-type scFv, and 100 times or more in HC_R50A / HC_R59A, which is a double mutant, and LC_R66G / HC_R50A / HC_R59A, which is a triple mutant.
  • the dissociation constant was increased by 1000 times or more, which revealed that the binding affinity to the antigen was lowered. From this, it was found that the binding ability of various scFv variants was reduced as compared to wild-type scFv, and in particular, the dissociation was faster. Moreover, it became clear that the binding ability was synergistically reduced by double mutation introduction of HC_R50A and HC_R59A and triple mutation introduction of LC_N30A, HC_R50A and HC_R59A.
  • the apparent dissociation constant K D of the antibody multimeric molecule was 2.0 ⁇ 10 ⁇ 2 (nM) in wt-dimer (wild-type), whereas it was 0.48 in HC_R59A-dimer. nM), HC_R50A / HC_R59A-dimer 5.9 (nM), LC_R66G / HC_R50A / HC_R59A-trimer 330 (nM), and it is apparent that the binding affinity of the antibody fragment of this example is lowered. (FIG. 16 (b)).
  • Example 4 (Binding specificity of scFv variant)
  • HER2-ECD-Fc and “EGFR of the same EGFR family”
  • An experiment for binding to EGFR-ECD was performed (FIG. 17 (a)).
  • HER2-ECD-Fc and EGFR-ECD were immobilized on the sensor chip in the same manner and in the same amount as in Example 3, and scFv variants at each concentration were added.
  • HER2-ECD-Fc As to HER2-ECD-Fc, the same as in Example 3 was used, and EGFR-ECD was prepared as follows. The measurement conditions are as follows. ⁇ Measurement equipment: Biaocre 3000 (GE Healthcare) ⁇ Chip: CM5 Ligand immobilization method: amine coupling method Ligand: HER2-ECD-Fc, EGFR-ECD ⁇ Immobilized amount of ligand: 600-700 RU Analyte concentration: wild type (wt) and single mutants (HC_Y33A, HC_R50A): 1 ⁇ M, double mutants (HC_R50A / HC_R59A): 5 mM Buffer: HBS-EP ⁇ Flow rate: 30 ⁇ L / min Temperature: 30 ° C
  • EGFR-ECD The preparation method of EGFR-ECD (FIG. 17 (b)) will be described. Prepared by a silkworm-baculovirus expression system (see http://www.pssj.jp/archives/protocol/expression/Bacmid_01/Bacmid_01.html). Sf9 (Insect cell)-Preparation example by baculovirus expression system (see also Kathryn M. Ferguson (2000). Extracellular domains drive homo-but not hetero-dimerization of erbB receptors. EMBO J, 19 (17), 4632-4643) did. 1) Preparation of a construct As shown in FIG.
  • a gene encoding a secretory signal sequence and EGFR extracellular domain (ECD, Ligand binding domain) from the full-length EGFR gene is pFastBac1 (Invitrogen) (FIG. 17 (c)) was inserted into the BglII-XbaI site of Using the PCR primers EGFR-F (5'-GGGGAAGATCTATGCGACCCTCCGGGACG-3 ': SEQ ID NO: 31) and EGFR-R (5'-GCGCTTCTAG ATTAGTGATGATGATGATGATGATGGCTGCTGCCTGTGGCACACATGGCCGGC-3': 3 'end of SEQ ID NO: 32) as shown in FIG.
  • ECD Ligand binding domain
  • the target region was amplified by adding 6xHis (histidine tag; for purification) to It can be expected that the target protein (EGFR-ECD) is secreted and expressed in the silkworm body fluid by the secretion signal.
  • 6xHis histidine tag; for purification
  • EGFR-ECD target protein
  • Tetracycline (final concentration 10 ⁇ g / mL) was added and cultured at 37 ° C. overnight.
  • Gentamicin (final concentration 7 ⁇ g / mL) was added and cultured at 37 ° C. for 2 hours.
  • the cells were seeded on LB agar medium containing Kanamycin (final concentration 50 ⁇ g / mL), Gentamicin (final concentration 7 ⁇ g / mL), IPTG (final concentration 200 ⁇ M), X-gal (final concentration 40 ⁇ g / mL).
  • the presence or absence of the target gene was confirmed by direct PCR (M13 Forward (-40): 5'-GTTTTCCCAGTCACGAC-3 ': SEQ ID NO: 33, M13 Reverse: 5'- CAGGAAACAGCTATGAC-3: SEQ ID NO: 34) for white colonies (purpose) If the gene is inserted into bacmid, a band of about 2,300 bp plus the size of the gene of interest is obtained). The colony having the recombinant BmNPV bacmid into which the target gene was inserted was inoculated in LB medium containing kanamycin (final concentration 50 ⁇ g / mL) and genamicin (final concentration 7 ⁇ g / mL), and cultured overnight at 37 ° C.
  • the recombinant BmNPV bacmid DNA into which the EGFR-ECD gene had been incorporated was purified from the grown E. coli using EndoFree Plasmid Midi Kit (QIAGEN). 3) Introduction of Recombinant BmNPV Bacmid DNA into Silkworm Larvae Recombinant BmNPV Bacmid DNA (1 ⁇ g) and DMRIE-C (Invitrogen) (3 ⁇ L) were mixed and allowed to stand at room temperature for 45 minutes.
  • the body fluid of the larva was collected, and the expression of the target protein was confirmed by Western blotting.
  • Sodium thiosulfate was added to the buffer to a final concentration of 0.5% to prevent melanization of the silkworm body fluid.
  • 5% sodium thiosulfate 50 ⁇ L (approximately 1/10 volume of body fluid) was placed, and the silkworm larva's foot was pierced with a needle of a syringe to make a hole, and the fluid was received in the tube.
  • About 500 ⁇ L of body fluid was collected per silkworm larva.
  • About 5 mL of body fluid was recovered from 10 silkworm larvae.
  • the mixture was centrifuged at 10,000 ⁇ g ⁇ 10 min at 4 ° C., and the supernatant containing the recombinant protein was recovered, and the next target protein was purified.
  • 5) Purification of Target Protein Purification of EGFR-ECD was carried out in 4 steps of ammonium sulfate precipitation, Ni affinity chromatography, 2 times gel filtration chromatography. In the ammonium sulfate precipitation, a saturated ammonium sulfate solution was added to the silkworm body fluid so that the final concentration was 80%, gently stirred at 4 ° C. for about 1 hour, and centrifuged at 10,000 ⁇ g for 20 minutes.
  • the pellet was collected and dissolved in a buffer (25 mM TrisHCl pH 8.0, 150 mM NaCl) used in the next experiment.
  • Ni-NTA (QIAGEN) resin was packed into the column and equilibrated with the above buffer. Thereafter, the pellet solution recovered by ammonium sulfate precipitation was applied to a column and eluted with a buffer containing 50, 100 and 300 mM imidazole.
  • Fractions containing EGFR-ECD were collected and subjected to gel filtration chromatography using a Superose 6 GL column.
  • the running buffer used 25 mM TrisHCl pH 8.0 and 150 mM NaCl as described above.
  • Fractions containing EGFR-ECD were collected and subjected to gel filtration chromatography using a Superdex 200 10/300 GL column. The same running buffer as above was used. The fraction containing EGFR-ECD was collected and used as a final purified product. The purity after each purification step and chromatography was confirmed by SDS-PAGE.
  • Example 5 Boding ability analysis of antibody fragment multimeric molecules
  • antigen binding experiments of wild-type multimeric molecules wt-dimer: dimer, wt-trimer: trimer, wt-pentamer: pentamer
  • multimers at each concentration were added to HER2-ECD-Fc immobilized on the chip to analyze the binding ability.
  • the measurement conditions are as follows.
  • BSA was used as a negative control.
  • HER2-ECD-Fc the same one as in Example 3 was used.
  • Example 6 Example 6 (Examination of cell surface retention using HER2 highly expressing cells) Subsequently, HER2 high expression cells were used to examine whether cell surface retention was improved by multimerization. The experiment was performed according to the cell surface retention test method conducted by Adams GP et al. Using ovarian cancer adenoma-derived SK-OV-3 as HER2-high expressing cells (Adams GP, et al. Br J Cancer 1998, vol. 77, 1405-1412.).
  • trastuzumab full-length antibody in which the N-terminal amino acid residue and surface lysine residue were biotinylated scFv was expressed in the same manner as in Example 1 from scFv (pET22b-scFv-LH (monomer) prepared in Example 1).
  • Proteins, wt-dimer and antibody fragment multimer molecule HC_R59A-dimer, HC_R50A / HC_R59A-dimer (HC_R50A / HC_R59A double mutation)
  • SK-OV-3 SK-OV-3
  • cells are recovered 0, 1, 2, 3 hours after addition of an antibody against SK-OV-3, and addition of fluorescently labeled streptavidin and FACS analysis are carried out to time-lapse the cell surface.
  • Antibody residual rate was evaluated.
  • the measurement conditions are as follows.
  • trastuzumab full-length antibody an antibody (Herceptin (registered trademark)) purchased from Roche was used.
  • ⁇ Cell SK-OV-3 (Ovarian cancer cell, HER2 overexpression) -Number of cells: 1.3 x 10 6 -Antibody: Biotinylated above antibody (full length 15 mg, other 5-6 mg)
  • Negative control human myeloma IgG kappa ⁇
  • Measurement equipment FACScalibur (Becton Dickinson) Buffer: FACS buffer (1 ⁇ PBS, 1% BSA, 0.1% NaN 3 )
  • Example 7 Using the antibody drug cetuximab as a model antibody, mutations were introduced into the variable region and its periphery, and multimerization was attempted by the addition of a multimerization peptide.
  • Cetuximab is a molecular targeting drug that targets the EGFR of the epidermal growth factor receptor family as an antigen, and is a humanized monoclonal antibody adapted for metastatic colorectal cancer or head and neck cancer in which EGFR overexpression has been confirmed.
  • Single-chain Fv antibody fragments were prepared from cetuximab, and scFv variants with intentionally reduced antigen binding capacity per molecule were multimerized by a protein engineering approach.
  • the amino acid sequence of cetuximab VL is shown in SEQ ID NO: 35
  • the amino acid sequence of VH is shown in SEQ ID NO: 36.
  • the method of mutagenesis is described.
  • pET22b-scFv-LH as a template DNA
  • 50 ⁇ L of the following reaction solution was prepared, and each plasmid was amplified by PCR.
  • the primer LC_Q27A_F (5'-GCATCAGCCTCGATTGGTACCAATATC-3 '(SEQ ID NO: 43)
  • the primer LC_Q27A_R (5'-GATATTGGTACCCAATCGAGGCTGATGC-3' (SEQ ID NO: 44)
  • the PCR amplification product was purified by ethanol precipitation and used for preparation of an E. coli expression plasmid.
  • the prepared plasmid was named pET22b-LC_Q27A.
  • the reaction solution is as follows. 1 ⁇ L of template DNA 10x PCR Buffer 5 ⁇ L 2 mM dNTP mix 5 ⁇ L 4 mM of 25 mM MgSO4 Primer 1 1.5 ⁇ L Primer 2 1.5 ⁇ L 1 ⁇ L of KOD Plus DNA polymerase Add sterile distilled water to make 50 ⁇ l
  • the thermal cycler was programmed as follows. (1) 94 ° C. 2 minutes (2) 98 ° C. 10 seconds (3) 55 ° C. 30 seconds (4) 68 ° C. 6 minutes 30 seconds (5) 68 ° C. 1 minute (2) to (4) were repeated 30 cycles.
  • each concentration of scFv variant and antibody fragmenter molecule was added, these sensorgrams were obtained, and kinetic parameters were calculated.
  • the measurement conditions are as follows. Bovine serum albumin (BSA) was used as a negative control.
  • BSA bovine serum albumin
  • EGFR-ECD a protein prepared by the same silkworm expression system as in Example 4 was used.
  • BIAcore 2000 and 3000 GE healthcare
  • HER2-ECD-Fc or BSA as a ligand is immobilized on the sensor chip CM5 for about 1200 RU by amine coupling method, and each scFv variant and antibody fragmented molecule molecule Dilution series (13, 25, 50, 100, 200 nM) as an analyte were prepared, and liquid delivery was performed sequentially from the low concentration side (FIG. 20 (c)).
  • the sensorgram (A) with each scFv mutant as a ligand and the sensorgram (B) with BSA as a ligand were subtracted (Sensogram A-Sensorgram B).
  • the scFv mutant of cetuximab prepared in this example is expected to have high target selectivity and high affinity, as the avidity effect due to the increase in valency number is obtained by multimerization. Also, for example, the trastuzumab scFv variants and heteromultimeric molecules produced in the previous example can be produced.
  • the fall of the antigen binding capacity per antibody fragment molecule, the improvement of the antigen binding ability by multimerization, and the improvement of retention property to a cell surface were seen.

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Abstract

La présente invention concerne une molécule multimère de fragment d'anticorps caractérisée en ce qu'elle est considérée comme un multimère d'au moins deux fragments d'anticorps Fv à chaîne unique, ayant une constante de dissociation apparente KD de 10-3–103 nM par rapport à un antigène, et par des fragments d'anticorps Fv à chaîne unique ayant une constante de vitesse de dissociation koff de 10-5–102 s-1 et une constante de dissociation KD de 10-1–105 nM par rapport à l'antigène.
PCT/JP2018/046869 2017-12-19 2018-12-19 Molécule multimère de fragment d'anticorps, agent pharmaceutique, agent antitumoral, agent de traitement de maladie auto-immune, et procédé de production de molécule multimère de fragment d'anticorps Ceased WO2019124461A1 (fr)

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JP2010119303A (ja) * 2008-11-17 2010-06-03 Tohoku Univ 多量体化低分子抗体
JP2011217760A (ja) * 2005-12-15 2011-11-04 Ind Technol Res Inst 組み換え三重足場ベースポリペプチド
JP2012522491A (ja) * 2009-04-03 2012-09-27 ベジニクス ピーティーワイ リミテッド 抗ｖｅｇｆ−ｄ抗体
WO2015166641A1 (fr) * 2014-05-02 2015-11-05 国立大学法人信州大学 Système d'expression-sécrétion de gène d'anticorps
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JP2011217760A (ja) * 2005-12-15 2011-11-04 Ind Technol Res Inst 組み換え三重足場ベースポリペプチド
JP2010119303A (ja) * 2008-11-17 2010-06-03 Tohoku Univ 多量体化低分子抗体
JP2012522491A (ja) * 2009-04-03 2012-09-27 ベジニクス ピーティーワイ リミテッド 抗ｖｅｇｆ−ｄ抗体
WO2015166641A1 (fr) * 2014-05-02 2015-11-05 国立大学法人信州大学 Système d'expression-sécrétion de gène d'anticorps
WO2016088376A1 (fr) * 2014-12-03 2016-06-09 株式会社アネロファーマ・サイエンス Plasmide de coexpression

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021047599A1 (fr) * 2019-09-13 2021-03-18 Beijing Xuanyi Pharmasciences Co., Ltd. Anticorps humanisés anti-claudine 18.2 (cldn18.2)

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