WO2021256524A1 - 免疫原性低減型低分子抗体とその製造法 - Google Patents

免疫原性低減型低分子抗体とその製造法 Download PDF

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WO2021256524A1
WO2021256524A1 PCT/JP2021/022968 JP2021022968W WO2021256524A1 WO 2021256524 A1 WO2021256524 A1 WO 2021256524A1 JP 2021022968 W JP2021022968 W JP 2021022968W WO 2021256524 A1 WO2021256524 A1 WO 2021256524A1
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antibody
amino acid
seq
molecular weight
small molecule
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元裕 野中
真也 大石
浩章 大野
啓輔 青木
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Kyoto University NUC
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Priority to US18/001,946 priority patent/US20230227536A1/en
Priority to CN202180056535.9A priority patent/CN116096747A/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the present invention relates to a low molecular weight antibody having reduced immunogenicity and a method for producing the same. More specifically, the present invention relates to a small molecule antibody composed of D-amino acid and achiral glycine and a method for producing the same, and screening of the small molecule antibody using a mirror image target protein corresponding to the target protein and an antibody library.
  • Monoclonal antibodies are one of the most attractive modality in the therapeutic field. High affinity, high selectivity, and long half-life in blood are the main advantages of this modality, but their size ( ⁇ 150 kDa) and structural complexity limit their scope of application.
  • Several antibody-like scaffolds have been developed to solve size and stability issues, such as adnectin, affibody, and afflin.
  • the drug candidate molecules derived from these scaffolds have good affinity and stability, even though they are much smaller than conventional antibodies.
  • their immunogenicity is often a problem, such as diminished effects and unexpected side effects due to the appearance of anti-drug antibodies (ADA).
  • Non-Patent Documents 1 and 2 Since the protease in the body is chiral and the structural difference between L- and D-protein can be distinguished, it is known that the degradation stability of D-protein is significantly improved.
  • the synthesis of VEGF-A inhibitors with high affinity and stability has been reported by applying a mirror image phage display to the library of D-protein scaffolds. This VEGF-A inhibitor composed of D-amino acids does not show immunogenicity (Non-Patent Documents 1 and 2).
  • Non-Patent Document 3-6 The inventors have established a screening process for a virtual mirror image library and reported the development of a new cancer treatment method (Non-Patent Document 3-6). In addition, several groups have reported mirror image screening of phage display peptide libraries (such as Non-Patent Documents 1 and 2 above).
  • VHH antibody which is a single domain antigen-binding fragment of a heavy chain-only antibody unique to camelids, is one of the most attractive scaffolds as a next-generation drug candidate (Non-Patent Document 7).
  • VHH antibodies Despite its small size ( ⁇ 15 kDa), VHH antibodies have the same high specificity and affinity as conventional antibodies ( ⁇ 150 kDa). Moreover, its high stability and solubility make it suitable for pharmaceutical development.
  • the first VHH antibody-based drug was approved by the FDA and clinical trials are underway for several candidate molecules.
  • VHH antibodies are said to have low immunogenicity, but the details are still unknown.
  • the VHH antibody has a refolding function, and the production of the VHH antibody by chemical synthesis has recently been reported (Non-Patent Document 8).
  • Non-Patent Document 8 there is a risk that anti-drug antibodies and neutralizing antibodies may be produced by degradation of the three-dimensional structure in immune cells.
  • VHH characterization Comparison of recombinant with chemically synthesized anti-HER2 VHH. Protein Sci. 28, 1865-1879. Papadopoulos, K.P., (2015) Unexpected Hepatotoxicity in a Phase I Study of TAS266, a Novel Tetravalent Agonistic Nanobody (R) Targeting the DR5 Receptor. Cancer Chemother. Kibria M.G., et al. (2020)
  • the immunogenicity of an anti-EGFR single domain antibody (V (HH)) is enhanced by misfolded amorphous aggregation but not by heat-aggregation.
  • An object of the present invention is to provide a new pharmaceutical modality with reduced immunogenicity.
  • mirror-image peptide proteins composed of D-amino acids, which are natural L-amino acid enantiomers, are less susceptible to substrate recognition by proteases in vivo.
  • the inventors investigated if it would be possible to reduce immunogenicity while maintaining its specificity by mirroring the antibody molecule.
  • the inventors focused on the VHH antibody known as the smallest domain having the function of the antibody, and used the Native Chemical Ligation (NCL) method to obtain D-amino acids and achiral glycine.
  • NCL Native Chemical Ligation
  • a low molecular weight antibody composed of D-amino acid and achiral glycine, wherein the low molecular weight antibody has a molecular weight of 60 kDa or less, more preferably 24KDa or less, still more preferably 15KDa or less.
  • the low molecular weight antibody is preferably 500 amino acids or less, more preferably 200 amino acids or less, still more preferably 120 amino acids or less, and specifically includes a single domain antibody, scFv, dsFV, Fab, Fab'and the like.
  • Single domain antibodies include VHH antibodies (including humanized VHH antibodies), IgNAR antibodies (VNAR antibodies), camelized VH antibodies and the like.
  • VHH antibodies including humanized VHH antibodies
  • IgNAR antibodies VNAR antibodies
  • camelized VH antibodies and the like camelized VH antibodies and the like.
  • Small molecule antibody (1) The amino acid sequences represented by SEQ ID NOs: 1 to 4, or the four framework regions represented by amino acid sequences having at least 60%, 70%, 80%, or 90% identity with the above sequences, respectively.
  • [5] A method for producing a low molecular weight antibody composed of D-amino acid and achiral glycine. 1) The amino acid sequence of the small molecule antibody is divided into a plurality of segments having a cysteine residue or an alanine residue as the N-terminal.
  • Each segment is chemically synthesized by the solid phase method.
  • a method comprising synthesizing a low molecular weight antibody composed of D-amino acid and achiral glycine by sequentially linking each synthesized segment by the NCL method.
  • the small molecule antibody is one selected from a single domain antibody, scFV, and Fab.
  • the small molecule antibody is a VHH antibody.
  • the segment is preferably divided into four segments with two cysteines present in FR1 and FR3 and a suitable cysteine or alanine residue present between them.
  • VHH antibody for example, the antibody described in [4] above can be used.
  • a method for producing a low molecular weight antibody composed of D-amino acid and achiral glycine against a target protein 1) To provide a mirror image target protein composed of D-amino acid and achiral glycine for the target protein. 2) An antibody library is screened using the mirror image target protein to obtain an antibody having an affinity for the mirror image target protein. 3) The above-mentioned method comprising synthesizing a small molecule antibody composed of D-amino acid and achiral glycine based on the amino acid sequence of the obtained antibody.
  • the library a phage display library or the like is suitable.
  • affinity matulation may be performed in order to enhance the affinity of the antibody obtained in step 2).
  • the method according to [8], wherein the synthesis of a low molecular weight antibody composed of D-amino acid and achiral glycine comprises the following steps. 1) The amino acid sequence of the small molecule antibody is divided into a plurality of segments having a cysteine residue or an alanine residue as the N-terminal. 2) Each segment is chemically synthesized by the solid phase method. 3) Each synthesized segment is sequentially linked by the NCL method to synthesize a low molecular weight antibody composed of D-amino acid and achiral glycine.
  • the small molecule antibody is one selected from a single domain antibody, scFV, and Fab.
  • the small molecule antibody is a VHH antibody.
  • the VHH antibody for example, the antibody described in [4] above can be used.
  • a multivalent or multispecific antibody obtained by ligating the low molecular weight antibody according to any one of [1] to [4].
  • a method for producing a multivalent or multispecific antibody composed of a D-amino acid which comprises ligating a small molecule antibody produced by the method according to any one of [5] to [11].
  • kits for producing a polyvalent or multispecific antibody composed of D-amino acid which is composed of D-amino acid and achiral glycine, each containing the amino acid sequence of (a) or (b) below.
  • Kits containing one or more polypeptides (a) The amino acid sequence set forth in any of SEQ ID NOs: 55-57, 61 and 63, (b) The amino acid sequence set forth in any of SEQ ID NOs: 55-57, 61 and 63 and at least 60, 70%, 80%, 90%, 95 (preferably in the portion corresponding to the framework region of said sequence). Amino acid sequence with%, 98%, or 99% identity.
  • the kit comprises the amino acid sequence set forth in SEQ ID NO: 55, or SEQ ID NO: 63, or at least 60, 70%, 80%, 90 with the sequence (preferably in the portion corresponding to the framework region of the sequence).
  • the sequence preferably in the portion corresponding to the framework region of the sequence.
  • the low molecular weight antibody composed of D-amino acid and achiral glycine according to the present invention has reduced immunogenicity while maintaining the same specificity and affinity as the full-length antibody. In addition, it has higher stability and solubility than a full-length antibody, and is suitable for drug development.
  • the small molecule antibody of the present invention has a completely mirror image relationship with the natural low molecular weight antibody composed of L-amino acids in terms of the structure of the target protein. Therefore, screening an antibody library using a mirror image target protein enables screening of a small molecule antibody composed of D-amino acid and achiral glycine specific to the target protein.
  • the concentration of the coated antigen is 1000 ng / mL.
  • the statistically significant differences calculated by the Mann-Whitney U test for ADA production in synthetic L-GFP-VHH antibody and synthetic D-GFP-VHH antibody-administered mice are shown. Sequence alignment of known VHH antibodies. The three CDR regions are shaded and the mutation sites in the FR regions are highlighted. As is clear from the figure, the FR region contains up to 16 mutations. Each sequence is shown in SEQ ID NOs: 21 to 51 of the sequence listing in order from the top. The synthesis process of PMP12A2h1 / D- PMP12A2h1 is shown.
  • the results of analysis of the binding affinity of pmp12a2h1 with the vWF A1 domain by surface plasmon resonance are shown.
  • the graph shows the data when PMP12A2h1 is flowed at 400nM, 200nM, 100nM, 50nM, 25nM, 13nM, 6.3nM, 3.1nM, 1.6nM, 0nM from the top.
  • Dissociation constant K D 39 ⁇ 2nM
  • Low-molecular-weight antibody of the present invention Low-molecular-weight antibody of the present invention
  • a "small molecule antibody” is an antibody, antibody fragment, or antibody-like antibody having a molecular weight lower than that of a full-length antibody (in the case of IgG, a molecular weight of about 150 kDa), but having affinity and specificity for an antigen. Means a molecule.
  • the target antigen (target protein) of the small molecule antibody of the present invention is not particularly limited, but in consideration of therapeutic and diagnostic applications, a tumor-related antigen, an antigen related to inflammation or allergy, an antigen related to viral or bacterial infection, or an antigen. It is preferably a low molecular weight antibody that recognizes an antigen associated with cardiovascular disease.
  • the molecular weight of the low molecular weight antibody of the present invention is preferably 60KDa or less, more preferably 24KDa or less, still more preferably 15KDa or less.
  • the amino length of the low molecular weight antibody of the present invention is preferably 500 amino acids or less, more preferably 200 amino acids or less, still more preferably 120 amino acids or less.
  • Examples of the low molecular weight antibody according to the present invention include a single domain antibody, scFv, dsFV, Fab, Fab'and the like.
  • the small molecule antibody is a single domain antibody, scFV, and Fab, more preferably a single domain antibody or scFV, and even more preferably a single domain antibody.
  • a multivalent antibody or a multispecific antibody such as deerbody (scFV dimer), F (ab') 2 (Fab'dimer) obtained by ligating and multimerizing these low molecular weight antibodies.
  • scFV dimer deerbody
  • F (ab') 2 (Fab'dimer) obtained by ligating and multimerizing these low molecular weight antibodies.
  • the average molecular weight and amino acid length of the monomers constituting the multimer are in the above ranges.
  • Single domain antibody A single domain antibody (SDAB) is an antibody molecule composed of a single domain (single variable domain) having antigen-binding property, and is, for example, a camel family animal (camera, llama, alpaca, etc.).
  • VHH antibody designed from heavy chain antibody including humanized VHH antibody
  • IgNAR antibody VNAR antibody
  • camelized VH antibody camelized VH antibody, etc.
  • VHH antibody The single domain antibody is preferably a VHH antibody.
  • VHH (Variable domain of Heavy chain of Heavy chain antibody) antibody is a low molecular weight antibody derived from the variable region of a heavy chain antibody composed of only H chains of camels, and is also called Nanobody (registered trademark).
  • the VHH antibody Since the VHH antibody has an average molecular weight of about 15 kDa and an amino acid length of about 110 to 120 amino acids, it can be recombinantly produced by a normal microbial expression system, and as mentioned above, production by chemical synthesis is also reported. Has been done.
  • the VHH antibody has high thermal stability and reversibility of the three-dimensional structure, and has the characteristics that it easily refolds and returns to the original three-dimensional structure even after heat denaturation. In addition, it is generally said to be safer because it has lower immunogenicity than a full-length antibody.
  • VHH antibodies are composed of three CDR regions (CDR1, CDR2, CDR3) that are important for antigen binding and four more conserved framework regions (FR1, FR2, FR3, FR4) that surround them.
  • FR1, FR2, FR3, FR4 have the following sequences (the framework region of the VHH antibody used in the examples).
  • FR1 QVQLVESGGALVQPGGSLRLSCAAS (SEQ ID NO: 1)
  • FR2 WYRQAPGKEREWVA (SEQ ID NO: 2)
  • FR3 YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCN (SEQ ID NO: 3)
  • FR4 WGQGTQVTVSS (SEQ ID NO: 4)
  • FR1, FR2, FR3, FR4 have the following sequences (framework region of Lama VHH antibody (sdAb_7047_Lg)).
  • FR1 QVQLQESGGGLVQAGGSLRLSCAAA (SEQ ID NO: 5)
  • FR2 WFRQAPGKEREFVG (SEQ ID NO: 6)
  • FR3 YADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCA (SEQ ID NO: 7)
  • FR4 WGQGTQVTVSS (SEQ ID NO: 8)
  • FR1, FR2, FR3, FR4 have the following sequences (framework region of alpaca VHH antibody (sdAb_6474_Vp)).
  • FR1 QVQLQESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 9)
  • FR2 WYRQAPGKERELVA (SEQ ID NO: 10)
  • FR3 DSVKGRYTISRDYAKNTVYLQMNSLKPEDTALYYCN (SEQ ID NO: 11)
  • FR4 WGQGTQVTVSS (SEQ ID NO: 12)
  • FR1, FR2, FR3, FR4 have the following sequences (framework region of alpaca VHH antibody (sdAb_0039_Vp)).
  • FR1 QVQLQESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13)
  • FR2 WFRQAPGKEREFVA (SEQ ID NO: 14)
  • FR3 YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA (SEQ ID NO: 15)
  • FR4 WGQGTQVTVSS (SEQ ID NO: 16)
  • FR1, FR2, FR3, FR4 have the following sequences (framework region of camel VHH antibody (sdAb_5835_Cd)).
  • FR1 QVQLVESGGGSVQAGGSLRLSCTAS (SEQ ID NO: 17)
  • FR2 WYHQAPGNECELVS (SEQ ID NO: 18)
  • FR3 YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAMYYCA (SEQ ID NO: 19)
  • FR4 WGQGTQVTVSS (SEQ ID NO: 20)
  • the framework regions can be altered without adversely affecting antibody binding properties, such as conservative substitutions (Mitchell LS. And Colwell LJ., Proteins (2016) 86 (7). : 697-706). Further, a technique for improving the stability and specificity of an antibody by modifying a framework residue important for stabilizing an antibody molecule or an antigen binding site is also known in the art.
  • a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with another amino acid of one or several amino acid residues to an amino acid sequence maintains its biological activity.
  • Well-known in the field Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. and Smith, M., Nucleic Acids Research ( 1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413 ).
  • amino acids classified in the same group in the amino acid sequence constituting a certain polypeptide may maintain the activity of the polypeptide even if they are replaced with each other.
  • substitutions between amino acids within such groups are called conservative substitutions.
  • Hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), Hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), Amino acids with aliphatic side chains (G, A, V, L, I, P), Amino acids with hydroxyl group-containing side chains (S, T, Y), Amino acids (C, M) with sulfur atom-containing side chains, Amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), Amino acids with base-containing side chains (R, K, H), Amino acids with aromatic-containing side chains (H, F, Y, W)
  • the framework regions are represented by SEQ ID NOs: 1 to 4, SEQ ID NOs: 5 to 8, SEQ ID NOs: 9 to 12, SEQ ID NOs: 13 to 16, or SEQ ID NOs: 17 to 20, respectively, as long as they have predetermined antigen specificity. It may be a combination of amino acid sequences having at least 60%, 70%, 80%, 90%, or 95% identity with FR1, FR2, FR3 and FR4.
  • the framework regions are represented by SEQ ID NOs: 1 to 4, SEQ ID NOs: 5 to 8, SEQ ID NOs: 9 to 12, SEQ ID NOs: 13 to 16, or SEQ ID NOs: 17 to 20, respectively, as long as they have predetermined antigen specificity. 1 to several, preferably 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2 missing from FR1, FR2, FR3 and FR4. It may be a combination of amino acid sequences with loss, substitution, addition, or insertion.
  • the CDR regions are determined according to the target protein.
  • a new VHH antibody can be designed by grafting the CDR regions of the known VHH antibody against the target protein or the VHH antibody against the target antigen searched for in the screening described later into the FR region.
  • the "target protein” is a protein to which a small molecule antibody (VHH antibody or the like) specifically binds, and its size does not matter.
  • VHH antibody small molecule antibody
  • the expression "target protein” may be used interchangeably with the terms target peptide, target polypeptide, target antigen.
  • D-amino acid The small molecule antibody of the present invention is composed of D-amino acids. However, since glycine has no asymmetric carbon and does not have D-type and L-type character isomers (achiral glycine), more accurately, the low molecular weight antibody of the present invention is composed of D-amino acid and achiral glycine. To. In the present specification, although it may be abbreviated as “composed of D-amino acid”, they include "composed of D-amino acid and achiral glycine".
  • the amino acids that make up the living body are L-type, and the D-amino acids are extremely limited, such as bacterial peptidoglycan.
  • a protein composed of D-amino acids (D-type, D-protein) has a mirror image structure with respect to a protein having the same sequence composed of L-amino acids (L-type, L-protein) (mandal, K, supra). .Et al., Uppalapati, M. et al.). It has been reported that D-protein has resistance to enzymatic degradation and is non-immunogenic because enzymes existing in living organisms distinguish between L-type and D-type and act stereospecifically on L-protein. ..
  • the inventors confirmed that the low molecular weight antibody composed of D-amino acid has a mirror image relationship with the low molecular weight antibody composed of L-amino acid.
  • various studies were conducted to apply the D-VHH antibody (Fig. 1) capable of acting on the target protein existing in the living body to drug discovery research. While establishing the chemical synthesis process of the D-VHH antibody, it was confirmed that the D-VHH antibody is non-immunogenic.
  • the low molecular weight antibody of the present invention can be chemically synthesized using D-amino acid and achiral glycine. Since there is a limit to the length of amino acids that can be synthesized by the solid phase method (SPPS), the small molecule antibody is divided into several segments, and each of the synthesized segments is sequentially linked and synthesized. Although the ligation of each segment can be carried out by a known peptide ligation method, it is desirable to use a native chemical ligation (NCL) method.
  • NCL native chemical ligation
  • NCL Native Chemical Liligation Method
  • the reaction proceeds only by mixing a peptide having ⁇ -thioester at the C-terminal and a peptide having unprotected cysteine at the N-terminal under mild conditions (pH 7, 20 ° C to 37 ° C), and the reaction proceeds.
  • a technique for binding protective peptide segments (Dawson, PE; Muir, TW; Clark-Lewis, I .; Kent, SBH Science, 1994, 266, 776.). Since this method utilizes the functional groups inherent in the peptide, no special activator is required, and the reaction proceeds in good yield even with a peptide having an unprotected side chain.
  • the NCL method requires the presence of ligation sites containing cysteine residues in the target amino acid sequence at appropriate intervals, but if the protein has very few cysteine residues or is not present at the appropriate position, Methods of using alanine residues in place of cysteine residues have also been developed (Johnson, ECB; Kent, SBHJ Am. Chem. Soc. 2006, 128, 6640). According to the NCL method, peptide chains up to about 200 residues can be chemically synthesized.
  • the method for producing a small molecule antibody of the present invention includes the following steps. 1) The amino acid sequence of the small molecule antibody is divided into a plurality of segments having a cysteine residue or an alanine residue as the N-terminal. 2) Each segment is chemically synthesized by the solid phase method. 3) Each synthesized segment is sequentially linked by the NCL method to synthesize a low molecular weight antibody composed of D-amino acid and achiral glycine.
  • Each segment is generally 50 amino acids or less, preferably 40 amino acids or less, and more preferably 30 amino acids or less due to the limitation of chemical synthesis by the solid phase method.
  • the solid-phase method to be used is not particularly limited, and known methods such as the Boc method and the Fmoc method can be used, but the Fmoc method is preferable.
  • the N-terminal of each segment is a cysteine residue, but if a cysteine residue does not exist at an appropriate position, an alanine residue is used as described above.
  • a soluble tag for example, a His tag is preferable.
  • two cysteine residues are present in the framework regions FR1 and FR3 (FIG. 2).
  • the length of the central segment of the three segments divided by these two cysteine residues exceeds 70 amino acids, and it is difficult to chemically synthesize them at one time. Therefore, by utilizing the alanine residue existing at an appropriate position between these or the alanine or cysteine residue in the CDR region in addition to the above two cysteine residues, the whole is four segments of 50 amino acids or less. It is divided into two parts, synthesized, and connected by the NCL method.
  • Cys 22 , Cys 96 , and Ala 54 are divided into four segments, but this is an example, and the second division point is particularly that of the VHH antibody. It is set appropriately according to the structure (array).
  • connection order of each segment is appropriately determined according to the antibody to be synthesized and the structure of each segment.
  • the segments divided into two by the Ala residue are connected in advance, but the segments at both ends may be connected and the Ala portion may be connected at the end.
  • the synthesized low-molecular-weight antibody is denatured using urea or the like, and then dialyzed or diluted to spontaneously refold and form a correct three-dimensional structure.
  • Drug discovery screening using a virtual mirror image library is possible because of the mirror image relationship between the substrate specificity of L-protein and D-protein. Specifically, (1) first chemically synthesize the D-type (D-protein) of the target protein molecule, (2) use the synthetic D-protein to screen for chiral natural products, and (3) hit compounds. Drug discovery candidates can be selected by synthesizing a mirror image (L-protein) and (4) evaluating the biological activity of the L-protein.
  • the low molecular weight antibody composed of D-amino acid has a complete mirror image relationship with the low molecular weight antibody composed of L-amino acid, and the target antigen also has a complete mirror image relationship. Therefore, by applying the screening by the virtual mirror image library, it becomes possible to screen a small molecule antibody composed of D-amino acid against a natural target protein.
  • the present invention also provides a method for producing a small molecule antibody composed of D-amino acid against a target antigen protein using the above strategy.
  • the method includes the following steps. 1) To provide a mirror image target protein composed of D-amino acid and achiral glycine for the target protein. 2) An antibody library is screened using the mirror image target protein to obtain an antibody having an affinity for the mirror image target protein. 3) Based on the amino acid sequence of the obtained antibody, a small molecule antibody composed of D-amino acid and achiral glycine is synthesized.
  • an antibody library known in the art such as a phage display library can be used, and it may be an immune library, a naive library, or a synthetic library.
  • Phage display libraries have already been constructed for small molecule antibodies such as scFv and VHH antibodies, and these can be suitably used, or may be prepared according to a conventional method.
  • phage selection can be performed using the D-target protein in the same manner as when using a normal target antigen. That is, the library is reacted with the immobilized target protein, the unbound phage is washed and removed, the bound phage is eluted, and the operation of growing in Escherichia coli is repeated to concentrate the phage specific to the target protein.
  • Immobilization can be immobilization by adsorption or immobilization using biotin-streptavidin.
  • the selected small molecule antibody may be subjected to affinity maturing in order to enhance the affinity, if necessary.
  • the affinity matulation may be in vivo or in vitro, but in vitro is preferable.
  • a secondary library is prepared by introducing mutations into antibody genes by known methods such as chain shuffling, random mutation introduction method by error prone PCR, and CDR walking, and screening for antibodies with high affinity from this is performed. May be good.
  • the sequence of the selected natural small molecule antibody is determined, and the small molecule antibody composed of D-amino acid is chemically synthesized based on the sequence. Chemical synthesis can be carried out by the NCL method according to the description in the previous section "3. Synthesis of low molecular weight antibody of the present invention.
  • the CDR regions are determined according to the target protein. Therefore, even if the antibody to be screened is not a small molecule antibody to be synthesized, the small molecule antibody can be designed by grafting the CDR region of the antibody into the FR region of the small molecule antibody to be synthesized.
  • the low molecular weight antibody of the present invention may be made into a polyvalent antibody or a multispecific antibody by ligating with a simple peptide bond or a linker to form a multimerization.
  • Such multivalent antibodies and multispecific antibodies are also within the scope of the present invention.
  • scFv can be linked with a peptide bond to make (scFv) 2.
  • scFV can be dimerized to form a deerbody, trimerized to form a triabomer, and tetramerized to form a tetrabody.
  • more complex tandem deer bodies and flexi bodies can be created.
  • single domain antibodies such as VHH can be tandemly linked or multimerized with simple peptide bonds or linkers.
  • the multivalent antibody thus constructed can be a multispecific antibody having binding properties to different target proteins by differentiating the variable domains of the constituent monomers.
  • a peptide linker composed of amino acids can be used, and for example, a linker composed of Gly and Ser can be used.
  • the length of the peptide linker is 1 to 50 amino acids, preferably 1 to 30 amino acids, more preferably 1 to 10 amino acids.
  • ⁇ may be linked using a chemical cross-linking agent (synthetic chemical linker).
  • synthetic chemical linker examples include NHS, DSS, BS3, DSP, DTSSP, EGS, sulfo-EGS, DST, sulfo-DST, BSOCOES, sulfo-BSOCOES and the like.
  • Modification of the low molecular weight antibody of the present invention The low molecular weight antibody of the present invention can be appropriately modified according to the purpose of use. Modifications include modifications with fluorescent labels, phosphorescent labels, chemiluminescent labels, bioluminescent labels, radioactive isotopes, metals, metal chelate, metal cations, chromophores, enzymes, lipids, hydrophilic polymers, and sugar chains. be able to.
  • Modifications include modifications for introducing linkers and spacers for compounding with drugs, and chemical modifications for stabilization. Therefore, even if one or several amino acids are replaced with unnatural amino acids other than D-amino acids or L-amino acids as long as they do not negatively affect the specificity and three-dimensional structure of the low molecular weight antibody. good.
  • the low molecular weight antibody of the present invention may be immobilized on an appropriate surface depending on the purpose of use.
  • the carrier to be immobilized include colloidal particles, magnetic particles, microplates and the like.
  • the low molecular weight antibody of the present invention having the same specificity as the natural antibody molecule but having further reduced immunogenicity is used as a drug alone or in combination with other drugs. be able to.
  • “Pharmaceutical composition” When the small molecule antibody of the present invention binds to a target protein and exerts a predetermined pharmacological action, the small molecule antibody is formulated together with a pharmacologically acceptable carrier or additive and used as a pharmaceutical composition. be able to.
  • Pharmacologically acceptable carriers and additives include, for example, surfactants, excipients, colorants, fragrances, preservatives, antioxidants, stabilizers, buffers, suspending agents, isotonic agents, etc. Examples thereof include, but are not limited to, binders, disintegrants, lubricants, fluidity promoters, and flavoring agents.
  • the aqueous carrier includes water, ethanol, polyol (glycerol, propylene glycol, polyethylene glycol, etc.), vegetable oil such as olive oil, and organic ester such as ethyloleic acid.
  • Non-aqueous carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride. , Polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts and the like.
  • Antibody drug conjugate Due to the specific target recognition function of the antibody, the small molecule antibody of the present invention can be used as an antibody drug conjugate (ADC) by complexing it with an appropriate drug via a linker.
  • the anticancer drug can be specifically used in the tumor cell.
  • Can act internalized ADC
  • an anticancer drug can be allowed to act in the vicinity of the tumor cells (extrinsic ADC). In some cases, non-cleaving linkers can be used.
  • Cleavable linkers can be designed on the basis of, for example, disulfides, hydrazone, peptides, non-cleavable linkers can be designed on the basis of thioethers (non-cleavable) and the like.
  • the linker can be appropriately designed according to the purpose, and such a technique is also known in the art.
  • the ADC containing the low molecular weight antibody of the present invention can be formulated together with a pharmacologically acceptable carrier or additive, and such carrier or additive is as described above.
  • the administration route of the pharmaceutical composition containing the low molecular weight antibody of the present invention or ADC is not particularly limited, but parenteral administration is preferable, and specific examples thereof include injection administration, nasal administration, pulmonary administration, and transdermal administration. Be done.
  • parenteral administration is preferable, and specific examples thereof include injection administration, nasal administration, pulmonary administration, and transdermal administration. Be done.
  • injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection.
  • the administration method can be appropriately selected depending on the age and symptoms of the patient.
  • the dose of the pharmaceutical composition or ADC containing the low molecular weight antibody of the present invention provides an optimum response (for example, therapeutic response) according to the desired therapeutic effect, administration method, treatment period, age and weight of the patient, and the like. Adjusted to bring, it is usually 10 ⁇ g / kg to 10 mg / kg as the active ingredient per day for adults.
  • Typical treatment methods include, for example, once-weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months. It may be administered or once every 3 to 6 months.
  • the low molecular weight antibody of the present invention has high in vivo stability and low immunogenicity, it can be used for diagnosis and imaging by modifying it with a detectable molecule.
  • Detectable molecules include, for example, radioactive nuclei: technetium 99m ( 99m Tc), iodine 123 ( 123 I), iodine 125 ( 125 I), indium 111 ( 111 In), fluorine 18 ( 18 F), gallium 67 ( 67 Ga), gallium 68 ( 68 Ga), copper 64 ( 64 Cu), etc .; fluorescent molecules: fluorescein, Alexa, cyanin, etc .; chemiluminescent molecules: luminol, etc .; bioluminescent molecules: luciferase, alkaline phosphatase, etc.; And so on.
  • Radioimmunoassay RIA
  • enzyme immunoassay EIA
  • fluorescent immunoassay FIA
  • luminescent immunoassay LIA
  • immunoprecipitation IP
  • TIA turbidimetric method
  • WB Western blotting
  • IHC immunohistochemistry
  • SRID immunoprecipitation method
  • Kits for the production of small molecule antibodies In VHH antibodies, CDR3 is the most diverse of the three CDR regions and is important for interaction with the target, while the other two CDRs have a small effect on the interaction. It can be regarded as a fixed array. Therefore, the segment before the cysteine (Cys 96 ) in the FR3 region (D-9 of Examples described later) or the partial segment constituting the segment is prepared in advance and provided as a kit for producing a VHH antibody kit. Can be done. The remaining segments can be designed and synthesized according to the target and linked to the segments of the kit by the above-mentioned NCL method to produce a target-specific VHH antibody.
  • the kit of the present invention comprises one or more polypeptides composed of D-amino acids and achiral glycine, each comprising the amino acid sequence of (a) or (b) below.
  • the sequence of the portion corresponding to the framework region may be used as the framework region of another known VHH antibody (for example, SEQ ID NO: 5 to 20 or SEQ ID NO: 21 to 54 (FIG. 8). ) Can be replaced with the corresponding sequence in).
  • VHH antibody for example, SEQ ID NO: 5 to 20 or SEQ ID NO: 21 to 54 (FIG. 8).
  • the kit of the present invention is (1) A polypeptide composed of D-amino acid and achiral glycine, which comprises the amino acid sequence shown by SEQ ID NO: 63 (D-9 of Example), which is the segment before cysteine (Cys 96) in the FR3 region. or, (2) A polypeptide composed of D-amino acid and achiral glycine, which comprises the amino acid sequence shown by SEQ ID NO: 55 (D-1 of Example) which is a part of the FR1 region which does not contain the CDR region.
  • Each polypeptide may be appropriately modified with a protective group, a histidine tag, a fluorescent label, or the like, or may be immobilized on a solid phase such as beads or a support.
  • a protective group such as a histidine tag, a fluorescent label, or the like
  • immobilized on a solid phase such as beads or a support.
  • modification and immobilization refer to the description in 5.
  • the kit may include reagents and buffers necessary for synthesis, D-amino acids, achiral glycine, instruction manuals, etc., in addition to the above-mentioned polypeptides which are essential components.
  • Example 1 In this example, a GFP-binding VHH antibody (L-GFP-VHH antibody) and an enantiomer thereof (D-GFP-VHH antibody) were synthesized by the process shown in FIG.
  • HPLC and MS High Performance Liquid Chromatography for analysis is performed on a Cosmosyl 5C18-AR300 column (4.6 ⁇ 250 mm, Nacalai Tesque) with a linear gradient of CH 3 CN containing 0.1% (v / v) TFA at a flow rate of 1 mL / min (25 ° C). Used in.
  • a Cosmosyl 5C4-AR300 column (4.6 x 150 mm, Nacalai Tesque) was used with a linear gradient of CH 3 CN containing 0.1% (v / v) TFA at a flow rate of 1 mL / min (40 ° C). Used in. The product was detected at 220 nm absorbance.
  • a Cosmocil 5C18-AR300 column (20 x 250 mm, Nacalai Tesque) or a Cosmocil 5C4-AR300 column (20 x 150 mm, Nacalai Tesque) with a flow rate of 8 mL / min (room temperature) and 0.1% (v / v) TFA.
  • a linear gradient of CH 3 CN For L-10 and D-10, a Cosmosyl 5C4-AR300 column (20 x 150 mm, Nacalai Tesque) was used with a linear gradient of CH 3 CN containing 0.1% (v / v) TFA at a flow rate of 12 mL / min (40 ° C). Used in.
  • Fmoc-SPPS Peptide solid phase synthesis (Fmoc-SPPS) by the Fmoc method was performed using an automatic peptide synthesizer (PSSM-8, Shimadzu). The following side chain protected amino acids were used: Arg (Pbf), Asn (Trt), Asp (OtBu), Cys (Trt), Gln (Trt), Glu (OtBu), His (Trt), Lys (Boc). ), Ser (tBu), Thr (tBu), Tyr (tBu).
  • L-2 A protective peptide was constructed from Fmoc-NH-SAL resin (45 mg, 0.02 mmol) according to the above standard procedure of Fmoc-SPPS.
  • coli was centrifuged (6,000 rpm, 20 minutes, 4 ° C.) and then resuspended in binding buffer (5 mM DTT in PBS and protease inhibitor [Nacalai Tesque]). After sonication and addition of Trition X-100, the cytolysate was centrifuged at 4 ° C. and 12,000 rpm for 30 minutes. The supernatant was incubated with glutathione-cepharose 4B (GE Healthcare) overnight at 4 ° C.
  • binding buffer 5 mM DTT in PBS and protease inhibitor [Nacalai Tesque]
  • the CD spectrum of the synthetic L-GFP-VHH antibody was consistent with that of the recombinant L-GFP-VHH antibody, suggesting the existence of a ⁇ -sheet structure (Fig. 4A).
  • the CD spectrum of the synthetic D-GFP-VHH antibody was the opposite of that of the recombinant L-GFP-VHH antibody.
  • the thermal stability of the synthetic L- and D-GFP-VHH antibodies was the same as that of the recombinant L-GFP-VHH antibody.
  • the D-VHH antibody scaffold was suggested to be stable under physiological conditions (Fig. 4B).
  • Enzyme-linked immunosorbent assay (ELISA) ELISA performed all wash and dilution processes on PBS (pH 7.4) containing 0.025% Tween 20. 96-well microtiter plates (Greiner, high binding) were coated overnight at 4 ° C. with GST-EGFP or GST-mCherry in 50 mM sodium carbonate buffer (pH 9.4) (50 ⁇ L / well; 30 nM). After coating, the wells were washed 3 times and blocked with PBS containing 3% BSA (150 ⁇ L / well) for 2 hours.
  • Quartz crystal microbalance (QCM) analysis was performed using Single-Q (SCINICS, Japan).
  • the gold surface of the sensor chip was washed with a piranha solution, 0.2 mM NTA-SAM forming reagent (Dojindo, Japan) dissolved in 10% EtOH was added, and the mixture was incubated overnight. After washing, 40 mM NiSO 4 solution was added and incubated for 1 hour. After washing and equilibration with 500 ⁇ L PBS (pH 7.4), 5 ⁇ L 1.0 mg / mL ligand solution (recombinant L-GFP-VHH antibody, synthetic L-GFP-VHH antibody or synthetic D-GFP-VHH in PBS) (Antibody) was added.
  • sample solutions of each concentration (final concentration; 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM and 300 nM) were repeatedly added.
  • the dissociation constant was evaluated in 3 assays.
  • the GFP-binding activity of the synthetic L-GFP-VHH antibody was confirmed. It was also found that the synthetic D-GFP-VHH antibody has a complete mirror image structure of the L-GFP-VHH antibody and has a different interaction surface.
  • 96-well microtiter plate (Greiner, high binding) in 50 mM sodium carbonate buffer (pH 9.4) with L-GFP-VHH antibody or D-GFP-VHH antibody (50 ⁇ L / well; 1.0 ng / mL, 10 ng / mL, Coated overnight at 4 ° C. with either 100 ng / mL or 1,000 ng / mL).
  • the wells were washed 3 times and blocked with PBS containing 3% BSA (150 ⁇ L / well) for 2 hours. After 3 washes, 1: 1,000 dilutions of immunized serum from each mouse were added (50 ⁇ L / well, days 0, 14, 21, and 28) and incubated for 1 hour.
  • VHH antibody PMP12A2h1 and its enantiomer (D-PMP12A2h1) were synthesized by the process shown in FIG.
  • PMP12A2h1 is a VHH antibody that constitutes a drug known by the common name of couplerizumab, and specifically binds to the A1 domain (vWF A1 domain) of von Willebrand factor, which is one of the blood coagulation factors, and suppresses platelet aggregation.
  • HPLC and MS High Performance Liquid Chromatography for analysis is performed on a Cosmosyl 5C18-AR300 column (4.6 ⁇ 250 mm, Nacalai Tesque) with a linear gradient of CH 3 CN containing 0.1% (v / v) TFA at a flow rate of 1 mL / min (25 ° C). Used in. The product was detected at 220 nm absorbance.
  • a Cosmocil 5C18-AR300 column (20 x 250 mm, Nacalai Tesque) or a Cosmocil 5C4-AR300 column (20 x 150 mm, Nacalai Tesque) with a flow rate of 8 mL / min (room temperature) and 0.1% (v / v) TFA.
  • a linear gradient of CH 3 CN used with a linear gradient of CH 3 CN.
  • Fmoc-protected amino acids (5 eq) were coupled twice in DMF for 60 minutes using Oxyma Pure (5 eq) and DIC (10 eq). The Fmoc protecting group was deprotected with 20% piperidine / DMF twice for 4 minutes.
  • the resin on which the peptide chain was constructed was treated with 50 mM 4-nitrophenylchloroformate in DCM (25 mL) for 1 hour and then treated with 0.5 M (i-Pr) 2 NEt in DMF (25 mL) for 15 minutes.
  • TFA / H 2 O / m-cresol / thioanisole / EDT 80: 5: 5: 5: 5) was used for 2 hours for deprotection and cleavage from the resin. After removing the resin by filtration, the crude product was precipitated and washed with cold Et 2 O.
  • PMP12A2h1 and D-PMP12A2h1 folding peptides L-20 or D-20 were dissolved in PBS (pH 7.4) containing 6M guanidine and 40 mM DTT to a pH of 1.0 mg and incubated at room temperature for 2 hours. .. This solution was diluted 100-fold with PBS (pH 7.4) and left overnight at room temperature. The solution was concentrated using a MWCO3000 centrifugal filtration membrane (Millipore, Amicon-Ultra3kDa). Subsequently, 10-fold doses of 5,5'-dithiobis (2-benzoic acid) (DTNB) were added and incubated at 37 ° C. for 5 hours. The target protein was purified using the MWCO3000 centrifugal filtration membrane.
  • CD spectra of PMP12A2h1 and D-PMP12A2h1 Folded pmp12a2h1 and D-PMP12A2h1 were diluted with PBS (pH 7.4) and their concentrations were adjusted to 10 ⁇ M respectively.
  • the CD spectrum of each protein was recorded at 20 ° C. using a circular dichroism spectrometer (JACSO J-720).
  • the CD spectrum of pmp12a2h1 is a spectrum with a minimum of 216 nm and a maximum of 203 nm, indicating the existence of a ⁇ -sheet-like structure (Fig. 10).
  • the CD spectrum of D-PMP12A2h1 is the opposite of the positive and negative signs of the CD spectrum of pmp12a2h1, suggesting that D-PMP12A2h1 was folded into a structure opposite to PMP12A2h1.
  • pmp12a2h1 binds to the vWFA1 domain with sufficient binding affinity (Fig. 11). It is suggested that synthetic pmp12a2h1 has appropriate biological activity.
  • Example 3 A T7 phage library with randomized CDR regions is used to attempt screening for VHH antibodies composed of D-amino acids specific for the target molecule.
  • Phage library A random library containing only CDR3 or a T7 phage library in which all three regions of CDR1, CDR2, and CDR3 are randomized from the sequences of the VHH antibody (PMP12A2h1) of coupler sizumab is used. At this time, use a library in which the lengths of the random amino acids of CDR3 are 7, 10, 13, 17, and 20-mer.
  • Target molecule As the target molecule, a molecule that can be chemically synthesized by the reported protocol (for example, cytokine or chemokine) is selected, and a mirror image protein synthesized using D-amino acid is used. At this time, a tag such as 6 ⁇ His is added to the N-terminal or C-terminal of the mirror image protein.
  • Phage screening Dissolve the target mirror-type protein in Tris buffer TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) to a concentration of 1 to 10 ⁇ g / ml, and use a Nickel plate (Thermo Scientific). ) At room temperature for 30 minutes, coating (100 ⁇ l / well). Then, block with a blocking agent (3% BSA or 4% BlockAce) at room temperature for 2 hours. After washing the wells with PBST three times , add 100 ⁇ l (10 9 to 10 10 pfu) of the above phage library and incubate at room temperature for 30 minutes. Wash the wells 10 times with wash solution (PBST) to remove unbound phage.
  • Tris buffer TBS 50 mM Tris-HCl pH 7.5, 150 mM NaCl
  • PBST wash solution
  • Sequence confirmation Clone is obtained from the phage lytic fraction in which enrichment is observed, and a random sequence of VHH is individually obtained by Sanger sequencing analysis (ABI 3130 Genetic analyzer). In addition, the sequence of the random region is comprehensively acquired using the next-generation sequencer (illumina's iSeq 100 system). VHH antibodies with sequences showing high homology in the random region are targeted for synthesis.
  • a D-VHH antibody having a CDR sequence specified as a target is synthesized according to the procedure of the present invention.
  • the affinity with the target molecule consisting of the original L-amino acid is analyzed using the Biacore system or the like.
  • the small molecule antibody of the present invention has the same specificity as the natural antibody molecule, but has reduced immunogenicity. Therefore, it is useful in the medical field such as medicine or diagnosis / imaging.

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US12359345B2 (en) 2022-02-17 2025-07-15 Rules Based Medicine Inc. Single domain antibody libraries with maximized antibody developability characteristics
US12442107B2 (en) 2022-02-17 2025-10-14 Rules-Based Medicine Inc. Single domain antibody libraries with maximized antibody developability characteristics

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