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WO2021136227A1 - Hétérodimère induit par modification de domaine structurel ch3, son procédé de préparation et son utilisation - Google Patents

Hétérodimère induit par modification de domaine structurel ch3, son procédé de préparation et son utilisation Download PDF

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WO2021136227A1
WO2021136227A1 PCT/CN2020/140595 CN2020140595W WO2021136227A1 WO 2021136227 A1 WO2021136227 A1 WO 2021136227A1 CN 2020140595 W CN2020140595 W CN 2020140595W WO 2021136227 A1 WO2021136227 A1 WO 2021136227A1
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mutated
heterodimer
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周易
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain

Definitions

  • the invention belongs to the field of antibody engineering, and specifically relates to a heterodimer induced by the transformation of a CH3 structural domain, and a preparation method and application thereof.
  • IgG type bispecific antibodies have similar structure, physicochemical properties and Fc segment functions to common antibodies.
  • an IgG bispecific antibody consists of two heavy chains with different amino acid sequences (ie, heavy chain HC_A against antigen A and heavy chain HC_B against antigen B) and two light chains with different amino acid sequences (ie light chain against antigen A).
  • IgG-type bispecific antibodies There are many ways to construct IgG-type bispecific antibodies, and one of the important methods is to modify Fc to form heterodimers. As early as the 1990s, Carter et al. used the "knob-into-hole" model to modify the Fc segment of antibodies, and successfully realized the preparation of bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001). Carter et al.
  • US2010286374A1 discloses a method for promoting the formation of heterodimers by using static electricity. Specifically, the charged amino acids in the CH3 domains of the two heavy chains are mutated to oppositely charged amino acids, so that the CH3 domain of one heavy chain is generally positively charged, and the CH3 domain of the other heavy chain is generally positively charged. It is negatively charged, and electrostatic repulsion of the same charge will inhibit the formation of homodimers. However, electrostatic effect cannot completely inhibit the formation of homodimers. The introduction of too many mutations will cause the decrease of protein expression level, reflecting the complexity of the interaction relationship between interface amino acids in the case of multiple amino acid mutations.
  • CN106883297A discloses a method. Specifically, on the basis of the "convex-concave” model of Carter et al., the F405K mutation is further introduced at one end of the “concave” to enhance the electrostatic repulsion between amino acids at the "concave-concave” interface. K409A mutation is introduced at the "convex” end to avoid electrostatic repulsion between amino acids at the "concave-concave” interface, thereby inhibiting the formation of "concave-concave” homodimers and maintaining the “convex-concave” heterodimer form. Although the method disclosed in this patent can increase the formation of heterodimers, one of the solutions can only increase the proportion of heterodimers up to 93%.
  • US20150307628A1 discloses another method that uses the "convex-concave” model and electrostatic interaction to promote the formation of heterodimers.
  • one of the schemes created a “convex” on the CH3 domain of the first heavy chain of Fc through K409W point mutations, and created a “convex” on the CH3 of the second heavy chain through F405T and D399V point mutations.
  • the "concave”; the CH3 domain of the first heavy chain was negatively charged by the K360E point mutation, and the CH3 on the second heavy chain was positively charged by the Q347R point mutation.
  • This scheme can increase the proportion of heterodimers up to 91.4 ⁇ 1.2% without introducing disulfide bonds.
  • the purpose of the present invention is to overcome the deficiencies in the prior art, and provide a heterodimer induced by the transformation of the CH3 structure domain, and a preparation method and application thereof.
  • the present invention combines the "convex-concave" model with electrostatic interaction through comprehensive consideration of intermolecular interactions, such as ionic interactions, steric interactions, and hydrophobic interactions, and selects preferred CH3 mutation sequences, which are more likely to form heterologous two.
  • Polymers, without forming homodimers, can increase the proportion of heterodimer molecules to more than 95% without introducing disulfide bonds.
  • the heterodimer in the present invention refers to an antibody molecule or fragment containing two heavy chains with different amino acid sequences, including but not limited to bispecific antibodies, monovalent antibodies, Fc fusion proteins and the like.
  • the first aspect of the present invention is to provide a heterodimer, which contains a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain each contains an antibody heavy chain constant
  • the CH3 domain of the region respectively named CH3_A domain and CH3_B domain.
  • the CH3_A and CH3_B domains Compared with the wild-type human antibody heavy chain constant region CH3 domain, the CH3_A and CH3_B domains contain the following amino acid mutations: CH3_A domain K409 and F405 are mutated, and the CH3_A domain and CH3_B domain are respectively at one or more amino acid positions selected from the group consisting of Q347, Y349, S354, E356, E357, K360, S364, L368, K370, K392, D399 and K439 With mutations.
  • the positions of the above-mentioned amino acids are determined according to the EU index of the KABAT number.
  • the CH3_A structural domain and the CH3_B structural domain have one or more selected from the following mutations:
  • the CH3_A structural domain and the CH3_B structural domain further have the following mutations:
  • the mutation is selected from one or more of the following mutations: Q347R, Y349C, S354C, E356K, E357K, E357S, K360E, S364R, S364K, L368D, K370D, K392D, D399K, K439E, F405E and K409F.
  • Q347R refers to the replacement of glutamine Gln347 with arginine (R).
  • Y349C means that Tyrosine Tyr349 is replaced with Cysteine (C).
  • S354C means that serine Ser354 is replaced with cysteine (C).
  • E356K means that glutamic acid Glu356 is replaced with lysine (K).
  • E357K means that glutamic acid Glu357 is replaced with lysine (K).
  • E357S means that glutamic acid Glu357 is replaced with serine (S).
  • K360E means that lysine Lys360 is replaced with lysine (K).
  • S364R means that serine Ser364 is replaced with arginine (R).
  • S364K means that serine Ser364 is replaced with lysine (K).
  • L368D refers to the replacement of leucine Leu368 with aspartic acid (D).
  • K370D means that lysine Lys370 is replaced with aspartic acid (D).
  • K392D means that lysine Lys392 is replaced with aspartic acid (D).
  • D399K means that Asp399 is replaced with Lysine (K).
  • K439E means that Lys439 is replaced with glutamic acid (E).
  • F405E means phenylalanine Phe405 is replaced with glutamic acid (E).
  • K409F means that lysine Lys409 is replaced with phenylalanine (F).
  • the CH3_A domain and CH3_B domain of the heterodimer contain mutations selected from the following group:
  • CH3_A domain F405E+K409F+K370D
  • CH3_B domain S364R+E357S
  • CH3_A domain F405E+K409F+K370D+S354C
  • CH3_B domain S364R+E357S+Y349C
  • CH3_A domain F405E+K409F+K370D+Y349C
  • CH3_B domain S364R+E357S+ S354C
  • CH3_A domain F405E+K409F+K392D
  • CH3_B domain D399K
  • CH3_A domain F405E+K409F+K392D+S354C
  • CH3_B domain D399K+Y349C
  • CH3_A domain F405E+K409F+K392D+Y349C
  • CH3_B domain D399K+S354C
  • CH3_A domain F405E+K409F+K439D
  • CH3_B domain E356K+E357K
  • CH3_A domain F405E+K409F+K439D+S354C
  • CH3_B domain E356K+E357K+Y349C
  • CH3_A domain F405E+K409F+K439D+Y349C
  • CH3_B domain E356K+E357K+S354C
  • CH3_A domain F405E+K409F+L368D+Y349C
  • CH3_B domain S364R+S354C
  • CH3_A domain F405E+K409F+L368D+S354C
  • CH3_B domain S364K+Y349C
  • CH3_A domain F405E+K409F+L368D+Y349C
  • CH3_B domain S364K+S354C
  • CH3_A domain F405E+K409F+K360E+S354C
  • CH3_B domain Q347R+Y349C
  • CH3_A domain F405E+K409F+K360E+Y349C
  • CH3_B domain Q347R+S354C
  • CH3_A domain F405E+K409F+K370D+K360E
  • CH3_B domain S364R+E357S+ Q347R;
  • CH3_A domain F405E+K409F+K370D+K360E+S354C
  • CH3_B domain S364R+E357S+ Q347R+Y349C;
  • CH3_A domain F405E+K409F+K370D+K360E+Y349C
  • CH3_B domain S364R+E357S+ Q347R+S354C.
  • the antibody constant region is derived from IgG (for example, IgG1, IgG2, IgG3, IgG4), IgA (for example, IgA1, IgA2), IgD, IgE or IgM.
  • IgG for example, IgG1, IgG2, IgG3, IgG4
  • IgA for example, IgA1, IgA2
  • IgD for example, IgE or IgM.
  • the second aspect of the present invention is to provide a composition
  • a composition comprising: (1) the heterodimer according to any one of claims 1-5, and (2) a pharmaceutically acceptable carrier and/ Or diluents and/or excipients.
  • the third aspect of the present invention is to provide a polynucleotide comprising: a nucleotide encoding the first polypeptide chain of the heterodimer according to any one of claims 1-5 Molecule A, and nucleotide molecule B encoding the second polypeptide chain of the heterodimer of any one of claims 1-5;
  • the fourth aspect of the present invention is to provide a vector combination comprising: a recombinant vector A containing the nucleotide molecule A and a recombinant vector B containing the nucleotide molecule B.
  • the expression vectors used in the recombinant vector A and the recombinant vector B are conventional expression vectors in the art, which means that they contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, and enhancers. Expression vectors for sequences, marker genes and/or sequences and other appropriate sequences.
  • the expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid.
  • Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.
  • the fifth aspect of the present invention is to provide a recombinant host cell containing the vector combination.
  • the original host cell of the recombinant host cell of the present invention can be various conventional host cells in the art, as long as it can make the above-mentioned recombinant vector stably replicate by itself, and the nucleotides carried can be effectively expressed. .
  • the original host cell may be a prokaryotic expression cell or a eukaryotic expression cell, and the host cell preferably includes: COS, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3) or TG1, more preferably E.coli TG1, BL21 (DE3) cells (expressing single-chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody).
  • the aforementioned expression vector is transformed into a host cell to obtain the preferred recombinant host cell of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
  • the original host cell is preferably a eukaryotic cell, and more preferably a CHO cell or 293E cell.
  • the sixth aspect of the present invention is to provide the heterodimer according to the first aspect of the present invention, the composition according to the second aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the present invention
  • the seventh aspect of the present invention provides a method for preparing the heterodimer according to the first aspect of the present invention, using the recombinant host cell according to the fifth aspect of the present invention to express the heterodimer .
  • the recombinant host cell contains both a recombinant vector A encoding the first polypeptide chain of the heterodimer and a recombinant vector B encoding the second polypeptide chain of the heterodimer, using the recombinant host The cells are expressed and recovered to obtain heterodimer molecules.
  • the heterodimer can be purified from the recombinant host cell by standard experimental means.
  • protein A can be used for purification. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration, or appropriate combinations of the above methods.
  • the transfection ratio of recombinant vector A and recombinant vector B in the recombinant host cell is 1:3 to 3:1, such as 1:2 to 2:1, such as 1:1.5 to 1.5:1, for example About 1:1.
  • the first polypeptide chain and the second polypeptide chain both contain the CH3 domain of the antibody Fc fragment, and the two polypeptide chains interact through the CH3 domain or the Fc fragment containing CH3 to form a dimer.
  • the two polypeptide chains can be in different combinations.
  • the first polypeptide chain is an antibody
  • the second polypeptide chain is a fusion protein
  • both polypeptide chains are fusion proteins
  • both polypeptide chains are antibodies.
  • the fusion protein contains the Fc portion of the antibody and the extracellular region of the cell adhesion molecule, it is also called an immunoadhesin.
  • the cell adhesion molecules mainly refer to molecules that can recognize specific ligand cell surface receptors, such as cadherins, selectins, immunoglobulin superfamily, integrins, and hyaluronic acid adhesives.
  • the CH3 is derived from an antibody Fc fragment, preferably a human antibody Fc fragment.
  • the CH3 domain of a human antibody Fc fragment is derived from a wild-type human antibody Fc fragment.
  • Wild-type human antibody Fc refers to the amino acid sequence that exists in the human population. Of course, there are some subtle differences in Fc fragments among individuals.
  • the human antibody Fc fragment of the present invention also includes individual amino acid changes to the wild-type human antibody Fc sequence, for example, including certain amino acid mutations at the glycosylation site, or other nonsense mutations.
  • the CH3 and CH2 domains may also contain other mutations that do not affect the function of the antibody, especially the Fc segment.
  • the hinge region is connected as a flexible segment between the two polypeptide segments to ensure the function of each segment of the polypeptide chain;
  • the length of the hinge region can choose the length of the hinge region as required, for example, the full-length sequence or a partial sequence thereof.
  • the numbering of amino acid positions in the Fc or its CH2, CH3 domain or hinge region is based on Kabat The position of the EU number index is determined. Those skilled in the art know that even if the amino acid sequence changes due to the insertion or deletion of amino acids or other mutations in the above-mentioned regions, according to Kabat The position number of each amino acid corresponding to the standard sequence determined by the EU numbering index remains unchanged.
  • the EU index is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991).
  • the present invention combines the "convex-concave” model with electrostatic interaction by comprehensively considering various interactions between interface amino acids, such as ionic interaction, hydrophobic interaction and steric interaction, and screens out the preferred CH3 mutation sequence. It tends to form heterodimers instead of homodimers, thus greatly increasing the yield of heterodimer molecules.
  • the "convex-concave" model construction method involved in the present invention is simpler, has fewer point mutations, and can increase the purity of the heterodimer without introducing disulfide bonds. To more than 95%.
  • Figure 1 is a schematic diagram of the crystal structure of the CH3 domain. The figure shows that K409 of CH3_A is located in the cavity surrounded by F405, D399 and K370 of CH3_B, and forms an ionic interaction with the side chain of D399.
  • Figure 2 is a schematic diagram of heterologous antibodies and homologous antibodies. From left to right are CH3_A/CH3_B heterodimer, CH3_A/CH3_A homodimer and CH3_B/CH3_B homodimer.
  • the square modules represent electrostatic interactions, and the circular modules represent spatial effects. There is repulsion between modules of different shapes. There are mainly electrostatic-hydrophobic repulsion between CH3_A/CH3_A homodimers, and electrostatic repulsion and electrostatic-hydrophobic repulsion exist between CH3_B/CH3_B homodimers.
  • Figure 3 shows the electrophoresis analysis of transiently expressing scFv-Fc/Fc heterodimer. 4-12% SDS-PAGE protein gel electrophoresis. The lanes from left to right are: protein molecular weight standard, combination 1, combination 2, combination 3 and combination 4. The homodimer and heterodimer contained in each group of products have different migration distances in gel electrophoresis due to the difference in molecular weight. The positions of different homodimer or heterodimer proteins are marked in the figure.
  • the homodimer and heterodimer contained in each group of products have different migration distances in gel electrophoresis due to the difference in molecular weight. The positions of different homodimer or heterodimer proteins are marked in the figure.
  • PBS purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117.
  • Citric acid purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Prime star HS DNA polymerase purchased from Takara company, catalog number R010A.
  • Endotoxin-free plasmid large-scale extraction kit purchased from TIANGEN company, item number DP117.
  • HiTrap MabSelectSuRe column purchased from GE Company.
  • AKTA-FPLC fast protein liquid chromatography system purchased from GE Company.
  • Chemidoc MP gel imager purchased from Bio-Rad.
  • G1600AX capillary electrophoresis instrument purchased from Agilent.
  • MicroCal PEAQ-DSC micro calorimeter scanning calorimeter purchased from Malvern Company.
  • IgG1 is preferably used as a template to design amino acid modifications of the CH3 domain, and these amino acid modifications are also applicable to IgG4 subtypes unless otherwise specified.
  • the CH3 domain will form a homodimer.
  • the crystal structure of the CH3 domain (pdb code: 4BSW) of the Fc segment of the IgG1 antibody is shown in Figure 1.
  • K409 of CH3_A is located in the empty space surrounded by F405, D399 and K370 of CH3_B. Inside the cavity, and form an ionic interaction with the side chain of D399.
  • the above-mentioned position is highly conserved on IgG4.
  • the only difference from IgG1 is that the amino acid with EU numbering at position 370 on the heavy chain of IgG4 is Arg.
  • the gene encoding scFv-Fc fusion protein (see SEQ ID: 1 for the sequence of scFv-Fc fusion protein) was artificially synthesized, where scFv refers to an anti-CD3 single-chain antibody. Then subcloned into mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express scFv-Fc fusion protein. The Fc fragment of the above gene (see SEQ ID NO: 2 for the Fc fusion protein sequence) was subcloned into the mammalian cell expression vector pTT5 to obtain a recombinant expression vector for mammalian cells to express the Fc fusion protein.
  • Each mutation combination includes its corresponding A chain (referring to the scFV-Fc fusion protein chain) and B chain (Refers to the Fc protein chain) recombinant expression vectors are co-transfected, and the co-transfection ratio of the A chain and B chain recombinant expression vectors is 1:1.
  • transient products all contain different ratios of homodimer proteins (scFv-Fc/scFv-Fc, Fc/Fc) and heterodimer proteins (scFv-Fc/Fc). Since the molecular weights of these three proteins (scFv-Fc/scFv-Fc, Fc/Fc, and scFv-Fc/Fc) are different, the homodimerization in each group of products can be detected by SDS-PAGE electrophoresis under non-reducing conditions
  • the composition of body protein (scFv-Fc/scFv-Fc, Fc/Fc) and heterodimer protein (scFv-Fc/Fc), the electrophoresis detection result is shown in Figure 3.
  • Combination 1 and combination 2 had significantly more homodimers than combination 3 and combination 4, indicating that the D399A mutation on CH3_B is not conducive to the formation of heterodimers.
  • Combination 4 has fewer homodimers than combination 3, indicating that the F405E mutation is better than F405D.
  • Example 1 by introducing positively charged amino acids into the CH3_B chain, electrostatic repulsion was used to inhibit the formation of homodimers (scFv-Fc/scFv-Fc).
  • scFv-Fc/scFv-Fc homodimers
  • it aims to further explore ways to reduce the mutual attraction between CH3_B chains and inhibit the formation of homodimer proteins.
  • the existing K409F in the CH3_A domain Based on the combination of F405E point mutations, the following charged amino acid mutation combinations were further introduced into the CH3_A domain and CH3_B domain:
  • CH3_A domain K392D
  • CH3_B domain D399K
  • CH3_A domain K439D
  • CH3_B domain E356K+E357K
  • CH3_A domain L368D, CH3_B domain: S364R;
  • CH3_A domain L368D
  • CH3_B domain S364K
  • CH3_A domain K360E
  • CH3_B domain Q347R
  • CH3_A domain K370D+K360E
  • CH3_B domain S364R+E357S+Q347R.
  • the purified protein fraction was analyzed by non-reducing capillary gel electrophoresis, and the percentage of peak area of each product component was calculated. The results are shown in Table 3.
  • Combinations 5-10 can obtain good heterodimer purity. Among them, the purity of the heterodimer obtained by combination 7 and combination 4 is equivalent, and combination 10 is the basis of combination 4. A new charged amino acid mutation was added to the top, and a better purity of the heterodimer was obtained.
  • the cotransformation expression vector used in the better mutation combination 4 was recombined with CH3_A.
  • the combined mutations of HC_A and HC_B encoding genes were carried out by overlap PCR, wherein the mutations for the CH3_A chain were located on the HC_A chain, and the mutations for the CH3_B chain were located on the HC_B chain.
  • the mutated genes were subcloned into pTT5, and finally recombinant expression vectors for expressing the mutations in mammalian cells were obtained.
  • the purified protein fraction was analyzed by LC-MS, and the percentage of each product component was calculated.
  • MicroCal PEAQ-DSC was used to measure the Tm value of the sample. The results are shown in Table 5.
  • Combination 11 was used as a control group, and combinations 12 and 13 introduced disulfide bonds on the basis of combination 11.
  • the obtained heterodimer ratios were 99.56% and 96.89%, which were similar to combination 11;
  • the Tm values of Fc of combination 12 and combination 13 were 69.21°C and 70.23°C, respectively, which was about 3°C higher than that of combination 11.
  • This example found that even though the control group (combination 11) already has a heterodimer ratio of greater than 95%, the introduction of disulfide bonds can still further promote the formation of heterodimers and improve the thermal stability of antibody molecules. Similarly, the introduction of disulfide bonds should also improve the heterodimer ratio and thermal stability of the combination 5-10.

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Abstract

Hétérodimère, son procédé de préparation et son utilisation. Par prise en considération complète de diverses interactions entre les acides aminés interfaciaux, telles qu'une interaction ionique, une interaction hydrophobe et une interaction stérique, un modèle « convexe-concave » est combiné à une interaction électrostatique pour filtrer la séquence de mutation CH3 préférable, ce qui tend à former l'hétérodimère plutôt qu'un homodimère, améliorant ainsi considérablement le rendement de molécules hétérodimères. Par rapport aux documents de référence CN106883297A et US20150307628A1, un procédé de construction du modèle « convexe-concave » est plus simple, des mutations ponctuelles sont moins importantes, et la pureté de l'hétérodimère peut être augmentée jusqu'à 95 % ou plus sans introduire de liaisons disulfure.
PCT/CN2020/140595 2019-12-31 2020-12-29 Hétérodimère induit par modification de domaine structurel ch3, son procédé de préparation et son utilisation Ceased WO2021136227A1 (fr)

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CN201911410315.2 2019-12-31
CN201911410315.2A CN113121697B (zh) 2019-12-31 2019-12-31 Ch3结构域改造诱导形成的异源二聚体及其制备方法和应用

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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025108310A1 (fr) * 2023-11-20 2025-05-30 江苏恒瑞医药股份有限公司 Procédé de préparation d'un multimère hétérologue au moyen d'une réaction de recombinaison

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104955953A (zh) * 2012-11-27 2015-09-30 亚洲大学校产学协力团 高效诱导抗体重链恒定区的异源二聚体形成的ch3域变体对,其制备方法及用途
WO2018148447A1 (fr) * 2017-02-08 2018-08-16 Adimab, Llc Domaines variables de chaînes lourdes d'anticorps ciblant le récepteur nkg2d
WO2018152547A1 (fr) * 2017-02-20 2018-08-23 Adimab, Llc Protéines de liaison à cd123, nkg2d et cd16
CN109790215A (zh) * 2016-09-29 2019-05-21 北京韩美药品有限公司 异源二聚体免疫球蛋白构建体和其制备方法
CN109897103A (zh) * 2011-11-04 2019-06-18 酵活有限公司 在Fc结构域中具有突变的稳定异源二聚的抗体设计
WO2019153200A1 (fr) * 2018-02-08 2019-08-15 北京韩美药品有限公司 Anticorps bispécifique de type structure d'anticorps naturel anti-pd -1/anti-her2 sous forme hétérodimère et sa préparation
WO2019154349A1 (fr) * 2018-02-11 2019-08-15 北京韩美药品有限公司 Anticorps bispécifique à forme hétérodimère de type similaire à une structure d'anticorps naturel anti-pd-1/anti-vegf et préparation associée
CN110573530A (zh) * 2017-02-20 2019-12-13 蜻蜓治疗公司 结合cd33、nkg2d和cd16的蛋白

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2569337A1 (fr) * 2010-05-14 2013-03-20 Rinat Neuroscience Corp. Protéines hétérodimériques et leurs procédés de production et de purification
CN106883297B (zh) * 2015-12-16 2019-12-13 苏州康宁杰瑞生物科技有限公司 基于ch3结构域的异二聚体分子、其制备方法及用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109897103A (zh) * 2011-11-04 2019-06-18 酵活有限公司 在Fc结构域中具有突变的稳定异源二聚的抗体设计
CN104955953A (zh) * 2012-11-27 2015-09-30 亚洲大学校产学协力团 高效诱导抗体重链恒定区的异源二聚体形成的ch3域变体对,其制备方法及用途
CN109790215A (zh) * 2016-09-29 2019-05-21 北京韩美药品有限公司 异源二聚体免疫球蛋白构建体和其制备方法
WO2018148447A1 (fr) * 2017-02-08 2018-08-16 Adimab, Llc Domaines variables de chaînes lourdes d'anticorps ciblant le récepteur nkg2d
WO2018152547A1 (fr) * 2017-02-20 2018-08-23 Adimab, Llc Protéines de liaison à cd123, nkg2d et cd16
CN110573530A (zh) * 2017-02-20 2019-12-13 蜻蜓治疗公司 结合cd33、nkg2d和cd16的蛋白
WO2019153200A1 (fr) * 2018-02-08 2019-08-15 北京韩美药品有限公司 Anticorps bispécifique de type structure d'anticorps naturel anti-pd -1/anti-her2 sous forme hétérodimère et sa préparation
WO2019154349A1 (fr) * 2018-02-11 2019-08-15 北京韩美药品有限公司 Anticorps bispécifique à forme hétérodimère de type similaire à une structure d'anticorps naturel anti-pd-1/anti-vegf et préparation associée

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN SHIXUE, ZHAO LEI;CUI YUEQIAN;JI SHIYU;HU YI: "Knobs-into-holes Structure of Bispecific Antibody based on Molecular Dynamics Simulation", JIEFANGJUN YIXUEYUAN XUEBAO - JOURNAL OF CHINESE PLA POSTGRADUATE MEDICAL SCHOOL, JIEFANGJUN ZONGYIYUAN, CN, vol. 38, no. 12, 1 December 2017 (2017-12-01), CN, pages 1155 - 1173, XP055828251, ISSN: 2095-5227, DOI: 10.3969/j.issn.2095-5227.2017.12.017 *
FANG SHIDI, MAO XIAO-YAN: "Progress on structure and application of bi-specific antibody", PROGRESS IN MICROBIOLOGY AND IMMUNOLOGY = WEI SHENG WU XUE MIAN YI XUE JIN ZHAN, CN, vol. 46, no. 2, 1 April 2018 (2018-04-01), CN, pages 2 - 78, XP055828254, ISSN: 1005-5673, DOI: 10.13309/j.cnki.pmi.2018.02.014 *

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