WO2023274104A1 - Variant de séquence d'extrémité c-terminale d'intéine et son utilisation - Google Patents

Variant de séquence d'extrémité c-terminale d'intéine et son utilisation Download PDF

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WO2023274104A1
WO2023274104A1 PCT/CN2022/101387 CN2022101387W WO2023274104A1 WO 2023274104 A1 WO2023274104 A1 WO 2023274104A1 CN 2022101387 W CN2022101387 W CN 2022101387W WO 2023274104 A1 WO2023274104 A1 WO 2023274104A1
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intein
antibody
terminal sequence
amino acid
sequence variant
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PCT/CN2022/101387
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Chinese (zh)
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张宝红
张敬仪
朱建伟
闵益佳
乔俣
韩雷
丁凯
宗会芳
边延林
江华
谢跃庆
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上海交通大学
美国杰科实验室有限公司
杰科（天津）生物医药有限公司
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Publication of WO2023274104A1 publication Critical patent/WO2023274104A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain

Definitions

  • This application relates to the field of biomedicine, in particular to an intein C-terminal sequence variant and its application.
  • Protein splicing is a post-translational maturation process in which an insertion sequence named "intein” can self-splice and connect the outer proteins on both sides with peptide bonds to form a mature protein.
  • antibody drugs have a certain curative effect and high activity specific targeting effect, the curative effect of antibody drugs on cancer, especially malignant tumors as a single drug is not satisfactory; at the same time, some highly active cytotoxic small molecule chemical drugs have Poor tropism, narrow therapeutic window, and/or large toxic side effects make it impossible to make medicines due to clinical failure.
  • ADC Antibody-drug conjugate
  • Antibody-drug conjugates currently on the market are mainly small molecules randomly coupled to lysine or cysteine of antibodies through covalent bonds.
  • the present application provides an intein C-terminal sequence variant and its application.
  • the intein C-terminal sequences described herein can retain cleavage activity under reducing conditions.
  • the intein C-terminal sequence variant can participate in the preparation of antibody-conjugated drugs together with the corresponding intein N-terminal sequence.
  • site-directed coupling of drugs to antibodies or antigen-binding fragments thereof can be achieved.
  • the intein C-terminal sequence variant can solve the uniformity and/or safety problems of antibody-conjugated drugs due to random conjugation.
  • the antibody-conjugated drug prepared by the intein C-terminal sequence variant compared with the original antibody or its antigen-binding fragment, can have the binding activity to the antigen and the target cell (such as a tumor cell) expressing the antigen.
  • the binding affinity and/or the killing ability of the target cells expressing the antigen are significantly improved.
  • the present application provides an intein C-terminal sequence variant, wherein compared with the amino acid sequence shown in SEQ ID NO.1, the intein C-terminal sequence variant comprises at least one position selected from the following Mutations at amino acid positions of the group: K2, K7, K11 and K28.
  • the intein C-terminal sequence variant comprises mutations at amino acid positions K2, K7, K11 and K28.
  • the K2 contains an amino acid substitution, wherein the amino acid substitution is a positively charged amino acid, or an uncharged but polar amino acid, or an amino acid with a size similar to that of the side group of K.
  • the K7 contains an amino acid substitution, wherein the amino acid substitution is a positively charged amino acid, or an uncharged but polar amino acid, or an amino acid with a size similar to that of the side group of K.
  • the K11 contains an amino acid substitution, wherein the amino acid substitution is a positively charged amino acid, or an uncharged but polar amino acid, or an amino acid with a size similar to that of the side group of K.
  • the K28 contains an amino acid substitution, wherein the amino acid substitution is a positively charged amino acid, or an uncharged but polar amino acid, or an amino acid with a size similar to that of the side group of K.
  • the K2 comprises an amino acid substitution, wherein the amino acid substitution is K2R, K2Q, K2M, K2E or K2G.
  • the K7 comprises an amino acid substitution, wherein the amino acid substitution is K7R, K7Q, K7M, K7E or K7G.
  • the K11 comprises an amino acid substitution, wherein the amino acid substitution is K11R, K11Q, K11M, K11E or K11G.
  • the K28 comprises an amino acid substitution, wherein the amino acid substitution is K28R, K28Q, K28M, K28E or K28G.
  • said intein C-terminal sequence variant comprises two or more of said amino acid substitutions to amino acids of the same type.
  • the intein C-terminal sequence variant comprises the amino acid sequence shown in any one of SEQ ID NO.2-6.
  • the present application provides a conjugate comprising the intein C-terminal sequence variant described in the present application.
  • the conjugates are used to prepare antibody-conjugated drugs.
  • the conjugate comprises an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof comprises a light chain.
  • the N-terminus of the intein N-terminal sequence is directly or indirectly linked to the C-terminus of the light chain.
  • the present application provides an isolated nucleic acid molecule encoding the intein C-terminal sequence variant described herein.
  • the present application provides a method for preparing antibody-conjugated drugs, which includes the following steps:
  • the first molecule comprises the intein C-terminal sequence variant described in this application and the drug, wherein the intein C-terminal sequence is directly or indirectly related to the drug connect.
  • the method comprises the steps of:
  • the second fusion protein comprises the intein N-terminal sequence corresponding to the intein C-terminal sequence variant described in the present application and an antibody or an antigen-binding fragment thereof, wherein the The N-terminal sequence of the intein is directly or indirectly linked to the antibody or its antigen-binding fragment.
  • the method comprises the steps of:
  • the second fusion protein comprises an antibody light chain, and the N-terminus of the intein N-terminal sequence is directly or indirectly linked to the C-terminus of the light chain.
  • the C-terminus of the light chain is directly or indirectly linked to the N-terminus of the drug.
  • said indirect linking comprises linking using a linker.
  • the antibody-drug conjugate specifically binds to a tumor-associated antigen.
  • the present application provides antibody-conjugated drugs prepared according to the method described in the present application.
  • the antibody or antigen-binding fragment thereof is directly or indirectly linked to the drug.
  • the C-terminus of the light chain of the antibody or antigen-binding fragment thereof is indirectly linked to the N-terminus of the drug.
  • said indirect linking comprises linking using a linker.
  • the antibody-drug conjugate specifically binds to a tumor-associated antigen.
  • the drug is site-specifically conjugated to the antibody or antigen binding protein thereof.
  • the antibody-drug conjugate has a drug/antibody ratio (DAR) of 2.
  • the present application provides the application of the intein C-terminal sequence variant described in the present application in the preparation of antibody-conjugated drugs.
  • the present application provides the application of the intein C-terminal sequence variant described in the present application in site-directed conjugation to antibodies or antigen-binding fragments thereof.
  • Figure 1 shows a map of plasmids expressing the second fusion protein described in the present application.
  • 2A-2C show the purification results of the second fusion protein described in the present application.
  • Figure 3A-3B shows the connection of the second fusion protein and his tag described in the present application.
  • Figure 4 shows the mass spectrometric detection results of the first molecule described in the present application.
  • Fig. 5 shows the results of SDS-PAGE electrophoresis detection of antibody-conjugated drugs described in this application.
  • 6A-6B show the results of affinity detection of antibody-drug conjugates described in the present application.
  • Figures 7A-7B show the ability of the antibody-drug conjugates described in this application to inhibit the growth of cells with high expression of HER2.
  • FIG. 8 shows the results of SDS-PAGE electrophoresis detection of the antibody-conjugated drug described in this application.
  • Figure 9 shows the process of using the intein C-terminal sequence variants described in this application to prepare antibody-drug conjugates.
  • intein generally refers to a polypeptide domain capable of self-processing.
  • the intein may be a class of protein capable of being excised from itself by protein splicing and joined to the remainder of the protein.
  • the inteins may include whole inteins and split inteins. Wherein the two splicing regions of the overall intein can co-exist on the same polypeptide fragment.
  • the two splice regions of the split intein can be split into two or more fragments. Wherein the two splicing regions may exist on different polypeptide fragments.
  • the intein N-terminal sequence and the intein C-terminal sequence can be called respectively according to the amino acid sequence of the intein.
  • the N-terminal of the N-terminal sequence of the intein can be connected to the C-terminal of a certain protein; wherein the C-terminal of the C-terminal sequence of the intein can be connected to the N-terminal of another protein.
  • the intein may participate in protein post-translational modification.
  • the intein gene is not an independent gene. It needs to be inserted into the extein gene to replicate and transcribe. It can be excised from the precursor protein and connected with the two exteins to form a mature protein.
  • the nucleotide sequence encoding the intein can be embedded in the corresponding nucleic acid sequence of the host protein, exist in the same open reading frame as the host protein gene, and perform synchronous transcription and translation with the host protein gene. After the protein precursor, the intein is cleaved from the host protein to form a mature active protein.
  • variant generally refers to a protein molecule having sequence homology to a native biologically active protein.
  • Polypeptide variants described herein include polypeptides having altered amino acid sequences by addition (including insertions), deletions, modifications and/or substitutions of one or more amino acid residues while retaining at least one of the therapeutic and and/or biological activity differs from the parental sequence.
  • a variant may have at least 0%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the parent protein.
  • Sequence identity generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical to those in a second reference polypeptide sequence, or a portion thereof, after alignment of the sequences, with gaps (GAPS) introduced if necessary to maximize The percent sequence identity and does not consider any conservative substitutions as part of the sequence identity.
  • Alignment for determining percent amino acid/nucleotide sequence identity can be achieved by various means known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Percent identity may be determined over the entire length of the defined polypeptide/polynucleotide sequence, or may be determined over shorter lengths, such as the length of fragments obtained from a larger defined polypeptide/polynucleotide sequence
  • Variants can be naturally occurring or non-naturally occurring. In this application, non-naturally occurring variants may be generated using techniques known in the art. Polypeptide variants may contain conservative or non-conservative amino acid substitutions, deletions or additions.
  • mutation generally refers to any type of change or modification of a sequence (nucleic acid or amino acid sequence), including deletion, truncation, inactivation, destruction, substitution or translocation of amino acids or nucleotides.
  • amino acid mutation may be amino acid substitution
  • substitution generally refers to replacing at least one amino acid residue in a predetermined parental amino acid sequence with a different "replacement” amino acid residue.
  • the one or more substituted amino acid residues may be "naturally occurring amino acid residues" (ie, encoded by the genetic code).
  • positively charged amino acid generally refers to an amino acid with a positively charged side chain.
  • the positively charged amino acids may include arginine (Arg, R), histidine (His, H) and lysine (Lys, K).
  • uncharged but polar amino acid generally refers to a polar amino acid with no charge in the side chain.
  • the uncharged but polar amino acids may include serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), asparagine (Asn, N), glutamine (Gln, E), cysteine (Cys, C) and glycine (Gly, G).
  • an amino acid having a side group similar in size to K generally refers to an amino acid having a side chain whose size is similar to that of lysine.
  • the amino acid with a side group size similar to that of K may include glutamine (Gln, Q) and/or methionine (Met, M).
  • antibody-drug conjugate generally refers to a binding protein (such as an antibody or antigen-binding fragment thereof) linked to one or more chemical drugs.
  • the chemical drug can be any therapeutic and/or cytotoxic drug.
  • the antibody-conjugated drug can have any number of drugs conjugated to the antibody from 1-8, for example, can include 2, 4, 6 or 8 drug loaded species.
  • the drugs may include mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, Hormones, antihormones, corticosteroids, phototherapeutics, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and/or radiosensitizers.
  • the antibody-conjugated drugs of the present application may include but not limited to antibody-conjugated drugs, antibody-conjugated supramolecular nanofibers (such as photothermal therapy ICG, etc.), antibody-coupled protein degradation agents (such as PROTAC molecules).
  • antibody-conjugated drugs such as photothermal therapy ICG, etc.
  • antibody-coupled protein degradation agents such as PROTAC molecules.
  • antibody generally refers to an immunoglobulin molecule composed of two identical pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.Mol.Biol.196:901-917 ; Definition by Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the term "antigen-binding fragment” generally refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (e.g., HER2) to which the antibody binds, competing with the intact antibody for binding to the antigen. specific binding. See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. or by enzymatic or chemical cleavage of intact antibodies to produce antigen-binding portions.
  • antigen e.g., HER2
  • antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, Single-chain antibodies (for example, scFv), chimeric antibodies, diabodies (diabodies) and such polypeptides, which comprise at least a portion of an antibody sufficient to confer polypeptide-specific antigen-binding ability.
  • CDR complementarity determining region
  • an antigen-binding portion of an antibody e.g., the antibody fragments described above
  • a given antibody e.g., monoclonal antibody 2E12
  • the method is to screen the antigen-binding portion of the antibody for specificity.
  • the term “drug/antibody ratio” generally refers to the number of drugs linked to the antibody of the ADC.
  • the DAR of an ADC can range from 1 to 8, or higher loadings (eg, 10), and the range of DAR can depend on the number of attachment sites on the antibody.
  • the DAR can be the number of drugs loaded on a single antibody.
  • the DAR can also be the average or average DAR of a group of ADCs.
  • linker generally refers to a chemical moiety for making a link.
  • the linker may comprise one coupling component, or may comprise multiple coupling components.
  • the linker can be a chemical moiety inserted between a first molecule (eg, the drug) and a second molecule (eg, the antibody or antigen-binding fragment thereof) for linking.
  • the linker may be a peptide linker.
  • the linker may not be immunogenic, and/or, not elicit an immune response.
  • the peptide linker can be a flexible polypeptide linker.
  • the linker may comprise a functional group that can covalently join two or more moieties.
  • tumor generally refers to the physiological condition in mammals that is often characterized by unregulated cell growth.
  • the tumor includes one or more cancerous cells.
  • the tumors may include solid tumors and/or non-solid tumors.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an intein C-terminal sequence variant, wherein compared with the amino acid sequence shown in SEQ ID NO.1, the intein C-terminal sequence variant comprises at least one position selected from the following Mutations at amino acid positions of the group: K2, K7, K11 and K28.
  • the intein can undergo self-splicing, so that proteins on both sides can be connected by peptide bonds to form a mature protein.
  • the gene encoding the intein can be broken at a specific site into an N-terminal fragment (for example, it can be called an intein N -terminal sequence, or IN for short) and a C-terminal fragment (for example, it can be called Intein C -terminal sequence, referred to as IC).
  • the intein N-terminal sequence and the intein C-terminal sequence may not be active when present alone.
  • the two can recognize each other (for example, by rebuilding the catalytic active center), and can mediate trans-cleavage of the protein.
  • the intein C-terminal sequence variant described in the present application may be a variant obtained on the basis of Npu DnaE intein.
  • the intein C-terminal sequence may comprise at least one (eg 1, 2, 3 or 4) mutation at an amino acid position selected from the group consisting of K2, K7, K11 and K28.
  • the amino acid position may be the amino acid position in the amino acid sequence shown in SEQ ID NO.1.
  • SEQ ID NO.1 starting from the N-terminus, the amino acid at the 2nd position is K, the amino acid at the 7th position is K, the amino acid at the 11th position is K, and/or the amino acid at the 28th position is K.
  • the intein C-terminal sequence variant may comprise mutations at amino acid positions K2, K7, K11 and K28.
  • the K2 may contain amino acid substitutions, wherein the amino acid substitutions may be positively charged amino acids, or uncharged but polar amino acids, or amino acids with a size similar to that of the side group of K.
  • the K7 may contain an amino acid substitution, wherein the amino acid substitution may be a positively charged amino acid, or an uncharged but polar amino acid, or an amino acid with a size similar to that of the side group of K.
  • the K11 may contain an amino acid substitution, wherein the amino acid substitution may be a positively charged amino acid, or an uncharged but polar amino acid, or an amino acid with a size similar to that of the side group of K.
  • the K28 may contain an amino acid substitution, wherein the amino acid substitution may be a positively charged amino acid, or an uncharged but polar amino acid, or an amino acid with a size similar to that of the side group of K.
  • the positively charged amino acid may include R, H and/or K.
  • the uncharged but polar amino acid may include S, T, Y, N, E, C and/or G.
  • the amino acids with a side group size similar to that of K may include Q and/or M.
  • the K2 may contain an amino acid substitution, wherein the amino acid substitution may be K2R, K2Q, K2M, K2E or K2G.
  • the K7 may contain an amino acid substitution, wherein the amino acid substitution may be K7R, K7Q, K7M, K7E or K7G.
  • the K11 may contain an amino acid substitution, wherein the amino acid substitution may be K11R, K11Q, K11M, K11E or K11G.
  • the K28 may contain an amino acid substitution, wherein the amino acid substitution may be K28R, K28Q, K28M, K28E or K28G.
  • the K2 may contain an amino acid substitution, wherein the amino acid substitution may be K2R, K2Q, or K2M.
  • the K7 may contain an amino acid substitution, wherein the amino acid substitution may be K7R, K7Q, or K7M.
  • the K11 may contain an amino acid substitution, wherein the amino acid substitution may be K11R, K11Q, or K11M.
  • the K28 may contain an amino acid substitution, wherein the amino acid substitution may be K28R, K28Q, or K28M.
  • said intein C-terminal sequence variant may comprise two or more (for example, may be 2, 3 or 4) substituted with the same type of amino acid Amino acid substitutions.
  • at least 2 (eg, may be 2, 3 or 4) amino acid substitutions may be all substituted as E, G, Q, M or R.
  • at least 2 (eg, may be 2, 3 or 4) amino acid substitutions may be all substituted as Q, M or R.
  • the intein C-terminal sequence variant may comprise the amino acid sequence shown in any one of SEQ ID NO.2-6.
  • the intein C-terminal sequence variant may comprise the amino acid sequence shown in any one of SEQ ID NO.4-6.
  • the present application provides a conjugate comprising the intein C-terminal sequence variant described in the present application.
  • the C-terminus of the intein C-terminal sequence variant may be directly or indirectly linked to a drug, thereby obtaining a first molecule comprising the intein C-terminal sequence variant and the drug.
  • the drug can be used to treat tumors.
  • the drug can be a small molecule drug.
  • the drugs may include chemotherapy drugs.
  • the N-terminus of the intein N-terminal sequence corresponding to the intein C-terminal sequence variant may be directly or indirectly linked to the antibody or its antigen-binding fragment (for example, may be linked to the antibody or its antigen-binding fragment). directly or indirectly connected to the C-terminus), thereby obtaining a second fusion protein comprising the N-terminal sequence of the intein and the antibody or antigen-binding fragment thereof.
  • the N-terminus of the intein N-terminal sequence may be directly or indirectly linked to the light chain of the antibody or antigen-binding fragment thereof (e.g., the C-terminus of the light chain).
  • the light chain can be covalently linked to the heavy chain.
  • the second fusion protein can comprise a complete antibody and the intein N-terminal sequence.
  • the N-terminus of the intein N-terminal sequence may be directly or indirectly linked to the C-terminus of the light chain in the intact antibody.
  • said indirect linking can include linking through a linker.
  • the antibody or antigen-binding fragment thereof can specifically bind to a tumor-associated antigen.
  • the antibody or antigen-binding fragment thereof can specifically bind tumor cells expressing the tumor-associated antigen.
  • the antibody or antigen-binding fragment thereof can specifically kill tumor cells expressing the tumor-associated antigen.
  • the tumor-associated antigen can include HER2.
  • the intein C-terminal sequence variant in the first molecule when the first molecule is in contact with the second fusion protein, the intein C-terminal sequence variant in the first molecule can be combined with the intein C-terminal sequence variant in the second fusion protein.
  • the N-terminal sequence of the intein undergoes trans-splicing.
  • an antibody-conjugated drug comprising the drug and the antibody or antigen-binding protein thereof can be obtained through the trans-splicing.
  • the conjugate can be used to prepare antibody-conjugated drugs.
  • the antibody-conjugated drugs of the present application may include antibody-conjugated drugs, antibody-coupled supramolecular nanofibers (such as photothermal therapy ICG, etc.), antibody-coupled protein degradation agents (such as PROTAC molecules)
  • the conjugate may comprise an antibody or an antigen-binding fragment thereof.
  • the present application provides an isolated nucleic acid molecule encoding the intein C-terminal sequence variant described herein.
  • the nucleic acid molecule described in the present application can adjust its nucleotide sequence according to the codon degeneracy and/or the expression preference of the host cell, as long as it can encode the intein C-terminal sequence Variants.
  • the nucleic acid molecule can be contained in a plasmid to facilitate transfection of cells and expression of the intein C-terminal sequence variant.
  • the nucleic acid molecule may further comprise a nucleic acid sequence that may encode the first molecule described herein.
  • the nucleic acid molecule may further comprise a nucleic acid sequence that may encode the second fusion protein described herein.
  • a plasmid comprising the nucleic acid sequence encoding the first molecule can be co-transfected with a plasmid comprising the nucleic acid sequence encoding the second fusion protein.
  • the nucleic acid molecule can be located on the same plasmid as the nucleic acid sequence encoding the second fusion protein.
  • the present application provides a method for preparing antibody-conjugated drugs, which includes the following steps:
  • the first molecule comprises the intein C-terminal sequence variant described in this application and the drug, wherein the intein C-terminal sequence is directly or indirectly related to the drug connect.
  • the method may include the following steps:
  • the second fusion protein comprises the intein N-terminal sequence corresponding to the intein C-terminal sequence variant described in the present application and an antibody or an antigen-binding fragment thereof, wherein the The N-terminal sequence of the intein is directly or indirectly linked to the antibody or its antigen-binding fragment.
  • the method may include the following steps:
  • the second fusion protein may include an antibody light chain, and the N-terminal of the intein N-terminal sequence may be directly or indirectly linked to the C-terminal of the light chain.
  • the C-terminal of the light chain may be directly or indirectly linked to the N-terminal of the drug.
  • the indirect connection may include connection using a linker.
  • the antibody-conjugated drugs can specifically bind to tumor-associated antigens.
  • the present application provides antibody-conjugated drugs prepared according to the method described in the present application.
  • antibodies or antigen-binding fragments thereof may be directly or indirectly linked to drugs.
  • the C-terminal of the light chain of the antibody or antigen-binding fragment thereof may be indirectly linked to the N-terminal of the drug.
  • the indirect connection includes connection using a linker.
  • the antibody-conjugated drugs can specifically bind to tumor-associated antigens.
  • the drug can be coupled to the antibody or its antigen-binding protein in a specific manner.
  • the drug can be site-directedly coupled to the C-terminus of the light chain of the antibody.
  • the drug/antibody ratio (DAR) of the antibody-conjugated drug can be 2. That is, in the antibody-conjugated drugs, two drugs can be site-specifically coupled to each antibody or antigen-binding fragment thereof (for example, they can be site-specifically coupled to the C-terminals of the two light chains of the antibody).
  • the present application provides the application of the intein C-terminal sequence variant described in the present application in the preparation of antibody-conjugated drugs.
  • the present application provides the application of the intein C-terminal sequence variant described in the present application in site-directed conjugation to antibodies or antigen-binding fragments thereof.
  • the intein C-terminal sequence variant described in the present application can be used to conjugate the drug at the specified position of the antibody or its antigen-binding fragment. Therefore, problems such as inconsistencies and safety defects caused by random coupling of drugs in the preparation of the antibody-coupled drugs can be solved.
  • the antibody-conjugated drug prepared by using the intein C-terminal sequence variant described in this application compared with the original antibody or its antigen-binding fragment, can produce a synergistic effect with the drug.
  • the ability to kill tumor cells is significantly improved; the ability to specifically bind to tumor cells expressing tumor-associated antigens can be maintained; and/or the ability to bind to tumor cells expressing tumor-associated antigens with high affinity can be maintained.
  • the intein C-terminal sequence variants described in this application have a wide range of application scenarios, and can be used for coupling various proteins, or proteins and other molecules.
  • said intein C-terminal sequence variant comprises at least one mutation at an amino acid position selected from the group : K2, K7, K11 and K28.
  • intein C-terminal sequence variant according to technical scheme 1, wherein compared with the amino acid sequence shown in SEQ ID NO.1, the intein C-terminal sequence variant comprises K2, K7, K11 and Mutation at amino acid position K28.
  • intein C-terminal sequence variant according to any one of technical schemes 3-10, which comprises two or more amino acid substitutions that are substituted with amino acids of the same type.
  • a conjugate comprising the intein C-terminal sequence variant described in any one of technical schemes 1-12.
  • conjugate according to technical scheme 13 which is used for preparing a site-specific conjugated antibody-conjugated drug (ADC).
  • a method for preparing a site-specific conjugated antibody-conjugated drug comprising the following steps:
  • the first molecule comprises the intein C-terminal sequence variant described in any one of technical schemes 1-12 and the drug, wherein the intein C-terminal sequence directly or indirectly linked to the drug.
  • the second fusion protein comprises the intein N-terminal sequence corresponding to the intein C-terminal sequence variant described in any one of technical schemes 1-12 and the antibody or its Antigen-binding fragment, wherein the intein N-terminal sequence is directly or indirectly linked to the antibody or its antigen-binding fragment.
  • the antibody-conjugated drug according to any one of technical schemes 26-29, which specifically binds to a tumor-associated antigen.
  • Embodiment 1 obtains the second fusion protein
  • the nucleic acid molecule encoding the N-terminal sequence of the intein is linked to the light chain of the encoding Herceptin
  • the 3' end of the nucleic acid molecules and insert these nucleic acid molecules into the pM09 plasmid to obtain the plasmid pM09-Her2-Lc-intein N capable of expressing the protein of interest. See Figure 1 for the plasmid map of plasmid Her2-Lc.
  • another nucleic acid molecule plasmid pM09-Her2-Hc encoding the heavy chain sequence of Herceptin was constructed.
  • FIG. 2A-2C the purification results of the second fusion protein are shown in Fig. 2A-2C.
  • Figure 2A shows the results of SDS-PAGE electrophoresis detection of the second fusion protein, numbers 2, 4, and 5 respectively represent the first 400 ⁇ L and last 400 ⁇ L of samples collected after purification and eluted with sodium hydroxide.
  • Control represents the sample before purification;
  • FT represents the flow-through sample; wherein, the size of the light chain of Herceptin is about 34kD; the size of the heavy chain of Herceptin is about 55kD.
  • Figure 2B shows the results of the OD 280 detection of the flow-through components during the purification process;
  • Figure 2C shows the enlarged schematic diagram of the elution peak in Figure 2B.
  • the wild-type intein C-terminal sequence (its amino acid sequence is shown in SEQ ID NO.1) and the C-terminal of the intein C-terminal sequence variant R (its amino acid sequence is shown in SEQ ID NO.6), respectively
  • the his tag is connected to obtain the wild-type intein C-terminal sequence connected to the his tag and the variant R of the intein C-terminal sequence connected to the his tag.
  • 1-5 are respectively: 1: the reaction result of the second fusion protein and the intein C-terminal sequence variant R connected to his tag; 2: the reaction result of the second fusion protein and the wild-type intein connected to his tag The reaction result of the peptide C-terminal sequence; 3: the detection result of the second fusion protein; 4: the detection result of the intein C-terminal sequence variant R connected to his tag; 5: the wild-type protein connected to his tag The detection results of the intein C-terminal sequence of the type.
  • the arrows in Figure 3A (incubated with anti-his-HRP secondary antibody and developed color) show from top to bottom that splicing has successfully dropped the light chain at the N-terminal of the intein and connected to the his tag, and the intein connected to the his tag
  • the band of the C-terminal sequence of the peptide; the arrows in Figure 3B (incubated with anti-HcLc-HRP secondary antibody and developed color) show the heavy chain of the antibody in sequence from top to bottom, the light chain connected to the N-terminal of the intein and the splicing were successfully dropped
  • the N-terminus of intein is connected to the light chain of his tag.
  • Embodiment 2 obtains the first molecule
  • the intein C-terminal sequence variant R (its amino acid sequence is shown in SEQ ID NO.6) was chemically modified with a linker and a small molecule drug (SMCC-DM1) (its CAS number is 1228105-51-8), The first molecule SMCC-DM1-R in which SMCC-DM1 is linked to the C-terminus of the intein C-terminal sequence variant R is obtained.
  • SMCC-DM1 small molecule drug
  • the molecular weight of the first molecule SMCC-DM1-R was detected by mass spectrometry, and the results are shown in FIG. 4 .
  • the molecular weight of the first molecule SMCC-DM1-R is 5686.97.
  • Figure 5 shows the results of SDS-PAGE electrophoresis detection of antibody-conjugated drugs.
  • the "flow-through" bands correspond to intein fragments after the reaction.
  • SKOV3 and SKBR3 cells were plated one day before the experiment, and the antibody-conjugated drugs prepared in Example 3 were administered at various concentration gradients for 5 days before detection, and the results are shown in Figures 7A-7B.
  • 7A-7B respectively show the effects of the antibody-drug conjugate prepared in Example 3 and the control antibody Herceptin on the cell viability of SKOV3 and SKBR3 cells.
  • the IC50 of the antibody-drug conjugate prepared in Example 3 and the control antibody Herceptin were 2.8nM and N.A.
  • Figure 7B the IC50 of the antibody-drug conjugate prepared in Example 3 and the control antibody Herceptin were 0.3205nM and 0.4896nM.
  • the intein C-terminal sequence variant E (whose amino acid sequence is shown in SEQ ID NO.2) is chemically modified with a linker and a small molecule drug (SMCC-DM1) (its CAS number is 1228105-51-8),
  • SMCC-DM1 small molecule drug
  • the intein C-terminal sequence variant G (its amino acid sequence is shown in SEQ ID NO.3) was chemically modified with a linker and a small molecule drug (SMCC-DM1) (its CAS number is 1228105-51-8), The first molecule SMCC-DM1-G in which SMCC-DM1 is linked to the C-terminus of the intein C-terminal sequence variant G is obtained.
  • SMCC-DM1 small molecule drug
  • the intein C-terminal sequence variant Q (its amino acid sequence is shown in SEQ ID NO.4) was chemically modified with a linker and a small molecule drug (SMCC-DM1) (its CAS number is 1228105-51-8), The first molecule SMCC-DM1-Q in which SMCC-DM1 is linked to the C-terminus of the intein C-terminal sequence variant Q is obtained.
  • SMCC-DM1 small molecule drug
  • the intein C-terminal sequence variant M (whose amino acid sequence is shown in SEQ ID NO.5) is chemically modified with a linker and a small molecule drug (SMCC-DM1) (its CAS number is 1228105-51-8),
  • SMCC-DM1 small molecule drug
  • the second fusion protein prepared in Example 1 and the first molecule SMCC-DM1-E, SMCC-DM1-G, SMCC-DM1-Q or SMCC-DM1-M were sheared
  • Antibody-conjugated drug-E, antibody-conjugated drug-G, antibody-conjugated drug-Q and antibody-conjugated drug-M were obtained respectively.
  • Figure 8 shows the results of SDS-PAGE electrophoresis detection of the above antibody-conjugated drugs.
  • 1-6 represent the antibody-conjugated drug prepared in Example 3, antibody-conjugated drug-Q, antibody-conjugated drug-G, antibody-conjugated drug-E, antibody-conjugated drug-M and the first antibody-conjugated drug prepared in Example 1.
  • Two fusion proteins wherein, Hc represents the heavy chain of the antibody; Lc represents the light chain of the antibody (about 26kD in size); Lc-IN represents the molecule comprising the N -terminal sequence of the intein and the light chain of Herceptin (about 37kD in size).

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Abstract

L'invention concerne un variant de séquence d'extrémité C-terminale d'intéine, par comparaison avec une séquence d'acides aminés telle que représentée dans SEQ ID NO. 1, le variant de séquence d'extrémité C-terminale d'intéine contient au moins une mutation positionnée à des positions d'acides aminés choisies dans le groupe constitué par K2, K7, K11 et K28. Le variant de séquence d'extrémité C-terminale d'intéine peut être utilisé pour participer à la préparation de médicaments conjugués à des anticorps, comprenant des conjugués anticorps-médicament, des conjugués anticorps-nanofibres supramoléculaires (tels qu'une thérapie photothermique ICG, etc.) et des conjugués anticorps-agent de dégradation de protéines (tels que des molécules PROTAC), de telle sorte que les médicaments sont soumis à une conjugaison spécifique à un site. L'invention concerne en outre un procédé de préparation d'un conjugué à l'aide du variant de séquence d'extrémité C-terminale d'intéine.
PCT/CN2022/101387 2021-06-28 2022-06-27 Variant de séquence d'extrémité c-terminale d'intéine et son utilisation WO2023274104A1 (fr)

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Citations (4)

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CN101899489A (zh) * 2009-05-27 2010-12-01 南京大学 利用内含肽反式剪接模式化生产融合蛋白质
CN102373234A (zh) * 2011-08-17 2012-03-14 华东理工大学 一种内含肽介导的类弹性蛋白纯化重组蛋白的方法
CN105925596A (zh) * 2016-02-23 2016-09-07 上海交通大学 基于内含肽的药用重组蛋白的合成方法
EP3421500A1 (fr) * 2016-02-23 2019-01-02 Shanghai Jiao Tong University Procédé d'expression et de préparation d'un anticorps polyvalent multi-spécifique et d'une protéine hybride immunitaire

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CN101899489A (zh) * 2009-05-27 2010-12-01 南京大学 利用内含肽反式剪接模式化生产融合蛋白质
CN102373234A (zh) * 2011-08-17 2012-03-14 华东理工大学 一种内含肽介导的类弹性蛋白纯化重组蛋白的方法
CN105925596A (zh) * 2016-02-23 2016-09-07 上海交通大学 基于内含肽的药用重组蛋白的合成方法
EP3421500A1 (fr) * 2016-02-23 2019-01-02 Shanghai Jiao Tong University Procédé d'expression et de préparation d'un anticorps polyvalent multi-spécifique et d'une protéine hybride immunitaire

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