WO2024055988A1 - 免疫球蛋白结合蛋白及其应用 - Google Patents

免疫球蛋白结合蛋白及其应用 Download PDF

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WO2024055988A1
WO2024055988A1 PCT/CN2023/118531 CN2023118531W WO2024055988A1 WO 2024055988 A1 WO2024055988 A1 WO 2024055988A1 CN 2023118531 W CN2023118531 W CN 2023118531W WO 2024055988 A1 WO2024055988 A1 WO 2024055988A1
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domain
igg
protein
amino acid
recombinant protein
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PCT/CN2023/118531
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French (fr)
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王超
廖华媛
柏琦
高伟
丁燕
江佩娟
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普米斯生物技术(珠海)有限公司
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • 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
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi

Definitions

  • the present invention relates to the field of protein purification. Specifically, the present invention provides an isolated polypeptide composed of a mutated B domain of Protein A, an immunoglobulin-binding protein comprising the mutated B domain, and an immunoglobulin-binding protein comprising the mutated B domain. Conjugates of B domains or non-natural immunoglobulin binding proteins, and related uses.
  • Staphylococcus aureus protein A Staphylococcus aureus protein A
  • Staphylococcus aureus protein A contains 5 highly homologous single domains, starting from the N-terminus, they are E, D, and A. , B and C, each single domain contains approximately 58 amino acid residues, and each domain forms a three-stranded anti-parallel ⁇ -helical spatial structure.
  • Each of the above single domains can specifically bind to the Fc (fragment crystallizable, crystallizable segment) segment and Fab (fragment of antigen binding, antigen-binding fragment) segment of the antibody IgG (Immunoglobulin G, immunoglobulin G) molecule, among which and
  • the Fc segment binding region is located at the junction of the second and third constant regions (CH2 and CH3), and the Fab segment binding region is located in the variable region of the heavy chain (VH, limited to the VH3 gene family).
  • recombinant Protein A As a ligand material for the purification of antibody molecules, recombinant Protein A has the following main advantages: 1) low expression cost and can be expressed in the E.coli prokaryotic system for large-scale expression; 2) strong specificity, and it has strong specificity for human IgG1 and IgG2 , IgG4 and mouse IgG2 all have strong affinity properties; 3) Good regeneration properties and can be repeatedly regenerated and recycled under alkaline conditions.
  • the immunoglobulin-binding protein comprising the mutated B domain of Protein A provided by the present invention has high binding activity to the Fc domain (for example, IgG Fc domain) and at the same time eliminates the interaction with the antibody heavy chain variable region.
  • Binding activity which can provide milder elution conditions during the purification process of bispecific or multispecific antibodies (for example, bispecific or multispecific antibodies containing IgG Fc domain), providing effective guarantee for sample quality , and reduce the steps and costs of later fine purification, thus effectively solving the harsh elution conditions (for example, lower pH value) faced when using traditional Protein A to purify bispecific or multispecific antibodies, and the quality of the antibody is greatly affected. issues such as large impacts.
  • the application provides an isolated polypeptide consisting of a mutated B domain of Protein A or its Variant composition; wherein, compared with the B domain of wild-type Protein A, the mutated B domain includes one or more (for example, 1, 2, 3) selected from N23T, D36V, D37R, Q40V or 4) amino acid substitution;
  • the variant has at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity compared to the mutated B domain; or, has Substitution (preferably conservative substitution), addition or deletion of one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acids, and retains the function of the mutated B domain; and;
  • the variant and mutated B domains are at positions corresponding to positions 1, 23, 29, 36, 37 and 40 of the B domain of wild-type Protein A.
  • the amino acid residues are the same.
  • the mutated B domain further comprises amino acid substitutions compared to the B domain of wild-type Protein A: (i) G29A, and/or, (ii) A1V.
  • the mutated B domain comprises amino acid substitutions compared to the B domain of wild-type Protein A: (a) N23T, G29A, D36V, D37R and Q40V, or, (b) A1V, N23T , D36V, D37R and Q40V.
  • the mutated B domain comprises amino acid substitutions compared to the B domain of wild-type Protein A: A1V, N23T, G29A, D36V, D37R, and Q40V.
  • the mutated B domain further comprises the amino acid substitution K58A compared to the B domain of wild-type Protein A.
  • the mutated B domain comprises amino acid substitutions compared to the B domain of wild-type Protein A: N23T, D36V, D37R, Q40V and K58A.
  • the mutated B domain comprises amino acid substitutions compared to the B domain of wild-type Protein A: (a) N23T, G29A, D36V, D37R, Q40V and K58A, or, (b) A1V , N23T, D36V, D37R, Q40V and K58A.
  • the mutated B domain comprises amino acid substitutions compared to the B domain of wild-type Protein A: A1V, N23T, G29A, D36V, D37R, Q40V and K58A.
  • the B domain of wild-type Protein A has the sequence set forth in SEQ ID NO: 4.
  • the mutated B domain has the amino acid sequence set forth in SEQ ID NO: 5 or 17.
  • the sequence shown here does not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus.
  • a start codon such as ATG
  • the mutated B domain of the present invention not only includes amino acid sequences that do not include the amino acid encoded by the start codon (such as Met) at its N terminus, but also includes amino acids encoded by the start codon (such as Met) at its N terminus. Amino acid sequence.
  • the N-terminus of the above-mentioned amino acid sequence further includes the amino acid encoded by the start codon (such as Met) are also within the protection scope of the present invention.
  • the isolated polypeptide specifically binds to an IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4); and/or, the isolated polypeptide The polypeptide dissociates from the IgG Fc domain in the range of pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).
  • pH 7.0-8.0 e.g., pH 7.0-7.6, pH 7.0-7.4
  • the polypeptide dissociates from the IgG Fc domain in the range of pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).
  • the present application also provides recombinant proteins comprising isolated polypeptides as described above.
  • the recombinant protein does not comprise the amino acid sequence of the B domain of wild-type Protein A.
  • the recombinant protein does not comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the recombinant protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) isolated polypeptides as described above . In certain embodiments, the recombinant protein comprises 3, 4, 5, or 6 isolated polypeptides as described above.
  • the recombinant protein contains multiple (e.g., 2, 3, 4, 5, 6, 7, or 8) isolated polypeptides as described above, and, Multiple isolated polypeptides are distinct, partially identical, or identical.
  • each of the isolated polypeptides is linked to each other, optionally by the same or different peptide linkers.
  • the peptide linkers each independently have the amino acid sequence set forth in SEQ ID NO: 6 or 18.
  • the recombinant protein further comprises a cysteine at its N-terminus or C-terminus. In certain embodiments, the recombinant protein further comprises a cysteine at its C-terminus. In certain embodiments, the cysteine is optionally linked to the isolated polypeptide through a linker (eg, a flexible peptide containing one or more glycines (G), such as GGGG).
  • a linker eg, a flexible peptide containing one or more glycines (G), such as GGGG).
  • the recombinant protein comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 2, 7, 11, 13, 15, 19-21.
  • the sequence shown here does not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus.
  • start codon such as ATG
  • the recombinant protein of the present invention not only includes amino acid sequences that do not include the amino acid encoded by the start codon (such as Met) at its N terminus, but also includes amino acid sequences that include the amino acid encoded by the start codon (such as Met) at its N terminus. Therefore, sequences that further include an amino acid (such as Met) encoded by a start codon at the N-terminus of the above amino acid sequence are also within the scope of the present invention.
  • the recombinant protein is further linked to an additional polypeptide selected from a tag, a restriction site, a signal or guide peptide, a detectable label, or any combination thereof.
  • protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how to use them according to the desired purpose (e.g., purification , Detection or tracing) Select appropriate protein tags.
  • the recombinant protein specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4).
  • the recombinant protein dissociates from the IgG Fc domain within the range of pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3).
  • the application also provides dimers or multimers comprising two or more (for example, 2, 3, 4, 5, 6, 7 or 8) as above The isolated polypeptide.
  • the dimer or multimer contains 3, 4, 5, or 6 isolated polypeptides as described above.
  • the two or more (e.g., 2, 3, 4, 5, 6, 7, or 8) isolated polypeptides are each different, partially the same, or completely identical .
  • the dimer or multimer does not comprise the amino acid sequence of the B domain of wild-type Protein A.
  • the dimer or multimer does not comprise the amino acid sequence set forth in SEQ ID NO:4.
  • the dimer or multimer comprises at least two polypeptide chains, and the two or more separate polypeptides are present in at least two polypeptide chains.
  • the isolated polypeptides are reversibly or irreversibly linked to each other through linkers to form dimers or multimers.
  • At least one polypeptide chain in the dimer or multimer further comprises a cysteine at its N-terminus or C-terminus.
  • the polypeptide chain further comprises a cysteine at its C-terminus.
  • the cysteine is optionally linked to the isolated polypeptide through a linker (eg, a flexible peptide containing one or more glycines (G), such as GGGG).
  • a linker eg, a flexible peptide containing one or more glycines (G), such as GGGG).
  • At least one polypeptide chain in the dimer or multimer is also connected to another polypeptide, and the other polypeptide is selected from the group consisting of tags, enzyme cleavage sites, signal peptides or guide peptides, and detectable labels. , or any combination thereof.
  • protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how to use them according to the desired purpose (e.g., purification , detection or tracing) to select the appropriate protein tag.
  • the dimer or multimer specifically binds to the IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4).
  • the dimer or multimer dissolves with an IgG Fc domain in the range of pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3). Leave.
  • pH 2.7-4.5 e.g., pH 2.7-4.0, pH 3.5-4.5, or pH 3.8-4.3.
  • the present application also provides a conjugate comprising a solid support and an isolated polypeptide, a recombinant protein, and/or a dimer or polypeptide as described above connected to the solid support. aggregate.
  • the isolated polypeptide, recombinant protein, or dimer or multimer is directly or indirectly, reversibly or irreversibly linked to a solid support.
  • the isolated polypeptide, recombinant protein, or dimer or multimer is linked to the solid support via covalent and/or non-covalent bonds.
  • the solid supports include those based on synthetic polymers, for example, polyvinyl ether, polyvinyl alcohol, polymethacrylate, polyacrylate, polystyrene, poly Acrylamide, polymethacrylamide and polycarbonate.
  • synthetic polymers for example, polyvinyl ether, polyvinyl alcohol, polymethacrylate, polyacrylate, polystyrene, poly Acrylamide, polymethacrylamide and polycarbonate.
  • the form of the solid support includes, but is not limited to, beads (spherical or irregular shapes), hollow fibers, solid fibers, pads, gels, membranes, boxes, columns, chips, slides , slab or integral column.
  • the solid support is selected from agarose microparticles, high molecular polymer microspheres, silica gel microspheres or magnetic beads.
  • Any suitable technology can be used to connect the polypeptide, recombinant protein or dimer or multimer of separation described herein to a solid support.
  • it can be connected to a solid support by conventional coupling techniques utilizing amino and/or carboxyl groups present in, for example, the polypeptide, recombinant protein or dimer or multimer.
  • conventional coupling techniques utilizing amino and/or carboxyl groups present in, for example, the polypeptide, recombinant protein or dimer or multimer.
  • bisepoxide, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) are known coupling agents.
  • a spacer can be introduced between a solid support and the polypeptide, recombinant protein or dimer or multimer of separation, so that it improves the availability of the polypeptide, recombinant protein or dimer or multimer of separation, or, promotes the polypeptide, recombinant protein or dimer or multimer of separation to be chemically coupled to a support.
  • more than one site on the isolated polypeptide, recombinant protein, or dimer or multimer can be linked to a solid support (ie, via multipoint linkage).
  • the isolated polypeptide, recombinant protein, or dimer or multimer is linked by a disulfide bond between the cysteine residues therein and the solid support.
  • the conjugates are used to purify immunoglobulins.
  • the conjugate is used to purify an IgG Fc domain-containing sample.
  • the IgG Fc domain-containing sample is an IgG antibody or a heavy chain thereof, an IgG Fc domain-containing bispecific or multispecific antibody, and/or, an IgG Fc domain-containing Nanoparticle Antibody.
  • the IgG Fc domain-containing sample is an IgG Fc domain-containing bispecific or multispecific antibody.
  • the conjugate is an affinity chromatography packing.
  • the conjugate specifically binds to an IgG Fc domain in the range of pH 7.0-8.0 (e.g., pH 7.0-7.6, pH 7.0-7.4).
  • the conjugate is at pH 2.7-4.5 (e.g., pH 2.7-4.0, pH 3.5-4.5, or, Dissociates from the IgG Fc domain within the range of pH 3.8-4.3).
  • an isolated polypeptide or recombinant protein or dimer or multimer as described above specifically binds to an Fc domain of an immunoglobulin (e.g., an IgG Fc domain) and does not bind (e.g., is undetectable binding) to the variable region of an immunoglobulin (e.g., Fab).
  • an immunoglobulin e.g., an IgG Fc domain
  • Fab immunoglobulin
  • the present application also provides an isolated nucleic acid molecule encoding an isolated polypeptide or recombinant protein or dimer or multimer as described above or a polypeptide chain comprised therein.
  • the application also provides a vector comprising a nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • the application also provides a host cell comprising a nucleic acid molecule or vector as described above.
  • Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).
  • prokaryotic cells such as bacterial cells (e.g., E. coli cells)
  • eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).
  • the host cell is a microorganism.
  • the isolated polypeptide, recombinant protein, dimer or multimer of the present invention or the polypeptide chain contained therein can be prepared by various methods known in the art, such as by chemical synthesis or genetic engineering recombinant technology.
  • a DNA molecule encoding the isolated polypeptide, recombinant protein, dimer or multimer of the present invention or the polypeptide chain contained therein is obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the isolated polypeptide, recombinant protein, dimer or multimer of the present invention or the polypeptide chain contained therein.
  • the present application also provides a method for preparing the isolated polypeptide or recombinant protein or dimer or multimer or the polypeptide chain contained therein as described above, which includes, under conditions that allow protein expression, Culturing the host cell as described above, and recovering the isolated polypeptide or the recombinant protein or the dimer or multimer or the polypeptide chain comprised therefrom from the cultured host cell culture.
  • the present application also provides a method for preparing a conjugate as described above, which includes combining the isolated polypeptide, recombinant protein, and/or dimer or multimer as described above with a solid support The steps in forming connections.
  • the method includes contacting the isolated polypeptide, recombinant protein, and/or dimer or multimer with the solid support, such that the isolated polypeptide , a connection is formed between the recombinant protein, and/or, the dimer or multimer and the solid support.
  • connection is direct or indirect, reversible or irreversible.
  • the linkage is a covalent linkage or a non-covalent linkage.
  • the solid support is selected from agarose microparticles, high molecular polymer microspheres, silica gel microspheres or magnetic beads.
  • the linkage includes a disulfide bond.
  • the application also provides the use of the isolated polypeptide, or recombinant protein, or dimer or multimer, or conjugate as described above for preparing a purification reagent for purification Immunoglobulin.
  • the purification reagents are used to purify a sample containing an IgG Fc domain.
  • the IgG Fc domain-containing sample is an IgG antibody or a heavy chain thereof, an IgG Fc domain-containing bispecific or multispecific antibody, and/or, an IgG Fc domain-containing Nanoparticle Antibody.
  • the IgG Fc domain-containing sample is an IgG Fc domain-containing bispecific or multispecific antibody.
  • the present application also provides a method of purifying an immunoglobulin, comprising using a conjugate as described above.
  • the purification is affinity chromatography.
  • the affinity chromatography involves using a conjugate as described above as the stationary phase.
  • the methods are used to purify a sample containing an IgG Fc domain.
  • the method includes the steps of:
  • the sample containing the IgG Fc domain is combined with the conjugate to form a complex
  • the pH of the first solvent is 7.0-8.0; for example, the pH of the first solvent is 7.0-7.6 or 7.0-7.4.
  • the first solvent is a buffer (eg, Tris buffer).
  • the IgG Fc domain-containing sample is provided in the first solvent.
  • the first solvent comprising the IgG Fc domain-containing sample as the mobile phase of affinity chromatography.
  • the pH of the second solvent is 2.7-4.5; for example, the pH of the second solvent is 2.7-4.0, 3.5-4.5, or 3.8-4.3.
  • the second solvent is a buffer (eg, glycine buffer).
  • the second solvent is provided in the mobile phase of affinity chromatography.
  • the present application also provides a kit comprising an isolated polypeptide, or a recombinant protein, or a dimer or multimer, or a conjugate as described above.
  • the kit further includes reagents for protein purification (eg, a first solvent as described above, and/or a second solvent as described above).
  • the purification is affinity chromatography.
  • the first solvent is a buffer (eg, Tris buffer).
  • the second solvent is a buffer (eg, glycine buffer).
  • the kit is used to purify immunoglobulins.
  • the kit is used to purify a sample containing an IgG Fc domain.
  • the sample containing an IgG Fc domain is an IgG antibody or a heavy chain thereof, containing an IgG Fc structure.
  • the IgG Fc domain-containing sample is an IgG Fc domain-containing bispecific or multispecific antibody.
  • the term "B domain of wild-type Protein A" and similar expressions shall include all such sequences, including for example the sequence shown in SEQ ID NO: 4 and natural variants thereof.
  • the term "B domain of wild-type Protein A” and similar expressions shall include all such sequences, including for example the sequence shown in SEQ ID NO: 4 and natural variants thereof.
  • a specific amino acid position of the B domain of wild-type Protein A it includes not only the specific amino acid position of SEQ ID NO: 4, but also the corresponding amino acid position in its natural variant.
  • amino acid residue at position 1 of the B domain of wild-type Protein A when expressing "the amino acid residue at position 1 of the B domain of wild-type Protein A", it includes the amino acid residue at position 1 of SEQ ID NO: 4, as well as its natural variation The amino acid residue at the corresponding position in the body.
  • amino acid residue at the corresponding position means that, when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percent identity, the sequences being compared are located at equivalent positions. Amino acid residues.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990;59:439-473).
  • K D , kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyomavacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. Host cells can include single cells or populations of cells.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions).
  • comparisons are made when two sequences are aligned to yield maximum identity.
  • alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0).
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • chromatography refers to a separation technique that uses mobile and stationary phases to separate one type of molecule (eg, immunoglobulins) from other molecules (eg, contaminants) in a sample.
  • a liquid mobile phase is a mixture containing the specific type of molecule to be separated and other molecules.
  • the specific types of molecules to be separated and other molecules in the mobile phase are transported across or through a stationary phase (such as a solid matrix). Because different molecules in the mobile phase interact differently with the stationary phase, the specific types of molecules to be separated in the mobile phase can be transported. types of molecules separated from other molecules.
  • affinity chromatography refers to chromatography methods that rely on specific binding between molecules, in which ligands coupled to the stationary phase interact with molecules in the mobile phase (sample) (e.g., immune globulin) interaction, that is, the ligand has specific affinity for the molecule to be purified.
  • affinity chromatography involves the addition of an immunoglobulin-containing sample to a sample containing a ligand (e.g., a mutated B domain of the invention, or comprising said mutation). B domain immunoglobulin binding protein) in the stationary phase.
  • the immunoglobulin-binding protein containing the mutated B domain of Protein A provided by the present invention has high binding activity to Fc and at the same time eliminates the binding activity to the variable region of the antibody heavy chain. It can be used in bispecific or Provides milder elution conditions during the purification process of multispecific antibodies, effectively guarantees sample quality, and reduces the steps and costs of later fine purification, thus effectively solving the problem of using traditional Protein A to purify bispecific or multispecific antibodies.
  • the elution conditions faced are harsh (for example, lower pH value), and the quality of the antibody is greatly affected.
  • the immunoglobulin-binding protein comprising the mutated B domain of Protein A provided by the invention has a reduced isoelectric point, so that the immunoglobulin-binding protein can react in a weakly alkaline environment. It is relatively stable and is conducive to subsequent coupling experiments, thereby effectively ensuring that protein A is coupled to the filler matrix and improving the utilization rate of Protein A filler.
  • Figure 1 shows the modification site of the Protein A mutant of the present application.
  • Figure 2 shows the SDS-Page protein electrophoresis results of the Protein A mutant of this application
  • lane M is the Marker protein sample
  • lane 1 is the Protein A mutant engineering bacterial sample before induction
  • lane 2 is the Protein A mutant after induction.
  • lane 3 is the supernatant sample of Protein A mutant bacterial lysate after induction
  • lane 4 is the precipitated sample of Protein A mutant bacterial lysate after induction.
  • FIG. 3 shows the HPLC-SEC results of the Protein A mutants of the present application.
  • Figure 4 shows the results of the affinity performance determination of the Protein A mutants of the present application and antibodies.
  • Figure 5 shows the results of the affinity determination between the chromatography medium prepared by the Protein A mutant of the present application and VHH-Fc, Fcalone and VHHalone.
  • Figure 6 shows that the chromatography medium prepared from the Protein A mutant of the present application is suitable for milder elution conditions.
  • FIG. 7 shows the HPLC-SEC results of Protein A mutants 2, 3 and 4 of the present application.
  • Figure 8 shows the results of determining the affinity performance of Protein A mutants 2, 3 and 4 of the present application to Fc.
  • the nucleotide sequence of the Protein A mutant was artificially synthesized using the method of DNA full sequence synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • the nucleotide sequence is SEQ ID NO:3, and its corresponding amino acid sequence is SEQ ID NO:2.
  • the theoretical size is about 30kDa
  • the nucleotide sequence is constructed into the pET-28a vector linearized by Nco I/Hind III double enzyme digestion to obtain the expression plasmid of the Protein A mutant.
  • the mutation site of the Protein A mutant relative to wild-type Protein A is shown in Figure 1.
  • the N-terminus of the Protein A mutant used in this example contains methionine, and the C-terminus is also fused with an 8 ⁇ His (HHHHHHHH) tag.
  • step 1 Take 1 ⁇ L of the plasmid in step 1 above, add it to 100 ⁇ L of commercial BL21 (DE3) competent cells, and incubate on ice for 30 minutes; then put the plasmid and competent cell mixture into a 42°C water bath for thermal shock for 90 seconds; then take Take it out of the ice bath for 2 minutes; then add 450 ⁇ L LB to the mixture, and incubate at 37°C and 220rpm for 45 minutes; finally, centrifuge to remove most of the culture medium, spread the bacteria on an LB plate containing kanamycin, and place it in a 37°C incubator Incubate overnight.
  • the grown transformants were identified as positive transformants by sequencing using the pET-28a vector universal primer.
  • Purify the bacterial lysate supernatant obtained in step 3 above through a Ni column using the AKTA system specifically: use a balancing solution (20mM Tris-HCl, 150mM NaCl, pH 7.4) to rinse the protein purifier and Ni column until the baseline Equilibrate; select the appropriate flow rate to use the lysate supernatant, so that the sample retention time in the purification column is 5 minutes, and use washing solution (20mM Tris-HCl, 150mM NaCl, 5mM imidazole, pH7.4) to wash out impurity proteins until Baseline balance; finally use eluent (20mM Tris-HCl, 150mM NaCl, 250mM imidazole, pH7.4) to elute the protein.
  • a balancing solution (20mM Tris-HCl, 150mM NaCl, pH 7.4
  • the eluted protein is reduced with 5mM DTT and its components are analyzed by liquid chromatography (SEC). check Measurement.
  • the HPLC-SEC detection results are shown in Figure 3. It can be seen from Figure 3 that the purity of the reduced protein is relatively high, close to 90%.
  • the non-specific adsorption between the His tag at the C-terminus of the protein and the chromatographic column results in a certain degree of drag on the characteristic peaks of the protein. tail.
  • characteristic peaks of the reducing agent DTT can be observed at high retention times.
  • VHH-Fc amino acid sequence is shown in SEQ ID NO:8
  • Fc Fc amino acid sequence is shown in SEQ ID NO:9
  • VHH VHH amino acid sequence is shown in SEQ ID NO:10
  • the target protein (see: Publication No. CN114195900A) is a bispecific antibody, a single molecule Contains 4 VHH regions (all VH3 gene family).
  • Use elution buffer (20mM glycine-HCl, pH4.0 or pH3.4) to sequentially elute the samples and collect the flow-through. After the UV value is stable, use 0.1M NaOH solution for regeneration.
  • the protein A affinity chromatography column (column volume 0.7 mL) of this application can recover 14.6 mg of protein under the elution condition of pH 4.0, and under the condition of pH 3.4 No additional protein flowed out, and the total recovery rate was 66.4%; only 1.1 mg of protein was recovered from the MabSelect PrismA chromatography column under pH 4.0 elution conditions.
  • mutant 2 mutates the C-terminal lysine of the mutated B domain (the amino acid sequence of the mutated B domain is shown in SEQ ID NO: 5) to alanine, and the amino acid sequence of mutant 2 is SEQ ID NO:11, the corresponding nucleotide sequence is SEQ ID NO:12;
  • Mutant 3 mutates the lysine on the linker (the linker amino acid sequence is shown in SEQ ID NO:6) to alanine, as described
  • the amino acid sequence of mutant 3 is SEQ ID NO:13, and the corresponding nucleotide sequence is SEQ ID NO:14; mutant 4 simultaneously mutates the lysine at the above two positions to alanine, and the mutant 4
  • the amino acid sequence is SEQ ID NO:15, and the corresponding nucleotide sequence is SEQ ID NO:16.
  • the theoretical isoelectric point of each mutant amino acid is shown in Table 2 (calculated by Expasy tool: https://web.expasy.org/cgi-bin/protparam/protparam). From the initial mutant (i.e., as shown in Example 1 The isoelectric point pI values of the mutants, the amino acid sequence is shown in SEQ ID NO: 2) respectively decreased from 7.90 to 5.91 (mutant 2, the amino acid sequence is shown in SEQ ID NO: 11), 6.17 (mutant 3, the amino acid sequence is shown The sequence is shown in SEQ ID NO: 13) and 5.37 (mutant 4, the amino acid sequence is shown in SEQ ID NO: 15), so that the immunoglobulin binding protein can be relatively stable in a weakly alkaline environment.
  • Example 7 For the construction, expression and purification process of each mutant, please refer to Example 1.
  • the final HPLC-SEC detection results of each mutant are shown in Figure 7.
  • the protein purity after reduction is relatively high, both greater than 90%.
  • a certain degree of tailing of the characteristic peaks may be caused by non-specific adsorption between the His tag at the C-terminus of the protein and the chromatographic column.

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Abstract

提供了一种由Protein A的突变的B结构域组成的分离的多肽，包含所述突变的B结构域的非天然免疫球蛋白结合蛋白，包含所述突变的B结构域或非天然免疫球蛋白结合蛋白的偶联物，以及相关用途。

Description

免疫球蛋白结合蛋白及其应用 技术领域
本发明涉及蛋白纯化领域，具体地，本发明提供了一种由Protein A的突变的B结构域组成的分离的多肽，包含所述突变的B结构域的免疫球蛋白结合蛋白，包含所述突变的B结构域或非天然免疫球蛋白结合蛋白的偶联物，以及相关用途。
背景技术
Protein A全称为staphylococcus protein A(金黄色葡萄球菌蛋白A)，来源于金黄色葡萄球菌的一个株系，它含有5个高度同源的单结构域，自N端起分别为E、D、A、B和C，每个单结构域约含58个氨基酸残基，各结构域形成三股反向平行排列的α-螺旋空间结构。上述各单结构域均可以和抗体IgG(Immunoglobulin G，免疫球蛋白G)分子的Fc(fragment crystallizable，可结晶段)段以及Fab(fragment of antigen binding，抗原结合片段)段特异性结合，其中和Fc段结合区位于第二及第三恒定区(CH2和CH3)连接处，和Fab段结合区位于重链的可变区(VH，仅限于VH3基因家族)。
重组Protein A作为抗体分子纯化的配基材料主要具有以下优点：1)表达成本低，可用E.coli(大肠杆菌)原核体系进行大规模表达；2)特异性强，其对于人源IgG1、IgG2、IgG4和鼠源IgG2都具有较强的亲和性能；3)良好的再生性，可在碱性条件下反复再生循环使用。
然而，目前市场上多数重组Protein A填料都是同时结合Fc与VH区域，对于双特异性或多特异性抗体而言，分子通常含有多个VH区，其与Protein A的结合强度明显高于单克隆抗体，这便导致抗体纯化过程的洗脱条件较为苛刻，例如较低的pH值。这类条件本身会对抗体质量造成潜在风险并增加后续纯化工艺步骤与成本。
发明内容
本发明提供的包含Protein A的突变的B结构域的免疫球蛋白结合蛋白在具备与Fc结构域(例如，IgG Fc结构域)的高结合活性的同时，消除了与抗体重链可变区的结合活性，其可在双特异性或多特异性抗体(例如，含IgG Fc结构域的双特异性或多特异性抗体)纯化过程中提供更温和的洗脱条件，对样品质量提供有效的保障，并减少后期精细纯化的步骤与成本，从而可有效解决使用传统Protein A纯化双特异性或多特异性抗体时所面临的洗脱条件苛刻(例如，较低的pH值)，抗体质量受到很大影响等问题。
因此，在一方面，本申请提供了分离的多肽，其由Protein A的突变的B结构域或其 变体组成；其中，所述突变的B结构域与野生型Protein A的B结构域相比包含选自N23T、D36V、D37R、Q40V中的一个或多个(例如1个、2个、3个或4个)氨基酸置换；
其中，所述变体与所述突变的B结构域相比，具有至少90％，例如至少95％，至少96％，至少97％，至少98％，至少99％的序列同一性；或者，具有一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，且保留了所述突变的B结构域的功能；并且；
所述变体与所述突变的B结构域在相应于野生型Protein A的B结构域的第1位、第23位、第29位、第36位、第37位和第40位的位置处的氨基酸残基相同。
在某些实施方案中，所述突变的B结构域与野生型Protein A的B结构域相比进一步包含氨基酸置换：(i)G29A，和/或，(ii)A1V。
在某些实施方案中，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：(a)N23T、G29A、D36V、D37R和Q40V，或者，(b)A1V、N23T、D36V、D37R和Q40V。
在某些实施方案中，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：A1V、N23T、G29A、D36V、D37R和Q40V。
在某些实施方案中，所述突变的B结构域与野生型Protein A的B结构域相比进一步包含氨基酸置换K58A。
在某些实施方案中，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：N23T、D36V、D37R、Q40V和K58A。
在某些实施方案中，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：(a)N23T、G29A、D36V、D37R、Q40V和K58A，或者，(b)A1V、N23T、D36V、D37R、Q40V和K58A。
在某些实施方案中，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：A1V、N23T、G29A、D36V、D37R、Q40V和K58A。
在某些实施方案中，所述野生型Protein A的B结构域具有如SEQ ID NO:4所示的序列。
在某些实施方案中，所述突变的B结构域具有如SEQ ID NO:5或17所示的氨基酸序列。此处所示序列在其N端不包含起始密码子(如ATG)编码的氨基酸(如甲硫氨酸(Met))。本领域技术人员理解，在通过基因工程制备蛋白的过程中，由于起始密码子的作用，所产生的多肽链第一位经常为起始密码子编码的氨基酸(如Met)。本发明的突变的B结构域不仅囊括在其N末端不包含起始密码子编码的氨基酸(如Met)的氨基酸序列，也囊括在其N末端包含起始密码子编码的氨基酸(如Met)的氨基酸序列。因此，在 上述氨基酸序列的N端进一步包含起始密码子编码的氨基酸(如Met)的序列也在本发明的保护范围内。
在某些实施方案中，所述分离的多肽在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合；和/或，所述分离的多肽在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或，pH 3.8-4.3)的范围内与IgG Fc结构域解离。
在另一方面，本申请还提供了重组蛋白，其包含如上所述的分离的多肽。
在某些实施方案中，所述重组蛋白不包含野生型Protein A的B结构域的氨基酸序列。
在某些实施方案中，所述重组蛋白不包含如SEQ ID NO:4所示的氨基酸序列。
在某些实施方案中，所述重组蛋白包含一个或多个(例如，1个，2个，3个、4个、5个、6个、7个或8个)如上所述的分离的多肽。在某些实施方案中，所述重组蛋白包含3个、4个、5个或6个如上所述的分离的多肽。
在某些实施方案中，所述重组蛋白包多个(例如，2个，3个、4个、5个、6个、7个或8个)如上所述的分离的多肽，并且，所述多个分离的多肽各不相同、部分相同或完全相同。
在某些实施方案中，各个所述分离的多肽之间任选地通过相同或不同的肽接头连接。在某些实施方案中，所述肽接头各自独立地具有如SEQ ID NO:6或18所示的氨基酸序列。
在某些实施方案中，所述重组蛋白在其N端或C端进一步包含半胱氨酸。在某些实施方案中，所述重组蛋白在其C端进一步包含半胱氨酸。在某些实施方案中，所述半胱氨酸任选地通过接头(例如包含一个或多个甘氨酸(G)的柔性肽，例如GGGG)与所述分离的多肽连接。
在某些实施方案中，所述重组蛋白包含如SEQ ID NO:2、7、11、13、15、19-21任一项所示的氨基酸序列或由其组成。此处所示序列在其N端不包含起始密码子(如ATG)编码的氨基酸(如甲硫氨酸(Met))。本领域技术人员理解，在通过基因工程制备蛋白的过程中，由于起始密码子的作用，所产生的多肽链第一位经常为起始密码子编码的氨基酸(如Met)。本发明的重组蛋白不仅囊括在其N末端不包含起始密码子编码的氨基酸(如Met)的氨基酸序列，也囊括在其N末端包含起始密码子编码的氨基酸(如Met)的氨基酸序列。因此，在上述氨基酸序列的N端进一步包含起始密码子编码的氨基酸(如Met)的序列也在本发明的保护范围内。
在某些实施方案中，所述重组蛋白还连接有另外的多肽，另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任何组合。
在本文中，蛋白标签是本领域熟知的，其实例包括但不限于His、Flag、GST、MBP、HA、Myc、GFP或生物素，并且本领域技术人员已知如何根据期望目的(例如，纯化、 检测或示踪)选择合适的蛋白标签。
在某些实施方案中，所述重组蛋白在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合。
在某些实施方案中，所述重组蛋白在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或，pH 3.8-4.3)的范围内与IgG Fc结构域解离。
在另一方面，本申请还提供了二聚体或多聚体，其包含两个或多个(例如，2个，3个、4个、5个、6个、7个或8个)如上所述的分离的多肽。在某些实施方案中，所述二聚体或多聚体包含3个、4个、5个或6个如上所述的分离的多肽。
在某些实施方案中，所述两个或多个(例如，2个，3个、4个、5个、6个、7个或8个)分离的多肽各不相同、部分相同或完全相同。
在某些实施方案中，所述二聚体或多聚体不包含野生型Protein A的B结构域的氨基酸序列。
在某些实施方案中，所述二聚体或多聚体不包含如SEQ ID NO:4所示的氨基酸序列。
在某些实施方案中，所述二聚体或多聚体包含至少两条多肽链，所述两个或多个分离的多肽存在于至少两条多肽链中。
在某些实施方案中，所述分离的多肽之间通过接头可逆或不可逆地连接形成二聚体或多聚体。
在某些实施方案中，所述二聚体或多聚体中至少一条多肽链在其N端或C端进一步包含半胱氨酸。在某些实施方案中，所述多肽链在其C端进一步包含半胱氨酸。在某些实施方案中，所述半胱氨酸任选地通过接头(例如包含一个或多个甘氨酸(G)的柔性肽，例如GGGG)与所述分离的多肽连接。
在某些实施方案中，所述二聚体或多聚体中至少一条多肽链还连接有另外的多肽，另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任何组合。
在本文中，蛋白标签是本领域熟知的，其实例包括但不限于His、Flag、GST、MBP、HA、Myc、GFP或生物素，并且本领域技术人员已知如何根据期望目的(例如，纯化、检测或示踪)选择合适的蛋白标签。
在某些实施方案中，所述二聚体或多聚体在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合。
在某些实施方案中，所述二聚体或多聚体在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或，pH 3.8-4.3)的范围内与IgG Fc结构域解离。
在另一方面，本申请还提供了偶联物，其包含固相支持物以及与所述固相支持物连接的如上所述的分离的多肽，重组蛋白，和/或，二聚体或多聚体。
在某些实施方案中，所述分离的多肽、重组蛋白或二聚体或多聚体直接或间接、可逆或不可逆地与固相支持物相连接。
在某些实施方案中，所述分离的多肽、重组蛋白或二聚体或多聚体通过共价和/或非共价键与所述固相支持物连接。
在某些实施方案中，所述固相支持物包括基于合成聚合物的那些固相支持物，例如，聚乙烯醚、聚乙烯醇、聚甲基丙烯酸酯、聚丙烯酸酯、聚苯乙烯、聚丙烯酰胺、聚甲基丙烯酰胺和聚碳酸酯。
在某些实施方案中，所述固相支持物的形式包括但不限于珠(球形或不规则的形状)、中空纤维、固体纤维、垫、凝胶、膜、盒、柱、芯片、玻片、板或整体柱。
在某些实施方案中，所述固相支持物选自琼脂糖微粒、高分子聚合物微球、硅胶微球或磁珠。
任何合适的技术均可以用于将本文所述的分离的多肽、重组蛋白或二聚体或多聚体连接至固相支持物。例如，在一些实施方案中，可以通过常规偶联技术利用例如所述分离的多肽、重组蛋白或二聚体或多聚体中存在的氨基和/或羧基基团将其连接至固相支持物。例如，双环氧化合物(bisepoxide)、表氯醇、CNBr、N-羟基琥珀酰亚胺(NHS)是公知的偶联试剂。在一些实施方案中，在固相支持物和所述的分离的多肽、重组蛋白或二聚体或多聚体之间可引入间隔，从而其提高所述分离的多肽、重组蛋白或二聚体或多聚体的可用性，或者，促进所述分离的多肽、重组蛋白或二聚体或多聚体化学偶联至支持物。
在某些实施方案中，可将所述分离的多肽、重组蛋白或二聚体或多聚体上一个以上的位点连接至固体支持物(即，通过多点连接)。
在某些实施方案中，所述分离的多肽、重组蛋白或二聚体或多聚体通过其中的半胱氨酸与所述固相支持物之间形成二硫键连接。
在某些实施方案中，所述偶联物用于纯化免疫球蛋白。
在某些实施方案中，所述偶联物用于纯化含IgG Fc结构域的样品。
在某些实施方案中，所述含IgG Fc结构域的样品为IgG抗体或其重链，含IgG Fc结构域的双特异性或多特异性抗体，和/或，含IgG Fc结构域的纳米抗体。
在某些实施方案中，所述含IgG Fc结构域的样品为含IgG Fc结构域的双特异性或多特异性抗体。
在某些实施方案中，所述偶联物是亲和层析填料。
在某些实施方案中，所述偶联物在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合。
在某些实施方案中，所述偶联物在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或， pH 3.8-4.3)的范围内与IgG Fc结构域解离。
在某些实施方案中，如上所述的分离的多肽或重组蛋白或二聚体或多聚体特异性结合免疫球蛋白的Fc结构域(如IgG Fc结构域)，并且不结合(例如不可检测的结合)免疫球蛋白的可变区(例如Fab)。
在另一方面，本申请还提供了分离的核酸分子，其编码如上所述的分离的多肽或重组蛋白或二聚体或多聚体或其所包含的多肽链。
在另一方面，本申请还提供了载体，其包含如上所述的核酸分子。在某些实施方案中，所述载体为克隆载体或表达载体。
在另一方面，本申请还提供了宿主细胞，其包含如上所述的核酸分子或载体。
此类宿主细胞包括但不限于，原核细胞例如细菌细胞(如大肠杆菌细胞)，以及真核细胞例如真菌细胞(例如酵母细胞)，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。在某些实施方案中，所述宿主细胞是微生物。
本发明的分离的多肽、重组蛋白、二聚体或多聚体或其所包含的多肽链可以本领域已知的各种方法来制备，例如通过化学合成或基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明分离的多肽、重组蛋白、二聚体或多聚体或其所包含的多肽链的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的分离的多肽、重组蛋白、二聚体或多聚体或其所包含的多肽链。
在另一方面，本申请还提供了制备如上所述的分离的多肽或重组蛋白或二聚体或多聚体或其所包含的多肽链的方法，其包括，在允许蛋白表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述分离的多肽或所述重组蛋白或所述二聚体或多聚体或其所包含的多肽链。
在另一方面，本申请还提供了制备如上所述的偶联物的方法，其包括将如上所述的分离的多肽，重组蛋白，和/或，二聚体或多聚体与固相支持物形成连接的步骤。
在某些实施方案中，所述方法包括：将所述分离的多肽，重组蛋白，和/或，二聚体或多聚体与所述固相支持物相接触，从而使所述分离的多肽，重组蛋白，和/或，二聚体或多聚体与所述固相支持物之间形成连接。
在某些实施方案中，所述连接是直接的或间接的，可逆或不可逆的。
在某些实施方案中，所述连接是共价连接或非共价连接。
在某些实施方案中，所述固相支持物选自琼脂糖微粒、高分子聚合物微球、硅胶微球或磁珠。
在某些实施方案中，所述连接包含二硫键。
在另一方面，本申请还提供了如上所述的分离的多肽，或重组蛋白，或二聚体或多聚体，或偶联物用于制备纯化试剂的用途，所述纯化试剂用于纯化免疫球蛋白。
在某些实施方案中，所述纯化试剂用于纯化含IgG Fc结构域的样品。
在某些实施方案中，所述含IgG Fc结构域的样品为IgG抗体或其重链，含IgG Fc结构域的双特异性或多特异性抗体，和/或，含IgG Fc结构域的纳米抗体。
在某些实施方案中，所述含IgG Fc结构域的样品为含IgG Fc结构域的双特异性或多特异性抗体。
在另一方面，本申请还提供了纯化免疫球蛋白的方法，其包括使用如上所述的偶联物。
在某些实施方案中，所述纯化是亲和层析。在某些实施方案中，所述亲和层析包括使用如上所述的偶联物作为固定相。
在某些实施方案中，所述方法用于纯化含IgG Fc结构域的样品。
在某些实施方案中，所述方法包含以下步骤：
(1)在第一溶剂中，使所述含IgG Fc结构域的样品与所述偶联物结合形成复合物；
(2)在第二溶剂中，使所述复合物解离；和，
(3)收集含有所述含IgG Fc结构域的样品的解离产物。
在某些实施方案中，所述第一溶剂的pH为7.0-8.0；例如，所述第一溶剂的pH为7.0-7.6或7.0-7.4。在某些实施方案中，所述第一溶剂为缓冲液(例如，Tris缓冲液)。
在某些实施方案中，所述含IgG Fc结构域的样品存在于所述第一溶剂中被提供。例如，通过将包含所述含IgG Fc结构域的样品的所述第一溶剂作为亲和层析的流动相被提供。
在某些实施方案中，所述第二溶剂的pH为2.7-4.5；例如，所述第二溶剂的pH为2.7-4.0、3.5-4.5或3.8-4.3。在某些实施方案中，所述第二溶剂为缓冲液(例如，甘氨酸缓冲液)。
在某些实施方案中，所述第二溶剂在亲和层析的流动相中被提供。
在另一方面，本申请还提供了试剂盒，其包含如上所述的分离的多肽，或重组蛋白，或二聚体或多聚体，或偶联物。
在某些实施方案中，所述试剂盒进一步包含蛋白纯化所用试剂(例如，如上所述的第一溶剂，和/或，如上所述的第二溶剂)。在某些实施方案中，所述纯化是亲和层析。
在某些实施方案中，所述第一溶剂为缓冲液(例如，Tris缓冲液)。
在某些实施方案中，所述第二溶剂为缓冲液(例如，甘氨酸缓冲液)。
在某些实施方案中，所述试剂盒用于纯化免疫球蛋白。
在某些实施方案中，所述试剂盒用于纯化含IgG Fc结构域的样品。
在某些实施方案中，所述含IgG Fc结构域的样品为IgG抗体或其重链，含IgG Fc结构 域的双特异性或多特异性抗体，和/或，含IgG Fc结构域的纳米抗体。
在某些实施方案中，所述含IgG Fc结构域的样品为含IgG Fc结构域的双特异性或多特异性抗体。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
如本文中所使用的，当提及野生型Protein A的B结构域的氨基酸序列时，其使用SEQ ID NO：4所示的序列来进行描述。然而，本领域技术人员理解，在野生型Protein A的B结构域的氨基酸序列中，可天然产生突变，而不影响其生物学功能。因此，在本发明中，术语“野生型Protein A的B结构域”及其类似表述应包括所有此类序列，包括例如SEQ ID NO：4所示的序列以及其天然变体。并且，当描述野生型Protein A的B结构域的某一特定氨基酸位置时，其不仅包括SEQ ID NO：4的特定氨基酸位置，还包括其天然变体中的相应氨基酸位置。例如，表述“野生型Protein A的B结构域的第1位的位置处的氨基酸残基”时，其包括，SEQ ID NO：4的第1位的位置处的氨基酸残基，以及其天然变体中的相应位置处的氨基酸残基。根据本发明，表述“相应位置处的氨基酸残基”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的氨基酸残基。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见 Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。宿主细胞可以包括单个细胞或细胞群体。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol. 48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文所使用的，术语“层析”指一种分离技术，其采用流动相和固定相以将样品中的一种类型的分子(例如免疫球蛋白)与其它分子(例如污染物)分开。液体流动相为包含待分离的特定类型的分子和其他分子的混合物。通过流动相将待分离的特定类型的分子和其他分子传送跨过或通过固定相(例如固体基质)，由于流动相中不同分子与固定相的相互作用不同，可以将流动相中待分离的特定类型的分子与其他分子分离开。
如本文所使用的，术语“亲和层析”是指依赖于分子间特异性结合的层析方法，其中，偶联到固定相的配体与流动相(样品)中的分子(例如，免疫球蛋白)相互作用，即配体对待纯化的分子具有特异性亲和力。如在本发明的上下文中所理解的，亲和层析涉及将含有免疫球蛋白的样品加入到包含配体(例如，本发明的突变的B结构域，或包含所述突变 的B结构域的免疫球蛋白结合蛋白)的固定相中。
发明的有益效果
本发明提供的包含Protein A的突变的B结构域的免疫球蛋白结合蛋白在具备与Fc的高结合活性的同时，消除了与抗体重链可变区的结合活性，其可在双特异性或多特异性抗体纯化过程中提供更温和的洗脱条件，对样品质量提供有效的保障，并减少后期精细纯化的步骤与成本，从而可有效解决使用传统Protein A纯化双特异性或多特异性抗体时所面临的洗脱条件苛刻(例如，较低的pH值)，抗体质量受到很大影响等问题。
并且，在某些实施方案中，本发明提供的包含Protein A的突变的B结构域的免疫球蛋白结合蛋白具备降低的等电点，使得所述免疫球蛋白结合蛋白可以在弱碱性环境下较为稳定，利于后续偶联试验的进行，从而有效保证protein A蛋白偶联到填料基质上，提高Protein A填料的利用率。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了本申请的Protein A突变体的改造位点。
图2显示了本申请的Protein A突变体的SDS-Page蛋白电泳结果；其中，泳道M为Marker蛋白样品，泳道1为诱导前Protein A突变体工程菌样品，泳道2为诱导后Protein A突变体全菌裂解液样品，泳道3为诱导后Protein A突变体菌裂解液上清样品，泳道4为诱导后Protein A突变体菌裂解液沉淀样品。
图3显示了本申请的Protein A突变体HPLC-SEC结果。
图4显示了本申请的Protein A突变体与抗体的亲和力性能测定结果。
图5显示了本申请的Protein A突变体所制备的层析介质与VHH-Fc、Fc alone及VHH alone的亲和力测定结果。
图6显示了本申请的Protein A突变体所制备的层析介质适用更为温和的洗脱条件。
图7显示了本申请的Protein A突变体2、3和4的HPLC-SEC结果。
图8显示了本申请的Protein A突变体2、3和4的与Fc的亲和力性能测定结果。
序列信息
本申请涉及的序列的描述提供于下表中。
表1：序列信息


具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
实施例1：Protein A突变体的表达及其性能
1.Protein A突变体表达载体的合成
采用DNA全序列合成的方法人工合成Protein A突变体的核苷酸序列(苏州金唯智生物科技有限公司)，核苷酸序列为SEQ ID NO:3，其对应的氨基酸序列为SEQ ID NO:2，理论大小约为30kDa，并将所述核苷酸序列构建到Nco I/Hind III双酶切线性化的pET-28a载体上，得到Protein A突变体的表达质粒。其中，所述Protein A突变体相对于野生型Protein A的突变位点如图1所示。
此外，为便于蛋白表达及纯化，本实施例所用Protein A突变体的N末端含甲硫氨酸，C末端还融合有8×His(HHHHHHHH)标签。
2.Protein A突变体工程菌的构建
取上述步骤1中的质粒1μL，加到100μL商品化的BL21(DE3)感受态细胞中，进行30min冰浴；然后将质粒、感受态细胞混合物放入42℃水浴锅中热冲击90s；再拿出来冰浴2min；接着向混合物中加450μL LB，于37℃、220rpm培养45min；最后离心去除大部分培养基，将细菌涂布在含卡那霉素的LB平板中，放入37℃恒温箱中过夜培养。长出来的转化子用pET-28a载体通用引物经测序鉴定为阳性转化子。
3.小规模表达Protein A突变体
挑取上述步骤2中的阳性单克隆于10mL含卡那霉素的LB中培养(37℃、220rpm)约14h，作为种子液。取10mL种子液于1000mL含卡那霉素的LB培养基中，于37℃、220rpm培养2h(OD600读数为0.6)；按培养体积的0.1％向培养基中加入1M IPTG，诱导表达条件为25℃、150rpm、16h。
将发酵液进行离心(6000rpm、4℃)30min去除培养基；用50mL Tris缓冲溶液(20mM Tris-HCl，150mM NaCl，pH7.4)将菌泥悬浮起来，进行超声波破碎，破碎条件为25％功率，开5s、关5s，工作30min；将裂解液进行离心，条件为12000rpm、4℃离心30min，分离上清与沉淀，并进行SDS-PAGE蛋白电泳。SDS-PAGE蛋白电泳结果如图2所示，由图2可以看出Protein A突变体被成功表达。
4.Protein A突变体的纯化
将上述步骤3中获得的细菌裂解液上清，使用AKTA系统经Ni柱纯化，具体为：使用平衡液(20mM Tris-HCl，150mM NaCl，pH7.4)冲洗蛋白纯化仪及Ni柱，直至基线平衡；选择合适的流速上裂解液上清，使样品在纯化柱中保留的时间为5min，并用洗杂液(20mM Tris-HCl，150mM NaCl，5mM imidazole，pH7.4)洗除杂蛋白，直至基线平衡；最后用洗脱液(20mM Tris-HCl，150mM NaCl，250mM imidazole，pH7.4)洗脱蛋白，洗脱后的蛋白使用5mM DTT进行还原并采用液相色谱(SEC)对其成分进行检 测。HPLC-SEC检测结果如图3所示，由图3可知，还原后的蛋白纯度较高，接近90％，蛋白C端的His标签与色谱柱间的非特异性吸附导致蛋白特征峰存在一定程度的拖尾。此外在高保留时间可以观察到还原剂DTT的特征峰。
5.Protein A突变体与抗体的亲和力测定
ForteBio亲和力测定按照现有的方法(参见：Estep,P等人，基于溶液的高通量抗体-抗原亲和力和表位分级的测量，MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载Biotin标记的双特异性抗体Ava-2GS-NSD(参见公开号CN114106190A)至SA传感器上。再将传感器放入100nM的Protein A突变体中作用110s，之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。
结果如图4所示，由图4可知，本申请的Protein A突变体与双特异性抗体有很强的结合活性，Kd值达到9.0×10-11M，可用于抗体的亲和纯化。
6.纯化用Protein A突变体亲和填料的制备
具体步骤如下：
(1)转移待偶联的层析介质到离心管中进行离心，去除上清并用0.1M磷酸盐缓冲溶液(pH8.0)平衡，离心去除磷酸盐缓冲溶液并重复3次该过程；
(2)将Protein A突变体蛋白换液至上述磷酸盐缓冲溶液并加入到上述介质中进行偶联，37℃，130rpm，24h；
(3)待偶联结束后离心去除上清并加入乙醇胺进行封闭，37℃，130rpm，6h；
(4)用0.1M磷酸盐缓冲溶液(pH7.0)清洗填料3次并将其在2-8℃保存待用。
7.Protein A突变体所制备的层析介质与VHH-Fc、Fc alone及VHH alone的亲和力测定
ForteBio亲和力测定按照现有的方法(参见：Estep,P等人，基于溶液的高通量抗体-抗原亲和力和表位分级的测量，MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载本发明中Biotin标记的Protein A突变体至SA传感器上。再将传感器放入100nM的VHH-Fc(VHH-Fc氨基酸序列如SEQ ID NO:8所示)、Fc(Fc氨基酸序列如SEQ ID NO:9所示)或VHH(VHH氨基酸的序列如SEQ ID NO:10所示)中作用5mins，之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。
结果如图5所示，由图5可知，由本申请Protein A突变体所制备的层析介质在保留与Fc的结合活性的同时，消除了与抗体重链可变区的结合活性。表明本申请Protein A突变体所制备的层析介质可有效避免抗体重链可变区与野生型Protein A的结合对抗体纯化 过程带来的不利影响。
8.Protein A突变体所制备的层析介质用于双特异性抗体纯化
采用Protein A亲和层析柱MabSelect PrismA(Cytiva，货号17519901)或本申请Protein A突变体填料纯化目的蛋白Bi-088，所述目的蛋白(参见：公开号CN114195900A)为双特异性抗体，单个分子含有4个VHH区域(均为VH3基因家族)。使用5到10个柱体积的平衡缓冲液(20mM Tris-HCl,150mM NaCl,pH7.4)平衡纯化柱，至流出液电导和pH值不变开始上样。上样结束后继续使用平衡缓冲液冲洗层析柱，至流出液UV值不再下降。使用洗脱缓冲液(20mM glycine-HCl,pH4.0或pH3.4)依次洗脱样品并收集流出液。待UV值稳定后用0.1M NaOH溶液进行再生。
结果如图6所示，由图6可知，由本申请Protein A突变体所制备的层析介质可在双特异性或多特异性抗体纯化过程中提供更温和的洗脱条件，对样品质量提供有效的保障，并减少后期精细纯化的步骤与成本。
具体来讲，同样上样量(22mg)情况下，本申请protein A亲和层析柱(柱体积0.7mL)在pH4.0洗脱条件下可以回收到14.6mg蛋白，在pH3.4条件下无额外蛋白流出，总回收率为66.4％；MabSelect PrismA层析柱在pH4.0洗脱条件下仅回收到1.1mg蛋白。
实施例2：Protein A突变体性能的进一步优化
为降低Protein A突变体的等电点，分别基于实施例1的Protein A突变体(即初始突变体)设计三个新的突变体。其中，突变体2为将突变的B结构域(突变的B结构域氨基酸序列如SEQ ID NO:5所示)C端的赖氨酸突变为丙氨酸，所述突变体2的氨基酸序列为SEQ ID NO:11，对应的核苷酸序列为SEQ ID NO:12；突变体3为将linker(Linker氨基酸序列如SEQ ID NO:6所示)上的赖氨酸突变为丙氨酸，所述突变体3的氨基酸序列为SEQ ID NO:13，对应的核苷酸序列为SEQ ID NO:14；突变体4为同时突变上述两个位置的赖氨酸为丙氨酸，所述突变体4的氨基酸序列为SEQ ID NO:15，对应的核苷酸序列为SEQ ID NO:16。
各突变体氨基酸理论等电点如表2所示(通过Expasy工具计算：https://web.expasy.org/cgi-bin/protparam/protparam)，从初始突变体(即，实施例1所示突变体，氨基酸序列如SEQ ID NO:2所示)的等电点pI值从7.90分别降为5.91(突变体2，氨基酸序列如SEQ ID NO:11所示)、6.17(突变体3，氨基酸序列如SEQ ID NO:13所示)和5.37(突变体4，氨基酸序列如SEQ ID NO:15所示)，使得免疫球蛋白结合蛋白可以在弱碱性环境下较为稳定。
表2各突变体的理论等电点值
各突变体的构建、表达与纯化过程可参考实施例1。最终所得各突变体的HPLC-SEC检测结果如图7所示。由图7可知，还原后的蛋白纯度较高，均大于90％。此外，特征峰存在一定程度的拖尾可能由蛋白C端的His标签与色谱柱间的非特异性吸附导致。
Protein A突变体与抗体的亲和力测定
ForteBio亲和力测定按照现有的方法(参见：Estep,P等人，基于溶液的高通量抗体-抗原亲和力和表位分级的测量，MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载Biotin标记的各突变体至SA传感器上。再将传感器放入100nM的Fc蛋白(SEQ ID NO:9)中作用200s，之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。
结果如图8所示，由图8可知，各突变体与Fc蛋白结合活性相当。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (17)

  1. 分离的多肽，其由Protein A的突变的B结构域或其变体组成，其中，所述突变的B结构域与野生型Protein A的B结构域相比包含选自N23T、D36V、D37R、Q40V中的一个或多个(例如1个、2个、3个或4个)氨基酸置换；
    其中，所述变体与所述突变的B结构域相比，具有至少90％，例如至少95％，至少96％，至少97％，至少98％，至少99％的序列同一性；或者，具有一个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸的置换(优选保守置换)、添加或缺失，且保留了所述突变的B结构域的功能；并且；
    所述变体与所述突变的B结构域在相应于野生型Protein A的B结构域的第1位、第23位、第29位、第36位、第37位和第40位的位置处的氨基酸残基相同。
  2. 权利要求1所述的分离的多肽，其中，所述突变的B结构域与野生型Protein A的B结构域相比进一步包含氨基酸置换：(i)G29A，和/或，(ii)A1V；
    优选地，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：(a)N23T、G29A、D36V、D37R和Q40V，或者，(b)A1V、N23T、D36V、D37R和Q40V；
    优选地，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：A1V、N23T、G29A、D36V、D37R和Q40V。
  3. 权利要求1或2所述的分离的多肽，其中，所述突变的B结构域与野生型Protein A的B结构域相比进一步包含氨基酸置换K58A；
    优选地，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：N23T、D36V、D37R、Q40V和K58A；
    优选地，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：(a)N23T、G29A、D36V、D37R、Q40V和K58A，或者，(b)A1V、N23T、D36V、D37R、Q40V和K58A；
    优选地，所述突变的B结构域与野生型Protein A的B结构域相比包含氨基酸置换：A1V、N23T、G29A、D36V、D37R、Q40V和K58A。
  4. 权利要求1-3任一项所述的分离的多肽，其中，所述野生型Protein A的B结构域具有如SEQ ID NO:4所示的序列。
  5. 权利要求1-4任一项所述的分离的多肽，其中，所述突变的B结构域具有如SEQ ID NO:5或17所示的氨基酸序列。
  6. 权利要求1-5任一项所述的分离的多肽，其中，所述分离的多肽在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合；和/或，所述分离的多肽在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或，pH 3.8-4.3)的范围内与IgG Fc结构域解离。
  7. 重组蛋白，其包含权利要求1-6任一项所述的分离的多肽；
    优选地，所述重组蛋白不包含野生型Protein A的B结构域的氨基酸序列；
    优选地，所述重组蛋白不包含如SEQ ID NO:4所示的氨基酸序列；
    优选地，所述重组蛋白包含一个或多个(例如，1个，2个，3个、4个、5个、6个、7个或8个)权利要求1-6任一项所述的分离的多肽；
    优选地，所述重组蛋白包多个(例如，2个，3个、4个、5个、6个、7个或8个)权利要求1-6任一项所述的分离的多肽，并且，所述多个分离的多肽各不相同、部分相同或完全相同；
    优选地，各个所述分离的多肽之间任选地通过相同或不同的肽接头连接；例如，所述肽接头各自独立地具有如SEQ ID NO:6或18所示的氨基酸序列；
    优选地，所述重组蛋白在其N端或C端进一步包含半胱氨酸；优选地，所述重组蛋白在其C端进一步包含半胱氨酸；优选地，所述半胱氨酸任选地通过接头(例如包含一个或多个甘氨酸(G)的柔性肽，例如GGGG)与所述分离的多肽连接；
    优选地，所述重组蛋白包含如SEQ ID NO:2、7、11、13、15、19-21任一项所示的氨基酸序列或由其组成；
    优选地，所述重组蛋白还连接有另外的多肽，另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任何组合；
    优选地，所述重组蛋白在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合；
    优选地，所述重组蛋白在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或，pH 3.8-4.3)的范围内与IgG Fc结构域解离。
  8. 二聚体或多聚体，其包含两个或多个(例如，2个，3个、4个、5个、6个、7个或8 个)权利要求1-6任一项所述的分离的多肽；
    优选地，所述两个或多个(例如，2个，3个、4个、5个、6个、7个或8个)分离的多肽各不相同、部分相同或完全相同；
    优选地，所述二聚体或多聚体不包含野生型Protein A的B结构域的氨基酸序列；
    优选地，所述二聚体或多聚体不包含如SEQ ID NO:4所示的氨基酸序列；
    优选地，所述二聚体或多聚体包含至少两条多肽链，所述两个或多个分离的多肽存在于至少两条多肽链中；
    优选地，所述分离的多肽之间通过接头可逆或不可逆地连接形成二聚体或多聚体；
    优选地，所述二聚体或多聚体中至少一条多肽链在其N端或C端进一步包含半胱氨酸；优选地，所述多肽链在其C端进一步包含半胱氨酸；优选地，所述半胱氨酸任选地通过接头(例如包含一个或多个甘氨酸(G)的柔性肽，例如GGGG)与所述分离的多肽连接；
    优选地，所述二聚体或多聚体中至少一条多肽链还连接有另外的多肽，另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任何组合；
    优选地，所述二聚体或多聚体在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合；
    优选地，所述二聚体或多聚体在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或，pH 3.8-4.3)的范围内与IgG Fc结构域解离。
  9. 偶联物，其包含固相支持物以及与所述固相支持物连接的权利要求1-6任一项所述的分离的多肽，权利要求7所述的重组蛋白，和/或，权利要求8所述的二聚体或多聚体；
    优选地，所述分离的多肽、重组蛋白或二聚体或多聚体直接或间接、可逆或不可逆地与固相支持物相连接；
    优选地，所述分离的多肽、重组蛋白或二聚体或多聚体通过共价和/或非共价键与所述固相支持物连接；
    优选地，所述固相支持物选自琼脂糖微粒、高分子聚合物微球、硅胶微球或磁珠；
    优选地，所述分离的多肽、重组蛋白或二聚体或多聚体通过其中的半胱氨酸与所述固相支持物之间形成二硫键连接；
    优选地，所述偶联物用于纯化免疫球蛋白；
    优选地，所述偶联物用于纯化含IgG Fc结构域的样品；优选地，所述含IgG Fc结构域的样品为IgG抗体或其重链，含IgG Fc结构域的双特异性或多特异性抗体，和/或，含IgG Fc结构域的纳米抗体；
    优选地，所述含IgG Fc结构域的样品为含IgG Fc结构域的双特异性或多特异性抗体；
    优选地，所述偶联物是亲和层析填料；
    优选地，所述偶联物在pH 7.0-8.0(例如，pH 7.0-7.6，pH 7.0-7.4)的范围内与IgG Fc结构域特异性结合；
    优选地，所述偶联物在pH 2.7-4.5(例如，pH 2.7-4.0，pH 3.5-4.5，或，pH 3.8-4.3)的范围内与IgG Fc结构域解离。
  10. 分离的核酸分子，其编码权利要求1-6任一项所述的分离的多肽或权利要求7所述的重组蛋白或权利要求8所述的二聚体或多聚体或其所包含的多肽链。
  11. 载体，其包含权利要求10所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  12. 宿主细胞，其包含权利要求10所述的核酸分子或权利要求11所述的载体。
  13. 制备权利要求1-6任一项所述的分离的多肽或权利要求7所述的重组蛋白或权利要求8所述的二聚体或多聚体或其所包含的多肽链的方法，其包括，在允许蛋白表达的条件下，培养权利要求12所述的宿主细胞，和从培养的宿主细胞培养物中回收所述分离的多肽或所述重组蛋白或所述二聚体或多聚体或其所包含的多肽链。
  14. 制备权利要求9所述的偶联物的方法，其包括将权利要求1-6任一项所述的分离的多肽，权利要求7所述的重组蛋白，和/或，权利要求8所述的二聚体或多聚体与固相支持物形成连接的步骤；
    优选地，所述方法包括：将所述分离的多肽，重组蛋白，和/或，二聚体或多聚体与所述固相支持物相接触，从而使所述分离的多肽，重组蛋白，和/或，二聚体或多聚体与所述固相支持物之间形成连接；
    优选地，所述连接是直接的或间接的，可逆或不可逆的；
    优选地，所述连接是共价连接或非共价连接；
    优选地，所述固相支持物选自琼脂糖微粒、高分子聚合物微球、硅胶微球或磁珠；
    优选地，所述连接包含二硫键。
  15. 权利要求1-6任一项所述的分离的多肽，或权利要求7所述的重组蛋白，或权利要求8所述的二聚体或多聚体，或权利要求9所述的偶联物用于制备纯化试剂的用途，所述纯 化试剂用于纯化免疫球蛋白；
    优选地，所述纯化试剂用于纯化含IgG Fc结构域的样品；
    优选地，所述含IgG Fc结构域的样品为IgG抗体或其重链，含IgG Fc结构域的双特异性或多特异性抗体，和/或，含IgG Fc结构域的纳米抗体；
    优选地，所述含IgG Fc结构域的样品为含IgG Fc结构域的双特异性或多特异性抗体。
  16. 纯化免疫球蛋白的方法，其包括使用权利要求9所述的偶联物；
    优选地，所述纯化是亲和层析；优选地，所述亲和层析包括使用权利要求9所述的偶联物作为固定相；
    优选地，所述方法用于纯化含IgG Fc结构域的样品；
    优选地，所述方法包含以下步骤：
    (1)在第一溶剂中，使所述含IgG Fc结构域的样品与所述偶联物结合形成复合物；
    (2)在第二溶剂中，使所述复合物解离；和，
    (3)收集含有所述含IgG Fc结构域的样品的解离产物；
    优选地，所述第一溶剂的pH为7.0-8.0；例如，所述第一溶剂的pH为7.0-7.6或7.0-7.4；
    优选地，所述第二溶剂的pH为2.7-4.5；例如，所述第二溶剂的pH为2.7-4.0、3.5-4.5或3.8-4.3。
  17. 试剂盒，其包含权利要求1-6任一项所述的分离的多肽，或权利要求7所述的重组蛋白，或权利要求8所述的二聚体或多聚体，或权利要求9所述的偶联物；
    优选地，所述试剂盒进一步包含蛋白纯化所用试剂(例如，如权利要求16中所定义的第一溶剂，和/或，如权利要求16中所定义的第二溶剂)；优选地，所述纯化是亲和层析；
    优选地，所述试剂盒用于纯化免疫球蛋白；
    优选地，所述试剂盒用于纯化含IgG Fc结构域的样品；
    优选地，所述含IgG Fc结构域的样品为IgG抗体或其重链，含IgG Fc结构域的双特异性或多特异性抗体，和/或，含IgG Fc结构域的纳米抗体；
    优选地，所述含IgG Fc结构域的样品为含IgG Fc结构域的双特异性或多特异性抗体。
PCT/CN2023/118531 2022-09-13 2023-09-13 免疫球蛋白结合蛋白及其应用 WO2024055988A1 (zh)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102516371A (zh) * 2002-03-25 2012-06-27 通用电气健康护理生物科学股份公司 一种突变的免疫球蛋白-结合蛋白
CN103037885A (zh) * 2010-07-02 2013-04-10 芝加哥大学 与蛋白A（SpA）变体相关的组合物和方法
US20150152195A1 (en) * 2012-06-14 2015-06-04 Daiso Co., Ltd. Carrier for antibody purification, manufacturing method for same, and application for same
CN105377880A (zh) * 2013-07-10 2016-03-02 通用电气健康护理生物科学股份公司 突变的免疫球蛋白结合多肽
CN107001432A (zh) * 2014-11-17 2017-08-01 通用电气医疗集团生物工艺研发股份公司 突变的免疫球蛋白结合多肽
CN107922483A (zh) * 2015-07-16 2018-04-17 纳维格蛋白质有限公司 新型免疫球蛋白结合蛋白及其在亲和纯化中的用途
CN109219613A (zh) * 2016-05-11 2019-01-15 通用电气医疗集团生物工艺研发股份公司 分离方法
CN110799521A (zh) * 2017-02-15 2020-02-14 拜奥普罗塞亚科技有限责任公司 具有温和洗脱pH的亲和色谱配体
CN112639099A (zh) * 2018-08-24 2021-04-09 Jsr株式会社 免疫球蛋白结合蛋白质以及使用其的亲和载体

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102516371A (zh) * 2002-03-25 2012-06-27 通用电气健康护理生物科学股份公司 一种突变的免疫球蛋白-结合蛋白
CN103037885A (zh) * 2010-07-02 2013-04-10 芝加哥大学 与蛋白A（SpA）变体相关的组合物和方法
US20150152195A1 (en) * 2012-06-14 2015-06-04 Daiso Co., Ltd. Carrier for antibody purification, manufacturing method for same, and application for same
CN105377880A (zh) * 2013-07-10 2016-03-02 通用电气健康护理生物科学股份公司 突变的免疫球蛋白结合多肽
CN107001432A (zh) * 2014-11-17 2017-08-01 通用电气医疗集团生物工艺研发股份公司 突变的免疫球蛋白结合多肽
CN107922483A (zh) * 2015-07-16 2018-04-17 纳维格蛋白质有限公司 新型免疫球蛋白结合蛋白及其在亲和纯化中的用途
CN109219613A (zh) * 2016-05-11 2019-01-15 通用电气医疗集团生物工艺研发股份公司 分离方法
CN110799521A (zh) * 2017-02-15 2020-02-14 拜奥普罗塞亚科技有限责任公司 具有温和洗脱pH的亲和色谱配体
CN112639099A (zh) * 2018-08-24 2021-04-09 Jsr株式会社 免疫球蛋白结合蛋白质以及使用其的亲和载体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIN ZIYU, DING YINGYING; ZHOU PENG: "Construction And Evolutional Selection of A Combinatorial Phage Library Displaying Randomly-Rearranged Mutant Binding Domains of SPA", ACTA UNIVERSITATIS MEDICINALIS ANHUI, vol. 50, no. 9, 30 September 2015 (2015-09-30), pages 1262 - 1267, XP093146253, ISSN: 1000-1492, DOI: 10.19405/j.cnki.issn1000-1492.2015.09.013 *
MEININGER, D. P. ET AL.: "Characterization of the binding interface between the E-domain of Staphylococcal protein A and an antibody Fv-fragment", BIOCHEMISTRY, vol. 39, no. 1, 11 January 2000 (2000-01-11), pages 26 - 36, XP001008600, DOI: 10.1021/bi9920174 *

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