WO2020177719A1 - 展示与分泌目的多肽的酵母展示系统及其用途 - Google Patents

展示与分泌目的多肽的酵母展示系统及其用途 Download PDF

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WO2020177719A1
WO2020177719A1 PCT/CN2020/077798 CN2020077798W WO2020177719A1 WO 2020177719 A1 WO2020177719 A1 WO 2020177719A1 CN 2020077798 W CN2020077798 W CN 2020077798W WO 2020177719 A1 WO2020177719 A1 WO 2020177719A1
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nucleic acid
acid molecule
polypeptide
saccharomyces cerevisiae
antibody
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PCT/CN2020/077798
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English (en)
French (fr)
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顾春银
王旭辉
康立山
刘军建
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信达生物制药(苏州)有限公司
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Priority to CN202080018092.XA priority Critical patent/CN113544275A/zh
Publication of WO2020177719A1 publication Critical patent/WO2020177719A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • 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
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the present invention relates to a yeast display system capable of displaying a target polypeptide on the surface of yeast cells and secreting the target polypeptide into a culture medium, and the use of the yeast display system.
  • Yeast display system of the present invention can be constructed of the order of 108 variant polypeptide stably display libraries, in a manner which enables the cell surface display by means of high-throughput screening to select a particular object of the polypeptide variant, secreted into the medium in such a way This enables biochemical characterization of polypeptide variants of interest; in particular, the yeast display system of the present invention can be used to construct antibody variant display libraries, so as to obtain high-affinity antibody variants.
  • the display The Pichia pastoris display system that secretes the target peptide cannot meet the requirements of library diversity for antibody discovery and antibody engineering.
  • the yeast display system in the prior art directly displays the Fc domain containing the hinge region, which causes the Fc domain containing the hinge region to easily form a homodimer by itself, and reduces the level of antibody display on the yeast surface.
  • the system can not only stably construct a large-scale target polypeptide variant display library, but also can easily perform the target polypeptide variant by testing the culture medium. Biochemical characterization.
  • the inventors have developed a cell display system of Saccharomyces cerevisiae, which can not only display the target polypeptide on the surface of Saccharomyces cerevisiae cells, but also secrete the target polypeptide into the culture medium.
  • yeast display system of the present invention it is possible to stably construct the order of 108 variant polypeptide display libraries, it is possible to meet the antibodies found engineered antibody of claim library diversity.
  • the present invention provides a yeast display system, which is a Saccharomyces cerevisiae cell into which a first nucleic acid molecule and a second nucleic acid molecule have been introduced, wherein the first nucleic acid molecule contains an encoding Saccharomyces cerevisiae
  • the first nucleic acid molecule and the second nucleic acid molecule are located on the same plasmid or on separate plasmids.
  • the first fusion protein comprising the anchor protein on the cell surface of Saccharomyces cerevisiae and the CH3 domain of the immunoglobulin Fc region is anchored on the cell surface of Saccharomyces cerevisiae through the anchor protein. on.
  • the yeast display system of the present invention expresses the second nucleic acid molecule, and immunizes a part of the second fusion protein containing the target polypeptide and the immunoglobulin Fc region or part thereof through the immunoglobulin Fc region or part thereof and the first fusion protein
  • the CH3 domain of the globulin Fc region associates stably to display the target polypeptide on the surface of Saccharomyces cerevisiae cells; the other part of the second fusion protein is kept in the culture medium.
  • the present invention provides such a yeast display system, which is a recombinant Saccharomyces cerevisiae cell, in which a first nucleic acid molecule is inserted at a target site in the genome of the Saccharomyces cerevisiae cell, the first nucleic acid molecule comprising Nucleotides encoding Saccharomyces cerevisiae cell surface anchor proteins and CH3 domains of immunoglobulin Fc regions, the recombinant Saccharomyces cerevisiae cells are used to introduce nucleotides containing polypeptides of interest and encoding immunoglobulin Fc regions or parts
  • the second nucleic acid molecule of nucleotides to display and secrete the polypeptide of interest on the cell surface.
  • the present invention provides such a yeast display system, wherein the first nucleic acid molecule is inserted into the URA3 locus in the Saccharomyces cerevisiae cell genome to obtain URA3 - auxotrophic marker Saccharomyces cerevisiae cells, It is used to introduce a second nucleic acid molecule comprising nucleotides encoding the polypeptide of interest and nucleotides encoding the Fc region of an immunoglobulin or a portion thereof to display and secrete the polypeptide of interest on the cell surface.
  • the first fusion protein containing the anchor protein on the cell surface of Saccharomyces cerevisiae and the CH3 domain of the immunoglobulin Fc region is anchored to the recombinant Saccharomyces cerevisiae cell through the anchor protein. On the surface.
  • the yeast display system of the present invention expresses the second nucleic acid molecule, and immunizes a part of the second fusion protein containing the target polypeptide and the immunoglobulin Fc region or part thereof through the immunoglobulin Fc region or part thereof and the first fusion protein
  • the CH3 domain of the globulin Fc region associates stably to display the target polypeptide on the surface of Saccharomyces cerevisiae cells; the other part of the second fusion protein is kept in the culture medium.
  • the Fc regions of the first nucleic acid molecule and the second nucleic acid molecule respectively comprise protrusions ("knobs") Or a hole ("hole”), whereby the first polypeptide chain expressed by the first nucleic acid molecule and the second polypeptide chain expressed by the second nucleic acid molecule can form a "knob-in -hole)” stable association.
  • one of the first polypeptide chain and the second polypeptide chain encoded by the first nucleic acid molecule and the second nucleic acid molecule contains an amino acid substitution T366W, and the first polypeptide chain and the second polypeptide chain
  • the other chain of the chain contains amino acid substitutions T366S, L368A and Y407V (according to Kabat’s "EU numbering"), whereby the bumps in one chain can be placed in the holes in the other chain, thereby promoting the first The association of the peptide chain and the second polypeptide chain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, preferably, the immunoglobulin is a human IgG1 immunoglobulin protein.
  • the second nucleic acid molecule comprises a nucleotide encoding a polypeptide of interest, optionally a hinge region encoding an immunoglobulin Fc region And nucleotides encoding the CH2 domain and CH3 domain of the immunoglobulin Fc region; preferably, the glycosylation site in the CH2 domain is eliminated, for example, the human IgG Fc region
  • the N297 residue in the CH2 domain of the CH2 domain is mutated to eliminate the glycosylation site, for example, the N297 residue is changed to Gly, Ala, Gln, Asp or Glu, preferably, the N297 residue is changed to Ala.
  • the Saccharomyces cerevisiae cell surface anchoring protein is Saccharomyces cerevisiae containing glycosylphosphatidylinositol (GPI) anchoring signal sequence Cell wall proteins, for example, ⁇ -lectin and ⁇ -lectin, Cwp1p protein and Flo1p protein.
  • GPI glycosylphosphatidylinositol
  • the Saccharomyces cerevisiae itself is an a-lectin-expressing aga1p subunit.
  • Saccharomyces cerevisiae such as Saccharomyces cerevisiae EBY100; the Saccharomyces cerevisiae cell surface anchoring protein expressed by the first nucleic acid molecule introduced into the Saccharomyces cerevisiae cell is the aga2p subunit, thus, the first polypeptide encoded by the first nucleic acid molecule comprises aga2p And the CH3 domain of the immunoglobulin Fc region, and the first polypeptide binds to the aga1p subunit that has been bound and presented on the surface of Saccharomyces cerevisiae cells.
  • the polypeptide of interest is an antibody or an antigen-binding fragment, for example, a Fab fragment, a VHH domain, scFv, sdAb.
  • the present invention provides such a yeast display system, wherein
  • the first nucleic acid molecule contains nucleotides encoding CH3 knob -optionally linker or tag-Aga2p from N-terminus to C-terminus, and the second nucleic acid molecule contains the encoding target polypeptide from N-terminus to C-terminus -Optionally the nucleotides of the hinge region-CH2(N297A)-CH3 hole ; or
  • the first nucleic acid molecule contains nucleotides encoding CH3 hole -optionally linker or tag-Aga2p from N-terminus to C-terminus
  • the second nucleic acid molecule contains the encoding target polypeptide from N-terminus to C-terminus -Optionally the hinge region-CH2 (N297A)-the nucleotides of the CH3 knob .
  • the linker comprises glycine (G) and serine (S) residues, for example, the linker is GS.
  • the tag is selected from Arg tag, Avi tag, His-Avi tag, His tag, Flag tag, 3xFlag tag, Strep tag, Nano tag, SBP tag, c-myc tag, S tag, calmodulin binding peptide , Cellulose binding domain, chitin binding domain, GST tag or MBP tag.
  • the present invention provides the use of the yeast display system of the first and second aspects of the present invention for expressing the target polypeptide displayed on the cell surface and secreted into the culture medium; preferably, for expression Antibodies displayed on the surface of cells and secreted into the culture medium.
  • the present invention provides the use of the yeast display system of the first and second aspects of the present invention to construct a variant display library of the target polypeptide, wherein the cell surface display method enables high-throughput screening
  • the method of selecting a specific target polypeptide variant and secreting it into the culture medium enables the biochemical characterization of the target polypeptide variant; preferably, it is used to construct an antibody variant display library to screen for high-affinity antibody variants.
  • Figure 1 A schematic diagram showing the display of antibodies by Saccharomyces cerevisiae on the cell surface through FC.
  • FIG. 1 shows the effect of different lengths of FC knobs on the display of antibodies by Saccharomyces cerevisiae on the cell surface.
  • Figure 3 A schematic diagram showing the flow cytometry staining of the antibody secreted into the culture fluid on the yeast cells displaying the antigen.
  • Figure 4 Shows the results of flow cytometry staining of antigen-displaying yeast cells with antibodies secreted into the culture medium.
  • Figure 5 A schematic diagram showing the modification of yeast to display CH3 knob on the cell surface.
  • Figure 6 shows the results of cell-surface display CH3 knob engineered yeast display CH3 knob IDY104 detection level.
  • Figure 7 Shows the flow cytometry detection of the level of antibodies displayed by yeast IDY104 transformed into plasmid pYDC042.
  • Figure 8 shows the results of the affinity determination of the culture supernatant sample of the yeast IDY104 transformed into the plasmid pYDC042.
  • Figure 9 shows the results of the determination of the level of the sdAb-Fc format antibody or scFv-Fc format antibody displayed by the yeast IDY104.
  • Figure 10 shows the results of the affinity determination of the culture supernatant of yeast IDY104 after 10 times of concentration.
  • Figure 11 shows the flow cytometry staining image showing the binding of different Spiking libraries of AmNB1613.36 and HzNB1613 to antigen.
  • Figure 12 shows the proportion of yeast displaying AmNB1613.36 after each round of screening of the Spiking library.
  • R0 represents the 0th round of screening
  • R1 represents the 1st round of screening
  • R2 represents the 2nd round of screening.
  • antibody is used in the broadest sense herein and refers to a protein containing an antigen-binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (for example, bispecific antibodies), single chain antibodies, whole antibodies, and antigen-binding fragments.
  • the terms “whole antibody”, “full-length antibody”, “full antibody” and “whole antibody” are used interchangeably herein to refer to at least two heavy chains (H) and two Light chain (L) glycoprotein.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • the VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDR)), with more conservative regions (framework regions (FR)) interposed between them.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four
  • the FR composition is arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but displays a variety of effector functions.
  • antigen-binding fragment refers to a part or segment of a complete or complete antibody that has fewer amino acid residues than a complete or complete antibody, which can bind to an antigen or a complete antibody (ie, the complete antibody from which the antigen-binding fragment is derived) Competition for antigen binding.
  • the antigen-binding fragment can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of the intact antibody.
  • Antigen-binding fragments include but are not limited to Fab, Fab', F(ab') 2 , Fv, single chain Fv, diabody, single domain antibody (sdAb).
  • the Fab fragment is a monovalent fragment composed of VL, VH, CL and CH1 domains.
  • the Fab fragment can be obtained by papain digestion of a complete antibody.
  • pepsin digests the complete antibody under the disulfide bond in the hinge region to produce F(ab') 2 , which is a dimer of Fab' and a bivalent antigen-binding fragment.
  • F(ab') 2 can be reduced by breaking the disulfide bond in the hinge region under neutral conditions, thus converting the F(ab') 2 dimer into Fab' monomer.
  • the Fab' monomer is basically a Fab fragment with a hinge region (for a more detailed description of other antigen-binding fragments, please refer to: Fundamental Immunology, edited by WEPaul, Raven Press, NY (1993)).
  • the Fv fragment is composed of the VL and VH domains of one arm of the antibody.
  • the two domains VL and VH of the Fv fragment are encoded by independent genes, using recombination methods, they can be connected by a synthetic linking peptide that can produce these two domains as a single protein chain.
  • the antigen-binding fragment can be obtained by chemical methods, recombinant DNA methods or protease digestion methods.
  • single domain antibody generally refers to an antibody in which a single variable domain (e.g., heavy chain variable domain (VH) or light chain variable domain (VL), derived from camelid heavy
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the heavy chain variable domain of the chain antibody, the VH-like single domain (v-NAR) derived from fish IgNAR) can confer antigen binding. That is, the single variable domain does not need to interact with another variable domain to recognize the target antigen.
  • single-domain antibodies include single-domain antibodies (WO 2005/035572) derived from camelids (llamas and camels) and cartilaginous fishes (such as nurse sharks).
  • the heavy chain variable domain of the camelid heavy chain antibody with high affinity to the target antigen can be obtained by genetic engineering methods (this region is also called VHH, and its molecular weight is one-tenth of that of a human IgG molecule, and has only A few nanometers of physical diameter). See U.S. Patent No. 5,759,808 issued on June 2, 1998.
  • the amino acid sequence of Camelidae VHH can be recombinantly changed to obtain a sequence that mimics the human sequence more realistically, that is, "humanization", thereby reducing the antigenicity of Camelidae VHH to humans.
  • the key elements derived from Camelidae VHH can also be transferred to the human VH domain to obtain a camelized human VH domain.
  • the human VH domain no longer needs to be paired with the VL domain to recognize the target antigen.
  • the modified human VH domain alone can confer antigen binding specificity.
  • the camelid VHH, humanized camelid VHH, and camelized human VH domains are all within the scope of the term "VHH domain".
  • immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
  • IgG immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide bonded. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable domain (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL).
  • VH heavy chain variable domain
  • CL light chain constant domain
  • the heavy chains of immunoglobulins can belong to one of five categories, called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG) or ⁇ (IgM), some of which can be further divided into sub-classes. Class, for example ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG 3 ), ⁇ 4 (IgG 4 ), ⁇ 1 (IgA 1 ), and ⁇ 2 (IgA 2 ).
  • the light chains of immunoglobulins can be divided into one of two types based on the amino acid sequence of their constant domains, called kappa and lambda.
  • An immunoglobulin basically consists of two Fab molecules and an Fc domain connected by the hinge region of an immunoglobulin.
  • Fc domain Fc region
  • FC FC
  • the terms "Fc domain”, “Fc region” or “FC” are used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the natural immunoglobulin "Fc domain” contains two or three constant domains, namely the CH2 domain, the CH3 domain and the optional CH4 domain.
  • the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes; or contains the source From the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of the two heavy chains of IgM and IgE antibodies.
  • the numbering of amino acid residues in the Fc region or the heavy chain constant region is based on, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also called the EU index) is numbered.
  • effector function refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with the isotype of the immunoglobulin.
  • immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) , Cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors) and B cell activation.
  • binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or biofilm layer interference technology or other conventional binding assays known in the art.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • biofilm layer interference technology or other conventional binding assays known in the art.
  • PD-L1 programmed cell death ligand 1
  • programmed cell death ligand 1 refers to any natural PD-L1 from any vertebrate source, including Mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • the term encompasses "full length", unprocessed PD-L1 and any form of PD-L1 produced by processing in the cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
  • the basic structure of PD-L1 includes 4 domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • anti-PD-L1 antibody refers to an antibody that can bind to PD with sufficient affinity -L1 protein or fragments thereof.
  • the degree of binding of the anti-PD-L1 antibody to the non-PD-L1 protein is less than about 10%, about 20%, about 30%, about 40%, about 50% of the binding of the antibody to PD-L1 , About 60%, about 70%, about 80%, or about 90% or more, for example, measured by radioimmunoassay (RIA) or bio-optical interferometry or SPR or biofilm layer interference.
  • RIA radioimmunoassay
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between the members of a binding pair.
  • the affinity of a molecule X to its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on, respectively ). Affinity can be measured by common methods known in the art.
  • variant refers to a modification in the parent amino acid sequence.
  • exemplary modifications include amino acid substitutions, insertions and/or deletions.
  • the polypeptide variant of interest is a substitution of the amino acid sequence of the parent polypeptide of interest.
  • Amino acid substitutions herein encompass substitutions with one or more naturally occurring and/or non-naturally occurring amino acid residues.
  • “Naturally occurring amino acid residues” are selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine Acid (Cys); Glutamine (Gln); Glutamic acid (Glu); Glycine (Gly); Histidine (His); Isoleucine (Ile): Leucine (Leu); Lysine ( Lys); Methionine (Met); Phenylalanine (Phe); Proline (Pro); Serine (Ser); Threonine (Thr); Tryptophan (Trp); Tyrosine (Tyr ); and valine (Val).
  • Non-naturally-occurring amino acid residues refer to residues that are capable of covalently binding adjacent amino acid residues in a polypeptide chain in addition to those naturally-occurring amino acid residues listed above.
  • Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, ⁇ -aminoisobutyric acid (aib) and other amino acid residue analogs, as described in Ellman et al., Those described in Meth. Enzym. 202 (1991) 301-336.
  • variable in relation to an antibody refers to herein, including at least one, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acid substitutions or deletions.
  • an antibody inserted into the target antibody region with amino acid changes for example, heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region
  • the variant basically retains the antibody molecule before the change Biological characteristics.
  • the variable region of the heavy chain or the variable region of the light chain, or each CDR region of an antibody can be changed individually or in combination. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 amino acid changes in one or more or all three heavy chain CDRs Or 10.
  • the amino acid changes are amino acid substitutions, preferably conservative substitutions.
  • the antibody variant has at least 80%, 85%, 90% or 95% or 99% or higher amino acid sequence identity with the parent antibody in the antibody sequence region of interest.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ( "Antigen contact point”) area.
  • CDR is mainly responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • CDR CDR
  • CDR sequence covers CDR sequences determined in any of the above-mentioned ways.
  • linker refers to a linking peptide composed of amino acids, such as glycine and/or serine residues used alone or in combination to link various regions in the fusion protein.
  • the linker is a Gly/Ser linking peptide, including an amino acid sequence (Gly 1-4 Ser) n, where n is a positive integer equal to or greater than 1, for example, n is a positive integer from 1 to 7.
  • the linker is GS. Also included in the scope of the present invention are the connecting peptides described in WO2012/138475, which is incorporated herein by reference.
  • the amino acid sequence tag refers to a sequence of amino acid residues connected to each other by peptide bonds with specific binding properties.
  • the amino acid sequence tag is an affinity or purification tag.
  • the amino acid sequence tag is selected from the group consisting of Arg tag, His tag, Avi tag, His-Avi tag, Flag tag, 3xFlag tag, Strep tag, Nano tag, SBP tag, c-myc tag, S tag, Calmodulin Protein binding peptides, cellulose binding domains, chitin binding domains, GST tags and MBP tags (see, for example, Amau, J. et al., Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins , Protein Expr. Purif., 2006, 48(1): 1-13).
  • fused with refers to a covalent bond formed between two parts, for example, a peptide bond.
  • first fusion protein has the same meaning and are used interchangeably herein to refer to the polypeptide product expressed by the first nucleic acid molecule.
  • second fusion protein has the same meaning and are used interchangeably herein to refer to the polypeptide product expressed by the second nucleic acid molecule.
  • N-terminal refers to the last amino acid of the N-terminal
  • C-terminal refers to the last amino acid of the C-terminal
  • introduction refers to any method suitable for introducing or transferring a polynucleotide (such as a plasmid) into a yeast cell by any technique known in the art, such as transformation, transduction, transfection.
  • transformation is used to describe the introduction of polynucleotides into yeast cells.
  • transduction refers to viral introduction or viral transfer of polynucleotides or genetic material into cells.
  • Any known viral system can be used to transduce the host cell of the present invention, such as an adenovirus-based system, an adeno-associated virus (AAV)-based system, a retroviral system, a lentiviral expression system, or a herpes simplex virus-based system (HSV) systems, or systems based on other viruses, such as systems based on vaccinia, Epstein-Barr virus, Sendai virus, Sindbis virus, polyoma virus and measles virus (see, for example, Mah C et al., Virus-based gene delivery systems, Clin .Pharmocokin, 2002, 41(12): 901-911).
  • viruses such as systems based on vaccinia, Epstein-Barr virus, Sendai virus, Sindbis virus, polyoma virus and measles virus (see, for example, Mah C et al., Virus-based gene delivery systems, Clin .Pharmocokin, 2002, 41(12): 901-911).
  • transfection when used in the methods of the present invention refers to the ingestion of nucleic acids by host cells by any suitable method known in the art, for example, as disclosed in Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories , New York, Davis, etc. (1986) Basic Methods in Molecular Biology, Elsevier's method, especially calcium phosphate co-precipitation, direct microinjection into cultured cells, ultrasound-mediated gene transfection, electroporation, lipofection Or nuclear transfection (nucleofection).
  • expression vector represents a natural or artificial DNA sequence comprising at least one nucleic acid molecule sequence encoding an amino acid sequence of a polypeptide, a promoter sequence, a terminator sequence, a selection marker and an origin of replication, and an optional secretion signal sequence.
  • Saccharomyces cerevisiae is a well-characterized eukaryotic host organism in the field. As a model organism for studying eukaryotic cell functions, it is also suitable for expressing heterologous polypeptides of interest. As a single-celled organism, Saccharomyces cerevisiae is less complicated than other eukaryotic systems, and it can be cultured in a defined medium. Therefore, its growth conditions can be well controlled and its culture cost can be reduced. The relatively short life cycle of about 90 minutes is one reason for preferential use of Saccharomyces cerevisiae.
  • Saccharomyces cerevisiae combines the advantages of the microbial expression system due to simple cultivation and the use of industrial fermentation methods and the advantages of the eukaryotic expression system due to the eukaryotic expression and secretion pathways existing in the cell, and can be selected on a large scale, therefore,
  • the present invention uses Saccharomyces cerevisiae to express and display heterologous polypeptides of interest.
  • the present invention provides a yeast display system prepared by the following steps:
  • the first nucleic acid molecule and the second nucleic acid molecule into the Saccharomyces cerevisiae cell, wherein the first nucleic acid molecule comprises nucleotides encoding the CH3 domain of the Saccharomyces cerevisiae cell surface anchor protein and the immunoglobulin Fc region, the first Two nucleic acid molecules include nucleotides encoding the polypeptide of interest and nucleotides encoding the immunoglobulin Fc region or part thereof,
  • the expressed first fusion protein is anchored on the surface of Saccharomyces cerevisiae cells through the anchoring protein; a part of the expressed second fusion protein is linked to the immunoglobulin Fc of the first fusion protein through the immunoglobulin Fc region or part thereof
  • the CH3 domain of the region associates stably to display the target polypeptide on the surface of Saccharomyces cerevisiae cells; the other part of the expressed second fusion protein remains in the medium.
  • the yeast display system of the present invention can be constructed of the order of 108 variant polypeptide stably display libraries, in a manner which enables the cell surface display by means of high-throughput screening to select a particular object of the polypeptide variant, secreted into the medium
  • the method enables the biochemical characterization of the polypeptide variants of interest.
  • the first nucleic acid molecule and the second nucleic acid molecule can be introduced into Saccharomyces cerevisiae cells using any method known in the art.
  • any method known in the art according to the method described by Gietz, RD et al. (Gietz, RD and Schiestl, RH, High-efficiency, yeast, transformation, using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2007, 2(1) ): 31-34)
  • Gietz, RD et al. Gaetz, RD and Schiestl, RH, High-efficiency, yeast, transformation, using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2007, 2(1) ): 31-34
  • the existence form of the first nucleic acid molecule and the second nucleic acid molecule and the sequence of introduction into the cell are not particularly limited, as long as the first nucleic acid molecule and the second nucleic acid molecule can be expressed after being introduced into the Saccharomyces cerevisiae cells.
  • the first nucleic acid molecule and the second nucleic acid molecule are located on the same plasmid, and the plasmid containing the first nucleic acid molecule and the second nucleic acid molecule is introduced into Saccharomyces cerevisiae cells.
  • first nucleic acid molecule and the second nucleic acid molecule are located on separate plasmids, and the plasmid containing the first nucleic acid molecule and the plasmid containing the second nucleic acid molecule are respectively introduced into the Saccharomyces cerevisiae cells. , Or simultaneously introduce Saccharomyces cerevisiae cells.
  • plasmids and/or expression vectors for expressing recombinant proteins in Saccharomyces cerevisiae such as the vector p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768).
  • a pYDC vector eg, pYDC011 is used to introduce the first nucleic acid molecule and/or the second nucleic acid molecule into Saccharomyces cerevisiae.
  • Suitable promoters expressed in yeast include GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidine alcohol dehydrogenase), etc.
  • GAL1 galactose
  • PGK phosphoglycerate kinase
  • ADH alcohol dehydrogenase
  • AOX1 alcohol oxidase
  • HIS4 histidine alcohol dehydrogenase
  • the secretion of the first fusion protein and/or the second fusion protein from the yeast host cell can be increased by using any available secretion signal sequence of the yeast protein.
  • a secretion signal sequence is the leader sequence of the precursor alpha factor of yeast conjugation pheromone, which is also used to direct the secretion of heterologous proteins in yeast (see, for example, Valenzuela, P. Ed., pages 269-280, Butterworths , London; Brake, AJ (1990) Meth. Enzymol. 185, 408-441).
  • Another example is the leader sequence from yeast invertase (MLLQAFLFLLAGFAAKISADAHKS).
  • leader sequence when entering the endoplasmic reticulum, the leader sequence will be cleaved from the nascent heterologous peptide.
  • signal sequence of yeast acid phosphatase which can also be used to direct the secretion of the fusion protein disclosed herein.
  • the Saccharomyces cerevisiae cell surface anchoring protein in the first fusion protein is usually an extracellular wall protein of Saccharomyces cerevisiae.
  • Many extracellular wall proteins of Saccharomyces cerevisiae can in principle display heterologous proteins and peptides as part of the first fusion protein of the present invention on the cell surface, such as ⁇ -lectin and ⁇ -lectin, Cwp1p protein and Flo1p protein.
  • a-lectin is used as the S. cerevisiae cell surface anchoring protein in the first fusion protein.
  • the cell wall protein a-lectin contains the subunits Aga1p and Aga2p.
  • the subunit Aga1p has a glycosylphosphatidylinositol (GPI) anchoring signal sequence, and the covalent binding of ⁇ -glucan mediates the immobilization of the protein on the cell wall.
  • the subunit Aga2p is also secreted by the cell and is bonded to Aga1p via two disulfide bonds.
  • the Saccharomyces cerevisiae is a Saccharomyces cerevisiae expressing the Agalp subunit of a-lectin, such as Saccharomyces cerevisiae EBY100 species, which expresses the agalp subunit through a chromosomal integrated galactose-inducible expression cassette.
  • the cell surface anchoring protein of S. cerevisiae is the aga2p subunit.
  • the CH3 domain of the immunoglobulin Fc region in the first fusion protein is a non-antigen binding region located at the C-terminus of the immunoglobulin heavy chain.
  • the IgG CH3 domain starts with Gly341. It is understood that the C-terminal Lys residue of human IgG may optionally be absent. It should also be understood that conservative amino acid substitutions of the CH3 domain of the Fc region are contemplated within the scope of the present invention without affecting the desired structure and/or stability of the Fc.
  • the CH3 domain of the immunoglobulin Fc region in the first fusion protein of the present invention and the immunoglobulin Fc region or part thereof in the second fusion protein use "knob-in-hole” "Technology (see, for example, John BBRidgway et al.,'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization.Protein Engineering, 1996, 9(7): p.617-21; Shane Atwell et al., Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol, 1997, 270: p.
  • this technology can be used between the first fusion protein and the second fusion protein of the present invention
  • the interface is modified to promote the correct association of the first fusion protein and the second fusion protein of the present invention.
  • this technique involves introducing a "protrusion" at the interface of one of the fusion proteins, and introducing a corresponding "cavity" at the interface of the other fusion protein to be paired with, so that the protrusion can be placed in the cavity.
  • a preferred interface contains the CH3 domain of one of the fusion proteins and the CH3 domain of the other fusion protein to be paired with.
  • the bulge can be constructed by replacing the small amino acid side chain from the interface of the CH3 domain of one of the fusion proteins with larger side chains, such as tyrosine or tryptophan.
  • a compensating void with the same or similar size as the bulge is constructed at the interface of the CH3 domain of another fusion protein to be paired. hole.
  • the preferred residues used to form knobs are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan ( W). The most preferred are tryptophan and tyrosine.
  • the original residue used to form the knot has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.
  • Exemplary amino acid substitutions in the CH3 domain for knot formation include, but are not limited to, T366W, T366Y, or F405W substitutions.
  • Preferred residues for forming a hole are generally naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V).
  • the original residue used to form the button has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.
  • Exemplary amino acid substitutions in the CH3 domain used to create buckles include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions.
  • the knot comprises a T366W substitution
  • the buckle comprises a T366S/L368A/Y407V substitution.
  • one of the first polypeptide chain and the second polypeptide chain encoded by the first nucleic acid molecule and the second nucleic acid molecule of the present invention contains the amino acid substitution T366W
  • the first polypeptide chain and The other chain of the second polypeptide chain contains amino acid substitutions T366S, L368A, and Y407V (according to Kabat’s "EU numbering"), so the protrusions in one chain can be placed in the holes in the other chain, thus Promote the association of the first polypeptide chain and the second polypeptide chain.
  • the target polypeptide in the second fusion protein is not particularly limited.
  • the target polypeptide can be any polypeptide to be expressed.
  • the polypeptide of interest is an antibody or antigen-binding fragment, for example, Fab fragment, VHH domain, scFv, sdAb.
  • the immunoglobulin Fc region or part thereof in the second fusion protein includes the CH2 domain and the CH3 domain of the immunoglobulin Fc region, and optionally the immunoglobulin Fc region hinge region.
  • the immunoglobulin Fc region or portion thereof in the second fusion protein is changed to reduce the degree of glycosylation of the Fc portion of the second fusion protein.
  • One or more glycosylation sites can be removed by changing the amino acid sequence to conveniently delete glycosylation sites from the protein.
  • the N297 residue in the CH2 domain of the IgG Fc region is mutated to eliminate the glycosylation site, for example, the N297 residue is changed to Gly, Ala, Gln, Asp or Glu, preferably, the N297 residue is changed to Into Ala.
  • N297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of residues in the Fc region); however, N297 can also be located approximately ⁇ 3 amino acids upstream or downstream of position 297, ie, position 294 and position Between 300, the reason is the tiny sequence variation in immunoglobulin.
  • the present invention also provides such a yeast display system for cell surface display and secretion of the polypeptide of interest, which is a recombinant Saccharomyces cerevisiae cell, wherein the A first nucleic acid molecule is inserted at the target site, and the first nucleic acid molecule contains nucleotides encoding Saccharomyces cerevisiae cell surface anchor protein and immunoglobulin Fc region CH3 domain, and the recombinant Saccharomyces cerevisiae cell is used for introduction A second nucleic acid molecule comprising a nucleotide encoding a polypeptide of interest and a nucleotide encoding an immunoglobulin Fc region or a portion thereof, to display and secrete the polypeptide of interest on the cell surface.
  • the first nucleic acid molecule is introduced into the target site in the genome of the Saccharomyces cerevisiae cell.
  • the target site in the Saccharomyces cerevisiae cell genome into which the first nucleic acid molecule is introduced is not particularly limited, as long as the recombined Saccharomyces cerevisiae cells can express the first fusion protein.
  • the target site is a nutrient synthesis gene possessed by wild-type Saccharomyces cerevisiae.
  • the introduction of the first nucleic acid molecule into the nutrition synthesis gene in the cell genome of Saccharomyces cerevisiae results in impaired ability of the nutrition synthesis gene to synthesize nutrition, so that the recombinant Saccharomyces cerevisiae cell is an auxotrophic yeast and has nutritional requirements.
  • the so-called "yeast has nutritional requirements” means that the wild-type yeast has a mutation in the nutritional synthesis gene for some reason, resulting in the loss of the nutritional synthesis ability, and the corresponding nutrients need to be added to the medium to maintain the auxotrophic yeast Growth.
  • auxotrophic yeast As specific examples of essential nutrients for Saccharomyces cerevisiae, methionine, tyrosine, isoleucine, phenylalanine, glutamic acid, threonine, aspartic acid, valine, serine, and arginine are known. Acid, uracil, adenine, lysine, tryptophan, leucine, histidine, etc. Examples of impaired nutrition synthesis genes possessed by auxotrophic yeast include the following examples.
  • ⁇ Methionine requirement met1, met2, met3, met4, met5, met6, met7, met8, met10, met13, met14, met20
  • ⁇ Arginine requirement arg1, arg3, arg4, arg5, arg8, arg9, arg80, arg81, arg82, arg84
  • ⁇ Adenine requirement ade1, ade2, ade3, ade4, ade5, ade6, ade8, ade9, ade12, ADE15
  • ⁇ Histidine requirement his1, his2, his3, his4, his5, his6, his7, his8.
  • the target site in the genome of the Saccharomyces cerevisiae cell into which the first nucleic acid molecule is introduced in the present invention preferably uses the genomic site where the aforementioned nutrient synthesis gene is located.
  • the first nucleic acid molecule is inserted into the URA3 locus in the Saccharomyces cerevisiae cell genome to obtain URA3 - auxotrophic marker Saccharomyces cerevisiae cells.
  • the first nucleic acid molecule is inserted into the TRP1 locus in the genome of the Saccharomyces cerevisiae cell to obtain TRP1 -an auxotrophic marker Saccharomyces cerevisiae cell.
  • the other characteristics of the first nucleic acid molecule and the characteristics of the second nucleic acid molecule are the same as described above.
  • the present invention provides a method for constructing a target polypeptide variant display library, including:
  • a polypeptide variant display library of interest wherein the second nucleic acid molecule library and the first nucleic acid molecule described in Part II above are introduced into Saccharomyces cerevisiae cells, or the second nucleic acid molecule library is introduced into the recombinant described in Part II above
  • the Saccharomyces cerevisiae cell wherein a first nucleic acid molecule has been inserted at a target site in the genome of the Saccharomyces cerevisiae cell, and the first nucleic acid molecule comprises a CH3 domain encoding a Saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region Of nucleotides.
  • the gene library of the polypeptide variant of interest is generated by methods well known in the art, such as using error-prone polymerase chain reaction, using random primer technology, or using computer technology.
  • the designed gene library of the target polypeptide variant has a suitable restriction enzyme cleavage site to combine the gene library of the target polypeptide variant with the immunoglobulin Fc region or part thereof of the second nucleic acid molecule described in Part II above.
  • the nucleotide connection is also known in the art, such as using error-prone polymerase chain reaction, using random primer technology, or using computer technology.
  • the target polypeptide variant library is displayed on Saccharomyces cerevisiae cells, and a target polypeptide variant display library of about 108 orders of magnitude is obtained.
  • the library of target polypeptide variants displayed by yeast is screened.
  • the target polypeptide variant library is an antibody library with different affinities
  • all bound antigens are immobilized on the surface of yeast cells.
  • flow cytometry that is, FACS detection
  • yeast cells that display antibodies with high affinity can be sorted out and expanded by cloning. Increase and obtain a clonal cell line.
  • Example 1 The role of FC knobs of different lengths in antibody display
  • This example compares the effects of FCs of different lengths on antibody display.
  • Plasmids pYDC039, pYDC040 and pYDC041 are used to express fusion proteins of Aga2p and FC of different lengths respectively (the amino acid sequence of "hinge region-CH2-CH3" expressed in pYDC039 is shown in SEQ ID NO:1;
  • amino acid sequence of "CH2-CH3" expressed in pYDC040 is shown in SEQ ID NO: 2;
  • amino acid sequence of "CH3" expressed in pYDC041 is shown in SEQ ID NO: 3;
  • the plasmid pYDC042 expresses the fusion protein of the heavy chain variable region (V H H) AmNB1613.36 (SEQ ID NO: 4) and FC (SEQ ID NO: 5) against the PD-L1 antigen.
  • the sequence of the fusion protein is as shown in SEQ. ID NO: 6 shown.
  • nucleotide sequence of the insert fragment of each plasmid in Table 1 was submitted to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize.
  • the specific amino acid sequence encoded by each nucleotide sequence is shown in SEQ ID NO: 1, 2, 3 and 6.
  • the nucleotide sequences encoding SEQ ID NOs: 1, 2 and 3 were digested with restriction enzyme BamHI and ligated into pYDC011 plasmid digested with restriction enzyme BamHI (the nucleotide sequence of pYDC011 plasmid is as SEQ ID NO : 7) between the two BamHI sites, replace the nucleotide sequence "GGaTCctgacatagtagggattataa" on the pYDC011 plasmid to obtain plasmids pYDC039, pYDC040 and pYDC041.
  • the plasmids are used to express fusion proteins of Aga2p and FC knobs of different lengths.
  • nucleotide sequence encoding SEQ ID NO: 6 was digested with the restriction enzyme BamHI, and ligated into the two BamHI sites of the pYDC011 plasmid digested with the restriction enzyme BamHI to replace the pYDC011 plasmid
  • the nucleotide sequence "GGaTCctgacatagtagggattataa” was used to obtain plasmid pYDC042, which was used to express the fusion protein of single domain antibody (sdAb) AmNB1613.36 and FC, that is, AmNB1613.36-hinge-CH2(N297A)-CH3 hole .
  • FC knobs of different lengths to display antibody staining of yeast cells
  • the induced cells were stained with PD-L1 antigen to analyze the level of antibodies displayed on the cell surface of Saccharomyces cerevisiae expressing FC knobs of different lengths.
  • the specific steps are as follows:
  • yeast cells transformed with the plasmid combination shown in Table 2 were transformed, by displaying an antibody CH3 knob significantly better than by a hinge region -CH2 (N297A) -CH3 knob or through the display of antibody CH2 (N297A) -CH3 knob Display antibody.
  • Example 2 The induced culture in Example 1 was centrifuged to obtain a supernatant.
  • the supernatant and yeast cells displaying PD-L1 (NP_054862.1, Phe 19-Arg 238) antigen (a plasmid encoding Aga2p-PD-L1) were transferred into the EBY100 strain, the encoding Aga2p -PD-L1 plasmid is obtained by digesting the nucleotide sequence encoding Aga2p-PD-L1 with restriction enzyme BamHI, and ligating it into pYDC011 plasmid digested with restriction enzyme BamHI), incubated and stained, as shown in Table 2.
  • the specific steps for the antibody content in the different supernatants of the yeast cells transformed with each plasmid combination after induction and centrifugation are as follows:
  • Example 2 Centrifuge the induced culture in Example 1 to obtain a supernatant. Take 100 ⁇ L of the supernatant and add it to the 96-well plate of step 1, and incubate at room temperature for 30 minutes;
  • the CH3 knob can significantly increase the amount of antibody secreted to the supernatant while displaying antibodies on the surface of yeast. From this, it can be determined that the CH3 knob is the most suitable length, which is used in the following examples.
  • Example 3 Modification and identification of yeast (IDY104) displaying CH3 knob
  • Example 1 From Example 1 and Example 2, it can be determined that the CH3 knob is the most suitable length. Therefore, as shown in FIG. 5, the nucleotide fragment encoding CH3 knob- Aga2p is inserted into the URA3 gene locus in the genome of Saccharomyces cerevisiae EBY100. Specific steps are as follows:
  • cerevisiae strain EBY100 available from ATCC
  • step 3 Pick the newly grown single colonies from each plate in step 2, and streak on the SD-Ura (Clontech, 630315) plate and on the YPD+G418 plate respectively, and culture at 30°C for 3 days;
  • mouse anti-Flag M2 (sigma, item number: F1804, 1:1000 dilution) to each sample, and incubate at room temperature for 30 minutes;
  • Example 4 Detection of antibody level displayed by yeast IDY104 and affinity determination of culture supernatant
  • the yeast IDY104 obtained in Example 3 has the ability to directly display the CH3 knob . Further, the plasmid pYDC042 (containing the nucleotide sequence encoding the AmNB1613.36-hinge region-CH2(N297A)-CH3 hole polypeptide shown in SEQ ID NO: 6 was transformed into yeast IDY104. The expressed fusion protein AmNB1613.36 -Hinge area-CH2(N297A)-CH3 hole interacts with the CH3 knob on the surface of yeast cells to indirectly display the anti-PD-L1 antibody AmNB1613.36 on the surface of yeast cells, and secrete a small amount of antibodies into the culture medium.
  • yeast cells displaying the anti-PD-L1 antibody AmNB1613.36 on the surface can be used for flow cytometry analysis and sorting, and the anti-PD-L1 antibody AmNB1613 is secreted
  • the .36 culture medium can be used to determine the affinity of the antibody. The specific steps are as follows:
  • step 2 Take a culture medium containing about 1 ⁇ 10 6 cells, centrifuge at 3000 rpm for 3 minutes, use the supernatant in step 7, wash the cell pellet with 1 ⁇ PBSA once and use it in step 3;
  • step 7 Place the supernatant collected by centrifugation in step 1 in an ultrafiltration concentration tube (Shanghai Tuokai Biotechnology Co., Ltd., MCPM02C67), concentrate the supernatant 10 times, and perform affinity detection on the obtained concentrated solution.
  • an ultrafiltration concentration tube Shanghai Tuokai Biotechnology Co., Ltd., MCPM02C67
  • the equilibrium dissociation constant (K D ) of the antibody and the antigen in the supernatant and the 10-fold concentrated sample was determined by the biofilm layer interference (BLI) technique.
  • the BLI method affinity determination is implemented according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013, 5(2): p.270-8).
  • yeast IDY104 can be used to effectively display antibodies on the cell surface, and the displayed antibodies can effectively bind to the corresponding antigen; in addition, yeast IDY104 can also be used to effectively secrete antibodies to culture
  • the culture medium containing the antibody can be directly used for affinity determination as it is, or it can be used for affinity determination after being concentrated 10 times.
  • This example shows that yeast stably transformed with CH3 knob can effectively work as a two-in-one system for antibody display and secretion on the yeast surface.
  • Example 5 Comparison of the ability of strain IDY104 to display and secrete different forms of antibodies on the surface
  • Example 4 it can be seen from Example 4 that the two-in-one yeast surface display and secretion system of the present invention can effectively display and secrete single domain antibodies, such as the anti-PD-L1 antibody AmNB1613.36. This example further implements the expression of other forms of antibodies by the two-in-one yeast surface display and secretion system of the present invention.
  • the anti-human PD-L1 monoclonal antibody Hz4485 is constructed into the scFv-Fc form, and its amino acid sequence is as SEQ ID NO: 9 (the amino acid sequence of Hz4485 scFv encoded by pYDC083 plasmid is shown in SEQ ID NO: 9), and the nucleotide sequence encoding SEQ ID NO: 9 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the nucleotide sequence encoding SEQ ID NO: 9 was digested with the restriction enzyme BamHI and ligated into the pYDC081 plasmid digested with the restriction enzyme BamHI (the nucleotide sequence of the pYDC081 plasmid is shown in SEQ ID NO: 10) Replace the nucleotide sequence "GGaTCctgacatagtagggattataa" on the pYDC081 plasmid between the two BamHI sites of the pYDC081 to obtain the plasmid pYDC083.
  • the plasmid pYDC083 was transformed into yeast IDY104 and spread on the SD-Trp plate (Clontech, 630309); the SD-Trp plate coated with the transformed yeast strain was cultured at 30°C for 3 days to obtain a monoclonal yeast. Yeast culture and expression induction were performed as described in Example 4. The display and secretion levels of sdAb and scFv-FC antibodies of the two-in-one yeast surface display and secretion system of the present invention were compared. The results are shown in Figure 9 and Figure 10.
  • yeast IDY104 can effectively display and secrete antibody forms such as single domain antibody and scFv-FC, and the supernatant sample can be used for affinity determination.
  • Example 6 Evaluation of the feasibility of antibody affinity maturation using the Spiking library for the display and secretion two-in-one system
  • the two-in-one system of yeast surface display and secretion of the present invention is very suitable for screening antibody affinity maturation libraries.
  • the system of the present invention enables the supernatant of mutant clones obtained by screening to be directly used for antibody affinity determination, which saves additional steps of plasmid construction and mammalian cell expression, and greatly accelerates the progress of related projects in antibody affinity research .
  • the plasmid pYDC042 (containing the nucleotide sequence encoding the AmNB1613.36-hinge region-CH2(N297A)-CH3 hole polypeptide shown in SEQ ID NO: 6 was transformed into yeast IDY104
  • the expressed fusion protein AmNB1613.36-hinge-CH2(N297A)-CH3 hole interacts with the CH3 knob on the surface of yeast cells to indirectly display the anti-PD-L1 antibody AmNB1613.36 on the surface of yeast cells and secrete them at the same time. Add a small amount of AmNB1613.36 antibody to the culture medium.
  • SPR Surface plasmon resonance
  • the K D is determined by the capture method. After the antibody is captured by the anti-human Fc antibody on the chip, the affinity and kinetic constant are obtained by detecting the binding and dissociation between the antigen and the captured antibody.
  • the method includes chip preparation and affinity detection. In the measurement process, 10xHBS-EP + (BR-1006-69, GE Healthcare) diluted by 10 times was used as the experimental buffer.
  • the chip preparation process uses the amino coupling kit (BR-1006-33, GE Healthcare), and the anti-human Fc antibody is coupled to the surface of the CM5 chip (29-1496-03, GE Healthcare).
  • the specific process is as follows: 50mM N-hydroxysuccinimide (NHS) and 200mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) are freshly mixed and injected into the dual channel of CM5 chip. Activate for 7 minutes. Then, the anti-human Fc antibody was diluted in 10 mM acetic acid (pH 5.0) and injected into the dual channel of the CM5 chip to make the protein covalently coupled to the surface of the chip channel with a coupling height of about 6000 RU. Finally, 1M ethanolamine was injected, and the remaining activated sites were blocked for 7 minutes.
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • Each cycle of affinity detection includes capturing antibody, binding a concentration of antigen, and chip regeneration. The specific operation is described below.
  • Capture antibody First, the antibodies AmNB1613.36 and HzNB1613 are diluted to 0.5 ⁇ g/mL, and captured in the second channel of the CM5 chip at a flow rate of 10 ⁇ L/min, and the capture time is 30s.
  • Binding antigen According to the optimal concentration range of SPR, dilute human PD-L1 (AcroBiosystems, catalog number: PD1-H5229) twice with experimental buffer to make it between 0.15nM-20nM, in the order of low concentration to high concentration , Injection into CM5 chip dual channel, binding time 180s, dissociation time 600s.
  • Chip regeneration Use 10mM Glycine pH 1.5 (BR-1003-54, GE Healthcare) to regenerate the chip before proceeding to the next antibody measurement.
  • the data result uses 1:1 combination model for dynamic analysis.
  • PD-L1 antibody HzNB1613 binding affinity (K D) of 3.9 nM both having significant The difference in affinity.
  • the yeast cells displaying the anti-PD-L1 antibody HzNB1613 on the cell surface and the yeast cells displaying the anti-PD-L1 antibody AmNB1613.36 on the cell surface were mixed in different ratios to prepare a Spiking library.
  • the display and yeast cell display HzNB1613 yeast cells AmNB1613.36 of 2 1:10, 1: 104 were mixed, 1% Spiking a library prepared, 0.01% Spiking library.
  • Yeast cells displaying only AmNB1613.36, yeast cells displaying only HzNB1613, 1% Spiking library, and 0.01% Spiking library were respectively inoculated into SD-Leu and cultured overnight, and 1 mL of bacterial solution was transferred to 5 mL of YPGP medium for 24 hours; each Take 1 ⁇ 10 6 cells and stain with 10nM PD-L1 biotin antigen.
  • the results of flow cytometry are shown in Figure 11.
  • the yeast cell population framed in Figure 11 was sorted by flow cytometry, inoculated into SD-Leu and cultured overnight, and 1 mL of bacterial solution was transferred to 5 mL of YPGP medium for 24 hours; each 1 ⁇ 10 6 cells were used for 10 nM PD-L1 biotin antigen staining was carried out for two rounds of screening by flow cytometry. In each round, 24 monoclonal yeast cells were randomly selected for sequencing. According to the sequencing results, the proportion of yeast cells displaying AmNB1613.36 in the Spiking library was observed. The result is shown in Figure 12.

Abstract

提供了能够在细胞表面展示目的多肽并能够分泌目的多肽至培养基中的酵母展示系统、以及所述酵母展示系统的用途。所述酵母展示系统能够稳定地构建10 8数量级的目的多肽变体展示文库，其中在细胞表面展示的方式使得能够借助高通量筛选选择特定的目的多肽变体，分泌至培养基中的方式使得能够进行目的多肽变体的生物化学表征；所述酵母展示系统能够用于构建抗体的变体展示文库，从而获得高亲和力的抗体变体。

Description

展示与分泌目的多肽的酵母展示系统及其用途 技术领域
本发明涉及能够在酵母细胞表面展示目的多肽并能够分泌目的多肽至培养基中的酵母展示系统、以及所述酵母展示系统的用途。本发明的酵母展示系统能够稳定地构建10 8数量级的目的多肽变体展示文库，其中在细胞表面展示的方式使得能够借助高通量筛选选择特定的目的多肽变体，分泌至培养基中的方式使得能够进行目的多肽变体的生物化学表征；特别地，本发明的酵母展示系统能够用于构建抗体的变体展示文库，从而获得高亲和力的抗体变体。
背景技术
现有技术中报导了在细菌系统中产生蛋白质，但是，即使进行广泛的工程化，细菌系统也不能产生完全的人糖基化的蛋白质，因此，使用原核表达蛋白质可能无法预测该蛋白质在具有翻译后修饰如糖基化修饰的真核宿主中的功能。
本领域也尝试了在哺乳细胞系统中展示多肽，例如IgG，这需要向IgG重链引入额外的异位序列来将IgG直接锚定到细胞膜上(Zhou C等人，Development of a novel mammalian cell surface antibody display platform，MAbs，2010，2：508-518)，导致了额外的转化步骤以及进一步选择以实现IgG的可溶性表达。
Shaheen HH等人描述了在低等真核系统巴斯德毕赤酵母种(Pichia pastoris)的酵母细胞表面展示和分泌单克隆抗体(Shaheen HH等人，A Dual-Mode Surface Display System for the Maturation and Production of Monoclonal Antibodies in Glyco-Engineered Pichia pastoris，PLoS ONE，2013，8(7):e70190)。由于包含单克隆抗体编码核酸的质粒必需插入巴斯德毕赤酵母的基因组中才能稳定存在并表达单克隆抗体，且质粒插入巴斯德毕赤酵母基因组的效率较低，因此，所述的展示与分泌目的多肽的巴斯德毕赤酵母展示系统不能满足抗体发现与抗体工程改造对文库多样性的要求。
另外，现有技术中的酵母展示系统直接展示包含铰链区的Fc结构域，这造成了包含铰链区的Fc结构域容易自身形成同源二聚体，并减弱了抗体在酵母表面展示的水平。
因此，本领域仍然需要新的展示与分泌目的多肽的酵母展示系统，所述系统不仅能够稳定地构建大数量级的目的多肽变体展示文库，而且能够容易地通过测试培养液来进行目的多肽变体的生物化学表征。
发明概述
本发明人通过研究，开发了一种酿酒酵母(Saccharomyces cerevisiae)细胞展示系统，所述系统不仅能够在酿酒酵母细胞表面展示目的多肽，而且能够将目的多肽分泌至培养基中。通过利用本发明的酵母展示系统，能够稳定地构建10 8数量级的目的多肽变体展示文库，由此能够满足抗体发现与抗体工程改造对文库多样性的要求。
因此，在第一方面，本发明提供了一种酵母展示系统，其是导入有第一核酸分子和第二核酸分子的酿酒酵母(Saccharomyces cerevisiae)细胞，其中所述第一核酸分子包含编码酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的核苷酸，所述第二核酸分子包含编码目的多肽的核苷酸和编码免疫球蛋白Fc区或其部分的核苷酸，优选地，所述第一核酸分子和第二核酸分子位于同一个质粒上或位于分开的质粒上。
本发明的该酵母展示系统通过表达第一核酸分子，将包含酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的第一融合蛋白通过所述锚定蛋白锚定在酿酒酵母细胞表面上。本发明的该酵母展示系统通过表达第二核酸分子，将包含目的多肽和免疫球蛋白Fc区或其部分的第二融合蛋白的一部分通过免疫球蛋白Fc区或其部分与第一融合蛋白的免疫球蛋白Fc区CH3结构域稳定缔合而将目的多肽展示在酿酒酵母细胞表面；将所述第二融合蛋白的另一部分保留在培养基中。
在第二方面，本发明提供了这样的酵母展示系统，其是重组的酿酒酵母细胞，其中在酿酒酵母细胞的基因组中的靶位点处插入有第一核酸分子，所述第一核酸分子包含编码酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的核苷酸，所述重组的酿酒酵母细胞用于导入包含编码目的多肽的核苷酸和编码免疫球蛋白Fc区或其部分的核苷酸的第二核酸分子，以在细胞表面展示和分泌目的多肽。在一个具体实施方案中，本发明提供了这样的酵母展示系统，其中所述第一核酸分子插入至酿酒酵母细胞基因组中的URA3基因座处，获得了URA3 －营养缺陷型标记的酿酒酵母细胞，用于导入包含编码目的多肽的核苷酸和编码免疫球蛋白Fc区或其部分的核苷酸的第二核酸分子，以在细胞表面展示和分泌目的多肽。
本发明的该酵母展示系统通过表达第一核酸分子，将包含酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的第一融合蛋白通过所述锚定蛋白锚定在重组酿酒酵母细胞表面上。本发明的该酵母展示系统通过表达第二核酸分子，将包含目的多肽和免疫球蛋白Fc区或其部分的第二融合蛋白的一部分通过免疫球蛋白Fc区或其部分与第一融合蛋白的免疫球蛋白Fc区CH3结构域稳定缔合而将目的多肽展示在酿酒酵母细胞表面；将所述第二融合蛋白的另一部分保留在培养基中。
在一些实施方案中，对于本发明的第一方面和第二方面的酵母展示系统，所述第一核酸分子和第二核酸分子各自的Fc区中分别包含凸起(“结(knob)”)或空穴(“扣(hole)”)，由此所述第一核酸分子表达的第一多肽链和第二核酸分子表达的第二多肽链彼此能够形成“结入扣(knob-in-hole)”的稳定缔合。优选地，所述第一核酸分子和第二核酸分子编码的第一多肽链和第二多肽链之一条链中包含氨基酸置换T366W，并且在所述第一多肽链和第二多肽链之另一条链中包含氨基酸置换T366S、L368A和Y407V(根据Kabat的“EU编号”)，由此一条链中的凸起能够置于另一条链中的空穴中，由此促进第一多肽链和第二多肽链的缔合。
在一些实施方案中，对于本发明的第一方面和第二方面的酵母展示系统，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个具体实施方案中，对于本发明的第一方面和第二方面的酵母展示系统，所述第二 核酸分子包含编码目的多肽的核苷酸、任选地编码免疫球蛋白Fc区的铰链区的核苷酸、以及编码免疫球蛋白Fc区的CH2结构域和CH3结构域的核苷酸；优选地，所述CH2结构域中的糖基化位点被消除，例如，将人IgG Fc区的CH2结构域中的N297残基突变以消除该糖基化位点，例如，将N297残基变成Gly、Ala、Gln、Asp或Glu，优选地，将N297残基变成Ala。
在一个具体实施方案中，对于本发明的第一方面和第二方面的酵母展示系统，所述酿酒酵母细胞表面锚定蛋白是含有糖基磷脂酰肌醇(GPI)锚定信号序列的酿酒酵母细胞壁蛋白，例如，α-凝集素和a-凝集素、Cwp1p蛋白和Flo1p蛋白。
在一些实施方案中，对于本发明的第一方面和第二方面的酵母展示系统，所述酿酒酵母在未导入第一核酸分子和第二核酸分子时，本身是表达a-凝集素的aga1p亚基的酿酒酵母，例如酿酒酵母EBY100；导入所述酿酒酵母细胞的第一核酸分子表达的酿酒酵母细胞表面锚定蛋白是aga2p亚基，由此，第一核酸分子编码的第一多肽包含aga2p和免疫球蛋白Fc区CH3结构域，且所述第一多肽结合至已经结合且呈递在酿酒酵母细胞表面的aga1p亚基。
在一些实施方案中，对于本发明的第一方面和第二方面的酵母展示系统，所述目的多肽是抗体或抗原结合片段，例如，Fab片段、VHH结构域、scFv、sdAb。
在一些具体实施方案中，本发明提供了这样的酵母展示系统，其中
(a)所述第一核酸分子从N端至C端包含编码CH3 knob-任选地接头或标签-Aga2p的核苷酸，且所述第二核酸分子从N端至C端包含编码目的多肽-任选地铰链区-CH2(N297A)-CH3 hole的核苷酸；或
(b)所述第一核酸分子从N端至C端包含编码CH3 hole-任选地接头或标签-Aga2p的核苷酸，且所述第二核酸分子从N端至C端包含编码目的多肽-任选地铰链区-CH2(N297A)-CH3 knob的核苷酸。
优选地，所述接头包含甘氨酸(G)和丝氨酸(S)残基，例如，所述接头是GS。
优选地，所述标签选自Arg标签、Avi标签、His-Avi标签、His标签、Flag标签、3xFlag标签、Strep标签、Nano标签、SBP标签、c-myc标签、S标签、钙调蛋白结合肽、纤维素结合结构域、几丁质结合结构域、GST标签或MBP标签。
在第三方面，本发明提供了本发明的第一方面和第二方面的酵母展示系统的用途，用于表达在细胞表面展示、且分泌至培养基中的目的多肽；优选地，用于表达在细胞表面展示、且分泌至培养基中的抗体。
在第四方面，本发明提供了本发明的第一方面和第二方面的酵母展示系统的用途，用于构建目的多肽的变体展示文库，其中细胞表面展示的方式使得能够借助高通量筛选选择特定的目的多肽变体，分泌至培养基中的方式使得能够进行目的多肽变体的生物化学表征；优选地，用于构建抗体的变体展示文库，以筛选高亲和力的抗体变体。
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通 过引用的方式完整地并入作为参考。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
附图简述：
图1：显示了酿酒酵母通过FC在细胞表面展示抗体的模式图。
图2：显示了不同长度FC knob对酿酒酵母在细胞表面展示抗体的影响。
图3：显示了分泌至培养液中的抗体对展示抗原的酵母细胞的流式细胞术染色示意图。
图4：显示了分泌至培养液中的抗体对展示抗原的酵母细胞的流式细胞术染色结果。
图5：显示了改造酵母菌使其细胞表面展示CH3 knob的示意图。
图6：显示了对经改造的细胞表面展示CH3 knob的酵母菌IDY104检测展示的CH3 knob水平的结果。
图7：显示了流式细胞术检测转入质粒pYDC042的酵母菌IDY104展示抗体的水平。
图8：显示了对转入质粒pYDC042的酵母菌IDY104的培养上清液样品测定亲和力的结果。
图9：显示了酵母菌IDY104展示sdAb-Fc形式抗体或scFv-Fc形式抗体的水平的测定结果。
图10：显示了酵母菌IDY104的培养上清液浓缩10倍后的亲和力测定结果。
图11：显示了展示AmNB1613.36和HzNB1613的不同Spiking文库与抗原结合的流式细胞术染色图。
图12：显示了Spiking文库每轮次筛选后的展示AmNB1613.36的酵母比例。图中R0表示第0轮筛选，R1表示第1轮筛选，R2表示第2轮筛选。
发明详述：
I.定义
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
术语“抗体”在本文中以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体和抗原结合片段。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
术语“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与该抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv、双体抗体(diabody)、单结构域抗体(sdAb)。所述Fab片段是一种由VL、VH、CL和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab') 2，其为Fab’的二聚体，是二价的抗原结合片段。F(ab') 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，因此将F(ab') 2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段(其它抗原结合片段的更详细的描述请参见：基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993))。所述Fv片段由抗体单臂的VL和VH结构域组成。另外，虽然Fv片段的两个结构域VL和VH由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性连接肽连接，在所述单条蛋白链中VL区和VH区配对以形成单链Fv。可以通过化学方法、重组DNA方法或蛋白酶消化法获得所述抗原结合片段。
术语“单结构域抗体”(sdAb)通常指这样的抗体，其中单个可变结构域(例如，重链可变结构域(VH)或轻链可变结构域(VL)、衍生自骆驼科重链抗体的重链可变结构域、衍生自鱼类IgNAR的VH样单结构域(v-NAR))即可赋予抗原结合。即，该单个可变结构域不需要与另一可变结构域相互作用以识别靶抗原。单结构域抗体的实例包括源自骆驼科(美洲驼和骆驼)和软骨鱼(例如护士鲨)的单结构域抗体(WO 2005/035572)。
可以通过基因工程方法获得骆驼科重链抗体的对靶抗原具有高亲和力的重链可变结构域(该区域也称为VHH，其分子量是人IgG分子的分子量的十分之一，并且具有仅数纳米的物理直径)。参见1998年6月2日授予的美国专利号5,759,808。与其他非人源抗体片段一样，骆驼科VHH的氨基酸序列可以重组地改变以获得更逼真模仿人序列的序列，即，“人源化”，由此降低骆驼科VHH对人类的抗原性。另外，也可以将衍生自骆驼科VHH的关键元件转移到人VH结构域上，获得骆驼化的人VH结构域，导致人VH结构域不再需要与VL结构域配对来识别靶抗原，经骆驼化的人VH结构域单独即可赋予抗原结合特异性。所述骆驼科VHH、人源化的骆驼科VHH、经骆驼化的人VH结构域均在术语“VHH结构域”的范围内。
术语“免疫球蛋白分子”指具有天然存在抗体的结构的蛋白质。例如，IgG类免疫球蛋白是由二硫键键合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体糖蛋白。从N端至C端，每条免疫球蛋白重链具有一个重链可变区(VH)，也称作重链可变结构域，随后是三个重链恒定结构域(CH1、CH2和CH3)。类似地，从N端至C端，每条免疫球蛋白轻链具有一个轻链可变区(VL)，也称作轻链可变结构域，随后是一个轻链恒定结构域(CL)。免疫球蛋白的重链可以归属5个类别之一，称作α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，其中某些类别可以进一步划分成亚类，例如γ 1(IgG1)、γ 2(IgG2)、γ 3(IgG 3)、γ 4(IgG 4)、α 1(IgA 1)和α 2(IgA 2)。免疫球蛋白的轻链可以基于其恒定结构域的氨基酸序列而划分成两种类型之一，称作κ和λ。免疫球蛋白基本上由借助免疫球蛋白铰链区连接的两个Fab分子和一个Fc结构域组成。
术语“Fc结构域”、“Fc区”或“FC”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如，在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的两条重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的两条重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明，否则Fc区或重链恒定区中的氨基酸残基编号根据如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述的EU编号体系(也称作EU索引)进行编号。
术语“效应子功能”指随免疫球蛋白同种型变动的归因于免疫球蛋白Fc区的那些生物学活性。免疫球蛋白效应子功能的例子包括：C1q结合和补体依赖的细胞毒性(CDC)、Fc受体结合作用、抗体依赖的细胞介导的细胞毒性(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞摄取抗原、下调细胞表面受体(例如B细胞受体)和B细胞活化。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、表面等离子共振(SPR)或生物膜层干涉技术或本领域已知的其他常规结合测定法测定。
如本文所用的术语“PD-L1”、“程序性细胞死亡配体1”、“程序性死亡配体1”是指来自任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。
本文所用的术语“抗PD-L1抗体”、“抗PD-L1”、“PD-L1抗体”或“结合PD-L1的抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合PD-L1蛋白或其片段。在一个实施方案中，抗PD-L1抗体与非PD-L1蛋白结合的程度低于所述抗体与PD-L1结合的约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％或约90％或以上，例如通过放射性免疫测定(RIA)或生物光干涉测定法或SPR或生物膜层干涉等测量的。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由解离常数(K D)代表，解离常数是解离速率常数和缔合速率常数(分别是k dis和k on)的比例。亲和力可以由本领域已知的常见方法测量。
术语“变体”指在亲本氨基酸序列中的修饰。示例性修饰包括氨基酸置换、插入和/或缺失。在一个实施方案中，目的多肽变体是对亲本目的多肽氨基酸序列的置换。本文的氨基酸置换涵盖用一种或多种天然存在的和/或非天然存在的氨基酸残基置换。“天然存在的氨基酸残基”(即由遗传密码编码)选自：丙氨酸(Ala)；精氨酸(Arg)；天冬酰胺(Asn)；天冬氨酸(Asp)；半胱氨酸(Cys)；谷氨酰胺(Gln)；谷氨酸(Glu)；甘氨酸(Gly)；组氨酸(His)；异亮氨酸(Ile)：亮氨酸(Leu)；赖氨酸(Lys)；甲硫氨酸(Met)；苯丙氨酸(Phe)；脯氨酸(Pro)；丝氨酸(Ser)；苏氨酸(Thr)；色氨酸(Trp)；酪氨酸(Tyr)；和缬氨酸(Val)。“非天然存在的氨基酸残基”指除上文所列的那些天然存在的氨基酸残基之外，能够共价结合多肽链中相邻氨基酸残基的残基。非天然存在的氨基酸残基的例子包括正亮氨酸、鸟氨酸、正缬氨酸、高丝氨酸、α-氨基异丁酸(aib)和其他氨基酸残基类似物，如在Ellman等人,Meth.Enzym.202(1991)301-336中描述的那些。
与抗体相关的术语“变体”在本文中指，包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的目标抗体区域(例如重链可变区或轻链可变区或重链CDR区或轻链CDR区)的抗体，其中变体基本上保持改变之前的抗体分子的生物学特性。可以理解的是，抗体的重链可变区或轻链可变区、或各CDR区可以单独改变或组合改变。在一些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。在一些实施方案中，在一个或多个或全部三个轻链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。在一些实施方案中，在一个或多个或全部6个CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。优选地，所述氨基酸改变为氨基酸取代，优选保守取代。在一些实施方案中，抗体变体与亲本抗体在目的抗体序列区域上具有至少80％、85％、90％或95％或99％或更高的氨基酸序列同一性。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位 于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standardconformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins ofImmunological Interest,第4版,U.S.Department of Health and Human Services,NationalInstitutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
术语“接头”是指由氨基酸组成的连接肽，例如单独或组合使用的甘氨酸和/或丝氨酸残基，以连接融合蛋白中的各个区域。在一个实施方案中，接头是Gly/Ser连接肽，包括氨基酸序列(Gly 1-4Ser)n，其中n是等于或大于1的正整数，例如，n是1－7中的正整数。在一个实施方案中，所述接头是GS。还包括在本发明范围内的是WO2012/138475中描述的连接肽，其通过引用并入本文。
术语“标签”指具有特定结合特性的通过肽键相互连接的氨基酸残基的序列。在一个实施方案中，氨基酸序列标签是亲和或纯化标签。在一个实施方案中，氨基酸序列标签选自Arg标签、His标签、Avi标签、His-Avi标签、Flag标签、3xFlag标签、Strep标签、Nano标签、SBP标签、c-myc标签、S标签、钙调蛋白结合肽、纤维素结合结构域、几丁质结合结构域、GST标签和MBP标签(参见，例如Amau,J.等人，Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins，Protein Expr.Purif.，2006，48(1)：1-13)。
术语“与…融合”指在两个部分之间形成的共价键，例如，肽键。
术语“第一融合蛋白”、“第一多肽”、“第一多肽链”具有相同的含义，在本文中可互换使用，是指由第一核酸分子表达的多肽产物。同样地，术语“第二融合蛋白”、“第二多肽”、“第二多肽链”具有相同的含义，在本文中可互换使用，是指由第二核酸分子表达的多肽产物。
术语“N端”指N端的最末氨基酸，术语“C端”指C端的最末氨基酸。
术语“导入”是指适于通过本领域已知的任何技术将多核苷酸(例如质粒)引入或转移至酵母细胞的任何方法，例如转化、转导、转染。术语“转化”用来描述将多核苷酸引入酵母细胞中。术语“转导”是指多核苷酸或遗传物质的病毒引入或病毒转移至细胞中。可使用任何已知的病毒系统用于转导本发明的宿主细胞，例如基于腺病毒的系统、基于腺伴随病毒(AAV)的系统、反转录病毒系统、慢病毒表达系统或基于单纯疱疹病毒(HSV)的系统，或基于其它病毒的系统例如基于痘苗、EB病毒、仙台病毒、辛德毕斯病毒、多瘤病毒和麻疹病毒的系统(参见例如Mah C等人，Virus-based gene delivery systems，Clin.Pharmocokin，2002，41(12)： 901-911)。术语“转染”在用于本发明的方法时是指通过本领域已知的任何合适方法使宿主细胞摄入核酸，例如公开于Sambrook等(1989)Molecular Cloning，a laboratory manual，Cold Spring Harbor Laboratories，New York，Davis等(1986)Basic Methods in Molecular Biology，Elsevier的方法，特别是磷酸钙共沉淀、直接显微注射入培养的细胞中、超声介导的基因转染、电穿孔、脂转染或核转染(nucleofection)。
术语“表达载体”代表包含至少一个编码多肽的氨基酸序列的核酸分子序列、启动子序列、终止子序列、选择标记和复制起点、以及任选的分泌信号序列的天然或人造DNA序列。
II.本发明的酵母展示系统
酿酒酵母是一种在本领域中被很好表征的真核宿主生物体，作为研究真核细胞功能的模式生物体，其同样适合于表达异源目的多肽。酿酒酵母作为单细胞生物体，与其他真核系统相比不那么复杂，且其能够在定义的培养基中培养，因此，能够良好控制其生长条件和降低其培养成本。具有约90分钟的相对短生命周期是优先使用酿酒酵母的一个原因。由于酿酒酵母组合了微生物表达系统由于培养简单和使用工业发酵方法带来的优点以及真核表达系统由于真核表达和存在细胞中的分泌途径带来的优点，且能够进行大规模选择，因此，本发明使用酿酒酵母表达和展示异源目的多肽。
在一个实施方案中，本发明提供了通过如下步骤制备的酵母展示系统：
i.将第一核酸分子和第二核酸分子导入酿酒酵母细胞，其中所述第一核酸分子包含编码酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的核苷酸，所述第二核酸分子包含编码目的多肽的核苷酸和编码免疫球蛋白Fc区或其部分的核苷酸，
ii.培养经转化的酿酒酵母细胞，表达包含酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的第一融合蛋白；以及表达包含目的多肽和免疫球蛋白Fc区或其部分的第二融合蛋白；
由此，表达的第一融合蛋白通过所述锚定蛋白锚定在酿酒酵母细胞表面上；表达的一部分第二融合蛋白通过免疫球蛋白Fc区或其部分与第一融合蛋白的免疫球蛋白Fc区CH3结构域稳定缔合而将目的多肽展示在酿酒酵母细胞表面；表达的另一部分第二融合蛋白则保留在培养基中。
使用本发明的该酵母展示系统能够稳定地构建10 8数量级的目的多肽变体展示文库，其中在细胞表面展示的方式使得能够借助高通量筛选选择特定的目的多肽变体，分泌至培养基中的方式使得能够进行目的多肽变体的生物化学表征。
可以使用本领域已知的任何方法将第一核酸分子和第二核酸分子导入酿酒酵母细胞。在一个具体实施方案中，根据Gietz,R.D.等人所述的方法(Gietz,R.D.和Schiestl,R.H.，High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.Nature Protocols,2007,2(1):31-34)实施所述第一核酸分子和第二核酸分子的导入。
不特别地限定所述第一核酸分子和第二核酸分子的存在形式和导入细胞中的先后顺序，只要所述第一核酸分子和第二核酸分子在导入酿酒酵母细胞后均能表达即可。
在一个实施方案中，所述第一核酸分子和第二核酸分子位于同一个质粒上，并将包含所述第一核酸分子和第二核酸分子的质粒导入酿酒酵母细胞。
在另一个实施方案中，所述第一核酸分子和第二核酸分子位于分开的质粒上，并将包含所述第一核酸分子的质粒和包含第二核酸分子的质粒分别地先后导入酿酒酵母细胞，或者同时地导入酿酒酵母细胞。
存在许多用于在酿酒酵母中表达重组蛋白质的质粒和/或表达载体，例如载体p426Met25或p426GAL1(Mumberg等人(1994)Nucl.Acids Res.,22,5767-5768)。在一个实施方案中，使用pYDC载体(例如，pYDC011)用于将第一核酸分子和/或第二核酸分子导入到酿酒酵母中。在酵母中表达的合适的启动子包括来自GAL1(半乳糖)、PGK(磷酸甘油酸激酶)、ADH(醇脱氢酶)、AOX1(醇氧化酶)、HIS4(组氨醇脱氢酶)等的启动子。在酵母表达中使用的合适的载体和启动子进一步描述于R.Hitzeman等人，EP073,657中。
可以通过使用酵母蛋白质的任一可用的分泌信号序列来增加第一融合蛋白和/或第二融合蛋白从酵母宿主细胞中分泌。分泌信号序列的一个实例是酵母接合信息素的前体α因子的前导序列，其也用于指导酵母中异源蛋白质的分泌(参见，例如，Valenzuela，P.编辑，第269-280页，Butterworths，London；Brake，A.J.(1990)Meth.Enzymol.185，408-441)。另一实例是来自酵母转化酶的前导序列(MLLQAFLFLLAGFAAKISADAHKS)。已经表明，当进入内质网时，该前导序列将从新生的异源肽中剪切。另外的实例是酵母酸性磷酸酶的信号序列，其也可以用于指导在本文中公开的融合蛋白的分泌。
第一融合蛋白中的酿酒酵母细胞表面锚定蛋白通常是酿酒酵母的细胞外壁蛋白。酿酒酵母的许多细胞外壁蛋白原则上能够将异源蛋白和肽作为本发明第一融合蛋白的一部分展示在细胞表面，例如α-凝集素和a-凝集素、Cwp1p蛋白和Flo1p蛋白。
在一个实施方案中，使用a-凝集素作为第一融合蛋白中的酿酒酵母细胞表面锚定蛋白。细胞壁蛋白a-凝集素包含亚单位Aga1p和Aga2p。亚单位Aga1p具有糖基磷脂酰肌醇(GPI)锚定信号序列，并且通过β-葡聚糖的共价结合介导该蛋白固定在细胞外壁。亚单位Aga2p同样由细胞分泌，并且经由两个二硫键键合至Aga1p。
在一个实施方案中，所述酿酒酵母是表达a-凝集素的Aga1p亚基的酿酒酵母，例如酿酒酵母EBY100物种，其通过染色体整合的半乳糖诱导型表达盒表达aga1p亚基。对于表达a-凝集素的aga1p亚基的酿酒酵母，酿酒酵母细胞表面锚定蛋白是aga2p亚基。当Aga1p和第一融合蛋白中的Aga2p结合后，异源目的多肽(例如抗体)实现在酵母细胞的表面上展示。
第一融合蛋白中的免疫球蛋白Fc区CH3结构域是位于免疫球蛋白重链的C端的非抗原结合区。在一些实施方案中，IgG CH3结构域始于Gly341。可以理解，人IgG的C端Lys残基可以任选地不存在。还应当理解，本发明的范围内构思了Fc区CH3结构域的保守性氨基酸置换而不影响Fc的所需结构和/或稳定性。
在一些实施方案中，本发明第一融合蛋白中的免疫球蛋白Fc区CH3结构域和第二融合蛋白中的免疫球蛋白Fc区或其部分使用了“结入扣(knob-in-hole)”技术(参见例如John  B.B.Ridgway等人，‘Knobs-into-holes’engineering of antibody CH3 domains for heavy chain heterodimerization.Protein Engineering,1996,9(7):p.617-21；Shane Atwell等人，Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library.J.Mol.Biol,1997,270:p.26-35)，该技术可在本发明的第一融合蛋白和第二融合蛋白之间改造界面，以促进本发明的第一融合蛋白和第二融合蛋白正确缔合。通常，该技术涉及在所述融合蛋白之一的界面引入“凸起”，在欲与之配对的另一融合蛋白的界面引入相应的“空穴”，使得凸起可置于空穴中。一个优选的界面包含融合蛋白之一的CH3结构域和欲与之配对的另一融合蛋白的CH3结构域。可通过将来自融合蛋白之一的CH3结构域的界面的小氨基酸侧链替换为较大的侧链(例如酪氨酸或色氨酸)来构建凸起。通过将大氨基酸侧链替换为较小的侧链(例如丙氨酸或苏氨酸)，在欲配对的另一融合蛋白的CH3结构域的界面构建与凸起相同或相似大小的补偿性空穴。
用于形成结(knob)的优选残基通常是天然存在的氨基酸残基并且优选地选自精氨酸(R)、苯丙氨酸(F)、酪氨酸(Y)和色氨酸(W)。最优选的是色氨酸和酪氨酸。在一个实施方案中，用于形成结的原始残基具有小的侧链体积，如丙氨酸、天冬酰胺，天冬氨酸、甘氨酸、丝氨酸、苏氨酸或缬氨酸。CH3结构域中用于形成结的示例性氨基酸置换包括但不限于T366W、T366Y或F405W置换。
用于形成扣(hole)的优选残基通常是天然存在的氨基酸残基并且优选地选自丙氨酸(A)、丝氨酸(S)、苏氨酸(T)和缬氨酸(V)。在一个实施方案中，用于形成扣的原始残基具有大的侧链体积，如酪氨酸、精氨酸、苯丙氨酸或色氨酸。CH3结构域中用于产生扣的示例性氨基酸置换包括但不限于T366S、L368A、F405A、Y407A、Y407T和Y407V置换。在某些实施方案中，结包含T366W置换，并且扣包含T366S/L368A/Y407V置换。在一些实施方案中，本发明的第一核酸分子和第二核酸分子编码的第一多肽链和第二多肽链之一条链中包含氨基酸置换T366W，并且在所述第一多肽链和第二多肽链之另一条链中包含氨基酸置换T366S、L368A和Y407V(根据Kabat的“EU编号”)，由此一条链中的凸起能够置于另一条链中的空穴中，由此促进第一多肽链和第二多肽链的缔合。
不特别地限定第二融合蛋白中的目的多肽。目的多肽可以是任何欲表达的多肽。在一些实施方案中，所述目的多肽是抗体或抗原结合片段，例如，Fab片段、VHH结构域、scFv、sdAb。
第二融合蛋白中的免疫球蛋白Fc区或其部分包含免疫球蛋白Fc区的CH2结构域和CH3结构域、以及任选的免疫球蛋白Fc区铰链区。
在一些实施方案中，改变第二融合蛋白中的免疫球蛋白Fc区或其部分以降低第二融合蛋白的Fc部分糖基化的程度。可以通过改变氨基酸序列从而移除一个或多个糖基化位点，便利地实现对蛋白质删除糖基化位点。例如，将IgG Fc区CH2结构域中的N297残基突变以消除该糖基化位点，例如，将N297残基变成Gly、Ala、Gln、Asp或Glu，优选地，将N297残基变成Ala。N297指位于Fc区内约位置297处的天冬酰胺残基(Fc区残基的EU编号)；然而， N297也可以位于位置297的约上游或下游±3个氨基酸，即，位置294和位置300之间，原因在于免疫球蛋白中的微小序列变异。
为了简化操作和省去额外的质粒构建步骤，本发明还提供了这样的用于细胞表面展示和分泌目的多肽的酵母展示系统，其是重组的酿酒酵母细胞，其中在酿酒酵母细胞的基因组中的靶位点处插入有第一核酸分子，所述第一核酸分子包含编码酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的核苷酸，所述重组的酿酒酵母细胞用于导入包含编码目的多肽的核苷酸和编码免疫球蛋白Fc区或其部分的核苷酸的第二核酸分子，以在细胞表面展示和分泌目的多肽。
为了制备重组的酿酒酵母细胞，向酿酒酵母细胞基因组中的靶位点处导入第一核酸分子。
对于导入第一核酸分子的酿酒酵母细胞基因组中的靶位点没有特别的限定，只要重组后的酿酒酵母细胞能够表达第一融合蛋白即可。
优选地，所述靶位点是野生型酿酒酵母具有的营养合成基因处。将第一核酸分子导入酿酒酵母细胞基因组中的营养合成基因处，导致营养合成基因合成营养的能力受损，从而重组的酿酒酵母细胞是营养缺陷型酵母，具有营养需求性。所谓“酵母具有营养需求性”是指野生型酵母具有的营养合成基因中由于某种原因发生变异，导致该营养的合成能力受损，需要向培养基中添加相应的营养素来维持营养缺陷型酵母的生长。
作为酿酒酵母所必需营养素的具体例子，已知有蛋氨酸、酪氨酸、异亮氨酸、苯丙氨酸、谷氨酸、苏氨酸、天冬氨酸、缬氨酸、丝氨酸、精氨酸、尿嘧啶、腺嘌呤、赖氨酸、色氨酸、亮氨酸、组氨酸等。作为营养缺陷型酵母所具有的受损营养合成基因，可以列举以下例子。
·蛋氨酸需求性：met1、met2、met3、met4、met5、met6、met7、met8、met10、met13、met14、met20
·酪氨酸需求性：tyr1
·异亮氨酸、缬氨酸需求性：ilv1、ilv2、ilv3、ilv5
·苯丙氨酸需求性：pha2
·谷氨酸需求性：glu3
·苏氨酸需求性：thr1、thr4
·天冬氨酸需求性：asp1、asp5
·丝氨酸需求性：ser1、ser2
·精氨酸需求性：arg1、arg3、arg4、arg5、arg8、arg9、arg80、arg81、arg82、arg84
·尿嘧啶需求性：ura1、ura2、ura3、ura4、ura6
·腺嘌呤需求性：ade1、ade2、ade3、ade4、ade5、ade6、ade8、ade9、ade12、ADE15
·赖氨酸需求性：lys1、lys2、lys4、lys5、lys7、lys9、lys11、lys13、lys14
·色氨酸需求性：trp1、trp2、trp3、trp4、trp5
·亮氨酸需求性：leu1、leu2、leu3、leu4、leu5
·组氨酸需求性：his1、his2、his3、his4、his5、his6、his7、his8。
因此，本发明中将第一核酸分子导入至的酿酒酵母细胞基因组中的靶位点优选使用上述 营养合成基因所在的基因组位点。
在一个实施方案中，将第一核酸分子插入至酿酒酵母细胞基因组中的URA3基因座处，获得了URA3 －营养缺陷型标记的酿酒酵母细胞。
在又一个实施方案中，将第一核酸分子插入至酿酒酵母细胞基因组中的TRP1基因座处，获得了TRP1 －营养缺陷型标记的酿酒酵母细胞。
关于第一核酸分子的其他特征和第二核酸分子的特征，同上文所述。
III.使用本发明的酵母展示系统构建目的多肽变体展示文库
本发明提供了构建目的多肽变体展示文库的方法，包括：
i.构建编码目的多肽变体的基因文库，将所述编码目的多肽变体的基因文库和编码免疫球蛋白Fc区或其部分的核苷酸连接产生第二核酸分子文库，
ii.构建目的多肽变体展示文库，其中将第二核酸分子文库和上述第II部分所述的第一核酸分子导入酿酒酵母细胞，或者将第二核酸分子文库导入上述第II部分所述的重组的酿酒酵母细胞，其中已在酿酒酵母细胞的基因组中的靶位点处插入有第一核酸分子，所述第一核酸分子包含编码酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的核苷酸。
在一些实施方案中，通过本领域熟知的方法，例如使用易错聚合酶链式反应、使用随机引物技术或者使用计算机技术产生目的多肽变体的基因文库。设计的目的多肽变体的基因文库具有合适的限制性酶切割位点，以将目的多肽变体的基因文库与上述第II部分所述的第二核酸分子的编码免疫球蛋白Fc区或其部分的核苷酸连接。
通过诱导本发明的酵母展示系统表达目的多肽变体的基因文库，将目的多肽变体文库展示于酿酒酵母细胞上，获得约10 8数量级的目的多肽变体展示文库。
使用本领域已知的生物淘洗方法，筛选酵母展示的目的多肽变体文库。例如，在目的多肽变体文库为亲和力不同的抗体文库的情形下，在允许特定浓度的抗原与抗体文库的成员特异结合的条件下与酵母展示文库接触。此时，所有结合的抗原都固定在酵母细胞的表面。通过使用流式细胞术，即FACS检测，将所有未结合特定浓度抗原的酵母细胞洗掉，经多轮这样的生物淘洗后，可以分选出展示亲和力高的抗体的酵母细胞，通过克隆扩增，获得克隆细胞系。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
一般方法：
除非另外说明，本发明的实施将利用本领域技术人员已知并可获得的细胞生物学、细胞培养、分子生物学(包括重组技术)、微生物学、生物化学、和免疫学的常规技术。这类技术在如下文献中描述：Molecular Cloning:A laboratory Manual,第3版(Sambrook等人,2001)Cold Spring Harbor Press；Oligonucleotide Synthesis(P.Herdewijn编著,2004)；Methods in  Enzymology(Academic Press,Inc.)；Current Protocols in Molecular Biology(F.M.Ausubel等人编著,1987)；PCR:The Polymerase Chain Reaction(Mullis等人编著,1994)；Current Protocols in Immunology(J.E.Coligan等人编著,1991)；和Short Protols in Molecular Biology(Wiley and Sons,1999)。除非另外规定，本文中所用的全部术语及科学术语具有与本发明所属领域的技术人员通常理解的相同含义。
实施例1：不同长度的FC knob在抗体展示中的作用
本实施例比较了不同长度的FC对于抗体展示的影响。
质粒构建
如下表1所示，构建了四种质粒，其中
质粒pYDC039、pYDC040和pYDC041分别用于表达Aga2p与不同长度FC的融合蛋白(pYDC039中表达的“铰链区-CH2-CH3”氨基酸序列如SEQ ID NO:1所示；
pYDC040中表达的“CH2-CH3”氨基酸序列如SEQ ID NO:2所示；
pYDC041中表达的“CH3”氨基酸序列如SEQ ID NO:3所示；
质粒pYDC042表达针对PD-L1抗原的重链可变区(V HH)AmNB1613.36(SEQ ID NO:4)与FC(SEQ ID NO:5)的融合蛋白，所述融合蛋白的序列如SEQ ID NO:6所示。在FC的设计中，根据基因泰克公司专利(WO9850431A2)公布的突变，考虑到CH3 knob(T366W)能有效降低CH3 knob自身的同源二聚体的水平，同时CH3 knob与CH3 hole(T366S,L368A,Y407V)形成异源二聚体占主要比例，并且CH3 hole的同源二聚体仍保持约20％的比例，在本实施例中将Knob-Hole突变引入FC的CH3中；进一步地，由于酿酒酵母有严重的过糖基化水平，对于抗体展示水平及抗体与抗原的结合会有影响，在本实施例中将FC的CH2进行N297A突变以去除糖基化位点。
表1：构建的质粒和其中插入的片段
质粒编号 质粒中所插入片段的描述
pYDC039 铰链区-CH2(N297A)-CH3 knob-GS接头-Aga2p
pYDC040 CH2(N297A)-CH3 knob-GS接头-Aga2p
pYDC041 CH3 knob-GS接头-Aga2p
pYDC042 AmNB1613.36-铰链区-CH2(N297A)-CH3 hole
将表1中各质粒的插入片段的核苷酸序列交由苏州金唯智生物科技有限公司合成，各核苷酸序列编码的具体氨基酸序列见SEQ ID NO:1、2、3和6。
分别将编码SEQ ID NO:1、2和3的核苷酸序列用限制性酶BamHI酶切，并连接入经限制性酶BamHI酶切的pYDC011质粒(pYDC011质粒的核苷酸序列如SEQ ID NO:7所示)的两个BamHI位点之间，以替换pYDC011质粒上的核苷酸序列“GGaTCctgacatagtagggattataa”，获得质粒pYDC039、pYDC040和pYDC041。所述质粒分别用于表达Aga2p与不同长度FC knob的融合蛋白。
另外，将编码SEQ ID NO:6的核苷酸序列用限制性酶BamHI酶切，并连接入经限制性酶BamHI酶切的pYDC011质粒的两个BamHI位点之间，以替换pYDC011质粒上的核苷酸序列“GGaTCctgacatagtagggattataa”，获得质粒pYDC042，用于表达单域抗体(sdAb)AmNB1613.36与FC的融合蛋白，即，AmNB1613.36-铰链区-CH2(N297A)-CH3 hole。
质粒转化与酵母细胞培养
根据Gietz,R.D.等人所述的方法(Gietz,R.D.和Schiestl,R.H.(2007).High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.Nature Protocols,2(1),31–34)，将构建后的质粒按表2所示的组合分别转入酿酒酵母菌株EBY100(购自ATCC)中，获得经转化的酿酒酵母。挑取单个的酿酒酵母克隆，接种于5mL的SD-Trp,Leu培养基(Takara货号：630316)，于30℃225转/分钟培养过夜；将培养物3000转/分钟离心3分钟弃上清；向细胞沉淀物加入5mL YPGP诱导培养基(2％半乳糖，2％蛋白胨，1％酵母提取物，0.54％Na 2HPO 4,0.86％NaH 2PO 4·H 2O)，于20℃225转/分钟诱导72小时，以表达Aga2p与不同长度FC knob的融合蛋白；以及融合蛋白AmNB1613.36-铰链区-CH2(N297A)-CH3 hole。
表2：质粒转化入酿酒酵母展示菌株EBY100
Figure PCTCN2020077798-appb-000001
用不同长度的FC knob来展示抗体的酵母细胞染色
将上述诱导后的细胞用PD-L1抗原染色，分析表达不同长度FC knob的酿酒酵母在其细胞表面展示抗体的水平，具体步骤如下：
1.各取1×10 6个细胞，3000转/分钟离心3分钟弃上清，用1×PBSA(1×PBS+1％BSA)洗一次，离心，获得细胞沉淀物待用；
2.向每个样品的细胞沉淀物中加入100μL 100nM PD-L1生物素(Acro货号：PD1-H82E5-200ug)，室温孵育30分钟；
3.加入1mL 1×PBSA洗一次，3000转/分钟离心3分钟弃上清；
4.加入100μL经1×PBSA稀释的链亲和素-PE稀释液(Thermo Fisher,货号：S21388 1:200稀释)，冰上避光孵育20分钟；
5.加入1mL 1×PBSA洗一次后，3000转/分钟离心3分钟弃上清，向细胞沉淀物加入500μL 1×PBSA重悬细胞，然后进行流式细胞术(FACS)检测，FL2的平均荧光信号强度反映了酿酒酵母表面所展示的抗体水平，结果如图2所示。
由图2可见，与未用质粒转化的EBY100阴性对照比较，用表2所示的质粒组合转化的酵母细胞中，PD-L1抗原染色的阳性细胞的比例及细胞整体的荧光信号强度均具有明显优势。另外，在用表2所示的质粒组合转化的酵母细胞中，通过CH3 knob展示抗体的效果明显好于通过铰链区-CH2(N297A)-CH3 knob展示抗体或通过CH2(N297A)-CH3 knob展示抗体。
实施例2：比较不同长度的FC knob在抗体分泌中的作用
基于展示抗原的酵母细胞染色流式细胞术分析
将实施例1中经诱导后的培养物进行离心，获得上清液。如图3所示，将所述上清液与展示PD-L1(NP_054862.1,Phe 19-Arg 238)抗原的酵母细胞(编码Aga2p-PD-L1的质粒转入EBY100菌株，所述编码Aga2p-PD-L1的质粒是将编码Aga2p-PD-L1的核苷酸序列用限制性酶BamHI酶切，并连接入经限制性酶BamHI酶切的pYDC011质粒而获得)孵育染色，比较表2中各质粒组合转化的酵母细胞经诱导后离心的不同上清液中抗体的含量，具体步骤如下：
1.取适量展示PD-L1抗原的酵母细胞，3000转/分钟离心3分钟，弃上清，将细胞沉淀物用1×PBSA(1×PBS+1％BSA)洗一次，以每孔1×10 6个细胞/100μL 1×PBSA铺于96孔2ml U型底深孔板(上海生工生物，货号：F600580-0001)中待用；
2.将实施例1中经诱导后的培养物进行离心，获得上清液。分别取100μL上清液加入步骤1的96孔板中，室温孵育30分钟；
3.离心板，弃上清，将细胞沉淀物加入1mL 1×PBSA洗一次；
4.加入100μL经1×PBSA稀释的二级抗体稀释液(小鼠抗V5-FITC，Invitrogen货号：R963-25,1:1000稀释；小鼠抗人IgG-APC,Biolegend货号：409306,1:200稀释)，冰上避光孵育20分钟；
5.加入1mL 1×PBSA洗一次后，加入500μL 1×PBSA重悬细胞，然后进行流式细胞术检测，抗V5-FITC抗体(FL1)检测酵母展示PD-L1抗原的水平；小鼠抗人IgG-APC(FL4)检测酵母上清液中单域抗体的含量。
结果如图4所示。由图4可见，在用表2所示的质粒组合转化的酵母细胞中，使用组合3时培养基中分泌的抗PD-L1抗体AmNB1613.36的量明显高于(高10倍-20倍)使用组合1或组合2时培养基中分泌的抗PD-L1抗体AmNB1613.36的量。
通过实施例1和实施例2可见，CH3 knob使得能够显著提高在酵母表面展示抗体的同时分泌抗体至上清液的量，由此可以确定CH3 knob为最适合的长度，在下述实施例中用于酵母展示与分泌的二合一模式。
实施例3：展示CH3 knob的酵母菌(IDY104)的改造与鉴定
由实施例1和实施例2可以确定CH3 knob为最适合的长度，因此如图5所示，将编码CH3 knob-Aga2p的核苷酸片段插入酿酒酵母EBY100的基因组中的URA3基因位点。具体步骤如下：
1.根据Gietz,R.D.等人所述的方法(Gietz,R.D.和Schiestl,R.H.,High-efficiency yeast  transformation using the LiAc/SS carrier DNA/PEG method.Nature Protocols,2007,2(1),31–34)，将编码P GAL1-CH3 Knob-FLAG-AGA2-TER MATα-P AgTEF-Kan R的核苷酸序列
(P GAL1-CH3 Knob-FLAG-AGA2-TER MATα-P AgTEF-Kan R:
ACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCGGACTACTAGCAGCTGTAATACGACTCACTATAGGGAATATTAAGCTAATTCCCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGGACAGCCTCGGGAGCCCCAGGTTTATACTCTCCCCCCCAGCCGGGACGAACTGACCAAGAATCAGGTGTCCCTCTGGTGCCTCGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAGTGGGAGAGCAATGGACAGCCCGAAAACAACTACAAGACCACACCCCCCGTCCTGGACTCCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTTAGCTGCAGCGTCATGCACGAGGCTCTCCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGAAAGGGaGGcGGtGGaTCcGATTACAAGGATGACGATGACAAGGGCGGAGGAGGCTCcCAGGAACTGACAACTATATGCGAGCAAATCCCCTCACCAACTTTAGAATCGACGCCGTACTCTTTGTCAACGACTACTATTTTGGCCAACGGGAAGGCAATGCAAGGAGTTTTTGAATATTACAAATCAGTAACGTTTGTCAGTAATTGCGGTTCTCACCCCTCAACgACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTTtaaTGAGTTTAAACCCGCTGATCTGATAACAACAGTGTAGATGTAACAAAATCGACTTTGTTCCCACTGTACTTTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGTTTTCCCATGTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATATAGCTATTCACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATTTCAATTTGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGAATCGAATCCTAAGAGAATTagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGA TGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA(SEQ ID NO:8))转化入酿酒酵母菌株EBY100(购自ATCC)中，涂布于含200μg/mL G418(上海生工，A600958-0001)的YPD平板(TAKARA,630410)，于30℃培养3天；
2.挑取8个单克隆，在新的YPD+G418平板划线，于30℃培养3天；
3.从步骤2中的各平板再挑取新长出的单菌落，分别在SD-Ura(Clontech,630315)平板上划线和在YPD+G418平板上划线，30℃培养3天；
4.挑取步骤3中Ura -G418 +的单克隆，YPD培养过夜、取1mL过夜培养菌液加入5mL YPGP诱导培养基中，20℃225rpm诱导24h；
5.离心，各取1×10 6个酵母细胞用1×PBSA洗一次；
6.每个样品加入100μL小鼠抗Flag M2(sigma,货号：F1804,1:1000稀释)，室温孵育30分钟；
7.加入1mL 1×PBSA洗一次；
8.加入100μL经1×PBSA稀释的Goat抗Mouse-647稀释液(Thermo Fisher,货号：A21235 1:200稀释)，冰上避光孵育20分钟；
9.加入1mL 1×PBSA洗一次后，加入500μL 1×PBSA重悬细胞，然后进行流式细胞术检测，FL4的平均荧光信号强度反映了各菌株的CH3 knob酵母表面展示水平，结果如图6所示。
由图6可知，各菌株均能有效将CH3 knob展示于酵母表面，在以下实施例中，随机挑取了其中的IDY104-7号克隆，用于后续实验。
实施例4：酵母菌IDY104展示抗体水平的检测以及培养上清液的亲和力测定
实施例3中获得的酵母菌IDY104具有直接展示CH3 knob的能力。进一步地，将质粒pYDC042(包含编码SEQ ID NO:6所示AmNB1613.36-铰链区-CH2(N297A)-CH3 hole多肽的核苷酸序列转入酵母菌IDY104中。表达的融合蛋白AmNB1613.36-铰链区-CH2(N297A)-CH3 hole通过与酵母细胞表面的CH3 knob的互相作用，间接地将抗PD-L1抗体AmNB1613.36展示于酵母细胞表面，同时分泌少量抗体至培养液中，由此实现细胞表面展示与分泌二合一的酵母展示技术，其中表面展示抗PD-L1抗体AmNB1613.36的酵母细胞可以用于流式细胞术分析与分选，且分泌有抗PD-L1抗体AmNB1613.36的培养液可用于测定抗体的亲和力，具体步骤如下：
1.将质粒pYDC042转化入菌株IDY104，30℃培养3天，挑取单克隆接种至SD-Leu培养(TAKARA货号：630310)过夜，取1mL菌液转接至5mL YPGP培养基诱导24h；
2.取包含约1×10 6个细胞的培养液，3000转/分钟离心3分钟，将上清液用于步骤7中，将细胞沉淀物用1×PBSA洗一次后用于步骤3中；
3.向细胞沉淀物中加入100μL 10nM PD-L1生物素，室温孵育30分钟；
4.离心，加入1mL 1×PBSA洗一次；
5.加入100μL经1×PBSA稀释的链亲和素-PE稀释液(Thermo Fisher,货号：S21388 1:200稀释)，冰上避光孵育20分钟；
6.加入1mL 1×PBSA洗一次后，加入500μL 1×PBSA重悬细胞，然后进行流式细胞术检测，FL2的平均荧光信号强度反映了酵母表面展示抗体的水平，结果如图7所示。
7.将步骤1的离心收集的上清液置于超滤浓缩管(上海拓开生物科技有限公司，MCPM02C67)中，将上清液浓缩10倍，对所获得的浓缩液也进行亲和力检测。
采用生物膜层干涉(BLI)技术分别测定上清液和浓缩10倍的样品中抗体与抗原的平衡解离常数(K D)。BLI法亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013，5(2):p.270-8)实施。传感器在分析缓冲液中预湿20分钟后，按照建立的方法，用Octet Red96测量抗体与PD-L1的亲和力：首先平衡基线120秒；然后将样品固化至AHC传感器(ForteBio,18-5060)；将已固化的传感器置于含100nM PD-L1(Acro，货号：PD1-H82E5-200ug)的溶液中直至平台期(100秒)，之后将传感器转移至分析缓冲液解离至少2分钟，分别测定结合及解离。实验结果使用1:1结合模型进行动力学的分析，结果如图8所示。
由图7和图8可见，酵母菌IDY104可以用于有效地在细胞表面展示抗体，并且展示的抗体能有效地与相应的抗原结合；此外，酵母菌IDY104还可以用于有效地分泌抗体至培养液中，且分泌有抗体的培养液可以原样地直接用于亲和力测定，也可以浓缩10倍后用于亲和力测定。
本实施例表明经CH3 knob稳定转化的酵母能作为抗体的酵母表面展示和分泌二合一的系统有效地工作。
实施例5：比较菌株IDY104表面展示和分泌不同形式抗体的能力
由实施例4可知，通过本发明的酵母表面展示和分泌二合一的系统能有效展示和分泌单域抗体，例如抗PD-L1抗体AmNB1613.36。本实施例进一步实施了本发明的酵母表面展示和分泌二合一的系统对其他形式抗体的表达，例如，将抗人PD-L1单克隆抗体Hz4485构建成scFv-Fc形式，其氨基酸序列如SEQ ID NO:9所示(pYDC083质粒编码的Hz4485 scFv的氨基酸序列如SEQ ID NO:9所示)，编码SEQ ID NO:9的核苷酸序列由苏州金唯智生物科技有限公司合成。将编码SEQ ID NO:9的核苷酸序列用限制性酶BamHI酶切，并连接入经限制性酶BamHI酶切的pYDC081质粒(pYDC081质粒的核苷酸序列如SEQ ID NO:10所示)的两个BamHI位点之间，以替换pYDC081质粒上的核苷酸序列“GGaTCctgacatagtagggattataa”，获得质粒pYDC083。
将质粒pYDC083转化入酵母菌IDY104，涂布于SD-Trp平板(Clontech,630309)；将涂布有转化的酵母菌株的SD-Trp平板于30℃培养3天，获得单克隆的酵母。如实施例4中所述进行酵母培养和诱导表达。比较了本发明的酵母表面展示和分泌二合一的系统对sdAb和scFv-FC两种形式抗体的展示和分泌水平。结果如图9和图10所示。
由图9和图10可知，酵母菌IDY104可以有效展示和分泌单域抗体、scFv-FC等抗体形式，上清液样品能用于亲和力测定。
实施例6：对展示和分泌二合一系统使用Spiking文库评估抗体亲和力成熟的可行性
本发明的酵母表面展示和分泌二合一的系统非常适用于抗体的亲和力成熟文库筛选。本发明的所述系统使得能将筛选获得的突变克隆上清液直接用于抗体亲和力测定，这省去了额外的质粒构建、哺乳动物细胞表达等步骤，大大加快了抗体亲和力研究的相关项目进度。
在本实施例中，构建了筛选抗体亲和力成熟文库的模拟文库。
具体而言，如实施例4所述，将质粒pYDC042(包含编码SEQ ID NO:6所示AmNB1613.36-铰链区-CH2(N297A)-CH3 hole多肽的核苷酸序列转入酵母菌IDY104中。表达的融合蛋白AmNB1613.36-铰链区-CH2(N297A)-CH3 hole通过与酵母细胞表面的CH3 knob的互相作用，间接地将抗PD-L1抗体AmNB1613.36展示于酵母细胞表面，同时分泌少量AmNB1613.36抗体至培养液中。
类似地，将质粒pYDC042中编码AmNB1613.36的核苷酸序列替换为编码亲本纳米抗体HzNB1613(HzNB1613的氨基酸序列:QVQLQESGGGLVQPGGSLRLSCAASAYTISRNSMGWFRQAPGKGLEGVAAIESDGSTSYSDSVKGRFTISLDNSKNTLYLEMNSLRAEDTAVYYCAAPKVGLGPRTALGHLAFMTLPALNYWGQGTLVTVSS(SEQ ID NO:11))的核苷酸序列，其他操作如实施例4所述实施，获得了细胞表面展示抗PD-L1抗体HzNB1613的酵母细胞，和分泌有少量抗体HzNB1613的培养液。
采用表面等离子共振(SPR)分别测定了抗体AmNB1613.36结合PD-L1的平衡解离常数(K D)和抗体HzNB1613结合PD-L1的K D。基于SPR原理，当一束偏振光以一定的角度入射到棱镜端面，在棱镜与金膜的界面将产生表面等离子波，引起金属膜内自由电子产生共振，即表面等离子共振。分析时，先在传感芯片表面固定一层生物分子识别膜，然后将待测样品流过芯片表面，若样品中有能够与芯片表面的生物分子识别膜相互作用的分子，会引起金膜表面折射率变化，最终导致SPR角变化，通过检测SPR角度变化，获得被分析物的亲和力、动力学常数等信息。
K D的测定采用捕获法，抗体被抗人Fc抗体捕获到芯片之后，通过检测抗原与被捕获的抗体之间的结合与解离获得亲和力及动力学常数。该方法包括芯片制备和亲和力检测。测定过程使用10倍稀释后的10xHBS-EP +(BR-1006-69,GE Healthcare)作为实验缓冲液。芯片制备过程使用氨基偶联试剂盒(BR-1006-33,GE Healthcare)，将其中的抗人Fc抗体偶联在CM5芯片(29-1496-03,GE Healthcare)表面，具体过程为：首先将50mM N-羟基琥珀酰亚胺(NHS)与200mM 1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)新鲜混匀并注入CM5芯片双通道，活化7分钟。然后将抗人Fc抗体稀释于10mM乙酸中(pH 5.0)中，注入CM5芯片双通道，使蛋白共价偶联在芯片通道表面，偶联高度约6000RU。最后注入1M乙醇胺，对剩余的活化位点进行封闭7分钟。
亲和力检测的每个循环包括捕获抗体、结合一种浓度抗原及芯片再生，具体操作见如下 所述。
捕获抗体：首先将抗体AmNB1613.36和HzNB1613稀释为0.5μg/mL，以10μL/min的流速，捕获在CM5芯片第二通道，捕获时间30s。
结合抗原：根据SPR的最佳浓度范围，用实验缓冲液两倍梯度稀释人PD-L1(AcroBiosystems，货号：PD1-H5229)，使之介于0.15nM-20nM，按低浓度到高浓度的顺序，注入CM5芯片双通道，结合时间180s，解离时间600s。
芯片再生：在进行下一个抗体测定前，使用10mM Glycine pH 1.5(BR-1003-54,GE Healthcare)对芯片进行再生。
数据结果使用1:1结合模型进行动力学的分析。在如以上测定法所述进行的实验中，抗体AmNB1613.36结合PD-L1的亲和力(K D)为0.1nM，抗体HzNB1613结合PD-L1的亲和力(K D)为3.9nM，两者具有显著的亲和力差别。
分别将细胞表面展示抗PD-L1抗体HzNB1613的酵母细胞与细胞表面展示抗PD-L1抗体AmNB1613.36的酵母细胞以不同的比例混合，制备Spiking文库。具体而言，将展示AmNB1613.36的酵母细胞与展示HzNB1613的酵母细胞按1:10 2、1:10 4分别混合，制备了1％Spiking文库、0.01％Spiking文库。
分别将仅展示AmNB1613.36的酵母细胞、仅展示HzNB1613的酵母细胞、1％Spiking文库、0.01％Spiking文库接种至SD-Leu培养过夜，取1mL菌液转接至5mL YPGP培养基诱导24h；各取1×10 6细胞用10nM PD-L1生物素抗原染色，流式细胞术的结果如图11所示.
由图11可知，在特定的抗原浓度下，酵母细胞展示的抗体亲和力越高，则在流式细胞术中该酵母细胞染色的平均荧光信号值就越高。
将图11中框出的酵母细胞群通过流式细胞术分选出，接种至SD-Leu培养过夜，取1mL菌液转接至5mL YPGP培养基诱导24h；各取1×10 6细胞用10nM PD-L1生物素抗原染色，通过流式细胞术进行二轮筛选，每一轮随机挑取24个单克隆酵母细胞测序，根据测序结果观察展示AmNB1613.36的酵母细胞在Spiking文库中的比例，结果如图12所示。
由图12可见，经过两轮次筛选，Spiking文库中表达AmNB1613.36的酵母细胞的比例富集非常明显；第0轮次(R0)为1％的Spiking文库经过两轮筛选后表达AmNB1613.36的酵母细胞的阳性率达到100％；R0为0.01％的Spiking文库经过两轮筛选后表达AmNB1613.36的酵母细胞的阳性率达到91％。由此，能够富集并获得表达亲和力成熟的酵母细胞株。
通过对Spiking文库的筛选，充分说明此项技术应用于亲和成熟文库筛选是可行的，并且获得的单克隆培养基上清直接测定亲和力；简而言之，应用本发明技术可以从亲和成熟文库中筛选出高亲和力突变克隆，并且单克隆培养基上清可以直接测定亲和力，省去了传统的质粒构建、表达的过程，即加快了速度又节省了成本，经济效益非常显著。
尽管已经出于说明本发明的目的显示了某些代表性实施方案和细节，但是本领域技术人员显而易见的是可以对它们进行多种变化和修改而不脱离主题发明的范围。在这个方面，本发明范围仅由以下权利要求限定。

Claims (11)

  1. 细胞表面展示和分泌目的多肽的酵母展示系统，其是导入有第一核酸分子和第二核酸分子的酿酒酵母(Saccharomyces cerevisiae)细胞，其中所述第一核酸分子包含编码酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的核苷酸，所述第二核酸分子包含编码目的多肽的核苷酸和编码免疫球蛋白Fc区或其部分的核苷酸，优选地，所述第一核酸分子和第二核酸分子位于同一个质粒上或位于分开的质粒上。
  2. 用于细胞表面展示和分泌目的多肽的酵母展示系统，其是重组的酿酒酵母细胞，其中在酿酒酵母细胞的基因组中的靶位点处插入有第一核酸分子，所述第一核酸分子包含编码酿酒酵母细胞表面锚定蛋白和免疫球蛋白Fc区CH3结构域的核苷酸，所述重组的酿酒酵母细胞用于导入包含编码目的多肽的核苷酸和编码免疫球蛋白Fc区或其部分的核苷酸的第二核酸分子，以在细胞表面展示和分泌目的多肽。
  3. 根据权利要求1或2的酵母展示系统，其中所述第一核酸分子和第二核酸分子各自的Fc区中分别包含凸起(“结(knob)”)或空穴(“扣(hole)”)，由此所述第一核酸分子表达的第一多肽链和第二核酸分子表达的第二多肽链彼此能够形成“结入扣(knob-in-hole)”的稳定缔合；优选地，所述第一核酸分子和第二核酸分子编码的第一多肽链和第二多肽链之一条链中包含氨基酸置换T366W，并且在所述第一多肽链和第二多肽链之另一条链中包含氨基酸置换T366S、L368A和Y407V(根据Kabat的“EU编号”)，由此一条链中的凸起能够置于另一条链中的空穴中，由此促进第一多肽链和第二多肽链的缔合；
    优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
  4. 根据权利要求1－3中任一项所述的酵母展示系统，其中所述第二核酸分子包含编码目的多肽的核苷酸、任选地编码免疫球蛋白Fc区的铰链区的核苷酸、以及编码免疫球蛋白Fc区的CH2结构域和CH3结构域的核苷酸；优选地，所述CH2结构域中的糖基化位点被消除，例如，将人IgG Fc区的CH2结构域中的N297残基突变以消除该糖基化位点，例如，将N297残基变成Gly、Ala、Gln、Asp或Glu，优选地，将N297残基变成Ala。
  5. 根据权利要求1－4中任一项所述的酵母展示系统，其中所述酿酒酵母细胞表面锚定蛋白是含有糖基磷脂酰肌醇(GPI)锚定信号序列的酿酒酵母细胞壁蛋白，例如，α-凝集素和a-凝集素、Cwp1p蛋白和Flo1p蛋白。
  6. 根据权利要求1－4中任一项所述的酵母展示系统，其中所述酿酒酵母是表达a-凝集素的aga1p亚基的酿酒酵母，例如酿酒酵母EBY100；所述酿酒酵母细胞表面锚定蛋白是aga2p亚基，由此，第一核酸分子编码的第一多肽包含aga2p和免疫球蛋白Fc区CH3结构域，且所述第一多肽结合至已经结合且呈递在酿酒酵母细胞表面的aga1p亚基。
  7. 根据权利要求2－4中任一项所述的酵母展示系统，其中第一核酸分子插入至酿酒酵母细胞基因组中的营养合成基因所在的基因组位点处，以破坏营养合成基因，例如，插入至酿酒酵母细胞基因组中的URA3基因座处，获得了URA3 －营养缺陷型标记的酿酒酵母细胞； 插入至酿酒酵母细胞基因组中的TRP1基因座处，获得了TRP1 －营养缺陷型标记的酿酒酵母细胞。
  8. 根据权利要求1－7中任一项所述的酵母展示系统，其中所述目的多肽是抗体或抗原结合片段，例如，Fab片段、VHH结构域、scFv、sdAb。
  9. 根据权利要求1－8中任一项所述的酵母展示系统，其中
    (a)所述第一核酸分子从N端至C端包含编码CH3 knob-任选地接头或标签-Aga2p的核苷酸，且所述第二核酸分子从N端至C端包含编码目的多肽-任选地铰链区-CH2(N297A)-CH3 hole的核苷酸；或
    (b)所述第一核酸分子从N端至C端包含编码CH3 hole-任选地接头或标签-Aga2p的核苷酸，且所述第二核酸分子从N端至C端包含编码目的多肽-任选地铰链区-CH2(N297A)-CH3 knob的核苷酸；
    优选地，所述接头包含甘氨酸(G)和丝氨酸(S)残基，例如，所述接头是GS；
    优选地，所述标签选自Arg标签、Avi标签、His-Avi标签、His标签、Flag标签、3xFlag标签、Strep标签、Nano标签、SBP标签、c-myc标签、S标签、钙调蛋白结合肽、纤维素结合结构域、几丁质结合结构域、GST标签或MBP标签。
  10. 根据权利要求1－9中任一项所述的酵母展示系统的用途，用于表达在细胞表面展示、且分泌至培养基中的目的多肽；优选地，用于表达在细胞表面展示、且分泌至培养基中的抗体，例如PD-L1抗体、PD-1抗体。
  11. 根据权利要求1－9中任一项所述的酵母展示系统的用途，用于构建目的多肽的变体展示文库，其中细胞表面展示的方式使得能够借助高通量筛选选择特定的目的多肽变体，分泌至培养基中的方式使得能够进行目的多肽变体的生物化学表征；优选地，用于构建抗体的变体展示文库，以筛选出高亲和力的抗体变体。
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