CN113544275A - Yeast display system for displaying and secreting target polypeptide and application thereof - Google Patents

Abstract

The present invention relates to a yeast display system capable of displaying a polypeptide of interest on the surface of a cell and capable of secreting the polypeptide of interest into a culture medium, and to the use of said yeast display system. The yeast display system of the present invention can be stably constructed 10⁸An order of magnitude of a polypeptide variant of interest display library, wherein the display on the cell surface is in a manner that enables selection of a particular polypeptide variant of interest by means of high throughput screening, and the secretion into the culture medium is in a manner that enables biochemical characterization of the polypeptide variant of interest; in particular, the yeast display system of the invention can be used to construct variants of antibodiesThe library is displayed in vivo, thereby obtaining high affinity antibody variants.

Description

Yeast display system for displaying and secreting target polypeptide and application thereof Technical Field

The present invention relates to a yeast display system capable of displaying a polypeptide of interest on the surface of a yeast cell and capable of secreting the polypeptide of interest into a culture medium, and to the use of said yeast display system. The yeast display system of the present invention can be stably constructed 10⁸An order of magnitude of a polypeptide variant of interest display library, wherein the display on the cell surface is in a manner that enables selection of a particular polypeptide variant of interest by means of high throughput screening, and the secretion into the culture medium is in a manner that enables biochemical characterization of the polypeptide variant of interest; in particular, the yeast display system of the invention can be used to construct variant display libraries of antibodies, thereby obtaining high affinity antibody variants.

Background

Production of proteins in bacterial systems has been reported in the prior art, but even with extensive engineering, bacterial systems do not produce fully human glycosylated proteins, and thus, the use of prokaryotic expression of proteins may not predict the function of the protein in eukaryotic hosts having post-translational modifications such as glycosylation modifications.

The art has also attempted to display polypeptides, such as IgG, in mammalian cell systems, which requires the introduction of additional ectopic sequences into the IgG heavy chain to anchor the IgG directly to the cell membrane (Zhou C et al, Development of a novel mammalin cell surface antibody display platform, MAbs, 2010, 2: 508-.

Shaheen HH et al describe the Display and secretion of Monoclonal Antibodies on the Surface of yeast cells of the lower eukaryotic Pichia pastoris species (Pichia pastoris) (Shaheen HH et al, A Dual-Mode Surface Display System for the mapping and Production of Monoclonal Antibodies in Glyco-Engineered Pichia pastoris, PLoS ONE, 2013, 8(7): e 70190). Because the plasmid containing the coding nucleic acid of the monoclonal antibody is required to be inserted into the genome of pichia pastoris to stably exist and express the monoclonal antibody, and the efficiency of inserting the plasmid into the genome of pichia pastoris is low, the pichia pastoris display system for displaying and secreting the target polypeptide cannot meet the requirement of antibody discovery and antibody engineering on library diversity.

In addition, the prior art yeast display systems directly display the Fc domain comprising the hinge region, which results in the Fc domain comprising the hinge region readily forming a homodimer by itself and reducing the level of antibody display on the yeast surface.

Thus, there remains a need in the art for new yeast display systems for displaying and secreting polypeptides of interest that not only allow for the stable construction of large-order display libraries of polypeptide variants of interest, but also allow for the easy biochemical characterization of polypeptide variants of interest by testing of culture broth.

Summary of The Invention

The present inventors have developed, through research, a Saccharomyces cerevisiae (Saccharomyces cerevisiae) cell display system capable of not only displaying a polypeptide of interest on the cell surface of Saccharomyces cerevisiae, but also secreting the polypeptide of interest into a culture medium. By using the yeast display system of the present invention, 10 can be stably constructed⁸Order of magnitude of polypeptide variants of interestDisplaying the library, thereby meeting the requirements of antibody discovery and antibody engineering on library diversity.

Thus, in a first aspect, the present invention provides a yeast display system which is a Saccharomyces cerevisiae (Saccharomyces cerevisiae) cell into which has been introduced a first nucleic acid molecule comprising nucleotides encoding a Saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain, and a second nucleic acid molecule comprising nucleotides encoding a polypeptide of interest and nucleotides encoding an immunoglobulin Fc region or part thereof, preferably, the first and second nucleic acid molecules are located on the same plasmid or on separate plasmids.

The yeast display system of the invention anchors a first fusion protein comprising a saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain, on the surface of a saccharomyces cerevisiae cell through the anchor protein by expressing a first nucleic acid molecule. The yeast display system of the invention displays a polypeptide of interest on the surface of a s.cerevisiae cell by expressing a second nucleic acid molecule, stably associating a portion of a second fusion protein comprising the polypeptide of interest and an immunoglobulin Fc region, or a portion thereof, with the immunoglobulin Fc region CH3 domain of the first fusion protein via the immunoglobulin Fc region, or a portion thereof; the other portion of the second fusion protein is retained in the culture medium.

In a second aspect, the present invention provides a yeast display system which is a recombinant s.cerevisiae cell in which a first nucleic acid molecule comprising nucleotides encoding a s.cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain is inserted at a target site in the genome of the s.cerevisiae cell for introduction of a second nucleic acid molecule comprising nucleotides encoding a polypeptide of interest and nucleotides encoding an immunoglobulin Fc region or a part thereof for display and secretion of the polypeptide of interest at the cell surface. In a specific embodiment, the present invention provides a yeast display system wherein said first nucleic acid molecule is inserted into the genome of a s.cerevisiae cell at the URA3 locus to obtainURA3 ^－An auxotrophic, tagged s.cerevisiae cell for introducing 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 for display and secretion of the polypeptide of interest at the cell surface.

The yeast display system of the invention anchors a first fusion protein comprising a saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain, on the recombinant saccharomyces cerevisiae cell surface through the anchor protein by expressing a first nucleic acid molecule. The yeast display system of the invention displays a polypeptide of interest on the surface of a s.cerevisiae cell by expressing a second nucleic acid molecule, stably associating a portion of a second fusion protein comprising the polypeptide of interest and an immunoglobulin Fc region, or a portion thereof, with the immunoglobulin Fc region CH3 domain of the first fusion protein via the immunoglobulin Fc region, or a portion thereof; the other portion of the second fusion protein is retained in the culture medium.

In some embodiments, for the yeast display systems of the first and second aspects of the invention, the first and second nucleic acid molecules each comprise a bulge ("knob") or a hole ("hole") in the Fc region, respectively, whereby the first polypeptide chain expressed by the first nucleic acid molecule and the second polypeptide chain expressed by the second nucleic acid molecule are capable of forming a stable association of "knob-in-hole" with each other. Preferably, the first and second nucleic acid molecules encode a first and second polypeptide chain comprising in one of the chains the amino acid substitution T366W and in the other of the first and second polypeptide chains the amino acid substitutions T366S, L368A and Y407V (according to Kabat' EU numbering "), whereby a protuberance in one chain is capable of being placed in a cavity in the other chain, thereby facilitating association of the first and second polypeptide chains.

In some embodiments, for the yeast display systems of the first and second aspects of the invention, the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, preferably the immunoglobulin is a human IgG1 immunoglobulin.

In a particular embodiment, for the yeast display system of the first and second aspects of the invention, the second nucleic acid molecule comprises nucleotides encoding the polypeptide of interest, optionally nucleotides encoding the hinge region of an immunoglobulin Fc region, and nucleotides encoding the CH2 and CH3 domains of the immunoglobulin Fc region; preferably, the glycosylation site in the CH2 domain is eliminated, e.g., the N297 residue in the CH2 domain of the human IgG Fc region is mutated to eliminate the glycosylation site, e.g., the N297 residue is changed to Gly, Ala, gin, Asp or Glu, preferably the N297 residue is changed to Ala.

In a particular embodiment, for the yeast display system of the first and second aspects of the invention, the s.cerevisiae cell surface anchor protein is a s.cerevisiae cell wall protein containing a Glycosylphosphatidylinositol (GPI) anchor signal sequence, e.g., alpha-lectin and a-lectin, Cwp1p protein, and Flo1p protein.

In some embodiments, for the yeast display system of the first and second aspects of the invention, the s.cerevisiae, when not introduced with the first and second nucleic acid molecules, is itself a s.cerevisiae expressing the aga1p subunit of a-lectin, e.g., s.cerevisiae EBY 100; the s.cerevisiae cell surface anchor protein expressed by the first nucleic acid molecule introduced into said s.cerevisiae cell is an aga2p subunit, whereby the first polypeptide encoded by the first nucleic acid molecule comprises aga2p and the immunoglobulin Fc region CH3 domain, and said first polypeptide binds to the aga1p subunit which has been bound to and presented on the s.cerevisiae cell surface.

In some embodiments, for the yeast display systems of the first and second aspects of the invention, the polypeptide of interest is an antibody or antigen binding fragment, e.g., a Fab fragment, a VHH domain, a scFv, a sdAb.

In some embodiments, the invention provides such a yeast display system, wherein

(a) The first nucleic acid molecule comprises an encoding CH3 from N-terminus to C-terminus_knob-optionally a linker or a tag-nucleotides of Aga2p, and the second nucleic acid molecule comprises, from N-terminus to C-terminus, a nucleotide encoding a polypeptide of interest-optionally a hingeThe region-CH 2(N297A) -CH3_holeThe nucleotide of (a); or

(b) The first nucleic acid molecule comprises an encoding CH3 from N-terminus to C-terminus_hole-optionally a linker or a tag-the nucleotides of Aga2p, and the second nucleic acid molecule comprises, from N-terminus to C-terminus, a nucleotide encoding a polypeptide of interest-optionally a hinge region-CH 2(N297A) -CH3_knobThe nucleotide of (a).

Preferably, the linker comprises glycine (G) and serine (S) residues, e.g., the linker is GS.

Preferably, the tag is selected from the group consisting of an Arg-tag, an Avi-tag, a His-tag, a Flag-tag, a3 XFlag-tag, a Strep-tag, a Nano-tag, an SBP-tag, a c-myc-tag, an S-tag, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, a GST-tag or an MBP-tag.

In a third aspect, the present invention provides the use of the yeast display system of the first and second aspects of the invention for expressing a polypeptide of interest displayed on the surface of a cell and secreted into the culture medium; preferably, for expression of the antibody displayed on the surface of a cell and secreted into the culture medium.

In a fourth aspect, the present invention provides the use of the yeast display system of the first and second aspects of the invention for constructing a variant display library of polypeptides of interest, wherein the cell surface display is in a manner that enables selection of a particular polypeptide variant of interest by means of high throughput screening, and secretion into culture medium in a manner that enables biochemical characterisation of the polypeptide variant of interest; preferably, variant display libraries for constructing antibodies are used to screen high affinity antibody variants.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Brief description of the drawings:

FIG. 1: a schematic diagram of saccharomyces cerevisiae displaying antibodies on the cell surface by FC is shown.

FIG. 2: shows different lengths FC_knobEffect on the display of antibodies on the cell surface of s.cerevisiae.

FIG. 3: a schematic representation of the flow cytometry staining of antigen displaying yeast cells with antibody secreted into the culture broth is shown.

FIG. 4: the results of flow cytometry staining of antigen-displaying yeast cells with antibody secreted into the culture broth are shown.

FIG. 5: shows that yeast is modified to show CH3 on the cell surface_knobSchematic representation of (a).

FIG. 6: shows the display of CH3 on the surface of the engineered cell_knobThe yeast IDY104 test showed CH3_knobHorizontal results.

FIG. 7: flow cytometry is shown to detect the levels of yeast IDY104 display antibody transferred into plasmid pYDC 042.

FIG. 8: the results of the affinity assay for a sample of the culture supernatant of yeast IDY104 transformed with plasmid pYDC042 are shown.

FIG. 9: results of the assay of yeast IDY104 displaying the levels of either the sdAb-Fc format antibody or the scFv-Fc format antibody are shown.

FIG. 10: the results of the affinity assay of the culture supernatant of yeast IDY104 after 10-fold concentration are shown.

FIG. 11: flow cytometry staining patterns showing antigen binding of different Spiking libraries of amnnb1613.36 and HzNB1613 are shown.

FIG. 12: the proportion of yeast displaying amnnb1613.36 after each round of screening of the Spiking library is shown. In the figure, R0 represents the 0 th round of selection, R1 represents the 1 st round of selection, and R2 represents the 2 nd round of selection.

Detailed description of the invention:

I. definition of

For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

As used herein, the term "and/or" means any one of the options or two or more of the options.

As used herein, the term "comprising" or "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps.

The term "antibody" is used herein in the broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, and antigen-binding fragments.

The terms "whole antibody", "full-length antibody", "whole antibody" and "intact antibody" are used interchangeably herein to refer to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions (as Complementarity Determining Regions (CDRs) with more conserved regions (as Framework Regions (FRs)) interposed between each VH and VL consisting of three CDRs and 4 FRs arranged in the order from amino to carboxyl, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. the constant regions are not directly involved in the binding of antibodies to antigens, but exhibit multiple effector functions.

The term "antigen binding fragment" is an amino acid of a more complete or complete antibodyA portion or fragment of a complete or complete antibody with a reduced number of residues that is capable of binding to an antigen or that competes with the complete antibody (i.e., the complete antibody from which the antigen-binding fragment is derived) for binding to an antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab ', F (ab')₂Fv, single chain Fv, diabody (diabody), single domain antibody (sdAb). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, and can be obtained, for example, by papain digestion of whole antibodies. In addition, complete antibody production F (ab') by pepsin digestion below the disulfide bond in the hinge region₂Which is a dimer of Fab' and is a bivalent antigen binding fragment. F (ab')₂Can be reduced under neutral conditions by disrupting disulfide bonds in the hinge region, thereby converting F (ab')₂The dimer is converted to Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antigen binding fragments see: basic Immunology, edited by W.E.Paul, Raven Press, N.Y. (1993)). The Fv fragment consists of the VL and VH domains of a single arm of an antibody. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, using recombinant methods, they can be joined by a synthetic linker peptide that enables the two domains to be produced as a single protein chain in which the VL and VH regions pair to form a single chain Fv. The antigen-binding fragment can be obtained by chemical methods, recombinant DNA methods, or protease digestion methods.

The term "single domain antibody" (sdAb) generally refers to antibodies in which a single variable domain (e.g., a heavy chain variable domain (VH) or light chain variable domain (VL), a heavy chain variable domain derived from a camelid heavy chain antibody, a VH-like single domain derived from a fish IgNAR (v-NAR)) can confer antigen binding. That is, the single variable domain need not interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include single domain antibodies derived from camelidae (llama and camel) and cartilaginous fish (e.g. nurse shark) (WO 2005/035572).

The heavy chain variable domain of camelid heavy chain antibodies with high affinity for the target antigen (this region is also called VHH, which has a molecular weight one tenth of that of human IgG molecules and has a physical diameter of only a few nanometers) can be obtained by genetic engineering methods. See U.S. patent No. 5,759,808 issued on 6/2/1998. Like other non-human antibody fragments, the amino acid sequence of a camelid VHH may be recombinantly altered to obtain a sequence that more closely mimics a human sequence, i.e. "humanised", thereby reducing the antigenicity of a camelid VHH to humans. Alternatively, key elements derived from camelid VHH may be transferred to a human VH domain to obtain a camelised human VH domain, resulting in the human VH domain no longer needing to pair with a VL domain to recognise the target antigen, the camelised human VH domain alone conferring antigen binding specificity. The camelid VHH, humanized camelid VHH, camelized human VH domain are all within the scope of the term "VHH domain".

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons composed of two light and two heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each immunoglobulin heavy chain has one heavy chain variable region (VH), also known as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH 3). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also known as a light chain variable domain, followed by a light chain constant domain (CL). Heavy chains of immunoglobulins can be assigned to one of 5 classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), wherein certain classes can be further divided into subclasses, e.g., γ₁(IgG1)、γ ₂(IgG2)、γ ₃(IgG ₃)、γ ₄(IgG ₄)、α ₁(IgA ₁) And alpha₂(IgA ₂). Immunoglobulin light chains can be divided into two types based on the amino acid sequence of their constant domainsOne, called κ and λ. An immunoglobulin essentially consists of two Fab molecules and one Fc domain connected by means of an immunoglobulin hinge region.

The terms "Fc domain", "Fc region" or "Fc" are used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in natural antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of IgG, IgA, and IgD class antibodies; or second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) derived from the two heavy chains of antibodies of the IgM and IgE classes. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al, Sequences of Proteins of Immunological Interes, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The term "effector functions" refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with the isotype of the immunoglobulin. Examples of 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 (e.g. B cell receptors) and B cell activation.

As used herein, the term "binding" or "specific 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 particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA), Surface Plasmon Resonance (SPR) or biofilm layer interference techniques or other conventional binding assays known in the art.

The terms "PD-L1", "programmed cell death ligand 1", "programmed death ligand 1" as used herein refer to any native 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, as well as any form of PD-L1 that results from processing in a cell. PD-L1 may be present 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: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain.

The terms "anti-PD-L1 antibody", "anti-PD-L1", "PD-L1 antibody", or "antibody that binds to PD-L1" as used herein refer to an antibody that is capable of binding to PD-L1 protein or a fragment thereof with sufficient affinity. In one embodiment, the anti-PD-L1 antibody binds to a non-PD-L1 protein to a lesser extent than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the binding of the antibody to PD-L1, as measured, for example, by Radioimmunoassay (RIA) or biophoto interferometry, or SPR or biofilm layer interference, or the like.

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y may be generally determined by the dissociation constant (K)_D) Typically, the dissociation constants are the dissociation and association rate constants (k, respectively)_disAnd k_on) The ratio of (a) to (b). Affinity can be measured by common methods known in the art.

The term "variant" refers to a modification in a parent amino acid sequence. Exemplary modifications include amino acid substitutions, insertions, and/or deletions. In one embodiment, the polypeptide variant of interest is a substitution to the amino acid sequence of a 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" (i.e., encoded by the genetic code) are selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (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 residue" refers to a residue that is capable of covalently binding to 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, α -aminoisobutyric acid (aib), and other amino acid residue analogs such as those described in Ellman et al, meth.enzym.202(1991) 301-336.

The term "variant" in relation to an antibody refers herein to an antibody comprising a region of the antibody of interest (e.g. a heavy chain variable region or a light chain variable region or a heavy chain CDR region or a light chain CDR region) that has been altered in amino acid by at least 1, e.g. 1-30, or 1-20 or 1-10, e.g. 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions and/or insertions, wherein the variant substantially retains the biological properties of the antibody molecule prior to the alteration. It is understood that the heavy chain variable region or the light chain variable region, or each CDR region of an antibody may be altered individually or in combination. In some embodiments, the amino acid change in one or more or all three heavy chain CDRs is no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the amino acid change in one or more or all three light chain CDRs is no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the amino acid change in one or more or all 6 CDRs is no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, the amino acid change is an amino acid substitution, preferably a conservative substitution. In some embodiments, the antibody variant has at least 80%, 85%, 90% or 95% or 99% or more amino acid sequence identity to the parent antibody over the region of the antibody sequence of interest.

"complementarity determining regions" or "CDR regions" or "CDRs" are regions of antibody variable domains that are mutated in sequence and form structurally defined loops ("hypervariable loops") and/or regions that contain antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et Al (1989) Nature 342:877-883, Al-Lazikani et Al, "Standard transformations for the structural construction of antibodies of immunological tissues", Journal of Molecular Biology,273,927-948(1997)), based on antibody sequence variations Kabat (Kabat et Al, Sequences of Proteins of immunological tissues, 4 th edition, U.S. Department of Health and Human Services, national instruments of Health (1987)), AbM (standardization of bath), reaction chemistry, cloning of Molecular tissues), and the topology of CDR loops (international patent GT), and based on the use of the generic domains of the general tissue. In the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the ways described above.

The term "linker" refers to a connecting peptide consisting of amino acids, such as glycine and/or serine residues, used alone or in combination, to connect the various regions in the fusion protein. In one embodiment, the linker is a Gly/Ser connecting peptide comprising the amino acid sequence (Gly)_1-4Ser) n, where n is a positive integer equal to or greater than 1, e.g., n is a positive integer from 1 to 7. In one embodiment, the linker is GS. Also included within the scope of the invention is WO2012The linker peptide described in/138475, which is incorporated herein by reference.

The term "tag" refers to a sequence of amino acid residues linked to each other by peptide bonds having specific binding properties. In one embodiment, the amino acid sequence tag is an affinity or purification tag. In one embodiment, the amino acid sequence tag is selected from the group consisting of an Arg tag, a His tag, an Avi tag, a His-Avi tag, a Flag tag, a 3xFlag tag, a Strep tag, a Nano tag, an SBP tag, a c-myc tag, an S tag, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, a GST tag, and an MBP tag (see, e.g., Amau, J. et al, Current protocols for the use of affinity tags and tag removalness for the purification of recombinant proteins, Protein Expr. purif., 2006, 48 (1): 1-13).

The term "fused to …" refers to a covalent bond, e.g., a peptide bond, formed between two moieties.

The terms "first fusion protein", "first polypeptide chain" have the same meaning and are used interchangeably herein to refer to the polypeptide product expressed by a first nucleic acid molecule. Likewise, the terms "second fusion protein", "second polypeptide chain" have the same meaning and are used interchangeably herein to refer to the polypeptide product expressed by a second nucleic acid molecule.

The term "N-terminal" refers to the last amino acid at the N-terminus, and the term "C-terminal" refers to the last amino acid at the C-terminus.

The term "introduction" refers to any method suitable for introducing or transferring a polynucleotide (e.g., a plasmid) into a yeast cell by any technique known in the art, e.g., transformation, transduction, transfection. The term "transformation" is used to describe the introduction of a polynucleotide into a yeast cell. The term "transduction" refers to viral introduction of a polynucleotide or genetic material or viral transfer into a cell. Any known viral system may be used for transducing host cells of the invention, such as adenovirus-based systems, adeno-associated Virus (AAV) -based systems, retrovirus systems, lentivirus expression systems or Herpes Simplex Virus (HSV) -based systems, or other Virus-based systems such as vaccinia, EB Virus, Sendai Virus, Sindbis Virus, polyoma Virus and measles Virus-based systems (see, e.g., Mah C et al, Virus-based gene delivery systems, Clin. Pharmocokin, 2002, 41 (12): 901-911). The term "transfection" as used in the Methods of the invention refers to the uptake of nucleic acid into a host cell by any suitable method known in the art, for example, the Methods disclosed in Sambrook et al (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al (1986) Basic Methods in Molecular Biology, Elsevier, in particular calcium phosphate co-precipitation, direct microinjection into cultured cells, ultrasound-mediated gene transfection, electroporation, lipofection, or nuclear transfection (nucleofection).

The term "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 selectable marker and an origin of replication, and optionally a secretion signal sequence.

The yeast display system of the invention

Saccharomyces cerevisiae is a well characterized eukaryotic host organism in the art as a model organism for studying eukaryotic cell function, and is also suitable for expressing heterologous polypeptides of interest. Saccharomyces cerevisiae, as a unicellular organism, is less complex than other eukaryotic systems and can be cultured in defined media, thus enabling good control of its growth conditions and reduction of its culture costs. Having a relatively short life cycle of about 90 minutes is one reason for the preference to use Saccharomyces cerevisiae. Since saccharomyces cerevisiae combines the advantages of microbial expression systems due to simple culture and the use of industrial fermentation methods and eukaryotic expression systems due to eukaryotic expression and the presence of secretory pathways in the cell, and enables large-scale selection, the present invention uses saccharomyces cerevisiae to express and display heterologous polypeptides of interest.

In one embodiment, the present invention provides a yeast display system prepared by the steps of:

i. introducing into a s.cerevisiae cell a first nucleic acid molecule comprising nucleotides encoding a s.cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain, and a second nucleic acid molecule comprising nucleotides encoding a polypeptide of interest and nucleotides encoding an immunoglobulin Fc region or a portion thereof,

culturing the transformed saccharomyces cerevisiae cell expressing a first fusion protein comprising a saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain; and expressing a second fusion protein comprising the polypeptide of interest and an immunoglobulin Fc region or portion thereof;

whereby the expressed first fusion protein is anchored on the surface of the s.cerevisiae cell by the anchor protein; expressing a portion of the second fusion protein to display the polypeptide of interest on the cell surface of s.cerevisiae by stable association of the immunoglobulin Fc region, or a portion thereof, with the immunoglobulin Fc region CH3 domain of the first fusion protein; the other portion of the second fusion protein expressed is retained in the culture medium.

The yeast display system of the present invention can be used to construct 10 stably⁸An order of magnitude of the polypeptide variant of interest display library, wherein the display on the cell surface is in a manner enabling the selection of a specific polypeptide variant of interest by means of high throughput screening, and the secretion into the culture medium is in a manner enabling the biochemical characterization of the polypeptide variant of interest.

The first nucleic acid molecule and the second nucleic acid molecule can be introduced into the s.cerevisiae cell using any method known in the art. In a specific embodiment, the introduction of the first nucleic acid molecule and the second nucleic acid molecule is performed according to the methods described by Gietz, R.D. et al (Gietz, R.D. and Schiestl, R.H., High-efficiency layer transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols,2007,2(1): 31-34).

The form in which the first nucleic acid molecule and the second nucleic acid molecule exist and the order of introduction into the cell are not particularly limited as long as both the first nucleic acid molecule and the second nucleic acid molecule are expressed after introduction into the s.cerevisiae cell.

In one embodiment, the first nucleic acid molecule and the second nucleic acid molecule are located on the same plasmid, and the plasmid comprising the first nucleic acid molecule and the second nucleic acid molecule is introduced into a s.cerevisiae cell.

In another embodiment, the first nucleic acid molecule and the second nucleic acid molecule are located on separate plasmids and the plasmid comprising the first nucleic acid molecule and the plasmid comprising the second nucleic acid molecule are introduced into the s.cerevisiae cell separately, sequentially or simultaneously.

There are many plasmids and/or expression vectors used for the expression of recombinant proteins in Saccharomyces cerevisiae, for example the vectors p426Met25 or p426GAL1(Mumberg et al (1994) nucleic acids Res.,22, 5767-. In one embodiment, a pYDC vector (e.g., pYDC011) is used for introducing the first nucleic acid molecule and/or the second nucleic acid molecule into saccharomyces cerevisiae. Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like. Suitable vectors and promoters for use in yeast expression are further described in r.hitzmann et al, EP073,657.

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 for the yeast protein. An example of a secretion signal sequence is the leader sequence of the precursor alpha factor of yeast junction pheromones, which is also used to direct secretion of heterologous proteins in yeast (see, e.g., Valenzuela, P. editor, p. 269, Butterworth, London; Brake, A.J. (1990) meth. enzymol.185, 408-441). Another example is a leader sequence from a yeast invertase (MLLQAFLFLLAGFAAKISADAHKS). It has been shown that this leader sequence will be cleaved from the nascent heterologous peptide when it enters the endoplasmic reticulum. An additional example is the signal sequence of yeast acid phosphatase, which can also be used to direct secretion of the fusion proteins disclosed herein.

The s.cerevisiae cell surface anchor protein in the first fusion protein is typically an extracellular wall protein of s.cerevisiae. Many of the extracellular wall proteins of s.cerevisiae are in principle capable of displaying heterologous proteins and peptides on the cell surface as part of the first fusion protein of the invention, such as alpha-and a-lectins, the Cwp1p protein and the Flo1p protein.

In one embodiment, a-lectin is used as the s.cerevisiae cell surface anchor protein in the first fusion protein. The cell wall protein a-lectin comprises the subunits Aga1p and Aga2 p. The subunit Aga1p has a Glycosylphosphatidylinositol (GPI) anchor signal sequence and mediates immobilization of the protein to the outer cell wall by covalent binding of β -glucan. The subunit Aga2p is also secreted by the cell and is bonded to Aga1p via two disulfide bonds.

In one embodiment, the saccharomyces cerevisiae is a saccharomyces cerevisiae expressing the Aga1p subunit of a-lectin, such as saccharomyces cerevisiae EBY100 species, which expresses the Aga1p subunit through a chromosomally integrated galactose-inducible expression cassette. For s.cerevisiae expressing the aga1p subunit of a-lectin, the s.cerevisiae cell surface anchoring protein is the aga2p subunit. When Aga1p is bound to Aga2p in the first fusion protein, the heterologous polypeptide of interest (e.g., an antibody) is displayed on the surface of the yeast cell.

The immunoglobulin Fc region CH3 domain in the first fusion protein is a non-antigen binding region located C-terminal to the immunoglobulin heavy chain. In some embodiments, the IgG CH3 domain begins at Gly 341. It is understood that the C-terminal Lys residue of human IgG may optionally be absent. It is also 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.

In some embodiments, the immunoglobulin Fc region CH3 domain in the first fusion protein of the invention and the immunoglobulin Fc region or portion thereof in the second fusion protein use the "knot-in-hole" technique (see, e.g., John B.B.Ridgway et al, 'Knobs-inter-holes' Engineering of antibodies CH3 domains for latent channel ligation. protein Engineering,1996,9(7): p.617-21; Shane Atwell et al, Stable carbohydrates for modulation of the domain interface of a modular user phase plate. J.mol.biol,1997,270: p.26-35) which can be engineered between the first and second fusion proteins of the invention to facilitate proper association of the first and second fusion proteins of the invention. In general, this technique involves introducing a "bump" at the interface of one of the fusion proteins and a corresponding "cavity" at the interface of the other fusion protein to be mated with it, so that the bump can be placed in the cavity. One preferred interface comprises the CH3 domain of one of the fusion proteins and the CH3 domain of the other fusion protein to which it is to be paired. 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 a larger side chain (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller side chain (e.g., alanine or threonine), compensatory cavities of the same or similar size to the projections are created at the interface of the CH3 domains of another fusion protein to be paired.

Preferred residues for forming the knot (knob) are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, 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 forming a junction include, but are not limited to, T366W, T366Y, or F405W substitutions.

Preferred residues for the formation of a knot (hole) are generally naturally occurring amino acid residues and are preferably selected from the group consisting of alanine (a), serine (S), threonine (T) and valine (V). In one embodiment, the original residue used to form the clasp has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain for generating clasps include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions. In certain embodiments, the knot comprises a T366W substitution and the clasp comprises a T366S/L368A/Y407V substitution. In some embodiments, the first and second nucleic acid molecules of the invention encode a first and second polypeptide chain comprising in one of the chains the amino acid substitution T366W and in the other of the first and second polypeptide chain the amino acid substitutions T366S, L368A and Y407V (according to Kabat' EU numbering "), whereby a protuberance in one chain is capable of being placed in a cavity in the other chain, thereby facilitating association of the first and second polypeptide chains.

The polypeptide of interest in the second fusion protein is not particularly limited. The polypeptide of interest may be any polypeptide to be expressed. In some embodiments, the polypeptide of interest is an antibody or antigen binding fragment, e.g., a Fab fragment, a VHH domain, a scFv, a sdAb.

The immunoglobulin Fc region or portion thereof in the second fusion protein comprises the CH2 domain and the CH3 domain of the immunoglobulin Fc region, and optionally an immunoglobulin Fc region hinge region.

In some embodiments, the immunoglobulin Fc region or portion thereof in the second fusion protein is altered to reduce the extent of glycosylation of the Fc portion of the second fusion protein. Deletion of glycosylation sites from a protein can be conveniently accomplished by altering the amino acid sequence so as to remove one or more glycosylation sites. For example, the N297 residue in the CH2 domain of the IgG Fc region is mutated to eliminate the glycosylation site, e.g., to change the N297 residue to Gly, Ala, Gln, Asp or Glu, preferably, to Ala. N297 refers to the asparagine residue at about position 297 within the Fc region (EU numbering of residues in the Fc region); however, N297 can also be located approximately upstream or downstream of position 297 by ± 3 amino acids, i.e., between position 294 and position 300, due to minor sequence variations in the immunoglobulin.

In order to simplify the handling and to omit additional plasmid construction steps, the invention also provides a yeast display system for cell surface display and secretion of a polypeptide of interest, which is a recombinant s.cerevisiae cell into which a first nucleic acid molecule comprising nucleotides encoding a s.cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain is inserted at a target site in the genome of the s.cerevisiae cell for introduction of a second nucleic acid molecule comprising nucleotides encoding a polypeptide of interest and nucleotides encoding an immunoglobulin Fc region or part thereof for display and secretion of a polypeptide of interest on the cell surface.

To prepare a recombinant s.cerevisiae cell, a first nucleic acid molecule is introduced at a target site in the genome of the s.cerevisiae cell.

The target site in the s.cerevisiae cell genome into which the first nucleic acid molecule is introduced is not particularly limited, as long as the recombinant s.cerevisiae cell is capable of expressing the first fusion protein.

Preferably, the target site is a nutrient synthesis gene possessed by a wild-type s.cerevisiae. The first nucleic acid molecule is introduced into the genome of the s.cerevisiae cell at a nutrient synthesis gene, resulting in an impaired ability of the nutrient synthesis gene to synthesize nutrients, such that the recombinant s.cerevisiae cell is an auxotrophic yeast having nutritional requirements. "the yeast is required to have a nutrient" means that the synthetic ability of the nutrient of the wild-type yeast is impaired by a mutation in the gene for synthesizing the nutrient due to some reason, and the growth of the auxotrophic yeast needs to be maintained by adding a corresponding nutrient to the medium.

Specific examples of nutrients essential to s.cerevisiae include methionine, tyrosine, isoleucine, phenylalanine, glutamic acid, threonine, aspartic acid, valine, serine, arginine, uracil, adenine, lysine, tryptophan, leucine, and histidine. Examples of the impaired biosynthetic gene of an auxotrophic yeast include the following.

Methionine requirement: met1, met2, met3, met4, met5, met6, met7, met8, met10, met13, met14, met20

Tyrosine requirement: tyr1

Isoleucine, valine requirement: ilv1, ilv2, ilv3, ilv5

Phenylalanine requirement: pha2

Glutamate requirement: glu3

Threonine requirement: thr1, thr4

Aspartic acid requirement: asp1, asp5

Serine requirement: ser1, ser2

Arginine requirement: arg1, arg3, arg4, arg5, arg8, arg9, arg80, arg81, arg82, arg84

Uracil requirement: ura1, ura2, ura3, ura4, ura6

Adenine requirement: ADE1, ADE2, ADE3, ADE4, ADE5, ADE6, ADE8, ADE9, ADE12, ADE15

Lysine requirement: lys1, lys2, lys4, lys5, lys7, lys9, lys11, lys13, lys14

Tryptophan requirement: trp1, trp2, trp3, trp4, trp5

Leucine requirement: leu1, leu2, leu3, leu4, leu5

Histidine requirement: his1, his2, his3, his4, his5, his6, his7, and his 8.

Therefore, in the present invention, the genomic site of the above-described nutrient synthesis gene is preferably used as the target site in the genome of the s.cerevisiae cell into which the first nucleic acid molecule is introduced.

In one embodiment, insertion of the first nucleic acid molecule into the genome of a s.cerevisiae cell at the URA3 locus results in URA3^－Auxotrophic, tagged Saccharomyces cerevisiae cells.

In yet another embodiment, insertion of the first nucleic acid molecule into the genome of a s.cerevisiae cell at the TRP1 locus results in TRP1^－Auxotrophic, tagged Saccharomyces cerevisiae cells.

With respect to the other features of the first nucleic acid molecule and the features of the second nucleic acid molecule, the same applies as above.

Construction of a polypeptide variant of interest display library Using the Yeast display System of the present invention

The present invention provides a method of constructing a display library of polypeptide variants of interest, comprising:

i. constructing a gene library encoding the polypeptide variant of interest, ligating said gene library encoding the polypeptide variant of interest with nucleotides encoding an immunoglobulin Fc region or a portion thereof to generate a second library of nucleic acid molecules,

constructing a library of displayed polypeptide variants of interest, wherein a library of second nucleic acid molecules comprising nucleotides encoding a Saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain, and a first nucleic acid molecule as described in section II above, have been inserted at a target site in the genome of a Saccharomyces cerevisiae cell, is introduced into a Saccharomyces cerevisiae cell, or a library of second nucleic acid molecules is introduced into a recombinant Saccharomyces cerevisiae cell as described in section II above.

In some embodiments, a gene library of polypeptide variants of interest is generated by methods well known in the art, for example, using error-prone polymerase chain reaction, using random primer technology, or using computer technology. The gene library of the polypeptide variant of interest is designed with suitable restriction enzyme cleavage sites for linking the gene library of the polypeptide variant of interest to the nucleotides encoding the immunoglobulin Fc region or portion thereof of the second nucleic acid molecule as described above in section II.

Expressing a library of polypeptide variants of interest by inducing the yeast display system of the invention to display the library of polypeptide variants of interest on Saccharomyces cerevisiae cells to obtain about 10⁸Orders of magnitude of polypeptide variants of interest display libraries.

The library of yeast-displayed polypeptide variants of interest is screened using biopanning methods known in the art. For example, where the library of polypeptide variants of interest is a library of antibodies of different affinities, it is contacted with a yeast display library under conditions that allow for specific binding of a specific concentration of antigen to the members of the antibody library. At this point, all bound antigens are immobilized on the surface of the yeast cell. By washing off all yeast cells not bound to an antigen at a specific concentration by flow cytometry, that is, FACS detection, yeast cells displaying an antibody with high affinity can be sorted out after a plurality of rounds of such biopanning, and a clonal cell line can be obtained by clonal expansion.

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed as, limiting the scope of the invention in any way.

Examples

The general method comprises the following steps:

the practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are known and available to those of skill in the art. Such techniques are described in the following documents: molecular Cloning, A laboratory Manual, 3 rd edition (Sambrook et al, 2001) Cold Spring Harbor Press; oligonucleotide Synthesis (p. herdewijn, 2004); methods in Enzymology (Academic Press, Inc.); current Protocols in Molecular Biology (F.M. Ausubel et al, 1987); PCR The Polymerase Chain Reaction (Mullis et al, 1994); current Protocols in Immunology (J.E.Coligan et al, 1991); and Short protocols in Molecular Biology (Wiley and Sons, 1999). Unless otherwise defined, all terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1: FC of different lengths_knobRole in antibody display

This example compares the effect of different lengths of FC on antibody display.

Plasmid construction

As shown in Table 1 below, four plasmids were constructed, among which

Plasmids pYDC039, pYDC040 and pYDC041 are respectively used for expressing fusion proteins of Aga2p and FCs of different lengths (the amino acid sequence of the "hinge region-CH 2-CH 3" expressed in pYDC039 is shown in SEQ ID NO: 1;

the amino acid sequence of 'CH 2-CH 3' expressed in the pYDC040 is shown as SEQ ID NO. 2;

the amino acid sequence of 'CH 3' expressed in pYDC041 is shown as SEQ ID NO 3;

plasmid pYDC042 expresses the heavy chain variable region (V) against the PD-L1 antigen_HH) AmNB1613.36(SEQ ID NO:4) and FC (SEQ ID NO:5), and the sequence of the fusion protein is shown in SEQ ID NO: 6. In the design of FC, according to the mutation published by the Gene Take corporation patent (WO9850431A2), CH3 was considered_knob(T366W) is effective in reducing CH3_knobLevel of homodimers of self, with CH3_knobAnd CH3_hole(T366S, L368A, Y407V) forms heterodimers in predominant proportion, and CH3_holeThe homodimer of (a) still maintained a ratio of about 20%, in this example the Knob-Hole mutation was introduced into CH3 of FC; further, since s.cerevisiae has a severe level of hyperglycosylation, which has an effect on the antibody display level and the binding of the antibody to the antigen, CH2 of FC was subjected to N297A mutation to remove the glycosylation site in this example.

Table 1: constructed plasmids and fragments inserted therein

Plasmid numbering	Description of the inserted fragment in the plasmid
pYDC039	Hinge region-CH 2(N297A) -CH3 knob-GS linker-Aga 2p
pYDC040	CH2(N297A) -CH3 knob-GS linker-Aga 2p
pYDC041	CH3 knob-GS linker-Aga 2p
pYDC042	AmNB1613.36-hinge region-CH 2(N297A) -CH3 hole >

The nucleotide sequences of the inserts of each of the plasmids in Table 1 were synthesized by Soviet Kirgiz Biotechnology, Inc., and the specific amino acid sequences encoded by each nucleotide sequence are shown in SEQ ID NOs 1, 2, 3 and 6.

The nucleotide sequences encoding SEQ ID NOS: 1, 2 and 3 were each cut with a restriction enzyme BamHI and ligated between two BamHI sites of a pYDC011 plasmid (the nucleotide sequence of the pYDC011 plasmid is shown in SEQ ID NO: 7) cut with the restriction enzyme BamHI to replace the nucleotide sequence "GGaTCctgacatagtagggattataa" on the pYDC011 plasmid, to obtain plasmids pYDC039, pYDC040 and pYDC 041. The plasmids were used to express Aga2p with FCs of different lengths, respectively_knobThe fusion protein of (1).

In addition, the nucleotide sequence encoding SEQ ID NO:6 was digested with the restriction enzyme BamHI and ligated between two BamHI sites of the pYDC011 plasmid digested with the restriction enzyme BamHI to replace the nucleotide sequence "GGaTCctgacatagtagggattataa" on the pYDC011 plasmid, to obtain the plasmid pYDC042 for expressing the fusion protein of the single domain antibody (sdAb) AmNB1613.36 with FC, i.e., AmNB1613.36-hinge region-CH 2(N297A) -CH3_hole。

Plasmid transformation and yeast cell culture

Transformed Saccharomyces cerevisiae strain EBY100 (purchased from ATCC) was obtained according to the methods described by Gietz, R.D. and Schiestl, R.H. (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method, Nature Protocols,2(1), 31-34, and the combinations shown in Table 2. Selecting individual Saccharomyces cerevisiae clones, inoculating in 5mL SD-Trp, Leu medium (Takara cat # 630316), culturing at 30 deg.C 225rpm overnight; centrifuging the culture at 3000 rpm for 3 minutes and discarding the supernatant; 5mL of YPGP Induction Medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na) was added to the cell pellet₂HPO ₄,0.86％NaH ₂PO ₄·H ₂O), was induced at 20 ℃ for 72 hours at 225rpm to expressAga2p with different lengths FC_knobThe fusion protein of (1); and the fusion protein AmNB1613.36-hinge region-CH 2(N297A) -CH3_hole。

Table 2: transformation of plasmids into the Saccharomyces cerevisiae display Strain EBY100

With FC of different lengths_knobTo display yeast cell staining of antibodies

The induced cells were stained with PD-L1 antigen and analyzed to express FC of different lengths_knobThe saccharomyces cerevisiae displays the level of the antibody on the cell surface, and the specific steps are as follows:

1. each is1 × 10⁶Centrifuging the cells at 3000 rpm for 3 minutes, discarding the supernatant, washing the cells once with 1 XPBSA (1 XPBS + 1% BSA), and centrifuging to obtain cell precipitates for later use;

2. to the cell pellet of each sample, 100. mu.L of 100nM PD-L1 biotin (Acro cat # PD1-H82E5-200ug) was added and incubated at room temperature for 30 minutes;

3. adding 1mL of 1 XPBSA for washing once, centrifuging at 3000 r/min for 3 min, and discarding the supernatant;

4. add 100. mu.L of streptavidin-PE diluted with 1 XPBSA (Thermo Fisher, cat # S213881: 200 dilution) and incubate for 20 min on ice in the dark;

5. after washing once with 1mL of 1 XPBSA, centrifuging at 3000 rpm for 3 minutes to discard the supernatant, adding 500. mu.L of 1 XPBSA to the cell pellet to resuspend the cells, followed by flow cytometry (FACS) detection, the mean fluorescence signal intensity of FL2 reflects the antibody level displayed on the surface of Saccharomyces cerevisiae, as shown in FIG. 2.

As seen from FIG. 2, the proportion of positive cells stained with PD-L1 antigen and the fluorescence signal intensity of the whole cells were significantly superior in yeast cells transformed with the plasmid combination shown in Table 2, compared with EBY100 negative control not transformed with the plasmid. In addition, yeast transformed with the plasmid combinations shown in Table 2In cells, by CH3_knobThe displayed antibody was significantly better than that of the antibody by the hinge region-CH 2(N297A) -CH3_knobDisplaying antibodies or by CH2(N297A) -CH3_knobDisplaying the antibody.

Example 2: comparing FC of different lengths_knobRole in antibody secretion

Display antigen based yeast cell staining flow cytometry analysis

The culture after induction in example 1 was centrifuged to obtain a supernatant. As shown in FIG. 3, the supernatants were incubated and stained with yeast cells displaying the PD-L1 (NP-054862.1, Phe 19-Arg 238) antigen (a plasmid encoding Aga2p-PD-L1, which was obtained by digesting the nucleotide sequence encoding Aga2p-PD-L1 with the restriction enzyme BamHI and inoculating the plasmid pYDC011 digested with the restriction enzyme BamHI) in EBY100 strain, and the antibody contents of the different supernatants centrifuged after induction of the yeast cells transformed with each plasmid combination in Table 2 were compared as follows:

1. an appropriate amount of yeast cells displaying PD-L1 antigen was taken, centrifuged at 3000 rpm for 3 minutes, the supernatant was discarded, and the cell pellet was washed once with 1 XPBSA (1 XPBS + 1% BSA) at 1X 10 per well⁶Each cell/100 μ L of 1 XPBSA was plated in a 96-well 2ml U-shaped bottom deep well plate (Shanghai Biotechnology, Cat: F600580-0001) for future use;

2. the culture after induction in example 1 was centrifuged to obtain a supernatant. Adding 100 mu L of supernatant into the 96-well plate in the step 1 respectively, and incubating for 30 minutes at room temperature;

3. centrifuging the plate, discarding the supernatant, and washing the cell precipitate once by adding 1mL of 1 XPBSA;

4. mu.L of a1 XPBSA diluted dilution of secondary antibody (mouse anti-V5-FITC, Invitrogen cat # R963-25,1:1000 dilution; mouse anti-human IgG-APC, Biolegend # 409306,1:200 dilution) was added and incubated on ice for 20 min protected from light;

5. after washing once by adding 1mL of 1 XPBSA, adding 500 uL of 1 XPBSA to resuspend cells, then carrying out flow cytometry detection, and detecting the level of the yeast display PD-L1 antigen by an anti-V5-FITC antibody (FL 1); mouse anti-human IgG-APC (FL4) was assayed for the content of single domain antibodies in yeast supernatants.

The results are shown in FIG. 4. As can be seen from fig. 4, in the yeast cells transformed with the plasmid combinations shown in table 2, the amount of the anti-PD-L1 antibody amnb1613.36 secreted in the medium when combination 3 was used was significantly higher (10-fold to 20-fold higher) than the amount of the anti-PD-L1 antibody amnb1613.36 secreted in the medium when combination 1 or combination 2 was used.

As can be seen from examples 1 and 2, CH3_knobSo that the amount of antibody secreted to the supernatant while displaying the antibody on the surface of yeast can be significantly increased, from which CH3 can be determined_knobFor optimal length, the two-in-one model for yeast display and secretion is used in the examples described below.

Example 3: exhibit CH3_knobTransformation and identification of yeast (IDY104)

From example 1 and example 2, CH3 was determined_knobIs the most suitable length, so as shown in FIG. 5, CH3 will be encoded_knobThe nucleotide fragment of Aga2p was inserted into the URA3 gene locus in the genome of Saccharomyces cerevisiae EBY 100. The method comprises the following specific steps:

1. the encoding P will be according to the methods described by Gietz, R.D. et al (Gietz, R.D. and Schiestl, R.H., High-efficiency layer transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols,2007,2(1), 31-34)_GAL1-CH3 _Knob-FLAG-AGA2-TER _MATα-P _AgTEF-Kan ^RNucleotide sequence of (A)

(P _GAL1-CH3 _Knob-FLAG-AGA2-TER _MATα-P _AgTEF-Kan ^R:

ACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCGGACTACTAGCAGCTGTAATACGACTCACTATAGGGAATATTAAGCTAATTCCCTACTTCATACATTTTCAATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTAGCAGGACAGCCTCGGGAGCCCCAGGTTTATACTCTCCCCCCCAGCCGGGACGAACTGACCAAGAATCAGGTGTCCCTCTGGTGCCTCGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAGTGGGAGAGCAATGGACAGCCCGAAAACAACTACAAGACCACACCCCCCGTCCTGGACTCCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTTAGCTGCAGCGTCATGCACGAGGCTCTCCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGAAAGGGaGGcGGtGGaTCcGATTACAAGGATGACGATGACAAGGGCGGAGGAGGCTCcCAGGAACTGACAACTATATGCGAGCAAATCCCCTCACCAACTTTAGAATCGACGCCGTACTCTTTGTCAACGACTACTATTTTGGCCAACGGGAAGGCAATGCAAGGAGTTTTTGAATATTACAAATCAGTAACGTTTGTCAGTAATTGCGGTTCTCACCCCTCAACgACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTTtaaTGAGTTTAAACCCGCTGATCTGATAACAACAGTGTAGATGTAACAAAATCGACTTTGTTCCCACTGTACTTTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGTTTTCCCATGTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATATAGCTATTCACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATTTCAATTTGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGAATCGAATCCTAAGAGAATTagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGA TGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA (SEQ ID NO:8)) into Saccharomyces cerevisiae strain EBY100 (purchased from ATCC), spread on YPD plates (TAKARA,630410) containing 200. mu.g/mL of G418 (Shanghai Biotech., A600958-0001), and cultured at 30 ℃ for 3 days;

2. 8 single clones were picked, streaked on a new YPD + G418 plate, and cultured at 30 ℃ for 3 days;

3. picking new single colonies from each plate in step 2, streaking on SD-Ura (Clontech,630315) plate and streaking on YPD + G418 plate, respectively, and culturing at 30 ℃ for 3 days;

4. picking Ura in step 3^-G418 ⁺The single clone of (1) is cultured overnight by YPD, 1mL of overnight culture liquid is added into 5mL of YPGP induction culture medium, and the mixture is induced for 24 hours at the temperature of 20 ℃ and the rpm of 225;

5. centrifuging to obtain 1 × 10 samples⁶Individual yeast cells were washed once with 1 x PBSA;

6. mu.L of mouse anti-Flag M2(sigma, cat # F1804,1:1000 dilution) was added to each sample and incubated at room temperature for 30 minutes;

7. adding 1mL of 1 XPBSA to wash once;

8. add 100. mu.L of Goat anti-Mouse-647 dilution (Thermo Fisher, cat # A212351: 200 dilution) diluted with 1 XPBSA and incubate for 20 min on ice in the dark;

9. after washing once with 1mL of 1 XPBSA, 500. mu.L of 1 XPBSA was added to resuspend the cells and then examined by flow cytometry, the mean fluorescence signal intensity of FL4 reflected the CH3 of each strain_knobThe yeast surface display levels and the results are shown in figure 6.

As can be seen from FIG. 6, each strain was effective in converting CH3_knobShown on the surface of yeast, clone IDY104-7 was randomly picked for subsequent experiments in the following examples.

Example 4: detection of yeast IDY104 display antibody level and affinity determination of culture supernatant

The yeast IDY104 obtained in example 3 has a direct display of CH3_knobThe ability of the cell to perform. Further, plasmid pYDC042 (containing a plasmid encoding AmNB1613.36-hinge region-CH 2(N297A) -CH3 shown in SEQ ID NO: 6)_holeThe nucleotide sequence of the polypeptide is transferred into the yeast IDY 104. Expressed fusion protein AmNB1613.36-hinge region-CH 2(N297A) -CH3_holeBy contact with CH3 on the surface of yeast cells_knobIndirectly displaying the anti-PD-L1 antibody AmNB1613.36 on the surface of the yeast cell, and simultaneously secreting a small amount of antibody into a culture solution, thereby realizing a yeast display technology integrating cell surface display and secretion, wherein the yeast cell displaying the anti-PD-L1 antibody AmNB1613.36 on the surface can be used for flow cytometry analysis and sorting, and the culture solution secreting the anti-PD-L1 antibody AmNB1613.36 can be used for determining the affinity of the antibody, and the specific steps are as follows:

1. transforming the plasmid pYDC042 into a strain IDY104, culturing for 3 days at 30 ℃, selecting a single clone, inoculating the single clone to SD-Leu culture (TAKARA cat # 630310) overnight, and transferring 1mL of bacterial liquid to 5mL of YPGP culture medium for induction for 24 h;

2. taking a composition comprising about 1 × 10⁶The culture solution of each cell was centrifuged at 3000 rpm for 3 minutes, the supernatant was used in step 7, and the cell pellet was washed once with 1 XPBSA and used in step 3;

3. adding 100 μ L of 10nM PD-L1 biotin to the cell pellet and incubating for 30 minutes at room temperature;

4. centrifuging, adding 1mL of 1 XPBSA to wash once;

5. add 100. mu.L of streptavidin-PE diluted with 1 XPBSA (Thermo Fisher, cat # S213881: 200 dilution) and incubate for 20 min on ice in the dark;

6. after washing once with 1mL of 1 XPBSA, 500. mu.L of 1 XPBSA was added to resuspend the cells and then flow cytometric assay was performed, and the mean fluorescence signal intensity of FL2 reflects the level of yeast surface-displayed antibody, as shown in FIG. 7.

7. The supernatant collected by centrifugation in step 1 was placed in an ultrafiltration concentration tube (MCPM 02C67, japan, ltd.) and the supernatant was concentrated 10-fold, and the obtained concentrate was also subjected to affinity detection.

The equilibrium dissociation constant (K) of the antibody and antigen in the supernatant and the 10-fold concentrated sample were measured by biofilm interference (BLI) technique_D). The BLI-method affinity assay is carried out according to the known method (Estep, P et al, High throughput solution Based measurement of affinity-affinity and affinity binding. MAbs,2013, 5(2): p.270-8). After the sensor was prewetted in assay buffer for 20 minutes, the affinity of the antibody to PD-L1 was measured with Octet Red96 as established: baseline equilibration first for 120 seconds; the sample was then cured to an AHC sensor (ForteBio, 18-5060); the cured sensors were placed in a solution containing 100nM PD-L1(Acro, cat # PD1-H82E5-200ug) until a plateau (100 seconds), after which the sensors were transferred to assay buffer for dissociation for at least 2 minutes, and binding and dissociation were determined separately. Experimental results analysis of kinetics was performed using the 1:1 binding model, and the results are shown in fig. 8.

As can be seen from fig. 7 and 8, the yeast IDY104 can be used to effectively display antibodies on the cell surface, and the displayed antibodies can effectively bind to the corresponding antigens; further, the yeast IDY104 can be used for efficiently secreting the antibody into the culture solution, and the culture solution secreting the antibody can be used for affinity measurement as it is or after being concentrated 10-fold.

This example shows the results obtained via CH3_knobStably transformed yeast can effectively work as a two-in-one system for yeast surface display and secretion of antibodies.

Example 5: comparison of the ability of Strain IDY104 to surface display and secrete different forms of antibodies

As can be seen from example 4, the yeast surface display and secretion two-in-one system of the present invention can effectively display and secrete single domain antibody, such as anti-PD-L1 antibody AmNB1613.36. This example further illustrates the expression of other forms of antibodies by the yeast surface display and secretion system of the present invention, for example, the anti-human PD-L1 monoclonal antibody Hz4485 was constructed as scFv-Fc form, the ammonia of which isThe amino acid sequence is shown as SEQ ID NO. 9 (Hz 4485 coded by pYDC083 plasmid_scFvThe amino acid sequence of (a) is shown as SEQ ID NO: 9), and the nucleotide sequence encoding SEQ ID NO:9 was synthesized by Jinzhi Biotech, Inc., Suzhou. The nucleotide sequence encoding SEQ ID NO:9 was digested with the restriction enzyme BamHI and ligated between two BamHI sites of a pYDC081 plasmid (the nucleotide sequence of pYDC081 plasmid is shown in SEQ ID NO: 10) digested with the restriction enzyme BamHI to replace the nucleotide sequence "GGaTCctgacatagtagggattataa" on the pYDC081 plasmid, to obtain plasmid pYDC 083.

Plasmid pYDC083 was transformed into yeast IDY104 and spread on SD-Trp plates (Clontech, 630309); the SD-Trp plates coated with the transformed yeast strain were cultured at 30 ℃ for 3 days to obtain a monoclonal yeast. Yeast cultures and induced expression were performed as described in example 4. The yeast surface display and secretion two-in-one system of the present invention was compared for the display and secretion levels of both forms of antibodies, sdAb and scFv-FC. The results are shown in FIGS. 9 and 10.

As can be seen from FIGS. 9 and 10, the yeast IDY104 can efficiently display and secrete antibody formats such as single domain antibody, scFv-FC, etc., and the supernatant samples can be used for affinity assay.

Example 6: evaluation of feasibility of antibody affinity maturation for a two-in-one display and secretion System Using Spiking libraries

The yeast surface display and secretion two-in-one system is very suitable for affinity maturation library screening of antibodies. The system of the invention enables the mutant clone supernatant obtained by screening to be directly used for antibody affinity determination, which saves additional steps of plasmid construction, mammalian cell expression and the like and greatly accelerates the progress of related projects of antibody affinity research.

In this example, a mock library was constructed that was screened for an antibody affinity maturation library.

Specifically, plasmid pYDC042 (comprising a plasmid encoding AmNB1613.36-hinge region-CH 2(N297A) -CH3 shown in SEQ ID NO: 6) was prepared as described in example 4_holeThe nucleotide sequence of the polypeptide is transferred into the yeast IDY 104. Expressed fusion protein AmNB1613.36-hinge region-CH2(N297A)-CH3 _holeBy contact with CH3 on the surface of yeast cells_knobIndirectly displaying the anti-PD-L1 antibody AmNB1613.36 on the surface of the yeast cell, and simultaneously secreting a small amount of the AmNB1613.36 antibody into the culture solution.

Similarly, the nucleotide sequence encoding AmNB1613.36 in plasmid pYDC042 was replaced with the nucleotide sequence encoding the parent nanobody HzNB1613 (amino acid sequence of HzNB 1613: QVQLQESGGGLVQPGGSLRLSCAASAYTISRNSMGWFRQAPGKGLEGVAAIESDGSTSYSDSVKGRFTISLDNSKNTLYLEMNSLRAEDTAVYYCAAPKVGLGPRTALGHLAFMTLPALNYWGQGTLVTVSS (SEQ ID NO:11)), and other manipulations were performed as described in example 4, to obtain yeast cells displaying the anti-PD-L1 antibody HzNB1613 on the cell surface, and a culture solution secreting a small amount of antibody HzNB 1613.

The equilibrium dissociation constant (K) of antibody AmNB1613.36 bound to PD-L1 was determined by Surface Plasmon Resonance (SPR), respectively_D) K binding to PD-L1 with antibody HzNB1613_D. Based on the SPR principle, when a beam of polarized light enters the end face of the prism at a certain angle, surface plasma waves are generated at the interface between the prism and the gold film, and free electrons in the metal film are caused to generate resonance, namely surface plasma resonance. During analysis, a layer of biomolecule recognition membrane is fixed on the surface of a sensing chip, then a sample to be detected flows on the surface of the chip, if molecules capable of interacting with the biomolecule recognition membrane on the surface of the chip exist in the sample, the refractive index of the surface of the gold membrane changes, and finally SPR angle changes are caused, and information such as affinity, kinetic constant and the like of an analyte is obtained by detecting the SPR angle changes.

K _DThe measurement of (2) is carried out by a capture method in which after an antibody is captured to a chip by an anti-human Fc antibody, affinity and kinetic constants are obtained by detecting binding and dissociation between an antigen and the captured antibody. The method comprises chip preparation and affinity detection. The 10XHBS-EP after 10 times dilution is used in the determination process⁺(BR-1006-69, GE Healthcare) was used as the experimental buffer. The chip preparation process comprises coupling anti-human Fc antibody on the surface of CM5 chip (29-1496-03, GE Healthcare) with amino coupling kit (BR-1006-33, GE Healthcare), and preparingComprises the following steps: first, 50mM N-hydroxysuccinimide (NHS) and 200mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were mixed fresh and injected into a CM5 chip for two channels to activate for 7 minutes. Then, the anti-human Fc antibody was diluted in 10mM acetic acid (pH 5.0) and injected into the CM5 chip for two channels, so that the protein was covalently coupled to the surface of the chip channel at a coupling height of about 6000 RU. Finally 1M ethanolamine was injected and the remaining activation sites were blocked for 7 min.

Each cycle of affinity detection involves capture of the antibody, binding of a concentration of antigen, and regeneration of the chip, as described below.

Capture antibody: antibodies AmNB1613.36 and HzNB1613 were first diluted to 0.5. mu.g/mL and captured on the second channel of the CM5 chip for 30s at a flow rate of 10. mu.L/min.

Binding to antigen: human PD-L1(Acrobiosystems, cat # PD1-H5229) was diluted with a two-fold gradient of assay buffer to 0.15nM-20nM, according to the optimal concentration range for SPR, and injected into CM5 chip double channels in the order of low to high concentration, with an association time of 180s and an dissociation time of 600 s.

Chip regeneration: the chip was regenerated using 10mM Glycine pH 1.5(BR-1003-54, GE Healthcare) before the next antibody assay was performed.

Data results analysis of kinetics was performed using a 1:1 binding model. In experiments performed as described in the above assay, antibody AmNB1613.36 binds to the affinity (K) of PD-L1_D) At 0.1nM, the affinity of the antibody HzNB1613 for binding to PD-L1 (K)_D) 3.9nM, with a significant difference in affinity.

Yeast cells displaying anti-PD-L1 antibody HzNB1613 on the cell surface and yeast cells displaying anti-PD-L1 antibody AmNB1613.36 on the cell surface are mixed in different proportions to prepare Spiking libraries. Specifically, yeast cells displaying AmNB1613.36 and yeast cells displaying HzNB1613 were mixed at a ratio of 1:10²、1:10 ⁴Separately, 1% Spiking library and 0.01% Spiking library were prepared.

Yeast cells displaying only AmNB1613.36, yeast cells displaying only HzNB1613Inoculating yeast cells, a 1% Spiking library and a 0.01% Spiking library to SD-Leu for overnight culture, taking 1mL of bacterial liquid, transferring to 5mL of YPGP culture medium, and inducing for 24 h; each is1 × 10⁶Cells were stained with 10nM PD-L1 biotin antigen and the results of flow cytometry are shown in FIG. 11.

As can be seen from FIG. 11, at a given antigen concentration, the higher the affinity of the antibody displayed by the yeast cells, the higher the average fluorescence signal value of the yeast cell staining in flow cytometry.

Separating the yeast cell population framed in the figure 11 by flow cytometry, inoculating the separated yeast cell population to SD-Leu for culture overnight, taking 1mL of bacterial liquid, transferring the bacterial liquid to 5mL of YPGP culture medium, and inducing for 24 h; each is1 × 10⁶Cells were stained with 10nM PD-L1 biotin antigen and screened by flow cytometry in two rounds, 24 monoclonal yeast cells were randomly picked for sequencing in each round, and the proportion of yeast cells displaying AmNB1613.36 in the Spiking library was observed based on the sequencing results, as shown in FIG. 12.

As can be seen from FIG. 12, the proportion enrichment of yeast cells expressing AmNB1613.36 in the Spiking library was very significant after two rounds of screening; the positivity of yeast cells expressing AmNB1613.36 after two rounds of screening of the Spiking library with the 0 th round (R0) being 1% reaches 100%; after two rounds of screening, the Spiking library with 0.01% R0 showed that the positive rate of yeast cells expressing AmNB1613.36 reached 91%. Thus, yeast cell strains expressing affinity maturation can be enriched and obtained.

Through screening of Spiking libraries, it is fully demonstrated that the application of this technique to affinity maturation library screening is feasible, and that the obtained monoclonal medium supernatant is directly assayed for affinity; in short, the high-affinity mutant clone can be screened from an affinity mature library by applying the technology of the invention, and the monoclonal culture medium supernatant can directly measure the affinity, so that the traditional plasmid construction and expression processes are omitted, the speed is increased, the cost is saved, and the economic benefit is very obvious.

While certain representative embodiments and details have been shown for the purpose of illustrating the subject invention, it will be apparent to those skilled in this art that various changes and modifications can be made therein without departing from the scope of the subject invention. In this respect, the scope of the invention is limited only by the following claims.

Claims (11)

1. A yeast display system for cell surface display and secretion of a polypeptide of interest, which is a Saccharomyces cerevisiae (Saccharomyces cerevisiae) cell introduced with a first nucleic acid molecule comprising nucleotides encoding a Saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain, and a second nucleic acid molecule comprising nucleotides encoding a polypeptide of interest and nucleotides encoding an immunoglobulin Fc region or part thereof, preferably the first and second nucleic acid molecules are located on the same plasmid or on separate plasmids.
2. Yeast display system for cell surface display and secretion of a polypeptide of interest, which is a recombinant saccharomyces cerevisiae cell in which a first nucleic acid molecule comprising nucleotides encoding a saccharomyces cerevisiae cell surface anchor protein and an immunoglobulin Fc region CH3 domain is inserted at a target site in the genome of the saccharomyces cerevisiae cell for introduction of a second nucleic acid molecule comprising nucleotides encoding a polypeptide of interest and nucleotides encoding an immunoglobulin Fc region or part thereof for display and secretion of a polypeptide of interest on the cell surface.
3. The yeast display system according to claim 1 or 2, wherein each of said first and second nucleic acid molecules comprises a bulge ("knob") or a cavity ("hole") in the Fc region, respectively, whereby the first polypeptide chain expressed by said first nucleic acid molecule and the second polypeptide chain expressed by said second nucleic acid molecule are capable of forming a stable association of "knob-in-hole" with each other; preferably, the first and second nucleic acid molecules encode a first and second polypeptide chain comprising in one of the chains the amino acid substitution T366W and in the other of the first and second polypeptide chains the amino acid substitutions T366S, L368A and Y407V (according to Kabat' EU numbering "), whereby a protuberance in one chain is capable of being placed in a cavity in the other chain, thereby promoting association of the first and second polypeptide chains;

   preferably, the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
4. The yeast display system of any one of claims 1-3, wherein the second nucleic acid molecule comprises nucleotides encoding a polypeptide of interest, optionally nucleotides encoding a hinge region of an immunoglobulin Fc region, and nucleotides encoding a CH2 domain and a CH3 domain of an immunoglobulin Fc region; preferably, the glycosylation site in the CH2 domain is eliminated, e.g., the N297 residue in the CH2 domain of the human IgG Fc region is mutated to eliminate the glycosylation site, e.g., the N297 residue is changed to Gly, Ala, gin, Asp or Glu, preferably the N297 residue is changed to Ala.
5. The yeast display system of any one of claims 1-4, wherein the Saccharomyces cerevisiae cell surface anchor protein is a Saccharomyces cerevisiae cell wall protein containing a Glycosylphosphatidylinositol (GPI) anchor signal sequence, e.g., alpha-lectin and a-lectin, Cwp1p protein, and Flo1p protein.
6. The yeast display system of any of claims 1-4, wherein the Saccharomyces cerevisiae is a Saccharomyces cerevisiae expressing the aga1p subunit of a-lectin, such as Saccharomyces cerevisiae EBY 100; the s.cerevisiae cell surface dockerin is an aga2p subunit, whereby the first polypeptide encoded by the first nucleic acid molecule comprises aga2p and the immunoglobulin Fc region CH3 domain, and binds to the aga1p subunit that has been bound and presented on the s.cerevisiae cell surface.
7. The yeast display system of any one of claims 2-4 wherein the first nucleic acid molecule is inserted into the genome of the s.cerevisiae cell at a genomic site where a nutrient synthesis gene is located to disruptA nutrient synthesis gene, for example, inserted at the URA3 locus in the genome of a s.cerevisiae cell, gives URA3^－Auxotrophic, tagged Saccharomyces cerevisiae cells; inserted into the TRP1 gene locus in the genome of a saccharomyces cerevisiae cell to obtain TRP1^－Auxotrophic, tagged Saccharomyces cerevisiae cells.
8. The yeast display system of any of claims 1-7, wherein the polypeptide of interest is an antibody or antigen binding fragment, e.g., a Fab fragment, a VHH domain, a scFv, a sdAb.
9. The yeast display system of any of claims 1-8, wherein

   (a) The first nucleic acid molecule comprises an encoding CH3 from N-terminus to C-terminus_knob-optionally a linker or a tag-the nucleotides of Aga2p, and the second nucleic acid molecule comprises, from N-terminus to C-terminus, a nucleotide encoding a polypeptide of interest-optionally a hinge region-CH 2(N297A) -CH3_holeThe nucleotide of (a); or

   (b) The first nucleic acid molecule comprises an encoding CH3 from N-terminus to C-terminus_hole-optionally a linker or a tag-the nucleotides of Aga2p, and the second nucleic acid molecule comprises, from N-terminus to C-terminus, a nucleotide encoding a polypeptide of interest-optionally a hinge region-CH 2(N297A) -CH3_knobThe nucleotide of (a);

   preferably, the linker comprises glycine (G) and serine (S) residues, e.g., the linker is GS;

   preferably, the tag is selected from the group consisting of an Arg-tag, an Avi-tag, a His-tag, a Flag-tag, a3 XFlag-tag, a Strep-tag, a Nano-tag, an SBP-tag, a c-myc-tag, an S-tag, a calmodulin-binding peptide, a cellulose-binding domain, a chitin-binding domain, a GST-tag or an MBP-tag.
10. Use of a yeast display system according to any one of claims 1-9 for expressing a polypeptide of interest displayed on the surface of a cell and secreted into the culture medium; preferably, for expression of an antibody displayed on the cell surface and secreted into the culture medium, e.g., PD-L1 antibody, PD-1 antibody.
11. Use of a yeast display system according to any one of claims 1-9 for constructing a variant display library of polypeptides of interest, wherein the cell surface display is in a manner enabling selection of a specific polypeptide variant of interest by means of high throughput screening, secretion into the culture medium in a manner enabling biochemical characterization of the polypeptide variant of interest; preferably, variant display libraries of antibodies are constructed to screen for high affinity antibody variants.

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