Triple-mode system for antibody maturation, surface display and secretion

Abstract

The present invention provides a triple-mode antibody display system that simultaneously matures, displays and secretes an antibody to a target of interest. An antibody in vivo-matured and complexed with membrane anchored bait can be expressed on the surface of the host cell, while complexed with a soluble bait the antibody is secreted from the host cell. Methods of using the system for identifying binders that bind specifically to an antigen of interest are also provided. Polypeptides, polynucleotides and host cells useful for making the protein binder display system are also provided along with methods of use thereof.

Claims

1. A eukaryotic cell providing a protein display, protein selection and soluble protein recovery system, comprising: a) recombinant DNA capable of expressing a mutagenic cytidine deaminase; b) recombinant DNA capable of expressing a membrane-bound version of a bait polypeptide; c) recombinant DNA capable of expressing a soluble version of the bait polypeptide; and d) recombinant DNA capable of expressing one or more polypeptides of a scaffold protein, with one or more said scaffold polypeptides operably fused to a prey polypeptide.

2. The eukaryotic cell of claim 1, wherein the cytidine deaminase is sea lamprey Petromyzon marinus cytidine deaminase CDA1 or functional fragment thereof.

3. The eukaryotic cell of claim 1, wherein the expressed bait polypeptide is whole Saccharomyces cerevisiae AGA1 or a functional fragment thereof, comprises a cell surface anchor, and is a membrane bound version.

4. The eukaryotic cell of claim 3, wherein the cell surface anchor comprises a polypeptide segment sufficient to incorporate into membrane, trigger a covalent association with lipid, or associate with another membrane component.

5. The eukaryotic cell of claim 1, wherein the bait polypeptide is a fragment of Saccharomyces cerevisiae AGA1 that lacks a cell surface anchor and is a soluble version.

6. The eukaryotic cell of claim 1, wherein the prey polypeptide comprises Saccharomyces cerevisiae AGA2 or a functional fragment thereof, capable to form heterodimeric complex with the bait.

7. The eukaryotic cell of claim 1, wherein the scaffold protein comprises an immunoglobulin heavy chain variable region, a light chain variable region, combinations of light and heavy chain regions, Anticalins, fibronectin type Ill domain, Designed Ankyrin Repeat Protein or Centyrin.

8. The eukaryotic cell of claim 1, wherein the expression of the membrane-bound version and the soluble version of a bait polypeptide are selectively inducible.

9. The eukaryotic cell of claim 1, wherein the cell is a Saccharomyces cell or Pichia cell.

10. The eukaryotic cell of claim 1, wherein the cell is a Chinese hamster ovary cell.

11. A method for maturing and identifying an antigen-binding variant of the scaffold protein comprising A. cultivating a culture of eukaryotic cells of claim 1; B. cultivating the culture such that the cytidine deaminase and the scaffold protein are expressed; C. contacting the culture with a mutagen; D. thereafter cultivating the culture such that the membrane-bound bait polypeptide and the scaffold protein are expressed; E. selecting a subset of the eukaryotic cells that express scaffold protein at the cell surface that binds the antigen more strongly than the rest; F. cultivating colonies of the selected cells such that soluble bait polypeptide and the scaffold protein are expressed; G. selecting a subset of one or more of the colonies that bind the antigen, with selection based one or more of apparent binding activity, binding affinity or protein stability.

12. The method of claim 11, wherein step D comprises contacting the culture with an inducer for the expression of the membrane-bound bait polypeptide.

13. The method of claim 12, wherein step F comprises contacting the culture with a second inducer, distinct from the first, which is for the expression of the soluble bait polypeptide.

14. The method of claim 11, wherein the cytidine deaminase is sea lamprey Petromyzon marinas cytidine deaminase CDA1 or functional fragment thereof.

15. The method of claim 11, wherein the expressed bait polypeptide is whole Saccharomyces cerevisiae AGA1 or a functional fragment thereof, comprises a cell surface anchor, and is a membrane bound version.

16. The method of claim 15, wherein the cell surface anchor comprises a polypeptide segment sufficient to incorporate into membrane, trigger a covalent association with lipid (e.g., GPI), or associate with another membrane component.

17. The method of claim 15, wherein the bait polypeptide is a fragment of Saccharomyces cerevisiae AGA1 that lacks a cell surface anchor and is a soluble version.

18. The method of claim 15, wherein the prey polypeptide comprises Saccharomyces cerevisiae AGA2 or a functional fragment thereof capable to form heterodimeric complex with the bait.

19. The method of claim 11, wherein the scaffold protein comprises an immunoglobulin heavy chain variable region, a light chain variable region, combinations of light and heavy chain regions, Anticalins, fibronectin type Ill domain, Designed Ankyrin Repeat Protein or Centyrin.

20. The method of claim 11, wherein the cell is a Saccharomyces cell or Pichia cell.

21. The method of claim 11, wherein the cell is a Chinese hamster ovary cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only illustrative embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

(2) FIG. 1A is a schematic representation of yeast-based triple mode system which shows three exemplary components;

(3) FIG. 1B shows two components for antibody display and secretion are described in more detail;

(4) FIGS. 2A, 2B, 2C, 2D and 2E present vectors for expressing polypeptide constructs;

(5) FIGS. 3A, 3B and 3C show various hypermutation tests. FIG. 3A and FIG. 3C patch test and spot test FIG. 3B of forward mutations in the yeast CAN1 gene induced by overexpression of full length cytosine deaminase PmCDA1 (FIG. 3A), various variants of PmCDA1 and PmCDA2 (FIG. 3C) and exposure to the replication fidelity compromising compound HAP (FIG. 3B). Left sidesno inducers are present; Right sideyeast are exposed to inducers. As shown in FIG. 3C, only PmCDA1 (SEQ ID NO:3), not PmCDA2 (SEQ ID NO:5) induces hypermutation.

(6) FIGS. 4A and 4B present data obtained in the double-mode system: anti-HER2 antibody Fab fragment secreted into the media detected by ELISA (FIG. 4A) and displayed on cell surface monitored by cytoflow (FIG. 4B).

(7) FIG. 5 shows graphs showing that the diversification and panning system of the invention efficiently sorts a cell population expressing camelid antibodies with high affinity binding to H5N1 neuraminidase target. The enriched library is labeled with anti-FLAG Dylight649 and NA-biotin/streptavidin-PE to monitor the VHH display efficiency and activity to NA. The double-positive sorted cell fraction is indicated.

(8) FIG. 6 presents a graph showing that the activity of antibodies secreted by yeast cells into the media can be detected by target-specific ELISA assay.

(9) FIG. 7 is a schematic diagram of the antibody discovery system of the present invention.

(10) To facilitate understanding, identical reference numerals have been used, where possible, to designate comparable elements that are common to the figures. The figures are not drawn to scale and may be simplified for clarity. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.

DETAILED DESCRIPTION

(11) In accordance with the present invention, a triple-mode display system that simultaneously matures, displays and secretes a binder is provided to produce protein scaffolds reactive to a specific target. In a preferred aspect of the invention, an antibody discovery method is provided which enables isolating target-specific antibodies starting from a scaffold library.

(12) FIG. 1A is a schematic representation of yeast-based triple mode system, shown with three exemplary components. Two exemplary components for antibody display and secretion are described in more detail in FIG. 1B. The third component can for example comprise expression of Petromyzon marinas sea lamprey PmCDA1 protein (Uniprot A5H718, SEQ ID NO:3), exposure to 6-N-hydroxylaminopurine (HAP) and a yeast host strain engineered to enhance the mutator effect of PmCDA1 and HAP. FIG. 1B is a schematic representation of an exemplary yeast dual-mode antibody display system. The exemplary membrane anchor is yeast full length AGA1 protein containing a GPI anchor to the cell wall. When co-expressed in the same host with a secretable AGA2-fused protein scaffold (e.g., antibody Fab fragment), the AGA1 (the anchored bait) heterodimerizes prior to secretion in the ER with the AGA2 portion of the scaffold constructs, forming two disulfide bridges. Thus, in this instance antibody Fab will be displayed on cell surface. The soluble bait is an N-terminal fragment of AGA1 (which lacks of the GPI-anchor amidated glycine at the position 699 of AGA1). When the soluble bait is co-expressed in the same host with a secretable AGA2-fused antibody scaffold, it heterodimerizes with AGA2-fused scaffolds, resulting in secretion of the antibody Fab fragment into the culture medium.

(13) FIGS. 2A, 2B, 2C, 2D and 2E present vectors for expressing the first polypeptide constructs. FIG. 2A is representative of one camelid VHH cloned in frame with yeast invertase secretory signal at the N-terminus and membrane anchor AGA2 at the C-terminus (SEQ ID NO:6; 1-19=secretory signal, 23-149=VHH, 190-258=anchor providing segment). A gene encoding a camelid heavy chain single domain variable was cloned directionally into Sfil-Sfil sites. The fusion construct is expressed under a galactose inducible promoter, Gal1/10. This yeast-E. coli centromeric shuttle plasmid contains TRP1 as a yeast transformation marker.

(14) SEQ ID NO:8: Nucleotide sequence of the yeast replicative vector for expression and display camelid VHH antibody on yeast cell surface as presented in FIG. 2A. SEQ ID NO:9: Nucleotide sequence of the yeast replicative vector for expression and display human heavy chain VH antibody on yeast cell surface as presented in FIG. 26. SEQ ID NO:10: Nucleotide sequence of the yeast replicative vector for expression and display the first construct, human antibody heavy chain, of human Fab fragment as presented in FIG. 2C. SEQ ID NO:11: Nucleotide sequence of the yeast replicative vector for expression and display the second construct, human antibody light chain kappa, of human Fab fragment as presented in FIG. 2D. SEQ ID NO:12: Nucleotide sequence of the yeast replicative vector for expression and display the second construct, human antibody light chain lambda, of human Fab fragment as presented in FIG. 2E.

(15) FIG. 2B represents an exemplary backbone vector for expression of human heavy chain variable domains where human VH can be cloned at Eagl-Sall sites in frame with yeast invertase secretory signal at the N-terminus and membrane anchor AGA2 at C-terminus.

(16) When the first construct is a heterodimeric protein such as an antibody Fab fragment two backbone vectors are used for co-expressionone vector (e.g., FIG. 2C) with TRP1 selection marker is for expressing antibody heavy chain; the heterodimeric partner, antibody light chain lambda or light chain kappa is expressed in vectors with HIS3 selection marker as presented for example in FIG. 2D and FIG. 2E, respectively.

(17) FIGS. 3A, 3B and 3C show various hypermutation tests. FIG. 3A and FIG. 3C show patch tests and FIG. 3B shows spot test of forward mutations in the yeast CAN1 gene induced by overexpression of full length cytosine deaminase PmCDA1 (FIG. 3A), various variants of PmCDA1 and PmCDA2 (FIG. 3C) and exposure to the replication fidelity compromising compound HAP (FIG. 3B). Left sidesno inducers are present; Right sideyeast are exposed to inducers. Various forms of cytidine deaminase mentioned in FIG. 3C are described in more detail in Tran et al., US20170183645A1, filed 15 Dec. 2016, the content of which, particularly as to protein sequence, is incorporated herein in its entirety.

(18) FIGS. 4A and 4B present data obtained in the double-mode system: anti-HER2 antibody Fab fragment secreted into the media detected by ELISA (FIG. 4A) and displayed on cell surface monitored by cytoflow (FIG. 4B).

(19) FIG. 5 shows graphs showing that the diversification and panning system of the invention efficiently sorts a cell population expressing camelid antibodies with high affinity binding to H5N1 neuraminidase (NA) target. The enriched library is labeled with anti-FLAG Dylight649 and NA-biotin/streptavidin-peroxidase to monitor the VHH display efficiency and activity to NA. The double-positive sorted cell fraction is indicated.

(20) FIG. 6 presents a graph showing that the activity of antibodies secreted by yeast cells into the media can be detected by target-specific ELISA assay.

(21) FIG. 7 is a schematic diagram of the antibody discovery system of the present invention.

I. Definition

(22) The following definitions are provided to facilitate an understanding of the present invention.

(23) A promoter is a DNA sequence located proximal to the start of transcription at the 5 end of an operably linked transcribed sequence. The promoter may contain one or more regulatory elements or modules that act together in coordinating and regulating transcription of the operably linked gene. An inducible promoter is a promoter that responds to the presence of different biochemical or physical stimuli. Such promoters include, but are not limited, to the CUP1 promoter, heat shock promoters, galactose-inducible promoters, glycolytic promoters such as alcohol dehydrogenase (ADH), glyceraldehyde phosphate dehydrogenase (GPD) and the like.

(24) Operably linked describes two macromolecular elements arranged such that modulating the activity of the first element induces an effect on the second element. In this manner, modulation of the activity of a promoter element may be used to alter and/or regulate the expression of an operably linked coding sequence. For example, the transcription of a coding sequence that is operably linked to a promoter element is induced by factors that activate the promoter's activity; transcription of a coding sequence that is operably-linked to a promoter element is inhibited by factors that repress the promoter's activity. Thus, a promoter region is operably linked to the coding sequence of a protein if transcription of such coding sequence activity is influenced by the activity of the promoter.

(25) Fusion construct refers generally to recombinant genes which encode fusion proteins. Such fusion constructs may include operably linked nucleic acids isolated from two or more different genes.

(26) A fusion protein is a hybrid protein, i.e., a protein that has been constructed to contain domains from at least two different proteins. An exemplary fusion protein, as described herein is a hybrid protein which possesses (a) a transcriptional regulatory domain from a transcriptional regulatory protein, or (b) a DNA binding domain from a DNA binding protein linked to a heterologous protein to be assayed for interaction. The structure of the fusion protein is such that the transcriptional regulatory domain and the DNA binding domain are arranged in a manner that allows both domains to be biologically active. The protein that is the source of the transcriptional regulatory domain is different from the protein that is the source of the DNA binding domain. In other words, the two domains are heterologous to each other. The transcriptional regulatory domain of the fusion protein may either activate or repress transcription of target genes, depending on the native biological activity of the domain.

(27) The term fusion protein gene refers to a DNA sequence that encodes a fusion protein. A fusion protein gene may further provide transcriptional and translational regulatory elements for the transcriptional and translational control thereof.

(28) A nucleic acid molecule, such as a DNA or gene is said to be capable of expressing a polypeptide if the molecule contains the coding sequences for the polypeptide operably linked to expression control sequences which, in the appropriate host environment, facilitate transcription, processing and translation of the encoded genetic information into a protein product.

(29) The phrase consisting essentially of when referring to a particular nucleotide or amino acid means a sequence having the properties of a given reference sequence. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

(30) As used herein, a cloning vector is any entity that is capable of delivering a nucleic acid sequence into a host cell for cloning purposes. Examples of cloning vectors include plasmids or phage genomes. A plasmid which replicates autonomously in a host cell is especially preferred. Alternatively, a nucleic acid molecule which stably integrates into the host cell's chromosomal DNA and is inherited by daughter cells may be employed. Optionally, such vectors include a number of endonuclease recognition sites to facilitate manipulation of the sequence in a controlled and targeted fashion. Cloning vectors of the invention may also comprise sequences conferring resistance to selection agents, often referred to herein as selectable marker genes. For example, a marker gene may be a gene which confers resistance to a specific antibiotic on a host cell.

(31) As used herein, an expression vector is a vehicle or vector similar to the cloning vector but is especially designed to provide an environment that facilitates expression of the cloned gene product after transformation into the host. Such vectors contain regulatory elements for expression in prokaryotic and/or eukaryotic hosts as well as sequences conferring selection properties of cells containing the expression vector. Optionally, enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites may be included.

(32) A host refers to any organism or cell line that is the recipient of a cloning or expression vector. In preferred embodiments, the host of the invention is a yeast cell or a cultured animal cell such as a mammalian or insect cell. Especially preferred is the yeast host Saccharomyces cerevisiae.

(33) A transformed cell is any cell into which (or into an ancestor of which) exogenous DNA has been introduced by means of recombinant DNA techniques or cell fusion, e.g. mating.

(34) The terms variant or derivative in relation to AGA1 polypeptide includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the original polypeptide sequence of AGA1. Preferably, nucleic acids encoding AGA1 are understood to comprise variants or derivatives thereof.

(35) Such modifications of AGA1 polypeptides include fusion proteins in which AGA1 polypeptide or a portion or fragment thereof is linked to or fused to another polypeptide or molecule.

(36) The term homologue as used herein with respect to the nucleotide sequence and the amino acid sequence of AGA1 may be synonymous with allelic variations in the AGA1 sequences and includes known homologues.

(37) The term expression refers to the transcription of a gene's DNA template to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product (i.e., a peptide, polypeptide, or protein). The tem activates gene expression refers to inducing or increasing the transcription of a gene in response to a treatment where such induction or increase is compared to the amount of gene expression in the absence of said treatment. Similarly, the terms decreases gene expression or down-regulates gene expression refers to inhibiting or blocking the transcription of a gene in response to a treatment and where such decrease or down-regulation is compared to the amount of gene expression in the absence of said treatment.

(38) A wide variety of proteins have been subject to random mutation procedures to generate proteins that selectively bind substances. Those of skill will recognize such scaffold proteins with a reasonable potential for generating such binding. As with many antibodies, scaffold proteins can be composed of subunit proteins. Scaffold proteins that have been used in the past include without limitation immunoglobulin heavy chain or light chain variable regions, combinations of light and heavy chains including Fab fragments, Anticalins, fibronectin type Ill domain (e.g., Adnectins), Designed Ankyrin Repeat Protein (DARPins), Centyrins, and the like.

(39) A scaffold protein library is a library of genetically diverse scaffold proteins. For example, the library can encode Adnectins.

(40) A surface, such as for example in a polystyrene multititer plate, has a substance bound thereto if its association with the surface is strong enough to allow cell panning. The binding can be, but is not necessarily, covalent.

(41) A bait polypeptide binds a prey polypeptide with sufficient binding affinity so that they can form a dimer. The dimer may be further stabilized by disulfide bond(s) between the bait polypeptide and the prey polypeptide. The prey polypeptide is a polypeptide fused with a scaffold protein, while the bait polypeptide is either fused with a cell membrane anchor or without a cell membrane anchor. Examples of potential pairs of proteins that can serve as preys and baits are presented in Table 1.

(42) TABLE-US-00001 TABLE 1 Exemplary prey/soluble bait pairs that can for heterodimer or homodimers Prey/Genebank Soluble bait/ Anchors number Genebank number (Genebank) AGA2 P32781 AGA1 P32323 Yeast anchors: (N-termini) AGA1 IL-12A P29459 IL-12B P29460 (P32323); CWP1 IL-12B P29460 IL-12A P29459 (P28319) SED1 uteroglobin Q9TS45 uteroglobin Q9TS45 (Q01589); GAS1 (P22146). IL-17A Q16552 IL-17F Q96PD4 Mammalian cell IL-17F Q96PD4 IL-17A Q16552 anchors: Folate IL-17A Q16552 IL-17A Q16552 receptor IL-17F Q96PD4 IL-17F Q96PD4 (P15328), CD14 (P08571); CD55 (P08174).

(43) Membrane anchored proteins are proteins located on the surface of the cell membrane that are covalently attached to lipids embedded within the cell membrane or cross the cell membrane. Overall, there are three main types of lipid-anchored proteins which include prenylated proteins, fatty acylated proteins and glycosylphosphatidylinositol-linked proteins (GPI). An example of yeast membrane anchored protein is AGA1anchorage subunit of a-agglutinin of a-cells; highly O-glycosylated protein with N-terminal secretion signal and C-terminal signal for addition of GPI anchor to cell wall, linked to adhesion subunit Aga2p via two disulfide bonds. Other yeast anchored proteins include CWP1, SED1, GAS1 and TIP1 (KIM et al. 2002). Examples of mammalian cell GPI-anchored proteins are GPI-proteins include cell surface receptors (e.g., folate receptor, CD14), cell adhesion molecules (e.g., NCAM isoforms, carcinoembryonic antigen variants, fasciclin I), cell surface hydrolases (e.g., 5-nucleotidase, acetylcholinesterase, alkaline phosphatase), complement regulatory proteins (e.g., decay accelerating factor (CD55)) (ORLEAN and MENON 2007).

(44) A dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules such as proteins or nucleic acids. A homodimer is formed by two identical molecules (a process called homodimerization). A heterodimer is formed by two different macromolecules (called heterodimerization). The dimer may be further stabilized by a disulfide bond between macromolecules.

(45) A disulfide bond, also called an SS bond, or disulfide bridge, is a covalent bond derived from two thiol groups. In proteins, these bonds form between the thiol groups of two cysteine amino acids. These bonds are responsible for the stabilizing the globular structure and are the strongest type of bond that a protein can possess intrinsically and are one of the major forces responsible for holding proteins in their respective conformations, and therefore have an important role in protein folding and stability.

(46) The mutation rate is the rate at which a particular mutation occurs, usually given as the number of events per gene per generation whereas mutation frequency is the frequency at which a particular mutant is found in the population.

(47) Hypermutation or increased mutation rate or increased mutation frequency refers to the mutation of a nucleic acid in a cell at a rate above background. Preferably, hypermutation refers to a rate of mutation of between 10.sup.5 to 10.sup.3/base/generation. This is greatly in excess of background mutation rates, which are of the order of 10.sup.9 to 10.sup.10/base/generation (DRAKE et al. 1998).

(48) The term constitutive hypermutation refers to the ability of certain cell lines to cause alteration of the nucleic acid sequence of one or more specific sections of endogenous or transgene DNA in a constitutive manner, that is without the requirement for external stimulation. Generally, such hypermutation is directed. In cells capable of directed constitutive hypermutation, sequences outside of the specific sections of endogenous or transgene DNA are not subjected to mutation rates above background mutation rates. The sequences which undergo constitutive hypermutation are under the influence of hypermutation-recruiting elements, as described further below, which direct the hypermutation to the locus in question. Thus in the context of the present invention, target nucleic acid sequences, into which it is desirable to introduce mutations, may be constructed, for example by replacing V gene transcription units in loci which contain hypermutation-recruiting elements with another desired transcription unit, or by constructing artificial genes comprising hypermutation-recruiting elements.

(49) As used herein, a mutator strain refers to a yeast strain having a higher than naturally occurring rate of spontaneous mutation. As used herein, mutator gene refers to a gene which inactivation or overexpression causes a higher than naturally occurring rate of spontaneous mutation. Culturing a host comprising a mutator gene will give rise to mutational events during genome replication. An example of mutator gene in the present invention is UNG1 encoding Uracil DNA-glycosylase required for repair of uracil in DNA formed by spontaneous or induced cytosine deamination. When uracil-DNA glycosylase (Ung-) is lacking, the deamination of cytosine becomes a significant source of mutations (DUNCAN and MILLER 1980). Inactivation of UNG1 in yeast results in increased mutation rates (MAYOROV et al. 2005a). Another example of mutator gene in the present invention is sea lamprey CDA1 encoding cytidine deaminase that induces cytosine deamination on single stranded-DNA in vivo. CDA1 is considered an active mutator gene as its overexpression causes a higher than naturally occurring rate of spontaneous mutation in the host (ROGOZIN et al. 2007). It has been shown that overexpression of cytosine deaminases in combination with inactivation of uracil-DNA glycosylase results in synergistic mutator effects (MAYOROV et al. 2005b).

(50) A color marker has optical density (in a frequency band) or fluorescence directly, has enzymatic activity that generates the same, or is adapted to selectively bind one or more substances (e.g., biotin) such that eventually in the binding substances directly have or enzymatically generate optical density or fluorescence changes.

(51) The meaning for identity for polypeptides is as follows: Polypeptide embodiments (including as components of methods or yeast cell systems) further include an isolated polypeptide comprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide Reference Sequence (e.g. SEQ ID NOs:1, 2, 3, 4 or 5), wherein said polypeptide sequence may be identical to the Reference Sequence or may include up to a certain integer number of amino acid alterations as compared to the Reference Sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the Reference Sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the Reference Sequence or in one or more contiguous groups within the Reference Sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in the Reference Sequence by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in the Reference Sequence, or:
n.sub.a<x.sub.a(x.sub.ay),

(52) wherein n.sub.a is the number of amino acid alterations, x.sub.a is the total number of amino acids in the Reference Sequence, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and is the symbol for the multiplication operator, and wherein any non-integer product of x.sub.a and y is rounded down to the nearest integer prior to subtracting it from x.sub.a.

(53) By way of example, a polypeptide sequence of the present invention may include a contiguous segment of sequence that is identical to the Reference Sequence, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the Reference Sequence such that the percent identity is less than 100% identity.

(54) All ranges recited herein include ranges there between, and can be inclusive or exclusive of the endpoints. Optional included ranges are from integer values there between (or inclusive of one original endpoint), at the order of magnitude recited or the next smaller order of magnitude. For example, if the lower range value is 0.2, optional included endpoints can be 0.3, 0.4, . . . 1.1, 1.2, and the like, as well as 1, 2, 3 and the like; if the higher range is 8, optional included endpoints can be 7, 6, and the like, as well as 7.9, 7.8, and the like. One-sided boundaries, such as 3 or more, similarly include consistent boundaries (or ranges) starting at integer values at the recited order of magnitude or one lower. For example, 3 or more includes 4 or more, or 3.1 or more. If there are two ranges mentioned, such as about 1 to 10 and about 2 to 5, those of skill will recognize that the implied ranges of 1 to 5 and 2 to 10 are within the invention.

(55) For all proteins or nucleic acids described herein with reference to a public database accession number, the database entry as of 22 May 2017 is incorporated herein in its entirety, particularly as to such sequence information and as to identification of functional domains.

II. Triple-Mode System for Maturation, Surface Display System and Secretion

(56) Cost effective and accelerated methods for antibody discovery will have broad impact on developing diagnostic, research and therapeutic antibodies. Currently ex vivo non-mammalian surface display techniques have several short comings such as either lack of post-translational modification (e.g., phage display (HAWKINS et al. 1992), ribosome display (HANES and PLUCKTHUN 1997; HE and TAUSSIG 1997), RNA display (REIERSEN et al. 2005) or the lack of protein secretion (e.g., yeast surface display (BODER and WITTRUP 1997; BODER and WITTRUP 2000), mammalian cell display (BEERLI et al. 2008)). Several other surface systems have been developed incorporating the secretion mode for antibody production (RAKESTRAW et al. 2011; RHIEL et al. 2014; SHAHEEN et al. 2013). All these systems are not intrinsically capable of affinity maturation because they lack the capacity to affect somatic hypermutation. Table 1 summarizes the advantages and disadvantages of the current display systems, Methods that may be potentially useful for antibody discovery are set forth in Table 1,

(57) TABLE-US-00002 TABLE 2 Comparison of the current antibody discovery platform with other technologies Display Technology Speed Cost Maturation Secretion Problems Triple-Mode High Low Yes Yes None System - Present Invention Phage display High Low No No (a) Yeast Display High Low No No (b) Ribosomal High Low No No (a) Display Mammalian Moder- Mod. No No (b) cell display ation AID + Mod. Mod. Yes No (b) Mammalian cell display Pichia pastoris High Low No Yes (c) Fc-bait Biotin/ High Low No Yes (d) Avidinated display Fc/ZZ-motive High Low No Yes (d) display (a) - lack of post-translational modification, secretion and maturation; (b) - lack of antibody maturation and secretion; (c) - lack of maturation; (d) - cross-clone contamination, lack of maturation.

(58) Numerous surface display techniques to generate antibodies were evaluated. As can be seen from Table 1 only the triple-mode display technology described herein meets the desired criteria of cost, speed, protein display and secretion, antibody in vivo maturation and ease of application. As antibody maturation and secretion mode is incorporated into the Triple-mode Display System, additional steps such as in vitro maturation by error-prone FOR, cloning of antibody-encoding genes into another expression system are not required.

(59) There is a continuing need in the art for improved antibody development platforms. This invention is a novel technology platform, that comprises three components (a) antibody maturation component; (b) antibody display component; and (c) antibody secretion component. The invented system can be used for polypeptide library diversification, protein maturation and rapid screening of binder proteins with modified affinity to another molecule. Advantages of this invention include low cost, rapid growth eukaryotic protein expression and a surface display system with ease of culture, culture maintenance, facile manipulation and genetic engineering. Moreover, yeast mating allows combination of antibody heavy chain and light chain libraries to form a single library with highly diverse random H/L combinations. The expression of sea lamprey ODAthe most powerful deaminase mutator in yeast-directed to a DNA target in combination with the chemical supermutagen HAP allows rapid library diversification, and finally the use of diploid and/or polyploid yeast strains protect yeast cells from detrimental genetic damage of the induced mutagenesis due to the presence of two or more copies of essential genes.

(60) In embodiments, in combination with Fluorescence Assisted Cell Sorting (FACS) followed by ELISA to measure activity of secreted antibodies in the cell culture media, yeast cells expressing functional binders can be quickly identified.

(61) In accordance with the present invention, a yeast-based genetic system and methods of use, thereof are provided to facilitate antibody discovery. The methods provided herein enable the rapid and efficient maturation and isolation of antibody clones to an antigen target of interest starting from a nave antibody library, or alternatively to improve the activity of a known protein, including antibodies.

III. Preparation of Nucleic Acid Molecules Encoding the Proteins of the Invention

A. Nucleic Acid Molecules

(62) Nucleic acid molecules encoding the expression vectors of the invention may be prepared by two general methods: (1) They may be synthesized from appropriate chemical starting materials, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.

(63) The availability of nucleotide sequence information, for the sea lamprey CDA1, yeast AGA1, AGA2 as well as for secretory signals from alpha-mating factor or yeast SUC2 gene (providing secretory signal) facilitates synthesis of DNA constructs containing such sequences, Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule encoding a construct of the present invention, must be synthesized in stages due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a 3 kilobase double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be ligated such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct the entire 3 kilobase double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

(64) In alternative embodiments of the invention, the sea lamprey CDA, yeast AGA1, AGA2 and yeast SUC2 secretory signal can be substituted with similar genes with functional homology from other biological sources. In the PmCDA1 example, suitable candidate genes for such substitution include, without limitation, lamprey cytosine deaminase mutated (modified or altered cytosine deaminases), derivatives such as a CDA1 hybrid with ER DNA binding domain (ER-DBD), which also has high mutator phentotype once expressed in yeast defective in Uracil-DNA glycosylase. In addition, one can replace PmCDA1 with cytosine deaminase from other species, including but not limited to human AID.

(65) In the example of yeast AGA1 and AGA2 heterodimerized via disulfide bond formation in ER, they can be substituted with components of a extracellular disulfide-bridged homodimer (e.g., 10 kDa uteroglobin, Uniprot # Q9TS45) or heterodimer protein such as human IL-12 encoded by two separate genes, IL-12A (Uniprot # P29459) and IL-12B (Uniprot # P29460).

(66) Yeast SUC2 secretory signal of the invention used for promoting protein secretion may be derived from different species not limited to S. pombe and K. lactis. It can be substituted with yeast alpha mating factor secretory signal that also functions as secretory signal.

(67) Nucleic acid sequences encoding the components of the expression plasmids of the invention may be isolated from appropriate biological sources using methods known in the art. For example, RNA isolated from a mammalian or insect cell may be used as a suitable starting material for the generation of cDNA molecules encoding the different receptor proteins.

(68) In accordance with the present invention, nucleic acids having the appropriate level of sequence homology with the protein coding region of the DNA molecules of the present invention may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed at 37 C. to 42 C. for at least six hours. Targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 C. Such a sequence would be considered substantially homologous to the sequences of the present invention.

(69) The nucleic acids of the invention may also be used as starting materials for the generation of sequence variants or truncation mutants of the nucleic acids of the invention using any number of synthetic and molecular biologic procedures well known in the art including, but not limited to, truncation at available restriction sites and site-directed mutagenesis techniques. Particular mutations may give rise to receptor proteins with altered characteristics such as increased or decreased ligand binding activity.

B. Fusion Proteins

(70) In one embodiment of the invention, the antibodies of the invention are expressed in yeast as fusion proteins having a yeast secretory signal at the N-terminus. Secretory signal fusion at the N-terminus of the protein facilitates extracellular secretion of the protein following translation. After translation of recombinant proteins, the secretory signals at the N-terminus are cleaved by the host secretory pathway proteases and native proteins are released. It is widely known that the presence of the secretory signals results in secretion of proteins of interest in yeast (KJAERULFF and JENSEN 2005).

(71) In one embodiment of the invention, the antibodies of the invention are expressed in yeast as fusion proteins with yeast AGA2 at their C-termini. As a result of heterodimerization of yeast AGA2 with yeast membrane anchor AGA1, secreted antibodies or polypeptides are retained on the yeast cell surface (BODER and WITTRUP 1997) therefore accessible to an antigen of interest located extracellularly.

(72) In another embodiment of the invention, the hybrid membrane anchor consists of fusion of the AGA1 N-terminal fragment (AA1-149) with yeast cell wall protein CWP1 (SEQ ID NO:7; 1-149=AGA1 fragment, 173-392=CWP1). As a result of heterodimerization of yeast AGA2 used with antibody scaffolds at their C-termini with yeast AGA1 N-terminal fragment (AA1-149) (SHEN et al. 2001), secreted antibodies or polypeptides are retained on the yeast cell surface therefore accessible to an antigen of interest located extracellularly.

(73) In another embodiment of the invention, the antibodies of the invention are expressed in yeast as fusion proteins with yeast AGA2 at their C-termini. As a result of heterodimerization of yeast AGA2 with yeast AGA1 N-terminal fragment (AA1-149) (SHEN et al. 2001) and the lack of membrane anchor, AGA2/antibodies complexed with the AGA1 fragment will be secreted extracellularly.

IV. Assay Methods and Kits

(74) In yet another embodiment of the invention, assays are provided wherein intact cells expressing a protein of interest are grown in cell culture media containing base analogue molecules and molecules to induce expression of cytosine deaminase. After a suitable time period, the diversification of a gene or protein of interest is measured. Such diversification of a gene or protein of interest may be quantitated in any number of ways. For example, such cell systems may utilize a reporter system in which the production of the reporter signal is dependent on enzymatic or fluorescence or binding activity of the protein of interest. Numerous reporters may serve equally well in this application including but not limited to, beta-galactosidase, alkaline phosphatase, green fluorescent protein, antibody, protein scaffold and the like. Inactivation or activation of the gene of interest can be also measured as forward and reverse mutation rates. For example, mutation in the arginine permease encoding gene CAN1 confers resistance to canavanine (LANG and MURRAY 2008). The gene diversification level can be measured by Next Generation Sequencing. Furthermore, the methods of the invention may be practiced in bacterial, fungal, insect, avian, mammalian or plant cells. However, yeast-based cell systems are preferred due to low cost and the feasibility of growing yeast cells in plastic devices.

(75) Assays for screening binders to a target of interest are also provided. Diversification of binders in the cell-based system may be followed by isolation of cells expressing modified binders reactive to a target by means of biological panning or fluorescence-activation cell sorting (FACS). Isolation of binders reactive to a target of interest can be performed as described previously (CHAO et al. 2006). In another aspect, the invention includes kits to facilitate the use of the compositions and methods disclosed herein. Exemplary kits include the expression plasmids, yeast-based scaffold libraries and yeast strains of the invention, and/or variants thereof. Also provided are cell culture media, compounds and protocols for use of the compositions of the invention for the particular application and the necessary reagents to carry out the application are also provided. Such reagents may include, but not be limited to, buffers, solvents, media and solutions.

(76) The following protocols are provided to facilitate construction of the expression plasmids for use in the methods and kits of the present invention.

Yeast Media, Strains, Plasmids, Antibody Library

(77) Standard yeast and E. coli media were prepared as described in detail (CHAO et al. 2006). For example, YPD composed of Yeast Extract (20 grams per liter), Peptone (20 g/liter), Dextrose (20 g/Liter) is preferred for use in the invention as most yeast strains grow in this media. Yeast selective media (complete-drop out) used to maintain plasmids is composed of yeast nitrogen base 1.7 g/L, ammonium sulfate 5 g/L, dextrose 20 g/L, different amino acids and other supplements depending on the requirements of the particular yeast strain. For example if the yeast plasmid contains the LEU2 marker, the leucine is dropped out from the media in order to select the plasmid. Other buffered selective media such as SDCAA glucose media and SGRCAA galactose media were prepared as described in detail (CHAO et al. 2006).

(78) Yeast strains suitable for use in the present invention include the yeast strains of opposite mating types presented in Table 2.

(79) TABLE-US-00003 TABLE 3 Yeast strains of opposite mating types used for developing the triple-mode sysem Strains Genotype Application A169x Mat alpha for Gal1p-AGA1/URA3 transformation opi1::[Bsd.sup.R/Cup1p- of DNA AGA1(AA1-149)] ura3-52 construct trp1::NatMX leu2-200 encoding light his3-200 lys2 pep4::Zeo.sup.R chain prb1.6R can1 ung1::HygB ham1::KanMX A170x MatA Gal1p-AGA1/URA3 for ura3-52 opi1::[Bsd.sup.R/Cup1p- transformation AGA1(AA1-149)] trp1-63 of DNA Gal1p-PmCDA1/LEU2 leu2 construct his3::Zeo.sup.R pep4-3 prb1-22 encoding prd-407 ung1::HygB heavy chain ham1::KanMX

(80) A series of yeast expression plasmids for expression and display of a protein of interest on the yeast cell surface were constructed based on the yeast-E. coli single copy shuttle plasmid pRS314 (SIKORSKI and H.sub.IETER 1989). All plasmid derivatives are yeast-E. coli centromeric shuttle plasmids containing TRP1 as a yeast transformation marker. FIG. 2A depicts an exemplary plasmid pRS314_Gal1/10p_VHH of the invention that is suitable for expression and display of camelid heavy chain variable domains on the yeast cell surface. pRS314_Gal1/10p_VHH is comprised of the centromeric plasmid pRS314 (SIKORSKI and HIETER 1989), S. cerevisiae bi-directional galactose inducible promoter Gal1/10, yeast Suc2 (invertase) secretory signal, Sfil-Sfil cloning sites, linker, membrane anchor AGA2 and FLAG tag at its C-terminus. pRS314-Gal1/10p-VH (FIG. 2B) used for expressing human VH has a similar structure containing the Gal1/10 promoter, yeast Suc2 (invertase) secretory signal, Eagl-Sall cloning sites, linker membrane anchor AGA2 and HA tag at its C-terminus. In another construct variable domains are expressed as a fusion at AGA2 C-terminus.

(81) Proteins of interest such as antibody heavy chain or light chain variable regions, protein scaffolds including, but not limited to Anticalins, fibronectin type III domainAdnectins, camelid antibodies, Designed Ankyrin Repeat Protein or DARPins and Centyrins can be expressed and displayed on the yeast cell surface using the two vectors mentioned above.

(82) When a protein of interest is a heterodimer such as an antibody Fab fragment, it requires two plasmids to express both partners simultaneously. For example, to express and display antibody Fab fragments, two yeast-E. coli centromeric plasmids have to be constructed to express both heavy chain and light chains. For expression of heavy chain, for example, the yeast-E. coli centromeric plasmid pRS314-Gal1/10p-Eagl-Sall plasmid (FIG. 2C) that contains the yeast Gal1/10 promoter, Suc2 invertase secretory signal, cloning sites Eagl and Sall for heavy chain VH cloning, CH1 constant domain, leucine zipper FOS followed with HA tag was constructed. The plasmid contains a TRP1 yeast transformation marker. For expression of light chain, for example, a series of plasmids derived from plasmid pRS316 (SIKORSKI and HIETER 1989) that contains a HIS3 transformation selection marker were constructed. pRS316-Gal1/10p-Eagl-BsrG1-Ck for kappa light chain (FIG. 2D) and pRS316-Gal1/10p-Eagl-Xhol-CL for lambda light chain (FIG. 2D) were constructed. These two plasmids each contain a yeast Gal1/10 promoter, Suc2 invertase secretory signal, cloning sites Eagl/BsrGl or Eagl/Xhol for light chain variable cloning, Kappa or Lambda light chain constant region, respectively, leucine zipper Jun to facilitate dimerization followed with FLAG tag for display and expression monitoring. Heteromeric formation is possible via formation of heavy chain and light chain constant domains facilitated by the leucine zipper Fos/Jun. The pRS316-based plasmid contains a HIS3 yeast transformation marker.

(83) To display and diversify Fab fragments, heavy chain and light chain variable domains are cloned in frame at the cloning sites in the respective vectors described above, Heavy chain vectors are introduced, for example, into the A169x yeast strain by yeast transformation selecting colonies growing in media without tryptophan, Light chain vectors are introduced, for example, into the A170x strain that already contains the integrated PmCDA1 selecting colonies growing in media without histidine. A diploid is obtained by mating Trp.sup.+ and His.sup.+ transformants and selecting Trp.sup.+ His.sup.+ colonies. Genes expressing heavy chain and light chain variable domains are diversified by growing diploids in media containing galactose as a sole carbon source to induce both antibody encoding genes and the CDA1 encoding gene.

(84) Also provided is a camelid VHH library constructed using the yeast-E. coli expression vector pRS314-Gal1/10-VHH (FIG. 2A). Complementary DNA (cDNA) derived from camelid leukocyte mRNA encoding heavy chain variable domains has been cloned in frame with the secretory signal and AGA2, cDNA library construction is well known in the art. The camelid VHH library has been introduced into the A169x yeast strains using Trp.sup.+ selection. The final self-diversifying camelid antibody library was obtained, for example, by mating A169x library with the A170x yeast strain containing the CDA gene integrated.

Exemplary Diversification Methods

(85) A diploid host cell containing a first DNA construct having a nucleic acid molecule encoding a protein that is subjected for diversification, a second DNA construct having a nucleic acid molecule encoding cytosine deaminase, a third DNA construct having a nucleic acid molecule encoding an anchored bait and a forth construct having a nucleic acid molecule encoding a soluble bait will undergo diversification by two means either performed separately or in combination.

(86) In the first means of diversification a host cell containing constructs of the invention is continuously grown in yeast selective media that contains promoter inducers including but not limited to (1) galactose (20 g/L) as a sole carbon source to induce the Gal1/10 promoter or (2) copper at concentrations of 100 micro molar to 1 mM to induce the Cup1 promoter. Under such conditions cytosine deaminase is expressed. Produced cytosine deaminases will convert C to U via deamination in transcriptionally active genes including a gene of interest.

(87) In the second means of diversification, a host cell containing constructs of the invention is continuously grown in yeast selective media containing base analogues including, but not limited to 6N-hydroxylamine purine or HAP. During replication the base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC.fwdarw.AT and AT.fwdarw.GC transitions (SHICHERBAKOVA and P.sub.AVLOV 1993).

(88) As gene diversification occurs via hypermutation that is active during cell division via DNA replication, the level of diversification obtained is directly related to numbers of mutations in a gene of interest that accumulate as cells grow. Therefore the longer cells undergo diversification, the more mutations will accumulate. The present invention, accordingly, includes the method comprising the following steps for diversifying an antibody or antigen-binding fragment thereof:

(89) (1) Grow in a liquid culture media host cells comprising: (i) sea lamprey cytidine deaminase or functional fragment thereof; (ii) and one or more AGA2 fused antibody scaffolds; (iii) and membrane anchor AGA1 bait or functional fragment thereof; (iv) and soluble AGA1 bait or functional fragment thereof.

(90) (2) Allow expression of antibody scaffolds and sea lamprey CDA1;

(91) (3) Optionally, contact cells with mutagen, such as base analog, 6N-hydroxyaminopurine.

Methods for Identifying Antibody to a Target of Interest

(92) In an embodiment of the invention, after the diversification step, the eukaryotic host cells expressing the anchored AGA1 bait dimerized with the AGA2/antigen-binding fragment are identified and sorted using fluorescence-activated cell sorting (FACS). For example, in an embodiment of the invention, cells expressing the anchored AGA1 bait dimerized with the AGA2/antigen-binding fragment on the cell surface are labeled with either a fluorescent antigen or biotinylated antigen/fluorescent streptavidin. The fluorescent label is detected during the FACS experiment and used as the signal for sorting. Labeled cells indicate the presence of a cell surface expressed AGA1/AGA2/antigen-binding fragment/antigen complex and are collected in one vessel whereas cells without signal are collected in a separate vessel. The present invention, accordingly, includes a method comprising the following steps for determining if an antibody or antigen-binding fragment thereof from a library specifically binds to an antigen:

(93) (1) Grow in a liquid culture media host cells comprising: (i) sea lamprey cytidine deaminase or functional fragment thereof; (ii) and one or more AGA2 fused antibody scaffolds; (iii) and membrane anchor AGA1 bait or functional fragment thereof; (iv) and soluble AGA1 bait of functional fragment thereof.

(94) (2) Allow expression of antibody scaffolds and the anchored AGA1 bait on the surface of the cells;

(95) (3) Optionally, enrich cells expressing binders to an antigen of interest as follows: (i) Label cells with biotinylated antigen; (ii) Collect labeled cells using streptavidin-coated magnetic particles for a one round; (iii) Regrow the labeled, enriched cells; (iv) Allow expression of antibody scaffolds and the anchored AGA1 bait on the surface of the cells; (v) Label cells with biotinylated antigen; (vi) Collect labeled cells using Avidin-coated magnetic particles for a second round. (vii) Regrow the labeled, enriched cells; (viii) Allow expression of antibody scaffolds and the anchored AGA1 bait on the surface of the cells; (ix) Label cells with biotinylated antigen; (x) Collect labeled cells using anti-biotin-coated magnetic particles for a third round

(96) (4) Regrow the enriched cells.

(97) (5) Allow expression of antibody scaffolds and the anchored AGA1 bait on the surface of the cells;

(98) (6) Label the cells with fluorescently labeled antigen or antigen bound to a fluorescently labeled secondary antibody;

(99) (7) Sort and isolate fluorescently labeled cells using FACS for one round;

(100) (8) Regrow the sorted cells;

(101) (9) Allow expression of antibody scaffolds and the anchored AGA1 bait on the surface of the cells;

(102) (10) Sort and isolate fluorescently labeled cells using FACS for a second round;

(103) (11) Regrow the labeled, sorted cells on solid culture medium so that individual cellular clones grow into discrete cellular colonies;

(104) (12) Identify colonies with affinity for the antigen using cytoflow analysis;

(105) (13) Grow cells from identified colonies in a liquid culture medium

(106) (14) Allow expression of a soluble AGA1 bait and antigen-binding fragments;

(107) (15) Isolate supernatant containing a soluble AGA1 bait complexed with secreted antigen-binding fragment thereof comprising the immunoglobulin light and heavy chains or VH1-1;

(108) (16) Determine affinity of secreted antibodies or antigen-binding fragments thereof, from the supernatant, for the antigen and identify clones with acceptable affinity;

(109) (17) Determine the nucleotide sequence of polynucleotides in the identified clones encoding the heavy and light chain immunoglobulins

NUMBERED EMBODIMENTS

(110) The invention is further described with reference to the following numbered embodiments;

(111) Embodiment 1. A eukaryotic cell providing a protein display, protein selection and soluble protein recovery system, comprising: a) recombinant DNA capable of expressing a mutagenic cytidine deaminase; b) recombinant DNA capable of expressing a membrane-bound version of a bait polypeptide; c) recombinant DNA capable of expressing a soluble version of the bait polypeptide; and d) recombinant DNA capable of expressing one or more polypeptides of a scaffold protein, with one or more said scaffold polypeptides operably fused to a prey polypeptide.

(112) Embodiment 2. The eukaryotic cell of a Cell Embodiment, wherein the cytidine deaminase is sea lamprey Petromyzon marinus cytidine deaminase CDA1 or functional fragment thereof.

(113) Embodiment 3. The eukaryotic cell of a Cell Embodiment, wherein the expressed bait polypeptide is whole Saccharomyces cerevisiae AGA1 or a functional fragment thereof, comprises a cell surface anchor, and is a membrane bound version.

(114) Embodiment 4. The eukaryotic cell of a Cell Embodiment, wherein the cell surface anchor comprises a polypeptide segment sufficient to incorporate into membrane, trigger a covalent association with lipid (e.g., GPI), or associate with another membrane component. [Examples include without limitation functional segments of yeast Saccharomyces AGA1, CWP1 SED1 or GAS1 or mammalian GPI-anchored proteins such as cell surface receptors (e.g., folate receptor, CD14, and the like), cell adhesion molecules (such as segments of NCAM isoforms, carcinoembryonic antigen variants, fasciclin I, and the like having such adhesion), cell surface hydrolases (e.g. 5-nucleotidase acetylcholinesterase, alkaline phosphatase, and the like), complement regulatory proteins (e.g., decay accelerating factor (CD55), and the like).]

(115) Embodiment 5. The eukaryotic cell of a Cell Embodiment, wherein the bait polypeptide is a fragment of Saccharomyces cerevisiae AGA1 that lacks a cell surface anchor [e.g., glycosylphosphatidylinisotol membrane anchor] and is a soluble version.

(116) Embodiment 6. The eukaryotic cell of a Cell Embodiment, wherein the prey polypeptide comprises Saccharomyces cerevisiae AGA2 or a functional fragment thereof, capable to form heterodimeric complex with the bait polypeptide.

(117) Embodiment 7. The eukaryotic cell of a Cell Embodiment, wherein the scaffold protein comprises an immunoglobulin heavy chain variable region, a light chain variable region, combinations of light and heavy chain regions, Anticalins, fibronectin type III domain, Designed Ankyrin Repeat Protein or Centyrin.

(118) Embodiment 8. The eukaryotic cell of a Cell Embodiment, wherein the expression of the membrane-bound version and the soluble version of a bait polypeptide are selectively inducible.

(119) Embodiment 9. The eukaryotic cell of a Cell Embodiment, wherein the cell is a Saccharomyces cell or Pichia cell.

(120) Embodiment 10. The eukaryotic cell of a Cell Embodiment, wherein the cell is a Chinese hamster ovary cell.

(121) Embodiment 11. A method for maturing and identifying an antigen-binding variant of the scaffold protein comprising: A, cultivating a culture of eukaryotic cells of Embodiment 1; B. cultivating the culture such that the cytidine deaminase and the scaffold protein are expressed; C. contacting the culture with a mutagen; D. thereafter cultivating the culture such that the membrane-bound bait polypeptide and membrane bound said scaffold protein are expressed; E. selecting a subset of the eukaryotic cells that express scaffold protein at the cell surface that binds the antigen more strongly than the rest; F. cultivating the selected cells (e.g., colonies thereof) such that soluble bait polypeptide and soluble said scaffold protein are expressed; G. selecting a subset of one or more of the selected cells that bind the antigen (e.g., colonies), with selection based one or more of apparent binding activity, binding affinity or protein stability, for example based on the soluble scaffold protein.

(122) Embodiment 12. The method of a Method Embodiment, wherein step D comprises contacting the culture with an inducer for the expression of the membrane-bound bait polypeptide.

(123) Embodiment 13. The method of a Method Embodiment, wherein step F comprises contacting the culture with a second inducer, distinct from the first, which is for the expression of the soluble bait polypeptide.

(124) Embodiment 14. The method of a Method Embodiment, wherein the cytidine deaminase is sea lamprey Petromyzon marinus cytidine deaminase CDA1 or functional fragment thereof.

(125) Embodiment 15. The method of a Method Embodiment, wherein the expressed bait polypeptide is whole Saccharomyces cerevisiae AGA1 or a functional fragment thereof, comprises a cell surface anchor, and is a membrane bound version.

(126) Embodiment 16. The method of a Method Embodiment, wherein the cell surface anchor comprises a polypeptide segment sufficient to incorporate into membrane, trigger a covalent association with lipid (e.g., GPI), or associate with another membrane component.

(127) Embodiment 17. The method of a Method Embodiment, wherein the bait polypeptide lacks a cell surface anchor [e.g., glycosylphosphatidylinisotol membrane anchor] and is a soluble version.

(128) Embodiment 18. The method of a Method Embodiment, wherein the prey polypeptide comprises Saccharomyces cerevisiae AGA2 or a functional fragment thereof capable to form heterodimeric complex with the bait.

(129) Embodiment 19. The method of a Method Embodiment, wherein the scaffold protein comprises an immunoglobulin heavy chain variable region, a light chain variable region, combinations of light and heavy chain regions, Anticalins, fibronectin type III domain, Designed Ankyrin Repeat Protein or Centyrin.

(130) Embodiment 20. The method of a Method Embodiment, wherein the expression of the membrane-bound version and the soluble version of a bait polypeptide are selectively inducible.

(131) Embodiment 21. The method of a Method Embodiment, wherein the cell is a Saccharomyces cell or Pichia cell.

(132) Embodiment 22. The method of a Method Embodiment, wherein the cell is a Chinese hamster ovary cell.

(133) Publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

(134) The citations below are incorporated herein in there entirety, and particularly with respect to the subject matter for which they are cited.

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