Multi-chain eukaryotic display vectors and uses thereof

<u style="single">Related application</u> This application claims the benefit of US Provisional Patent Application No. 60 / 326,320 filed October 1, 2001. The entire teachings of the above application are incorporated herein by reference.

<u style="single">Background of the invention</u> The development of phage display technology, in which a non-natural (heterologous) polypeptide or protein is expressed and fixed (displayed) on the surface of a bacteriophage, is high for a molecule having the desired biological activity, for example, a certain target molecule. It provides a powerful tool for identifying peptide ligands that bind with specificity and / or affinity. A library of synthetic oligonucleotides can be cloned in-frame into the coding sequence of a gene encoding a phage surface protein, such as the III or VIII gene of phage M13. When expressed, these clones are "displayed" on the surface of the phage as multiple peptide-capsid fusion proteins due to the diversity in the sequences of the oligonucleotides used. These peptide display libraries are then screened for binding to the target molecule, usually by affinity selection or "biopanning" (Ladner, R. et al., 1993; Kay et al., 1996; Hoogenboom, H. et al., 1997).

Phage display library screening involves a huge number of different polypeptides (typically 1x10).⁹(Beyond) can be contained in a single phage display library, which is a great advantage over other screening methods. This allows screening of a wide variety of libraries in a single screening process. Displaying small peptides or single-chain proteins on phage is advantageous unless intracellular processing or post-translational modification (which a phage or prokaryotic host cannot do) is required or desired. For example, an effective display of a heterologous polypeptide transports various post-translational modifications, intracellular structures, and the display polypeptide thereof, glycosylates, conforms, assembles, and is suitable for the surface of the host cell. Neither of these methods can be achieved by bacteriophage or prokaryotic cell processes, although special enzymes and chaperone protein complements required for fixation may be required.

Displaying more complex eukaryotic proteins such as immunoglobulins and their functional fragments (eg Fabs), or multi-stranded polypeptides containing the extracellular domain of MHC or T cell receptor molecules presents additional problems to overcome. Exists: Synchronously expressing component chains at a level of expression sufficient to produce a multi-chain product, transporting and secreting each chain while achieving association with a functional multi-chain polypeptide, and Appropriate assembly and function of the multi-stranded polypeptide product outside the host cell, while immobilizing (fixing) at least one strand of the multi-stranded polypeptide to the surface of the host cell (ie, for display). It is to maintain sex.

A display system utilizing eukaryotic cells such as yeast for expressing and displaying single-chain polypeptides has been reported (Boder, E. and Wittrup, K., 1998; Horwitz, A. et al. , 1988; Kieke, M. et al., 1997; Kieke, M. et al., 1999; WO94 / 18330; WO99 / 36569). However, there is a need for improved eukaryotic systems for the expression and functional display of multi-chain polypeptides, especially immunoglobulins and fragments thereof. In addition, there is a need in the field of polypeptide display technology for systems that take advantage of the ability of phage display and the processing advantages of eukaryotic host cells. For example, in contrast to phage display libraries, the actual size, or "diversity" of a library that can be expressed and displayed on the surface of a eukaryotic host cell is at most about 10.⁶~10⁷That is.

The above and other technical challenges have hampered advances in biological tools and techniques useful in identifying novel molecules possessing the desired biological activity. Due to these technical challenges, materials or methods for the successful construction of multi-chain eukaryotic display vectors, of eukaryotic host cells (such as antibodies or Fab fragments) of multi-chain polypeptides (such as antibodies or Fab fragments). Successful display on the surface, creation of a multi-strand polypeptide display library in a eukaryotic host cell, or detection and isolation of a particular multi-stranded polypeptide of interest (eg, binding specificity or affinity for a target molecule) No successful use of these libraries for (based on) has been reported to date.

<p><patcit num="1"><text>WO94 / 18330</text></patcit><patcit num="2"><text>WO99 / 36569</text></patcit></p>

<p><nplcit num="1"><text>Ladner, R. et al., 1993</text></nplcit><nplcit num="2"><text>Kay et al., 1996</text></nplcit><nplcit num="3"><text>Hoogenboom, H. et al., 1 997</text></nplcit><nplcit num="4"><text>Boder, E. and Wittrup, K., 1998</text></nplcit><nplcit num="5"><text>Horwitz, A. et al., 1988</text></nplcit><nplcit num="6"><text>Kieke, M. et al., 1997</text></nplcit><nplcit num="7"><text>Kieke, M. et al., 1999</text></nplcit></p>

<u style="single">Outline of the invention</u> The above and other deficiencies in the art provide improved display vectors, cells containing display libraries, and methods of using such libraries and vectors, according to the invention described herein. Overcome. In particular, the present invention provides a eukaryotic expression vector capable of displaying a multi-chain polypeptide on the surface of a host cell so that the biological activity of the multi-chain polypeptide appears on the surface of the host cell. Such vectors allow the display of biologically active polypeptides, such as biologically active multi-stranded polypeptides, which are more complex than those available by conventional phage display techniques.

The present invention relates to the display and isolation of biologically active polypeptides. In particular, the present invention is directed to the design and use of novel multi-chain display vectors. The present invention describes and enables successful display of multi-chain polypeptides on the surface of eukaryotic host cells. For vectors preferred for expressing the multi-stranded polypeptide chain separately and independently in the host cell (eg, under a separate vector control sequence and / or with a separate expression vector, a compatible vector set is produced). Describe. The use of such a matching vector set is a multi-stranded polypeptide by recombination of vectors expressing a variety of individual strands of a multi-stranded polypeptide, eg, a level of flexibility and versatility in the production of display libraries. Provides the ability to produce and display. These vector sets can be used to design the entire repertoire of novel chain combinations.

In addition, the present invention combines the capabilities of phage display technology (with its ease of operation and magnitude of diversity) with the potential complexity and versatility of multi-strand eukaryotic display vectors (or vector sets). provide. The special method described herein allows the practitioner to efficiently transfer sequence information of a peptide library (or selected member of the library) between a phage display system and a eukaryotic display system. This allows for physical transfer of sequence information from one display vector to the other (using conventional genetic engineering techniques) or can operate in both eukaryotic and phage display systems. Achieved by the use of a novel dual display vector (with necessarily prokaryotic expression).

The present invention presents a multi-chain polypeptide on the surface of a host cell such that in a eukaryotic host cell, the biological activity of the multi-chain polypeptide, eg, the binding activity of the multi-chain polypeptide, appears on the surface of the host cell. Directed to useful novel vectors. One preferred embodiment of the vector of the invention is that of a single replicable gene package, but the multi-stranded eukaryotic display vector exists as a single vector or as a number of independent vectors in a vector set. It's okay. As used herein, "vector" means either a single vector molecule or a set of vectors. In one embodiment, the display vector is a shuttle vector or, more precisely, a dual display vector, wherein the vector transforms a biologically active multi-stranded polypeptide with the vector. It can be displayed on the surface of eukaryotic host cells or on the surface of bacteriophage produced as a result of prokaryotic expression. In another aspect of the invention, the vector may be present as a vector set, where each strand of the multi-stranded polypeptide is encoded by one of the matching pairs of the vector, so that the vector pair is single. When present in eukaryotic cells, the multi-chain polypeptide chains associate and the biological activity of the multi-chain polypeptide appears on the surface of the eukaryotic cell.

The eukaryotic multi-chain display vector of the present invention contains a polynucleotide encoding a polypeptide chain of a multi-chain polypeptide. The first polynucleotide encodes the first strand of a multi-stranded polypeptide that links to an anchor protein. Other polynucleotides in this vector (or vector set) encode other strands of a multi-stranded polypeptide. Since all of the polynucleotides in the display vector are operably located in the display vector, host eukaryotic cells transformed with that vector (or vector set) will have the biological activity of the multi-stranded polypeptide on the surface of the cell. The multi-stranded polypeptide is displayed on the surface of the host cell so that it appears.

Preferably, the multi-stranded polypeptide encoded by the multi-stranded display vector of the present invention exists as any of two, three, four, or multi-stranded polypeptides. More preferably, the multi-stranded polypeptide is a double-stranded or quadruplex polypeptide consisting of two different strands. More preferably, the multi-chain polypeptide is selected from the group of multi-chain polypeptides consisting of T cell receptors, MHC class I molecules, MHC class II molecules, and immunoglobulin Fab fragments. More preferably, the multi-chain polypeptide is IgA, IgD, IgE, IgG, IgM, or a biologically active fragment thereof. Most preferably, the multi-stranded polypeptide is a Fab fragment, where the first polynucleotide of the multi-stranded display vector is the Ig heavy chain V._HDomain and C_HThe second polynucleotide contains the segment encoding one domain, the Ig light chain (V)._LDomain and C_LContains the segment that encodes the domain).

According to the present invention, the first polynucleotide encoding the first strand of a multi-stranded polypeptide is linked to the anchor protein. Preferably, the anchor protein is a cell surface protein of eukaryotic cells, or a functional fragment thereof. More preferably, the anchor protein is α-aglutinine, a-aglutinine, Aga1p, Aga2p, or FLO1. As disclosed herein, ligation of a first chain polypeptide to an anchor protein can be achieved by a variety of molecular biology techniques. Preferably, the first polynucleotide encoding the first strand of the multi-stranded polypeptide is expressed in a eukaryotic host cell as a first strand-anchor fusion protein, most preferably as a first strand: Aga2p fusion protein. To.

In one embodiment, one or more strands of a multi-stranded polypeptide expressed by a vector in a host cell are linked to a reporter gene or tag. Preferably, the tag is an epitope tag selected from the group consisting of 6 × His tag, HA tag, and myc tag. Most preferably, each strand of the multi-stranded polypeptide is linked to a different tag.

Preferably, the multi-stranded display vector of the present invention provides a cloning site that facilitates the transfer of the polynucleotide sequence encoding the multi-stranded polypeptide chain. Such cloning sites include restriction endonuclease recognition sites (ie, restriction sites) that are located to facilitate cleavage and insertion of the polynucleotide encoding one or more strands of the multi-stranded polypeptide. For example, the restriction site is preferably located at the 5'and 3'ends of the polynucleotide encoding the multi-stranded polypeptide chain. The vectors of the invention can contain only two restriction sites at the end of a polynucleotide segment, including all segments encoding a multi-chain polypeptide chain, or preferably the restriction sites are multi-chain polypeptides. It is located at the end of each polynucleotide segment encoding one strand of (Figs. 1 and 2). Each restriction endonuclease recognition site is preferably a unique recognition site in the vector.

The vectors (or vector sets) of the invention can be operable in various eukaryotic host cells and, in some cases, in prokaryotic cells (eg, bacteria). Preferably, the multi-strand display vector of the present invention is an animal cell display vector, a plant cell display vector, a fungal cell display vector, or a prokaryotic cell display vector. More preferably, the display vector is a yeast display vector. Most preferably, the yeast display vector is operational in Saccharomyces cerevisiae.

In another aspect, the invention is directed to a method of using a vector (or vector set) described and taught herein to display a multi-chain polypeptide on the surface of a eukaryotic host cell. Here, the vector (or vector set) is introduced into the eukaryotic cell, where the host cell expresses, transports, and associates the multi-stranded polypeptide chain such that the biological activity of the multi-stranded polypeptide appears on the surface of the host cell. It is cultured under the conditions suitable for. As described herein, the polynucleotide encoding a multi-stranded polypeptide chain can be introduced into a host cell by one or more vectors. Methods for introducing a vector into a host cell include any method known in the art for introducing a genetic material into a cell. Preferred methods include, but are not limited to, transformation techniques known in the art, including, but not limited to, electroporation, microinjection, virus transfer, bullet insertion, and the like.

Another preferred method of introducing a eukaryotic display vector into a host cell is to fuse two haploid eukaryotic cells, each expressing at least one of the multiplex polypeptide chains, and both ( Includes producing diploid host cells that express all) chains. The biological activity of the multi-chain polypeptide appears on the surface of this resulting diploid host cell. For example, the biological activity of a multi-stranded polypeptide is multiplied because each of the two haploid cells can contain one (or more) of the vectors of the vector set (as described herein). Appears on the surface of diploid host cells resulting from body / haploid fusion. Preferably, the haploid host cell pair is of the opposite mating type, which facilitates the fusion (conjugation) of the two eukaryotic haploid cells.

Another object of the present invention is directed to eukaryotic host cells that reveal the biological activity of multi-chain polypeptides on the surface of the cell. As described herein, eukaryotic host cells are preferably animal cells, plant cells, fungal cells, or prokaryotic cells. More preferably, the eukaryotic host cell is a yeast cell. Preferably, the yeast host cell is selected from the genera Saccharomyces, Pichia, Hanzenula, Sizosaccalomyces, Kluyveromyces, Yarrowia, and Candida. Most preferably, the eukaryotic host cell is S. cerevisiae. The eukaryotic host cell of the present invention may be of any gene construct, but is preferably haploid or diploid.

One aspect of the invention is directed towards eukaryotic haploid cell pairs (preferably of the opposite mating type). Here, the first haploid cell expresses at least the first polynucleotide encoding the first strand of a biologically active multi-stranded polypeptide that ligates to an anchor protein, and the second haploid cell. Expresses at least a second polynucleotide encoding the second strand of a multi-stranded polypeptide. As discussed above, this fusion of haploid cell pairs results in diploid cells that reveal the biological activity of the multi-chain polypeptide on the surface of the cell.

Further, the present invention is directed to a set of various aspects described herein. This forms a novel library of multi-stranded polypeptides or polynucleotides encoding them. The library of the present invention contains a plurality of vectors encoding a multi-chain polypeptide, which constitutes the expression and secretion of the multi-chain polypeptide chain and the biological activity of the multi-chain polypeptide in a eukaryotic host cell. Such chain associations and the biological activity of the multi-chain polypeptide can be actuated to direct the attachment of at least one strand of the multi-chain polypeptide such that it appears on the surface of the eukaryotic host cell. Preferably, the library of the invention consists of library members encoding a number of different multichain polypeptides. Most preferably, the library consists of library members encoding a large number of mutant multi-chain polypeptides (designed and produced by modification of the multi-chain polypeptide template). The novel multi-strand library set of the present invention includes a vector library, a vector set library, a host cell library, and a host cell pair library as described and taught herein.

A related aspect of the invention is directed to a method of transferring nucleic acid sequence information encoding a biologically active multi-strand polypeptide between a phage display vector and a eukaryotic display vector. One transfer method involves inserting a polynucleotide sequence encoding a multi-stranded polypeptide chain obtained from a phage display vector into a eukaryotic multi-stranded display vector described and taught herein. The transfer of nucleic acid sequence information encoding a multi-stranded polypeptide chain can occur as a single transfer event or as a separate and independent transfer event of the nucleic acid sequence information encoding each of the multi-stranded polypeptide chains. May be good. Similarly, the sequence information encoding each of the multi-stranded polypeptide chains may be transferred from one display vector or from many different display vectors.

Another method of transferring nucleic acid sequence information encoding a biologically active multi-strand polypeptide between a phage display vector and a eukaryotic display vector (and vice versa) is described herein. It involves inserting into a phage display vector a polynucleotide sequence encoding a multi-stranded polypeptide chain obtained from a eukaryotic multi-stranded display vector described and taught. The phage display-eukaryotic display transition method of the present invention is bidirectional. That is, it can occur from a phage display vector to a eukaryotic display vector, or from a eukaryotic display vector to a phage display vector.

The transfer of nucleic acid sequence information between the phage display vector and the eukaryotic vector of the present invention can be achieved by various gene transfer methods known in the art (for example, genetic engineering techniques such as recombinant DNA techniques). it can. Preferred migration methods include restriction digestion, PCR amplification, or homologous recombination techniques (see, eg, Liu, Q. et al., 2000; Walhout, A. et al., 2000).

The present invention is also directed to a method of detecting and isolating a multi-chain polypeptide that exhibits the biological activity of interest to the practitioner. The methods of the invention include desirable interactions between a multi-stranded polypeptide and another molecular species, preferably protein-protein interactions, more preferably with the multi-stranded polypeptide and its ligand / substrate (ie, the target molecule). Allows detection of interactions. Preferably, the nature of this interaction involves non-covalent association (ie, binding) between molecular species, but the nature of this binding is high, even if it is transient (eg, enzyme-substrate binding). It may be of affinity / avidity (eg, for affinity ligands useful for isolation methods, diagnostic agents, and / or therapeutic agents).

In one embodiment, the methods of the invention are by detecting a library of multi-chain polypeptides (displayed on the surface of a eukaryotic host cell) and members of the library that exhibit the biological activity of interest to the practitioner. Useful for screening. In a particularly preferred embodiment, a host cell displaying a multi-stranded polypeptide exhibiting the biological activity of interest is isolated. The isolated host cells may then be screened for repeated rounds or engineered to characterize or utilize the polypeptide sequences of the displayed multi-chain polypeptides. In addition, the screening methods of the present invention can be combined with (preliminary) phage display screening and the transfer of selected phage display isolates to the eukaryotic display system for eukaryotic display screening described herein. ..

In a further aspect of the invention, a library of multi-stranded polypeptides displayed on the surface of a diploid eukaryotic host cell, where the diploid cells are described and taught herein. (Containing the set) can be screened to detect (and optionally, to isolate) a ploid polypeptide that exhibits the biological activity of interest to the practitioner. Preferably, the diploid eukaryotic host cell is the product of a fusion of a pair of haploid eukaryotic host cells described and taught herein. In one particularly preferred embodiment, screened diploid cells displaying a multi-stranded polypeptide exhibiting the biological activity of interest may, after isolation, undergo meiosis, thereby the daughter ( Haploid) cells express distinct strands of selected multi-stranded polypeptides. The daughter cells may then optionally be fused with other haploid cells expressing a multi-stranded polypeptide chain (eg, other daughter cells from the same subpopulation of isolated diploid cells) to multiplex. It produces a recombinant population of diploid eukaryotic host cells that display chain polypeptides on their surface. Additional rounds of screening and repeated recombination of individual strands of the selected multi-chain polypeptide may be performed, and finally, the polypeptide sequence of the displayed multi-chain polypeptide is characterized as discussed above. Can be decided or utilized. Recombination of selected diploid daughter cells can also be recombined (by cell fusion) with other obligate or non-obligate eukaryotic display vectors to produce a novel multi-stranded display host cell library. Can be done.

Eukaryotic display vectors can be used to create eukaryotic display libraries, such as yeast display libraries, that include multiple such eukaryotic display vectors. Preferably, the plurality of eukaryotic display vectors encode a heterologous population of multi-chain polypeptides and the displayed repertoire of multi-chain polypeptides, eg, at least 10.⁴, Preferably at least 10⁵, More preferably at least 10⁶, More preferably at least 10⁷, More preferably at least 10⁸, Most preferably at least 10⁹Produces different polypeptides.

In certain aspects of the invention, the anchor is a polypeptide that can act as an anchor on the surface of eukaryotic cells and as an anchor on the surface of phage. In another embodiment, the anchor is part of a surface protein that adheres to the cell surface of the eukaryotic host cell and the surface of the phage.

In a preferred embodiment of the invention, the anchor and one strand of the multi-stranded polypeptide are expressed as a fusion protein. In another embodiment, the anchor and one strand of the multi-stranded polypeptide are linked at expression, preferably by indirect linkage such as Jun / Fos linkage.

In another aspect, the invention is directed to a method of displaying a biologically active multi-chain polypeptide containing at least two polypeptide chains on the surface of a eukaryotic host cell. The method comprises a first eukaryotic vector comprising a first polynucleotide encoding a first polypeptide chain of a biologically active multi-stranded polypeptide linked to a cell surface anchor (where the vector is It can act to direct the expression and secretion of the first strand in a eukaryotic host cell); and a second comprising a second polynucleotide encoding the second polypeptide strand of the multi-stranded polypeptide. The step of introducing a eukaryotic vector, where the vector is capable of directing the expression and secretion of the second strand in the eukaryotic host cell, into the eukaryotic host cell (where the first eukaryotic vector is described). The eukaryotic host cell transformed with the nuclear vector and the second eukaryotic vector of the multi-chain polypeptide on the surface of the eukaryotic host cell upon expression of the first and second polynucleotides. (Exhibits biological activity); and comprises culturing the host cell under conditions suitable for the expression of the first and second polynucleotides.

In a further aspect, the invention is directed to a method of displaying a biologically active multi-chain polypeptide containing at least two polypeptide chains on the surface of a eukaryotic host cell. The method is a step of introducing a eukaryotic display vector, a set of eukaryotic display vectors, or a dual display vector as described above into a eukaryotic host cell, and conditions suitable for the host cell to express the polynucleotide. Includes the step of culturing under.

Furthermore, the present invention provides eukaryotic host cells comprising a eukaryotic display vector, a set of eukaryotic display vectors, or a dual display vector as described herein. Suitable eukaryotic host cells can be animal cells, plant cells, or fungal cells. Preferably, the eukaryotic host cell will be a mammalian cell, an insect cell, and a yeast cell. Most preferably, the eukaryotic host cell is a yeast cell, for example, a yeast cell selected from the genus Saccharomyces, Pichia, Hanzenula, Saccharomyces, Cryberomyces, Yarrowia, Devaliomyces, or Candida. Will. Preferred yeast hosts include Saccharomyces cerevisiae, Hansenula polymorpha, Cryberomyces lactis, Pichia pastoris, Saccharomyces cerevisiae and Yarrowvia lipolytica. The most preferred yeast host cell is Saccharomyces cerevisiae.

Such and other objects, features, and advantages of the present invention are illustrated in the accompanying drawings, where similar reference symbols mean the same parts throughout different drawings, in the following preferred embodiments of the present invention. It will be clear from the more specific description of. This drawing is not necessarily a correct measure, but rather the emphasis is on exemplifying the principles of the invention.<figref num="1">FIG. 1 is a schematic diagram illustrating a phage display-eukaryotic display transition system. The genetic information encoding the Fab polypeptide chain is transferred from the phage display vector to the multi-stranded eukaryotic vector of the invention as a single cleaved nucleic acid. It then replaces the unwanted intervening genetic elements (if any).</figref><figref num="2">FIG. 2 is a schematic diagram illustrating a phage display / eukaryotic display transition system, in which the genetic information encoding the Fab polypeptide chain is independently transferred from the phage display vector to the multi-stranded eukaryotic vector of the present invention. And it will be migrated separately.</figref><figref num="3">FIG. 3 is a schematic representation of the multi-chain yeast display vector, pTQ3, described in the present invention, which is unique for the insertion of at least two strands of a multi-stranded polypeptide (eg, the light and heavy chain components of Fab). It has a unique cloning site with additional elements arranged so that the two strands are independently expressed by the induction of the tandem GAL1 promoter. In this vector, the first strand (eg, Ig light chain) is inserted as an ApaLI / AscI fragment, expressed as a soluble secretory protein using the Aga2p signal sequence (Aga2p / ss), and fused to the HA epitope tag. The second strand (eg, the Ig heavy chain fragment) is inserted as an SfiI / NofI fragment and expressed as a cell surface binding fusion protein using Aga2p / ss and a sessile protein subunit (mature Aga2p). Similarly, the second strand fuses with the myc epitope tag. Other elements useful for plasmid replication (eg pMB1-ori and Cent6 / ARSH4) and other elements useful as selectable markers (ie ampR and TRP) are also shown.</figref><figref num="4A">It is a representative example of data exemplifying the independent expression of a fusion protein. Figure 4A shows 45 kD Aga2p-V in yeast host cells EBY100pTQ3-F2 and EBY100pTQ3-PH1._H-C_H1 Shows the expression of the fusion protein. No fusion product was detected in a vector control in which either one was empty. For each host cell, samples were prepared both before (-) and after (+) induction of galactose by the GAL1 promoter, which can be actuated in the yeast display vector.</figref><figref num="4B">It is a representative example of data exemplifying the independent expression of a fusion protein. Figure 4B shows 30 kD V in yeast host cells EBY100pTQ3-F2 and EBY100pTQ3-PH1._L-C_LShows chain expression. No fusion product was detected in a vector control in which either one was empty. For each host cell, samples were prepared both before (-) and after (+) induction of galactose by the GAL1 promoter, which can be actuated in the yeast display vector.</figref><figref num="4C">It is a representative example of data exemplifying the independent expression of a fusion protein. FIG. 4C is a representative example of immunofluorescence detection of Fab antibody assembled on the surface of yeast cells. (a) Phase difference (b) HC detection (c) LC detection.</figref><figref num="5">Figures 5A-5C represent a series of cytometric plots. FIG. 5A shows transformed with pTQ3-F2 (left panel) and pTQ3-PH1 (right panel) constructs, either untreated (dotted line) or induced at 20 ° C for 48 hours (light gray line). ) Yeast cells are illustrated. Flow cytometry was used to analyze heavy chain (a), light chain display (b), and antigen binding (c).</figref><figref num="6">FIG. 6 is a histogram plot illustrating whole cell ELISA of three different anti-streptavidin Fabs displayed on the surface of yeast host cells EBY100pTQ3-F2, EBY100pTQ3-A12, and EBY100pTQ3-4C8. The antigen binding, LC display, and HC display are shown, respectively.</figref><figref num="7">FIG. 7 is a cytometric plot of the yeast cell mixture. EBY100pTQ3-F2, EBY100pTQ3-A12, and EBY100pTQ3-A12 / pESC were double labeled for both antigen binding and LC display. The LC display plot for antigen binding and the normalized antigen binding gate are shown.</figref><figref num="8A">It is a representative example of data showing binding to yeast repertoire and individually selected yeast clones at different antigen concentrations. FIG. 8A shows a histogram series of antigen binding and Fab displays for the non-selected library (a) and the polyclonal products of selection rounds 1, 2, and 3 (b, c, d). A diversified anti-streptavidin yeast repertoire was subjected to 3 rounds of FACS. Shows the sorting gate used to select each library.</figref><figref num="8B">It is a representative example of data showing binding to yeast repertoire and individually selected yeast clones at different antigen concentrations. FIG. 8B shows polyclonal FACS analysis at various antigen concentrations of the FACS affinity selection campaign of the anti-streptavidin repertoire. A series of bimodal cytometry plots labeled for both antigen binding and Fab display shows an increase in the ratio of antigen binding to Fab display, indicating an increase in yeast cell population.</figref><figref num="8C">It is a representative example of data showing binding to yeast repertoire and individually selected yeast clones at different antigen concentrations. Figure 8C shows wild-type F2 (represented by "") and mutants R2E10 (represented by triangles), R3B1 (represented by squares), and R3H3 (represented by diamonds) labeled with anti-HA mAb and streptavidin-PE. ) Indicates data obtained from yeast cells. The average fluorescence of streptavidin binding was monitored over time. The dissociation rate constant is calculated from the slope of a straight line.</figref><figref num="8D">It is a representative example of data showing binding to yeast repertoire and individually selected yeast clones at different antigen concentrations. Figure 8D shows a cytometric plot series of two selection campaigns using either Kingfisher (left column) in combination with FACS or FACS alone (right column). This cytometric plot shows that the proportion of antigen-binding cells increases through selection of non-selection (a), round 1 (b), and round 2 (c).</figref><figref num="9">FIG. 9 is a schematic diagram of pTQ5-HC, a heavy chain yeast display vector according to the present invention, having heavy chain fragment insertion under the control of an inducible GAL1 promoter. The Ig heavy chain fragment is located as an SfiI / NotI insertion fragment and is expressed as a cell surface binding fusion protein using the Aga2p signal sequence (Aga2p / ss) and the sticking protein subunit (Aga2p protein). The heavy chain fragment (HC) fuses into the myc epitope tag. Other elements required for plasmid replication (ie, pMB1-ori and Cent6 / ARSH4), other elements required for yeast conjugation (ie, Matα terminator), and other elements useful as selectable markers (ie, ampR and TRP). ) Is also shown.</figref><figref num="10">FIG. 10 shows the monoploid parent yeast cell EBY100pTQ5-HC (lane 2) compared to the (control) empty vector yeast host cell EBY100pTQ5 (lane 1) and the (standard) Fab display vector yeast host cell EBY100pTQF2 (lane 3). It is a representative example of Western blot exemplifying the expression of 45 kD Aga2p-HC fusion product detected by anti-c-Myc antibody in.</figref><figref num="11">FIG. 11 is a series of cytometric plots showing HC displays on the surface of yeast cells in the absence of light chains at time equal to 0 (ie, background; solid black line) and 48 hours after induction (dotted line). Is. Anti-C yeast cell EBY100pTQ5-HC and control yeast cell EBY100pTQ5_HThe presence of HC was detected by labeling with 1 and rabbit anti-mouse IgG FITC, and the antigen-binding activity was detected on the yeast surface by labeling with streptavidin FITC (strep-FITC). Only HC is visible on the yeast cell surface, but in the absence of a pair of LCs, it has no antigen-binding activity.</figref><figref num="12">FIG. 12 is a schematic representation of the light chain yeast display vector pTQ6-LC according to the present invention, which has a light chain insertion under the control of an inducible GAL1 promoter. Ig light chain is positioned as an ApaLI / AscI insert fragment, it is expressed as a soluble protein using Aga2p / ss. The light chain fragment (LC) also fuses with the HA epitope tag. Other elements useful for plasmid replication (ie, pUC1-ori and Cent6 / ARSH4) and other elements useful as selectable markers (ie, ampR and Blasticidin®) are also shown.</figref><figref num="13">FIG. 13 is detected in culture supernatant with anti-HA antibody in haploid parent yeast cell W303pTQ6-LC (lane S2) compared to (control) empty vector yeast host cell W303pYC6 (lane S1) 60. This is a representative example of Western blot exemplifying the expression of kD light chain polypeptide.</figref><figref num="14">FIG. 14 shows parent haploid yeast cells compared to induced diploid yeast cells (DIPLOID LC / HC) and control free vector haploid yeast host cells W303pYC6 and standard Fab display vector yeast host cells EBY100pTQ3-F2. It is a histogram plot exemplifying the whole cell ELISA quantification of streptavidin binding activity on the cell surface of W303pTQ6-LC and EBY100pTQ5-HC).</figref><figref num="15">Figures 15A-15C show anti-streptavidin haploid HC parent (A), diploid control (B) containing free LC and HC expression plasmid, and positive streptavidin-specific Fab on its surface. A series of FACS histograms showing antigen binding and light chain display in diploid (C).</figref><figref num="16">FIG. 16 shows EBY100pTQ5-HC compared to W303pYC6, a diploid yeast cell (lane 2) produced by mating EBY100pTQ5 with W303pYC6 and a parent LC vector yeast host cell W303pTQ6-LC (lane 1). -A representative example of Western blot exemplifying the expression of a 30 kD LC polypeptide detected by an anti-HA antibody in diploid yeast cells (lane 3) produced by mating with LC.</figref><figref num="17">Figure 17 shows (control) diploid yeast cells (lane 4) produced by mating EBY100pTQ5 with W303pYC6, parent HC vector yeast host cells EBY100pTQ5-HC (lane 3), standard Fab display vector yeast host cells EBY100pTQ3F2 (lane 4). Anti-c-Myc antibody in diploid yeast cells (lane 5) produced by mating EBY100pTQ5-HC with W303pYC6-LC compared to lane 2) and (control) empty vector yeast host cells EBY100pTQ5 (lane 1). It is an example of a Western blot exemplifying the expression of the 45 kD Aga2p-HC fusion product detected in.</figref><figref num="18">FIGS. 18A-18C are typical examples of immunofluorescence detection of Fab antibody combinatorially assembled on the surface of yeast diploid cells. (A) LC display (B) HC display (C) Antigen binding. The upper row shows immunofluorescence and the lower row shows phase difference.</figref>

<u style="single">Detailed description of the invention</u> A description of preferred embodiments of the present invention follows. The invention disclosed in this application is the successful expression, transport, assembly, and immobilization (or "or") of a functional heterologous multichain polypeptide (eg, Fab antibody fragment) on the surface of a eukaryotic host cell (eg, yeast). Describe that the display ) was illustrated for the first time. The present invention allows the construction of vector libraries and eukaryotic host cell libraries, where the cells display a highly variable repertoire of multi-stranded polypeptides, which are highly advanced within that repertoire. It inevitably exhibits a highly variable range of biological activity, such as sequence diversity and target (eg, antibody) specificity. Those skilled in the art will appreciate that by following the teachings of the present invention, numerous multichain molecules can be stably expressed on the surface of eukaryotic host cells such as yeast.

<u style="single">Definition</u> Unless otherwise defined herein, the terms and terms used in the description of the present invention are used in accordance with the plain meaning of the terms and terms as are generally understood and accepted by those skilled in the art. To avoid potential confusion or ambiguity, special elements or features relating to the present invention are described below.

As used herein, a "multi-chain polypeptide" is one or more distinct polypeptide elements (ie, "chains"" that are covalently or non-covalently linked together by molecular associations other than peptide bonds. ") Means a functional polypeptide consisting of. The multi-chain polypeptide chain may be the same or different. A prominent example of a multi-chain polypeptide typically consists of four chains, two heavy chains and two light chains, the chains of which are linked by several disulfide (covalent) bonds. Immunoglobulins (eg, IgA, IgD, IgE, IgG and IgM) that are assembled into. Active immunoglobulin Fab fragments, including combinations of light chain (LC) and heavy chain (HC) domains, give rise to particularly important types of multichain polypeptides. In addition to forming disulfide bonds, Fab LCs and HCs are also known to associate effectively (non-covalently) in the absence of disulfide bridges. Other examples of multi-chain polypeptides include, but are not limited to, the extracellular domain of T cell receptor (TCR) molecules (including α and β chains, or γ and δ chains), MHC class I molecules. Includes α1, α2, and α3 domains that are non-covalently associated with β2 microglobulin, and MHC class II molecules (including α and β chains). Of particular consideration herein is the expression of TCR and MHC binding domains in eukaryotic host cells, where at least one strand is attached to a non-naturally occurring (heterologous) anchor on the surface of the host cell.

The term "biologically active" means, for example, when associated with a multi-chain polypeptide, that the polypeptide exhibits functionality or properties that are useful with respect to any biological process, pathway, or reaction. .. Biological activity may mean, for example, the ability to interact with or associate (eg, bind) with another polypeptide or molecule, or it may refer to the interaction of another protein or molecule (eg, binding). May mean the ability to catalyze or regulate (enzymatic reactions). Biological activity includes quadruplex conformation of naturally occurring immunoglobulin γ (IgG) molecules, α and β chains of T cell receptor molecules, or antigen presentation of major histocompatibility complex (eg, MHC peptide groove). It may also mean the ability to bring about the physical conformations characteristic of naturally occurring structures, such as structural conformations.

As used herein, "vector" means an element that can serve as a carrier for gene transfer, gene expression, or replication or integration of a heterologous polynucleotide in a host cell. The vector may be an artificial chromosome or plasmid, integrated into the host cell genome or present as an independent genetic element (eg, episome, plasmid). The vector may exist as a single polynucleotide or as two or more separate polynucleotides. The "multi-stranded display vector" of the present invention can direct the expression of at least one strand of a multi-stranded polypeptide in a suitable host and process it for display on the surface of said host. Is. The vector described in the present invention may be a single copy vector or a multicopy vector (typically indicating the number of copies of the vector maintained in the host cell). Preferred vectors of the present invention include yeast expression vectors, especially 2μ vectors and centromere vectors. A "shuttle vector" (or bifunctional vector) is known in the art as a vector that can replicate in one or more species of organisms. For example, a shuttle vector that can replicate in both Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) was constructed by ligating a sequence from the E. coli plasmid with a sequence from the yeast 2μ plasmid. can do. A particularly preferred embodiment of the invention is a "dual display vector", which is capable of replicating in two different species as well as expressing and displaying heterologous polynucleotides in two or more host species. It is a shuttle vector that is possible.

As used herein, "secretory" is associated with a peptide that has a secretory signal and is processed in the endoplasmic reticulum. A peptide is said to be "displayed" if the secreted peptide contains an anchor sequence or associates with the outside of the cell surface. As used herein, "display" and "surface display" (used interchangeably herein) are heterologous polypeptides that adhere to or "stick" to the outer surface of a phage or host cell. It means a phenomenon in which the adhered polypeptide is exposed to the extracellular environment. The present invention presents the present invention on the surface of a eukaryotic host cell of a multi-stranded polypeptide by expression of each strand of the multi-stranded polypeptide in the host cell and attachment of at least one strand of the multi-stranded polypeptide to the surface of the host cell. Especially aimed at the display of. "Display vector" means a vector capable of expressing a polypeptide in a host cell or phage, and the expressed polypeptide is displayed on the surface of the host cell or phage. The display vector of the present invention directs the expression of a multi-stranded polypeptide in a host cell or phage so that the biological activity of the displayed polypeptide appears on the surface of the host cell or phage. The dual display vector of the present invention is a multi-stranded polypeptide in at least two different hosts (preferably, for example, a prokaryotic host cell and a eukaryotic host cell) so that the biological activity of the polypeptide appears on the surface of each host. Command the expression of.

The term "repertoire" means a group of diverse molecules, eg, nucleic acid molecules with different nucleotide sequences, or polypeptides with different amino acid sequences. According to the present invention, a repertoire of polypeptides is preferably designed to carry a diverse population of molecules with different binding sites for the target molecule. The polypeptides of this repertoire are designed to have common structural elements, for example, in the Fab repertoire, a well-recognized double-stranded structure (Ig light chain and V of Ig heavy chain associated with it)._HDomain and C_HIt has one domain), but exhibits various binding specificities due to mutations in each variable region of this component chain.

The term "library" means a heterogeneous polypeptide or mixture of polynucleotides. The library consists of members with similar polypeptide or polynucleotide sequences. If the library is a polynucleotide library, it encodes a repertoire of polypeptides, especially, for example, the repertoire of multi-chain polypeptides in the present invention. Sequence differences between library members contribute to the diversity that exists in the library. The library may take the form of a simple mixture of polypeptides or polynucleotides, or organisms or cells transformed with the library of polynucleotides, such as bacteria, viruses, animals or plant cells, etc. It may be in the form. A library is a "display library" when a heterologous polypeptide is expressed and manifested on the surface of the cells or organisms that form the library. Advantageously, the polynucleotide is incorporated into an expression vector, allowing expression of the polynucleotide encoded by this polynucleotide. Thus, in a preferred aspect, the library may take the form of a population of host organisms, each organism having a single member of a library of polynucleotides that can be expressed to produce the corresponding polypeptide member. Contains one or more copies of the expression vector it contains. Thus, the host organism population has the potential to encode a large repertoire of genetically diverse polypeptide variants.

The present invention is directed to novel multi-chain display vectors. In one embodiment of the invention, the polynucleotide encoding a multi-stranded polypeptide chain is present in a separate (ie, two or more) expression vector, and its compilation provides a functional display "vector set". Occurs (the general term "vector" includes a set of vectors). For example, if the multi-chain polypeptide is a double-stranded polypeptide consisting of a biologically active Fab light chain and a heavy chain, the LC-encoding polynucleotide is incorporated into a single expression vector and the HC-encoding polynucleotide is incorporated. The nucleotide can be incorporated into a second, separate expression vector (most preferably expressed as an HC-anchor fusion protein). Each vector can individually express its respective polypeptide chain; the two vectors form a matching vector set, which sets the biologically active multi-stranded polypeptide chain. Code. Similarly, separate host cells transformed with different vectors of the vector set collectively form a compatible host cell set (or, in the case of a 2-vector set, a compatible "cell pair"). The vector and vector set preferably also include one or more selectable markers (eg, TRP, ampR, etc.) to promote the selection and proliferation of successfully transformed hosts.

"Host cell" means any cell (prokaryote or eukaryote) transformed to contain a vector. According to the invention, preferred host cells are bacterial cells and eukaryotic cells, including, but not limited to, prokaryotic cells, fungal cells, plant cells, and animal cells. The host cell of the present invention may be any gene construct, but is preferably haploid, diploid, or polyploid (eg, an immortalized cell line in culture is typical). Preferred host cells include insect cells (eg Sf9), mammalian cells (eg CHO cells, COS cells, SP2 / 0 and NS / 0 myeloma cells, human embryonic kidney (HEK) cells, infant hamster kidney (BHK)). Cells, human B cells, human cell lineage PER.C6TM (Crucell)), seed plant cells, and ascospore cells (eg, Neurospora and yeast cells; in particular, Saccharomyces, Pikia, Hanzenula, Sizosaccalomyces , Cryberomyces, Yarrowia, and Candida yeasts). Preferred exemplary yeast species include S.I. Includes Saccharomyces cerevisiae, Hansenula polymorpha, Cryberomyces lactis, Pichia pastoris, Sizosaccaromis ponbe, and Yarrowvia lipolytica. A particularly preferred yeast host cell is S. cerevisiae.

The term "phage" means "bacteriophage", which is a bacterial virus containing a nucleic acid core and a proteinaceous protective shell. The terms "bacteriophage" and "phage" are used interchangeably herein. Unless otherwise stated, the terms "bacteriophage" and "phage" also include "phagemid" (ie, a bacteriophage whose genome contains a plasmid that can be packaged by co-infection of the host with helper phage). Is done. In a preferred embodiment of the invention, the phage is an M13 phage.

The terms "anchor", "cell surface anchor", and "anchor polypeptide", upon expression in a host cell, are on the outer surface of the host cell or, in the case of a phage display system, on the surface of phage particles (eg, one of the capsids). Means a polypeptide moiety that adheres or otherwise associates (as part or as part of a filament). The anchor polypeptide may be a coat protein moiety, a transmembrane polypeptide moiety, or a polypeptide moiety that is otherwise linked to the cell surface (eg, by post-translational modification, such as by phosphatidylinositol or disulfide crosslinks). May be. The term includes proteins that are native to the host cell or phage, or foreign proteins that have been introduced for the purpose of anchoring to the host cell wall or phage coat. Anchors include naturally occurring anchors that retain the ability to adhere to the surface of host cells or phage particles, even when artificially modified or truncated. Preferred anchor protein moieties are contained, for example, in eukaryotic cell surface proteins. Effective anchors include portions of cell surface proteins sufficient to provide surface anchors when fused to another polypeptide, such as the multi-stranded polypeptide chains described in the present invention. The use of protein pairs that are separately encoded and expressed but associate on the surface of the cell by covalent (eg, disulfide) or non-covalent bonds is also considered as a suitable anchor, and special mention is made in this regard. Yeast α-aglutinine components, Aga1p and Aga2p, which give rise to glycan-immobilized, disulfide-linked complexes on the surface of yeast cells. Another protein pair that can be used as an anchor is a protein that produces "leucine zipper" interactions, etc., such as the nuclear proteins Jun and Fos (which yield "jun / fos linkage"). For example, the display vector is designed in accordance with the present invention to direct the expression of the first strand of the multi-stranded polypeptide fused to the leucine zipper portion of Jun in the host cell. The second vector can be designed to direct the independent expression of the leucine zipper portion of Fos that fuses with the host surface protein. Upon expression of this vector structural gene, the leucine zipper of Jun and Fos becomes the link between the first-strand polypeptide and the host cell surface protein fused to the Fos portion of the zipper, so that the first-strand polypeptide is jun / fos linked. It associates (ie, adheres) to the surface of the host cell via. Any suitable protein binding pair of this type may be used. A preferred example of a polypeptide anchor is, in a phage display system, a polIII-coated protein of linear phage or a fragment thereof (see, eg, pIII anchor domain or "stump", US Pat. No. 5,658,727). And in the yeast display system, FLO1 (a protein associated with the cotton-like phenotype of S. cerevisiae), α-aglutinine, and a-aglutinine (eg, Aga1p and Aga2p subunits) and their functional fragments are included.

As used herein, the term "fusion protein" means a hybrid polypeptide consisting of an amino acid sequence from more than one source and linked together to yield a non-naturally occurring single polypeptide. Fusion proteins are prepared, for example, by operably linking the coding sequences of the component amino acid sequences in-frame so that they are produced as a single polypeptide upon expression. Alternatively, the fusion protein may be assembled synthetically, for example, by creating a peptide bond between two or more distinct polypeptides.

As used herein, "linkage" means a functional and structural connection between two or more elements. As used herein, linking elements typically relate to operable linkages between two or more polynucleotide or polypeptide elements. For example, as discussed above, a polypeptide can ligate to an anchor protein (via a peptide bond or via a peptide linker), thereby resulting in a fusion protein. Similarly, the polynucleotide encoding this polypeptide and anchor protein may be ligated such that the fusion protein is transcribed and translated as a single RNA message. Polypeptides may also be indirectly linked to anchors via intermediate associations, one example using the high affinity interaction of Jun and Fos leucine zippers (ie, "jun / fos ligation"). The polypeptide is effectively linked to the surface of the phage or host cell (Crameri, R. and Blaser, K., 1996). Any suitable heterodimer or homodimer molecule pair may be used (Chang, H. et al., 1994; Moll, J. et al., 2001; Pu, W. and Struhl, K., 1993).

As will be appreciated by those skilled in the art, polynucleotides encoding one or more strands of a multi-stranded polypeptide expressed and displayed in a phage display or host cell display system are directed to the promoter (promoting transcription). It can be operably linked (to) or operably linked to a signal sequence or leader peptide (to facilitate cell processing or transport to the surface). There are many such gene regulators and their functional links to them, well known in the art, and the invention is not limited by its use. However, preferred promoters include inducible promoters. Particularly preferred promoters (for eukaryotic systems) include those useful in yeast vectors, such as pGAL1, pGAL1-10, pGAl104, pGal10, pPGK, pCYC1, and pADH1. Other preferred promoters include the LacZ promoter (for non-eukaryotic systems). Particularly preferred signal sequences include the Aga2p signal sequence (for eukaryotic systems) and the pIII signal sequence (for non-eukaryotic systems).

Another useful tool known to practitioners in the art is molecular labels or "tags" (eg, epitope tags, reporter genes, radioisotopes, fluorescent or chemiluminescent moieties, etc.), which are these. For example, it facilitates the practitioner's ability to detect the presence of a polypeptide linked to it. Epitope tags (eg, peptide segments known to be recognized by a particular antibody or binding moiety) are those with one or more strands of a multi-stranded polypeptide in the single or multiple vectors described in the invention. It is particularly useful herein in that it allows detection of the expression of one or more strands that are co-expressed as a fusion partner and the tag is co-expressed. As known and used in the art, tags are typically placed under the same gene control as the gene of interest (preferably as a component of the expressed fusion protein). If the gene product of interest is not readily detectable, the tag provides a signal that is easily detectable and often quantifiable, indicating the presence of the gene product of interest. By linking the tag to the polypeptide gene product of interest, the practitioner can monitor processes such as gene expression, polypeptide transfer, extracellular display, and protein-protein interactions (Fields, S. and Sternglanz, R., 1994; Phizicky, E. and Fields, S., 1995).

Thus, the multi-stranded polypeptide chain can optionally be linked to one or more tags individually or together. A variety of tags are known and commercially available in the art (Amersham Pharmacia Biotech, Piscataway, NJ; Applied Biosystems, Foster City, California; Promega, Madison, Wisconsin; Roche Molecular. Biochemicals, Indianapolis, Indiana; Stratagene, La Jolla, CA). Preferably, this ligation is achieved via peptide bonds (thus creating a fusion protein), where the polynucleotide encoding the multi-stranded polypeptide chain ligates to a tag (eg, an epitope tag). Preferred tags include polyHis tags, HA tags, and myc tags.

As used herein, the term "recombination" is a non-naturally modified or engineered nucleic acid, a host cell transfected with a foreign nucleic acid, or by manipulation of isolated DNA and transformation of the host cell. Used to describe non-naturally expressed polypeptides. "Recombinant" is a term that specifically embraces DNA molecules constructed in vitro using genetic engineering techniques and is an acronym for describing molecules, constructs, vectors, cells, polypeptides, or polynucleotides. The use of the term "recombination" as in particular excludes naturally occurring molecules.

Similarly, the term "transformation" generally refers to an artificial (ie, practitioner-controlled) method of introducing a genetic material into a cell or phage, not limited to the method of insertion. Numerous methods are known in the art and are described in the references cited and incorporated herein. In particular, the term "transformant" as applied herein refers to a transformed host cell and is adapted, for example (as in the case of controlled conjugation of opposite zygotic haploid yeast spores). Includes diploid cells, the product of controlled fusion of haploid cell pairs.

The method of "transferring" nucleic acid sequence information from one vector to another is not limited in the present invention and includes any of the various genetic engineering or recombinant DNA techniques known in the art. Again, a vast number of methods are known in the art and are described in the references cited and incorporated herein. Particularly preferred migration techniques include, but are not limited to, restriction digestion and ligation techniques (using unique cloning sites), PCR amplification protocols (using specific primer sequences), and homologous recombination techniques (homologous). Utilizes a sexual polynucleotide region).

Utilization of genetic engineering techniques inevitably brings recombinant host cells (transformants) under a variety of special conditions, as determined by the requirements of the organism and the special cellular conditions desired by the practitioner. Needs to grow. For example, if an organism possesses specific nutritional requirements (as determined by its genetic predisposition) or special resistance or susceptibility to physical (eg, temperature) and / or chemical (eg, antibiotic) conditions. There is. In addition, specific culture conditions are required to induce or suppress the expression of the desired gene (eg, use of an inducible promoter) or to initiate a special cellular state (eg, yeast cell junction or sporulation). May become. These variable conditions and the requirements for satisfying these conditions are understood and evaluated by practitioners in the art.

Therefore, the practice of various aspects of the invention requires culturing host cells under "suitable" or "sufficient conditions" to achieve or induce a particular cellular condition. .. Such desirable cellular states include, but are not limited to, cell proliferation and reproduction; expression of haploid polypeptide such that the biological activity of the haploid polypeptide appears on the surface of the host cell (or phage particle). Secretion or transport, and association; fusion of haploid cells that give rise to diploid cells (eg fertilization, conjugation, conjugation of opposite mating cells); and diploid cells that give rise to haploid daughter cells Ploidy division (eg, haploid formation, spore formation) is included. The present invention is not limited by the physical and chemical variables of these "favorable conditions", but these conditions are determined by the organism and vector used to carry out the invention and by the preference of the practitioner.

<u style="single">Multichain polypeptide eukaryotic display vector</u> As outlined above, the present invention is a novel genetic vector useful for displaying a multi-stranded polypeptide on the surface of a cell so that the biological activity of the multi-stranded polypeptide appears on the surface of the cell in eukaryotic cells. Directed to. According to the present invention, a multi-stranded polypeptide can be encoded in a single vector, or individual strands of a multi-stranded polypeptide can be encoded in a vector set. For example, in one aspect of the invention, the vector may exist as a vector set, where each strand of the multi-stranded polypeptide is encoded by one of the matching pairs of vectors, so that the vector set is single. When present in eukaryotic cells, the multi-stranded polypeptide chain associates on the surface of the eukaryotic cell. In another aspect of the invention, the display vector may be a dual display vector, wherein the vector (i) expresses a biologically active multi-stranded polypeptide in a eukaryotic cell. It is possible to display on the surface of the bacteriophage, and (ii) to express the biologically active multi-strand polypeptide in eukaryotic cells and display on the surface of the bacteriophage.

The multi-chain polypeptide can be any polypeptide consisting of two or more separate polypeptide elements, called a multi-chain polypeptide chain, which covalently or non-covalently (other than peptide bonds). Link to yield a biologically active polypeptide. Preferably, the multi-stranded polypeptide encoded by the multi-stranded display vector of the present invention exists as either a two-, three-, or four-stranded polypeptide. The polypeptide chains may be the same (eg, homodimer, trimer or tetramer) or different (eg, heterodimer, trimer or tetramer). Preferably, the multi-stranded polypeptide is a double- or quadruplex polypeptide consisting of two different strands. More preferably, the multi-chain polypeptide is a group of multi-chain polypeptides consisting of a T cell receptor, MHC class I molecule, MHC class II molecule, immunoglobulin, and a biologically active immunoglobulin fragment (eg, Fab). Will be selected. More preferably, the multi-chain polypeptide is IgA, IgD, IgE, IgG, IgM, or a biologically active fragment thereof. Most preferably, the multi-chain polypeptide is a Fab fragment of Ig, where the first polynucleotide of the multi-chain display vector is the V of the Ig heavy chain._HDomain and C_HThe second polynucleotide contains the segment encoding one domain, the Ig light chain (ie, V)._LAnd C_LContains the segment that encodes the domain).

A multi-stranded polypeptide chain (eg, first chain, second chain, third chain, etc.) is a polynucleotide in the expression vector (eg, first polynucleotide, second polynucleotide, third polynucleotide, etc., respectively). ). As evaluated and understood by those skilled in the art, the polynucleotide sequence encoding this strand does not necessarily have to be inserted into the same plasmid to produce a functional multi-stranded polypeptide. , It does not have to be under the same gene expression control. For example, the polynucleotides encoding the light and heavy chains of Ig Fab are located on separate plasmids and directly host the same host cell for co-expression and co-processing into a functional multi-chain polypeptide. It may be transformed.

Also, as evaluated by those skilled in the art, the sequences of the polynucleotides encoding the multi-stranded polypeptide chains need not be derived from the same or the same source. For example, the same variable domain (V) as a monoclonal antibody molecule with the desired specificity._HAnd V_L) And constant domains (C) from different monoclonal antibodies with the desired properties_H1 and C_L) Can be produced (eg, to provide human compatibility or to provide a special complement fixation site).

In addition, heterologous polynucleotides encoding the strands of multi-stranded polypeptides (eg, Ig domains) may produce a family of polynucleotide homologues encoding polypeptide chains having slightly different amino acid sequences but the same overall structure. Can be changed to. In this way, when this homologue is integrated and expressed in different host cells, a library of multi-chain polypeptides of various sequences is displayed, eg, homologous multi-chain polypeptides with modified biological activity. Provided is a peptide display library suitable for screening for discovering. Such modifications in the amino acid sequence can be achieved by suitable mutations in the appropriate regions of the corresponding polynucleotide coding sequences, or partial synthesis and substitution, or partial or total substitution thereof. Alternative constant domain moieties can be obtained from compatible recombinant DNA sequences.

With proper selection of expression vector components and compatible host cells, multi-stranded polypeptide chains will appear on the surface of eukaryotic host cells. Those skilled in the art will appreciate that this can be achieved using any of several variable expression vector constructs, and that the invention is not limited thereby. The display vector itself is a number of genetic vectors and genetic regulatory sequences known and commercially available in the art (eg, Invitrogen (Carlsbad, CA); Stratagene (La Jolla, CA); American Type. It may be constructed or modified from any of the Cultural Collection (Manassas, VA). In essence, the vector constructs of the invention are multi-stranded polys fully assembled on the surface of eukaryotic cells transformed with the vector so that the biological activity of the multi-stranded polypeptide appears on the surface of the host cell. Express the polypeptide chain to effectively display the peptide.

To achieve effective cellular expression of a multi-stranded polypeptide, the polynucleotide encoding each strand of the multi-stranded polypeptide is preferably linked to a transcriptional promoter that regulates the expression of the polypeptide chain. A valid promoter must be functional in the eukaryotic system and, in some cases (especially in the case of dual display vectors), also effective as a prokaryotic promoter. In a special dual display vector, the eukaryotic and prokaryotic promoters selected to regulate the expression of heterologous polypeptide chains of a multi-stranded polypeptide are as long as they are properly functional in the intended host organism. May be the same or different promoters. Alternatively, they may be independently selected for expression of each strand in a particular host. The eukaryotic promoter may be a constitutive promoter, but is preferably an inducible promoter. To achieve balanced expression and ensure simultaneous induction of expression, vector constructs utilizing the same promoter for each strand are preferred.

Several eukaryotic promoters useful in the present invention are known in the art. Particularly preferred promoters (for eukaryotic systems) include the galactose-inducing promoter, pGAL1, pGAL1-10, pGal4, and pGal10; phosphoglycerate kinase promoter, pPGK; cytochrome c promoter, pCYC1; and alcohol dehydrogenase I promoter, pADH1. In addition, those useful in yeast expression vectors are included.

Preferably, each polynucleotide encoding a multi-stranded polypeptide chain is also linked to a signal sequence (or leader peptide sequence). The signal sequence acts to direct the transport (sometimes referred to as secretion) of the nascent polynucleotide into or through the cell membrane. The multi-stranded polypeptide chains expressed from the vectors of the invention in eukaryotic cells are transported to the endoplasmic reticulum (ER) for assembly and to the cell surface for extracellular display. A valid signal sequence should be functional in the eukaryotic system, and in some cases (especially in the case of a dual display vector), the signal sequence should also be valid in the prokaryotic system. The polynucleotide encoding the multi-stranded polypeptide chain is typically linked directly to the signal sequence in frame (immediately adjacent to the polynucleotide or, optionally, via a linker or spacer sequence). , Thereby producing a polypeptide chain-signal sequence peptide fusion protein. Preferably, each strand of the multi-stranded polypeptide fuses into a separate signal peptide.

The signal sequence encoding the signal peptide may be the same or different for each strand of the multi-stranded polypeptide. The signal sequence may be native or heterologous to the host as long as it is operable and results in extracellular transport of the polypeptide to which it fuses. Several operational signal sequences in the present invention are known to those of skill in the art (eg, Mfα1 prepro, Mfα1 pre, acid phosphatase Pho5, invertase SUC2 signal sequences that are operational in yeast; E. PIII, PelB, OmpA, PhoA signal sequences that can be actuated in coli; gp64 readers that can be actuated in insect cells; IgK readers, bee meltin secretion signal sequences that can be actuated in mammalian cells). The signal sequence is preferably derived from the host cell's native secretory protein. Particularly preferred eukaryotic signal sequences include yeast α-conjugating factor, yeast α-aglutinine, saccharomyces invertase, clyberomyces inulinase, and most preferably the signal peptide of the Aga2p subunit of a-aglutinine (particularly, In an embodiment where the sticking polypeptide used is an Aga2p polypeptide).

In a particularly preferred embodiment where the multi-chain polypeptide is Fab, the first polynucleotide is the V of the Ig heavy chain._HArea and C_HThe segment encoding one region and the in-frame contain the Aga2p signal sequence, and the second polynucleotide contains the segment encoding the Ig light chain and the in-frame Aga2p signal sequence.

The multi-chain eukaryotic display vector of the present invention operates in a eukaryotic host cell such that the multi-chain polypeptide encoded by the vector is displayed on the surface of the host cell. Adhesion (tethering or display) on the surface of the host cell is achieved by linking at least one strand of the multi-stranded polypeptide to a molecular moiety attached to the host cell wall. One or more strands of a multi-chain polypeptide may be linked to an anchor, but a fully assembled multi-chain polypeptide requires only one attachment point to the host cell surface and thus adheres to the cell. The point requires only one multi-stranded polypeptide chain. Display on the surface of the cell can be achieved by linking at least one of the polypeptide chains to its anchor protein or functional fragment (part). Effective anchors should be functional in the eukaryotic system, and in some cases (especially in the case of dual display vectors), anchors should also be effective as anchors on the surface of bacteriophage. Preferably, the anchor is a surface-expressing protein native to the host cell, eg, a transmembrane protein, or a protein linked to the cell surface via a glycan crosslink. Several operable anchor proteins in the present invention are known to those of skill in the art (eg, pIII, pVI, pVIII, LamB, PhoE, Lpp-OmpA, Flagellin (FliC), or prokaryotic / phage operable. , At least its transmembrane portion; platelet-derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor; gp64 anchor in insect cells, etc.). Preferably, when yeast is the host, the anchor protein is α-aglutinine, a-aglutinin (having subcomponents Aga1p and Aga2p), or FLO1, which inherently form a link to the yeast cell surface. To do.

Linkage of polypeptide chains to anchors can be achieved directly or indirectly by a variety of molecular biology techniques. The present invention is not limited by the method of chain-anchor ligation, and the functional need for the ligated polypeptide chain to be immobilized on the surface of the host cell (or optionally bacteriophage) as a result of such ligation. Limited to.

The preferred method of chain-anchor linkage is by constructing a chain-anchor fusion protein. Similar to and preferably in concert with the chain-signal peptide fusion protein, the polynucleotide encoding the multi-chain polypeptide chain is in-frame to the anchor (immediately adjacent to or optionally adjacent to the polynucleotide). Directly linked (linked via a linker or spacer sequence), thereby producing a signal peptide-polypeptide chain-anchor fusion protein.

Alternative peptide-peptide linkage schemes are known in the art and are available to achieve effective chain-anchor linkages of the present invention. For example, and as already cited, the multi-stranded polypeptide chain is indirectly linked to the anchor through intermediate associations such as the high affinity interaction of Jun and Fos leucine zippers (jun / fos ligation). , Phage or host cell anchors can be covalently linked to a polypeptide chain (Crameri, R. and Suter, M., 1993; Crameri, R. and Blaser, K., 1996).

In a particularly preferred embodiment where the multi-chain polypeptide is an Ig Fab fragment, the first polynucleotide is in the segment and in-frame encoding the Aga2p anchor, as well as the V of the Ig heavy chain._HDomain and C_HOne domain-encoding segment and in-frame contains the Aga2p signal sequence; the second polynucleotide contains the Ig light chain-encoding segment and in-frame the Aga2p signal peptide.

Preferably, the multi-stranded display vector of the present invention provides a cloning site that facilitates the transfer of the polynucleotide sequence encoding the multi-stranded polypeptide chain. Such vector cloning sites include at least one restriction endonuclease recognition site located to facilitate cleavage and insertion of the polynucleotide segment in the reading frame. Any restriction site known in the art may be utilized in the vector constructs of the present invention. Most commercial vectors already contain a multiple cloning site (MCS) or polylinker region. In addition, genetic engineering techniques useful for incorporating new and unique restriction sites into vectors are known and routinely practiced by those of skill in the art. The cloning site may contain only one restriction endonuclease recognition site that allows the insertion or cleavage of a single polynucleotide fragment. More typically, two or more restriction sites are utilized, for example, stronger control of insertion (eg, direction of insertion) or more flexible manipulation (eg, directed migration of one or more polynucleotide fragments). I will provide a. Many restriction sites may be the same or different recognition sites.

The multi-stranded eukaryotic display vector of the present invention preferably contains a restriction site located at the end of the coding sequence of the multi-stranded polypeptide chain. The restriction site may be located at the cutting edge, i.e. at the 5'end and 3'of the polynucleotide segment containing all of the coding sequence (on a single vector) of the multi-stranded polypeptide chain; or more preferably. , Restriction sites may be located at the 5'and 3'ends of the respective polynucleotide segments encoding the multi-stranded polypeptide chain. More preferably, each of the restriction sites is unique in the vector and is different from the other restriction sites. This particularly useful vector construct provides flexibility and control over the modular translocation of individual polynucleotide sequences encoding multi-stranded polypeptide chains.

In a particularly preferred vector construct in which the multi-stranded polypeptide is Fab, the first polynucleotide is the segment and in-frame encoding the Aga2p anchor, as well as the V of the Ig heavy chain._HDomain and C_HThe segment encoding one domain and the in-frame contain the Aga2p signal sequence, where the Ig heavy chain region borders on unique restriction sites (eg SfiI and NotI); and the second polynucleotide contains the Ig light chain. It contains the Aga2p signal peptide in the encoding segment and in-frame, where the Ig light chain region is bounded by unique restriction sites (eg, ApaLI and AscI).

In a preferred embodiment of a multi-stranded eukaryotic display vector, one or more strands of a multi-stranded polypeptide expressed in a host cell by the vector are linked to a molecular tag or reporter gene. Preferably, the link is a peptide bond that links the polypeptide tag to the multi-chain polypeptide chain. One or more strands of a multi-stranded polypeptide may be tagged with the same, similar, or different tags. Preferred tags include epitope tags (Munro, S. and Pelham, H., 1987). Preferred epitope tags include polyHis tags, HA tags, and myc tags, preferably fusing each strand to a different tag.

Based on the particularly preferred vector constructs exemplified herein, where the multi-chain polypeptide is a Fab fragment of an immunoglobulin, the first polynucleotide is the Ig heavy chain in the segment and in-frame encoding the Aga2p anchor. V_HDomain and C_HIt contains the Aga2p signal sequence in the segment and in-frame encoding one domain, and in the segment and in-frame encoding the myc tag, where the Ig heavy chain region is bounded by unique restriction sites (eg SfiI and NotI); The second polynucleotide contains the Aga2p signal peptide in the segment and in-frame encoding the HA tag, as well as in the segment and in-frame encoding the Ig light chain, where the Ig light chain region is a unique restriction site ( For example, ApaLI and AscI) are the boundaries.

<u style="single">Eukaryotic cell display of multi-chain polypeptide</u> For the first time, a method of displaying a biologically active multi-chain polypeptide on the surface of a eukaryotic host cell using the vectors described and taught herein will be illustrated. The method of displaying a multi-stranded polypeptide on the surface of a eukaryotic host cell is to introduce a vector (perhaps as a vector set) into the eukaryotic host cell (ie, the host cell), and the biological activity of the multi-stranded polypeptide. It involves culturing the host cell under conditions suitable for expression, transport, and association of the multi-stranded polypeptide chain on the surface of the host cell so that it appears on the surface of the host cell.

The method for introducing the vector of the present invention into a host cell is not limited to the present invention, and includes all methods known in the art for introducing a genetic substance into a cell. These methods are known, but not limited to, transfection, transformation, electroporation, liposome-mediated translocation, biolistic translocation, conjugation, cell fusion, and nuclear microinjection. Is included. Transformation techniques known in the art are preferred methods of gene transfer.

<u style="single">Multichain Polypeptide Display Host Cell (and Host Cell Pair)</u> The vectors of the invention are operable in eukaryotic host cells, resulting in surface expression of eukaryotic host cells to display multi-stranded polypeptides. In some cases, especially in the case of dual display vectors, the vectors of the invention are also operable in prokaryotic host cells, resulting in expression in bacterial host cells, displaying the multi-chain polypeptide on the surface of the bacteriophage. The eukaryotic host cell may be any eukaryotic cell of any genotype, differentiated or undifferentiated, single cell or multicellular, depending on the special interests and requirements of the practitioner. Particularly useful eukaryotic cells include mammalian cells, plant cells, fungal cells, and prokaryotic cells. Preferably, the host cell is an undifferentiated, unicellular, haploid or diploid cell organism. Preferred host cells are fungi, especially because of the ease and variety of culture conditions, the variety of biochemical and cellular mutants available, their short generation time, and their life cycle (see below). , A species of Ascomycota (Ascomycete). Preferred fungal host cells include those of the genus Neurospora and various yeasts such as Saccharomyces, Pichia, Hanzenula, Sizosaccalomyces, Cryberomyces, Yarrowia, Devariomis, and Candida. The most preferred species is probably the most well-known, characterized and utilized eukaryotic host cell line in molecular biology research, Saccharomyces cerevisiae (baker's yeast).

In a particular embodiment, eukaryotic host cells are suitable for cell fusion (see below). For example, opposite mating yeast cells can "match" to produce fused diploid cells. Further, yeast protoplasts or spheroplasts suitable for cell fusion are also eukaryotic host cells suitable for the object of the present invention. Alternatively, cells that proliferate in culture (eg, mammalian cells, insect cells, etc.) may also be fused by methods known in the art (eg, using Sendai virus or electrical current).

<u style="single">Phage Display-Eukaryotic Display Transition System</u> The technological advances of the present invention that display complex multi-chain polypeptides on the surface of eukaryotic host cells can be combined with the power of phage display technology. For example, by utilizing the phage display-eukaryotic display transition system described herein, practitioners can take advantage of the vast variety provided by the phage display library and phage display technology to the multiple chain eukaryotes described above. It can be combined for the first time with cell expression, processing, assembly, and display provided by display technology. The transfer of nucleic acid sequence information between the phage display vector and the eukaryotic vector of the present invention is achieved by various gene transfer methods known in the art (eg, genetic engineering techniques such as recombinant DNA techniques). be able to. Preferred migration methods include restriction digestion, PCR amplification, or homologous recombination techniques.

In one embodiment, the eukaryotic / prokaryotic multiplex display shuttle vector described and taught herein is utilized. The gene regulatory element of the dual display vector of the present invention is a true nuclear host cell in which the expression, processing, assembly, and display of a biologically active multi-stranded polypeptide are transformed with the dual display vector. In addition to providing to the surface of the prokaryotic host cell, within the prokaryotic host cell, the expression, processing, assembly, and display of the biologically active multi-chain polypeptide is provided to the surface of the infected bacteriophage in the prokaryotic host cell. To do.

In another embodiment, the phage display-eukaryotic display transition system is a conventional phage display vector known in the art (ie, a bacterium engineered to display an exogenous polypeptide on the surface of a phage particle. A strand coding polynucleotide segment cleaved from a phage) is inserted into the multi-chain eukaryotic display vector of the present invention, thereby expressing the strand coding segment and at the surface of a eukaryotic host cell transformed with the eukaryotic display vector. Enables eukaryotic processing, assembly, and display of biologically active multi-strand polypeptides. As described above, the transfer of polynucleotide sequences from phage display vectors to multi-strand eukaryotic display vectors may be accomplished by any genetic engineering technique known in the art. Two particularly preferred methods include a single cut / insert transition method and a multiple (or modular) cut / insert transition method.

In the single cleavage / insertion transition method, the polynucleotide segment encoding the multi-strand polypeptide chain is cleaved from the phage display vector as a single single nucleic acid (eg, by restriction digestion) and subsequently inserted into the multi-stranded display vector. Once inserted into a eukaryotic display vector, it replaces the unwanted prokaryotic gene regulators located between the strand coding polynucleotides with eukaryotic gene regulators (if any). This method is illustrated in FIG. 1 for an Ig Fab multi-chain polypeptide transferred from a phage display vector to a particularly preferred multi-chain yeast display vector of the present invention.

Alternatively, the polynucleotide segment encoding the multi-stranded polypeptide chain is individually cleaved from the phage display vector and subsequently inserted into the multi-stranded display vector in a separate and independent manner. This approach provides greater control and flexibility than migrating individual strands of a multi-stranded polypeptide separately or collectively. In fact, only selected strands of the multi-stranded polypeptide should be migrated, depending on the practitioner's interests and needs. This method is illustrated in Figure 2 for Ig Fab multi-chain polypeptides.

Practitioners in the art can use the phage display-eukaryotic display transition system described and taught herein to go from a phage display vector to a multi-stranded eukaryotic display vector, or from a multi-stranded eukaryotic display vector to a phage display. It will be appreciated that the transfer of sequence information to the vector is equally functional, i.e., the phage display-eukaryotic display transfer system of the present invention is effectively bidirectional. A particularly preferred phage display library for use in the phage display-eukaryotic display transition system described in the present invention is the Mass Human Fab Fragment Library (de Haard, H. et al., 1999).

<u style="single">Multi-chain eukaryotic display library and its screening protocol</u> The multi-stranded eukaryotic display vector of the present invention and the host cell transformed with these vectors are used to produce a display library so that the biologically active multi-stranded polypeptide is displayed on the surface of the host cell. It is useful. These display libraries also screen for a variety of biological activities of interest to the practitioner, eg, for any of a variety of target molecules to identify binding polypeptides that are specific to that target. Therefore, it is useful.

There are several ways to express a large number of diverse molecules on the surface of a host cell or phage. Phage display libraries and their screening represent powerful research and development tools. Methods for producing and screening phage display libraries are well known and used in the art (Hoogenboom, H. et al., 1997; Kay et al., 1996; Ladner, R. et al). ..

The multi-stranded eukaryotic display vector of the present invention can be used to de novo a novel peptide library similar to a known phage display library. However, the vectors described herein allow for more efficient expression of properly folded, assembled, glycosylated, and displayed multi-chain polypeptides that can only be achieved in eukaryotic systems. To. These multi-stranded eukaryotic display libraries can then be used in screening assays. One of ordinary skill in the art will evaluate display library screening protocols known in the art (eg phage display screening assays) and readily apply them to the multi-strand eukaryotic display libraries of the invention.

In addition to producing a novel multi-chain eukaryotic display library, the present invention further presents the present invention to a multi-chain eukaryotic display system in which the practitioner discloses and teaches an existing phage display library herein. Allows migration. In particular, a phage display-eukaryotic display transition system allows the phage display library to be constructed for the display of a very large repertoire of multichain polypeptides (eg Fabs with light and heavy chain components). To. Phage display library is in the library&gt; 1x10⁸(Preferably&gt; 1x10⁹, More preferably&gt; 1x10¹⁰) Can have a diversity of different multi-chain polypeptides, but after initial screening, about 1 × 10⁷Less than (preferably 1x10)⁵~1×10⁶During) may give rise to a subpopulation of phage display isolates. The polynucleotide encoding the strand of the multi-stranded polypeptide isolate may then be "batch-translocated" to the multi-stranded eukaryotic display vector of the invention for transformation into a eukaryotic host. The multi-chain polypeptides displayed in eukaryotic host cells are the culture conditions and expression characteristics of the eukaryotic host system as discussed above (eg, protein folding, proper association of distinct strands in the multi-chain protein, glycosylation). , Secretion, and post-translational modifications such as phosphatidylinositol ligation to cell membranes) can be further screened and manipulated. In addition, once inserted into a multi-strand eukaryotic display vector, the multi-strand polypeptide library (or preselected isolate thereof) is further diversified for additional rounds of screening (eg, set of polypeptide chains). It may be replaced, reshuffled, or remixed).

In a particularly preferred embodiment: Ig light chain cloning site, defined by the ApaLI and AscI restriction sites, 3'aligned with respect to the signal sequence (eg, the pIII signal sequence), and under transcriptional control of the LacZ promoter; and Demarcated by an SfiI restriction site and a NotI restriction site, 3'orientated with respect to a signal sequence (eg, pIII signal sequence), under transcriptional control of the LacZ promoter, and a fixed portion of the sequence encoding mature pIII or pIII (disruption). An M13 phage expression vector with an Ig heavy chain fragment cloning site that is 5'oriented with respect to the end) is provided.

The multi-stranded eukaryotic display vector in this preferred embodiment is: Ig light chain cloning sites defined by ApaLI and AscI restriction sites, 3'oriented to Aga2p secretory signals, and under transcriptional control of the GAL promoter (preferably GAL1 or GAL1-10); Demarcated by SfiI and NotI restriction sites, 3'orientated for Aga2p secretory signals, under transcriptional control of the GAL promoter (preferably GAL1 or GAL1-10), and for sequences encoding mature Aga2p. A yeast vector with a 3'oriented Ig heavy chain fragment cloning site.

This yeast expression vector is used to transform yeast host cells for expression of antibodies or Fab fragments displayed on the yeast cell surface. The light and heavy chain coding sequences are cleaved individually (by ApaLI / AscI digestion and SfiI / NotI digestion, respectively) or together (by ApaLI / NotI digestion) from the phage display vector and multi-chain yeast by batch transfer. Insert into a display vector to obtain multiple LC / HC chain pairs for expression and display in yeast. A yeast display vector particularly preferred for Fab yeast display is pTQ3 (described below). A particularly preferred phage display is the massive human Fab fragment library (de Haard, H. et al., 1999).

One of ordinary skill in the art can select the method described above via various detectable properties (eg, catalytic activity, peptide interactions, thermostability, desired expression levels) or surface expression of the indicated multi-chain polypeptide. It will be appreciated for its usefulness in identifying and isolating multi-chain polypeptides that possess some other improvement.

In addition, it will be appreciated that the present invention can be used in immunopurification, immunoassay, cytochemical labeling, and the production of antibodies or antibody fragments useful for targeting methods, and in diagnostic or therapeutic methods. For example, this antibody or fragment may bind to interferon or a therapeutically effective protein such as a blood coagulation factor such as factor VIII, and therefore affinity chromatography used for immunopurification or assay of the protein. It can be used to produce a vehicle.

<u style="single">Multi-chain polypeptide display as a product of cell fusion</u> The basic life cycle of eukaryotic cells is between the diploid (two copies of the organism's chromosome or genome in the cell) and the haploid (one copy of the organism's chromosome or genome in the cell) state. Accompanied by modification of. Modifications between these two states include fusion of two haploid cells that give rise to a single diploid cell (typically, but not necessarily necessary, opposite mating fertilization) and numerous. Achieved by meiosis of diploid cells that give rise to haploid (daughter) cells. Biologists say that this basic life cycle (ie, modification of the haploid and diploid generations) is an important natural mechanism of biological recombination of genetic information (ie, sexual reproduction). I understand that it will bring.

In most animals, the diploid state is the predominant stage of the life cycle, which is the opposite mating type of two haploid cells (usually called gametes); produced by sperm and egg fusion. .. Haploid cell division (gametogenesis) of diploid cells produces a haploid cell state for sexual reproduction.

The life cycle of the plant kingdom provides a more general generational modification, where haploid and diploid states may exist as more pronounced generations, depending on the particular plant species. In "lower" (ie, more primitive) plants, the generation of haploid cells ("gametophytes") predominates (eg, mosses, mosses, and hornworts), whereas "higher" (ie, hornworts). In (more evolved) plants, the generation of diploid cells (sporophytes) predominates (eg, ferns, conifers, and flowering plants).

In many fungi and protists, the haploid stage of life cycle predominates. Fertilization leads to the diploid stage, which often undergoes meiosis almost immediately (depending on environmental conditions) to give rise to haploid cells. Importantly, with the meiosis of diploid cells that produce haploid cells, regardless of which genus of organisms are being discussed, or which stage predominates in the life cycle of the organism, (new Natural recombination and remixing of genetic material resulting from the cell fusion of individual haploid cells that produce diploid cells (of a diploid cell) is a powerful method available for biological research. .. This powerful mechanism, first described and taught herein, is utilized in combinatorial protein studies to produce unique multi-stranded peptide display libraries.

In a further aspect of the invention, the method of introducing a eukaryotic multi-strand display vector into a host cell involves fusion of two eukaryotic cells, preferably haploids, each of which is a multi-stranded polypeptide organism. It expresses at least one of the multi-stranded polypeptide chains so that it appears on the surface of the host cell in which the activity occurs, preferably diploid. For example, each of the two haploid cells can contain one of the vectors of the vector set (described above), so that once bound (eg, via cell fusion of the host cell), the resulting diploid When co-expressed in a host cell, the biological activity of the multi-stranded polypeptide appears on the surface of the host cell. Using these methods, antibodies or Fab displays live, including diploid cells displaying a novel multi-stranded polypeptide library as described above (eg, a multi-stranded polypeptide having a greater diversity than the repertoire of sources). Rally) can be prepared.

Alternatively, an Ig Fab light chain (V) in the form of a large number (eg, repertoire or library) of one eukaryotic expression vector population._LAnd C_LNumerous forms of Ig Fab heavy chain (V) expressing a domain) and a second eukaryotic expression vector population fused to a yeast anchor protein (eg Aga2p)_HDomain and C_HA population of matching vector sets may be constructed to express (including one domain). Each of these vector populations is used to transform opposite mating haploid yeast cells, one vector construct being one mating type and the second vector construct being the opposite mating type. .. The two haploid yeast populations are co-cultured under conditions sufficient to induce two mating yeast mating (ie, cell fusion). The resulting population of diploid yeast host cells carries both vector constructs and expresses and displays a fully formed and assembled Ig Fab.

However, as discussed above, any eukaryotic cell capable of cell fusion can be used in the present invention. Cell fusion can occur sexually or artificially by conjugation, such as in tissue culture or other artificial conditions. In the case of sexual cell fusion, any eukaryotic cell is suitable as long as it can exist in both haploid and diploid states (no matter how short). In artificial cell fusion, cells are not limited by ploidy, as in sexual fusion. For example, diploid mammalian cells maintained in tissue culture may be induced to fuse, thereby yielding tetraploid host cells. In the present invention, the actual ploidy of the fused host cell is not limiting as long as it can be fused. An important feature of cells is that a cell partner contains a vector or vector set containing a special strand of the multi-stranded polypeptide and a particular selectable marker, and the partner host cell selects the second strand of the multi-stranded polypeptide. It is to contain a vector or vector set containing possible markers. Thus, when cells fuse, the resulting fused cells contain a vector encoding two or more strands of the multi-stranded polypeptide in the cells that are easily identified by selectable markers.

Fungi, especially ascomycetes (ascomycetes; eg, neurospora and yeast), are particularly preferred eukaryotic host cells. Ascomycetes are so named because they produce meiotic haploid spore products in microscopic sac, which are easily harvested, isolated, analyzed, and manipulated. (Neurospora is of particular interest because its ascomycete size and shape maintain the order of meiotic haploid cell products). In addition, the above fungi, especially S. cerevisiae, are stable in both haploid and diploid states, both of which are easily induced and maintained (eg, yeast haploid state). Is typically induced and maintained under some form of nutritional stress, namely starvation). Finally, in many fungi (again, particularly preferred yeasts), haploid cells exist as two sexes (the mating form of α and a), and then only the opposite mating forms fuse (mute) and double. Causes physical condition. Under laboratory conditions operable by one of ordinary skill in the art, alpha cells fuse with a cells, thereby creating fused diploid cells.

As mentioned above, artificial methods of fusing cells are known in the art. Therefore, the present invention is suitable for eukaryotic cells such as mammalian, insect, or plant cells that are propagated in culture, for example. In addition, yeast protoplasts or spheroplasts can be manipulated to cause cell fusion, even if they are of the same mating type. Such artificial cell fusion methods are known in the art and may be suitable for the purposes of the present invention.

Finally, practitioners in the art will recognize that the products and methods illustrated herein are not limited to special polyploid eukaryotic host cells. In fact, it expresses other polyploid organisms (eg, the rarer triploid and tetraploid forms) and higher order multi-stranded polypeptides (eg, triple-stranded and quadruplex polypeptides, respectively). May be specially used as a host for a compatible vector set.

<u style="single">Multi-chain polypeptide screening using eukaryotic cell fusion</u> The multi-strand polypeptide library displayed on the eukaryotic host cell can be screened and manipulated in a manner similar to procedures and techniques known in the art, such as phage display library screening, but the eukaryotic host. It also allows the practitioner to utilize the culture conditions and expression characteristics of the system. As discussed above, eukaryotic display screening can be started with the first round of phage display screening, after which the display library can be transferred from the phage display vector to the multi-strand eukaryotic display vector. Once inserted into a multi-strand eukaryotic display vector, this multi-strand polypeptide library (or preselected isolate) can be screened for one or more additional rounds in the eukaryotic display system.

As a further aspect of the screening method of the invention, and unique to the method of the invention, the multi-stranded eukaryotic display library utilizes several generations of modifications characteristic of the eukaryotic system as discussed above. Following the assay, it may undergo further (biased or unbiased) diversification. A diploid containing a multi-strand eukaryotic display vector in which different strands of the multi-strand are expressed from different vectors (eg, the diploid host cell is the product of haploid junction or cell fusion, as described above). Populations of eukaryotic host cells can be induced to undergo meiosis (eg, spore formation in yeast). The haploid yeast cells (spores) can be isolated and / or selected depending on the screening conditions, and different eukaryotic expression vectors with the desired preferred properties can be combined into separate haploid daughter cells. Can be isolated. Then the daughter cells, in some cases: Mutagenesis (deformation) in vitro (eg, isolated DNA manipulation) or in vivo (eg, UV light) to provide multiple homologues of preselected strands (co-expression of these homologous strands). When displayed, homologous multi-chain polypeptides with greater affinity for the same target molecule can be selected); or Fuse back with other daughter host cells, thereby recombining individual preselected strands of the multistranded polypeptide isolate between them; or Fuse with the first multi-strand library host cell population (thus recombining the preselected strand of the multi-strand polypeptide isolate with the original source of the multi-strand mutation); or Fuse with a new multi-strand library host cell population, thereby combining preselected strands of the multi-strand polypeptide isolate with a new source of multi-strand variability; or Any of the above steps can be combined as appropriate.

Once this recombination, reshuffling, or remixing of the preselected strands of the multi-chain polypeptide to be screened between them or with another source of multi-chain diversity is complete, this new mix. The library population can be subjected to additional rounds of new or repeated screening.

The present invention incorporates well-known techniques in the field of molecular biology by reference as they are. These techniques include, but are not limited to, the techniques described in the following publications: Supervised by Ausubel, F. et al., "Short Protocols In Molecular Biology" (4th edition, 1999), John Willy and Sons, New York, New York (ISBN 0-471-) 32938-X).

Supervised by Fink and Guthrie, Guide to Yeast Genetics and Molecular Biology (1991), Academic Press, Boston, Massachusetts (ISBN 0-12-182095-5).

Kay et al., Phage Display of Peptides and Proteins: A Laboratory Manual (1996), Academic Press, San Diego, CA.

Kabat, E. et al., "Sequences of Proteins of Immunological Interest" (5th edition, 1991), US Department of Health and Human Services, Bethesda, Maryland.

Supervised by Lu and Weiner, Cloning and Expression Vectors for Gene Function Analysis (2001), Biotechnique Press, Westborough, Massachusetts (ISBN 1-881299-21-X) ..

Old, R. and Primrose, S., "Principles of Gene Manipulation: An Introduction to Genetic Engineering" (3rd edition, 1985) Blackwell Scientific Publications, Boston ,Massachusetts. "Studies in Microbiology"; Volume 2: 409 (ISBN 0-632-01318-4).

Supervised by Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual" (2nd edition, 1989), Cold Spring Harbor Laboratory Press, New York, NY, Volumes 1-3 ( ISBN 0-87969-309-6).

Winnacker, E., "From Genes to Clones: Introduction to Gene Technology" (1987) VCH Publishers, NY, NY (Translated by Horst Ibelgaufts) (ISBN 0-89573-614) -Four).

<u style="single">References</u>Boder, E. and Wittrup, K. 1998. Biotechnol. Prog., 14: 55-62. Chant, H. et al., 1994. Proc. Natl. Acad. Sci. USA, 91: 11408-12. Crameri, R. and Blaser, K., 1996. Int. Arch. Allergy Immunol., 110: 41-45. Crameri, R. and Suter, M., 1993. Gene, 137: 69-75. de Haard, H. et al., 1999. J. Biol. Chem., 274: 18218-18230. Fields, S. and Stemglanz, R., 1994, Trends Genet., 10: 286-292. Gietz, D. et al., 1992. Nucleic Acids Res., 20: 1425. Hoogenboom, H. et al., 1997. Trends Biotechnol., 15: 62-70. Horwitz, A. et al., 1988. Proc. Natl. Acad. Sci. USA, 85: 8678-8682. Kieke, M. et al., 1997. Protein Eng., 10: 1303-1310. Kieke, M. et al., 1999. Proc. Natl. Acad. Sci. USA, 96: 5651-5656. Ladner, R. et al., 1993, US Pat No. 5,223,409. Liu, Q. et al., 2000. Methods Enzymol., 328: 530-549. Moll, J. et al., 2001. Protein Sci., 10: 649-55. Munro, S. and Pelham, H., 1987. Cell, 48: 899. Phizicliy, E. and Fields, S., 1995. Microbiol. Rev., 59: 94-123. Pu, W. and Struhl, K., 1993. Nucleic Acids Res., 21: 4348-55. Walhout, A. et al., 2000. Methods Enzymol., 328: 575-593. Wittrup et al., WO 99/36569.

<p> The present invention will be further illustrated by the following examples. These should by no means be construed as limiting the invention.</p><p><u style="single">Example 1 Construction of multi-stranded eukaryotic display vector: pTQ3</u> The materials and techniques described above and incorporated by reference were used to construct multi-chain eukaryotic display vectors; in particular, yeast display vectors that were effective in host yeast cells transformed with the vector. The vector is useful for expressing, transporting, assembling, and displaying biologically active multi-chain polypeptides (eg, Ig Fab) on the surface of host yeast cells.</p><p> In this example, a commercially available vector, pYD1 (Invitrogen, Carlsbad, Calif.), A 5.0 kb expression vector designed for surface expression, secretion, and display of single-chain proteins in S. cerevisiae cells. Was used as a starting eukaryotic expression vector template. pYD1 includes: the aga2 gene, which encodes one of the subunits of the α-agglutinin receptor; the GAL1 promoter for regulatory expression of the Aga2 / polypeptide fusion; the HA epitope tag for the detection of display proteins; Polyhistidine (6 × His) tag for purification; CEN6 / ARS4 for stable episome replication in yeast; and TrpI gene for S. cerevisiae subunit selection, ampicillin resistance gene (ampR) and in E. coli Includes pMB1 origin for selection and duplication.</p><p> The pYD1 plasmid was modified for expression of Ig light and heavy chain fragments from two tandem galactose-inducible promoters, display of intact Fab antibody fragments. One GAL1 promoter directs the expression of the light chain and the other GAL1 promoter directs the expression of the heavy chain fragment that fuses to the C-terminus of the Aga2p yeast anchor protein.</p><p> A unique restriction site was created as part of this vector construct to effectively transfer the multi-stranded polypeptide chain to the display vector. The restriction endonuclease recognition sequence (ie, restriction site) selected for this vector construct includes ApaLI, AscI, SfiI, as unique cloning sites for the strand of the double-stranded polypeptide (Ig Fab in this case). And NotI, and NheI to facilitate phage display-eukaryotic display migration with existing phage display libraries.</p><p> Several vector sequence modifications were made to ensure the effective use of ApaLI as a unique restriction site. ApaLI sites located on the pYD1 plasmid (supplied by Invitrogen) starting at positions 1393, 3047, and 4293 are listed below by localized mutagenesis (using QUICKCHANGE (Stratagene, La Jolla, CA)). Removed as shown:</p><p><chemistry num="1"><img file="JP5111558B2_D0001.tif" /></chemistry></p><p>The ApaLI site starting at position 3047 was present in ampR and required a silent mutation to keep the amino acid coding sequence of this gene unchanged. The multi-chain yeast display vector construct is known in the art for compatibility with other pre-existing phage display libraries known in the art (Dyax, Cambridge, Mass.). PCR-positioned mutagenesis techniques were used to introduce unique restriction sites into the aga2p signal sequence of the pYD1 vector without modifying the coding sequence. In particular, by replacing codon TCA with codon AGC, a NheI site was created beyond the terminal serine codon of the aga2p signal sequence.</p><p> A vector thus modified with a unique ApaLI site near 3'near the pre-existing GAL1 promoter-aga2p signal sequence-HA tag segment, followed by the AscI site, and the NheI site incorporated into the aga2p signal sequence. Named pTQ2.</p><p> A polylinker compatible with existing phage display libraries for cleaving / inserting structural genes of the light chain component of Fab into a multi-chain yeast display vector using assembly PCR techniques known in the art. Was built. The resulting intermediate multi-stranded eukaryotic display vector segments that cover the apa2p signal sequence through the designed polylinker site are as follows (* indicates a stop codon):</p><p><chemistry num="2"><img file="JP5111558B2_D0002.tif" /></chemistry></p><p> The MATα transcription terminator sequence was amplified by PCR from the pYD1 plasmid and attached with BamHI and Pst1 restriction sites to facilitate cloning into the above plasmid pTQ2. The MATα terminator was then digested with BamHI and Pst1 and inserted into the BamHI / Pst1 site of plasmid pTQ2.</p><p> A DNA construct containing the GAL1 promoter, aga2p signal sequence, Aga2p protein coding sequence, and glycine / serine linker (in 5'-3') was amplified from plasmid pYD1. A DNA linker segment containing SfiI and NotI restriction sites and a segment encoding the myc tag were added at the 3'end of the amplified pYD1 segment. The myc tag was included to allow detection of fixed strands (of multi-stranded polypeptides) on the yeast cell surface. The sequence of this linker-myc segment is as follows:</p><p><chemistry num="3"><img file="JP5111558B2_D0003.tif" /></chemistry></p><p> This linker-myc segment was inserted into EcoRI and PacI digested pTQ2. The resulting plasmid had a unique cloning site for insertion / cleavage of multi-stranded polypeptide chains, especially the light and heavy strand fragments of Fab, and was named pTQ3 (Fig. 3). The plasmid pTQ3 is a 5810 bp multi-chain yeast display plasmid containing the following vector sequences in the relevant part:</p><p><chemistry num="4"><img file="JP5111558B2_D0004.tif" /></chemistry></p><p><chemistry num="5"><img file="JP5111558B2_D0005.tif" /></chemistry></p><p> A 6 × His tag for purification of soluble Fab antibody was inserted into the above vector, and a stop codon (TAA) was rearranged at the end of the myc tag, before the PstI site to create a post-modification. Amino acids were eliminated. Other modifications included localization mutagenesis removal of endogenous XbaI restriction sites within Trp selectable markers. This was done to facilitate cloning and manipulation of read antibodies from the Cj library set (Dyax Corporation, Cambridge, Mass.).</p><p><u style="single">Example 2 Phage Display-Eukaryotic Translocation and Eukaryotic Host Cell Expression of Streptavidin, Mutin-1, and Cytotoxic T Lymphocyte-Related Antigen 4 Specific Multichain Fab Polypeptides</u> The usefulness of the phage display-eukaryotic display transition system and the ability to express the multi-stranded polypeptide of the multi-stranded eukaryotic vector of the present invention by migrating different phage display Fabs from the phage display vector to the multi-stranded eukaryotic display vector. Illustrated. The vector was then inserted into eukaryotic host cells and the transformed host cells were grown under conditions suitable for Fab expression.</p><p> Anti-streptavidin Fab antibodies, F2, A12, and 4C8, from a large native human Fab library (de Haard, H. et al., 1999), paired light chains (V)._LC_L) And heavy chains (V)_HC_HAs 1), they were cloned into the multi-chain yeast display vector pTQ3 constructed in Example 1. In addition, the anti-mucin Fab antibody, PH1, from the same Fab library, paired light chains (V)._LC_L) And heavy chains (V)_HC_HAs 1), it was cloned into the multi-chain yeast display vector pTQ3 constructed in Example 1. In addition, four antibodies specific for cytotoxic T lymphocyte-related antigen 4 (CTLA-4), E7, E8, A9, A11, were added from the same Fab library to a pair of light chains (V)._LC_L) And heavy chains (V)_HC_HAs 1), it was cloned into the multi-chain yeast display vector pTQ3 constructed in Example 1.</p><p> Fab strands were cloned into a multi-chain yeast display vector using the single cleavage / insertion transfer method described above and illustrated in FIG. LC-HC polynucleotides from this Fab library were inserted as a single ApaLI / NotI fragment. Intervening in the coding region of this LC and HC fragment, the unwanted prokaryotic gene regulatory element defined by the AscI / SfiI restriction fragment from the Fab library was replaced with the AscI / SfiI fragment derived from pTQ3.</p><p> The resulting plasmids were named pTQ3-F2, pTQ3-A12, pTQ3-4C8, pTQ3-PH1, pTQ3-E7, pTQ3-E8, pTQ3-A9, and pTQ3-A11 and were named Gietz, D. et al., 1992. According to the method, S. cerevisiae strain, EBY100 (Invitrogen, Carlsbad, CA) was transformed separately. EBY100 was also, as a control, a transformant, pTQ3, that did not contain a multi-strand insert. Transformants were selected by selecting the vector tryptophan auxotrophic markers (tryptophan (-), 2% (w / v) glucose, 2% agar (SDCAA + GA) synthetic medium).</p><p> Successful transformants (correspondingly, "EBY100pTQ3-F2", "EBY100pTQ3-A12", "EBY100pTQ3-4C8", "EBY100pTQ3-PH1", "EBY100pTQ3-E7", "EBY100pTQ3-E8", "EBY100pTQ3-A9" , "EBY100pTQ3-A11", and the control "EBY100pTQ3") were grown overnight at 30 ° C with shaking in 10 mL SDCAA + G. OD₆₀₀When reaches 1.0, a sample of two cells (eg, OD)₆₀₀2 mL culture with 1.0) at the point where the induction time is equal to 0 (T)₀) Was immediately removed as a protein lysate preparation. The next day, the culture was centrifuged and the pelleted yeast cells were resuspended in 10 mL SDCAA, 2% (w / v) galactose for OD.₆₀₀Was set to 1. Cells were grown at 20 ° C and induced light and heavy chain vector expression for 48 hours. The cultured cells were then centrifuged, washed twice with 1 mL sterile water and transferred to an Eppendorf tube for centrifugation.</p><p> The cell pellet was resuspended in 250 mL SDS-PAGE buffer + dithiothreitol (DTT). 425-600 micron glass beads (Sigma, St. Louis, Missouri) were added beneath the meniscus and the suspension was vigorously stirred 4 times for 1 minute. The suspension was kept on ice during vigorous agitation. The supernatant was transferred to a fresh tube and heated to 100 ° C for 5 minutes.</p><p> Protein samples were separated on SDS-PAGE gels and transferred to nitrocellulose membranes for Western blot. Detection of light chain polypeptides was performed using anti-HA antibody (1 μg / mL) (Dako, Carpinteria, CA). Detection of the heavy chain-Aga2p fusion polypeptide was performed using an anti-c-Myc antibody (1 μg / mL) with a secondary rabbit anti-mouse HRP antibody (Dako, Carpinteria, CA). Immunodetection was performed by enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ). Approximately 30 kD LC products and approximately 45 kD HC-Aga2p fusion products (F2 and PH1; Figures 4A and 4B) of the displayed Fab were detected. No detectable LC or HC-Aga2p fusion product was detected prior to induction with galactose (see Figures 4A and 4B).</p><p><u style="single">Example 3 Functional surface display of a multi-chain polypeptide in a eukaryotic host cell</u> As an illustration of the ability of the multi-chain eukaryotic vectors of the invention to express, assemble, and properly display biologically active multi-chain polypeptides on the surface of eukaryotic host cells, the multi-chain eukaryotic display vector is used. Inserted into eukaryotic host cells, transformed host cells were grown under conditions suitable for surface expression and display of Fab host cells.</p><p> As described in Example 2 above, yeast clones EBY100pTQ3-F2, EBY100pTQ3-PH1, EBY100pTQ3-E7, EBY100pTQ3-E8, EBY100pTQ3-A9, and EBY100pTQ3-A11 were prepared, cultured, and induced antibody expression. .. OD prior to induction with galactose₆₀₀T of three 0.2 mL aliquots of yeast cells that are 1.0₀Removed as a point.</p><p> After inducing expression with galactose (Example 2), OD₆₀₀Three more 0.2 mL aliquots of cells with a value of 1.0 were removed. Yeast samples were centrifuged and cell pellet was resuspended in PBS containing 1 mg / mL BSA.</p><p> The two samples were centrifuged again and the cell pellet was resuspended in either 100 mL anti-c-Myc antibody (2.5 μg per sample) or 100 mL anti-HA antibody (2.0 μg antibody per sample). The sample was then incubated at room temperature for 1 hour, the cells were pelleted and washed once with 0.5 mL PBS / BSA. This sample was then incubated with FITC-conjugated rabbit anti-mouse antibody (1:40 dilution) for 1 hour in the dark.</p><p> Cell samples were labeled with streptavidin-FITC (1:20 dilution) in PBS / 1% (w / v) BSA and incubated overnight at room temperature in the dark. All samples were centrifuged and the cell pellet was washed once with 0.5 mL PBS and then resuspended in 500 mL PBS.</p><p> The presence of Fab-antigen binding bound to the cell surface was detected by flow cytometry. Pre-induction cells showed no display of light chain, heavy chain, or functional streptavidin-binding Fab antibody. After induction of Fab expression, yeast cells were LC, HC, and further functional by immunofluorescence (FIGS. 4C), FACS (FIGS. 5A-C), and yeast whole cell ELISA (see Figure 6, Example 7). It was possible to detect that a typical streptavidin-binding Fab antibody was also displayed. A functional display of anti-CTLA-4 Fab antibody was also demonstrated (data not shown).</p><p> In the case of EBY100pTQ3-F2 and EBY100pTQ3-PH1, antigen binding as detected by FACS could be completed with unlabeled soluble antigen. Competitive binding demonstrated the complete specificity of the combinatorially assembled Fab antibody displayed on the yeast cell surface (Fig. 5C).</p><p><u style="single">Example 4 Selective Concentration of Fab Display Yeast Cells: Detection by Magnetic Bead Selection</u> Model selection experiments were performed using an automated magnetic bead selector to illustrate that yeast cells displaying antigen-specific Fab antibodies can be enriched against excess unrelated yeast cells.</p><p> Fab-labeled yeast cells, EBY100pT-F2 (with a tryptophan auxotrophic selectable marker), were mixed with non-specific yeast cells in various proportions. This non-specific yeast cell is EBY100p UR3867 (Unilever Research, V)_LIt consisted of aardingen (Netherlands), encoded a mucin-1 (PH1) -specific scFv antibody, and was a leucine auxotrophic selectable marker. Leu before and after selection⁺/ Trp⁺The cell ratio was used to calculate the enrichment rate after one round of selection.</p><p> As described in Example 2, yeast clones were grown and antibody expression was induced with galactose. The two yeast clones were mixed in the proportions shown above and in a final volume of 1 mL of 2% phosphate buffered saline (eg, 2% MARVEL-PBS or "MPBS", Premier Brands, UK). Incubated for 1 hour with 100 μL of streptavidin paramagnetic beads (Dynal M280, Dynal Biotech, Oslo, Norway).</p><p> After incubation of the yeast-bead mixture, the cell-bead complex was washed in 2% MPBS for 11 cycles by transferring the complex from one well to the next in an automated magnetic bead selector. did. After the 2% MPBS wash, two more wash steps were performed with PBS. In the final well of the automatic magnetic bead selector, this cell-bead complex was resuspended in 1 mL PBS and plate-cultured on SDCAA + G agar plates or with 2% leucine dropout (w / w /). v) The titer was determined using a synthetic medium containing glucose + 2% agar (SD-Leu + G agar plate). For selection by magnetically activated cell preparative (MACS), yeast cells were incubated with 500 μL streptavidin microbeads (Miltenyi Biotec, Cologne, Germany) in 6 mL PBS + 2 mM EDTA for 1 hour at room temperature. The cell / bead mixture was loaded onto a washed LC column (Miltenyi Biotec, Cologne, Germany) in the presence of a magnet and the column was PBS + 2 mM. Washed twice with EDTA. After removing the magnet, bound yeast cells were eluted with 6 mL PBS buffer.</p><p> For yeast selection using a capillary washer (CWD), the yeast cell mixture and 100 μL streptavidin-coated paramagnetic beads (Dynal M280) were blocked in 1 mL of 2% PBS for 1 hour. The paramagnetic beads were resuspended in 1 mL yeast cell suspension and gently spun in an Eppendorf tube at room temperature for 1 hour. After incubation of the yeast cells with streptavidin-coated paramagnetic beads, the mixture was introduced into the capillaries of CWD (1 mL was used to load into one capillary in 5 200 μL steps). After automatic washing and resuspension of the yeast-bead mixture, a final wash was performed with PBS and the yeast / bead complex was harvested by adjusting the magnets.</p><p> The use of two selectable markers allows identification of specific yeasts (which can grow on tryptophan (-) selective agar plates) from non-specific yeasts (which can grow on leucine (-) selected agar plates). I made it. The number of colony forming units (CFU) for each titer was recorded.</p><p> The enrichment rate was calculated by dividing the ratio of specific yeast cells before and after selection by the ratio of non-specific yeast before and after selection.</p><p><tables num="1"><img file="JP5111558B2_D0006.tif" /></tables></p><p>As shown in Table 1, specific yeast cells displaying Fab antibodies against streptavidin are selected by one round of selection in a Kigfisher, capillary washer, or automated magnetic bead selector such as Magnetically Activated Cell Sorting (MACS). , Can be concentrated in 2nd to 6th order sizes for unrelated yeast cells.</p><p><u style="single">Example 5 Selective Concentration of Fab-Displayed Yeast Cells: Detection by Flow Cytometry</u> As an alternative to the magnetic bead detection method of Example 4 above, enrichment of antigen-specific Fab antibodies against excess unrelated yeast cells was illustrated using fluorescent labeling cell fractionation (FACS) technology.</p><p> Fab-labeled yeast cells, EBY100pTQ3-F2 (with tryptophan auxotrophic marker), non-specific yeast cells carrying the Leu auxotrophic marker, EBY100 (pUR3867-PH1) and 1: 100, 1: 1000, and 1: Mixed at a ratio of 10,000. The yeast cell mixture was incubated with 1 μM streptavidin-FITC (Dako, Carpinteria, CA) and equilibrated at room temperature for 30 minutes.</p><p> 3000 cells were sorted by free cytometry and 6.5% of the cells with the highest fluorescence signal were collected. Yeast cells before and after selection were plate-cultured on SDCAA + G agar plates and SD-Leu + G agar plates to determine the number of CFUs. The enrichment rate was calculated as the ratio obtained by dividing the output ratio of EBY100pTQ3-F2 and EBY100pUR3867-PH1 by the input ratio.</p><p> After one round of FACS, EBY100pTQ3-F2 was enriched 10-fold relative to EBY100pUR3867-PH1 (data not shown).</p><p><tables num="2"><img file="JP5111558B2_D0007.tif" /></tables></p><p><u style="single">Example 6 Batch transfer of phage display antibody library to multi-stranded eukaryotic display vector</u> Prepared using techniques known in the art as an illustration of the usefulness of a phage display / eukaryotic display transfer system that collectively transfers the phage display peptide library to the multi-stranded eukaryotic display vector of the present invention. The phage display Fab library was transferred to the multi-chain yeast display vector pTQ3 produced as described in Example 1 above.</p><p> To transfer the phage display repertoire to the multi-chain yeast display vector, the single cleavage / insertion transfer method described above and illustrated in FIG. 1 was used (see also Example 1).</p><p> 50 mL of TYAG culture (TY, ampicillin 100 μg / mL, glucose 2%) was inoculated with 10 μL of glycerol stock from a round of streptavidin selection in the native Fab library cloned into phage (de Haard, de Haard, H. et al., 1999). The culture was grown overnight at 37 ° C to prepare plasmid DNA (QIAGEN plasmid purification system, Qiagen, Valencia, CA).</p><p> The Fab antibody repertoire was digested with ApaLI and NotI, and approximately 1.5 kb of Fab antibody fragments were recovered and purified by extraction from 1.0% TBE ethidium bromide agarose gel (QIAEX gel extraction kit, Qiagen, Valencia, CA).</p><p> Similarly, the multi-chain yeast display vector, pTQ3, was digested with ApaLI and NotI, and a fragment of approximately 4.6 kb was purified by extraction from 1.0% TBE ethidium bromide agarose gel.</p><p> Ligation of Fab antibody inserts recovered from the Fab library to ApaLI and NotI digested pTQ3 plasmids in a 4: 1 (insert-vector) ratio of 100 μL using a 1 μg Fab fragment and a 0.7 μg pTQ3 vector. The reaction was carried out overnight at 16 ° C. The ligation mixture was purified by phenol, chloroform, and isoamyl alcohol (PCI) extraction and subsequently precipitated with 100% ethanol.</p><p> E. coli strain, TG1 (Netherlands Bacterial Culture Collection, PC4028, Utrecht, Netherlands) transformed into a purified ligation mixture by electroporation using BioRad Pulser (BioRad, CA) at 2.5 kV, 25 mF and 200 W. Converted. This library is a 2 × TY agar plate containing 100 μg / mL ampicillin and 2% w / v glucose (Bact-tryptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L, Bact- Placed on agar 15 g / L) (TYAG plate). After overnight growth at 37 ° C, this repertoire was recovered in 2 x TY medium + ampicillin (100 μg / mL) by pouring a large amount of water into the plate and aliquoted to 15% (w / v) glycerol. Frozen in.</p><p> This library is 5.6x10⁶Contained an independent clone of. 5.4 × 10¹⁰15 μL of the cell / mL library suspension was inoculated into 100 mL of TYAG and the culture was grown overnight at 37 ° C. The plasmid DNA was recovered as described above.</p><p> The intermediate pTQ3-Fab repertoire was then digested with AscI and SfiI. A fragment of about 6.1 kb was purified as described above. The source vector pTQ3 was similarly digested with AscI and SfiI to purify a fragment of approximately 1150 bp.</p><p> The above purified 1150 bp fragment was ligated with the AscI and Sfi digested pTQ3-Fab repertoire in a 6: 1 (insert-vector) ratio using 1.6 μg inserts and 1 μg vectors. This ligation mixture was purified and transformed with E. coli strain, TG1 as described above, 1 × 10⁶The final pTQ3-Fab library consisting of independent clones of was obtained.</p><p> The library was removed from the plate as described above, 10 mL was inoculated into 50 mL TYAG and grown overnight at 37 ° C. Plasmid DNA was prepared from the pTQ3-Fab library by the method of Gietz, D. et al., (1992), and the yeast strain, EBY100, was transformed and 2 × 10⁶The final library size of yeast from independent yeast clones was obtained.</p><p><u style="single">Example 7 Batch-migrated eukaryotic display Fab library selection: Detection by magnetic bead selection</u> Multiple selection experiments were performed using an automatic magnetic bead selection device to illustrate that the yeast display Fab library can make selections from a population of yeast cells displaying a diverse repertoire of Fab antibodies.</p><p> The yeast repertoire prepared in Example 6 was grown in SDCAA + G at 30 ° C to induce antibody expression in galactose (as in Example 4). A pool of yeast cells was incubated with 100 μL streptavidin paramagnetic beads (Dynal M280, Dynal Biotech, Oslo, Norway) for 1 hour in a final volume of 1 mL 2% MPBS.</p><p> After incubation of the yeast-bead mixture, the cell-bead complex was washed in 2% MPBS for 11 cycles by transferring the complex from one well to the next in an automated magnetic bead selector. did. After the 2% MPBS wash, two more wash steps were performed with PBS. Yeast colony titers were determined before and after selection by resuspending this cell-bead complex in 1 mL PBS and plate culturing on SDCAA + G agar plates in the final well of an automatic magnetic bead selection device. .. The selected yeast cells were then inoculated into a fresh culture of 10 mL SDCAA + G and a second round of selection was performed as described above.</p><p> After the first round of selection and after the second round of selection, the proportion of positive and negative clones was determined by yeast whole cell ELISA. Cells were proliferated and induced in 100 mL SDCAA + 2% (w / v) galactose on 96-well plates (Corning Coster, Cambridge, Mass.).</p><p> After induction, cells were washed with PBS for one cycle and divided equally into two plates for antigen binding and detection of heavy chain display. In the first plate, cells were resuspended in 100 μL PBS containing anti-streptavidin-HRP (0.87 μg / mL) to detect antigen binding. The cells in the second plate were resuspended in 100 μL 2% MPBS containing anti-c-Myc (1 μg / mL) to detect heavy chain displays.</p><p> After 1 hour of incubation, cells were washed with PBS for 2 cycles and the cells were resuspended in 100 μL TMB solution to quantify specific binding. After coloration, the reaction was stopped by adding 50 μL 2 N sulfuric acid. Cells were pelleted by centrifugation and 100 μL of supernatant was transferred to a flexible 96-well plate (Falcon, BD Biosciences, Bedford, MA) and absorbance at 450 nm recorded. For heavy chain detection, 100 μL 2% MPBS containing rabbit anti-mouse HRP (1: 1000) was added to each well. After 1 hour of incubation, cells were washed for 2 cycles as described above to detect heavy chain displays. The results are presented in Table 3.</p><p><tables num="3"><img file="JP5111558B2_D0008.tif" /></tables></p><p>After one round of selection, 20% of yeast clones screened for antigen binding were found to be positive, and after the second round of selection, the number of antigen-reactive yeast clones was 100%.</p><p><u style="single">Example 8 Anti-streptavidin display yeast cell affinity selection: detection by flow cytometry</u> In another affinity discrimination experiment, the clones EBY100pTQ3-F2 and EBY100pTQ3-A12 / pESC contain a free vector, pECS (Stratagene, La Jolla, CA), which carries the Leu auxotrophic marker. The anti-streptavidin antibody F2 has an affinity of 54 nM as quantified by plasmon resonance (BIAcore), and the anti-streptavidin antibody A12 has an affinity of about 500 nM. These two clones were grown overnight and had an OD of 1.0 in SDCAA + 2% (w / v) galactose.₆₀₀Dilute to and grow at 20 ° C for 48 hours. High affinity antibody-containing clones (EBY100pTQ3-F2) and low-affinity antibody-containing clones (EBY100pTQ3-A12 / pESC) were mixed at a ratio of about 1: 100. EBY100pTQ3-A12 / pESC (which can be grown on tryptophan (-), leucine (-) selective agar plates) by using different selectable markers present in each clone is EBY100pTQ3-F2 (tryptophan (-)). It can be identified from (which can only grow on selected agar plates). The cell mixture was labeled as before, except with a serial diluent of 500 nM, 100 nM, 50 nM, 25 nM, and 10 nM streptavidin-FITC. EPIC based on both LC display and antigen binding Cells were harvested by flow cytometry at ALTRA (Beckman Coulter, Fullerton, CA). The preparative rate was set to 2000 cells / sec and the preparative gate was set to harvest 1% of the cell population with the highest FITC / PE ratio (typical FACS histogram is shown in Figure 7). Input and output cells after selection at different antigen concentrations were titrated on a selection plate and the number of colonies was totaled to calculate enrichment and recovery of higher affinity clones (Table 4). The above results show that clones with higher affinity can be preferentially recovered by flow cytometric preparative methods._d= 54 nM and about 500 nM K_dIllustrates that the optimum antigen concentration is between 100 nM and 25 nM in a mixture of the two antibodies.</p><p><tables num="4"><img file="JP5111558B2_D0009.tif" /></tables></p><p><u style="single">Example 9 Construction of a yeast labeling library diversified by error-prone PCR</u> To illustrate its ability to produce a novel multi-strand display vector library, streptavidin-specific Fab antibody F2 was subjected to error prone PCR. Separate LC, HC and total Fab antibodies were cloned into a yeast display vector. The error-prone PCR, 2.25 mM MgCl₂And 0.375 mM MnCl₂Was performed for 30 cycles in the presence of. The purified product was cloned into a pTQ3 yeast display vector as an ApaL1 / AscI fragment, Sfi1 / Not1 fragment, or ApaL1 / Not1 fragment corresponding to LC, HC, and all Fab fragments, as in Example 2. E. coli was transformed with this ligation mixture and grown on a selective agar plate containing 100 μg / mL ampicillin, 5 × 10⁶LC repertoire (pTQ3F2-LC^epNamed), 5.6 × 10⁸HC repertoire (pTQ3F2-HC)^epNamed), and pTQ3F2-Fab^epObtained the entire Fab repertoire of. These repertoires were collected to prepare 200 mL of inoculum (sufficient to contain at least 10-fold library diversity). The plasmid DNA was isolated from a 200 mL culture and transformed into a yeast strain, EBY100, as described in Example 2. The generated repertoire, EBY100-pTQ3F2-LC^ep(Size = 5 × 10⁶); EBY100-pTQ3F2-HC^ep(Size = 1.7 × 10⁶); EBY100-pTQ3F2-Fab^ep(Size = 10⁶). The frequency of mutations at the nucleotide level was 1.5% for LC and 0.8% for HC. The frequency of mutations at the amino acid level was 3% for LC and 1.3% for HC.</p><p><u style="single">Example 10 Affinity Selection of Anti-Streptavidin Display Yeast Cell Library: Detection by Flow Cytometry</u> To illustrate the affinity selection of the multi-chain yeast display library, the library EBY100-pTQ3F2-LC^ep; EBY100-pTQ3F2-HC^ep, And EBY100-pTQ3F2-Fab^epOvernight cultures were prepared as in Example 2 and 1.0 OD in selective medium containing SDCAA + 2% (w / v) galactose.₆₀₀Dilute to and grow at 20 ° C for 48 hours. A second label of this repertoire with anti-HA mAb (25 μg / mL) at room temperature for 1 hour followed by rabbit anti-mouse Ig-FITC (1:40 dilution) and 6 nM streptavidin PE at room temperature for 1 hour. The incubation process was continued. Following each incubation step, cells were washed once with 0.5 mL PBS and after the final wash the cells were kept on ice to prevent antigen dissociation. Samples were fractionated at a preparative rate of 2000 cells / sec with an EPIC ALTRA flow cytometer. The first preparative round was performed in concentrated form and the preparative gate was set to collect the population of cells entering the gate based on both LC display and antigen binding. The proportion of cells harvested decreased with continued selection rounds, explaining the decline in repertoire diversity (Figure 8A). Then, as in Example 2, the collected cells were subjected to SDCAA + (w / v) glucose at 30 ° C with an OD of 1.0.₆₀₀After growing to, it was induced with galactose. Selections were repeated in rounds 2 and 3 in a pureity mode with fewer preparative gates (Table 5). Polymeric FACS analysis was performed at different antigen concentrations and FACS histograms of both LC display and antigen binding activity are shown in Figure 8B.</p><p><tables num="5"><img file="JP5111558B2_D0010.tif" /></tables></p><p><u style="single">Example 11 Analysis of Selected Fab Antibodies</u> Repertoire, EBY100-pTQ3F2-LC^ep; EBY100-pTQ3F2-HC^ep; EBY100-pTQ3F2-Fab^epThe yeast clones edited by affinity selection were subjected to affinity screening to quantify the improvement in affinity for the starting wild-type antibody. Selected antibodies were also sequenced to determine mutations that correlate with improved affinity.</p><p> Yeast colonies were removed and resuspended in 25 μL of lyticase solution (2.5 mg / mL; Sigma, St. Louis, Missouri) at 37 ° C for 1 hour, then 2 μL was taken and used in the PCR reaction. .. Separate LCs and HCs were amplified and sequenced using the ABI-PRISM sequencer. Sequence alignment was used to determine mutations from wild type and are shown in Table 6.</p><p><tables num="6"><img file="JP5111558B2_D0011.tif" /></tables></p><p> The off rate of the selected Fab was determined as a decrease in fluorescent signal over time by measuring the dissociation rate in FACS, and the clone, R2H10, gave the greatest improvement in affinity (10.7 fold, 3.2 nM). This dissociation rate is fitted to the exponential decay model and k_dWas calculated. Yeast cells were labeled with anti-HA to detect LC and also with streptavidin PE for antigen detection. Approximately 2 × 10 by growing and inducing yeast cultures as described in Example 2.⁷Cells were harvested and washed with PBS. The cells were then incubated with 100 μL anti-HA Mab (20 μg / mL) for 1 hour and then washed with 0.5 mL PBS. The cells were then incubated with rabbit anti-mouse FITC (1:40) and streptavidin PE (1:40 dilution of 1 μg / mL stock) on ice for 1 hour. The cell pellet was then resuspended in excess non-fluorescent ligand at room temperature. The concentration of the non-fluorescent label was set to be 10-100 times higher than the molar concentration of Fab antibody displayed on yeast, assuming that there were approximately 100,000 copies of Fab antibody per yeast cell. The decrease in fluorescence intensity was monitored by flow cytometry for 1.5 to 30 minutes. Background fluorescence was set using unlabeled yeast cells. Then k_dBy adapting the dissociation rate to the model of exponential decay to calculate k_dWas calculated. FIG. 10C shows FACS off-rate determination for clones of wild-type F2 and mutants R2E10, R3B1 and R3H3.</p><p> The affinity of soluble Fabs was determined by subcloning the selective Fab antibody into the E. coli expression vector, pCES1, as in Example 2. Soluble Fabs were purified and affinity tested by BIAcore (de Haard, H. et al.). Table 7 shows the affinity of the selected Fab.</p><p><tables num="7"><img file="JP5111558B2_D0012.tif" /></tables></p><p><u style="single">Example 12 Rapid selection of yeast display Fab repertoire using a combination of Kingfisher and FACS selection</u> To speed up affinity selection in the yeast display repertoire, and also 10⁸Kingfisher as a selection in the first round (as in Example 4) and FACS for subsequent selection rounds (as in Example 5) to develop a methodology that allows selection of a larger repertoire beyond ) Both combinations were used. The LC repertoire constructed in Example 9 was grown overnight to induce antibody expression as in Example 2. This yeast cell population was incubated with streptavidin-coated magnetic particles and selected with Kingfisher as in Example 4. In parallel, the same repertoire was selected by FACS as in Example 5. A pool of yeast cells from a Round 1 selection campaign using Kingfisher and FACS was grown overnight to induce antibody expression as in Example 5. Yeast cells were labeled as in Example 2 and selected by FACS as the second round. Analysis of a selective pool of yeast displaying Fabs was performed using polyclonal FACS (see Example 10). The proportion of antigen-binding cells is seen to increase faster in preference to FACS when using Kingfisher as the first round of selection (Fig. 8D).</p><p><u style="single">Example 13 Construction of Ig heavy chain eukaryotic display vector: pTQ5-HC</u> As an illustration of an alternative embodiment of the multi-strand eukaryotic display vector of the invention, in particular a multi-stranded eukaryotic display vector in which the strands of the multi-stranded are encoded into separate vectors, thereby resulting in separate components of the vector set. A yeast display vector effective in host yeast cells transformed with a vector that expresses, transports, and displays Ig heavy chain fragments was constructed as one of the matching vector sets.</p><p> The HC fragment display vector was constructed by further modifying the vector pTQ3 produced according to Example 1. The display vector, TQ3, was digested with BseRI to determine the design restriction site of the vector, located within each of the two tandem GAL1 promoters (see Example 1, SEQ ID NO: 5, designated bases 990-995). thing). The 924 bp fragment (Figure 3) that straddled one of the cloning sites of this multi-strand display vector was removed and the remaining 4,868 bp vector skeleton was gel-purified using techniques known in the art (particularly GFR). PCR and Gel Band Purification Kit, by Amersham-Pharmacia, Piscataway, NJ). This vector skeleton was reconnected and transformed into E. coli. The resulting vector (named "pTQ5") was validated using restriction analysis.</p><p> The anti-streptavidin Fab antibody, HC of F2, was restricted and purified as an SfiI / NotI fragment from pTQ3-F2 to 709 bp, purified and cloned into the SfiI / NotI digested vector, pTQ5. The resulting HC display vector was named "pTQ5-HC" (Fig. 9).</p><p> Subsequent modifications to this vector were performed by inserting a 6 × His tag for purification of the soluble Fab antibody and rearranging the stop codon (TAA) at the end of the myc tag, before the Pac1 site, to remove excess amino acids. It disappeared. Other modifications included removal of the endogenous XbaI restriction site within the Trp selectable marker by localization mutagenesis. This was done to facilitate cloning and manipulation of read antibodies from the CJ Library Set (Dyax Corporation, Cambridge, Mass.).</p><p><u style="single">Example 14 Eukaryotic host cell expression of Ig heavy chain eukaryotic display vector: HC expression in haploid yeast cells</u> To illustrate the usefulness of the independent vector of the multi-strand eukaryotic display vector set, a yeast display vector (of the vector set) encoding an Ig heavy chain fragment was inserted into the eukaryotic host cell and the transformed host cell was subjected to. It was grown under conditions suitable for the expression of heavy chain components of Ig Fab.</p><p> The yeast strain EBY100 (Invitrogen, Carlsbad, CA) was transformed with the vector pTQ5-HC (of Example 13) and separately with pTQ5 as a control according to the transformation procedure described above. Successful transformants were named EBY100pTQ5-HC and EBY100pTQ5, respectively, and grown overnight at 30 ° C in 10 mL SDCAA + G.</p><p> The next day, the culture was centrifuged and the pelleted yeast cells were resuspended in 10 mL SDCAA + 2% (w / v) galactose for OD.₆₀₀Was set to 1. The cell culture was then grown at 20 ° C for 24 hours to induce expression of the Aga2p-heavy chain fusion product. The cells were centrifuged, washed twice with 1 mL sterile water and transferred to an Eppendorf tube.</p><p> Cell pellets were resuspended in 200 mL SDS-PAGE sample buffer + DTT and glass beads (425-600 microns) were added beneath the meniscus. The suspension of cells and beads was vigorously stirred 4 times for 1 minute and the suspension was kept on ice while vigorously stirring. The supernatant was transferred to a fresh tube and heated to 100 ° C for 5 minutes.</p><p> Protein samples were separated on SDS-PAGE gels and transferred to nitrocellulose membranes for Western blot. Detection of the Aga2p-HC fusion polypeptide was performed using an HRP-binding anti-c-Myc monoclonal antibody (1 μg / mL, Roche Molecular Biochemicals, Indianapolis, Illinois). Immunodetection was performed by enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ). Approximately 45 kD of Aga2p-HC fusion polypeptide was detected. No detectable Aga2p-HC fusion product was detected in the (empty) control vector clone, EBY100pTQ5 (Fig. 10).</p><p><u style="single">Example 15 Eukaryotic Host Cell Display of Ig Heavy Chain Eukaryotic Display Vector: HC Display on the Surface of Uniplex Yeast Cells</u> Encoding an Ig heavy chain fragment (of the vector set) to illustrate the ability of a vector from a multi-chain eukaryotic display vector set to display the adherent strand of a multi-chain polypeptide on the surface of a monoploid eukaryotic cell. A yeast display vector was inserted into a eukaryotic host cell and the transformed host cell was grown under conditions suitable for expression and display of the heavy chain component of Ig Fab.</p><p> EBY100pTQ5-HC (from Example 14) was grown and antibody expression was induced as described above. HC expression was induced by 48 hours of growth with shaking at 20 ° C. Yeast samples were centrifuged and cell pellet was resuspended in PBS containing 1 mg / mL BSA. The two samples were centrifuged again and the cell pellet was each 100 μL of anti-human C._HSeparately resuspended in 1 (25 μg / mL; Zymed, San Francisco, CA) followed by 1 hour incubation at room temperature. The cells were pelleted and washed once with 0.5 mL PBS / 1% (w / v) BSA. Cell samples were then incubated with rabbit anti-mouse FITC (1:50 dilution; Dako, Carpinteria, CA) for 1 hour in the dark.</p><p> Label cells with streptavidin-FITC (1:25 dilution; Dako, Carpinteria, CA) in PBS / 1% (w / v) BSA to detect antigen binding and at room temperature in the dark. Incubated overnight. All samples were centrifuged and the cell pellet was washed once with 0.5 mL PBS and then resuspended in 500 mL PBS.</p><p> The presence of Fab-antigen binding bound to the cell surface was detected by flow cytometry. Pre-induction cells showed no display of heavy chains or functional streptavidin binding. After induction of HC expression, it could be detected that yeast cells displayed only heavy chains, but functional streptavidin binding was not detected as expected (Fig. 11).</p><p><u style="single">Example 16 Construction of Ig light chain eukaryotic display vector: pTQ6-LC</u> A light chain yeast display vector was constructed to provide a multi-chain eukaryotic display vector set when used with the heavy chain yeast display vector described above (see Example 13 above).</p><p> An LC yeast expression vector was constructed by amplifying a fragment containing an anti-streptavidin LC fused to the HA epitope tag and the Aga2p signal sequence. The amplification product was gel purified using GFX PCR and a gel band purification kit (Amersham-Pharmacia, Piscataway, NJ) and digested with HindIII and PmeI. This 783 bp LC fragment was purified on a 1.2% TAE-agarose gel with a 4,323 bp vector backbone of the HindIII / PmeI digested pYC6 / CT vector (Invitrogen, Carlsbad, CA). The LC fragment was ligated together with the pYC6 / CT vector and the ligation mixture was transformed into E. coli strain, TG1. The resulting LC expression vector was named "pTQ6-LC" (Fig. 12).</p><p><u style="single">Example 17 Eukaryotic host cell expression of Ig light chain eukaryotic display vector: Soluble LC expression in monopolyyeast cells</u> To illustrate the usefulness of the independent vector of the multi-chain eukaryotic display vector set, a yeast display vector (of the vector set) encoding the Ig light chain fragment was inserted into the eukaryotic host cell and the transformed host cell was subjected to. It was grown under conditions suitable for the expression of the soluble light chain component of Ig Fab.</p><p> Yeast strain obtained from P. Slonimski, W303-1B (a / alpha ura3-1 / ura3-1 leu2-3, 112 / leu2-3, 112 trp1-1 / trp1-1) according to the transformation procedure described above. his3-11, 15 / his3-11, 15 ade2-1 / ade2-1 can1-100 / can1-100) were transformed with pTQ-LC (of Example 16) and separately with pYC6 / CT as a control. Successful transformants were named W303pTQ6-LC and W303pYC6 / CT, respectively, and cultured overnight at 30 ° C in 10 mL SD-G + 300 μg / mL blasticidin® (SD-G + Bls).</p><p> The next day, the culture was centrifuged and the pelleted yeast cells were resuspended in 10 mL SD + Bls + 2% (w / v) galactose for OD.₆₀₀Was set to 0.4. The cell culture was then grown at 20 ° C. for 24 hours to induce expression of the soluble light chain polypeptide. Cells were centrifuged and concentrated 10-fold using a centrifuge filter unit (CENTRICON YM-10; Millipore, Bedford, MA).</p><p> Wash cell pellet and break breaking buffer (50 mM sodium phosphate, pH 7.4, 1 mM EDTA, 5% (w / v) glycerol + protease inhibitor cocktail; Roche Molecular Biochemicals, Indianapolis , Illinois) resuspended in OD₆₀₀Was set to 50, and glass beads (425 to 600 microns) were added directly under the meniscus. The suspension of cells and beads was vigorously stirred 4 times for 1 minute and the suspension was kept on ice while vigorously stirring. The supernatant was transferred to a fresh tube and the aliquot was heated to 100 ° C for 5 minutes in SDS-PAGE sample buffer + DTT.</p><p> Protein samples were separated on SDS-PAGE gels and transferred to nitrocellulose membranes for Western blot. Detection of LC polypeptides was performed using an anti-HA monoclonal antibody (1 μg / mL) in combination with HRP (1/1000) -bound rabbit anti-mouse. Immunodetection was performed by enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ). 30 kD and 60 kD polypeptide products were detected in the culture supernatant. No detectable LC product could be detected on the empty vector control W303pYC6 / CT (Fig. 13).</p><p><u style="single">Example 18 Surface Display of Multichain Polypeptides in Eukaryotic Host Cells: Products of Cell Fusion of Uniplex Host Cell Pairs</u> Through cell fusion of two haploid eukaryotic cells, each carrying a different vector from a compatible multiploid eukaryotic display vector set, the biologically active polyploid eukaryotic polypeptide of the diploid eukaryotic cell To illustrate the operability of a novel method of labeling on the surface, haploid yeast cells containing a vector expressing a soluble Ig light chain fragment are labeled expressing an Ig heavy chain-anchor fusion polypeptide. Mating to haploid yeast cells containing the vector produced diploid yeast cells displaying a functional Fab polypeptide on the surface of host cells.</p><p> Yeast clones, W303pTQ6-LC (from Example 17) and EBY100pTQ5-HC (from Example 14), tryptophan® (Invitrogen, Carlsbad, CA; 300 μg / mL; SD + G + Bls agar It was grown on an agar plate supplemented with either (plate) or tryptophan dropout medium (SD-Trp + G agar plate). These plates were then replicated on a double-selection plate containing synthetic medium for tryptophan dropout + 300 μg / mL blasticidin® (SD-Trp + G + Bls). The diploid yeast cells in the resulting cell layer were line-cultured to form a single colony. 7 Trp + / Bls^RColonies were selected and grown overnight in 100 mL SD + G-Trp + Bls on a 96-well plate with shaking at 30 ° C.</p><p> The next day, centrifuge the culture and pellet the yeast cells into 10 mL SD-Trp + Bls + 2% (w / v) galactose or 10 mL YP medium + Bls + 2% (w / v) galactose. Resuspended at 20 ° C for 24 hours in any of the above. Cells were washed in PBS and divided equally into three 96-well plates. First plate cells resuspended in 100 μL streptavidin-HRP (0.87 μg / mL), second plate cells resuspended in 100 μL anti-c-Myc-HRP (1 μg / mL), third plate cells Was resuspended in 100 μL anti-HA (1 μg / mL) and further labeled with rabbit anti-mouse HRP (1/1000).</p><p> Whole yeast ELISA was performed (as in Example 7) and FACS was performed (as in Example 15) to detect antigen binding and HC display. All diploid cells tested bound to streptavidin and displayed light chains in whole cell ELISA (FIGS. 14) and FACS (FIGS. 15A-C). In particular, streptavidin-binding activity was detected on diploid yeast cells displaying a combinatorially assembled Fab antibody (diploid LC / HC) on its surface, whereas only LC (W303pTQ6-LC) was detected. Alternatively, haploid parent cells expressing either HC alone (EBY100pTQ5-HC) showed no binding activity. Standard haploid yeast cells displaying Fab antibodies (EBY100pTQ3-F2) showed streptavidin-binding activity. Also, haploid parent yeast cells (W303pTQ6-LC) expressing only LC (as expected) did not show HC display, whereas standard haploid yeast cells (EBY100pTQ3) displaying Fab antibody. -F2) showed the HC display.</p><p> Five yeast clones were selected and cultured overnight at 30 ° C with shaking in 10 mL SD + G-Trp + Bls. The next day, centrifuge the cell culture and add pelleted yeast cells to 10 mL SD-Trp + Bls + 2% (w / v) galactose with 0.4 OD.₆₀₀Vector expression was induced by resuspending for 24 hours. Alternative protocols include resuspension in 10 mL YP medium + Blasticidin® + 2% (w / v) galactose.</p><p> After a 24-hour induction incubation, one aliquot from each of the five diploid yeast cultures was pelleted, washed, resuspended in disruption buffer and OD.₆₀₀Was set to 50. Glass beads (425-600 microns) were added beneath the meniscus and the cell-bead suspension was vigorously agitated 4 times for 1 minute and the suspension was kept on ice during the vigorous agitation. The supernatant was transferred to a fresh tube and the aliquot was heated to 100 ° C for 5 minutes in SDS-PAGE sample buffer + DTT.</p><p> Protein samples were separated on SDS-PAGE gels and transferred to nitrocellulose membranes for Western blot. Anti-HA antibody (1 μg / mL) was used in combination with HRP-conjugated rabbit anti-mouse against one membrane to perform light chain polypeptide detection. Detection of the heavy chain-Aga2p fusion polypeptide was performed using an anti-c-Myc antibody (1 μg / mL, Roche Molecular Biochemicals, Indianapolis, Illinois) directly bound to HRP. Immunodetection was performed by enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ). In the diploid yeast lysate, both an LC product of about 30 kD and an HC-Aga2p fusion product of about 45 kD were detected (FIGS. 16 and 17). No detectable LC or HC-Aga2p fusion product was detected in the control diploid clones containing the two free vectors, pTQ5 and pYC6 / CT.</p><p> Also, after 24-hour inducible incubation, second aliquots from each of the five diploid yeast cultures were analyzed by flow cytometry. 5 x 10 per detector⁶The cells were washed with PBS for one cycle, and the cells were put into 100 μL PBS containing anti-c-Myc (25 μg / mL) for heavy chain detection, and anti-streptavidin-FITC (1:40) for antigen binding detection. ), And for light chain detection, resuspended in 100 μL PBS containing anti-HA (25 μg / mL). The cells were incubated in the dark for 1 hour and then washed again with PBS for 1 cycle. After washing, they were resuspended in 100 μL PBS containing rabbit anti-mouse-FITC (1:40) and incubated again in the dark for 1 hour.</p><p> During the second incubation step, cells were treated with anti-streptavidin-FITC for one step labeling. After incubation, cells were washed for an additional cycle, resuspended in 500 μL PBS and analyzed by flow cytometry. All five samples were shown to bind to the antigen and display HC and LC (FIGS. 15A-C).</p><p> After 24 hours of (induction) incubation, tertiary aliquots from 5 diploid yeast cultures were also labeled for immunofluorescence. Streptavidin-FITC (30 μg / mL, Dako) or rabbit anti-human λ chain (1:40; Dako, Carpinteria, CA) and monoclonal anti-C_H10 to 100 μL of any mixture of 1 (25 μg / mL, Zymed, San Francisco, USA)⁸The cells were resuspended. The first sample is further incubated with rabbit anti-FITC (1:40; Dako, Carpinteria, CA) and finally with FITC-bound porcine anti-rabbit (1:20; Dako, Carpinteria, CA). Incubated. The second sample was FITC-conjugated pig anti-rabbit (1:20; Dako, Carpinteria, CA) for the light chain and tetramethylrhodamine isothiocyanate-conjugated rabbit anti-mouse (TRITC, 1: 20) for the heavy chain. 30, Sigma, St. Louis, Missouri) double-labeled (Figs. 18A-C).</p><p> This diploid displayed light and heavy chains on the cell surface and was shown to bind to streptavidin as expected. Ploidy parents expressing only HC were stained only with the heavy chain TRITC label. The LC of the haploid parent was negative in all cases.</p><p><u style="single">Example 19 Mating efficiency of haploid host yeast cell pairs</u> A biologically active multi-chain polypeptide is displayed on its surface to illustrate the efficiency of cell fusion of two haploid yeast cells, each carrying a different vector from a compatible multi-stranded eukaryotic display vector set. As a viable process for producing diploid yeast cells, the mating efficiency was determined for the host yeast cell pair described in the present invention. The quantitative determination of the efficiency of the junction reaction was evaluated as follows.</p><p> Each haploid parent, EBY100pTQ5 (from Example 14) and W303pYC6 / CT (from Example 17), at 30 ° C in their respective appropriate selection media, SD + G + Trp and SD + G + Bls. It was grown late. 3x10 from two fresh haploid cultures⁷Cells were mixed and harvested on a 45 mm nitrocellulose filter (microfiller, Millipore, Bedford, Mass.). The filter was incubated on a non-selective concentrated medium plate (YPD) at 30 ° C. for 4 hours. The cells were then resuspended in YPD medium and titrated with two parental selective medium and double-selective medium (allowing only diploid growth) SD + G-Trp + Bls. Spontaneous reversion or tolerance was assessed by treating each haploid parent separately in the same manner and plate-culturing them undiluted on double-selective medium.</p><p> The conjugation efficiency of the haploid parent EBY100pTQ5 was calculated as follows: (Number of diploids growing on SD + G-Trp + Bls-Number of naturally tolerated EBY100pTQ5 growing on SD + G-Trp + Bls) Divide by (total number of cells from conjugation reaction showing proliferation on SD + G-Trp).</p><p> The conjugation efficiency of the haploid parent W303 (pYC6) was calculated as follows: (Number of diploids proliferating with SD + G + Trp + Bls-Haploid cells proliferating with SD + G-Trp + Bls (Number of W303pYC6 / CT) divided by (total number of cells proliferating with SD + G + Bls).</p><p> 3x10 of each joint type⁷One haploid cell contains both pTQ5 and pYC6 yeast expression vectors 1.5 × 10⁷Produced diploid cells. The results of conjugation efficiency revealed that 51% of haploid parents containing the pTQ plasmid give rise to diploids, and 64% of haploid parents containing the pYC plasmid give rise to diploids. ..</p><p><u style="single">Example 20 Selective enrichment of diploid yeast cells displaying combinatorially assembled Fab antibodies: detection by flow cytometry</u> Fluorescence-labeled cell fractionation (FACS) was used to determine the ability to select yeast cells displaying antigen-specific Fab antibodies against excess unrelated yeast cells. Positive diploid yeast cells displaying a combinatorially assembled Fab antibody specific for streptavidin were used. This diploid yeast cell is Trp⁺/ Leu^-/ Bls^RIt was able to carry the phenotypic marker of tryptophan (-) and grow on a selective agar plate containing Blasticidin®. Bls this diploid^RIt was named diploid. Trp⁺/ Leu⁺Unrelated yeast diploid cells carrying the phenotypic marker of leucine (-) were used and could grow on a tryptophan-containing selective agar plate of leucine (-). Leu this diploid⁺It was named diploid. Positive (Bls^R) And unrelated (Leu)⁺) Both diploid yeast cells were grown overnight in SD + 2% (w / v) glucose medium under selective conditions of -Trp / + Leu / + Bls medium and -Trp / -Leu medium, respectively. .. Yeast cultures were induced in YP medium containing 2% (w / v) of galactose. OD of this yeast culture₆₀₀Determine and 1 OD₆₀₀Is 4x10⁶After using conversions equal to cell / mL, a mixture of positive cells to unrelated yeast cells was prepared with an approximation of 1: 10000. The yeast cell mixture was labeled with 500 nM streptavidin PE for selection by FACS and selected as in Example 8. In Kingfisher, the yeast cell mixture was selected by incubation with streptavidin-coated beads as in Example 4. For selection by MACS, the induced diploids were incubated with 500 μL streptavidin microbeads (Miltenyi Biotec, Cologne, Germany) in 6 mL PBS + 2 mM EDTA for 1 hour at room temperature. The cell / bead mixture was loaded onto a washed LC column (Miltenyi Biotec, Cologne, Germany) in the presence of a magnet and the column was washed twice again with PBS + 2 mM EDTA. After removing the magnet, the cells held in the column were eluted with 6 mL PBS buffer.</p><p> Collect yeast cells and Bls^RPhenotype or Leu⁺Titering was performed on a selective agar plate of either phenotype. Bls before and after selection^R/ Leu⁺The colony ratio was used to calculate the enrichment rate and the recovery rate of positive yeast cells.</p><p><tables num="8"><img file="JP5111558B2_D0013.tif" /></tables></p><p> In the above example of streptavidin-specific antibodies, Kingfisher appeared to give a higher enrichment rate than MACS. However, the recovery rate of positive yeast cells was significantly low. Using FACS, first-order magnitude enrichment was observed from a round of selection for anti-streptavidin Fab antibody.</p><p><u style="single">Example 21 LC and HC recombination and affinity selection by cell fusion of haploid host cell pairs: detection by flow cytometry</u> To illustrate the usefulness of fusion of two haploid eukaryotic cells, each carrying a different vector from a compatible multi-stranded eukaryotic display vector set, a vector expressing multiple soluble Ig light chain fragment variants was used. Opposite haploid haploid haploid containing a vector that expresses and displays multiple Ig heavy chain-anchor fusion polypeptide variants of the containing haploid yeast cell population (ie, a library of LC variants). A novel diploid yeast cell population (ie, a novel Fab library) that ligates to a body yeast cell population (ie, a library of HC variants) and displays multiple functional Fab polypeptides on the surface of host cells. ) Is produced.</p><p> Using Fab phage display isolates preselected from the Fab repertoire for target molecules, the genetic information of the phage display isolates (as illustrated in Example 6) was batched into a multi-chain yeast display vector set. As illustrated in Example 18, a source of heavy and light chain components for migration is provided and a multi-chain yeast display vector set (as described in Examples 14 and 17) is used. ) Provide novel recombination of light and heavy chain isolates via host cell fusion of two monoploid eukaryotic cells, each carrying a different vector from a compatible multi-chain eukaryotic display vector set.</p><p> A phage display antibody library (de Haard, H. et al., 1999) was subjected to one round of selection on streptavidin-coated magnetic particles using a protocol familiar to those of skill in the art. This repertoire was used as the starting repertoire for the transition to the yeast display system. Inputs for this library are 5x10¹²It is a phage particle, and the output after one round of selection is 3.75 × 10.⁵It was a phage particle.</p><p> The HC fragment was isolated as an SfiI / NotI fragment from the Round 1 selective phage display library, cloned into pTQ5 of the Ig heavy chain yeast display vector, and digested with SfiI and NotI (Example 13). This ligation mixture was transformed into E. coli and 1 × 10⁸I got the library. This library was then transformed into yeast strain EBY100 to 4x10.⁷Get the library of EBY100-pTQ5-HC^repI named it.</p><p> The LC fragment was isolated as an ApaL1 / AscI fragment from the Round 1 selective phage display library and cloned into pTQ6 (Example 16) of the Ig light chain yeast display vector digested with ApaL1 and AscI. This ligation mixture was transformed into E. coli and 1 × 10⁸I got the library. This library was then transformed into yeast strain BJ5457 to 8x10.⁷Get the library of BJ5457-pTQ6-LC^repI named it. The HC and LC repertoires in yeast are both 3.75 × 10 in phage.⁵It contained enough diversity to cover its departure repertoire. DNA fingerprint analysis of individual clones showed diverse restriction modes, suggesting that different germline segments appear in separate LC and HC libraries.</p><p> In the first joining method, the LC repertoire (BJ5457-pTQ6-LC)^rep) 7.25 × 10⁸3.4 × 10 of EBY100-pTQ5-F2HC derived from clone F2 containing a single HC specific for streptavidin⁸Joined with individual cells. This conjugation condition was under selective pressure (tryptophan auxotrophy and blastidin resistance) to maintain both LC and HC expression plasmids. 1.9 x 10 with 55% bonding efficiency⁸Obtained a diploid library. Analysis of individual clones from this library by yeast whole cell ELISA showed that 100% of the clones displayed HC and 100% of the clones displayed LC.</p><p> In the second joining method, the HC repertoire (EBY100-pTQ5-HC)^rep) 3.6 × 10⁸3 × 10 cells of BJ5457-pTQ6-F2LC derived from clone F2 containing a single LC specific for streptavidin⁸Joined with individual cells. This conjugation condition was under selective pressure (tryptophan auxotrophy and blastidin resistance) to maintain both LC and HC expression plasmids. 8x10 with 27% bonding efficiency⁷Obtained a diploid library. Analysis of individual clones from this library by yeast whole cell ELISA showed that 89% of the clones displayed HC and all of these clones displayed LC.</p><p> In the third joining method, the HC repertoire (EBY100-pTQ5-HC)^rep) 2.0 × 10¹⁰LC repertoire (BJ5457-pTQ6-LC)^rep) 5.6 × 10⁹Joined with individual cells. This conjugation condition was under selective pressure to maintain both LC and HC expression plasmids (tryptophan auxotrophy and blastidin® resistance). 4x10 with 68% bonding efficiency⁹Obtained a diploid library. Analysis of individual clones from this library by yeast whole cell ELISA showed that 94% of clones displayed HC and 53% of clones displayed LC.</p><p> This series of joining experiments shows that joining of individual repertoires of LC and HC can be used to create large libraries. These repertoires contain diverse V gene germline segments and can be expressed and displayed on the yeast cell surface. Kingfisher was used to select these repertoires with the antigen streptavidin (see Example 7). After two rounds of selection, 97% of the edited clones showed antigen-binding activity in yeast whole-cell ELISA (see Example 7).</p><p><u style="single">Example 22 Construction of LC and HC repertoire diversified by error-prone PCR</u> To illustrate the fusion of two repertoires of haploid yeast cells, each repertoire carrying different vectors from a compatible multi-strand vector set may be used for affinity maturation in pTQ5 (Example 13). The HC repertoire diversified by error-prone PCR (Example 9) and another LC repertoire diversified by error-prone PCR (Example 9) in pTQ6 (Example 16) are combined with the opposite yeast. Constructed in haploid cells.</p><p> The HC repertoire was constructed by amplifying the anti-streptavidin F2 antibody under erroneous conditions (Example 9). This amplified fragment was digested with SfiI and NotI, purified, and cloned into pTQ5 (Example 13), an HC-only expression vector already digested with SfiI and NotI. The resulting ligation mixture was transformed into E. coli and 7 × 10⁷I got the library. Transform the yeast strain EBY100 with this library 9 × 10⁶Get the library of EBY100pTQ5-HC^*I named it.</p><p> The LC repertoire was constructed by amplifying the LC of the anti-streptavidin F2 antibody under erroneous conditions (Example 9). This amplified fragment was digested with ApaL1 and AscI, and cloned into an LC-only expression vector (Example 16) that had already been digested with ApaL1 and AscI. The resulting ligation mixture was transformed into E. coli and 4 × 10⁷Obtained the library of, and continued to transfer it to the yeast strain BJ5457, 1.8 × 10⁷Library (BJ5457pTQ6-LC)^*Named).</p><p> The frequency of mutations at the nucleotide level that occurred was 0.8% in the HC repertoire and 1.5% in the LC repertoire. These frequencies correspond to mutation frequencies of 1.3% and 3% at the amino acid level, respectively. This haploid cell repertoire, EBY100pTQ5-HC^*And BJ5457pTQ6-LC^*Was inoculated into 10 μL and 30 μL glycerol stocks, respectively, to represent at least 10 copies of each independent clone and grown overnight in selective medium (Example 18). BJ5457pTQ6-LC^*Approximately 1.6 x 10 corresponding to⁶Haploid cells and EBY100p TQ5-HC^*3x10 corresponding to¹⁰When haploid cells were mated (Example 19) and grown in selective medium, 5 × 10⁹Obtained a diploid repertoire of (EBY100pTQ5-HC)^*/ BJ5457pTQ6-LC^*Named). Ten clones were removed and tested for the presence of LC and HC-containing vectors by yeast colony PCR (Example 11), all of which yielded the expected LC and HC products. EBY100pTQ5-HC, a diploid repertoire that displays HC products and also shows binding to the antigen streptavidin^*/ BJ5457pTQ6-LC^*Whole yeast ELISA was performed to determine the fraction of the yeast (Example 7). 68% (15/22) of the diploids tested displayed HC and 18% (4/22) of the diploid clones tested showed binding to streptavidin.</p><p> To emphasize the versatility of this procedure, a similar hierarchical junction experiment was performed in which either wild-type HC or wild-type LC was kept steady and only the corresponding opposite strands changed. did. EBY100-pTQ5-F2HC and BJ5457pTQ6-LC using anti-streptavidin F2 Fab as a model antibody^*A diploid repertoire was prepared from the joints of. This diploid repertoire has stationary HC and variable LC. This conjugation resulted in 100% of diploids displaying HC and 30% showing antigen binding by yeast whole cell ELISA. Similarly, BJ5457pTQ6-F2LC to EBY100pTQ5-HC^*A diploid repertoire was prepared by joining with. This diploid repertoire has steady LC and variable HC. This conjugation resulted in 70% of diploids displaying HC and 45% exhibiting antigen binding activity by yeast whole cell ELISA.</p><p><u style="single">Example 23 Affinity Selection of Combinatorially Assembled Fab Repertoire</u> Kingfisher selection and affinity flow cytometry to illustrate the ability to affinity-select a repertoire of yeast cells displaying Fab antibodies assembled in multiple combinatriators diversified by error-prone PCR. Optimal affinity clones were harvested using a combination of methods.</p><p> Diploid repertoire, EBY100pTQ5-HC^*/ BJ5457pTQ6-LC^*An overnight culture of (Example 22) was prepared (Example 18). Induce this culture as in Example 18 for a total of 10¹⁰The cells were subjected to one round of Kingfisher selection (Example 7). Antigen-binding yeast diploid cells were edited and subjected to FACS affinity selection (see Example 20). The proportion of antigen-binding clones increased during selection as determined by yeast whole cell ELISA (Example 7). The proportion of antigen-binding clones also increased, and the average antigen fluorescence intensity also increased during selection, as determined by FACS (Table 9).</p><p><tables num="9"><img file="JP5111558B2_D0014.tif" /></tables></p><p> Progress of this selection campaign was monitored using polyclonal FACS analysis, where overnight cultures of the selection repertoire from each round of selection were prepared to induce antibody expression as in Example 18. Yeast cells were labeled as in Example 20 and analyzed by FACS for both LC display (FITC labeling) and antigen binding (PE labeling).</p><p> The selected clones were sequenced and the mutations in variable LC and variable HC are shown in Table 10. FACS by either off-rate screening assay (Example 10) or nonlinear least squares analysis was used to determine the affinity of the selected Fab (data not shown).</p><p><tables num="10"><img file="JP5111558B2_D0015.tif" /></tables></p><p><u style="single">Example 24 Re-shuffling of selective pools of LC and HC</u> Combinatorial EBY100pTQ5-HC to illustrate the versatility of this procedure and the ability to select and reshuffle iterative cycles.^*/ BJ5457pTQ6-LC^*The pool of selected LCs and HCs from the output of the third selection round of the repertoire (Example 23) was reshuffled.</p><p> Plasmid DNA was prepared using lytic enzyme treatment (Example 11) and pTQ5-HC containing the selected LC and HC.^{* sel}And pTQ6-LC^{* sel}DNA extracts containing both expression plasmids were directly transformed into fresh EBY100 and BJ5457 cells, respectively. This transformant mixture, BJ5457-pTQ6-LC^{* sel}Selection plate (selective agar plate containing blastsidin) or EBY100pTQ5-HC in which only colonies can grow^{* sel}It was grown on a selection plate (tryptophan (-) selection agar plate) in which only can be grown. BJ5457pTQ6-LC^{* sel}250 colonies were obtained by transformation, EBY100pTQ5-HC^{* sel}Twenty-five colonies were obtained by transformation. These two mini repertoires were harvested and grown overnight and joined as in Example 18. This junction reaction covers the theoretical combinatorial variety of 6250 different LC / HC combinations, EBY100pTQ5-HC.^{* sel}/ BJ5457pTQ6-LC^{* sel}Obtained a diploid repertoire of. Fab antibody expression was induced in this diploid culture and selected using AutoMACS. This represented the choice of the fourth round. Diploid cultures from this fourth selection round were edited. Antibody expression was induced, after which streptavidin PE was labeled with 0.5 nM and selected using FACS (Example 20).</p><p><tables num="11"><img file="JP5111558B2_D0016.tif" /></tables></p><p><u style="single">Example 25 Construction of native HC repertoire yeast display vector and haploid host cell</u> To produce a novel heavy-chain eukaryotic display vector useful as a component of a multi-chain eukaryotic vector set, the native repertoire of HC is cloned into the vector pTQ5 (Example 13).</p><p> An antibody HC fragment is isolated from a peripheral blood lymphocyte source of the V gene and isolated by an antibody PCR method known in the art. The HC library was captured in a phage display vector according to a technique known in the art, then transferred to pTQ5 as an SfiI / NotI fragment, transformed into E. coli, and approximately 1 × 10.⁸Produces a library of. The yeast strain was then transformed into EBY100 in this library, approximately 1x10.⁷Library, EBY100pTQ5-HC^{* rep}To produce.</p><p><u style="single">Example 26 Construction of native LC repertoire yeast display vector and haploid host cell</u> To produce a novel light chain eukaryotic display vector useful as a component of a multi-chain eukaryotic vector set, the native repertoire of LCs is cloned into the vector pTQ6 (Example 16).</p><p> An antibody LC fragment is isolated from a peripheral blood lymphocyte source of the V gene and isolated by an antibody PCR method known in the art. After capturing the LC library in a phage display vector according to techniques known in the art, it is translocated to pTQ6 as an ApaLI / AscI fragment, transformed into E. coli, and approximately 1 × 10⁸Produces a library of. This library was then transformed into yeast strain W303, approximately 1 × 10.⁷Library, W303pTQ5-LC^{* rep}To produce.</p><p><u style="single">Example 27 LC / HC recombinant library via cell fusion of haploid host cell pairs and subsequent affinity selection: detection by flow cytometry</u> Contains two (haploid) host cell populations, a first population containing a repertoire of light chain fragments and a repertoire of heavy chain fragments to produce a novel Fab (diploid) yeast display library. The second population) is co-cultured under conditions sufficient to allow cell fusion and the resulting diploid population allows expression and display of recombinant Fab (LC / HC) libraries. It was grown under sufficient conditions.</p><p> According to the procedure outlined in Example 18, about 10¹⁰EBY100pTQ5-HC^{* rep}About 10 yeast cells (from Example 26)¹⁰W303p TQ6-LC^{* rep}Join with yeast cells (from Example 22). Approximately 10 with 10% bonding efficiency⁹(Therefore, if the haploid parent has a starting diversity of the individual component LC and HC repertoires, up to 10 possible¹⁴Approximately 10 of the street combinatorial LC / HC diversity⁹To capture LC / HC combinations). This diploid repertoire is cultured to induce LC and HC expression (see Example 15). This diploid culture is incubated with streptavidin-FITC and affinity selection is performed using a flow cytometric fractionation method (see Example 8). Affinity variants are screened by off-rate determination using flow cytometry (see Example 9) and, in addition, surface plasmon resonance techniques known in the art using soluble Fab antibodies.</p><p><u style="single">Example 28 Multiple Chain Display Host Cell vs. Library via Diploid Spore Formation: Production of LC and HC Ploidy Yeast Cell Repertoire</u> A cell population expresses multiple variants of a single strand of a biologically active multi-stranded polypeptide that ligates to an anchor protein; a second cell is a plurality of soluble second strands of a multi-stranded polypeptide. As an example of a novel host cell-to-library expressing the polypeptide of, diploid Fab-labeled yeast isolates resulting from streptavidin selective screening as described in Example 23, under nitrogen starvation conditions (Guthrie). Inducing spore formation by culturing this isolate under (as described in and Fink, 1991). Spore-formed diploids are harvested, treated with zymolase, sonicated and plate-cultured on concentrated plates.</p><p> Separation of monoploid colonies into two subsamples; the first subsample promotes loss of LC expression vector, but grows under conditions of choice for HC display vectors, and the second subsample is HC. It promotes display vector loss, but grows LC expression vectors under selective conditions (for 2μ derived plasmids under non-selective conditions, plasmid loss is 2-6% per generation). After several generations, the non-selective chain expression vector was effectively eliminated from each yeast subculture and contained only the selected (LC or HC) expression vector, thereby resulting in two biases (ie, proto-preliminary). Selection) Produces single-chain expression monopoly yeast cells, "HAPLOID pTQ6-LC^{* sel}And "HAPLOID pTQ5-HC"^{* sel}I named it. From these two haploid yeast populations, each containing either the light chain of the preselective Fab or the heavy chain of the preselective Fab, three mating schemes are established as follows: In the first joining method, 10⁹Yeast HAPLOIDpTQ6-LC^{* sel}10⁹Yeast EBY100pTQ5-HC^{* rep}It is back-conjugated (from Example 21) and grown under selective conditions that maintain both LC and HC yeast expression plasmids. Approximately 10 with 10% bonding efficiency⁸Produces a diploid of. This diploid repertoire is cultured to induce the expression of LC and HC (Example 18). The resulting diploid culture represents an obligate repertoire containing a unique combination of the original HC repertoire relative to the preselective LC repertoire, which is further described, for example, by the flow cytometric fractionation method (Examples 8 and 11). And / or can be screened by surface plasmon resonance techniques known in the art using soluble Fab antibodies.</p><p> In the second joining method, 10⁹Yeast HAPLOIDpTQ6-HC^{* sel}10⁹Yeast W303pTQ6-LC^{* rep}It is back-conjugated with (Example 22) and grown under selective conditions that maintain both LC and HC yeast expression plasmids. Approximately 10 with 10% bonding efficiency⁸Produces a diploid of. This diploid repertoire is cultured to induce the expression of LC and HC (Example 18). The resulting diploid culture represents an obligate repertoire containing a unique combination of the original LC repertoire relative to the preselected HC repertoire, which is further described, for example, by flow cytometric fractionation (Examples 8 and 11). And / or can be screened by surface plasmon resonance techniques known in the art using soluble Fab antibodies.</p><p> Finally, in the third joining method, 10⁹Yeast HAPLOIDpTQ6-LC^{* sel}10⁹Yeast HAPLOIDpTQ6-HC^{* sel}And grow under selective conditions that maintain both LC and HC yeast expression plasmids. Approximately 10 with 10% bonding efficiency⁸Produces a diploid of. This diploid repertoire is cultured to induce LC and HC expression (see Example 18). The resulting diploid culture represents an obligate recombinant repertoire containing a unique combination of preselected LC repertoires relative to the preselected HC repertoire, which is further described, for example, by the flow cytometric fractionation method (Example 8). And 11) and / or can be screened by surface plasmon resonance techniques known in the art using soluble Fab antibodies.</p><p><u style="single">Example 29 Affinity maturation by restriction-based diversification of Fab antibodies</u> To illustrate the usefulness of restriction-based diversification and shuffling of Fab antibodies for affinity maturation using yeast display and selection, a Fab antibody library was prepared from read-target-specific Fabs and restriction-based cloning. To diversify either the total LC or HC fragments using. In one preferred method, multiple antibody gene fragments for cloning are prepared using an antibody library that bundles the antibody V gene sequences and constructs them with restriction sites internal to the V gene sequences. This leads to diversification of lead antibodies.</p><p> Read antibodies isolated from one such antibody library (eg, CJ Library Set, Dyax, Cambridge, Mass.) Can be affinity matured by this approach. For example, antibodies containing the CJ phagemid library have LCs bundled by unique ApaL1 and AcsI restriction sites and HCs bundled by unique SfiI and NotI restriction sites. This HC also contains an internal unique XbaI restriction site between the CDR2 and CDR3 sequences.</p><p> To diversify LC in either a single antigen-specific read antibody or a pool of antigen-specific read antibodies, the Fab antibody gene was first cloned into the yeast display vector pTQ3 as in Example 2 and pTQ3- Produces Fab. Multiple LCs are isolated from the DNA preparation of the CJ phagemid library by restriction digestion with ApaL1 and AscI restriction enzymes. pTQ3-Fab is also digested with ApaL1 and AscI, replacing endogenous LC with multiple LCs, repertoire, pTQ3-LC^cj-repProduces. This repertoire was then transferred to the yeast strain EBY100 and EBY100pTQ3-LC.^cj-repTo get.</p><p> V in either a pool of antigen-specific read antibodies or antigen-specific read antibodies_HTo diversify CDR1-2, first clone the Fab antibody gene into the yeast display vector pTQ3 as in Example 2 to obtain pTQ3-Fab. Multiple Vs from the CJ Phagemid Library by Restriction Digest with SfiI and XbaI_HIsolate the CDR1-2 fragment. pTQ3-Fab is also digested with SfiI and XbaI, endogenous V_HCDR1-2 Fragment with multiple Vs_HReplaced with CDR1-2 fragment, repertoire, pTQ3-V_HCDR1-2^cj-repProduces. This repertoire was then transferred to the yeast strain EBY100 and EBY100pTQ3-V._HCDR1-2^cj-repTo get.</p><p> For those skilled in the art, this cloning procedure can be performed in several different ways, eg, first V._HAfter constructing the repertoire of CDR1-2, antigen-specific V_HCDR3 or V_HIt will be clear that this can be done by cloning in the pool of CDR3.</p><p> EBY100pTQ3-V_HCDR1-2^cj-repAnd EBY100p TQ3-LC^cj-repThe culture of is prepared as in Example 2. The yeast culture is then labeled for antigen binding to the LC display as in Example 10 and affinity selected by flow cytometric fractionation. Then, as in Example 10, the improvement in DNA sequence and affinity of the selected clone is analyzed.</p><p><u style="single">Example 30 Affinity maturation by combinatorial shuffling of gene fragments using yeast conjugation</u> Whether to rediversify the selected LC or pool of LCs to illustrate that yeast conjugation can be used for combinatorial gene diversification and affinity maturation of antigen-specific read antibodies or antigen-specific read antibody groups. , Or the V of the selected HC or pool of HC_HRediversify CDR1-2 fragments. Antibodies containing the CJ phagemid library are applicable to such approaches. They have LCs aggregated by unique ApaL1 and AscI restriction sites and HCs aggregated by unique SfiI and NotI restriction sites. This HC also contains an internal unique XbaI restriction site between the CDR2 and CDR3 sequences. Since this LC and HC are present in opposite mating yeast cells, yeast mating is used to generate multiple Vs of antigen-specific LC._HThe CDR1-2 fragment, or antigen-specific HC, is combined with multiple LCs, thereby eliminating the need for restriction-based cloning to pair the LCs with the HCs.</p><p> In one preferred method of diversifying the pool of antigen-specific read antibodies or antigen-specific read antibodies, the component HC antibody gene is cloned into the yeast display vector pTQ5 as in Example 13 to obtain pTQ-HCAg. Multiple Vs by restriction digestion of HC fragments from the CJ phagemid library with SfiI and XbaI restriction enzymes_HPrepare a CDR1-2 fragment. Then this multiple V_HCDR1-2 fragment has already been digested with SfiI and XbaI and endogenous V_HRemove CDR1-2 fragments and multiple Vs_HReplaced with CDR1-2 fragment, antigen-specific V_HCDR3 is cloned into the retained DNA prepared from pTQ5-HCAg. As a result, pTQ5-V_HA library named CDR1-2 (CDR3Ag) was obtained and introduced into the yeast strain EBY100, repertoire, EBY100pTQ5-V._HObtain CDR1-2 (CDR3Ag). Multiple LCs are isolated from the DNA preparation of the CJ phagemid library by restriction digestion with ApaL1 and AscI. Cloning these multiple LCs into pTQ6 and repertoire, pTQ6-LC^repObtain and serve as one master repertoire for affinity maturation of other antibodies specific for other targets. This repertoire was then transferred to the opposite mating yeast strain, BJ5457, to BJ5457pTQ6-LC.^repTo get.</p><p> LC and V_HEBY100pTQ5-V in one mating method that allows simultaneous diversification of both CDR1-2 gene fragments_HCDR1-2 (CDR3Ag) and BJ5457pTQ6-LC^repThe culture of is prepared as in Example 22. The two repertoires are joined together (see Example 19) and the diploid repertoire is EBY100pTQ5-V._HCDR1-2 (CDR3Ag) / BJ5457pTQ6-LC^repTo get. Induce Fab antibody expression (see Example 18) and affinity select the diploid repertoire as in Example 20. Affinity improvement is analyzed for the selected clones as in Example 23.</p><p> Although the present invention has been specifically shown and described with reference to its preferred embodiments, those skilled in the art will make various changes in form and detail without departing from the scope of the invention contained in the accompanying claims. It will be understood that what can be done in the invention.</p><p><u style="single">Specific aspect</u>[Aspect 1] A multi-chain polypeptide eukaryotic display vector: (a) A first polynucleotide encoding a polypeptide comprising the first strand of a biologically active multi-strand polypeptide that ligates to a cell surface anchor; and (b) A second polynucleotide encoding the second strand of a multi-stranded polypeptide; Including It can act to direct the expression and secretion of a multi-chain polypeptide chain in a eukaryotic host cell, where the multi-chain polypeptide chain is such that the biological activity of the multi-chain polypeptide appears on the surface of the eukaryotic host cell. The vector associated with. [Aspect 2] (c) A third polynucleotide encoding the third strand of a multi-stranded polypeptide, The eukaryotic display vector according to aspect 1, further comprising. [Aspect 3] (d) A fourth polynucleotide encoding the fourth strand of a multi-stranded polypeptide, The eukaryotic display vector according to aspect 2, further comprising. [Aspect 4] The eukaryotic display vector according to aspect 1, wherein the multi-stranded polypeptide is a double-stranded polypeptide. [Aspect 5] The eukaryotic display vector according to aspect 1, wherein the multi-stranded polypeptide is a quadruplex polypeptide, wherein the quadruplex polypeptide comprises two first strands and two second strands. [Aspect 6] The double-stranded polypeptide according to embodiment 1, wherein the multi-stranded polypeptide is a double-stranded polypeptide selected from the group consisting of immunoglobulin Fab fragments, extracellular domains of T cell receptors, MHC class I molecules, and MHC class II molecules. Eukaryotic display vector. [Aspect 7] The eukaryotic display vector according to aspect 1, wherein the multi-chain polypeptide is an immunoglobulin (Ig) or an Ig fragment. [Aspect 8] The eukaryotic display vector according to aspect 7, wherein the multi-chain polypeptide is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG, and IgM. [Aspect 9] The eukaryotic display vector according to aspect 8, wherein the multi-chain polypeptide is IgG. [Aspect 10] The eukaryotic display vector according to aspect 7, wherein the multi-chain polypeptide is Fab. [Aspect 11] The eukaryotic display vector according to aspect 1, wherein the anchor is a cell surface protein of a eukaryotic cell. [Aspect 12] The eukaryotic display vector according to aspect 1, wherein the anchor is a part of a cell surface protein of the eukaryotic cell that adheres to the cell surface of the eukaryotic host cell. [Aspect 13] The eukaryotic display vector according to aspect 1, wherein the anchor is selected from the group consisting of α-aglutinine, a-aglutinine components Aga1p and Aga2p, and FLO1. [Aspect 14] The eukaryotic display vector according to aspect 1, wherein at the time of expression, the first strand and the cell surface anchor are expressed as a fusion protein in a eukaryotic host cell. [Aspect 15] The eukaryotic display vector according to embodiment 1, wherein at the time of expression, the first strand is linked to the cell surface anchor by Jun / Fos linkage. [Aspect 16] The eukaryotic display vector according to embodiment 14, wherein upon expression, the first strand of the multi-stranded polypeptide fuses to Aga2p, Aga2p covalently binds to Aga1p, which in turn ligates to the surface of the eukaryotic host cell. [Aspect 17] The eukaryotic display vector according to aspect 1, wherein the first polynucleotide is operably linked to the Aga2p signal sequence and the second polynucleotide is operably linked to the Aga2p signal sequence. [Aspect 18] The first embodiment is in-frame linked to a polynucleotide encoding a first epitope tag, and a second polynucleotide is in-frame linked to a polynucleotide encoding a second epitope tag. True core display vector. [Aspect 19] The eukaryotic display vector according to aspect 1, further comprising a restriction endonuclease recognition site located at the 5'and 3'ends of a polynucleotide segment comprising all of the polynucleotides encoding a multi-stranded polypeptide chain. [Aspect 20] The eukaryotic display vector according to aspect 1, further comprising a restriction endonuclease recognition site located at the 5'and 3'ends of the polynucleotide encoding the multi-stranded polypeptide chain, respectively. [Aspect 21] A vector library comprising the eukaryotic display vector according to aspect 1. [Aspect 22] The vector library according to aspect 21, comprising a heterologous population of multi-chain polypeptides. [Aspect 23] A method for displaying a biologically active multi-chain polypeptide on the surface of a eukaryotic host cell, comprising utilizing the vector according to embodiment 1. [Aspect 24] The eukaryotic display vector according to aspect 1, which is a vector selected from the group consisting of a vector set, a binary display vector, and a yeast display vector. [Aspect 25] A eukaryotic display vector in which the multi-stranded polypeptide is a double-stranded polypeptide, a set of vectors containing a first eukaryotic vector and a second eukaryotic vector, each vector being one of the double-stranded polypeptides. The eukaryotic display vector according to aspect 24, comprising a polynucleotide encoding a book strand. [Aspect 26] A set of vectors is a eukaryotic display vector, the multi-stranded polypeptide is a triple-stranded polypeptide, and this vector set is a first eukaryotic vector, a second eukaryotic vector, and a third eukaryote. The eukaryotic display vector of aspect 24, wherein each vector comprises a vector encoding a single strand of a triple-stranded polypeptide. [Aspect 27] At least the following three vectors: (a) Contains a first polynucleotide encoding the first strand of a four-stranded polypeptide that connects to a cell surface anchor and can act to direct expression and secretion of the first strand in a eukaryotic host cell. First eukaryotic vector; (b) A second eukaryotic vector containing a first polynucleotide encoding the first strand of a four-stranded polypeptide and capable of directing expression and secretion of the first strand in a eukaryotic host cell. ; And (c) A third eukaryotic vector containing a second polynucleotide encoding the second strand of a four-stranded polypeptide and capable of directing expression and secretion of the second strand in a eukaryotic host cell. , Is a vector set that contains, thereby forming a vector set. The eukaryotic host cell transformed with the vector set exhibits the biological activity of the quadruplex polypeptide on the surface of the eukaryotic host cell upon expression of the first and second polynucleotides, according to aspect 24. Nuclear display vector. [Aspect 28] The eukaryotic display vector according to aspect 24, which is a dual display vector, wherein the anchor is a polypeptide capable of acting as an anchor on the surface of eukaryotic cells and as an anchor on the surface of phage. [Aspect 29] 28. The dual display vector according to aspect 28, wherein the anchor is part of a cell surface protein that adheres to the cell surface of a eukaryotic host cell and the surface of a phage. [Aspect 30] A dual display vector, the first polynucleotide operably linked to the polynucleotide encoding the first signal sequence and the second polynucleotide operably linked to the polynucleotide encoding the second signal sequence. The eukaryotic display vector according to aspect 24, wherein the first signal sequence and the second signal sequence are operable in bacterial cells and operable in eukaryotic cells. [Aspect 31] A dual display vector, the first polynucleotide operably linked to the first promoter and the second polynucleotide operably linked to the second promoter, where the first and second promoters are: The eukaryotic display vector according to aspect 24, both operable in bacterial cells and operable in eukaryotic cells. [Aspect 32] Yeast Fab display vector: (a) A first polynucleotide encoding a first polypeptide containing the VH and CH1 regions of the Ig heavy chain that links to activable cell surface anchors in yeast; and (b) A second polynucleotide encoding a second polypeptide containing an Ig light chain, Including Said vector, which can act to direct the expression and secretion of a multi-chain polypeptide chain in a yeast host cell, and the multi-chain polypeptide chain associates to form a Fab on the surface of the yeast host cell. [Aspect 33] The yeast Fab display vector according to aspect 32, wherein the anchor is selected from the group consisting of α-aglutinine, a-aglutinine components Aga1p and Aga2p, and FLO1. [Aspect 34] The yeast Fab display vector according to embodiment 32, wherein the VH region and the CH1 region are linked to the cell surface anchor by Jun / Fos linkage at the time of expression. [Aspect 35] The yeast Fab display vector according to aspect 33, wherein upon expression, the VH and CH1 regions fuse to Aga2p, Aga2p covalently binds to Aga1p, which in turn ligates to the yeast host cell surface. [Aspect 36] The yeast Fab display vector according to aspect 32, further comprising a restriction endonuclease recognition site located at the 5'and 3'ends of the polynucleotide encoding the multi-stranded polypeptide chain. [Aspect 37] The Fab yeast display vector according to aspect 32, which is a vector set. [Aspect 38] The Fab yeast display vector according to aspect 32, which is a dual display vector. [Aspect 39] The Fab yeast display vector according to aspect 38, wherein the dual display vector is operable in phage and yeast. [Aspect 40] Fab yeast display library comprising the yeast display vector according to aspect 32. [Aspect 41] The Fab yeast display library according to aspect 40, which comprises a heterologous population of multi-chain polypeptides. [Aspect 42] A method for displaying a biologically active multi-chain polypeptide on the surface of a eukaryotic host cell, comprising utilizing one or more of the vectors described in embodiment 32. [Aspect 43] A method of displaying a biologically active multi-chain polypeptide containing at least two polypeptide chains on the surface of a eukaryotic host cell, the following steps: (a) Steps of introducing the following (i) and (ii) into eukaryotic host cells, (i) Containing a first polynucleotide encoding the first polypeptide chain of a biologically active multi-stranded polypeptide that ligates to a cell surface anchor, expressing and secreting the first chain in a eukaryotic host cell. A first eukaryotic vector that is operational to command; and (ii) A second true that contains a second polynucleotide encoding a second polypeptide chain of a multi-stranded polypeptide and is capable of directing expression and secretion of the second strand in a eukaryotic host cell. Nuclear vector (Here, eukaryotic host cells transformed with the first eukaryotic vector and the second eukaryotic vector show the biological activity of the multi-stranded polypeptide upon expression of the first and second polynucleotides. On the surface of eukaryotic host cells), and (b) A step of culturing the host cell under conditions suitable for expression of the first polynucleotide and the second polynucleotide. The method described above. [Aspect 44] The method of aspect 43, wherein the eukaryotic host cell is selected from the group consisting of animal cells, plant cells, and fungal cells. [Aspect 45] The method of aspect 43, wherein the eukaryotic host cell is selected from the group consisting of mammalian cells, insect cells, and yeast cells. [Aspect 46] The method of aspect 43, wherein the eukaryotic host cell is a yeast cell. [Aspect 47] 46. Aspect 46, wherein the yeast cell is of a genus selected from the group consisting of the genus Saccharomyces, Pichia, Hanzenula, Sizosaccalomises, Cryberomyces, Yarrowia, Devariomis, and Candida. Method. [Aspect 48] The method according to aspect 47, wherein the yeast cells are selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Cryberomyces lactis, Pichia pastoris, Saccharomyces cerevisiae and Yarrowvia lipolytica. [Aspect 49] 47. The method of aspect 47, wherein the yeast cell is Saccharomyces cerevisiae. [Aspect 50] A method of displaying a biologically active multi-chain polypeptide containing at least two polypeptide chains on the surface of diploid eukaryotic cells: (a) To provide a first diploid eukaryotic cell containing a first polynucleotide encoding a polypeptide comprising the first strand of a biologically active multi-stranded polypeptide that ligates to a cell surface anchor. ; (b) To provide a second diploid eukaryotic cell containing a second polynucleotide encoding a polypeptide containing the second strand of a multi-stranded polypeptide; (c) Contacting the first haploid eukaryotic cells and the second haploid eukaryotic cells under conditions sufficient to fuse these cells to produce diploid eukaryotic cells. ;as well as (d) The diploid eukaryotic cells are cultured under conditions sufficient to allow expression and association of the multi-chain polypeptide chain (where the biological activity of the multi-chain polypeptide is diploid. Appears on the surface of eukaryotic cells), The method comprising. [Aspect 51] The method of aspect 50, wherein the first haploid eukaryotic cell and the second haploid eukaryotic cell are opposite mating types. [Aspect 52] The method of aspect 50, wherein the first haploid eukaryotic cell and the second haploid eukaryotic cell are opposite mating types. [Aspect 53] A method of displaying Fab on the surface of diploid yeast cells in the following steps: (a) A step of providing a first diploid yeast cell containing a first polynucleotide encoding a first polypeptide containing a VH region and a CH1 region of an IgG heavy chain linked to a cell surface anchor; (b) A step of providing a second haploid yeast cell containing a second polynucleotide encoding a second polypeptide containing an Ig light chain; (c) The step of contacting the first haploid yeast cells and the second haploid yeast cells under conditions sufficient for these cells to fuse to produce diploid yeast cells; (d) A step of culturing the diploid yeast cells under conditions sufficient to allow expression and association of the first and second polypeptides (where Fab refers to diploid yeast). Appears on the surface of cells), The method comprising. [Aspect 54] 53. The method of aspect 53, wherein the first haploid yeast cell and the second haploid yeast cell are opposite mating types. [Aspect 55] 54. The method of aspect 54, wherein the first haploid yeast cell and the second haploid yeast cell are cells of Saccharomyces cerevisiae. [Aspect 56] A method of detecting a biologically active multi-chain polypeptide containing at least two polypeptide chains from a multi-chain polypeptide library: (a) First monoplex eukaryote containing multiple first polynucleotides, each encoding a polypeptide containing a first-strand variant of a biologically active multi-stranded polypeptide that ligates to a cell surface anchor. Providing a cell population; (b) A conjugated second haploid opposite to the first haploid eukaryotic cell population, each containing multiple second polynucleotides encoding a second strand variant of a multi-stranded polypeptide. Providing a eukaryotic cell population; (c) Sufficient to fuse the first haploid eukaryotic cell population and the second haploid eukaryotic cell population with different mating individual cells to produce a diploid eukaryotic cell population Contact under various conditions; (d) The diploid eukaryotic cells are cultured under conditions sufficient to allow expression and association of the multi-chain polypeptide chain (where the biological activity of the multi-chain polypeptide is doubled). Appears on the surface of eukaryotic cells); (e) To detect a particular biological activity of interest, The method comprising. [Aspect 57] (f) The method of aspect 56, further comprising the step of isolating diploid eukaryotic cells displaying biological activity. [Aspect 58] (G) The method of aspect 57, further comprising repeating steps (d), (e) and (f). [Aspect 59] (g) The isolated diploid eukaryotic cells of step (f) are cultured under conditions sufficient for the isolated diploid eukaryotic cells to undergo meiosis to produce haploid eukaryotic cells. Process to do; (h) A step of contacting the haploid eukaryotic cells from step (g) under conditions sufficient to fuse different mating eukaryotic cells to produce a population of diploid eukaryotic cells. ; And (i) Steps (d), steps (e) and (f) are repeated, 57. [Aspect 60] (g) The isolated diploid eukaryotic cells of step (f) are cultured under conditions sufficient for the isolated diploid eukaryotic cells to undergo meiosis to produce haploid eukaryotic cells. Process to do; (h) The diploid eukaryotic cells from step (g) are fused with one or more diploid eukaryotic cell populations selected from the group consisting of the following and different mating type eukaryotic cells. Step of contact under conditions sufficient to produce a population of diploid eukaryotic cells: (i) First haploid eukaryotic cell population in step (a); (ii) Second haploid eukaryotic cell population in step (b); (iii) A second haploid eukaryote containing multiple first polynucleotides encoding a polypeptide comprising a first-strand variant of a biologically active multi-stranded polypeptide that ligates to a cell surface anchor. A conjugative third haploid eukaryotic cell population opposite to the cell population; and (iv) A conjugated fourth haploid opposite to the first haploid eukaryotic cell population, each containing multiple second polynucleotides encoding a second strand variant of a multi-stranded polypeptide. Eukaryotic cell population; (i) Steps (d), steps (e) and (f) are repeated, 57. [Aspect 61] A method of detecting and isolating one or more multi-stranded polypeptides that exhibit the desired biological activity: (a) To provide eukaryotic cells that display a multi-stranded polypeptide on its surface upon expression of a multi-stranded eukaryotic display vector; (b) Culturing the eukaryotic cells under conditions sufficient to allow expression of the multi-chain polypeptide; (c) Bringing the cell into contact with the molecule of interest; (d) Select and isolate cells that exhibit a special interaction with the molecule of interest, The method comprising. [Aspect 62] 16. The method of aspect 61, wherein a host cell displaying a multi-stranded polypeptide exhibiting the biological activity of interest is isolated and optionally subjected to at least one additional selection round. [Aspect 63] The method of aspect 61, further comprising screening the library using phage display screening. [Aspect 64] The method of aspect 61, wherein the molecule of interest is a protein. [Aspect 65] The method of aspect 64, wherein the biological activity of interest is the interaction of a multi-chain polypeptide with another molecular species, comprising non-covalent association between the molecular species. [Aspect 66] 65. The method of aspect 65, wherein the interaction is transient. [Aspect 67] 65. The method of aspect 65, wherein the interaction is a covalent interaction. [Aspect 68] A method of transferring a nucleic acid sequence encoding a biologically active multi-strand polypeptide between a phage display vector and a eukaryotic display vector: (a) Obtain a phage display vector containing: (i) A first polynucleotide encoding a polypeptide comprising the first strand of a biologically active multi-strand polypeptide that ligates to a cell surface anchor; and (ii) A second polynucleotide encoding the second strand of a multi-stranded polypeptide, (Here, the phage display vector can act to direct the expression of the multi-chain polypeptide chain in the bacterial host cell, and the multi-chain polypeptide chain is the biological activity of the multi-chain polypeptide, which causes the phage display vector. Associate to appear on the surface of phages that contain and proliferate in bacterial host cells); (b) Inserting the first and second polynucleotides encoding a multi-stranded polypeptide chain into a eukaryotic display vector (where the eukaryotic display vector is a multi-stranded polypeptide chain in a eukaryotic host cell. Can be actuated to direct the expression and secretion of, and the multi-chain polypeptide chains associate such that the biological activity of the multi-chain polypeptide appears on the surface of the eukaryotic host cell), The method comprising. [Aspect 69] 28. The method of aspect 68, wherein the polynucleotide sequences encoding each of the multi-stranded polypeptide chains are inserted together into a eukaryotic display vector as a single polynucleotide. [Aspect 70] 28. The method of aspect 68, wherein the polynucleotide sequences encoding each of the multi-stranded polypeptide chains are independently inserted into the eukaryotic display vector as separate polynucleotides. [Aspect 71] 28. The method of aspect 68, wherein the transition step (b) comprises a gene transfer technique selected from the group consisting of restriction digestion, PCR amplification, homologous recombination, and a combination of these techniques. [Aspect 72] A method of transferring a nucleic acid sequence encoding a biologically active Fab between a phage display vector and a eukaryotic display vector: (a) Obtain a phage display vector containing: (i) A first polynucleotide encoding a first polypeptide containing the VH and CH1 regions of the Ig heavy chain that connects to the cell surface anchor; and (ii) A second polypeptide encoding a second polypeptide containing an Ig light chain (where the phage display vector directs expression of the first and second polypeptides in a bacterial host cell. And the polypeptide associates so that the biological activity of the multi-stranded polypeptide appears on the surface of the phage that is transfected with the phage display vector and proliferates in the bacterial host cell); (b) Inserting the first and second polynucleotides encoding the first and second polypeptides into a eukaryotic display vector (where the eukaryotic display vector is in a yeast host cell). It can act to direct the expression and secretion of the first and second polypeptides, which associate so that the biological activity of the Fab appears on the surface of the yeast host cell), The method comprising. [Aspect 73] 28. The method of aspect 72, wherein the polynucleotide sequences encoding each of the polypeptides are inserted together into a eukaryotic display vector as a single polynucleotide. [Aspect 74] 28. The method of aspect 72, wherein the polynucleotide sequence encoding each of the polypeptides is independently inserted into the eukaryotic display vector as a separate polynucleotide. [Aspect 75] 28. The method of aspect 72, wherein the transition step (b) comprises a gene transfer technique selected from the group consisting of restriction digestion, PCR amplification, homologous recombination, and a combination of these techniques. [Aspect 76] A eukaryotic host cell containing a vector, wherein the vector is: (a) A first polynucleotide encoding a polypeptide comprising the first strand of a biologically active multi-strand polypeptide that ligates to a cell surface anchor; and (b) A second polynucleotide encoding the second strand of a multi-stranded polypeptide, Including Here, the vector can act to direct the expression and secretion of a multi-chain polypeptide chain in a eukaryotic host cell, and the multi-chain polypeptide chain is a eukaryotic host cell in which the biological activity of the multi-chain polypeptide is eukaryotic. Meet to appear on the surface of The eukaryotic host cell. [Aspect 77] The eukaryotic host cell according to aspect 76, selected from the group consisting of animal cells, plant cells, and fungal cells. [Aspect 78] The eukaryotic host cell according to aspect 76, selected from the group consisting of mammalian cells, insect cells, and yeast cells. [Aspect 79] The eukaryotic host cell according to aspect 76, which is a yeast cell. [Aspect 80] The eukaryotic host cell according to aspect 79, wherein the yeast cell is haploid. [Aspect 81] The eukaryotic host cell according to aspect 79, wherein the yeast cell is diploid. [Aspect 82] (a) First monoplex eukaryotic cells containing a first polynucleotide encoding a polypeptide containing the first strand of a biologically active multi-stranded polypeptide that ligates to a cell surface anchor; and (b) A second haploid eukaryotic cell containing a second polynucleotide encoding the second strand of a multi-stranded polypeptide, A pair of haploid eukaryotic cells containing. [Aspect 83] The monoploid eukaryotic cell pair according to aspect 82, wherein the first diploid eukaryotic cell and the second diploid eukaryotic cell are opposite mating types. [Aspect 84] The haploid eukaryotic cell pair according to aspect 82, wherein the multi-stranded polypeptide is a double-stranded polypeptide. [Aspect 85] The haploid eukaryotic cell pair according to aspect 82, wherein the multi-stranded polypeptide is a quadruplex polypeptide, wherein the quadruplex polypeptide comprises two first strands and two second strands. [Aspect 86] 28. Aspect 82, wherein the multi-stranded polypeptide is a double-stranded polypeptide selected from the group consisting of immunoglobulin Fab fragments, extracellular domains of T cell receptors, MHC class I molecules, and MHC class II molecules. A pair of monoploid eukaryotic cells. [Aspect 87] The haploid eukaryotic cell pair according to aspect 82, wherein the anchor is a cell surface protein of eukaryotic cells. [Aspect 88] A eukaryotic host cell library comprising a plurality of diploid cells, which is a fusion product of the plurality of eukaryotic host cell pairs according to aspect 82. [Aspect 89] The eukaryotic host cell library according to aspect 88, wherein the plurality of diploid cells display a heterologous population of multistranded polypeptides. [Aspect 90] (a) A first polynucleotide encoding a polypeptide comprising the first strand of a biologically active multi-strand polypeptide that ligates to an operable cell surface anchor in yeast; and (b) A second polynucleotide encoding the second strand of a multi-stranded polypeptide, A yeast cell transformed with a heterologous display vector comprising, the vector can act to direct the expression and secretion of a multi-chain polypeptide chain in the yeast cell, and the multi-chain polypeptide chain is: The yeast cell in which the biological activity of the multi-chain polypeptide is associated to appear on the surface of the yeast cell. [Aspect 91] (a) First monoplex yeast cells containing a first polynucleotide encoding a polypeptide containing the first strand of a biologically active multi-stranded polypeptide that ligates to a cell surface anchor; and (b) A second haploid yeast cell containing a second polynucleotide encoding the second strand of a multi-stranded polypeptide, A pair of haploid yeast cells containing. [Aspect 92] The haploid yeast cell pair according to aspect 91, wherein the first haploid yeast cell and the second haploid yeast cell are opposite mating types. [Aspect 93] A yeast display library containing a population of yeast cells that collectively displays a repertoire of at least 107 polypeptides. [Aspect 94] A yeast display library containing a population of yeast cells that collectively displays a heterologous population of at least 104 multi-chain polypeptides. [Aspect 95] The yeast display library according to aspect 94, comprising a population of yeast cells collectively displaying a heterologous population of at least 107 multi-chain polypeptides. [Aspect 96] The yeast display library according to aspect 95, comprising a population of yeast cells collectively displaying a heterologous population of at least 108 multi-chain polypeptides. [Aspect 97] A yeast display library comprising a plurality of diploid cells, which is a fusion product of the plurality of yeast cell pairs according to aspect 91. [Aspect 98] The yeast display library according to aspect 97, wherein the plurality of diploid cells display a heterologous population of Fab.</p>