JP2005529607A - Recombination of nucleic acid library members - Google Patents

Abstract

The present invention provides, among other things, a method for preparing a nucleic acid sequence encoding a polypeptide displayed on the surface of a heterologous cell. The present invention generally includes recombining donor and acceptor nucleic acids to form a recombinant nucleic acid encoding the indicated polypeptide. The recombination reaction is typically an in vivo reaction, in which at least resolving of the recombination intermediate occurs within the cell. Both site-specific recombination and homologous recombination can be used.

Description

REFERENCE TO RELATED APPLICATIONS This application is June 14, 2002 U.S. Patent Application No. 60 / 389,032, filed, the entire disclosure of which is incorporated herein in claims priority.
TECHNICAL FIELD The present invention relates to a method for altering a nucleic acid sequence by recombination. Nucleic acid recombination can generally be classified homologously or site-specifically.

  Homologous recombination generates a new nucleic acid sequence that incorporates the sequence from the parent strand such that the 5 'end of one parent strand is linked to the 3' end of another parent strand. Typically, but not necessarily, homologous recombination proceeds through an intermediate termed a Holiday junction where the four-way junction of the substrate nucleic acid is split and recombined. A chain is formed. An essential feature of homologous recombination is that, if the substrate nucleic acids are homologous, recombination is primarily dependent on the underlying sequence.

In contrast, site-specific recombination results in nucleic acid exchange at a specific site. An enzyme that mediates site-specific recombination, site-specific recombinase, acts to recognize these sites and recombines sequences at those sites. In many natural cases, site-specific recombination results in sequence integration and / or removal. For example, the bacteriophage lambda genome encodes an enzyme that uses site-specific recombination to specifically integrate or excise the lambda genome into and out of the bacterium. In another example, the yeast FLP-FRT system uses recombinase to split the replication intermediate.
Both types of recombination have been used in a variety of applications. For example, homologous recombination has been used to insert sequences into the homologous context, such as in target genetic recombination. Meiotic recombination has been used to generate new sequence combinations, such as in broad breeding of crop and farm animal lines. Site-specific recombination has been used in vitro for cloning nucleic acids (see, eg, US Pat. No. 5,888,732).

SUMMARY OF THE INVENTION In one aspect, the invention features the following: providing a first number of yeast cells, each cell of the first number of yeast cells being on the surface of the cell. Each cell expresses an associated antibody protein, and each cell comprises an antibody-encoding nucleic acid sequence that encodes the antibody protein, wherein the antibody protein comprises a light chain variable immunoglobulin domain and a heavy chain variable immunoglobulin domain. And wherein the first number of yeast cells express a unique antibody protein; a subset comprising one or more yeast cells that interact with the target is selected from the first number; Introducing one or more Ig segment-encoding nucleic acids into one or more subsets of cells, wherein each of the one or more Ig segment-encoding nucleic acids Comprises a sequence encoding a segment of an immunoglobulin variable domain or its complement; and allows the introduced Ig segment-encoding nucleic acid to recombine with the antibody-encoding nucleic acid sequence of a subset of cells. Under conditions that maintain a cell or a subset of cells, thereby producing one or multiple cells capable of expressing an antibody protein having a modified immunoglobulin variable domain. Clones and other cells can be present in addition to the first number of cells.

In one aspect, the invention features a method that includes the following steps:
Each provides a yeast cell comprising a heterologous nucleic acid comprising a promoter and a coding region, wherein the coding region comprises a nucleic acid homologous to an immunoglobulin variable domain coding nucleic acid, and the promoter is operable to the coding region. Connected in a way;
Introducing one or more Ig segment-encoding nucleic acids into a yeast cell, wherein each of the one or more Ig segment-encoding nucleic acids comprises a sequence encoding a segment of an immunoglobulin variable domain or its complement. And maintaining the yeast cell under conditions that allow the introduced Ig segment-encoding nucleic acid to recombine with the coding region, whereby the coding region encodes a polypeptide comprising an immunoglobulin variable domain A recombinant nucleic acid is produced, and the variable domain is encoded, at least in part, by a sequence introduced by an Ig segment-encoding nucleic acid. This method can be used, for example, to alter the sequence of an antibody protein.

  In one embodiment, the method further comprises expressing the recombinant nucleic acid such that a polypeptide comprising an immunoglobulin variable domain is displayed on the cell surface and is accessible to the probe.

  In one embodiment, prior to recombination, the coding region is a non-functional coding region. In one embodiment, the coding region includes a non-immunoglobulin sequence (eg, a stop codon), an inserted coding sequence for a non-immunoglobulin protein (eg, a marker gene, eg, a counter selectable marker gene). . For example, in the absence of recombination, non-immunoglobulin sequences can prevent the production of polypeptides that contain immunoglobulin variable domains. In one embodiment, the coding region includes a frameshift, inversion, or other modification that prevents production of a polypeptide comprising an immunoglobulin variable domain.

  In one embodiment, the method further comprises recombining with a yeast cell comprising the recombinant nucleic acid such that the fused cell comprises a nucleic acid encoding a polypeptide comprising Ig LC and a polypeptide comprising Ig HC. Fusing other yeast cells containing a nucleic acid encoding an immunoglobulin variable domain that is compatible with the immunoglobulin variable domain encoded by the encoded nucleic acid.

  In one embodiment, providing a yeast cell includes introducing one or more Ig segment-encoding nucleic acids into a yeast cell that contains the heterologous nucleic acid. In one aspect, providing a yeast cell simultaneously introduces one or more Ig segment-encoding nucleic acids and one or more nucleic acids that are homologous to a region of an immunoglobulin variable domain of a heterologous nucleic acid into the yeast cell. Including doing. For example, a yeast cell can be cotransduced with one or more Ig segment-encoding nucleic acids and a linearized nucleic acid that includes an interruption in an immunoglobulin variable domain coding sequence, eg, a CDR coding sequence.

  The method can further include selecting one or more recombinant nucleic acids on one basis and repeating the method by providing additional yeast cells, wherein further yeast The coding region of the cell is prepared from one or more selected recombinant nucleic acids.

  In one embodiment, each Ig segment-encoding nucleic acid comprises a sequence encoding an CDR of an immunoglobulin variable domain or its complement, eg, each encodes a single CDR of an immunoglobulin variable domain. Sequence or its complement. In one embodiment, one or more Ig segment-encoding nucleic acids are obtained from human nucleic acids.

  In one embodiment, the one or more Ig segment-encoding nucleic acids include multiple nucleic acids that encode different variants of the same CDR. For example, yeast cells are contacted with nucleic acids containing variants that differ only in CDR1.

  In one embodiment, the one or more Ig segment-encoding nucleic acids include multiple nucleic acids encoding different variants of a number of different CDRs. For example, a yeast cell is contacted with a nucleic acid comprising a different variant of LC CDR1 and a different variant of HC CDR2.

  In one embodiment, the one or more Ig segment-encoding nucleic acids comprise nucleic acids encoding different variants of CDR1, CDR2 and CDR3 of the immunoglobulin light or heavy chain variable domains.

In one embodiment, each Ig segment-encoding nucleic acid is less than 300, 250, 200, 150, or 100 nucleotides in length.
In one embodiment, the introducing contacts at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁸ , 10 ⁹ , or 10 ¹⁰ different Ig segment-encoding nucleic acids to one or more yeast cells Including that. In one embodiment, the one or more Ig segment-encoding nucleic acids are single stranded. The method further includes inserting a counter-selectable marker into the nucleic acid membrane of the subset of cells prior to the introduction, and after the introduction, the counter-selectable marker is replaced with an Ig segment-encoding nucleic acid. Including selecting the cells that are present. The invention can further include introducing a mutation into the coding region prior to the introduction, wherein the mutation prevents expression of a functional immunoglobulin variable domain.

In one embodiment, the mutation comprises a stop codon, marker gene, frameshift, or site-specific endonuclease site.
In one embodiment, the polypeptide expressed from the recombinant nucleic acid interacts with the cell such that recovery of the polypeptide results in recovery of the cell containing nucleic acid encoding the polypeptide. Contains sequences that allow for action. For example, sequences that allow interaction with cells encode anchor domains, including transmembrane domains or domains that become GPI-linked to the yeast cell surface.

  In one embodiment, the nucleic acid member can be integrated into a yeast cell chromosome (eg, an endogenous chromosome or an artificial chromosome), eg, as described herein, or is present on a plasmid. It is possible.

The method can include other features described herein.
In another aspect, the invention features a method comprising the following steps: providing a number of yeast cells, each cell of the number of yeast cells comprising an antibody encoding nucleic acid sequence encoding a unique antibody protein. Wherein the antibody protein comprises a light chain variable immunoglobulin domain and a heavy chain variable immunoglobulin domain; into one or more cells from a number of yeast cells, one or more Ig segment-encoding nucleic acids Wherein each of the one or more Ig segment-encoding nucleic acids comprises a sequence encoding a segment of an immunoglobulin variable domain or its complement; and cells from multiple yeast cells or Recombining multiple cells with the introduced Ig segment-encoding nucleic acid with the antibody-encoding nucleic acid sequence of a subset of cells It maintained under conditions that permit, thereby, capable of expressing the antibody protein having an altered immunoglobulin variable domain, producing one or a number of cells. This method can be used, for example, to alter the sequence of an antibody protein.

  Multiple Ig segment-encoding nucleic acids are introduced into the cells of multiple yeast cells separately (eg, at different times), in parallel (eg, in separate reaction mixtures or in the same reaction mixture) it can.

In another aspect, the invention features a method that includes the following steps:
A number of yeast cells are provided, the number of yeast cells being capable of encoding an antibody protein comprising a light chain variable immunoglobulin domain and a heavy chain variable immunoglobulin domain, or recombining with an Ig segment-encoding nucleic acid to produce an antibody protein. Including cells each containing an antibody coding sequence capable of forming a functional coding sequence encoding;
Perform one or more of the following cycles:
(I) introducing a nucleic acid each comprising a segment encoding one CDR of an immunoglobulin variable domain, or a complement thereof, into a cell comprising at least a portion of an antibody coding sequence from a subset of cells;
(Ii) maintaining the cell contacted with the nucleic acid in (i) under conditions that allow the introduced nucleic acid to recombine with the antibody coding sequence of the cell, thereby providing one or more modified immunoglobulin variable Producing a modified cell comprising a modified antibody coding sequence having a domain;
(Iii) expressing the altered coding sequence in the modified cell;
(Iv) contacting the target with the modified cell; and (v) selecting a further subset of cells from the modified cell, wherein the further subset comprises one or more yeast cells that interact with the target. Yes.

For example, at least 2, 3, 4 or 5 cycles are performed.
In one embodiment, contacting in step (iv) comprises contacting the cell with the target under different conditions during different cycles. In one embodiment, the selection of step (v) includes requiring improved binding to the target as compared to the previous selection step. For example, higher stringency washes can be used in later cycles, or higher concentrations of competitor can be used.

In one embodiment, a marker sequence is inserted into the antibody coding sequence prior to introducing the nucleic acid of step (i).
In one embodiment, the method further comprises recovering the antibody coding sequence from an additional subset of cells from one or more cycles. In one embodiment, the present invention further comprises sequencing at least the CDR coding region of the antibody coding sequence in a further subset of cells from one or more cycles. In one embodiment, the nucleic acid is introduced into a subset of cells.

  In one embodiment, the method further comprises, in one or more cycles, sporulating the subset of cells prior to (i), and after (ii) joining the cells into which the nucleic acid has been introduced. Contains.

  In one embodiment, providing a large number of yeast cells includes amplifying the original cells so that the large number of yeast cells are substantially identical. In one embodiment, providing a large number of yeast cells comprises amplifying a large number of native cells, wherein the multiple native cells interact with the same target, different antibodies Contains the antibody coding sequence for. For example, the method can be used to affinity mature a single antibody, or multiple antibodies, where the antibody or antibodies are of any origin.

  In one embodiment, providing a large number of yeast cells comprises selecting one or more nucleic acids from a phage display library and rearranging the one or more nucleic acids from the phage display system into a yeast expression system. Is included.

The method can be used to select cells that exhibit antibody protein. The method can include other features described herein.
In another aspect, the invention features a method for altering a subunit of an antibody protein displayed on a yeast cell. The method includes the following steps:
A first haploid yeast cell is provided comprising a first nucleic acid member encoding a first subunit of an antibody protein, wherein the first subunit comprises an immunoglobulin variable domain. Contains;
A nucleic acid introduced into a first haploid cell or clone thereof into a first haploid cell or clone thereof, each comprising one or more nucleic acids comprising a segment encoding one CDR of an immunoglobulin variable domain or its complement. Haploid cells or a first nucleic acid member thereof in a clone thereof, thereby producing one or more modified first haploid cells; and one or more modifications The produced first haploid cells are joined with one or more second haploid yeast cells to provide one or more diploid cells, wherein a second haploid yeast cell Contains a second nucleic acid member encoding the second subunit of the antibody protein, the second subunit comprising an immunoglobulin variable domain complementary to the immunoglobulin variable domain of the first subunit And Each diploid cell can then express an antibody protein containing first and second subunits on its cell surface.

  In one embodiment, the method further comprises inserting a non-immunoglobulin sequence into the first nucleic acid member prior to introduction, thereby disrupting the code of the first subunit. .

In one embodiment, the non-immunoglobulin coding sequence includes a counter-selectable marker. The method can further include other features described herein.
In another aspect, the method includes the following steps:
Providing a first haploid yeast cell comprising a first nucleic acid member capable of encoding a polypeptide comprising a first subunit of an antibody protein, the first subunit comprising an immunoglobulin variable domain Where the region of the nucleic acid member encoding the immunoglobulin variable domain has been disrupted;
A nucleic acid introduced into a first haploid cell or clone thereof, wherein one or a number of nucleic acids, each comprising a segment encoding one CDR of an immunoglobulin variable domain or its complement, is Introduced into one haploid cell or a first nucleic acid member in a clone thereof, thereby producing one or more modified first haploid cells; and one or more The modified first haploid cell is conjugated with one or more second haploid cells to provide one or more diploid cells, wherein a second haploid yeast The cell includes a second nucleic acid member that encodes a polypeptide that includes a second subunit of the antibody protein, the second subunit being complementary to an immunoglobulin variable domain of the first subunit. Variable immunoglobulin Each diploid cell contains a domain and is capable of expressing an antibody protein containing first and second subunits on its cell surface. The method can further include amplifying the one or more modified first haploid cells, eg, by one or more rounds of cell division prior to conjugation. The method can include other features described herein.

  In another aspect, the invention features a method that includes the following steps: a first nucleic acid member that encodes a polypeptide that includes a first subunit of an antibody protein; Ploidy yeast cells, the first subunit comprises an immunoglobulin light chain variable domain and a second nucleic acid member encoding a polypeptide comprising a second subunit of the antibody protein A second haploid yeast cell, wherein the second subunit comprises an immunoglobulin heavy chain variable domain; a segment each encoding one CDR of an immunoglobulin variable light chain domain or One or multiple nucleic acids, including their complements, are introduced into the first haploid cell or clone thereof and the introduced nucleic acid is the first haploid cell or clone thereof. In a recombination with a first nucleic acid member therein, thereby producing one or more modified first haploid cells; each encoding one CDR of an immunoglobulin variable heavy chain domain The nucleic acid introduced into the second haploid cell or clone thereof is the second haploid cell or clone thereof in the second haploid cell or clone thereof. Introducing into a nucleic acid member, thereby producing one or more modified second haploid cells; and one or more modified first haploid cells and one One or more two or more 2 haploid cells that are joined together, each capable of expressing an antibody protein having an altered immunoglobulin light chain variable domain and an altered immunoglobulin heavy chain variable domain on the cell surface Provide ploidy cells That.

In one embodiment, providing the first and second haploid cells includes the following steps:
(I) a first nucleic acid member encoding the first subunit of the antibody protein, the first subunit comprises an immunoglobulin light chain variable domain, and (ii) a second subunit of the antibody protein Providing a diploid yeast cell comprising: a second nucleic acid member encoding the unit, wherein the second subunit comprises an immunoglobulin heavy chain variable domain; and causing the diploid yeast cell to sporulate A first haploid cell comprising a first nucleic acid member (but not comprising a second nucleic acid member), and a second comprising a second nucleic acid member (but not comprising a first nucleic acid member) Haploid cells are provided.

  In one embodiment, the method further amplifies the one or more modified first or second haploid cells, eg, by one or more rounds of cell division prior to joining. Is included.

  In one embodiment, the method further amplifies the one or more modified first and second haploid cells, eg, by one or more rounds of cell division prior to joining. Is included.

In one embodiment, the method further comprises sporulating one or more diploid cells formed by joining.
In one embodiment, the method further comprises selecting a subset of one or more diploid cells by contacting one or more diploid cells or clones thereof with a target. For example, the subset includes one or more cells that interact with the target or one or more cells that do not interact with the target. A subset of cells can sporulate.

The method can include other features described herein.
In another aspect, the invention features a method that includes the following steps:
Providing a large number of diverse nucleic acids comprising a protein coding sequence or its complement;
Provide a large number of eukaryotic cells, each cell of the large number of eukaryotic cells comprising a heterologous nucleic acid comprising an acceptor sequence that is recombinable with homologous sequences present in one or more diverse nucleic acids. Wherein recombination of the acceptor sequence and the various nucleic acids produces a recombinant nucleic acid that encodes a polypeptide comprising a segment encoded by the various nucleic acids or a complement thereof;
Introducing nucleic acids from a number of diverse nucleic acids into the cells of a number of cells;
Recombining one or more introduced nucleic acids with a heterologous nucleic acid in each cell, thereby each encoding a polypeptide comprising a segment encoded by the one or more introduced nucleic acids, or a complement thereof. Producing recombinant nucleic acid;
Expressing the recombinant nucleic acid in the cell such that the polypeptide encoded by the recombinant nucleic acid associates with the surface of the cell;
The target is contacted with a cell that expresses the recombinant nucleic acid; and one or more cells are selected for their function of interaction with the target. The method can also be used to select cells that display binding protein.

  In one embodiment, the heterologous nucleic acid includes a non-functional immunoglobulin domain coding sequence that provides a accepting sequence for recombination. For example, a non-functional immunoglobulin domain coding sequence contains a mutation (eg, a stop codon or frameshift) prior to the 3 'end of the immunoglobulin domain coding sequence. In one embodiment, the mutation is in a region encoding a CDR.

  In one embodiment, the non-functional immunoglobulin domain coding sequence includes a marker gene in the region encoding CDR and the sequence encoding the FR region of the immunoglobulin domain is intact. For example, a marker gene includes a counter-selectable marker.

  In one embodiment, the recombinant nucleic acid encodes an antibody light chain and expressing further associates the antibody heavy chain and light chain and an anchor domain anchors the antibody heavy chain to the cell surface. Thus, further comprising expressing a nucleic acid encoding an antibody heavy chain comprising a VH domain and a CH1 domain and an anchor domain.

  In one embodiment, the heterologous nucleic acid includes a sequence that allows interaction with the cell such that recovery of the polypeptide results in recovery of the cell containing nucleic acid encoding the polypeptide. . For example, the sequence encodes an anchor domain (eg, a transmembrane domain) or a domain that becomes GPI-linked to the yeast cell surface.

In one embodiment, the eukaryotic cell is a yeast cell, e.g. Cerevisiae cells.
In one embodiment, the target is a protein. In another embodiment, the target is a cell, eg, a mammalian cell, eg, a cancer cell or a differentiated cell.

  In one embodiment, the heterologous nucleic acid does not encode a functional protein prior to recombination. In one embodiment, prior to recombination, a counter-selectable marker is located between the acceptor sequences of the heterologous nucleic acid and recombination removes the counter-selectable marker.

  In one embodiment, recombination is facilitated by cleaving the heterologous nucleic acid in each cell. For example, cleaving creates a double stranded break in the heterologous nucleic acid. In one embodiment, prior to recombination, a site recognized by a site-specific endonuclease that can be expressed in a yeast cell is located between the accepting sequences of the heterologous nucleic acid and This is facilitated by providing a site-specific endonuclease in each cell. For example, the site-specific endonuclease is a HO endonuclease and providing includes transcription of a gene encoding the HO endonuclease. Typically, if the endonuclease is expressed, the endonuclease is selected so as not to cause lethality.

In one embodiment, the recombinant nucleic acid includes a sequence encoding an immunoglobulin variable domain.
The method can include other features described herein.

  In another aspect, the invention features a method of modifying the sequence of an antibody protein. The method includes the following steps: providing a first number of yeast cells (eg, a yeast cell display library or a replica of the original cell), each cell of the first number of yeast cells being Expressing an antibody protein associated with the surface of the cell, and each cell comprises an antibody-encoding nucleic acid sequence encoding the antibody protein, wherein the antibody protein comprises a light chain variable immunoglobulin domain and a heavy chain variable immunity Wherein each cell of the first multiple yeast cells is capable of expressing a unique antibody protein; optionally, a subset comprising one or more yeast cells that interact with the target. Selecting from a first number of yeast cells; a subset, or a first number of yeast cells (eg, using replicas of the original cells) ) One or more Ig segment-encoding nucleic acids, wherein each of the one or more Ig segment-encoding nucleic acids encodes a segment of an immunoglobulin variable domain A sequence or complement thereof; and a cell or cells of a subset or of the first multiple yeast cells (eg when using replicas of the original cell) are introduced into the introduced Ig segment— One or many cells that can be maintained under conditions that allow the encoding nucleic acid to recombine with an antibody-encoding nucleic acid sequence of a subset of cells, thereby expressing an antibody protein having a modified immunoglobulin variable domain Produce. Clones or other cells can be present in addition to the first number of cells.

  In one embodiment, each Ig segment-encoding nucleic acid comprises a sequence encoding a CDR of an immunoglobulin variable domain or a complement thereof. For example, they can include a single CDR, for example, not spanning a complete FR domain, eg, include a single CDR and a portion of an adjacent FR region. In another embodiment, each Ig segment-encoding nucleic acid comprises a sequence encoding an FR of an immunoglobulin variable domain or its complement.

In one embodiment, one or more Ig segment-encoding nucleic acids are obtained from human nucleic acids.
One or more Ig segment-encoding nucleic acids may be different variants of the same CDR (eg, different variants of CDR1), or multiple different CDRs (eg, CDR1 of an immunoglobulin light chain or heavy chain variable domain). , CDR2 and CDR3) can contain multiple nucleic acids encoding different variants.

In one embodiment, each Ig segment-encoding nucleic acid is less than 300, 250, 200, or 150 nucleotides in length. In one embodiment, introducing comprises contacting at least 10 ³ , 10 ⁵ , or 10 ⁷ different Ig segment-encoding nucleic acids with one or more cells of the subset. One or more Ig segment-encoding nucleic acids can be single-stranded or double-stranded. Multiple Ig segment-encoding nucleic acids can be introduced into a subset of cells in parallel or in the same reaction mixture.

  The method can further comprise inserting a non-immunoglobulin coding sequence, eg, a counter-selectable marker, into the nucleic acid member of the subset of cells, and after being introduced, the counter-selectable marker is Cells that are replaced by the Ig segment-encoding nucleic acid are selected.

The method can further include contacting the target with cells from a pool of cells and selecting a subset of cells that interact with the target.
In one embodiment, at least a portion of an antibody protein (eg, an immunoglobulin heavy chain comprising VH and CH1 domains) is fused to an anchor domain (eg, a transmembrane domain or a GPI-linked polypeptide on the surface of a yeast cell). The The immunoglobulin heavy chain can further comprise, for example, CH2 and CH3 domains. The immunoglobulin light chain can associate to the surface of the yeast cell by covalent or non-covalent association with an immunoglobulin heavy chain. In another embodiment, the immunoglobulin light chain is fused to an anchor domain (eg, a transmembrane domain or a GPI-linked polypeptide on the yeast cell surface).

  The nucleic acid member can be integrated into a yeast cell chromosome (eg, an endogenous chromosome or an artificial chromosome) or can be present on a plasmid. For example, the LC and HC coding sequences are integrated into different yeast chromosomes (eg, different homologous chromosomes or different heterologous chromosomes).

  For example, an antibody-encoding nucleic acid sequence includes an LC coding sequence that encodes a polypeptide that includes an LC variable immunoglobulin domain, and an HC coding sequence that encodes a polypeptide that includes an HC variable immunoglobulin domain.

  In one embodiment, at least at the time of selection, the yeast cell is a diploid cell, and the LC-coding sequence and the HC-coding sequence are sequestered into different spores when the diploid cell sporulates. Thus, LC-coding sequences and HC-coding sequences are integrated into loci on homologous chromosomes. For example, the sequence can be incorporated, for example, at a position linked to a MAT locus, for example within 20 kb, of the MAT locus, for example within the MAT locus, so that at least the MATα gene encoding the MATα2 protein is not destroyed. For example, it may be desirable to perform in vivo recombination after sporulation to modify the same CDR, another CDR, another set of CDRs, and the like. For example, one can walk along a CDR by using an oligonucleotide that mutagenizes a different set of amino acids in the CDR, eg, a different set of four amino acids in the CDR.

In one embodiment, the antibody protein is Fab. The method can include other features described herein.
In another aspect, the invention features a method comprising the following steps: providing a first number of yeast cells, each of the first number of yeast cells being associated with a cell surface, different antibody proteins And an antibody coding sequence encoding an antibody protein, wherein the antibody protein comprises a light chain variable immunoglobulin domain and a heavy chain variable immunoglobulin domain; from a first number of yeast cells A subset of cells, wherein the subset comprises one or more yeast cells that interact with the target; perform one or more of the following cycles:
(I) introducing a nucleic acid comprising a segment each encoding one CDR of an immunoglobulin variable domain, or a complement thereof, into a cell comprising at least a portion of an antibody coding sequence from a subset of cells;
(Ii) maintaining the cell contacted with the nucleic acid in (i) under conditions that allow the introduced nucleic acid to recombine with the antibody coding sequence of the cell, thereby providing one or more modified immunity Producing a modified cell comprising a modified antibody coding sequence having a globulin variable domain;
(Iii) expressing the altered coding sequence in the modified cell;
(Iv) contacting the target with the modified cell; and (v) selecting a further subset of cells from the modified cell, wherein the further subset comprises one or more yeast cells that interact with the target. Yes. The method can be used to select cells that exhibit antibody protein. In one embodiment, at least two cycles are performed. In one embodiment, the contacting of step (iv) comprises contacting the cell with the target under different conditions during different cycles. In one embodiment, the selection of step (v) includes requiring improved binding to the target as compared to the previous selection step. In one embodiment, the sequence is inserted into the antibody coding sequence prior to introducing the nucleic acid of step (i). The inserted sequence can be one or more of: a stop codon, a marker gene (eg, a counter-selectable marker), or a site-specific endonuclease (eg, an endonuclease that can be expressed without lethal consequences to yeast cells, eg, A site for cleavage by an endonuclease that does not cleave so that endogenous genes in yeast cells cannot be repaired can be included.

  The method further recovers antibody coding sequences from the additional subset of cells from the one or more cycles, and at least the antibody coding sequence in the additional subset of cells from the one or more cycles. Sequencing of the CDR coding region.

In one embodiment, the nucleic acid is introduced into a subset of cells.
The method can further comprise sporulating the subset of cells prior to (i) and joining cells into which the nucleic acid has been introduced after (ii) in one or more cycles. The method can include other features described herein.

  In another aspect, the invention features a method that includes the following steps: providing a first number of yeast cells, each cell of the first number of yeast cells being an antibody associated with the surface of the cell. Expressing the protein, and each cell comprises a nucleic acid sequence encoding the antibody protein, wherein the antibody protein comprises a light chain variable immunoglobulin domain and a heavy chain variable immunoglobulin domain, and wherein Wherein each cell of the first number of yeast cells expresses a unique antibody protein; comprising a sequence encoding a segment of an immunoglobulin variable domain (eg, CDR or FR) or its complement. Providing one or multiple immunoglobulin-encoding nucleic acids; one or more interacting with a target from a first multiple yeast cells Selecting a subset comprising a mother cell; introducing one or more immunoglobulin-encoding nucleic acids into one or more cells of the subset; and Maintain cells or subsets of cells under conditions that allow them to recombine with an encoding nucleic acid sequence; thereby producing one or many cells capable of expressing an antibody protein having a modified immunoglobulin variable domain To do. Clones and other cells can be present in addition to the first number of cells. This method can be used to modify the sequence of an antibody protein. The method can include other features described herein.

  In another aspect, the invention features a method of identifying a modified polypeptide. The method comprises the following steps: (a) (1) a number of encoding nucleic acids, each comprising a segment encoding a polypeptide domain that becomes bound to the surface of a yeast cell, The polypeptide domain is heterologous to the yeast cell, and (2) provides a number of diverse nucleic acids; (b) for each of a number of yeast cell encoding nucleic acids, one of the various nucleic acids and the coding Recombining the nucleic acid in a yeast cell to form a modified nucleic acid encoding the modified polypeptide domain; (c) expressing the modified nucleic acid in the yeast cell, wherein the modified polypeptide The domain is bound to the surface of the yeast cell, and each modified nucleic acid contains an immunoglobulin variable domain; (d) target the yeast cell (eg, target compound or target). Contacting the cells); and (e) recovering the one or more cells that bind to the target, thereby identifying one or more polypeptides encoded by a modified nucleic acid of the recovered cells. The method can further comprise modifying (eg, by repeating the method) one or more modified nucleic acids of the recovered cells.

In one embodiment, each of the various nucleic acids includes a segment encoding a CDR or its complement.
The present invention can further include rearranging the modified nucleic acid of the recovered cell for mammalian cell expression and expressing the rearranged nucleic acid in the mammalian cell. The method also assesses the biological properties (eg, properties involving mobilization of cytotoxic cells or complement proteins) of a polypeptide (eg, a polypeptide comprising an immunoglobulin domain) expressed by a mammalian cell. Can be included.

  In one embodiment, recombination further inserts a cassette (eg, containing a counter-selectable marker (eg, URA3 or kanR)) into the plasmid to form a recipient nucleic acid, and then Replacing the cassette with a donor nucleic acid can be included. In one embodiment, inserting the cassette removes the target-binding sequence.

  In one embodiment, each modified polypeptide is shared, for example, by being fused to a cell-associated anchor domain, eg, a membrane-associated domain, such as a transmembrane domain or GPI-tethered domain. Joined by joining. In one embodiment, the anchor domain comprises the yeast Aga1p, Aga2p, or a fragment thereof.

In one embodiment, the various donor nucleic acids comprise at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ different donor nucleic acids. For example, the various donor nucleic acids can comprise between 10 ³ -10 ¹² , 10 ³ -10 ¹⁰ or 10 ⁴ -10 ⁹ different donor nucleic acids. In one embodiment, each diverse donor nucleic acid is less than 2000, 500, 200, 120, 80, 50 or 40 nucleotides in length. The various donor nucleic acids can be at least about 20, 40, 100, 500, 1000 or 2000 nucleotides in length. Each diverse donor nucleic acid can be at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% identical to at least another diverse donor nucleic acid. For example, a diverse donor nucleic acid can have 1, 2, 3, or at least 4 mismatched base pairs with respect to another diverse donor nucleic acid.

  In one embodiment, each of the various donor nucleic acids is equal in length to the other or within 30, 20, 15 or 10% of the average length of the various donor nucleic acids. In one embodiment, the various donor nucleic acids of multiple yeast cells all have a length within 8, 6, 4, 3, 2, or 1 nucleotides of each other. Each of the diverse donor nucleic acids comprises at least 6 base pairs of 3 ′ and / or 5 ′ terminal regions that are identical (or at least 70% identical) to the corresponding terminal regions of each of the other diverse donor nucleic acids. Can do. In preferred embodiments, the terminal region is exactly complementary to the corresponding site on the template nucleic acid. In one embodiment, each of the various donor nucleic acids includes a sequence corresponding (eg, partially complementary) to a common region (eg, at least 5 or 10 nucleotides) of the template. Each diverse donor nucleic acid can include naturally occurring sequences or artificial sequences.

  In one embodiment, each diverse donor nucleic acid encodes a CDR or fragment thereof, eg, a fragment comprising at least 5 amino acids. In one embodiment, the various donor nucleic acids are further 3 ′ and / or 5 ′ end regions annealed to sequences flanking the CDR encoding sequence (or its complement), eg, a framework region. Sequence (or its complement), eg, at least 1, 2, 3, 4 or 5 nucleotides thereof. The terminal region is preferably less altered than the sequence between the terminal regions in the various donor nucleic acids. The CDR can be a heavy chain CDR (eg, heavy chain CDR1, CDR2 and CDR3) or a light chain CDR (eg, light chain CDR1, CDR2 and CDR3). In a preferred embodiment, the various donor nucleic acids preferably do not include the entire sequence of the framework regions adjacent to the CDR, eg, less than 2, 5, 8, 10 or 15 amino acids in each adjacent framework region. Contains.

  In another aspect, the invention features a method comprising the following steps: (a) (1) a receiving nucleic acid comprising a first recombination site and a receiving sequence, and (2) a second recombination site and A donor nucleic acid comprising a segment encoding a subject polypeptide, or a complement thereof; (b) a first and second set of each recipient and donor nucleic acid in a cell (eg, a yeast cell). The recombination site is recombined to form a recombinant nucleic acid that encodes a polypeptide of interest and includes a receptor sequence; and (c) so that the polypeptide of interest is detectable on the surface of a cell ( For example, the polypeptide of interest is covalently or non-covalently bound to the cell surface) and expresses the recombinant nucleic acid in the cell. In one embodiment, the donor nucleic acid includes a sequence encoding a CDR.

  In another aspect, the invention features a method of expressing a modified polypeptide. The method comprises the following steps: (a) (1) encoding nucleic acid comprising a segment encoding a heterologous polypeptide domain detectable on the cell surface, and (2) a modifying nucleic acid. (B) recombining the modifying nucleic acid and the encoding nucleic acid in a cell to form a modified nucleic acid encoding the modified polypeptide domain; and (c) detecting the modified nucleic acid on the surface of the cell; As possible, the modified nucleic acid is expressed in cells. The polypeptide can be bound directly or indirectly on the cell surface. For example, a polypeptide can be fused to a transmembrane protein and bound by a covalent bond (eg, a disulfide bond to a transmembrane protein) or by a non-covalent interaction to a transmembrane protein. A polypeptide can also bind to another membrane (eg, a non-transmembrane protein) or cell-associated protein.

The cell may be a microorganism, such as a yeast cell, such as S. cerevisiae. Cerevisiae or S. cerevisiae. It can be a pombe.
The method further includes assessing the properties of the modified nucleic acid, eg, binding to a target (eg, target compound or target cell). Assessing can include contacting the cell with the target and assessing the interaction between the cell and the target, eg, between the modified polypeptide domain and the target. Assessing can further include assessing a quantitative measurement of binding affinity.

  In one embodiment, the cell is a yeast cell, and the method further comprises rearranging the modified nucleic acid for mammalian cell expression and expressing the rearranged nucleic acid in the mammalian cell. Can be included. The method also provides for the biological properties (eg, properties involving mobilization of cytotoxic cells or complement proteins) of modified polypeptides expressed by mammalian cells (eg, polypeptides comprising immunoglobulin domains). It can include evaluating.

  In another aspect, the invention features a method of expressing a modified polypeptide. The method comprises the following steps: (a) (1) a number of encoding nucleic acids, each comprising a segment encoding a polypeptide domain that is detectable on the surface of a cell. The peptide domain is heterologous to the yeast cell, and (2) provides a number of diverse nucleic acids; (b) for each of a number of yeast cell encoding nucleic acids, one of the various nucleic acids and the encoding nucleic acid. Recombined in the cell to form a modified nucleic acid encoding the modified polypeptide domain; and (c) modified so that the modified polypeptide domain is detectable on the surface of the cell. Nucleic acids are expressed in cells.

The cell may be a microorganism, such as a yeast cell, such as S. cerevisiae. Cerevisiae or S. cerevisiae. It can be a pombe.
The method further includes assessing the properties of the modified nucleic acid, eg, binding to the target target compound. Assessing can include contacting the cell with the target compound and recovering a cell that binds to the target compound, eg, a cell that exhibits a modified polypeptide domain that binds to the target compound on its surface. . Assessing can further include assessing a quantitative measurement of binding affinity.

  The method can further include, after recombination, joining the cell to another cell in which, for example, the second donor nucleic acid and the second recipient nucleic acid have been recombined. If cells are allowed to divide prior to conjugation, multiple combinations of recombinant products can be generated.

  In one embodiment, each modified polypeptide is shared, for example, by being fused to a cell-associated anchor domain, eg, a membrane-associated domain, such as a transmembrane domain or GPI-tethered domain. Joined by joining. In one embodiment, the anchor domain comprises the yeast Aga1p, Aga2p, or a fragment thereof.

  In one embodiment, the cell is a yeast cell, and the method further comprises rearranging the modified nucleic acid for mammalian cell expression and expressing the rearranged nucleic acid in the mammalian cell. be able to. The method also assesses the biological properties (eg, properties involving the mobilization of cytotoxic cells or complement proteins) of a polypeptide expressed by a mammalian cell (eg, a polypeptide comprising an immunoglobulin domain). Can be included.

In one embodiment, the various donor nucleic acids comprise at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ different donor nucleic acids. In one embodiment, each diverse donor nucleic acid is less than 2000, 500, 200, 120, 80, 50 or 40 nucleotides in length. The various donor nucleic acids can be at least about 20, 40, 100, 500, 1000 or 2000 nucleotides in length. Each diverse donor nucleic acid can be at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% identical to at least another diverse donor nucleic acid. For example, a diverse donor nucleic acid can have 1, 2, 3, or at least 4 mismatched base pairs with respect to another diverse donor nucleic acid.

  In one embodiment, each of the various donor nucleic acids is equal in length to the other or within 30, 20, 15 or 10% of the average length of the various donor nucleic acids. In one embodiment, the various donor nucleic acids of multiple yeast cells all have a length within 8, 6, 4, 3, 2, or 1 nucleotides of each other. Each of the diverse donor nucleic acids comprises at least 6 base pairs of 3 ′ and / or 5 ′ terminal regions that are identical (or at least 70% identical) to the corresponding terminal regions of each of the other diverse donor nucleic acids. Can do. In preferred embodiments, the terminal region is exactly complementary to the corresponding site on the template nucleic acid. In one embodiment, each of the various donor nucleic acids includes a sequence corresponding (eg, partially complementary) to a common region (eg, at least 5 or 10 nucleotides) of the template. Each diverse donor nucleic acid can include naturally occurring sequences or artificial sequences.

  In one embodiment, each diverse donor nucleic acid encodes a CDR or fragment thereof, eg, a fragment comprising at least 5 amino acids. In one embodiment, the various donor nucleic acids further encode 3 ′ and / or 5 ′ terminal regions, eg, framework regions, annealed to sequences flanking the CDR-encoding sequence (or its complement). It includes a sequence (or its complement), eg, at least 1, 2, 3, 4 or 5 nucleotides thereof. The terminal region is preferably less altered than the sequence between the terminal regions in the various donor nucleic acids. The CDR can be a heavy chain CDR (eg, heavy chain CDR1, CDR2 and CDR3) or a light chain CDR (eg, light chain CDR1, CDR2 and CDR3). In a preferred embodiment, the various donor nucleic acids preferably do not include the entire sequence of the framework regions adjacent to the CDR, eg, less than 2, 5, 8, 10 or 15 amino acids in each adjacent framework region. Contains.

The method can include other features described herein.
In another aspect, the invention features a method comprising the following steps: (a) (1) a receiving nucleic acid comprising a first recombination site and a receiving sequence, and (2) a second recombination site and A donor nucleic acid comprising a segment encoding a polypeptide of interest or a complement thereof; (b) recombining the first and second recombination sites of each recipient and donor nucleic acid in a cell, Forming a recombinant nucleic acid that encodes a peptide and includes a receptor sequence; and (c) the polypeptide of interest is detectable on the surface of a cell (eg, the polypeptide of interest is cellular Recombinant nucleic acid is expressed in the cell (covalently or non-covalently bound to the surface).

  The cell may be a microorganism, e.g., a eukaryotic microorganism such as a yeast cell, e.g. Cerevisiae or S. cerevisiae. It can be a pombe or a prokaryotic microorganism, for example a bacterial cell, for example E. coli.

  In one embodiment, the method further includes introducing a donor nucleic acid into the cell and / or introducing a recipient nucleic acid into the cell prior to recombination. For example, the donor nucleic acid is single stranded and / or the acceptor nucleic acid is single stranded. In one embodiment, the receiving nucleic acid is circular.

  In one embodiment, the method further comprises generating an interruption (eg, a nick or duplex interruption) in the recipient nucleic acid. The interruption can occur in vitro or in the cell. For example, the interruption is generated by induced expression of a cleavage enzyme (eg, HO endonuclease). In another example, the interruption is generated by a cleaved-directed oligonucleotide.

  The method can further include inserting a cassette into the plasmid to form a recipient nucleic acid, wherein the cassette includes a marker. Recombination can replace the cassette with the donor nucleic acid. In one embodiment, inserting the cassette removes the target-binding sequence. In one embodiment, the receiving nucleic acid includes a counter-selectable marker (eg, URA3 or kanR) between the first recombination sites.

  In one embodiment, the donor nucleic acid is single stranded, eg, an oligonucleotide or a single stranded plasmid, or is double stranded, eg, a double stranded plasmid. The donor nucleic acid can include a sequence encoding a CDR, or a segment thereof. The donor nucleic acid can be a member of a diverse nucleic acid pool. The recombinant nucleic acid can encode a polypeptide that includes an immunoglobulin domain. In another embodiment, the recombinant nucleic acid can encode an enzyme or fragment thereof. For example, the donor nucleic acid can encode an active site residue, eg, a residue within 2 angstroms of the bound substrate or cofactor.

  In one embodiment, the first and / or second recombination sites include sites that include site-specific recombination sites, such as yeast FLP recognition sites or lambda attB sites. The method can further include inducing a site-specific recombinase.

  The method can include fusing a first cell comprising a donor nucleic acid and a second cell comprising a recipient nucleic acid. Fusion can be yeast mating. The method can further include inducing a site-specific recombinase or generating an interruption (eg, a double-stranded interruption) in the donor or acceptor nucleic acid, eg, using a HO endonuclease. It is.

  In one embodiment, the acceptor sequence includes a sequence that encodes a first subunit of a multimeric protein and a subject sequence that encodes a fragment of at least a second subunit of the multimeric protein. For example, the multimeric protein is an antibody or a T cell receptor. In one embodiment, the cassette removes sequences encoding CDRs or proteins thereof.

The acceptor and / or donor nucleic acid can be bound (eg, coated) by a nucleic acid binding protein, eg, a single stranded binding protein, eg, recA.
In one embodiment, the recipient and donor nucleic acids are combined in vitro prior to recombination. It is possible to introduce the mixture formed by binding into the cell.

  Providing can include selecting a member of a display library, eg, a cell display library, eg, a yeast display library, with a predetermined property. Providing can further include isolating a recipient nucleic acid from a selected member of the display library and / or introducing a donor nucleic acid into the selected member. Providing a recipient nucleic acid can include amplifying the recipient nucleic acid by PCR and / or isolating a tagged or untagged nucleic acid strand, or oligonucleotide-mediated cleavage. is there.

  The method can further include one or more of the following steps: contacting the cell with the ligand, assessing the binding of the cell to the ligand, or treating the cell based on its ability to bind to the ligand. And after recombination, the cell is conjugated to another cell in which, for example, the second donor nucleic acid and the second recipient nucleic acid have been recombined.

  In one embodiment, the polypeptide of interest is fused to a cell-surface anchor domain, eg, a membrane-associated domain, such as a transmembrane domain or GPI-tethering domain. In one embodiment, the anchor domain comprises: yeast Aga1p, Aga2p or a fragment thereof.

  The method can further comprise ligating the first and second nucleic acids in vitro and introducing the ligated nucleic acid into a cell, wherein the assembly in the cell. The replacement circularizes the nucleic acid.

  In one embodiment, the donor nucleic acid comprises a first non-functional allele of the marker gene and the acceptor nucleic acid comprises a second non-functional allele of the marker gene, wherein the first And the second allele can bind to generate a functional allele of the marker gene.

The method can include other features described herein.
In another aspect, the invention features a method that includes the following steps: (a) providing a first number of yeast cells, each of the first number of yeast cells being a different nucleic acid member, and a cell; A cell comprising a hetero-oligomeric protein detectable on the surface, wherein the nucleic acid member encodes a polypeptide; (b) contacting a first multiple yeast cell with a first target; (c) Selecting a subset comprising one or more first multiple yeast cells, the one or more yeast cells comprising a nucleic acid member expressing a hetero-oligomeric protein that binds to the first target; and (d) The nucleic acid members of the subset of cells are recombined with a diverse pool of donor nucleic acids. At least one recombination reaction phase occurs in the yeast cell to obtain a second large number of yeast cells. The second large number of yeast cells contain cells that each contain a variant corresponding to a subset of cell nucleic acid members. The donor nucleic acid encodes a segment of the functional domain and includes a region that is homologous to the region of the sequence that encodes the hetero-oligomeric protein of one selected cell.

  The method further comprises (e) contacting a cell from a second multiple yeast cell with a second target, and (f) a second library member encoding a polypeptide that binds the second target. Selecting one or more yeast cells comprising:

The second target can be the same as the first target.
The method can further include inserting a marker gene into each nucleic acid member region of the subset of cells prior to recombination, wherein the region encodes a segment of the expressed polypeptide. doing. In one embodiment, the insertion removes the sequence encoding the binding determinant.

Each nucleic acid member of the subset of cells can encode a polypeptide comprising an immunoglobulin variable domain. In such cases, the binding determinant can comprise a CDR or fragment thereof. For example, the first number of yeast cells can comprise at least 10 ³ 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ or 10 ⁸ different cells. The subset can include at least 10, 10 ³ , 10 ³ , 10 ⁵ , 10 ⁶ , 10 ⁷ different cells. The second large number of yeast cells can comprise 10, 10 ³ , 10 ³ , 10 ⁵ , 10 ⁶ , 10 ⁷ different cells.

  The method further includes automatically processing members of the selected subset for recombination, eg, robotic selection of members of the selected subset and members in a common container for recombination. Or placing each in a separate container for recombination. The method can further include automatically evaluating the second number of yeast cell members with characteristics conferred by expression of the corresponding mutant nucleic acid. For example, the method can include electronically recording information obtained by automatically evaluating.

The method can include other features described herein.
In another aspect, the invention includes a method of altering the sequence of a polypeptide. The method includes: (a) providing a first number of yeast cells, the first number of yeast cells comprising a segment each encoding at least a portion of a polypeptide, and a first marker gene. Comprising a different nucleic acid member; (b) providing a second multiple yeast cell, wherein the second multiple yeast cell is a segment each encoding at least a portion of a polypeptide; and Optionally, comprising a different nucleic acid member comprising a second marker gene, wherein the first and second multiple yeast cell nucleic acid members are the first multiple yeast cell members are second. Contains a region of homology so that it can recombine with a number of yeast cell members; (c) form conjugated cells, each conjugated cell being the first number of cells. One parent cell And (d) a nucleic acid member from the parent cell is recombined to produce a first marker gene (and optionally a second marker gene). A conjugated cell is selected that is excluded and in which a recombinant nucleic acid is formed that contains a sequence from a nucleic acid member from the parent cell.

  In one embodiment, the recombinant nucleic acid encodes an immunoglobulin variable domain. In addition, the parent nucleic acids can each encode a portion of an immunoglobulin variable domain. The method can include other features described herein.

In another aspect, the invention features a method comprising the following steps: providing a host bacterial cell comprising a host nucleic acid encoding the first and second subunits of restriction activity and hetero-oligomeric receptor Infecting host bacterial cells with phage particles comprising unmethylated nucleic acids that are cleavable by restriction activity and that encode different first and second subunits of a hetero-oligomeric receptor; Unmethylated nucleic acids are cleaved after infection; recombined cleaved nucleic acids and host nucleic acids at sites in the region encoding the first and second subunits of the hetero-oligomeric receptor; and recombined nucleic acids A phage display particle that exhibits a hetero-oligomeric receptor encoded by is isolated. The unmethylated nucleic acid can further include a first non-functional allele of the marker gene, and the host nucleic acid includes a second non-functional allele of the marker gene, wherein the first and A second non-functional allele can be combined to generate a functional allele.

The method can further comprise selecting with an activity that depends on the functional allele of the marker gene. The method can include other features described herein.
In another aspect, the invention features a method comprising the following steps: (i) a first nucleic acid member encoding a first subunit of a hetero-oligomeric protein, wherein the first subunit Comprises an immunoglobulin light chain variable domain and (ii) a second nucleic acid member encoding the second subunit of the hetero-oligomeric protein, wherein the second subunit is an immunoglobulin heavy chain variable Providing a diploid yeast cell comprising a domain and (iii) a hetero-oligomeric protein detectable on the surface of the cell; sporulating the diploid yeast cell to obtain a first nucleic acid member A first haploid cell comprising (but not comprising a second nucleic acid member) and a second nucleic acid member (only A second haploid cell (not including the first nucleic acid member); one or more comprising each segment encoding a CDR of an immunoglobulin variable light chain domain, or a complement thereof. Is introduced into the first haploid cell or clone thereof such that the introduced nucleic acid recombines with the first nucleic acid member in the first haploid cell or clone thereof, thereby Producing one or more modified first haploid cells; one or a plurality of nucleic acids each comprising a segment encoding the CDR of an immunoglobulin variable heavy chain domain or its complement The introduced nucleic acid is introduced into the second haploid cell or clone thereof such that it recombines with the second nucleic acid member in the second haploid cell or clone thereof; Above A second modified haploid cell; and one or more modified first haploid cells are fused (joined) to one or more modified second haploid cells. To provide cells (eg, diploids) each capable of expressing a hetero-oligomeric protein having an altered immunoglobulin variable light chain domain and an immunoglobulin variable heavy chain domain on the cell surface. The method can include other features described herein. In addition, any polyploid cell that can sporulate to produce a spore that can be fused to another spore can be used. For example, tetraploid cells that produce antibiotic diploid cells can be used. In addition, mating type locus deletions can be used or generated to facilitate mating.

  The methods described herein can be used in a continuous screening method, eg, for yeast cell display. For example, a library of yeast cells is screened for cells displaying proteins that bind to the target. Nucleic acids encoding the indicated proteins are modified by the methods described herein (eg, recombination, conjugation, counter-selectable markers including the introduction of various nucleic acids by transformation) And other combinations), and form a separate display library. For example, the cycle can be repeated until it is identified in the protein to bind to the target with at least a predetermined affinity and / or specificity. The method can be used to generate protein variants from each selected member of the display library without purifying and / or cloning nucleic acids.

  In another aspect, the present invention also provides a yeast cell display library whose nucleic acids comprise members modified by the methods described herein, yeasts modified and isolated by the methods described herein. Also featured are immunoglobulin proteins, including cell display members and immunoglobulins modified and isolated by the methods described herein.

  In another aspect, the invention features an isolated nucleic acid comprising a 5 'recombination sequence, a marker, and a 3' recombination sequence. Each recombination sequence can be at least 10, 20, 30 or 50 nucleotides in length (eg, between 10-50 or 20-50 or 30-100 nucleotides in length). Each recombination sequence can be homologous or identical to a different immunoglobulin framework region. For example, a 5 'recombination sequence can be homologous to FR1, and a 3' recombination sequence can be homologous to FR2, and vice versa. The marker can be a selectable marker, a dominant marker, or a counter selectable marker (eg, URA3). Typically, a marker is a marker that allows cells that have lost the marker to be selectively identified, for example, by growth or other phenotype. The marker can be functional in yeast cells; for example, the marker can be a yeast gene.

  In another aspect, the invention features a yeast cell comprising a heterologous nucleic acid comprising a coding region for encoding an immunoglobulin variable domain, wherein the coding region encodes a functional immunoglobulin variable domain. Such that the coding region is revived upon recombination with one or more other fragments from an immunoglobulin variable domain coding sequence and that encodes a functional immunoglobulin variable domain. It includes one or more portions of a globulin variable domain and a sequence encoding a non-immunoglobulin coding sequence. In one embodiment, the non-immunoglobulin coding sequence comprises a genetic marker, such as a selectable marker or a detectable marker. In one embodiment, the selectable marker is a counter-selectable marker. In one embodiment, the yeast cell further comprises a second coding region for encoding a second immunoglobulin variable domain (eg, the other chain of the antibody). The second coding region can also be disrupted, eg, with a non-immunoglobulin coding sequence, eg, a different genetic marker. At least one of the coding regions is such that an antibody formed by expression of a functional body of the coding region is expressed on the surface of a yeast cell (eg, bound to the plasma membrane, eg, membrane Can be formed (through penetrating domains or GPI anchors). Yeast cells can be yeast cells treated with the nucleic acids described herein (eg, those described above) and can include other features described herein. .

  A “heterologous” sequence refers to a sequence that has been introduced into the cell or in association with a nucleic acid using techniques. The heterologous sequence may be a copy of the endogenous gene, but is inserted, for example, into an exogenous plasmid or into a chromosomal location other than its endogenous location.

  The term “fusion” refers to a single polypeptide chain comprising fused components. Examples of fusion proteins include an immunoglobulin domain and a transmembrane domain or GPI-linked domain. The fused components need not be directly adjacent to each other. For example, one or more other sequences (eg, a linker polypeptide or functional domain) can be located between the fused elements.

  The term “coding region” includes one or more mutations that can encode a polypeptide or prevent coding of a polypeptide, but can be repaired, for example, by recombination, particularly homologous recombination. , Refers to the nucleotide sequence. Examples of mutations include non-coding sequences, separate coding sequences (eg, marker genes), stop codons, frameshift mutations, and the like.

  An “immunoglobulin domain” refers to a domain from the variable or constant domain of an immunoglobulin molecule. Immunoglobulin domains typically contain two beta sheets formed from about seven beta chains, and conserved disulfide bonds (see, eg, AF Williams and AN Barclay 1988 Ann. Rev Immunol.6: 381-405). A variable domain can typically be described as a light chain variable domain (VL) or a heavy chain variable domain (VH). The VH and VL regions are also more conserved, interspersed, termed “complementarity determining regions” (“CDRs”), hypervariable regions, “framework regions” (FRs). Can be subdivided into areas. Framework regions and CDR ranges have been accurately determined (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242. , And Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917). For the definitions herein, the Kabat reference for describing CDRs was used. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A variable domain can include one or more human or humanized framework regions and / or one or more human complementarity determining regions (CDRs).

The term “antibody” or “antibody protein” refers to at least one pair of VH and VL domains, or a sufficiently large VH and VL fragment, in which a VH and VL fragment pair forms an antigen binding site. Thus, the term “antibody” refers to a full-length antibody (eg, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA (eg, IgA1, IgA2), IgD and IgE, but preferably IgG), one Antigen binding fragments lacking the above constant immunoglobulin domains (eg, Fab, F (ab ′) ₂ or single chain antibodies (eg, scFv fragments)) and other structures comprising at least one pair of VH and VL domains. Including, but not limited to. The antibody can comprise two heavy chain immunoglobulin polypeptides and two light chain immunoglobulin polypeptides; one heavy chain immunoglobulin polypeptide and one light chain immunoglobulin polypeptide; It can also be an antibody. The antibody optionally comprises a constant region selected from kappa, lambda, alpha, gamma, delta, epsilon or mu constant region genes. Examples of antibodies include heavy and light chain constant regions substantially from human antibodies, eg, human IgG1 constant regions or portions thereof.

  The antibody is preferably monospecific, eg, a monoclonal antibody or antigen-binding fragment thereof. The term “monospecific antibody” refers to an antibody that exhibits a single binding specificity and affinity for a particular target, eg, an epitope. The term includes “monoclonal antibodies” or “monoclonal antibody components”, which are used herein to refer to a preparation of antibodies or fragments thereof of a single molecular composition.

  In one embodiment, the antibody is a recombinant antibody. The term “recombinant” antibody, as used herein, is isolated from an antibody, recombinant, combinatorial antibody library expressed using a recombinant expression vector transfected into a host cell. Any antibody, splicing human immunoglobulin gene sequences to other DNA sequences, or antibodies isolated from animals (eg, mice, goats or cows) that are transgenic for human immunoglobulin genes It includes all antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies prepared, expressed, produced or isolated by other means.

  The term “indicated antibody” refers to an antibody present on a genetic package such that the indicated antibody is physiologically associated with the genetic package, and the genetic package is at least variable of the indicated antibody. Contains nucleic acid encoding the region. Examples of genetic packages include cells (eg, yeast cells) and phage particles.

  “Antigen binding site” refers to the antigen binding region of a pair of VH and VL. A particular antigen binding region may or may not have a known ligand bound by an antigen binding site.

  The term “recombination” refers to the process of exchanging genetic information between nucleic acid strands. In one example, the process results in the formation of a new nucleic acid strand that includes a region from the donor strand and a region from the acceptor strand. In another example, the process results in the insertion of one strand, such as a donor nucleic acid sequence, into another strand, such as a target or receptor strand.

  As used herein, the term “homologous” is sufficiently related to a reference sequence so that the two sequences recombine with each other in a suitable host cell, particularly in a yeast cell. Points to an array. The number of differences allowed between an almost identical sequence and its reference sequence is defined by the ability of the two sequences to recombine with each other in the host cell. A homologous sequence will be at least 70, 75, 80, 85, 90, 91, 92, 95, 96, 97, 98 or 99% identical, nearly identical, or identical to the reference sequence.

As used herein, the term “recombinase” refers to a polypeptide that stimulates, eg catalyzes, the exchange of genetic information between agents, preferably nucleic acids.
The term “single-stranded DNA binding protein” refers to a polypeptide that binds to single-stranded DNA of a variable sequence with an affinity greater than, for example, 10 μM, 100 nM or 10 nM. However, E. coli SSB protein (single-stranded DNA binding protein) refers to a specific polypeptide (ie SWISSPROT: P02339).

  The details of one or more aspects of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. The contents of all references, pending patent applications and published patents cited throughout this application are specifically incorporated herein.

DETAILED DESCRIPTION The present invention provides, among other things, a method for producing a nucleic acid sequence encoding a polypeptide displayed on the surface of a heterologous cell. In many implementations, a population of nucleic acid sequences is produced (eg, forming a nucleic acid library). The library can be screened using, for example, cell-based display and selection.

  The method generally recombines the donor and acceptor nucleic acids to form a recombinant nucleic acid encoding the indicated polypeptide. A recombination reaction is an in vivo reaction, meaning that resolution of at least the recombination intermediate (in some implementations it may be formed outside the cell) occurs within the cell. Both site-specific recombination and homologous recombination can be used.

  A variety of cells, including yeast, bacteria and mammalian cells, support homologous recombination with heterologous nucleic acids. In particular, yeast cells have been shown to be highly effective in recombining nucleic acid with free ends and homologous counterparts. See, for example, Orr-Weaver et al. (1981) Proc. Natl. Acad. Sci. USA 78: 6354-8. In yeast, sequences with homology of about 100 nucleotides and even sequences with as little as 15 nucleotides can be successfully used for homologous recombination. Oligonucleotides approximately 80 nucleotides in length have been shown to be incorporated at homologous positions and to truncate counter selectable markers (Storici et al. (2001) Nature Biotechnol. 19: 773).

First, various exemplary embodiments are shown.
1. In the first example, the donor and acceptor nucleic acids are combined in vitro and then introduced into the cell.

  A vector for yeast display includes a sequence encoding a promoter and signal sequence, a first coding sequence encoding the N-terminal constant region of an immunoglobulin variable domain (eg, VL), and an immunoglobulin constant domain ( For example, it includes a second coding sequence encoding the N-terminal constant region of CL). A unique restriction enzyme site is located between the first and second coding sequences. At this stage, the CDR regions and intervening framework regions have been removed from the vector so that the vector does not encode a functional immunoglobulin chain.

  In some implementations, the vector can also include an anchor protein or fragment thereof. The anchor protein can be, for example, Aga1p or Aga2p.

  The vector nucleic acid is digested with restriction enzymes and then combined with a diverse pool of nucleic acids encoding the immunoglobulin variable domain and at least the N-terminus of the constant domain. Pooled nucleic acids are homologous to the ends of the linearized vector. Electroporation of a linearized vector and a mixture of various nucleic acids into yeast cells. Intracellular homologous recombination results in circularization of the vector and the introduction of one diverse nucleic acid into each copy of the vector. After recombination, the sequence encoding the immunoglobulin variable domain is complete. At this stage, the vector encodes a polypeptide having a signal sequence and a fully variable domain and a constant domain (eg, VL-CL).

  2. In the second example, a cell display library is screened to identify candidate ligands. Candidate ligands are diversified by recombination to form a secondary cell display library.

  A yeast display library showing Fab fragments is screened for binding to the target molecule. A yeast cell encoding a Fab that binds to the target is selected. A library plasmid encoding the Fab is isolated from yeast cells. The plasmid is digested with a restriction enzyme that uniquely cuts on either side of the region containing one or more CDRs of one of the immunoglobulin chains. A larger fragment of the plasmid (ie, main chain fragment) is purified.

  The linearized plasmid is combined with a diverse pool of nucleic acids encoding one or more excised CDRs. The various nucleic acids may be double stranded or single stranded. One requirement is that the various nucleic acids overlap in sequence with the terminal region of the linearized plasmid. The overlap will be at least 15, 20 or 50 nucleotides at each end. The mixture is transformed with a yeast cell that ensures recombination and introduces the excised CDR coding sequence in association with the antigen binding site selected in the first screen.

  The transformed yeast cells can be used as a cell display library. For example, cells can be rescreened for binding to the target molecule. This strategy allows the polypeptides identified in the initial screen to be diversified in specific regions such as one or more CDRs. As described further below, a variety of recombinantly incorporated nucleic acids can be from a variety of sources; for example, the same population of molecules (thus mixing a combination of specific regions). Combined), synthetic sequences, and sequences of natural origin.

  3. In the third example, as in the second example, candidate ligands from the cell display library are diversified by recombination to form a cell display library. The selected ligand is diversified without isolating the library nucleic acid from the selected cells.

  A yeast display library showing Fab fragments is screened for binding to the target molecule. A yeast cell encoding a Fab that binds to the target is selected. Each cell is transformed with a nucleic acid cassette containing a URA3 marker. The end of the cassette is homologous to one sequence of the Fab chain. The two ends can represent non-contiguous or contiguous sequences.

  If the cassette ends correspond to non-contiguous sequences, transformation and selection of Ura + cells will result in a population of cells in which a portion of the immunoglobulin-encoding sequence has been replaced with a URA3 marker. The URA3 marker can be used to delete one or more CDRs. If the cassette ends correspond to flanking sequences, transformation and selection of Ura + cells will yield a population of cells in which part of the immunoglobulin-encoding sequence has been disrupted by the URA3 marker. The destruction can be, for example, a location within the CDR.

  A nucleic acid that encodes a cell that encodes at least a portion of an immunoglobulin gene sequence that has been deleted or destroyed by a URA3 marker and a region that allows for homologous recombination with the immunoglobulin coding sequence ( Transformation with various pools (single stranded or double stranded). After transformation, cells are grown on 5-fluoroorotic acid (5-FOA) to select ura-cells. Such cells arise when a diverse pool of nucleic acids has been recombined with the Fab coding sequence and replaced with a cassette. As a result, immunoglobulin coding sequences (eg, donor sequences) from various pools are combined with other immunoglobulin sequences (eg, acceptor sequences) isolated from the display library.

  The method can be used to change one strand of a Fab while retaining the sequence for another strand. Furthermore, if the disruption or deletion by the URA3 marker is localized in one or two CDRs rather than all three CDRs in one immunoglobulin chain, the new CDRs in the chain Combinations can be generated.

  4). In the fourth example, two libraries, each encoding a different Fab chain, are constructed in different mating types of yeast cells. The libraries are combined by conjugation and recombination to form the Fab locus, an artificial locus that includes sequences encoding the light chain and sequences encoding the heavy chain.

  Prior to recombination, a library of light chains is constructed using a single conjugated cell. A light chain library (plasmid-carrying or chromosome) contains a polypeptide coding region that encodes the light chain and a site-specific recombination site. The marker gene can be placed such that it is separated from the coding region by the recombination site. Similarly, prior to recombination, a library of heavy chains is constructed using reverse mating cells. The heavy chain library contains polypeptide coding regions that encode heavy chains and site-specific recombination sites. Different marker genes can be placed again, separated from the coding region by the recombination site.

  Cells from the two libraries are mated together. A gene encoding a recombinase specific for the site-specific recombination site is induced. Recombinase activates chain exchange between heavy and light chains in diploid cells produced by conjugation. The result of recombination is the Fab locus. For example, in the case of a chromosomal library, the heavy and light chains are recombined onto the same chromosome.

  The construction of a single locus containing both heavy and light chains is particularly useful for recovering Fabs selected in bulk. If the coding sequence was on a separate strand, after nucleic acid purification from a population of cells expressing different Fabs, the sequences encoding the single Fab heavy and light chains would be Will be randomly selected. In contrast, a single nucleic acid fragment encoding both strands will retain the linkage even when many different Fab coding sequences are manipulated in the same reaction mixture.

  5). In a fifth example, in conjunction with the second example, a cell display library is screened to identify candidate ligands. Candidate ligands are diversified by recombination to form a second cell display library.

  A yeast display library showing Fab fragments is screened for binding to the target molecule. Yeast cells that encode Fabs that bind to the target are selected. A single stranded nucleic acid encoding one or both of the immunoglobulin chains of the Fab is isolated. An oligonucleotide is annealed to a sequence encoding the framework region at one end of one of the CDRs (or its complement). These oligonucleotides form a double stranded region that is cleaved with a restriction enzyme. The result is a linear single stranded nucleic acid from which the CDR coding sequence has been excised.

  This cleaved single stranded nucleic acid is combined with a diverse pool of nucleic acids encoding a CDR coding sequence (or its complement). If the pool nucleic acid is single stranded, the sense of the pool nucleic acid is opposite to the sense of the cleaved single stranded nucleic acid. The mixture is transformed with a yeast cell that ensures recombination and introduces the excised CDR coding sequence in association with the antigen binding site selected in the first screen.

6). In a sixth example, the cell display library is a mammalian cell display library.
A first mammalian cell display library is constructed from a first Fab expression cassette (eg From episomal or integrated). The Fab light chain gene and Fab heavy chain gene are separated by site-specific recombination sites (eg, Cre / lox). The encoded Fab fragment is shown on the surface of a mammalian cell with one of the chains (typically the heavy chain) attached to the eukaryotic transmembrane domain. A second Fab antibody display library comprising a second functional selectable marker (eg, neomycin) and a non-functional selectable marker (eg, a mutated blasticidin resistance gene). Similarly constructed from (eg, episomal or integrated). The non-functional selectable marker of the second cassette is a different allele of the non-functional selectable marker of the first cassette. Recombination between two alleles regenerates a functional allele. Two non-functional alleles can be engineered. Similar to the first cassette, the light and heavy chain genes are separated by site-specific recombination sequences (eg, Cre / lox).

  Two cassettes of Fab antibodies from two different mammalian cell display libraries are combined by cell fusion (eg, using PEG or Sendai virus). The two cassettes enable the generation of recombination intermediates that can be split by strand exchange and homologous recombination between two defective alleles of non-functional selectable markers (eg, two different blasticidin resistant alleles) It is possible to contact each other so that site-specific recombination can occur. Recombination generates a functional blasticidin resistance marker gene. Selection with hygromycin and blasticidin, or neomycin and blasticidin, generates a pool of cells that contain sequences encoding a novel combination of heavy and light chain genes that are not present in the two original libraries.

  7). The seventh example features a phage display repertoire diversified by in vivo recombination. The first pool of Fab antibodies is cloned into a phagemid fused to a p3 phage anchor protein, but with a unique EcoRI restriction site, the first non-functional allele of the first antibiotic resistance marker ( From the Tet gene carrying the first mutation). The phagemid vector also carries a functional and selectable second antibiotic resistance marker (AmpR). A phagemid Fab display library is packaged using helper phage in restriction-negative and modification-negative host cells (RM-) and contains nucleic acids that are not methylated by the selected restriction-modification system. Produces rally phage.

  These phage infect F + E. coli cells, which are restriction positive and modification positive host cells (R + M +). In particular, these cells also harbor nucleic acids from a second pool of constructs encoding Fab antibodies. The second pool of Fab-encoded constructs is cloned into a second expression plasmid carrying a third functional antibiotic resistance marker (eg, chloramphenicol resistance) and the first antibiotic resistance marker A second non-functional allele (eg, a tet resistance gene carrying a second mutation different from the first mutation, but it can complement each other to produce a functionally active Tet gene ) Is separated.

  E. coli strains are restriction and modification plus, so unmethylated DNA is cleaved at the EcoRI restriction site, so the first unprotected phagemid Fab pool is cleaved at the appropriate unique EcoRI restriction site and linearized. Produce a vector. The second pool of Fab antibodies contained in the second expression plasmid is protected and remains uncut. Homologous recombination (or site-specific recombination) is two defects between Fab antibody genes (ie, light and heavy chain genes) and recombined to generate functional resistance markers (eg, tetR). Guaranteed at sites of homology between antibiotic resistance genes (eg, two tet genes with different complementary mutations). This recombined phagemid vector is then rescued by helper phage and used to produce viral particles that show the recombined Fab gene.

  8). In the eighth example, a yeast cell display library is screened to identify candidate ligands. Candidate ligands are diversified by recombination to form a second display library as follows.

  Referring also to FIG. 3, the first yeast display library includes a gene encoding a Fab light chain, a gene encoding a Fab heavy chain, and an auxotrophic marker gene (eg, URA3). Shaped on a yeast plasmid. Two unique incompatible restriction sites, R1 and R2, are located between the Fab heavy chain gene and the auxotrophic marker. The second yeast display library is a gene encoding a Fab light chain, a gene encoding a Fab heavy chain, and an auxotrophic marker gene different from the corresponding marker in the first library. Formed on a yeast plasmid. For example, the auxotrophic marker of the second library would be TRP1 if the corresponding marker in the first library is URA3. Two unique incompatible restriction sites, R2 'and R3, are located between the auxotrophic marker and the Fab light chain gene. The R2 'site is compatible with the R2 restriction site of the first yeast display library. For example, R2 'and R2 can be cleaved by the same restriction endonuclease. In another example, they are cleaved by different restriction endonucleases but nevertheless have compatible attachment overhangs.

  The first and second yeast display libraries are screened to identify Fabs that have at least minimal binding activity against the target molecule. A pool of plasmids encoding the identified Fabs is isolated from each library. The first pool is digested with a restriction endonuclease that cuts at R1 and R2. The second pool is digested with restriction endonucleases that cut at R2 'and R3. Appropriate fragments from each pool (as observed in FIG. 5) are ligated together using the compatible ends formed by R2 and R2 '. The ligation product is a linear DNA containing genes encoding two Fab antibodies and two different auxotrophic markers (eg, URA3 and TRP1).

  Yeast cells are transformed with these linear DNA molecules. In the cell, the DNA molecule is circularized by intramolecular homologous recombination. Recombination can occur at any homologous site, for example within the two light chain genes and the two heavy chain genes. Depending on the execution, recombination can also occur in the intervening region between the heavy and light chain genes. However, it is possible to engineer this region to be heterologous in order to reduce recombination in this region. In another implementation, the heavy and light chain genes lack CH domains (eg, CH1 and CL), eg, they are arranged like scFv. CH domain loss is advantageous for recombination within variable domains where diversity is observed. Following transformation, cells can be grown under conditions selective for both auxotrophic markers, for example, in media lacking uracil and tryptophan.

  9. In the ninth example, the cell display library is diversified by V gene shuffling using a combination of site-specific recombination and homologous recombination. A diversified library is formed by joining cells from two different pools, introducing site-specific recombination, and selecting for gene shuffling events.

  Referring also to FIG. 4, the first pool of yeast cells (FIG. 4, top left) shows that the gene encoding the Fab light chain, the gene encoding the Fab heavy chain, the non-functional first And an expression plasmid having a first allele of the auxotrophic marker (eg, leu2-701) and a functional second auxotrophic marker (eg, TRP1). A site-specific recombination site (eg, a lox site) is located between the light and heavy chain genes. leu2-701 is a non-functional allele of LEU2 and contains a stop codon within the 5 'region (eg, within the first 20 codons) of the LEU2 coding region. Such alleles can be created by genetic engineering. The first pool of yeast cells has a first mating type.

  The second pool of yeast cells (FIG. 4, top right) shows the gene encoding the Fab light chain, the gene encoding the Fab heavy chain, the second non-function of the first auxotrophic marker. And an expression plasmid having a sex allele (eg, leu2-702) and a functional third auxotrophic marker (eg, URA3). The functional third marker is typically different from the functionality of the first pool, the second marker. A site-specific recombination site (eg, a lox site) is located between the light and heavy chain genes. leu2-702 is a non-functional allele of LEU2 and contains a stop codon in the 3 'region of the LEU2 coding region. Such alleles can be created by genetic engineering. The second pool of yeast cells has a second mating type.

The two pools of cells are combined so that the two pools of cells join, for example, in multiple combinations. Appropriate site-specific recombinases are expressed during or after conjugation. For example, a gene encoding recombinase can be controlled by a pheromone-inducible promoter such as FUS1, or a diploid-specific promoter. The recombinase induces site-specific recombination and can form a large plasmid containing sequences from the first pool of plasmids and the second pool of plasmids. Selection of the functional allele of the first auxotrophic marker requires homologous recombination between the first and second alleles of the marker. After conjugation and growth under any non-selective conditions, the conjugated cells require first and second markers (eg, Trp + Leu + cells), or require first and third markers. It is possible to select with medium (eg Ura + Leu + cells). The resulting cells contain a diverse library that encodes Fab antibodies with V gene shuffling such that the light and heavy chains are present in a new combination compared to the combination in the starting pool. Forming.
Site-specific recombination site-specific recombinases have both endonuclease and ligase properties. They recognize the specific sequence of bases in the DNA and exchange the DNA segments adjacent to these segments. A number of site-specific recombination systems have been described from a variety of organisms. Examples include the integrase / att system from bacteriophage lambda (Landy, A. (1993) Current Opinions in Genetics and Dev. 3: 699-707), the Cre / loxP system from bacteriophage P1 (Hoess and Abremski ( 1990), in Nucleic Acids and Molecular Biology, Volume 4: Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and LP of Saccharomyces cerevisiae 2μ circular a system RT / ro F et al. Cell 29: 227-234 (1982)). As exemplified herein, site-specific recombinases can be used to generate nucleic acid combinations of nucleic acid sequences residing on the same strand from parent material residing on different strands. .

Transposases can also be used to transfer nucleic acid segments between strands. Transposases are typically encoded by genes with mobile genetic elements known as transposons. Incorporation of transposons may be random or highly specific. Representatives such as Tn7, which are highly site specific, have been applied to in vivo movement of DNA segments between replicons (Lucklow et al., J. Virol. 67: 4566-4579 (1993)). Artificial transposons can be used to give a nucleic acid sequence mobility from one strand to another. For example, a library of protein coding sequences can be generated within a transposon.
Counter-selectable marker Counter-selectable marker is a marker whose absence allows cell growth or survival, typically under specialized conditions. Counter-selectable markers can be used to select for recombination events that excise the marker. These markers can be used in many cell types, including yeast and mammalian cells.

  For example, URA3 and CAN1 are A counter that works with cerevisiae-a selectable marker. CAN1 confers sensitivity to canavanine. Loss of the CAN1 marker is selected by growing the cells in canavanine. URA3 is particularly useful if the host cell is ura-, because both its presence and absence can be selected. The presence of URA3 can be selected by growth on minimal medium lacking uracil, while its absence can be selected by growth on medium containing 5-FOA.

For mammalian cells, the thymidine kinase gene can be used as a counter-selectable marker. The loss of the tk gene can be selected by growth in a medium containing ganciclovir. Many other counter-selectable markers are known for a variety of systems.
Cell-based display library In yet another manner, the library is a cell display library.

  The protein is shown on the surface of a cell, eg, a eukaryotic or prokaryotic cell. Examples of prokaryotic cells include E. coli cells, Subtilis cells, spores are included (see, eg, Lu et al. (1995) Biotechnology 13: 366). Examples of eukaryotic cells include yeast (eg, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula, or Pichia pastors). One implementation of yeast surface display is described by Boder and Wittrup (1997) Nat. Biotechnol. 15: 553-557.

  WO 03 / 029,456 and U.I. S. S. N. 60 / 326,320, among others, describes a yeast display system that can be used to display immunoglobulin proteins such as Fab fragments, and the use of conjugation to generate heavy and light chain combinations.

  In one embodiment, the nucleic acid encoding the immunoglobulin variable domain is cloned into a vector for yeast display. Cloning encodes a nucleic acid encoding at least one of the variable domains and a fragment of a yeast cell surface protein, eg, Flo1, a-agglutinin, α-agglutinin or a fragment thereof, eg, Aga2p, Aga1p. The nucleic acids that are present are joined together. The domains of these proteins are encoded by diverse nucleic acid sequences by GPI-anchors (eg, a-agglutinin, α-agglutinin or fragments thereof, eg, Aga2p, Aga1p), by transmembrane domains (eg, Flo1). It is possible to tether the polypeptides. A vector can be formed to express two polypeptide chains on the cell surface such that one of the chains is linked to a yeast cell surface protein. For example, the two chains will be immunoglobulin chains.

A number of methods are available for introducing nucleic acids into yeast cells, including LiAc / PEG methods, electroporation and spheroplast methods.
Examples of cells for mammalian cell display include: COS cells, CV-1 cells, OVCAR cells, lymphocytes, lymphocyte cell lines (eg, NS0 myeloma cells and SP2 cells, and Chinese hamster ovary cells (CHO cells)). Is included.

Furthermore, as has been seen before, some implementations are also suitable for other display methods, such as phage display.
Examples of execution of the yeast mating the method, S. Utilizes conjugation of cerevisiae cells. The first cell has a first mating type, eg, MATa; the second cell has a second mating type different from the first, eg, MATα. When two cells are brought into contact with each other, yeast mating produces a single cell (eg, MATa / α) with a nucleus formed by fusion of the genomes of both the first and second cells. The fusion phenomenon directs the first cell display library nucleic acid and the second nucleic acid into the same nucleus and provides an opportunity for them to recombine. Recombination between two similar loci (eg, two artificial Fab loci) can result in a new Fab coding sequence that includes CDRs from both parental loci. For example, homologous recombination crossover can occur in the region between CDR2 and CDR3, resulting in a recombinant sequence having sequences encoding CDR2 from one parental locus and CDR3 from another parent .

  In another example, site-specific recombination is induced. For example, see Example 4. A variation of this example is that site-specific recombination is induced between two complete Fab loci.

  The nucleic acid of the second cell can also be a display library nucleic acid, eg, a nucleic acid selected simultaneously with or separately from the first nucleic acid. The nucleic acid can be a non-display library nucleic acid, eg, a linear nucleic acid such as a PCR amplification product or a synthetic oligonucleotide.

The method provides a large number of first cells, all in the same first mating type, each first cell being a polypeptide having a first property (encoded by a first nucleic acid). Providing a large number of second cells, all in the same second mating type, each second cell having a second characteristic and in at least some areas Homologous to the first nucleic acid. Cells are fused, for example, in multiple pairs of combinations. Induces or enables recombination. The polypeptide is then displayed with the changed properties, eg, the first or second properties, or the new properties. This method is used to “breed” the protein.
Recombinant stimulants A variety of agents can be used to stimulate recombination. For embodiments that occur completely intracellularly, such agents can be provided, for example, by inducing gene expression, typically using a heterologous expression cassette. For embodiments initiated outside the cells, the agents can be, for example, those that can be contacted with the nucleic acid prior to their introduction into the cell, can be introduced simultaneously with the nucleic acid, or can be expressed from within the cell. Is possible.

  For example, donor nucleic acids for homologous recombination can be treated in vitro with agents that stimulate recombination. It is possible to coat nucleic acids with agents. The agent can be a polypeptide such as a recombinase or a single stranded DNA binding protein.

  A recombinase can be a recA-like protein that multiplies like a filament on the incoming ssDNA, by showing one of the annealed strands from the duplex with the incoming strand Induces strand exchange. Such proteins include eukaryotic proteins such as Rad51, Rad52, Rad54, DMC1 and mei3; and E. coli recA (eg recA-803 and wild type recA), E. coli SSB (single chain binding protein) and T4 bacteriophage gene 32. Prokaryotic proteins such as In addition, many organisms have a recA-like chain transport protein (eg, Fugisawa et al. (1985) Nucl. Acids Res. 13: 7473; Hsieh et al. (1986) Cell 44: 885; Hsieh et al., ( 1989) J. Biol.Chem.264: 5089; Fishel et al., (1988) Proc.Natl.Acad.Sci.USA 85: 3683; Cassuto et al., (1987) Mol.Gen.Genet.208: 10; (1987) Mol. Cell Biol.7: 3124; Moore et al., (1990) J. Biol.Chem.19: 11108; Keene et al., (1984) Nucl. Acids Res.12: 3057; Kimiec (1984) Cold. spring Harbor Symp. 48: 675; Kimeic (1986) Cell 44: 545; Korodner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5560; Sugino et al. (1985) Proc. Natl. Acad. USA 85: 3683; Halbrook et al., (1989) J. Biol. Chem. 264: 21403; Eisen et al., (1988) Proc. Natl. Acad. Sci. USA 85: 7481, McCarthy et al., (1988) Proc. Acac.Sci.USA 85: 5854; Lowenhaupt et al. (1989) J. Biol. Chem. 264: 20568). Additional examples include the following: sep1 (Koldner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5560; Tishkoff et al., Molec. Cell. Biol. 11: 2593. ), RuvC (Danderdale et al., (1991) Nature 354: 506), DST2, KEM1, XRN1 (Dykstra et al., (1991) Molec. Cell. Biol. 11: 2583), STPα / DST1 (Clark et al., (1991) Molec Cell.Biol.11: 2576), HPP-1 (Moore et al. (1991) Proc. Natl. Acad. Sci. USA 88: 9067), and uvsX. The recA-803 protein is a highly active mutant of recA. In addition, functionally active fragments of these proteins can be used.

A substantially pure preparation of the polypeptide can be prepared by standard biochemical purification, eg, from E. coli overexpressing the recombinase using methods known in the art, for example, See McEntee et al. (1980) Proc Natl Acad Sci USA 77: 857-861. Alternatively, recA protein can also be purchased from, for example, Pharmacia (Piscataway, NJ). The nucleic acid can be incubated with the purified polypeptide, for example, in a buffer at room temperature for a time sufficient for the polypeptide to attach to or coat the nucleic acid. One method of coating nucleic acid with recA is as follows. Briefly, the nucleic acid is denatured in aqueous solution at 95 ° C. for 5 minutes, immediately cooled to 4 ° C. in 1 minute, and then rapidly centrifuged at 0 ° C. for 20 seconds. The denatured nucleic acid is then mixed with a reaction buffer containing non-hydrolyzable nucleotides, such as ATPγS. Reaction buffer also can include 2mM of Mg ²⁺ C1 _2, 50 mM Tris-HCl pH 7.5, and 0.5mM dithiothreitol 0.5. Coating can be initiated by adding purified recA for 10 minutes at 37 ° C. The concentration of recA can be varied in a ratio of 1: 3 to 3: 1, depending on the concentration of individual bases in the denatured nucleic acid.

  The coated nucleic acid can be isolated, for example, by gel filtration, for example using a spin column. The degree of coating can be evaluated by electrophoresis under native conditions.

Endonucleases Endonucleases that cleave nucleic acids in the phosphate backbone can be used to generate termini that are substrates for recombination. The ends can be made at least partially single stranded, for example, by endogenous exonucleases.

  In Saccharomyces cerevisiae, the HO endonuclease cleaves only three sites in the genome. The cleavage phenomenon digests a single site, the HO cleavage site at the MAT locus. The other two sites are in silent junction cassettes HMRa and HMLα and are resistant to cleavage. The cleavage of the MAT locus triggers a mating type conversion, which is a recombination phenomenon between the MAT locus and one of the two silent junction cassettes. Yeast strains express HO, but can be engineered to avoid conjugative conversion due to deletion of the MAT locus. The HO endonuclease can be used to stimulate recombination between the display library vector and the donor nucleic acid in yeast cells.

  In one embodiment, the display library vector is targeted with a selectable marker cassette that includes a HO cleavage site. Selectable markers, such as URA3, can be both selectable and counter-selectable. In combination with HO endonuclease-mediated cleavage, a donor nucleic acid or a pool of donor nucleic acids can be introduced into a cell. The donor nucleic acid contains ends that are homologous to the vector.

  In another embodiment, an intron, such as a yeast MATa intron, is inserted into the sequence encoding the indicated ligand. For the example of an immunoglobulin display, the intron can be located within the framework coding region between the two CDR coding regions. Introns contain a HO endonuclease site. Since intron sequences are spliced out, this site does not disrupt the translation of the immunoglobulin domain. Recombination in the immunoglobulin coding sequence is induced by expression of the HO endonuclease. The HO gene can be controlled by, for example, an inducible GAL promoter. See, for example, Herskowitz et al. (1991) Methods Enzymol. 194: 132-46. Endonucleases can be induced, for example, during or after transformation with diverse nucleic acid pools, or during or after conjugation to cells containing homologous sequences.

Of course, any endonuclease can be expressed ectopically in the cell. For example, a rare cutter can be used without harmful effects.
Construction of diverse nucleic acids In many implementations, recombinant methods use a pool of diverse nucleic acids as a donor sequence for incorporation into one or more different acceptor sequences. Such diverse pools of nucleic acids can be obtained from a variety of sources.

The construction of the initial design diverse pool is based in part on the intended use, eg, the recipient sequence and the cell type on which recombination occurs. One example of a design includes regions of high (or complete) homology at the 3 ′ and 5 ′ ends of various nucleic acids, and a central segment. In this case, the various oligonucleotides vary to the greatest extent in the central segment and to a lesser extent in the region of homology.

Homologous regions can be designed to be sufficiently homologous to the intended template nucleic acid or consensus sequence to ensure recombination.
PCR amplification PCR can be used to amplify a variety of nucleic acids. The forward and reverse primers are designed to anneal to a region of diversity, eg, a conserved region adjacent to the homologous region. Any mixture containing potential diversity can be a template for amplification. For example, the mixture may be an environmental sample that includes different microorganisms or heterogeneous cell populations (eg, spleen).

Oligonucleotide-directed cleavage A variety of nucleic acids can be obtained by oligonucleotide- directed cleavage . This method results in precise excision of single stranded nucleic acids. In general, synthetic oligonucleotides are single-stranded by the formation of a duplex (eg, a homo- or hetero-2 heavy chain) between the single-stranded region of the oligonucleotide and the region of the nucleic acid from which diversity is derived. Directs cleavage of the target nucleic acid.

  Some example methods for such cutting are described in USSN 09 / 837,306, filed April 11, 2001. In one particular embodiment, the method is used to cleave homo- or hetero-2 heavy chains formed by a large number of diverse single-stranded nucleic acids and one or more cleavage-directed oligonucleotides. It was. Therefore, the method provides easy access to a variety of natural sources. For example, a diverse population of single stranded segments can be excised from a source and used for recombination with a recipient nucleic acid. Examples of application of this method to nucleic acids encoding immunoglobulin domains are described in USSN 60 / 343,954 and WO 03 / 035,842.

Automated oligonucleotide synthesis A variety of nucleic acids can be synthesized , for example, using an automated oligonucleotide synthesizer. The synthesizer can be programmed to generate oligonucleotides that include: specific nucleotides, a mixture of nucleotides, a mixture of trinucleotides (or other oligomers), or artificial nucleotides. Synthesizers typically use 3 ′ phosphoramide-activated and 5′-protected nucleotides to continuously add nucleotides (or oligomers) to a solid support. Oligonucleotides are also described, for example, in photographic plates (see, eg, US Pat. No. 5,143,854, Fodor et al. (1991) Science 251: 767-773; Fodor et al. (1993) Nature 364: 555-556). Can also be synthesized on flat solid supports using jet ink printing (eg, US Pat. No. 5,474,796). These oligonucleotide synthesis methods can be programmed to produce a large number of diverse individual oligonucleotides. After synthesis, the oligonucleotide is released from the array using, for example, chemical treatment or enzymes. The released oligonucleotides are pooled for the diversification methods described herein.
A variety of nucleic acid source display libraries contain mutations at one or more positions in the indicated polypeptides. Mutations at a given position can be synthetic or natural. Some libraries contain both synthetic and natural diversity.

Synthetic diversity libraries can contain regions of diverse nucleic acid sequences that originate from artificially synthesized sequences. Typically, these are formed from a degenerate oligonucleotide population that includes a distribution of nucleotides at each predetermined position. The default sequence inclusion is random in terms of distribution. One example of a degenerate source of synthetic diversity is an oligonucleotide containing NNN, where N is any of the four nucleotides in equal proportions.

  Synthetic diversity can also be made more constrained, for example, by limiting the number of codons in a nucleic acid sequence in a given trinucleotide to a distribution less than NNN. For example, such a distribution can be constructed using fewer than 4 nucleotides at some positions of the codon. In addition, trinucleotide addition techniques can be used to further constrain the distribution.

  The so-called “trinucleotide addition technology” is described, for example, in Virnekas et al. (1994) Nucleic Acids Res. 22 (25): 5600-7. Oligonucleotides are synthesized simultaneously on a solid support with one codon (ie, trinucleotide). The support contains many functional groups for synthesis so that many oligonucleotides are synthesized in parallel. The support is first exposed to a solution containing a mixture of codon sets for the first position. The unit is protected from additional units. The solution containing the first mixture is washed away and the solid support is deprotected so that the second mixture containing the codon set for the second position can be added to the first unit. This process is repeated to assemble a large number of codons continuously. Trinucleotide addition technology allows the synthesis of nucleotides that can encode multiple amino acids at predetermined positions. The frequency of these amino acids can be controlled by the ratio of codons in the mixture. Furthermore, the selection of amino acids at a given position is not limited to quadrants of the codon table, as is the case when a mixture of single nucleotides is added during synthesis.

Natural diversity libraries can include regions of diverse nucleic acid sequences that originate from (or are synthesized on) different natural occurring sequences. An example of natural diversity that can be included in a display library is sequence diversity present in immune cells (see also below). Nucleic acids are prepared from these immune cells and manipulated in a manner for polypeptide display. Another example of natural diversity is sequence diversity between different species of organisms. For example, a variety of nucleic acid sequences can be amplified from environmental samples such as soil and used to construct a display library.
Antibody Display Library In one embodiment, the display library displays a diverse pool of polypeptides, each containing an immunoglobulin domain, eg, an immunoglobulin variable domain. Display libraries are particularly useful, for example, to identify human or “humanized” antibodies that recognize human antigens. Such antibodies can be used as therapeutic agents for treating human disorders such as cancer. Since the constant and framework regions of the antibody are human, these therapeutic antibodies can avoid recognizing and targeting their confidence as antigens. The constant region is also optimized to recruit effector functions of the human immune system. The in vitro display selection process has overcome that the normal immune system is unable to generate antibodies against self-antigens.

  A typical antibody display library shows a protein comprising a VH domain and a VL domain. Display libraries can show antibodies as Fab fragments (eg, using two polypeptide chains) or single chain Fv (eg, using a single polypeptide chain). Other styles can also be used.

  As in the case of Fab and other modalities, the antibodies shown can include constant regions as part of the light or heavy chain. In one embodiment, each chain contains one constant region, as for example in the case of Fab. In another embodiment, additional constant regions are indicated.

  Within a library, for example, recombination can be used to generate mutations in a single immunoglobulin domain (eg, VH or VL) or in multiple immunoglobulin domains (eg, VH and VL). It is. Mutations can occur within an immunoglobulin variable domain, eg, one or more regions of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4 in relation to either or both such heavy and light chain variable domains. It is possible to introduce inside. In one embodiment, mutations are introduced into all three CDRs of a given variable domain. In another desirable embodiment, mutations are introduced into, for example, CDR1 and CDR2 of the heavy chain variable domain. Any combination can be performed.

  Antibody libraries for cell-based displays can be constructed by a number of processes (see, eg, USS 60 / 326,320 and WO 03 / 029,456). Wanna)

  The recombinant methods described herein are diverse, including synthetic nucleic acids, natural nucleic acids, patients (eg, immunized or ill human subjects), and animals (eg, immunized animals). It can be used to capture diversity from sources.

Natural Immune Source In one embodiment, immune cells can be used as a natural source of diversification for mutations in antibodies, MHC-complexes and T cell receptors. Some examples of immune cells are B cells and T cells. Immune cells can be obtained from, for example, humans, primates, mice, rabbits, camels or rodents. Cells can be selected with specific characteristics. For example, T cells that are CD4 ⁺ and CD8 ⁻ can be selected. B cells can be selected at various stages of maturity.

  In another embodiment, fluorescence-activated cell sorting is used to sort B cells that express IgM, IgD, or IgG molecules bound to the surface. In addition, B cells expressing different isotypes of IgG can be isolated. In another embodiment, B or T cells are cultured in vitro. For example, by culturing with feeder cells, or mitogens or other regulatory reagents such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin or American pokeweed mitogen In addition, it is possible to stimulate in vitro.

  In yet another aspect, from a subject having an immunological disorder, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren's syndrome, systemic sclerosis or antiphospholipid syndrome, Isolate cells. The subject may be a human or an animal (eg, an animal model of a human disease or an animal having a similar disorder). In yet another embodiment, cells are isolated from a transgenic non-human animal that contains a human immunoglobulin locus.

  In one embodiment, the cell has an activated somatic hypermutation program. Cells can be stimulated to progress somatic mutagenesis of immunoglobulin genes, for example, by treatment with anti-immunoglobulin, anti-CD40 and anti-CD38 antibodies (see, eg, Bergthorsdottil et al. (2001) J Immunol. 166: 2228). In another embodiment, the cell remains intact.

  Of course, a variety of sequences can be obtained as cDNA generated from mRNA isolated from cells, and samples described herein. Full length (ie, capped) mRNA is isolated (eg, by cleaving uncapped RNA with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and reverse transcription is used to generate cDNA. The reverse transcription of the first (antisense) strand can be performed with any suitable primer and by any method. For example, de Haard et al. (1999) J. MoI. Biol. See Chem 274: 18218-30. The primer binding region will not change between different immunoglobulins, eg, to reverse transcribe different isotypes of immunoglobulin. The primer binding region can also be specific for a particular isotype of immunoglobulin. Typically, a primer is specific for a region that is 3 'to a sequence encoding at least one CDR. Poly-dT primers (eg, for heavy chain genes) or synthetic primers that hybridize to synthetic sequences linked to mRNA strands can also be used.

  The cDNA can be amplified, modified, fragmented or cloned into a vector to form an antibody library. For example, WO 00/70023 and de Haard et al. (1999), which describe a method of cleaving cDNA using oligonucleotide directed cleavage and incorporating immunological diversity into the template immunoglobulin sequence. The above references, and U.S. S. S. N. See 60 / 343,954 and WO 03 / 035,842.

Mouse-derived human immunoglobulin pools of diverse nucleic acids can be obtained from immune cells of the immunized animal. In one embodiment, the immunized animal is a transgenic animal (eg, a mouse) having a human immunoglobulin gene. See, for example, US Pat. No. 6,150,584; Fishwild et al. (1996) Nature Biotechnol. 14: 845-85; Mendez et al. (1997) Nature Genet. 15: 146-156; Nicholson et al. (1999) J. MoI. Immunol. 163: 6898. One such transgenic mouse has a YAC for human heavy chain locus, such as yH1C (1020 kb), and a human light chain locus YAC, such as yK2 (880 kb), as described in WO 94/02602. It is possible to build using. yH1C contains the 870 kb human variable region, all D and JH regions, human μ, δ, γ2 constant regions, and the mouse 3 ′ enhancer. yK2 contains Cκ together with the 650 kb human kappa chain proximal variable region (Vκ), the entire Jκ region, and its flanking sequences. Administration of antigen to such mice causes the generation of human antibodies against the antigen. The spleen of such mice is isolated. MRNA encoding the human antibody gene is extracted and used to generate a nucleic acid library encoding an antibody against the antigen. In some implementations, the library is mutagenized in vitro (eg, affinity is mature) prior to selection and screening.
Cell-Display Library Screening Cell-display technology can be used to obtain ligands that bind to a target. As illustrated, display technology can be used at multiple points during the recombination process. In one example, a cell-display library is screened to identify sequences that are then diversified by recombination. In another example, a cell-display library is constructed recombinantly and then screened. In yet another example, the cell-display library is screened, the cells encoding the binding ligand are recombined to generate a secondary cell-display library, and then the secondary library is screened. .

In general, a ligand can be identified from a cell-display library by one or more cycles of selection. Some examples of selection are as follows.
The panning target molecule is immobilized on a solid support such as a microtiter well, matrix, particle or bead. Contact the display library with the support. A library member having affinity for the target is bound. Non-specifically or weakly bound members are washed from the support. The bound members are then collected for further analysis (eg, screening) or pooled for additional rounds of selection.

One example of magnetic particle processor automation selection uses magnetic particles and magnetic particle processors. In this case, the target is immobilized on magnetic particles as described below, for example. A magnetic particle processor, KingFisher® system from Thermo LabSystems (Helsinki, Finland) was used to select display library members for the target. A display library is contacted with magnetic particles in a tube. Mix beads and library. Next, the magnetic particles are collected with a magnetic pin covered with a disposable sheath and transferred to another tube containing the wash solution. Wash particles with wash solution. In this manner, a magnetic particle processor can be used to continuously transfer magnetic particles to multiple tubes in order to wash nonspecific or weakly bound library members from the particles. After washing, the specifically and / or strongly bound library members are transferred to tubes containing elution buffer to release them from the particles. These eluted library members can be individually isolated for analysis (eg, screening) or pooled for additional rounds of selection.

Fluorescent cell sorting can also be used to sort cells from the FACS cell display library. The cells can be contacted with a target that is fluorescently labeled. Cells that interact with the target can be detected by a FACS sorter and deflected into the container. In addition, cells can be contacted with an unlabeled label to form a cell-label complex. The cell-label complex can then be labeled using, for example, a fluorescent reagent that is specific for the target.

Capillary device for cleaning magnetic particles S. S. N. 60 / 337,755 describes an apparatus and method that can be used to clean magnetic particles in a capillary tube in one implementation. One example of an apparatus features a capillary that contains magnetic particles. A chamber is located between the first magnet and the second magnet. The magnet is attached to a frame that can be moved from a first position to a second position. When the frame moves, the magnetic particles in the capillary are agitated.

  To use an apparatus for display library screening, library members are contacted with magnetic particles having bound targets. Place particles in capillary (before, during, or after contact). The particles are then washed in the capillaries with a cycle of agitation and liquid flow to remove non-specifically or weakly bound library members from the particles. After washing, the bound library members can be eluted or dissociated from the particles and recovered. To amplify the bound cells, for example, the cells can be grown during selection.

  Screening for bound ligands can include measurements to identify ligands that are bound to a specific epitope of the target or have a specific specificity. This can be done, for example, by using a competitive non-target molecule that lacks a particular epitope or is mutated within the epitope (eg, with alanine). These non-target molecules bind the display library to the target in a negative selection method as described below, for example, to capture display library members that are not target specific in the dissociated wash solution. Can be used as a competing molecule or as a pre-eluting agent.

Iterative Selection In one embodiment, display library technology is used in an iterative fashion. The first display library is used to identify one or more ligands for the target. These identified ligands are then diversified by recombination to form a second display library. Higher affinity ligands are selected from the second library, for example by using higher stringency or more competitive binding and wash conditions.

  As discussed, recombination can target a known or likely region at the binding interface. For example, if the identified ligand is an antibody, then recombination can be directed to the heavy or light chain CDR regions as described herein. Similarly, if the identified ligand is an enzyme, recombination can be directed to and near the active site.

  In one example of iterative selection, a method described herein binds at least to a target with minimal binding specificity or activity (eg, an equilibrium dissociation constant for binding greater than 1 nM, 10 nM or 100 nM), The protein ligand is used to initially identify from the display library. A second protein ligand having an enhanced property (eg, binding affinity, kinetics or stability) compared to the first protein ligand, eg, a nucleic acid sequence encoding the first identified protein ligand Is used as a template nucleic acid for the introduction of mutations.

Since the off-rate selective slow dissociation rate would be predictive of high affinity (especially with respect to the polypeptide and its target), the methods described herein can be used for binding to a target. It can be used to isolate ligands that have a desirable (ie, slow) kinetic dissociation rate for action.

  In order to select a slowly dissociating ligand from the display library, the library is contacted with an immobilized target. The immobilized target is then washed with a first solution that removes nonspecifically or weakly bound biomolecules. The immobilized target is then eluted with a second solution that contains a saturating amount of free target, ie, replication of the target that is not bound to the particle. The free target binds biomolecules that have dissociated from the target. Rebinding is effectively prevented by a saturating amount of free target for a lower concentration of immobilized target.

  The second solution can have solution conditions that are substantially physiological or stringent. Typically, the solution conditions of the second solution are the same as the solution conditions of the first solution. The fractions of the second solution are collected in chronological order to distinguish the early to late fractions. The late fraction contains biomolecules that dissociate at a slower rate from the target than the biomolecules of the early fraction.

  Furthermore, it is possible to recover display library members that remain bound to the target even after extended incubation. These are targeted under conditions that support cell division, for example, by cleaving the target from the support or by allowing the bound cells to divide and outnumber the available binding sites on the solid support. Can be dissociated by incubation.

Selection or Screening for Specificity The display library screening methods described herein can include a selection or screening process that discards display library members bound to non-target molecules. In one implementation, a so-called “negative selection” process is used to distinguish between targets and related non-target molecules, and related but different non-target molecules. A display library or a pool thereof is contacted with a non-target molecule. Sample members that do not bind to the non-target molecule are collected and used for subsequent selection for binding to the target molecule or subsequent negative selection. The negative selection step can be prior to or after selecting library members that bind to the target molecule.

After selecting candidate display library members that bind to the screening target of individual library members , each candidate display library member can be further analyzed, for example, to further characterize its binding properties to the target. is there. Each candidate display library member can be subjected to one or more secondary screening assays. Assays can be of binding properties, catalytic properties, physiological properties (eg, cytotoxicity, renal clearance, immunogenicity), structural properties (eg, stability, conformation, oligomerization state) or other functional properties It is possible for that. For example, the same assay can be used repeatedly with varying conditions to determine pH, ionicity, or heat sensitivity.

  Where appropriate, the assay can be performed by synthesizing display library members directly, recombinant polypeptides produced from nucleic acids encoding the indicated polypeptide, based on the sequence of the indicated peptide. Peptides can be used. Examples of assays for binding properties include:

Polypeptides encoded by an ELISA display library can also be screened for binding properties using an ELISA assay. For example, each polypeptide is contacted with a microtiter plate whose bottom surface is coated with a target (eg, a limited amount of target). The plate is washed with buffer to remove non-specifically bound polypeptides. Next, the amount of polypeptide bound to the plate is determined by searching the plate with an antibody capable of recognizing the polypeptide, eg, a tag or constant portion of the polypeptide. The antibody is linked to an enzyme, such as alkaline phosphatase, that produces a colorimetric product when provided with the appropriate substrate. Polypeptides can be purified from cells or can be assayed in display library formats, eg, as cell surface proteins. In another variation of the ELISA assay, each polypeptide of the diversity chain library is used to coat different wells of a microtiter plate. Next, to examine each well, an ELISA is started using a universal target molecule.

The binding interaction of a candidate polypeptide with a homogeneous binding assay target can be analyzed using a homogeneous assay (ie, no additional liquid handling is required after adding all components of the assay). . For example, fluorescence resonance energy transfer (FRET) can be used as a homogeneous assay (see, eg, Lakowicz et al., US Pat. No. 5,631,169; Stavrianopoulos et al., US Pat. No. 4,868,103). I want to be) The fluorophore label on the first molecule (eg, the molecule identified in the fraction) causes the emitted fluorescent energy to be reduced if the second molecule is present in close proximity to the first molecule. Is selected so that it can be absorbed by a fluorophore label on the molecule (eg, target) The fluorescent label on the second molecule fluoresces when it absorbs the transferred energy. Since the efficiency of energy transfer between labels is related to the distance that the molecules are separated, it is possible to evaluate the spatial relationship between the molecules. In situations where binding occurs between molecules, the fluorescence emission of the “receptor” molecule label in the assay should be maximal. Binding events shaped for monitoring by FRET can be conveniently measured by standard fluorometric detection means well known in the art (eg, using a fluorimeter). By titrating the amount of the first or second binding molecule, it is possible to generate a binding curve for estimating the equilibrium binding constant.

  Another example of a homogeneous assay is Alpha Screen (Packard Bioscience, Meriden CT). Alpha Screen uses two labeled beads. One bead generates singlet oxygen when excited with a laser. The other bead generates an optical signal when singlet oxygen is diffused from the first bead and collides with it. The signal is generated only when the two beads are in close proximity. The signal is measured to determine the degree of binding.

A homogeneous assay can be performed while the candidate polypeptide is bound to the cell surface of the presenting cell.
Cell Array In yet another embodiment, a recombinantly generated cell display library is formatted as a cell array. Individual cells or small pools of the library are placed on a grid and cultured. A labeled target can be contacted to the lattice to identify library members that bind to the target. Cell arrays can also be screened with appropriate detectable activity.

Among these, U. S. S. N. 10 / 309,391 describes methods and automation that can be used in connection with the methods described herein.
Examples of Eukaryotic Display Vectors Gene vectors useful for displaying multi-chain polypeptides on the surface of eukaryotic cells will be as described below. A multi-chain polypeptide may be encoded on a single vector, or individual chains of a multi-chain polypeptide may be encoded on another vector. In one example, the vector may exist as a set of vectors, where when the vector pair is present in a single eukaryotic cell, the chains of the multi-chain polypeptide associate and Each chain of a multi-chain polypeptide is encoded in one of the corresponding pair of vectors so that the biological activity of the peptide is shown on the surface of eukaryotic cells. In another example, the display vector may be a dual display vector, wherein the vector (i) expresses a biologically active multi-chain polypeptide in a eukaryotic cell, and Can be displayed on the surface of eukaryotic cells, and (ii) biologically active multi-chain polypeptides can be expressed in prokaryotic cells and displayed on the surface of bacteriophage (or prokaryotic cells) .

  A multi-chain polypeptide is a biologically active polypeptide in which two or more separate polypeptide elements (referred to as chains of a multi-chain polypeptide), which chains are covalently or non-covalently linked. Which may form a polypeptide. For example, a multi-chain polypeptide encoded by multi-chain display vector (s) can form a protein comprising two, three, or four polypeptide chains. Polypeptide chains may be the same (eg, homodimer, homotrimer or homotetramer) or different (eg, heterodimer, heterotrimer or heterotetramer). . In one embodiment, the multi-chain polypeptide is a two-chain or four-chain polypeptide consisting of two different chains. For example, 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, immunoglobulins and biologically active immunoglobulin fragments (eg, Fab). Is done. The multi-chain polypeptide can be IA, IgD, IgE, IgG, IgM or biologically active fragments thereof. The multi-chain polypeptide can be an Fab fragment of Ig, wherein the first polynucleotide of the multi-chain display vector comprises segments encoding the VH and CH domains of the Ig heavy chain and The two polynucleotides comprise segments encoding Ig light chains (ie, VL and CL domains).

  A chain of a multi-chain polypeptide (eg, first strand, second strand, third strand, etc.) is linked to a polynucleotide (eg, each first polynucleotide, second polynucleotide, third polynucleotide, etc.) in an expression vector. Nucleotides, etc.). As previously mentioned, the polynucleotide sequence encoding the chain must be inserted into the same plasmid or under the same gene expression control in order to produce a functional multi-chain polypeptide. There is no sex. For example, the polynucleotides encoding the light and heavy chains of an Ig Fab may be located on separate plasmids and are identical for co-expression and processing into functional multi-chain polypeptides. As such, it is transformed into the host cell.

  The sequence of the polynucleotide encoding the chain of the multi-chain polypeptide need not originate from the same source. For example, the Ig molecule has the desired specificity, the same variable domains (VH and VL) as those from the monoclonal, and the desired properties (eg, to provide human compatibility or specific Can be produced to have constant regions (CH and CL) from different monoclonals. In addition, heterologous polynucleotides encoding chains of multi-chain polypeptides (eg, Ig domains) can be variegated and have the same overall structure, while the amino acid sequences of each other are slightly A family of polynucleotide homologues that encode the altered polypeptide chain is produced. Thus, when homologues are taken up into different host cells and expressed, a large number of polypeptides with altered (chain) sequences are presented, eg, having enhanced biological activity Provide a display library suitable for screening, such as to find homologous multi-chain polypeptides. Such amino acid sequence alterations (or variegations) can be achieved by suitable mutations, or partial synthesis and exchange, or by partial or complete substitution of suitable regions of the corresponding polynucleotide coding sequence. The replacement constant domain portion can be obtained from a compatible recombinant DNA sequence. Given the reasonable selection of expression vector components and compatible host cells, the chains of the multi-chain polypeptide will be presented on the surface of eukaryotic host cells. This can be accomplished, for example, using any number of variable expression vector constructs. Display vectors themselves are known in the art and commercially available (eg, from Invitrogen (Carlsbad, CA); Stratagene (La Jolla, CA); American Type Culture Collection (Manassas, VA)), optional Can be constructed or modified with a number of gene vectors and gene regulatory elements.

  Typically, a vector construct is a fully assembled polymorphism on the surface of a eukaryotic cell transformed with the vector such that the biological activity of the multi-chain polypeptide is demonstrated on the surface of the host cell. To obtain efficient transport and tethering of the chain polypeptide, it is designed to express the polypeptide chain.

  In order to achieve effective cellular expression of a multi-chain polypeptide, a polynucleotide encoding each of the chains of the multi-chain polypeptide functions, for example, in a transcription promoter to regulate the expression of the polypeptide chain. Connected as possible. An effective promoter must function in a eukaryotic system and is optionally effective as a prokaryotic promoter as well (especially in the case of dual display vectors). In dual display vectors, the eukaryotic and prokaryotic promoters selected to regulate the expression of the heterologous polypeptide chain of the multi-chain polypeptide may be the same or different promoters (the host for which they are intended). As long as it is properly functional in the organism) or may be independently selected for expression of each strand in a particular host. The eukaryotic promoter can be a constitutive promoter or an inducible promoter. Vector constructs that utilize the same promoter for each strand are preferred to achieve balanced expression and to ensure simultaneous induction of expression. A number of eukaryotic promoters can be used. Examples of eukaryotic promoters include yeast promoters such as galactose inducible promoters, pGAL1, pGAL1-10, pGa14 and pGa110; phosphoglycerate kinase promoter, pPGK; cytochrome c promoter, pCYC1; and alcohol dehydrogenase I promoter, pADH1 Is included. If T7 RNA polymerase is expressed in the cell, the T7 promoter can also be used in prokaryotes and eukaryotes.

  Each of the polypeptides encoding the chains of the multi-chain polypeptide can also be linked to a signal sequence (or leader peptide sequence). The signal sequence serves to direct the transport of nascent polypeptides (often referred to as secretion) into or across the cell. Chains of multi-chain polypeptides expressed in eukaryotic cells from vectors can be transported to the endoplasmic reticulum (ER) for transport to the cell surface for assembly and extracellular display. Effective signal sequences function in eukaryotic systems. Optionally (eg in the case of dual display vectors) the signal sequence is equally effective in prokaryotic systems. The polypeptide encoding the chain of a multi-chain polypeptide is typically linked directly to the signal sequence in frame (immediately adjacent to the polynucleotide or optionally via a linker or spacer sequence). Therefore, a polypeptide chain signal peptide fusion protein is generated. Preferably, each chain of the multi-chain polypeptide is fused to another signal peptide. The signal sequence encoding the signal peptide may be the same or different for each chain of the multi-chain polypeptide. The signal sequence can be native to the host or heterologous so long as it is feasible to achieve extracellular transport of the polypeptide to which it is fused. Several signal sequences are known to those skilled in the art (eg, Mfα1 prepro, Mfα1 prepro, acid phosphatase Pho5, invertase SUC2 signal sequence that can function in yeast; pIII, PelB, OmpA, PhoA signals that can function in E. coli. Sequence; gp64 leader capable of functioning in insect cells; IgK leader capable of functioning in mammals, honeybee melittin secretion signal sequence). The signal sequence can be derived from the host cell's natural secreted protein. Other examples of eukaryotic signal sequences include yeast α-mating factor (eg, S. cerevisiae), yeast α-agglutinin (eg, S. cerevisiae), yeast invertase (eg, S. cerevisiae). ), The signal sequence of Kluyveromyces inulinase, and the signal sequence of the Aga2p subunit of α-agglutinin (eg, of S. cerevisiae) (particularly in embodiments used to tether polypeptides) , An Aga2p polypeptide).

  For example, if the multi-chain polypeptide is a Fab, the first polynucleotide can include an Aga2p signal sequence in frame with segments encoding the VH and CH regions of the Ig heavy chain and a second The polynucleotide comprises an Aga2p signal sequence in frame with a segment encoding an Ig light chain.

  A multi-chain eukaryotic display vector can act in a eukaryotic host cell such that the multi-chain polypeptide encoded by the vector is displayed on the surface of the host cell. Immobilization on the surface of a host cell (“tethering” or “indication”) can be accomplished, for example, by attaching at least one chain of a multi-chain polypeptide to a molecular moiety immobilized on the host cell wall or host cell plasma membrane. It can be achieved by linking. Binding may be covalent or non-covalent. Typically, the linkage is covalent (eg, a peptide, disulfide, amide or other linkage). More than one chain of a multi-chain polypeptide can be linked to an anchor. In general, a fully assembled multi-chain polypeptide has only one point of attachment to the host cell surface. Although more than one attachment can be used, only one chain of a multi-chain polypeptide is required to be attached to a cell. Display on the surface of the cell can be achieved by linking at least one polypeptide chain to an anchor protein or functional fragment thereof. An effective anchor must be functional in a eukaryotic system, and optionally (especially in the case of dual display vectors) the anchor should be equally effective as an anchor on the surface of the bacteriophage . The anchor can be a surface expressed protein native to the host cell (eg, a transmembrane protein or a protein that is linked to the cell surface via a glycan bridge). Several functional anchor proteins can be used (eg, ~ 111, pVI, pVII1, LamB, PhoE, Lpp-OmpA, flagellin (FliC) or their transmembranes functional in prokaryotes / phages Partial; platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosyl phosphatidylinositol (GPI) anchor; gp64 anchor in insect cells, etc. that can function in mammalian cells). When yeast is the host, the anchor protein can be α-agglutinin (having the accessory components Aga1p and Aga2p) or FLO, which naturally forms a bond to the yeast cell surface. is there.

  Binding of the polypeptide chain to the anchor can be accomplished directly or indirectly, by a variety of biological techniques, and by other indirect linkage engineering techniques (eg, disulfide bonds, or non-covalent bonds). Interaction).

  One example method of chain-anchor binding is through the construction of a chain: anchor fusion protein. The polypeptide encoding the chain of the multi-chain polypeptide is linked directly to the anchor in frame (immediately adjacent to the polynucleotide, or optionally via a linker or spacer sequence), and Thus, a polypeptide comprising a signal peptide and a chain: anchor fusion protein has been generated. Other modes of peptide-peptide bonds are known and can be used to achieve effective chain-anchor bonds. For example, a chain of a multi-chain polypeptide may be an intermediate, such as a high affinity interaction of Jun and Fos leucine zipper c-jun / fos, to covalently link the polypeptide chain to a phage or host cell anchor. It can be indirectly linked to the anchor via association (see, eg, Crameri and Suter, 1993; Crameri and Blaser, 1996).

  A multi-chain display vector can include a cloning site that facilitates transfer of a polynucleotide sequence encoding a chain of the multi-chain polypeptide. Such vector cloning sites can include at least one restriction endonuclease recognition site located in the reading frame to facilitate excision and insertion of the polynucleotide segment. Any restriction site known in the art can be utilized in the vector construct; most commercially available vectors already contain multiple cloning sites or polylinkers. In addition, genetic engineering techniques useful for incorporating new and unique restriction sites into the vector are known and routinely practiced by those skilled in the art.

  The cloning site can contain a minimum of one restriction endonuclease recognition site that allows insertion or excision of a single polynucleotide fragment. More typically, two or more restriction sites can be defined, for example, greater control of insertion (eg, orientation of insertion), and greater flexibility of manipulation (eg, more than one polynucleotide fragment). Used to provide directed transport).

  In one example of the vector construct disclosed in US Serial No. 60 / 326,320 and WO 03/029456, the multi-chain polypeptide is a Fab and the first polynucleotide is an Aga2p anchor in frame. Together with a segment encoding the VH and CH regions of the Ig heavy chain in frame, together with an Aga2p signal sequence, wherein the Ig heavy chain region has unique restriction sites (eg, SfiI and NotI And the second polynucleotide comprises an Aga2p signal sequence with a segment encoding the Ig light chain in frame, wherein the Ig light chain region has a unique restriction site (eg, ApaLI and AscI)

  In examples of multi-chain eukaryotic display vectors, one or more multi-chain polypeptide chains expressed by the vector in a host cell are linked to a molecular tag or reporter gene. Binding can be accomplished via a fusion protein comprising a polypeptide tag and a chain of multi-chain polypeptides. Examples of tags include epitope tags (eg, tags generated by fusion peptide sequences that are recognized as antigenic determinants for the polypeptide of interest) and metal chelating tags (eg, poly-His tags). included. Examples of epitope tags include HA tags and myc12CA5 tags.

  As previously described, an example of a vector construct for encoding a multi-chain polypeptide such as an immunoglobulin Fab fragment is illustrated below: a first polynucleotide is a segment encoding an Aga2p anchor in frame Together with a segment encoding the VH and CH regions of the Ig heavy chain in frame and with a segment encoding the myc tag in frame, wherein the Ig heavy chain region is a unique restriction. And the second polynucleotide comprises an Aga2p signal sequence with a segment encoding the HA tag in frame and with a segment encoding the Ig light chain in frame. Comprising, where Ig Chain regions are spread by unique restriction sites (e.g., ApaLI, and AscI).

The following are examples of methods for affinity matured Fab antibodies. The method uses repetitive combinatorial junctions of LC and HC repertoires. These repertoires are diversified by in vivo recombination with various DNA fragments containing CDR coding sequences.
Construction of LC Expression Cassette in Matα1 Ploidy Cell Chromosome Referring to FIG. 5, the LC expression cassette is haploid Matα S. cerevisiae. It is constructed in the yeast chromosome of cerevisiae cells. When the LC of the target-specific lead antibody was cloned into the yeast display vector pTQ6 as an ApaL1 / Asc1 fragment, the result was that the N-terminus was fused to the Aga2p signal sequence and an added HA epitope tag. LC driven by expression of the inducible GAL1 promoter. The orientation of the expression cassette is: GAL1: Aga2p signal sequence (SS): LC: HA epitope tag: CYCtt transcription terminator sequence. The PCR fragment corresponding to this expression cassette is amplified using primers specific for the vector sequence 5 'adjacent to the GAL1 promoter and 3' adjacent to the CYCtt transcription terminator sequence.

  The LEU2 marker gene is amplified by PCR from a host plasmid (eg, pESC) and added to the sequence at the 5 'end, which is homologous to the 3' end adjacent to the CYCtt transcription terminator sequence. PCR product GAL1: SS: LC: HA: CYCtt and LEU2 PCR products are mixed together and a 5 ′ sequence adjacent to the GAL1 promoter and a 3 ′ specific pull through primer adjacent to the Leu marker. When assembled using, the PCR product of GAL1: SS: LC: HA: CYCtt: LEU2 is given.

  Referring also to FIG. 5, the sequences appended to both ends of the PCR product GAL1: SS: LC: HA: CYCtt: LEU2 are two incompatible unique restrictions for cloning into the appropriate yIP plasmid. Contains parts. The PCR product is digested with appropriate restriction enzymes and cloned into the yIP plasmid. Yeast strain BJ5457 (Matα) is then transformed with a yIP vector containing an LC expression vector and selected for integration into the yeast chromosome and Leu + phenotype. This method produces the haploid yeast strain BJ5457-LC.

After successful construction of this strain, it is possible to replace the LC with a counter selectable marker such as URA3 to create a strain for direct cloning of antibody LC using in vivo recombination (FIG. 7A, see top left). LC expression strains can be constructed by amplification of the target LC with primers carrying a 5 ′ flanking sequence homologous to the SS: HA sequence and a 3 ′ flanking sequence to the CYCtt gene.
Construction of the HC expression cassette in the Mata haploid cell chromosome Referring also to FIG. It is constructed in the yeast chromosome of cerevisiae cells. When the target-specific lead antibody HC was cloned as a Sfi1 / Not1 fragment into the yeast display vector pTQ7, the result was that the N-terminus was the signal sequence alpha agglutinin yeast cell surface anchor protein (SS), and its C-terminus. Is an HC driven by expression of an inducible GAL1 promoter fused to an alpha agglutinin yeast cell surface anchor gene (αAGG). The construct further added a c-Myc epitope tag and a CYCtt transcription terminator sequence. The orientation of the expression cassette was: GAL1: SS: HC: c-Myc: αAGG: CYCtt. The PCR fragment corresponding to this expression cassette is amplified using primers specific for the vector sequence 5 'adjacent to the GAL1 promoter and 3' adjacent to the CYCtt transcription terminator sequence.

  The TRP1 marker gene is amplified by PCR from a host plasmid (eg, pYD1) and added to the sequence at the 5 'end, which is homologous to the 3' end adjacent to the CYCtt transcription terminator sequence. PCR product GAL1: SS: HC: c-Myc: αAGG: CYCtt and TRP1 PCR product mixed together, 5 ′ sequence adjacent to GAL1 promoter and 3 ′ specific pull-through primer adjacent to TRP1 marker To give a PCR product of GAL1: SS: HC: c-Myc: αAGG: CYCtt: Trp.

When the sequence is added 3 'adjacent to the end of the PCR product GAL1 promoter and the TRP1 marker:
GAL1: SS: HC: c-Myc: αAGG: CYCtt: TRP1
A PCR product is produced.

  This product contains two unique restriction sites for cloning into the appropriate yIP plasmid. The PCR product is digested with appropriate restriction enzymes and cloned into the yIP plasmid. The yeast strain BJ5459 (Mata) is then transformed with a yIP vector containing an HC expression vector and selected for integration into the yeast chromosome and the Trp + phenotype. This method produces the haploid yeast strain BJ5459-HC-AGG (FIG. 6).

After successful construction of this strain, it is possible to replace HC with a counter-selectable marker such as URA3 in order to create a strain for direct cloning of antibody HC using in vivo recombination. HC expression strains can be constructed by amplification of the target HC with primers carrying a 5 ′ flanking sequence homologous to the SS: cMyc sequence and a 3 ′ flanking sequence for the αAGG gene.
Counter LC-HC targeted disruption haploid yeast with a counter-measurable marker is transformed with diversified oligonucleotides to diversify the CDR3 loop of HC and the CDR3 loop of LC. These oligonucleotides contain a region of diversity (encoding CDR3) flanked by sequences homologous to framework region 3 (FR3) and framework region 4 (FR4) of the target LC and HC. It is out. These homologous sequences allow recombination into chromosomal LC and HC expression cassettes. The counter-measurable marker URA3 or LYS2 is used to select recombinants. These are introduced by targeted deletion of the VH-CDR3 or VL-CDR3 sequence. Subsequent replacement of the URA3 marker by the recombinant oligonucleotide is selected by growth on 5-FOA (FIG. 3). This counter selection reduces or eliminates the presence of wild-type antibody after recombination.

  To make a targeted deletion of the LC-CDR3 sequence, it is homologous at its 5 'end to framework region 3 of antigen-specific LC (FR3) and at its 3' end, framework region 4 (FR4 URA3 cassette carrying the translation region of the URA3 gene sandwiched between sequences homologous to). The FR3-URA3-FR4 PCR product is constructed using PCR and oligonucleotides carrying homology to the URA3 gene and the accompanying FR3 and FR4 homologous sequences. When the yeast strain tethering the LC expression cassette (BJ5457-LC) is transformed with the FR3-URA3-FR4 PCR product and the targeted deletion of LC-CDR3 is selected on a Ura3-selective plate, Yeast strain BJ5457-LC / URA3 is obtained. To determine if recombination has occurred at the correct location in the yeast chromosome, diagnostic PCR with primers specific for the URA3 gene and LC is used to confirm the location of the insertion.

  FIG. 7A illustrates a method for creating a targeted deletion of the HC-CDR3 sequence. URA3 flanked by sequences homologous to the antigen-specific HC framework region 3 (FR3) at its 5 ′ end and homologous to framework region 4 (FR4) at its 3 ′ end A URA3 cassette carrying the gene translation region is constructed. The FR3-URA3-FR4 PCR product is constructed using PCR and oligonucleotides carrying homology to the URA3 gene and the accompanying FR3 and FR4 homologous sequences. A yeast strain tethering the HC expression cassette (BJ5459-HC-αAGG) is transformed with the FR3-URA3-FR4 PCR product and the targeted deletion of HC-CDR3 is selected on a Ura3-selective plate Then, yeast strain BJ5459-HC / URA3 is obtained. To determine whether recombination has occurred at the correct location in the yeast chromosome, diagnostic PCR with primers specific for the URA3 gene and HC is used to confirm the location of the insertion.

Several representative clones are sequenced to determine library size and mutation frequency in the HC-CDR3 sequence.
Construction of a haploid LC-CDR3 and HC-CDR3 repertoire diversified by in vivo recombination A pool of oligonucleotides corresponding to the LC-CDR3 sequence of the target LC is constructed. For oligonucleotide synthesis, spiked through the length of the CDR and synthesized at a ratio of 90% wild type nucleotide and 2.5% A, C, G and T, respectively, FR3-LC / CDR3 ^* -FR4 An oligonucleotide pool is obtained. The region of diversity is flanked by 20 bp sequences that are homologous to FR3 and FR4 of the target LC. Yeast strain BJ5457-LC / URA3 is transformed with FR3-LC / CDR3 ^* -FR4 and homologous recombination is selected by loss of URA3 gene and exchange with mutated FR3-LC / CDR3 ^* -FR4 oligonucleotides . This creates a haploid Matα repertoire BJ5457-LC / CDR3 ^* , where the LC is diversified in the CDR3 loop using mutagenic oligonucleotides recombined in vivo. This repertoire will be about 10 ⁶ in size, or larger or smaller.

A pool of oligonucleotides corresponding to the LC-CDR3 sequence of the target HC is constructed. For oligonucleotide synthesis, spiked through the length of the CDR and synthesized at a ratio of 90% wild type nucleotide and 2.5% A, C, G and T, respectively, FR3-HC / CDR3 ^* -FR4 An oligonucleotide pool is obtained. The region of diversity is flanked by 20 bp sequences that are homologous to FR3 and FR4 of the target LC. Yeast strain BJ5457-LC / URA3 was transformed with FR3-LC / CDR3 ^* -FR4 to perform homologous recombination and growth on 5-FOA (5′-fluoroorotic acid; As required), and by exchange with a mutated FR3-HC / CDR3 ^* -FR4 oligonucleotide. This creates a haploid Mata repertoire BJ5457-HC / CDR3 ^{*, in} which HC is diversified in the CDR3 loop using mutagenic oligonucleotides that are recombined in vivo. This repertoire will be about 10 ⁶ in size, or larger or smaller.

Several representative clones are sequenced to determine library size and mutation frequency in the HC-CDR3 sequence.
Combinatorially conjugated novel Fab (diploid) yeast display of BJ5457-LC / CDR3 ^* and BJ5457-HC / CDR3 ^* repertoires to generate Fab repertoires comprising antibodies with mutated LC-CDR3 and HC-CDR3 To generate a library, two (haploid) host cell populations: a first population containing a repertoire of LC fragments diversified in the CDR3 loop, and HC fragments diversified in the CDR loop A second population containing a repertoire of cells was co-cultured under conditions sufficient to allow cell fusion, and the resulting diploid population was obtained from a Fab (LC-CDR3 ^* / HC-CDR3 ^* ) library. Are grown under sufficient conditions to allow expression and display of

About 10 ¹⁰ BJ5457-LC / CDR3 ^* cells are joined to about 10 ¹⁰ BJ5457-HC / CDR3 ^* cells. A conjugation efficiency of 10% yields about 10 ⁹ diploids (eg, obtaining 10 ⁹ LC / HC combinations of 10 ¹² possible combinatorial LC / HC diversity is haploid parent Giving the maximum starting diversity of the individual components LC and HC repertoire in it) (FIG. 7).
Combinatorial [BJ5457-LC / CDR3 ^* ] [BJ5457-HC / CDR3 ^* ] Affinity-selected diploid repertoires of Fab-displayed repertoires are cultured to induce LC and HC expression. The diploid culture is then incubated with the antigen and selected using a combination of magnetic bead-based selection methods (eg MACS) for the first round of selection followed by flow cytometry sorting. The first round of MACS selection is performed with excess antigen to ensure that all functional clones are enriched, and is approximately 5 times lower than the starting Kd of the target antibody to be affinity matured Affinity-driven selection at concentration follows.

Affinity variants are screened for off-rate using, for example, FACS to quantify affinity improvements after one or more rounds of combinatorial yeast conjugation and in vivo recombination.
Selected diploids comprising Fab antibodies with improved recurrent gene diversification affinity by splitting the selected diploid Fab into LC and HC1 haploid yeast cells and in vivo recombination are Incubation of the isolate under conditions induces sporulation to occur (Guthrie and Fink, 1991). Sporulated diploids were collected, treated with zymolase, sonicated, and plated on rich plates. Haploid colonies were subsequently transferred to selective plates to select either haploid HC cells (-Try selection) or haploid LC cells (-Leu selection). This yields cells BJ5457-HC / CDR3 ^sel and BJ5457-LC / CDR3 ^sel , which contain HC and LC expression cassettes with recombined and optimized CDR3, respectively. Haploid colonies anchoring the improved LC-CDR3 ^* or LC-CDR3 ^* are then subjected to a second round of diversification by in vivo recombination by one of several strategies:
(I) Haploid cells BJ5457-HC with mutagenic oligonucleotides corresponding to FR3-HC / CDR3 ^* -FR4 for HC diversification or FR3-LC / CDR3 ^* -FR4 for LC diversification / CDR3 ^sel and BJ5457-LC / CDR3 ^sel transformation.

  (Ii) Targeted removal of the LC-CDR1 loop and the HC-CDR1 loop using a URA3 expression cassette flanked by sequences homologous to the target LC and HC FR1 and FR2, respectively. Subsequent transformation with mutagenic oligos in which the CDR1 loop is diversified and flanked by sequences homologous to LC or HC FR1 and FR2.

  (Iii) Repetitive targeted removal of the LC-CDR3 loop and the HC-CDR3 loop using a URA3 expression cassette flanked by sequences homologous to the target LC and HC FR1 and FR2, respectively. Subsequent transformation with mutagenic oligos in which the CDR3 loop is diversified and flanked by sequences homologous to the FR1 and FR2 of LC or HC.

A second round of combinatorial yeast conjugation and affinity selection is then performed.
A number of aspects of the invention have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other aspects are within the scope of the claims.

FIG. 1 shows an example of a method for recombining immunoglobulin proteins in yeast cells. FIG. 2 shows an example of a circulation method for recombining immunoglobulin proteins in yeast cells. FIG. 3 shows an example of a method for recombining immunoglobulin proteins in yeast cells for yeast cell display. FIG. 4 shows an example of a method for recombining immunoglobulin proteins in yeast cells for yeast cell display. In FIG. 4, the plasmid in haploid cells contains sites 42 and 44 for site-specific recombination 46. Homologous recombination 48 occurs between two different LEU2 alleles. FIG. 4 shows an example of a method for recombining immunoglobulin proteins in yeast cells for yeast cell display. In FIG. 4, the plasmid in haploid cells contains sites 42 and 44 for site-specific recombination 46. Homologous recombination 48 occurs between two different LEU2 alleles. FIG. 5 shows an example of a method for assembling an immunoglobulin light chain expression nucleic acid into an integrating yeast vector. FIG. 6 shows an example of a method for assembling an immunoglobulin heavy chain expression nucleic acid into an integrating yeast vector. Figure 7 alters immunoglobulin heavy and light chain coding sequences in separate haploid cells and joins cells containing the altered coding sequence to produce a diploid Fab repertoire and bind from the repertoire FIG. 6 shows an example of a method in which sex Fabs are affinity selected, cells that display binding Fabs are sporulated, and optionally additional rounds of affinity maturation are performed. Figure 7 alters immunoglobulin heavy and light chain coding sequences in separate haploid cells and joins cells containing the altered coding sequence to produce a diploid Fab repertoire and bind from the repertoire FIG. 6 shows an example of a method in which sex Fabs are affinity selected, cells that display binding Fabs are sporulated, and optionally additional rounds of affinity maturation are performed.

Claims (77)

A method for altering the sequence of an antibody protein comprising:
Provided are yeast cells comprising heterologous nucleic acids, each comprising a promoter and an operably linked coding region, wherein the coding region comprises a nucleic acid homologous to an immunoglobulin variable domain coding nucleic acid. Comprising;
Introducing one or more Ig segment-encoding nucleic acids into a yeast cell, wherein each of the one or more Ig segment-encoding nucleic acids is a sequence encoding a segment of an immunoglobulin variable domain or a complement thereof. And maintaining the yeast cell under conditions that allow the introduced Ig segment-encoding nucleic acid to recombine with the coding region, whereby the coding region comprises a polypeptide comprising an immunoglobulin variable domain. The variable domain that produces the encoded recombinant nucleic acid is at least partially encoded by a sequence introduced by an Ig segment-encoding nucleic acid,
Said method comprising.   2. The method of claim 1, wherein the polypeptide comprising an immunoglobulin variable domain further comprises expressing the recombinant nucleic acid such that it is displayed on the cell surface and is accessible to the probe.   2. The method of claim 1, wherein the coding region is a non-functional coding region prior to recombination.   Yeast cells containing the recombinant nucleic acid and immunoglobulin variable encoded by the recombinant nucleic acid such that the fused cell contains a nucleic acid encoding a polypeptide comprising Ig LC and a polypeptide comprising Ig HC 2. The method of claim 1, further comprising fusing another yeast cell comprising a nucleic acid encoding an immunoglobulin variable domain that is compatible with the domain.   The method of claim 1, wherein providing a yeast cell comprises introducing one or more Ig segment-encoding nucleic acids into a yeast cell containing a heterologous nucleic acid.   Providing a yeast cell comprises simultaneously introducing into the yeast cell one or more Ig segment-encoding nucleic acids and one or more nucleic acids that are homologous to a region of an immunoglobulin variable domain of a heterologous nucleic acid. The method according to claim 1.   5. The method of claim 4, wherein the fusing comprises yeast mating. A method for altering the sequence of an antibody protein comprising:
A number of yeast cells are provided, each cell of the number of yeast cells comprising an antibody-encoding nucleic acid sequence encoding a unique antibody protein, wherein the antibody protein comprises a light chain variable immunoglobulin domain and a heavy chain variable. Comprising an immunoglobulin domain;
Introducing one or more Ig segment-encoding nucleic acids into one or more cells from a number of yeast cells, wherein each of the one or more Ig segment-encoding nucleic acids comprises a segment of an immunoglobulin variable domain. The coding sequence or its complement; and cells or cells from multiple yeast cells, the introduced Ig segment-encoding nucleic acid recombines with the antibody-encoding nucleic acid sequence of the subset of cells. Producing one or multiple cells that can be maintained under conditions that allow the expression of antibody proteins having modified immunoglobulin variable domains,
Said method comprising.   2. The method of claim 1, wherein each Ig segment-encoding nucleic acid comprises a sequence encoding a CDR of an immunoglobulin variable domain or a complement thereof.   2. The method of claim 1, wherein each Ig segment-encoding nucleic acid comprises a sequence encoding a single CDR of an immunoglobulin variable domain, or a complement thereof.   2. The method of claim 1, wherein the one or more Ig segment-encoding nucleic acids are obtained from a human nucleic acid.   2. The method of claim 1, wherein the one or more Ig segment-encoding nucleic acids comprises multiple nucleic acids encoding different variants of the same CDR.   2. The method of claim 1, wherein the one or more Ig segment-encoding nucleic acids comprise multiple nucleic acids encoding different variants of multiple different CDRs.   11. The method of claim 10, wherein the one or more Ig segment-encoding nucleic acids comprise nucleic acids encoding different variants of immunoglobulin light chain or heavy chain variable domains CDR1, CDR2 and CDR3. .   2. The method of claim 1, wherein each Ig segment-encoding nucleic acid is less than 300 nucleotides in length. 2. The method of claim 1, wherein introducing comprises contacting at least 10 < ³ > different Ig segment-encoding nucleic acids with one or more yeast cells.   2. The method of claim 1, wherein the one or more Ig segment-encoding nucleic acids are single stranded.   9. The method of claim 8, wherein multiple Ig segment-encoding nucleic acids are introduced in parallel into the cells of multiple yeast cells.   9. The method of claim 8, wherein multiple Ig segment-encoding nucleic acids are introduced into the cells of multiple yeast cells in the same reaction mixture.   Prior to the introduction, a counter-selectable marker is inserted into the nucleic acid member of the subset of cells, and after introduction, the cells in which the counter-selectable marker has been replaced by an Ig segment-encoding nucleic acid. The method of claim 1, further comprising selecting.   2. The method of claim 1, further comprising inserting a mutation into the coding region prior to introducing, wherein the mutation prevents expression of a functional immunoglobulin variable domain.   24. The method of claim 21, wherein the mutation comprises a stop codon, marker gene, frameshift, or site-specific endonuclease site.   The polypeptide expressed from the recombinant nucleic acid contains sequences that allow interaction with the cell, such that recovery of the polypeptide results in recovery of cells that contain the nucleic acid encoding the polypeptide. The method of claim 2, comprising:   24. The method of claim 23, wherein the sequence that allows interaction with the cell encodes an anchor domain comprising a transmembrane domain or a domain that becomes GPI-linked to the yeast cell surface.   The antibody-encoding nucleic acid sequence comprises an LC coding sequence encoding a polypeptide comprising an LC variable immunoglobulin domain and an HC coding sequence encoding a polypeptide comprising an HC variable immunoglobulin domain. 9. The method according to 8.   At least at the time of selection, the yeast cell is a diploid cell and the LC-coding sequence so that when the diploid cell sporulates, the LC-coding sequence and the HC-coding sequence are segregated into different spores. 26. The method of claim 25, wherein the and HC-coding sequences are integrated into loci on homologous chromosomes.   27. The method of claim 26, wherein the sitting position is linked to the MAT sitting position.   9. The method of claim 8, wherein the antibody-encoding nucleic acid sequence is integrated into the yeast chromosome.   26. The method of claim 25, wherein the LC and HC coding sequences are integrated into different yeast chromosomes.   7. The method of claim 6, wherein the antibody-encoding nucleic acid sequence comprises a single chain coding sequence that encodes a polypeptide comprising an LC variable immunoglobulin domain and an HC variable immunoglobulin domain.   The method of claim 6, wherein the antibody protein is Fab. A method for selecting cells exhibiting antibody protein comprising:
A number of yeast cells are provided, the number of yeast cells being capable of encoding an antibody protein comprising a light chain variable immunoglobulin domain and a heavy chain variable immunoglobulin domain, or recombinantly with an Ig segment encoding nucleic acid. Each contains an antibody coding sequence capable of forming a functional coding sequence encoding the antibody protein;
Perform one or more of the following cycles:
(I) introducing a nucleic acid comprising a segment encoding a CDR of an immunoglobulin variable domain, or a complement thereof, into a cell comprising at least a portion of an antibody coding sequence from a subset of cells;
(Ii) maintaining the cell in contact with the nucleic acid in (i) under conditions that allow the introduced nucleic acid to recombine with the antibody coding sequence of the cell, thereby providing one or more modified immunoglobulins Producing a modified cell comprising a modified antibody coding sequence having a variable domain;
(Iii) expressing the altered coding sequence in the modified cell;
(Iv) contacting the target with the modified cell; and (v) selecting a further subset of cells from the modified cell, wherein the further subset comprises one or more yeast cells that interact with the target. Become,
Said method comprising.   35. The method of claim 32, wherein at least two cycles are performed.   34. The method of claim 33, wherein the contacting of step (iv) comprises contacting the cell with the target under different conditions during different cycles.   34. The method of claim 33, wherein selecting in step (v) comprises requiring improved binding to the target compared to the previous selection step.   35. The method of claim 32, wherein a marker sequence is inserted into the antibody coding sequence prior to introducing the nucleic acid of step (i).   35. The method of claim 32, further comprising recovering the antibody coding sequence from an additional subset of cells from one or more cycles.   35. The method of claim 32, further comprising sequencing at least the CDR-coding region of the antibody coding sequence in the further subset of cells from one or more cycles.   35. The method of claim 32, wherein the nucleic acid is introduced into a subset of cells.   35. The method of claim 32, further comprising sporulating a subset of cells prior to (i) and joining cells into which nucleic acid has been introduced after (ii) in one or more cycles. the method of.   35. The method of claim 32, wherein providing a large number of yeast cells comprises amplifying the original cells such that the large number of yeast cells are substantially identical.   Providing a large number of yeast cells comprises amplifying a large number of native cells, wherein the native cells contain antibody coding sequences for different antibodies that interact with the same target. 40. The method of claim 32, comprising.   Providing a large number of yeast cells comprises selecting one or more nucleic acids from a phage display library and rearranging the one or more nucleic acids from the phage display system into a yeast expression system. 35. The method of claim 32. A method for altering the subunit of an antibody protein shown on yeast cells, comprising:
Providing a first haploid yeast cell comprising a first nucleic acid member encoding a first subunit of an antibody protein, the first subunit comprising an immunoglobulin variable domain;
A nucleic acid introduced into a first haploid cell or clone thereof into one or more nucleic acids each comprising one CDR-encoding segment of an immunoglobulin variable domain or its complement. Introduced into one haploid cell or a first nucleic acid member in a clone thereof, thereby producing one or more modified first haploid cells; and one or more The modified first haploid cell is conjugated with one or more second haploid cells to provide one or more diploid cells, wherein a second haploid yeast The cell comprises a second nucleic acid member encoding a second subunit of the antibody protein, the second subunit being an immunoglobulin variable domain complementary to the immunoglobulin variable domain of the first subunit. Not including And each diploid cell can express on its cell surface an antibody protein comprising first and second subunits,
Said method comprising.   45. The method of claim 44, comprising inserting a non-immunoglobulin sequence into the first nucleic acid member prior to the introduction, thereby destroying the code of the first subunit.   46. The method of claim 45, wherein the non-immunoglobulin coding sequence comprises a counter-selectable marker. A method for altering the subunit of an antibody protein shown on yeast cells, comprising:
A first haploid yeast cell comprising a first nucleic acid member capable of encoding a polypeptide comprising a first subunit of an antibody protein is provided, wherein the first subunit is an immunoglobulin variable domain Wherein the region of the nucleic acid member encoding the immunoglobulin variable domain is disrupted;
A nucleic acid introduced into a first haploid cell or clone thereof, one or multiple nucleic acids each comprising a segment encoding one CDR of an immunoglobulin variable domain or its complement In combination with the first nucleic acid member in the first haploid cell or clone thereof, thereby producing one or more modified first haploid cells; and The above modified first haploid cells are joined with one or more second haploid yeast cells to provide one or more diploid cells, wherein the second 1 The ploidy yeast cell comprises a second nucleic acid member encoding a polypeptide comprising a second subunit of the antibody protein, the second subunit being an immunoglobulin variable of the first subunit. Complementary immunity to the domain Each diploid cell is capable of expressing an antibody protein comprising first and second subunits on its cell surface.
Said method comprising.   48. The method of claim 47, further comprising amplifying the one or more modified first haploid cells by one or more rounds of cell division prior to joining. A method for altering the subunit of an antibody protein shown on yeast cells, comprising:
A first haploid yeast cell comprising a first nucleic acid member encoding a polypeptide comprising a first subunit of an antibody protein, the first subunit comprising an immunoglobulin light chain variable domain And a second haploid yeast cell comprising a second nucleic acid member encoding a polypeptide comprising a second subunit of the antibody protein, wherein the second subunit is an immunoglobulin heavy Comprising a chain variable domain;
A nucleic acid introduced into a first haploid cell or clone thereof, one or multiple nucleic acids, each comprising a segment encoding the CDR of an immunoglobulin variable light chain domain or its complement Introduced to recombine with the first nucleic acid member in the first haploid cell or clone thereof, thereby producing one or more modified first haploid cells;
A nucleic acid introduced into a second haploid cell or clone thereof into one or more nucleic acids each comprising a segment encoding a CDR of an immunoglobulin variable heavy chain domain or its complement. Introducing into a second haploid cell or a second nucleic acid member in a clone thereof, thereby producing one or more modified second haploid cells; and
An antibody that joins one or more modified first haploid cells and one or more second haploid cells and has an altered immunoglobulin light chain variable domain and an altered immunoglobulin heavy chain variable domain Providing one or more diploid cells each capable of expressing a protein on the cell surface;
Said method comprising. Providing the first and second haploid cells comprises:
(I) a first nucleic acid member encoding a first subunit of an antibody protein, the first subunit comprising an immunoglobulin light chain variable domain, and (ii) a second of a hetero-oligomeric protein Providing a diploid yeast cell comprising a second nucleic acid member encoding the subunit, the second subunit comprising an immunoglobulin heavy chain variable domain; and sporulating the diploid yeast cell A first haploid cell comprising a first nucleic acid member but not a second nucleic acid member, and a second haploid comprising a second nucleic acid member but not the first nucleic acid member Providing cells,
50. The method of claim 49, comprising the steps.   52. The method of claim 49, further comprising amplifying the one or more modified first or second haploid cells by one or more rounds of cell division prior to joining. Method.   50. The method of claim 49, further comprising sporulating one or more diploid cells formed by joining.   50. The method of claim 49, further comprising selecting a subset of one or more diploid cells by contacting one or more diploid cells or clones thereof with a target.   54. The method of claim 53, wherein the subset comprises one or more cells that interact with the target.   54. The method of claim 53, wherein the subset comprises one or more cells that do not interact with the target.   54. The method of claim 53, further comprising sporulating the subset of cells. A method for selecting cells exhibiting a binding protein comprising:
Providing a large number of diverse nucleic acids comprising a protein coding sequence or its complement;
Providing a number of eukaryotic cells, each cell of the number of eukaryotic cells comprising a heterologous nucleic acid comprising a recombination sequence recombinable with homologous sequences present in one or more diverse nucleic acids. Wherein recombination of the acceptor sequence and the various nucleic acids produces a recombinant nucleic acid encoding a polypeptide comprising a segment encoded by the various nucleic acids or its complement;
Introducing nucleic acids from a number of diverse nucleic acids into the cells of a number of cells;
Recombining one or more introduced nucleic acids with a heterologous nucleic acid in each cell, whereby a polypeptide comprising a segment encoded by one or more introduced nucleic acids, or a complement thereof, each Producing a recombinant nucleic acid encoding;
Expressing the recombinant nucleic acid in the cell such that the polypeptide encoded by the recombinant nucleic acid associates with the surface of the cell;
Contacting the target with a cell expressing the recombinant nucleic acid; and selecting one or more cells by function of the interaction with the target;
Said method comprising.   58. The method of claim 57, wherein the heterologous nucleic acid comprises a non-functional immunoglobulin domain coding sequence that provides a accepting sequence for recombination.   59. The method of claim 58, wherein the non-functional immunoglobulin domain coding sequence comprises a stop codon prior to the 3 'end of the immunoglobulin domain coding sequence.   60. The method of claim 59, wherein the stop codon is in a region encoding a CDR.   59. The non-functional immunoglobulin domain coding sequence comprises a marker gene in the region encoding CDR and the sequence encoding the FR region of the immunoglobulin domain is intact. Method.   62. The method of claim 61, wherein the marker gene comprises a counter-selectable marker.   The recombinant nucleic acid encodes and expresses the antibody light chain such that the antibody heavy and light chains associate and the anchor domain anchors the antibody heavy chain to the cell surface. 59. The method of claim 58, further comprising expressing a nucleic acid encoding an antibody heavy chain comprising an anchor domain.   58. The method of claim 57, wherein the heterologous nucleic acid comprises a segment encoding an anchor domain.   58. The method of claim 57, wherein the eukaryotic cell is a yeast cell.   58. The method of claim 57, wherein the target is a protein.   58. The method of claim 57, wherein the target is a mammalian cell.   65. The method of claim 64, wherein the anchor domain comprises a transmembrane domain.   65. The method of claim 64, wherein the anchor domain mediates GPI-linkage.   58. The method of claim 57, wherein the heterologous nucleic acid does not encode a functional protein prior to recombination.   72. The method of claim 70, wherein prior to recombination, the counter-selectable marker is located between the accepting sequences of the heterologous nucleic acid and recombination removes the counter-selectable marker.   58. The method of claim 57, wherein recombination is facilitated by cleaving the heterologous nucleic acid in each cell.   73. The method of claim 72, wherein the cleaving creates a double stranded break in the heterologous nucleic acid.   Prior to recombination, a site recognized by a site-specific endonuclease that can be expressed in yeast cells is located between the acceptor sequences of the heterologous nucleic acid and recombination 75. The method of claim 72, wherein the method is facilitated by providing a site-specific endonuclease in each cell.   75. The method of claim 74, wherein the site-specific endonuclease is a HO endonuclease and providing comprises transcribing a gene encoding the HO endonuclease.   58. The method of claim 57, wherein the recombined nucleic acid comprises a sequence encoding an immunoglobulin variable domain.   The method further comprises selecting one or more recombinant nucleic acids on one basis and repeating the method by providing additional yeast cells, wherein the additional yeast cell coding region is one 2. The method of claim 1, wherein the method is prepared from the selected recombinant nucleic acid.

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