Surface display of recombinant proteins in lower eukaryotes
09845464 · 2017-12-19
Assignee
Inventors
Cpc classification
C12N15/1037
CHEMISTRY; METALLURGY
C07K2317/51
CHEMISTRY; METALLURGY
C12N15/1055
CHEMISTRY; METALLURGY
C40B50/06
CHEMISTRY; METALLURGY
C40B40/08
CHEMISTRY; METALLURGY
C40B40/02
CHEMISTRY; METALLURGY
International classification
C40B40/08
CHEMISTRY; METALLURGY
C40B50/06
CHEMISTRY; METALLURGY
C12N15/10
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
Abstract
Methods for display of recombinant proteins or protein libraries on the surface of lower eukaryotes such as yeast and filamentous fungi are described. The methods are useful for screening libraries of recombinant proteins in lower eukaryotes to identify particular proteins with desired properties from the array of proteins in the libraries. The methods are particularly useful for constructing and screening antibody libraries in lower eukaryotes.
Claims
1. An expression vector comprising a nucleic acid encoding a fusion protein comprising an antibody heavy chain immunoglobulin fused at its C-terminus to the N-terminus of a polypeptide that includes a binding moiety which is selected from the group consisting of: GABAB-R1; GABAB-R2; GR1; GR2; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 1; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 2; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 3; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 4; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 5; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 6; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 7; a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 8; wherein the nucleic acid includes a single stop codon in frame between the nucleotide sequence encoding the antibody heavy chain immunoglobulin and the nucleotide sequence encoding the polypeptide that includes a binding moiety, wherein the expression vector is capable of expressing the fusion protein in a host cell, wherein the host cell is Pichia pastoris.
2. The vector of claim 1 wherein the polypeptide that includes a binding moiety is GABAB-R1.
3. The vector of claim 1 wherein the polypeptide that includes a binding moiety is GABAB-R2.
4. The vector of claim 1 wherein the polypeptide that includes a binding moiety is GR1.
5. The vector of claim 1 wherein the polypeptide that includes a binding moiety is GR2.
6. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 1.
7. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 2.
8. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 3.
9. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 4.
10. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 5.
11. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 6.
12. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 7.
13. The vector of claim 1 wherein the polypeptide that includes a binding moiety is a leucine zipper comprising the amino acid sequence set forth in SEQ ID NO: 8.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(24) The present invention provides a protein display system that is capable of displaying diverse libraries of proteins on the surface of a eukaryote host cell such as a lower eukaryote host cell (e.g., yeast or filamentous fungal cells). The compositions and methods are particularly useful for the display of collections of proteins in the context of discovery (that is, screening) or molecular evolution protocols. A salient feature of the method is that it provides a display system in which proteins of interest can be displayed on the surface of a host cell without having to express the protein of interest as a fusion protein in which the protein of interest is fused to a surface anchor protein.
(25) In general, provided is a method for selecting proteins for displayability on a lower eukaryote cell surface, comprising providing a host cell that expresses a capture moiety comprising a cell surface anchoring protein fused to a first binding moiety; transforming the host cell with a nucleic acid encoding proteins fused to a second binding moiety that is capable of specifically interacting with the first binding moiety fused to the cell surface anchoring protein, wherein mutagenesis is used to generate a plurality of host cells encoding a variegated population of mutants of the proteins; contacting the plurality of host cells with a detection means that specifically binds to proteins that are displayed on the surface of the host cell and does not bind to proteins that are not displayed on the surface of the host cell; and isolating the host cells with which the detection means is bound, wherein the presence of the detection means bound to a protein on the surface of the host cells indicates the proteins are displayable on the lower eukaryote cell surface.
(26) Further provided is a method for selecting recombinant lower eukaryote host cells that display a desired protein on the surface of the host cells, comprising providing host cells that expresses a capture moiety comprising a cell surface anchoring protein fused to a first binding moiety; transforming the host cells with nucleic acids encoding proteins fused to a second binding moiety that is capable of specifically interacting with the first binding moiety fused to the cell surface anchoring protein to produce a plurality of host cells wherein at least one host cell is suspected of displaying the desired protein on the cell surface; contacting the transformed host cells with a detection means that specifically binds to the desired proteins that are displayed on the cell surface; and isolating the host cells with which the detection means is bound to select the host cells that display the desired protein.
(27) Thus, as shown in
(28) Both of the components can be provided in vectors which integrate the nucleic acids into the genome of the host cell by homologous recombination. Homologous recombination can be double crossover or single crossover homologous recombination. Roll-in single crossover homologous recombination has been described in Nett et al., Yeast 22: 295-304 (2005). Each component can be integrated in the same locus in the genome or in separate loci in the genome. Alternatively, one or both components can be transiently expressed in the host cell.
(29) The method enables selection of proteins with desirable binding properties including but not limited to antibodies or fragments thereof (e.g., Fab fragments) of a desired affinity or avidity, enzymes with a particular enzymatic activity or substrate specificity, including catalytic antibodies, receptors with a particular specificity for particular ligands, and fusion proteins including but not limited to those comprising the Fc region of antibody fused to a heterologous protein. In general, the method comprises transforming lower eukaryote host cells with a first nucleic acid expressing a host cell wall binding protein fused at its N- or C-terminus to a first binding moiety such as an adapter peptide capable of pairwise binding to the second adapter peptide and a second nucleic acid expressing a protein to be tested fused at its N- or C-terminus to a second binding moiety such as an adapter peptide capable of pairwise binding to the first adapter peptide. The first and second nucleic acids can be operably linked to the same promoter or to different promoters that are separately inducible. Preferably, the protein of interest is fused to a cellular signal peptide that facilitates shuttle of the fusion protein through the secretory pathway to the cell surface. Expression of first nucleic acids results in the production of the cell wall binding fusion protein, which is transported to the cell surface where it then binds to the surface of the cell with the first binding moiety exposed to the extracellular environment. Expression of the second nucleic acid results in the production of the protein of interest fusion protein, which is transported through the secretory pathway and secreted from the cell. However, as the protein of interest fusion protein is secreted, it is retained on the cell surface because the second binding moiety fused to the protein of interest forms a specific interaction with the first binding moiety fused to the cell wall binding protein.
(30) Further provided is a library method for identifying and selecting cells that produce a particular member of a specific binding pair including but not limited to antibodies and Fab fragments. Therefore, in further aspects, a method of producing a protein that is a member of a specific binding pair, wherein the specific binding pair member is an antibody or antibody fragment, comprising an antibody VH domain and an antibody VL domain, and having an antigen binding site with binding specificity for an antigen of interest. The method comprises providing a library of lower eukaryote host cells displaying on their surface a specific binding pair member, which specific binding pair member is an antibody or antibody fragment comprising a synthetic human antibody VH domain and a human antibody VL domain. The library is created by providing lower eukaryote host cells that express a capture moiety comprising a cell surface anchoring protein fused to a first binding moiety and providing a library of nucleic acid sequences encoding a genetically diverse population of the specific binding pair member, wherein the VH domains of the genetically diverse population of the specific binding pair member are biased for one or more VH gene families and wherein the specific binding pair member includes a second binding moiety that is capable of specifically interacting with the first binding moiety fused to the cell surface anchoring protein. The library of nucleic acid sequences is expressed in the lower eukaryote host cells so that each specific binding pair member is displayed at the surface of a lower eukaryote host cell. Then, cells that produce one or more specific binding pair members having a binding specificity for the antigen of interest are selected by binding the one or more specific binding pair members with the antigen of interest.
(31) The further aspects, the specific binding pair member comprises a synthetic human antibody VH domain and a synthetic human antibody VL domain and wherein the synthetic human antibody VH domain and the synthetic human antibody VL domain comprise framework regions and hypervariable loops, wherein the framework regions and first two hypervariable loops of both the VH domain and VL domain are essentially human germ line, and wherein the VH domain and VL domain have altered CDR3 loops. In further still aspects in addition to having altered CDR3 loops, the human synthetic antibody VH and VL domains contain mutations in other CDR loops. In further aspects, each human synthetic antibody VH domain CDR loop is of random sequence. In further still aspects, the human synthetic antibody VH domain CDR loops are of known canonical structures and incorporate random sequence elements. The binding pair member can be a full-sized or whole antibody or a fragment such as a single-chain Fv antibody fragment.
(32) Detection of host cells that express the desired protein of interest can be achieved by labeling the host cells with a first label, wherein the first label associates with or binds to the protein of interest and does not associate with or bind to host cells which do not express the protein of interest. For example, in the case when the protein of interest is an antibody, the first label can be an antigen that is specifically recognized by the antibody of interest. The host cells with which the first label is associated are selected and the amount of first label associated with the host cell is quantitated. A high occurrence of the first label indicates the protein of interest has desirable binding properties and a low occurrence of the first label indicates the protein of interest does not have desirable binding properties.
(33) A further aspect includes the steps of labeling the above host cells with a second label, wherein the second label associates with or binds to host cells expressing an epitope tag fused to the protein of interest and does not associate with or bind to host cells which do not express the epitope tag. The amount of second label associated with the host cells is quantitated. The amount of the second label associated with the host cell indicates a number of expressed copies of the epitope-tagged protein of interest on the host cell surface and by comparing the quanititation of the first label to the quantitation of the second label enables the amount of the first label normalized for the amount of the second label, wherein a high occurrence of first label relative to the occurrence of the second label indicates the protein to be tested has desirable binding properties.
(34) Another aspect includes the steps of labeling the above host cells with a third label that competes with the first label for binding to the protein of interest. In this aspect, the host cells are labeled with the first label and the amount of first label associated with host cells is quantitated. Then the host cells are labeled with the second label and the amount of second label associated with host cells is quantitated. Comparing the quantitation of the first label to the quantitation of the second label is performed to determine the occurrence of the first label normalized for the occurrence of the second label, wherein a low occurrence of the first label relative to the occurrence of the second label indicates the protein of interest has desirable binding properties.
(35) In further aspects, the first label is a fluorescent label attached to a ligand specific for the protein of interest and the second label is a fluorescent label attached to an antibody specific for the protein of interest. When the labels are fluorescent, the quantitation step is performed by flow cytometry or confocal fluorescence microscopy. In a further still aspect, the first label is a fluorescent label attached to a ligand specific for the protein of interest and fluorescence-activated cell sorting (FACS) is used to separate the host that express the protein of interest from host cells that do not produce the protein of interest.
(36) Further provided is a method for selecting antibodies and fragments thereof with desirable binding properties, performed as described above using a vector in which a single stop codon is place between the nucleic acid encoding the antibody sequence and the nucleic acid encoding the second adapter peptide. The vector is transformed into lower eukaryote host cells comprising nucleic acids expressing a host cell wall binding protein fused at its N- or C-terminus to a first adapter peptide that is capable of pairwise binding to the second adapter peptide. Translation of mRNAs encoded by the vector is performed under conditions that increases translational readthrough through the stop codon thereby producing antibodies that are fused to the second adapter. Labeling the host cells with a first label, wherein the first label associates with or binds to host cells expressing the desired antibodies and does not associate with or bind to host cells Which do not express the desired antibodies enables identification and selection of those host cells that produce the desired antibodies. After the host cells that produce the desired antibodies have been selected and isolated, the host cells are grown under conditions that do result in an increase in translational readthrough through the stop codon. Under the second conditions, the host cells produce antibodies or fragments thereof that are not fused to the second adapter peptide.
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(38) I. General Characteristics of the Adapters
(39) A further consideration in constructing the display system is to select a pair of adapter peptides that encode two adapters capable of pairwise interaction. Whereas a nucleic acid encoding one of the adapter peptides is inserted in-frame with the nucleic acid encoding an exogenous protein of interest carried by the vector, a nucleic encoding the other is fused in-frame with a nucleic acid encoding a cell surface anchoring protein capable of attaching to the outer wall or membrane of the host cell. By “pairwise interaction” is meant that the two adapters can interact with and bind to each other to form a stable complex. The stable complex must be sufficiently long-lasting to permit detecting the protein of interest on the outer surface of the host cell. The complex or dimer must be able to withstand whatever conditions exist or are introduced between the moment of formation and the moment of detecting the displayed polypeptide, these conditions being a function of the assay or reaction which is being performed. The stable complex or dimer may be irreversible or reversible as long as it meets the other requirements of this definition. Thus, a transient complex or dimer may form in a reaction mixture, but it does not constitute a stable complex if it dissociates spontaneously and yields no detectable polypeptide displayed on the outer surface of a genetic package.
(40) The pairwise interaction between the first and second adapters may be covalent or non-covalent interactions. Non-covalent interactions encompass every exiting stable linkage that do not result in the formation of a covalent bond. Non-limiting examples of noncovalent interactions include electrostatic bonds, hydrogen bonding, Van der Waal's forces, steric interdigitation of amphiphilic peptides. By contrast, covalent interactions result in the formation of covalent bonds, including but not limited to disulfide bond between two cysteine residues, C—C bond between two carbon-containing molecules, C—O or C—H between a carbon and oxygen- or hydrogen-containing molecules respectively, and O—P bond between an oxygen- and phosphate-containing molecule.
(41) Adapter peptides applicable for constructing the expression and helper vectors of the display system can be derived from a variety of sources. Generally, any protein sequences involved in the formation of stable multimers are candidate adapter peptides. As such, these peptides may be derived from any homomultimeric or heteromultimeric protein complexes. Representative homomultimeric proteins are homodimeric receptors (e.g., platelet-derived growth factor homodimer BB (PDGF), homodimeric transcription factors (e.g. Max homodimer, NF-kappaB p65 (RelA) homodimer), and growth factors (e.g., neurotrophin homodimers). Non-limiting examples of heteromultimeric proteins are complexes of protein kinases and SH2-domain-containing proteins (Cantley et al., Cell 72: 767-778 (1993); Cantley et al., J. Biol. Chem. 270: 26029-26032 (1995)), heterodimeric transcription factors, and heterodimeric receptors.
(42) Currently used heterodimeric transcription factors are α-Pal/Max complexes and Hox/Pbx complexes. Hox represents a large family of transcription factors involved in patterning the anterior-posterior axis during embryogenesis. Hox proteins bind DNA with a conserved three alpha helix homeodomain. In order to bind to specific DNA sequences, Hox proteins require the presence of hetero-partners such as the Pbx homeodomain. Wolberger et al. solved the 2.35 Å crystal structure of a HoxB1-Pbx1-DNA ternary complex in order to understand how Hox-Pbx complex formation occurs and how this complex binds to DNA. The structure shows that the homeodomain of each protein binds to adjacent recognition sequences on opposite sides of the DNA. Heterodimerization occurs through contacts formed between a six amino acid hexapeptide N-terminal to the homeodomain of HoxB1 and a pocket in Pbx1 formed between helix 3 and helices 1 and 2. A C-terminal extension of the Pbx1 homeodomain forms an alpha helix that packs against helix 1 to form a larger four helix homeodomain (Wolberger et al., Cell 96: 587-597 (1999); Wolberger et al., J Mol Biol. 291: 521-530).
(43) A vast number of heterodimeric receptors have also been identified. They include but are not limited to those that bind to growth factors (e.g. heregulin), neurotransmitters (e.g. γ-Aminobutyric acid), and other organic or inorganic small molecules (e.g. mineralocorticoid, glucocorticoid). Currently used heterodimeric receptors are nuclear hormone receptors (Belshaw et al., Proc. Natl. Acad. Sci. U.S.A 93:4604-4607 (1996)), erbB3 and erbB2 receptor complex, and G-protein-coupled receptors including but not limited to opioid (Gomes et al., J. Neuroscience 20: RC110 (2000)); Jordan et al. Nature 399: 697-700 (1999)), muscarinic, dopamine, serotonin, adenosine/dopamine, and GABA.sub.B families of receptors. For majority of the known heterodimeric receptors, their C-terminal sequences are found to mediate heterodimer formation.
(44) Peptides derived from antibody chains that are involved in dimerizing the L and H chains can also be used as adapters for constructing the subject display systems. These peptides include but are not limited to constant region sequences of an L or H chain. Additionally, adapter peptides can be derived from antigen-binding site sequences and its binding antigen. In such case, one adapter of the pair contains antigen-binding site amino acid residues that is recognized (i.e. being able to stably associate with) by the other adapter containing the corresponding antigen residues.
(45) Based on the wealth of genetic and biochemical data on vast families of genes, one of ordinary skill will be able to select and obtain suitable adapter peptides for constructing the subject display system without undue experimentation.
(46) Where desired, sequences from novel hetermultimeric proteins can be employed as adapters. In such situation, the identification of candidate peptides involved in formation of heteromultimers can be determined by any genetic or biochemical assays without undue experimentation. Additionally, computer modeling and searching technologies further facilitates detection of heteromultimeric peptide sequences based on sequence homologies of common domains appeared in related and unrelated genes. Non-limiting examples of programs that allow homology searches are Blast (http://www.ncbi.nlm.nih.gov/BLAST/), Fasta (Genetics Computing Group package, Madison, Wis.), DNA Star, Clustlaw, TOFFEE, COBLATH, Genthreader, and MegAlign. Any sequence databases that contains DNA sequences corresponding to a target receptor or a segment thereof can be used for sequence analysis. Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS.
(47) The subject adapters that are derived from heterodimerization sequences can be further characterized based on their physical properties. Current heterodimerization sequences exhibit pairwise affinity resulting in predominant formation of heterodimers to a substantial exclusion of homodimers. Preferably, the predominant formation yields a heteromultimeric pool that contains at least 60% heterodimers, more preferably at least 80% heterodimers, more preferably between 85-90% heterodimers, and more preferably between 90-95% heterodimers, and even more preferably between 96-99% heterodimers that are allowed to form under physiological buffer conditions and/or physiological body temperatures. In certain embodiments of the present invention, at least one of the heterodimerization sequences of the adapter pair is essentially incapable of forming a homodimer in a physiological buffer and/or at physiological body temperature. By “essentially incapable” is meant that the selected heterodimerization sequences when tested alone do not yield detectable amounts of homodimers in an in vitro sedimentation experiment as detailed in Kammerer et al., Biochemistry 38: 13263-13269 (1999)), or in the in vivo two-hybrid yeast analysis (see e.g. White et al., Nature 396: 679-682 (1998)). In addition, individual heterodimerization sequences can be expressed in a host cell and the absence of homodimers in the host cell can be demonstrated by a variety of protein analyses including but not limited to SDS-PAGE, Western blot, and immunoprecipitation. The in vitro assays must be conducted under a physiological buffer conditions, and/or preferably at physiological body temperatures. Generally, a physiological buffer contains a physiological concentration of salt and at adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5.
(48) An illustrative adapter pair exhibiting the above-mentioned physical properties is GABA.sub.B-R1/GABA.sub.B-R2 receptors. These two receptors are essentially incapable of forming homodimers under physiological conditions (e.g. in vivo) and at physiological body temperatures. Research by Kuner et al. and White et al. (Science 283: 74-77 (1999)); Nature 396: 679-682 (1998)) has demonstrated the heterodimerization specificity of GABA.sub.B-R1 and GABA.sub.B-R2 in vivo. In fact, White et al. were able to clone GABA.sub.B-R2 from yeast cells based on the exclusive specificity of this heterodimeric receptor pair. In vitro studies by Kammerer et al. supra has shown that neither GABA.sub.B-R1 nor GABA.sub.B-R2 C-terminal sequence is capable of forming homodimers in physiological buffer conditions when assayed at physiological body temperatures. Specifically, Kammerer et al. have demonstrated by sedimentation experiments that the heterodimerization sequences of GABA.sub.B receptor 1 and 2, when tested alone, sediment at the molecular mass of the monomer under physiological conditions and at physiological body temperatures (e.g., at 37° C.). When mixed in equimolar amounts, GABA.sub.B receptor 1 and 2 heterodimerization sequences sediment at the molecular mass corresponding to the heterodimer of the two sequences (see Table 1 of Kammerer et al.). However, when the GABA.sub.B-R1 and GABA.sub.B-R2 C-terminal sequences are linked to a cysteine residue, homodimers may occur via formation of disulfide bond.
(49) Adapters can be further characterized based on their secondary structures. Current adapters consist of amphiphilic peptides that adopt a coiled-coil helical structure. The helical coiled coil is one of the principal subunit oligomerization sequences in proteins. Primary sequence analysis reveals that approximately 2-3% of all protein residues form coiled coils (Wolf et al., Protein Sci. 6: 1179-1189 (1997)). Well-characterized coiled-coil-containing proteins include members of the cytoskeletal family (e.g., α-keratin, vimentin), cytoskeletal motor family (e.g., myosine, kinesins, and dyneins), viral membrane proteins (e.g. membrane proteins of Ebola or HIV), DNA binding proteins, and cell surface receptors (e.g. GABA.sub.B receptors 1 and 2). Coiled-coil adapters of the present invention can be broadly classified into two groups, namely the left-handed and right-handed coiled coils. The left-handed coiled coils are characterized by a heptad repeat denoted “abcdefg” with the occurrence of apolar residues preferentially located at the first (a) and fourth (d) position. The residues at these two positions typically constitute a zig-zag pattern of “knobs and holes” that interlock with those of the other stand to form a tight-fitting hydrophobic core. In contrast, the second (b), third (c) and sixth (f) positions that cover the periphery of the coiled coil are preferably charged residues. Examples of charged amino acids include basic residues such as lysine, arginine, histidine, and acidic residues such as aspartate, glutamate, asparagine, and glutamine. Uncharged or apolar amino-acids suitable for designing a heterodimeric coiled coil include but are not limited to glycine, alanine, valine, leucine, isoleucine, serine and threonine. While the uncharged residues typically form the hydrophobic core, inter-helical and intra-helical salt-bridge including charged residues even at core positions may be employed to stabilize the overall helical coiled-coiled structure (Burkhard et al (2000) J. Biol. Chem. 275:11672-11677). Whereas varying lengths of coiled coil may be employed, the subject coiled coil adapters preferably contain two to ten heptad repeats. More preferably, the adapters contain three to eight heptad repeats, even more preferably contain four to five heptad repeats.
(50) In designing optimal coiled-coil adapters, a variety of existing computer software programs that predict the secondary structure of a peptide can be used. An illustrative computer analysis uses the COILS algorithm which compares an amino acid sequence with sequences in the database of known two-stranded coiled coils, and predicts the high probability coiled-coil stretches (Kammerer et al., Biochemistry 38:13263-13269 (1999)).
(51) While a diverse variety of coiled coils involved in multimer formation can be employed as the adapters in the subject display system. Currentcoiled coils are derived from heterodimeric receptors. Accordingly, the present invention encompasses coiled-coil adapters derived from GABA.sub.B receptors 1 and 2. In one aspect, the subject coiled coils adapters comprise the C-terminal sequences of GABA.sub.B receptor 1 and GABA.sub.B receptor 2. In another aspect, the subject adapters are composed of two distinct polypeptides of at least 30 amino acid residues, one of which is essentially identical to a linear sequence of comparable length depicted in SEQ ID NO:13 (GR1), and the other is essentially identical to a linear peptide sequence of comparable length depicted in SEQ ID NO:11 (GR2).
(52) Another class of current coiled coil adapters are leucine zippers. The leucine zipper have been defined in the art as a stretch of about 35 amino acids containing 4-5 leucine residues separated from each other by six amino acids (Maniatis and Abel, Nature 341:24 (1989)). The leucine zipper has been found to occur in a variety of eukaryotic DNA-binding proteins, such as GCN4, C/EBP, c-fos gene product (Fos), c-jun gene product (Jun), and c-Myc gene product. In these proteins, the leucine zipper creates a dimerization interface wherein proteins containing leucine zippers may form stable homodimers and/or heterodimers. Molecular analysis of the protein products encoded by two proto-oncogenes, c-fos and c-jun, has revealed such a case of preferential heterodimer formation (Gentz et al., Science 243: 1695 (1989); Nakabeppu et al., Cell 55: 907 (1988); Cohen et al., Genes Dev. 3: 173 (1989)). Synthetic peptides comprising the leucine zipper regions of Fos and Jun have also been shown to mediate heterodimer formation, and, where the amino-termini of the synthetic peptides each include a cysteine residue to permit intermolecular disulfide bonding, heterodimer formation occurs to the substantial exclusion of homodimerization.
(53) The leucine-zipper adapters of the present invention have the general structural formula known as the heptad repeat (Leucine-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6).sub.n, where X may be any of the conventional 20 amino acids, but are most likely to be amino acids with alpha-helix forming potential, for example, alanine, valine, aspartic acid, glutamic acid, and lysine, and n may be 2 or greater, although typically n is 3 to 10, preferably 4 to 8, more preferably 4 to 5. Currently, the sequences are the Fos or Jun leucine zippers.
(54) As used herein, a linear sequence of peptide is “essentially identical” to another linear sequence, if both sequences exhibit substantial amino acid or nucleotide sequence homology. Generally, essentially identical sequences are at least about 60% identical with each other, after alignment of the homologous regions. Generally, the sequences are at least about 70% identical; more specifically, they are at least about 80% identical; more specifically, they are at least about 90% identical; more specifically, the sequences are at least about 95% identical; still more specifically, the sequences are 100% identical.
(55) In determining whether polypeptide sequences are essentially identical, a sequence that preserves the functionality of the polypeptide with which it is being compared is particularly preferred. Functionality may be established by different criteria, such as ability to form a stable complex with a pairing adapter, and ability to facilitate display of polypeptides fused in-frame with the adapter.
(56) The subject adapters include modified leucine zippers and GABA.sub.B heterodimerization peptide sequences which are functionally equivalent to the polypeptide sequences exemplified herein. In particular embodiments, modified polypeptides providing improved stability to the paired adapters and/or display efficiency are used. Examples of modified polypeptides include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids which do not significantly deleteriously alter the heterodimerization specificity. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the pairwise interaction is maintained. Amino acid substitutions, if present, are preferably conservative substitutions that do not deleteriously affect folding or functional properties of the peptide. Groups of functionally related amino acids within which conservative substitutions can be made are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tryosine/tryptophan. Polypeptides of this invention can be in glycosylated or unglycosylated form, can be modified post-translationally (e.g., acetylation, and phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group).
(57) One c-fos zipper is: LQAETDQLEDEKSALQTEIANLLKEKEKL (SEQ ID NO: 1). One c-Jun zipper is LEEKVKTLKAQNSELASTANMLREQVAQL (SEQ ID NO: 2). Longer forms of these zippers are as follows: c-fos: LTDTLQAETDQLEDEKSALQ TEIANLLKEKEKLEFILA (SEQ ID NO: 3). c-Jun: RIARLEEKVKTLKAQNSELAS TANMLREQVAQLKQKVMN (SEQ ID NO: 4).
(58) Alternative c-Jun zippers may also be used. These zippers have reduced ability to form homodimers, but still heterodimerize with c-Fos (Smeal et al. (1989) Genes & Development 3:2091-2100).
(59) Some c-Jun zippers with reduced heterodimerization ability include:
(60) TABLE-US-00001 LEEKVKTLKAQNSELASTFNMLREQFAQL; (SEQ ID NO: 5) LEEKVKTLKAQNSELASTANMLREQVAQF; (SEQ ID NO: 6) LEEKVKTFKAQNSELASTANMLREQVAQF; (SEQ ID NO: 7) LEEKVKSFKAQNSEHASTANMLREQVAQL (SEQ ID NO: 8)
(61) The adapter sequences of the present invention can be obtained using conventional recombinant cloning methods and/or by chemical synthesis. Using well-established restriction and ligation techniques, the appropriate adapter sequences can be excised from various DNA sources and integrated in-frame with the exogenous gene sequences and the outer-surface sequences to generate the expression and helper vectors, respectively.
(62) Preferably, the second adapter sequence is inserted into the expression vector in such a way to minimize structural interference, if any, on the resulting exogenous fusion polypeptide. Whereas the first adapter can be fused to the 5′ or 3′ of the exogenous gene sequence,
(63) Similarly, the first adapter peptide sequence is inserted into the second vector in a position where the integrity of the cell surface anchoring protein is not undermined. The adapter sequence can be fused to the 5′ or 3′ end of an outer-surface sequence without disrupting the coding region.
(64) II. Host Cells
(65) In general, lower eukaryotes such as yeast are used for expression of the proteins, particularly glycoproteins because they can be economically cultured, give high yields, and when appropriately modified are capable of suitable glycosylation. Yeast particularly offers established genetics allowing for rapid transformations, tested protein localization strategies and facile gene knock-out techniques. Suitable vectors have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
(66) While the invention has been demonstrated herein using the methylotrophic yeast Pichia pastoris, other useful lower eukaryote host cells include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum and Neurospora crassa. Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha are particularly suitable for cell culture because they are able to grow to high cell densities and secrete large quantities of recombinant protein. Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp, Neurospora crassa and others can be used to produce glycoproteins of the invention at an industrial scale. In the case of lower eukaryotes, cells are routinely grown from between about 1.5 to 3 days under conditions that induce expression of the capture moiety. The induction of immunoglobulin expression while inhibiting expression of the capture moiety is for about 1 to 2 days. Afterwards, the cells are analyzed for those cells that display the immunoglobulin of interest.
(67) Lower eukaryotes, particularly yeast and filamentous fungi, can be genetically modified so that they express glycoproteins in which the glycosylation pattern is human-like or humanized. In this manner, glycoprotein compositions can be produced in which a specific desired glycoform is predominant in the composition. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or genetically engineering the host cells and/or supplying exogenous enzymes to mimic all or part of the mammalian glycosylation pathway as described in US 2004/0018590. If desired, additional genetic engineering of the glycosylation can be performed, such that the glycoprotein can be produced with or without core fucosylation. Use of lower eukaryotic host cells is further advantageous in that these cells are able to produce highly homogenous compositions of glycoprotein, such that the predominant glycoform of the glycoprotein may be present as greater than thirty mole percent of the glycoprotein in the composition. In particular aspects, the predominant glycoform may be present in greater than forty mole percent, fifty mole percent, sixty mole percent, seventy mole percent and, most preferably, greater than eighty mole percent of the glycoprotein present in the composition.
(68) Lower eukaryotes, particularly yeast, can be genetically modified so that they express glycoproteins in which the glycosylation pattern is human-like or humanized. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or supplying exogenous enzymes as described by Gemgross et al., US 20040018590. For example, a host cell can be selected or engineered to be depleted in 1,6-mannosyl transferase activities, which would otherwise add mannose residues onto the N-glycan on a glycoprotein.
(69) In one embodiment, the host cell further includes an α1,2-mannosidase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target the α1,2-mannosidase activity to the ER or Golgi apparatus of the host cell. Passage of a recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a Man.sub.5GlcNAc.sub.2 glycoform, for example, a recombinant glycoprotein composition comprising predominantly a Man.sub.5GlcNAc.sub.2 glycoform. For example, U.S. Pat. No. 7,029,872 and U.S. Published Patent Application Nos. 2004/0018590 and 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a Man.sub.5GlcNAc.sub.2 glycoform.
(70) In a further embodiment, the immediately preceding host cell further includes a GlcNAc transferase I (GnT I) catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target GlcNAc transferase I activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a GlcNAcMan.sub.5GlcNAc.sub.2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAcMan.sub.5GlcNAc.sub.2 glycoform. U.S. Pat. No. 7,029,872 and U.S. Published Patent Application Nos. 2004/0018590 and 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a GlcNAcMan.sub.5GlcNAc.sub.2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a hexaminidase to produce a recombinant glycoprotein comprising a Man.sub.5GlcNAc.sub.2 glycoform.
(71) In a further embodiment, the immediately preceding host cell further includes a mannosidase II catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target mannosidase II activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a GlcNAcMan.sub.3GlcNAc.sub.2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAcMan.sub.3GlcNAc.sub.2 glycoform. U.S. Pat. No. 7,029,872 and U.S. Published Patent Application No. 2004/0230042 discloses lower eukaryote host cells that express mannosidase II enzymes and are capable of producing glycoproteins having predominantly a GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a hexaminidase to produce a recombinant glycoprotein comprising a Man.sub.3GlcNAc.sub.2 glycoform.
(72) In a further embodiment, the immediately preceding host cell further includes GlcNAc transferase II (GnT II) catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target GlcNAc transferase II activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform. U.S. Pat. No. 7,029,872 and U.S. Published Patent Application Nos. 2004/0018590 and 2005/0170452 disclose lower eukaryote host cells capable of producing a glycoprotein comprising a GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a hexaminidase to produce a recombinant glycoprotein comprising a Man.sub.3GlcNAc.sub.2 glycoform.
(73) In a further embodiment, the immediately preceding host cell further includes a galactosyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target galactosyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2 or Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform, or mixture thereof for example a recombinant glycoprotein composition comprising predominantly a GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform or Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform or mixture thereof. U.S. Pat. No. 7,029,872 and U.S. Published Patent Application No. 2006/0040353 discloses lower eukaryote host cells capable of producing a glycoprotein comprising a Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform. The glycoprotein produced in the above cells can be treated in vitro with a galactosidase to produce a recombinant glycoprotein comprising a GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform, for example a recombinant glycoprotein composition comprising predominantly a GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform.
(74) In a further embodiment, the immediately preceding host cell further includes a sialyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target sialytransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising predominantly a NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform or NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNA.sub.2 glycoform or mixture thereof. For lower eukaryote host cells such as yeast and filamentous fungi, it is useful that the host cell further include a means for providing CMP-sialic acid for transfer to the N-glycan. U.S. Published Patent Application No. 2005/0260729 discloses a method for genetically engineering lower eukaryotes to have a CMP-sialic acid synthesis pathway and U.S. Published Patent Application No. 2006/0286637 discloses a method for genetically engineering lower eukaryotes to produce sialylated glycoproteins. The glycoprotein produced in the above cells can be treated in vitro with a neuraminidase to produce a recombinant glycoprotein comprising predominantly a Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform or GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform or mixture thereof.
(75) Any one of the preceding host cells can further include one or more GlcNAc transferase selected from the group consisting of GnT III, GnT IV, GnT V, GnT VI, and GnT IX to produce glycoproteins having bisected (GnT III) and/or multiantennary (GnT IV, V, VI, and IX) N-glycan structures such as disclosed in U.S. Published Patent Application Nos. 2004/074458 and 2007/0037248.
(76) In further embodiments, the host cell that produces glycoproteins that have predominantly GlcNAcMan.sub.5GlcNAc.sub.2 N-glycans further includes a galactosyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target Galactosyltransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising predominantly the GalGlcNAcMan.sub.5GlcNAc.sub.2 glycoform.
(77) In a further embodiment, the immediately preceding host cell that produced glycoproteins that have predominantly the GalGlcNAcMan.sub.5GlcNAc.sub.2 N-glycans further includes a sialyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain and selected to target sialytransferase activity to the ER or Golgi apparatus of the host cell. Passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell produces a recombinant glycoprotein comprising a NANAGalGlcNAcMan.sub.5GlcNAc.sub.2 glycoform.
(78) Various of the preceding host cells further include one or more sugar transporters such as UDP-GlcNAc transporters (for example, Kluyveromyces lactis and Mus musculus UDP-GlcNAc transporters), UDP-galactose transporters (for example, Drosophila melanogaster UDP-galactose transporter), and CMP-sialic acid transporter (for example, human sialic acid transporter). Because lower eukaryote host cells such as yeast and filamentous fungi lack the above transporters, it is preferable that lower eukaryote host cells such as yeast and filamentous fungi be genetically engineered to include the above transporters.
(79) Host cells further include lower eukaryote cells (e.g., yeast such as Pichia pastoris) that are genetically engineered to eliminate glycoproteins having α-mannosidase-resistant N-glycans by deleting or disrupting one or more of the β-mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and BMT4)(See, U.S. Published Patent Application No. 2006/0211085) and glycoproteins having phosphomannose residues by deleting or disrupting one or both of the phosphomannosyl transferase genes PNO1 and MNN4B (See for example, U.S. Pat. Nos. 7,198,921 and 7,259,007), which in further aspects can also include deleting or disrupting the MNN4A gene. Disruption includes disrupting the open reading frame encoding the particular enzymes or disrupting expression of the open reading frame or abrogating translation of RNAs encoding one or more of the α-mannosyltransferases and/or phosphomannosyltransferases using interfering RNA, antisense RNA, or the like. The host cells can further include any one of the aforementioned host cells modified to produce particular N-glycan structures.
(80) Host cells further include lower eukaryote cells (e.g., yeast such as Pichia pastoris) that are genetically modified to control O-glycosylation of the glycoprotein by deleting or disrupting one or more of the protein O-mannosyltransferase (Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase genes) (PMTs) (See U.S. Pat. No. 5,714,377) or grown in the presence of Pmtp inhibitors and/or an alpha-mannosidase as disclosed in Published International Application No. WO 2007061631, or both. Disruption includes disrupting the open reading frame encoding the Pmtp or disrupting expression of the open reading frame or abrogating translation of RNAs encoding one or more of the Pmtps using interfering RNA, antisense RNA, or the like. The host cells can further include any one of the aforementioned host cells modified to produce particular N-glycan structures.
(81) Pmtp inhibitors include but are not limited to a benzylidene thiazolidinediones.
(82) Examples of benzylidene thiazolidinediones that can be used are 5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; 5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; and 5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid.
(83) In particular embodiments, the function or expression of at least one endogenous PMT gene is reduced, disrupted, or deleted. For example, in particular embodiments the function or expression of at least one endogenous PMT gene selected from the group consisting of the PMT1, PMT2, PMT3, and PMT4 genes is reduced, disrupted, or deleted; or the host cells are cultivated in the presence of one or more PMT inhibitors. In further embodiments, the host cells include one or more PMT gene deletions or disruptions and the host cells are cultivated in the presence of one or more Pmtp inhibitors. In particular aspects of these embodiments, the host cells also express a secreted alpha-1,2-mannosidase.
(84) PMT deletions or disruptions and/or Pmtp inhibitors control O-glycosylation by reducing O-glycosylation occupancy, that is by reducing the total number of O-glycosylation sites on the glycoprotein that are glycosylated. The further addition of an alpha-1,2-mannsodase that is secreted by the cell controls O-glycosylation by reducing the mannose chain length of the O-glycans that are on the glycoprotein. Thus, combining PMT deletions or disruptions and/or Pmtp inhibitors with expression of a secreted alpha-1,2-mannosidase controls O-glycosylation by reducing occupancy and chain length. In particular circumstances, the particular combination of PMT deletions or disruptions, Pmtp inhibitors, and alpha-1,2-mannosidase is determined empirically as particular heterologous glycoproteins (Fabs and antibodies, for example) may be expressed and transported through the Golgi apparatus with different degrees of efficiency and thus may require a particular combination of PMT deletions or disruptions, Pmtp inhibitors, and alpha-1,2-mannosidase. In another aspect, genes encoding one or emore endogenous mannosyltransferase enzymes are deleted. This deletion(s) can be in combination with providing the secreted alpha-1,2-mannosidase and/or PMT inhibitors or can be in lieu of providing the secreted alpha-1,2-mannosidase and/or PMT inhibitors.
(85) Thus, the control of O-glycosylation can be useful for producing particular glycoproteins in the host cells disclosed herein in better total yield or in yield of properly assembled glycoprotein. The reduction or elimination of O-glycosylation appears to have a beneficial effect on the assembly and transport of whole antibodies and Fab fragments as they traverse the secretory pathway and are transported to the cell surface. Thus, in cells in which O-glycosylation is controlled, the yield of properly assembled antibodies or Fab fragments is increased over the yield obtained in host cells in which O-glycosylation is not controlled.
(86) In addition, O-glycosylation may have an effect on an antibody or Fab fragment's affinity and/or avidity for an antigen. This can be particularly significant when the ultimate host cell for production of the antibody or Fab is not the same as the host cell that was used for selecting the antibody. For example, O-glycosylation might interfere with an antibody's or Fab fragment's affinity for an antigen, thus an antibody or Fab fragment that might otherwise have high affinity for an antigen might not be identified because O-glycosylation may interfere with the ability of the antibody or Fab fragment to bind the antigen. In other cases, an antibody or Fab fragment that has high avidity for an antigen might not be identified because O-glycosylation interferes with the antibody's or Fab fragment's avidity for the antigen. In the preceding two cases, an antibody or Fab fragment that might be particularly effective when produced in a mammalian cell line might not be identified because the host cells for identifying and selecting the antibody or Fab fragment was of another cell type, for example, a yeast or fungal cell (e.g., a Pichia pastoris host cell). It is well known that O-glycosylation in yeast can be significantly different from O-glycosylation in mammalian cells. This is particularly relevant when comparing wild type yeast o-glycosylation with mucin-type or dystroglycan type O-glycosylation in mammals. In particular cases, O-glycosylation might enhance the antibody or Fab fragments affinity or avidity for an antigen instead of interfere. This effect is undesirable when the production host cell is to be different from the host cell used to identify and select the antibody or Fab fragment (for example, identification and selection is done in yeast and the production host is a mammalian cell) because in the production host the O-glycosylation will no longer be of the type that caused the enhanced affinity or avidity for the antigen. Therefore, controlling O-glycosylation can enable use of the materials and methods herein to identify and select antibodies or Fab fragments with specificity for a particular antigen based upon affinity or avidity of the antibody or Fab fragment for the antigen without identification and selection of the antibody or Fab fragment being influenced by the O-glycosylation system of the host cell. Thus, controlling O-glycosylation further enhances the usefulness of yeast or fungal host cells to identify and select antibodies or Fab fragments that will ultimately be produced in a mammalian cell line.
(87) Yield of antibodies and Fabs can in some situations be improved by overexpressing nucleic acid molecules encoding mammalian or human chaperone proteins or replacing the genes encoding one or more endogenous chaperone proteins with nucleic acid molecules encoding one or more mammalian or human chaperone proteins. In addition, the expression of mammalian or human chaperone proteins in the host cell may control O-glycosylation in the cell. Thus, further included are the host cells herein wherein the function of at least one endogenous gene encoding a chaperone protein has been reduced or eliminated, and a vector encoding at least one mammalian or human homolog of the chaperone protein is expressed in the host cell. Also included are host cells in which the endogenous host cell chaperones and the mammalian or human chaperone proteins are expressed. In further aspects, the lower eukaryotic host cell is a yeast or filamentous fungi host cell. Examples of the use of chaperones of host cells in which human chaperone proteins are introduced to improve the yield and reduce or control O-glycosylation of recombinant proteins has been disclosed in U.S. Provisional Application Nos. 61/066,409 filed Feb. 20, 2008 and 61/188,723 filed Aug. 12, 2008. Like above, further included are lower eukaryotic host cells wherein, in addition to replacing the genes encoding one or more of the endogenous chaperone proteins with nucleic acid molecules encoding one or more mammalian or human chaperone proteins or overexpressing one or more mammalian or human chaperone proteins as described above, the function or expression of at least one endogenous gene encoding a protein O-mannosyltransferase (PMT) protein is reduced, disrupted, or deleted. In particular embodiments, the function of at least one endogenous PMT gene selected from the group consisting of the PMT1, PMT2, PMT3, and PMT4 genes is reduced, disrupted, or deleted.
(88) Therefore, the methods disclosed herein can use any host cell that has been genetically modified to produce glycoproteins that have no N-glycan compositions wherein the predominant N-glycan is selected from the group consisting of complex N-glycans, hybrid N-glycans, and high mannose N-glycans wherein complex N-glycans are selected from the group consisting of Man.sub.3GlcNAc.sub.2, GlcNAC.sub.(1-4)Man.sub.3GlcNAc.sub.2, Gal.sub.(1-4GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2, and NANA.sub.(1-4)Gal.sub.(1-4)Man3GlcNAc.sub.2; hybrid N-glycans are selected from the group consisting of GlcNAcMan.sub.5GlcNAc.sub.2, GalGlcNAcMan.sub.5GlcNAc.sub.2, and NANAGalGlcNAcMan.sub.5GlcNAc.sub.2; and high Mannose N-glycans are selected from the group consisting of Man.sub.5GlcNAc.sub.2, Man.sub.6GlcNAc.sub.2, Man.sub.7GlcNAc.sub.2, Man.sub.8GlcNAc.sub.2, and Man.sub.9GlcNAc.sub.2. In particular aspects, the composition of N-glycans comprises about 39% GlcNAC.sub.2Man.sub.3GlcNAc.sub.2; 40% Gal.sub.1GlcNAC.sub.2Man.sub.3GlcNAc.sub.2; and 6% Gal.sub.2GlcNAC.sub.2Man.sub.3GlcNAc.sub.2 or about 60% GlcNAC.sub.2Man.sub.3GlcNAc.sub.2; 17% Gal.sub.1GlcNAC.sub.2Man.sub.3GlcNAc.sub.2; and 5% Gal.sub.2GlcNAC.sub.2Man.sub.3GlcNAc.sub.2, or mixtures in between.
(89) In the above embodiments in which the yeast cell does not display 1,6-mannosyl transferase activity (that is, the OCH1 gene encoding och1p has been disrupted or deleted or the activity of Och1p has been disabled), the host cell is not capable of mating. Thus, depending on the efficiency of transformation, the potential library diversity of light chains and heavy chains appears to be limited to a heavy chain library of between about 103 to 10.sup.6 diversity and a light chain library of about 10.sup.3 to 10.sup.6 diversity. However, in a yeast host cell that is capable of mating, the diversity can be increased to about 10.sup.6 to 10.sup.12 because the host cells expressing the heavy chain library can be mated to host cells expressing the light chain library to produce host cells that express heavy chain/light chain library. Therefore, in particular embodiments, the host cell is a yeast cell such as Pichia pastoris that displays 1,6-mannosyl transferase activities (that is, has an OCH1 gene encoding a functional och1p) but which is modified as described herein to display antibodies or fragments thereof on the cell surface. In these embodiments, the host cell can be a host cell with its native glycosylation pathway.
(90) In embodiments that express whole antibodies or the Fc region of an antibody heavy chain (e.g., Fab fragments), the nucleic acid molecule encoding the antibody or heavy chain fragment thereof is modified to replace the codon encoding an asparagine residue at position 297 of the molecule (the glycosylation site) with a codon encoding any other amino acid residue. Common replacements include but are not limited to alanine, glutamine, and aspartate. Thus, the antibody or fragment thereof that is produced in the host cell is not glycosylated at asparagine-297. In this embodiment, the host cell displaying the heavy chain library is mated to the host cell displaying the light chain library and the resulting combinatorial library is screened as taught herein. Because the antibodies or fragments thereof lack N-glycosylation at asparagine-297, the non-human yeast N-glycans of the host cell linked to asparagine-297 which might interfere with antibody affinity for a desired antigen are not present on the recombinant antibodies or fragments thereof. Cells producing antibodies or fragments that have desired affinity for an antigen of interest are selected. The nucleic acid molecules encoding the heavy and light chains of the antibody or fragments thereof are removed from the cells and the nucleic acid molecule encoding the heavy chain is modified to reintroduce an asparagine residue at position 297. This enables appropriate human-like glycosylation at position 297 of the antibody or fragment thereof when the nucleic acid molecule encoding the antibody or fragment thereof is introduced into a mammalian cell line (e.g., CHO or the like) or lower eukaryote (e.g., Pichia pastoris) host cell that has been engineered to make glycoproteins that have human-like N-glycans (e.g., high mannose, hybrid, or complex N-glycans as discussed previously.
(91) While in general the host cells used to practice the present invention are lower eukaryote host cells (e.g., yeast or filamentous fungal cells), it is envisioned that the methods herein can be adapted to use higher eukaryote cells. Thus, in particular embodiments, the cell systems used for recombinant expression and display of the immunoglobulin can also be any higher eukaryote cell, tissue, organism from the animal kingdom, for example transgenic goats, transgenic rabbits, CHO cells, insect cells, and human cell lines. Examples of animal cells include, but are not limited to, SC-I cells, LLC-MK cells, CV-I cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0, NSO cells, and derivatives thereof. Insect cells include cells of Drosophila melanogaster origin. In addition, these cells can be genetically engineered to render the cells capable of making immunoglobulins that have particular N-glycans or predominantly particular N-glycans. For example, U.S. Pat. No. 6,949,372 discloses methods for making glycoproteins in insect cells that are sialylated. Yamane-Ohnuki et al. Biotechnol. Bioeng. 87: 614-622 (2004), Kanda et al., Biotechnol. Bioeng. 94: 680-688 (2006), Kanda et al., Glycobiol. 17: 104-118 (2006), and U.S. Pub. Application Nos. 2005/0216958 and 2007/0020260 disclose mammalian cells that are capable of producing immunoglobulins in which the N-glycans thereon lack fucose or have reduced fucose.
(92) In particular embodiments, the higher eukaryote cell, tissue, organism can also be from the plant kingdom, for example, wheat, rice, corn, tobacco, and the like. Alternatively, bryophyte cells can be selected, for example from species of the genera Physcomitrella, Funaria, Sphagnum, Ceratodon, Marchantia, and Sphaerocarpos. Exemplary of plant cells is the bryophyte cell of Physcomitrella patens, which has been disclosed in WO 2004/057002 and WO2008/006554. Expression systems using plant cells can further manipulated to have altered glycosylation pathways to enable the cells to produce immunoglobulins that have predominantly particular N-glycans. For example, the cells can be genetically engineered to have a dysfunctional or no core fucosyltransferase and/or a dysfunctional or no xylosyltransferase, and/or a dysfunctional or no β1,4-galactosyltransferase. Alternatively, the galactose, fucose and/or xylose can be removed from the immunoglobulin by treatment with enzymes removing the residues. Any enzyme resulting in the release of galactose, fucose and/or xylose residues from N-glycans which are known in the art can be used, for example α-galactosidase, β-xylosidase, and α-fucosidase. Alternatively an expression system can be used which synthesizes modified N-glycans which can not be used as substrates by 1,3-fucosyltransferase and/or 1,2-xylosyltransferase, and/or 1,4-galactosyltransferase. Methods for modifying glycosylation pathways in plant cells has been disclosed in U.S. Published Application No. 2004/0018590.
(93) The methods disclosed herein can be adapted for use in mammalian, insect, and plant cells. The regulatable promoters selected for regulating expression of the expression cassettes in mammalian, insect, or plant cells should be selected for functionality in the cell-type chosen. Examples of suitable regulatable promoters include but are not limited to the tetracycline-regulatable promoters (See for example, Berens & Hillen, Eur. J. Biochem. 270: 3109-3121 (2003)), RU 486-inducible promoters, ecdysone-inducible promoters, and kanamycin-regulatable systems. These promoters can replace the promoters exemplified in the expression cassettes described in the examples. The capture moiety can be fused to a cell surface anchoring protein suitable for use in the cell-type chosen. Cell surface anchoring proteins including GPI proteins are well known for mammalian, insect, and plant cells. GPI-anchored fusion proteins has been described by Kennard et al., Methods Biotechnol. Vo. 8: Animal Cell Biotechnology (Ed. Jenkins. Human Press, Inc., Totowa, N.J.) pp. 187-200 (1999). The genome targeting sequences for integrating the expression cassettes into the host cell genome for making stable recombinants can replace the genome targeting and integration sequences exemplified in the examples. Transfection methods for making stable and transiently transfected mammalian, insect, plant host cells are well known in the art. Once the transfected host cells have been constructed as disclosed herein, the cells can be screened for expression of the immunoglobulin of interest and selected as disclosed herein.
(94) III. Glycosylphosphatidylinositol-Anchored (GPI) Proteins
(95) Lower eukaryotic cells have systems of GPI proteins that are involved in anchoring or tethering expressed proteins to the cell wall so that they are effectively displayed on the cell wall of the cell from which they were expressed. For example, 66 putative GPI proteins have been identified in Saccharomyces cerevisiae (See, de Groot et al., Yeast 20: 781-796 (2003)). GPI proteins which may be used in the methods herein include, for example Saccharomyces cerevisiae CWP1; CWP2; SED1; GAS1; Pichia pastoris SP1; GAS1; and H. polymorpha TIP1. Additional GPI proteins may also be useful. Suitable GPI proteins Can be identified using the methods and materials of the invention described and exemplified herein.
(96) The selection of the appropriate GPI protein will depend on the particular recombinant protein to be produced in the host cell and the particular post-translation modifications to be performed on the recombinant protein. For example, production of antibodies or fragments thereof with particular glycosylation patterns will entail the use of recombinant host cells that produce glycoproteins having particular glycosylation patterns. The GPI protein most suitable in a system for producing antibodies or fragments thereof that have predominantly Man.sub.5GlcNAc.sub.2 N-glycosylation many not necessarily be the GPI protein most suitable in a system for producing antibodies or thereof having predominantly Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycosylation. In addition, the GPI most suitable in a system for producing antibodies or fragments thereof specific for one epitope or antigen may not necessarily be the most suitable GPI protein in a system for producing antibodies or fragments thereof specific for another epitope or antigen. Furthermore, the GPI most suitable in a system for producing antibody fragments such as scFv or the like may not necessarily be the most suitable GPI protein in a system for producing full-length antibodies.
(97) Therefore, further provided is a library method for constructing the host cell that is to be used for producing a particular recombinant protein. In general, the host that is desired to produce the recombinant proteins is selected based on the desired characteristics that will be imparted to the recombinant protein produced by the host cell. For example, a host cell that produces glycoproteins having predominantly Man.sub.5GlcNAc.sub.2 or Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycosylation is selected. A library of vectors encoding GPI proteins fused to one or more adapters is then provided. A library of host cells is then constructed wherein each host cell to make up the library is transfected with one of the vectors in the library of vectors encoding GPI-adapter fusion proteins such that each host cell species in the library will express one particular GPI-adapter fusion protein. Each host cell species of the library is then transformed with a vector encoding the desired protein or a protein similar in function or structure to the desired protein. The host cell that results in the best presentation of recombinant protein on the surface of the host cell is selected as the host cell for producing the desired recombinant protein.
(98) In general, the GPI protein used in the methods disclosed herein is a chimeric protein or fusion protein comprising the GPI protein fused at its N-terminus to the C-terminus of a binding moiety or adapter peptide. The N-terminus of the binding moiety or adapter peptide is fused to the C-terminus of a signal sequence that enables the GPI fusion protein to be transported through the secretory pathway to the cell surface where the GPI fusion protein is secreted and then bound to the cell surface. In some aspects, the GPI fusion protein comprises the entire GPI protein and in other aspects, the GPI fusion protein comprises the portion of the GPI protein that is capable of binding to the cell surface.
(99) V. Regulatory Sequences
(100) Regulatory sequences which may be used in the practice of the methods disclosed herein include signal sequences, promoters, and transcription terminator sequences. It is generally preferred that the regulatory sequences used be from a species or genus that is the same as or closely related to that of the host cell or is operational in the host cell type chosen. Examples of signal sequences include those of Saccharomyces cerevisiae invertase; the Aspergillus niger amylase and glucoamylase; human serum albumin; Kluyveromyces maxianus inulinase; and Pichia pastoris mating factor and Kar2. Signal sequences shown herein to be useful in yeast and filamentous fungi include, but are not limited to, the alpha mating factor presequence and preprosequence from Saccharomyces cerevisiae; and signal sequences from numerous other species.
(101) Examples of promoters include promoters from numerous species, including but not limited to alcohol-regulated promoter, tetracycline-regulated promoters, steroid-regulated promoters (e.g., glucocorticoid, estrogen, ecdysone, retinoid, thyroid), metal-regulated promoters, pathogen-regulated promoters, temperature-regulated promoters, and light-regulated promoters. Specific examples of regulatable promoter systems well known in the art include but are not limited to metal-inducible promoter systems (e.g., the yeast copper-metallothionein promoter), plant herbicide safner-activated promoter systems, plant heat-inducible promoter systems, plant and mammalian steroid-inducible promoter systems, Cym repressor-promoter system (Krackeler Scientific, Inc. Albany, N.Y.), RheoSwitch System (New England Biolabs, Beverly Mass.), benzoate-inducible promoter systems (See WO2004/043885), and retroviral-inducible promoter systems. Other specific regulatable promoter systems well-known in the art include the tetracycline-regulatable systems (See for example, Berens & Hillen, Eur J Biochem 270: 3109-3121 (2003)), RU 486-inducible systems, ecdysone-inducible systems, and kanamycin-regulatable system. Lower eukaryote-specific promoters include but are not limited to the Saccharomyces cerevisiae TEF-1 promoter, Pichia pastoris GAPDH promoter, Pichia pastoris GUT1 promoter, PMA-1 promoter, Pichia pastoris PCK-1 promoter, and Pichia pastoris AOX-1 and AOX-2 promoters. For temporal expression of the GPI-IgG capture moiety and the immunoglobulins, the Pichia pastoris GUT1 promoter operably linked to the nucleic acid molecule encoding the GPI-IgG capture moiety and the Pichia pastoris GAPDH promoter operably linked to the nucleic acid molecule encoding the immunoglobulin are shown in the examples herein to be useful.
(102) Examples of transcription terminator sequences include transcription terminators from numerous species and proteins, including but not limited to the Saccharomyces cerevisiae cytochrome C terminator; and Pichia pastoris ALG3 and PMA1 terminators.
(103) VI. Nucleic Acid Sequences Encoding the Protein of Interest
(104) The methods of the present invention can be employed with any gene of interest for further study. Because of the particular advantages afforded by the methods disclosed herein, the methods and materials will utilize genes encoding glycoproteins. Of particular interest are human glycoproteins with known therapeutic utility, including but not limited to monoclonal antibodies and functional fragments thereof such as Fab fragments; immunoglobulins including but not limited to IgG, IgM, IgD, antibody fragments such as scFv, Fab fragments, or the like; Fc fusion proteins; catalytic antibodies, camel or lama antibodies; erythropoietin; cytokines such as interferon-alpha, interferon-beta, interferon-gamma, interferon-omega, and granulocyte-CSF; coagulation factors such as factor VIII, factor IX, and human protein C; soluble IgE receptor alpha-chain; urokinase; chymase and urea trypsin inhibitor; IGF-binding protein; epidermal growth factor; growth hormone-releasing factor; annexin V fusion protein; angiostatin; vascular endothelial growth factor-2; myeloid progenitor inhibitory factor-1; and osteoprotegerin.
(105) Nucleic acids encoding desired glycoproteins can be obtained from several sources. cDNA sequences can be amplified from cell lines known to express the glycoprotein using primers to conserved regions (See, e.g., Marks et al., J. Mol. Biol. 581-596 (1991)). Nucleic acids can also be synthesized de novo based on sequences in the scientific literature. Nucleic acids can also be synthesized by extension of overlapping oligonucleotides spanning a desired sequence (See, e.g., Caldas et al., Protein Engineering, 13: 353-360 (2000)). Production of active glycoproteins requires proper folding of the protein when it is produced and secreted by the cells. The presence of effective molecular chaperone proteins may be required, or may enhance the ability of the cell to produce and secrete properly folded proteins.
(106) Nucleic acid molecules encoding immunoglobulins can be obtained from any suitable source including spleen and liver cells and antigen-stimulated antibody producing cells, obtained from either in vivo or in vitro sources. Regardless of source, the cellular VH and VL mRNAs are reverse transcribed into VH and VL cDNA sequences. Reverse transcription may be performed in a single step or in an optional combined reverse transcription/PCR procedure to produce cDNA libraries containing a plurality of immunoglobulin-encoding DNA molecules. (See, for example, Marks et al., J. Mol. Biol. 222: 581-596 (1991)). Nucleic acid molecules can also be synthesized de novo based on sequences in the scientific literature. Nucleic acid molecules can also be synthesized by extension of overlapping oligonucleotides spanning a desired sequence (See, e.g., Caldas et al., Protein Engineering, 13: 353-360 (2000)). Humanized immunoglobulin-encoding cDNA libraries can be constructed by PCR amplifying the complementary-determining regions (CDR) from the cDNAs in one or more libraries from any source and integrating the PCR amplified CDR-encoding nucleic acid molecules into nucleic acid molecules encoding a human immunoglobulin framework to produce a cDNA library encoding a plurality of humanized immunoglobulins (See, for example, U.S. Pat. Nos. 6,180,370; 6,632,927; and, 6,872,392). Chimeric immunoglobulin-encoding cDNA libraries can be constructed by PCR amplifying the variable regions from the cDNAs in the cDNA library from one species and integrating the nucleic acid molecules encoding the PCR-amplified variable regions onto nucleic acid molecules encoding immunoglobulin constant regions from another species to produce a cDNA library encoding a plurality of chimeric immunoglobulins (See, for example, U.S. Pat. No. 5,843,708). Various methods that have been developed for the creation of diversity within protein libraries, including random mutagenesis (Daugherty et al., Proc. Natl Acad. Sci. USA, 97: 2029-2034 (2000); Boder et al., Proc. Natl Acad. Sci. USA, 97: 10701-10705 (2000); Holler et al., Proc. Natl Acad. Sci. USA, 97: 5387-5392 (2000)), in vitro DNA shuffling (Stemmer, Nature, 370: 389-391 (1994); Stemmer, Proc. Natl Acad. Sci. USA, 91: 10747-10751 (1994)), in vivo DNA shuffling (Swers et al., Nucl. Acid Res. 32: e36 (2004)), and site-specific recombination (Rehberg et al., J. Biol. Chem., 257: 11497-11502 (1982); Streuli et al., Proc. Natl Acad. Sci. USA, 78: 2848-2852 (1981); Waterhouse et al., (1993) Nucl. Acids Res., 21: 2265-2266 (1993); Sblattero & Bradbury, Nat. Biotechnol., 18: 75-80 (2000)) can be used or adapted to produce the plurality of host cells disclosed herein that express immunoglobulins and the capture moiety comprising a cell surface anchoring protein fused to a binding moiety that is capable of specifically binding an immunoglobulin.
(107) Production of active immunoglobulins requires proper folding of the protein when it is produced and secreted by the cells. In E. coli, the complexity and large size of an antibody presents an obstacle to proper folding and assembly of the expressed light and heavy chain polypeptides, resulting in poor yield of intact antibody. The presence of effective molecular chaperone proteins may be required, or may enhance the ability of the cell to produce and secrete properly folded proteins. The use of molecular chaperone proteins to improve production of immunoglobulins in yeast has been disclosed in U.S. Pat. No. 5,772,245; U.S. Pat. Nos. 5,700,678 and 5,874,247; U.S. Application Publication No. 2002/0068325; Toman et al., J. Biol. Chem. 275: 23303-23309 (2000); Keizer-Gunnink et al., Martix Biol. 19: 29-36 (2000); Vad et al., J. Biotechnol. 116: 251-260 (2005); Inana et al., Biotechnol. Bioengineer. 93: 771-778 (2005); Zhang et al., Biotechnol. Prog. 22: 1090-1095 (2006); Damasceno et al., Appl. Microbiol. Biotechnol. 74: 381-389 (2006); Huo et al., Protein Express. Purif. 54: 234-239 (2007); and copending application Ser. No. 61/066,409, filed 20 Feb. 2008.
(108) As used herein, the methods can use host cells from any kind of cellular system which can be modified to express a capture moiety comprising a cell surface anchoring protein fused to a binding moiety capable of binding an immunoglobulin and whole, intact immunoglobulins. Within the scope of the invention, the term “cells” means the cultivation of individual cells, tissues, organs, insect cells, avian cells, reptilian cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, stem cells, plant cells, yeast cells, filamentous fungal cells, and/or genetically engineered cells, such as recombinant cells expressing and displaying a glycosylated immunoglobulin.
(109) VII. Uses of the Adapter-Directed Display Systems
(110) The adapter-directed display systems disclosed herein allows the display of monomeric and multimeric polypeptides on the surface of suitable lower eukaryote host cells. The subject display systems also can be used to create libraries of random or predetermined polypeptides, full-length proteins, and protein domains for a variety of purposes. For instance, the displayed libraries can be employed for mapping epitopes and mimotopes, identifying antagonists and agonists of various target proteins, engineering antibodies, optimizing antibody specificities and creating novel binding activities.
(111) Accordingly, provided is a method of detecting the presence of a specific interaction between a test agent and an exogenous polypeptide that is displayed on the surface of a suitable lower eukaryote host cell. The method involves the steps of: (a) providing a lower eukaryote host cell of the subject display system that presents the exogenous polypeptide; (b) contacting the lower eukaryote host cell with the test agent under conditions suitable to produce a stable polypeptide-agent complex; and (c) detecting the formation of the stable polypeptide-agent complex on the surface of the lower eukaryote host cell, thereby detecting the presence of the specific interaction.
(112) The term “test agent” is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic molecule, a protein, carbohydrate, lipid, polynucleotide or combinations thereof. A vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term “agent.” In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. It should be understood, although not always explicitly stated that the agent is used alone or in combination with another agent, having the same or different biological activity as the agents identified by the inventive screen. In particular embodiments, the agents are candidate diagnostics and/or therapeutics, such as those capable of modulating the signal transduction pathways of a cell.
(113) In a separate embodiment, the present invention provides a method of obtaining a polypeptide with desired property. The method comprises the steps of (a) providing a selectable library of the subject display system; and (b) screening the selectable library to obtain at least one lower eukaryote host cell displaying a polypeptide on its surface with the desired property. The method may further comprise the step of isolating the lower eukaryote host cell that displays a polypeptide having the desired property. Such isolation of the lower eukaryote host cell may involve obtaining a nucleotide sequence from the lower eukaryote host cell that encodes the desired polypeptide. The desired property encompasses the ability of the polypeptide to specifically bind to an agent of interest. The selected polypeptide with the desired property may fall within one or more classes of the following molecules, namely antigen-binding unit, cell surface receptor, receptor ligand, cytosolic protein, secreted protein, nuclear protein, and functional motif thereof. The choice of specific agent to be tested and the libraries of exogenous polypeptides to by displayed will depend on the intended purpose of the screening assay.
(114) VIII. Isolating Antibodies Exhibiting Desired Binding Specificity or Affinity
(115) One of the most powerful applications of display system herein is its use in the arena of antibody engineering. It has been shown that scFv antigen-binding units can be expressed on the surface of lower eukaryote host cells with no apparent loss of binding specificity and affinity (See for example, U.S. Pat. No. 6,300,065). It has also been shown that full-length antibodies can be captured and bound to the surface of hybridomas and CHO cells, for example (See U.S. Pat. Nos. 6,919,183 and 7,166,423). While antibodies and fragments thereof to many diverse antigens have been successfully isolated using phage display technology, there is still a need for a robust display system for producing antibodies and fragments thereof in lower eukaryotic host cell. It is particularly desirable to have a robust display system for producing antibodies and fragments thereof that have human-like glycosylation patterns. Genetically engineered lower eukaryotes that produce glycoproteins that have various human-like glycosylation patterns has been described in U.S. Pat. No. 7,029,872 and for example in Choi et al., Hamilton, et al., Science 313; 1441 1443 (2006); Wildt and Gerngross, Nature Rev. 3: 119-128 (2005); Bobrowicz et al., Glyco Biol. 757-766 (2004); Li et al., nature Biotechnol. 24: 210-215 (2006); Chiba et al., J. Biol. Chem. 273: 26298-26304 (1998); and, Mara et al., Glycoconjugate J. 16: 99-107 (1999).
(116) The subject display system is particularly suited for this application because the system allows presentation of a vast diverse repertoire of antibodies having particular glycosylation patterns. In many respects the subject display system mimics the natural immune system. Antigen-driven stimulation can be achieved by selecting for high-affinity binders from a display library of cloned antibody H and L chains. The large number of chain permutations that occur during recombination of H and L chain genes in developing B cells can be mimicked by shuffling the cloned H and L chains as DNA, and protein and through the use of site-specific recombination (Geoffory et al. Gene 151: 109-113 (1994)). The somatic mutation can also be matched by the introduction of mutations in the CDR regions of the H and L chains.
(117) Antibodies or fragments thereof with desired binding specificity or affinity can be identified using a form of affinity selection known as “panning” (Parmley and Smith (1988) Gene 73:305-318). The library of Antibodies or fragments thereof is first incubated with an antigen of interest followed by the capture of the antigen with the bound antibodies or fragments thereof. The antibodies or fragments thereof recovered in this manner can then be amplified and again gain selected for binding to the antigen, thus enriching for those antibodies or fragments thereof that bind the antigen of interest. After one or more rounds of selection isolation will enable isolation of antibodies or fragments thereof with the desired specificity or avidity. Thus, rare host cells expressing a desired antibody or fragment thereof can easily be selected from greater than 10.sup.4 different individuals in one experiment. The primary structure of the binding Antibody or fragment thereof is then deduced by nucleotide sequence of the individual host cell clone. When human V.sub.H and V.sub.L regions are employed in the displayed antibodies or fragments thereof, the subject display systems allow selection of human antibodies without further manipulation of a non-human antibodies or fragments thereof.
(118) IX. Generating Novel Proteins Including Antibodies and Fragments Thereof with Improved Binding Specificity or Affinity
(119) Using the subject display systems, one can obtain a replicable host cells that displays a polypeptide, such as an antibody or fragment thereof, having high affinity and specificity for a target protein. Such a host cells carries a first polynucleotide encoding the antibody or fragment thereof fused to a second adapter peptide and a second polynucleotide encoding the cell surface anchoring protein fused to a first adapter peptide that is capable of pairwise interaction with the second adapter peptide. The presence of the first polynucleotide facilitates recombinant expression and subsequent manipulation of the binding protein. For instance, the first polynucleotide can be mutagenized by cassette mutagenesis, error-prone PCR, or shuffling to generate a refined repertoire of altered sequences that resemble the parent polynucleotide. Upon screening the refined repertoire of novel antibodies or fragments thereof, those exhibiting improved binding specificity or affinity can be identified.
(120) X. Mapping Antigenic Epitopes
(121) Traditionally, epitope mapping of an antigen has relied heavily on physical chemical analysis. These approaches have included: (1) fragmenting the purified antigen with various proteases, identifying reactive fragments, and sequencing them; (2) chemical modification experiments in which residues interaction with the antigen-binding unit are protected from modification; (3) synthesizing a series of peptides corresponding to the primary structure of the antigen; and (4) direct physical characterization using NMR or X-ray crystallography. All of these methods are labor intensive and generally not amenable to high-throughput analyses. Lower eukaryote display as disclosed herein provides a highly efficient and robust alternative for localizing the antigenic epitope. Fragments of DNA that encode portions of the antigen can be expressed as the exogenous polypeptides by the subject expression vectors. The lower eukaryote host cells can then be tested with the antibody to determine which displayed fragments react with the antibody. This application of display technology has been widely used in the art and has been shown to be successful for determining the antigenic epitopes of a variety of molecules.
(122) XI. Mapping Binding Epitopes
(123) The subject display system also can be used to present random peptide libraries for mapping the specificity of the antigen-binding sites. Random peptide libraries represent a source of sequences from which epitopes and mimotopes can be operationally defined. With such a library, one can identity and obtain peptide competitors for antigen-antibody interactions, and thus map accessible and/or functional sites of numerous antibodies or fragments thereof.
(124) XII. Kits Comprising the Vectors of the Present Invention
(125) The present invention also encompasses kits containing the expression and helper vectors of this invention in suitable packaging. Each kit necessarily comprises the reagents which render the delivery of vectors into a host cell possible. The selection of reagents that facilitate delivery of the vectors may vary depending on the particular transfection or infection method used. The kits may also contain reagents useful for generating labeled polynucleotide probes or proteinaceous probes for detection of exogenous sequences and the protein product. Each reagent can be supplied in a solid form or dissolved/suspended in a liquid buffer suitable for inventory storage, and later for exchange or addition into the reaction medium when the experiment is performed. Suitable packaging is provided. The kit can optionally provide additional components that are useful in the procedure. These optional components include, but are not limited to, buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.
(126) In the following examples, heterologous human proteins are expressed in host cells of the species Pichia pastoris. The following examples are intended to promote a further understanding of the present invention.
Example 1
(127) The objective was to develop a novel yeast display method especially designed for Pichia pastoris strains genetically engineered to produce glycoproteins with various mammalian glycosylation patterns. In this example, a nucleic acid encoding the N-terminus of a cell surface anchoring protein that inherently contains an attached glycophosphotidylinositol (GPI) post-translational modification that anchors the protein in the cell wall was linked to a nucleic acid that encodes a first coiled coil peptide that is capable of forming a heterodimer with a second coiled coil peptide fused to a test protein. The specific cell surface anchoring protein that was used was Sed1p, which had been identified by screening a panel of cell wall or plasma membrane proteins that had been identified using GPI protein prediction software.
(128) Expression cassettes encoding the GPI protein and the test antibodies and Fab fragments were constructed using as the adapter peptides the coiled coil peptides GABAB-R2 (AEQ ID NO:19) fused to the N-terminus of the GPI protein and the GABAB-R1 (SEQ ID NO:21) fused to the C-terminus of the antibody or Fab fragment. GABAB-R1 (GR1) and GABAB-R2 (GR2) are derived from the γ-Aminobutyric acid (GABA) receptors GABAB-R1 and GABAB-R2. Heterodimerization of GABAB-R1 and GABAB-R2 subunits is a prerequisite for the formation of a functional GABAB receptor. Each individual subunit contains one stretch of 30 amino acid residues within its intracellular C-terminal domain that mediates heterodimer formation. (Kammerer et al., J. Biochem. 38:13263-9 (1999)). Heterodimerization of a functional GABAB receptor is mediated by parallel coiled-coil alpha-helices. Three additional amino acid residues, Gly, Gly, and Cys were attached at the end of GR1. The Cys at the end of the GR1 creates a disulfide bond with the Cys at the end of GR2, which is fused at the C-terminal of the display Fab fragment CH1. The two Glys are believed to increase the flexibility of the heterodimer.
(129) Construction of expression cassettes encoding the cell surface anchoring protein library was as follows. Candidate cell surface anchoring proteins were selected from S. cerevisiae, P. pastoris and H. polymorpha according to the literature and further identified as cell surface anchoring proteins using GPI protein prediction software available at IMP (Research Institute of Molecular Pathology), Bioinformatics Group, Dr. Bohr-Gasse 7, 1030 Vienna, Austria. Ten proteins were selected for analysis.
(130) Table 1 below shows the amino acid sequences for the relevant portion of ten GPI proteins and truncated variants of the proteins that were selected for analysis. Because highly expressed genes are desirable, truncation of the 3′ end of the candidate nucleic acid sequences was made for several of the proteins in an attempt to improve expression. For all of the GPI proteins, the nucleic acid encoding the endogenous signal sequence for the GPI protein was removed. Therefore, the amino acid sequences shown in Table 1 do not include the amino acid sequences for the endogenous signal peptides. The bold-faced amino acids in the amino acid sequences shown in Table 1 signify the omega site. The omega site is the region at which GPI is attached to the protein. The GPI proteins were separated into two types based upon site of anchoring: GPI-anchored plasma membrane proteins (GPI-PMP) and GPI-dependent cell surface anchoring proteins (GPI-CWP).
(131) TABLE-US-00002 TABLE 1 GPI SEQ pro- ID tein Source Type Sequence NO: CWP2 S. CWP VDESAAAISQITDGQIQATTTATT 9 cerevisiae EATTTAAPSSTVETVSPS STETI SQQTE NGAAKAAVGMGAGALAAA AMLL CWP2* S. CWP VDTTEATTTAAPSSTVETVSPSST 10 cerevisiae ETISQQTENGAAKAAVGMGAGALA Truncated AAAMLL SED1 S. CWP VDQFSNSTSASSTDVTSSSSISTS 11 cerevisiae SGSVTITSSEAPESDNGTSTAAPT ETSTEAPTTAIPTNGTSTEAPTTA IPTNGTSTEAPTDTTTEAPTTALP TNGTSTEAPTDTTTEAPTTGLPTN GTTSAFPPTTSLPPSNTTTTPPYN PSTDYTTDYTVVTEYTTYCPEPTT FTTNGKTYTVTEPTTLTITDCPCT IEKPTTTSTTEYTVVTEYTTYCPE PTTFTTNGKTYTVTEPTTLTITDC PCTIEKSEAPESSVPVTESKGTTT KETGVTTKQTTANPSLTVSTVVPV SSSASSHSVVINSNGANVVVPGAL GLAGVAMLFL SED1* S. CWP VDLTVSTVVPVSSSASSHSVVINS 12 cerevisiae NGANVVVPGALGLAGVAMLFL Truncated SPI1 P.pastoris CWP VDLVSNSSSSVIVVPSSDATIAGN 13 DTATPAPEPSSAAPIFYNSTATAT QYEVVSEFTTYCPEPTTFVTNGAT FTVTAPTTLTITNCPCTIEKPTSE TSVSSTHDVETNSNAANARAIPGA LGLAGAVMMLL GAS1 S. PMP VDDVPAIEVVGNKFFYSNNGSQFY 14 cerevisiae IRGVAYQADTANETSGSTVNDPLA NYESCSRDIPYLKKLNTNVIRVYA INTTLDHSECMKALNDADIYVIAD LAAPATSINRDDPTWTVDLFNSYK TVVDTFANYTNVLGFFAGNEVTNN YTNTDASAFVKAAIRDVRQYISDK NYRKIPVGYSSNDDEDTRVKMTDY FACGDDDVKADFYGINMYEWCGKS DFKTSGYADRTAEFKNLSIPVFFS EYGCNEVTPRLFTEVEALYGSNMT DVWSGGIVYMYFEET NKYGLVSIDGNDVKTLDDFNNYSS EINKISPTSANTKSYSATTSDVAC PATGKYWSAATELPPTPNGGLCSC MNAANSCVVSDDVDSDDYETLFNW ICNEVDCSGISANGTAGKYGAYSF CTPKEQLSFVMNLYYEKSGGSKSD CSFSGSATLQTATTQASCSSALKE IGSMGTNSASGSVDLGSGTESSTA SSNASGSSSKSNSGSSGSSSSSSS SSASSSSSSKKNAATNVKANLAQV VFTSIISLSIAAGVGFALV GAS1 P. CWP VDADFPTIEVTGNKFFYSNNGSQF 15 pastoris YIKGVAYQKDTSGLSSDATFVDPL ADKSTCERDIPYLEELGTNV1RVY AVDADADHDDCMQMLQDAGIYVIA DLSQPNNSIITTDPEWTVDLYDGY TAVLDNLQKYDNILGFFAGNEVIT NKSNTDTAPFVKAAIRDMKTYMED KGYRSIPVGYSANDDELTRVASAD YFACGDSDVKADFYGINMYEWCGK ATFSNSGYKDRTAEFKNLSIPVFF SEYGCNEVQPRLFTEVQSLYGDDM TDVWSGGIVYMYFEETNNYGLVTI KSDGDVSTLEDFNNLKTELASISP SIATQSEVSATATEIDCPATGSNW KASTDLPPVPEQAACQCMADALSC VVSEDVDTDDYSDLFSYVCENVSS CDGVSADSESGEYGSYSFCSSKEK LSFLLNLYYSENGAKSSACDFSGS ATLVSGTTASECSSILSAAGTAGT GSITGITGSVEAATQSGSNSGSSK SSSASQSSSSNAGVGGGASGSSWA MTGLVSISVALGMIMSF GAS1* P. CWP VDSILSAAGTAGTGSITGITGSVE 16 pastoris AATQSGSNSGSSKSSSASQSSSSN Truncated AGVGGGASGSSWAMTGLVSISVAL GMIMSF TIP1 H. CWP VDAAATSSVAAAASEVSSSSAAAS 17 polymorpha STQAAAAASTSAAASTEATTSAAA AATSSSEAASSSAHVHSHAAESTS AVESTSAAHSHAAESSSAAHSHAV ESSSAAHVHSHAAESSSAAHSHAA GSSSAASNSSGHISTFSGAGAKLA VGAGAGIVGLAALLM TIP1 H. CWP VDSSAAHSHAVESSSAAHVHSHAA 18 polymorpha ESSSAAHSHAAGSSSAASNSSGHI Truncated STFSGAGAKLAVGAGAGIVGLAAL LM
(132) The nucleic acids encoding each of the anchoring proteins was codon-optimized according to Pichia pastoris codon usage. A nucleic acid encoding a valine and aspartic acid dipeptide (VD) was added to the 5′ end of the nucleic acid encoding the proteins to create a SalI restriction site at the 5′ end of the nucleic acid. The endogenous signal peptides of each of these GPI proteins was replaced with the Aspergillus niger alpha-amylase signal peptide. The DNA encoding the signal peptide is ATGGTTGCTT GGTGGTCCTT GTTCTTGTAC GGATTGCAAG TTGCTGCTCC AGCTTTGGCT (SEQ ID NO:33) and the signal peptide has the amino acid sequence MVAWWSLFLY GLQVAAPALA (SEQ ID NO:34).
(133) Further optimization of anchor protein expression and cell surface localization may be achieved through screening a library of N-terminal signal peptides fused to the n-terminus of the anchoring proteins to identify signal peptides that best localize the GPI protein to the cell surface. For each construct, a nucleic acid encoding a GR2 coiled coil peptide having the amino acid sequence TSRLEGLQSE NHRLRMKITE LDKDLEEVTM QLQDVGGC (SEQ ID NO:19) was inserted between the nucleic acid encoding the signal peptide and the nucleic acid encoding the GPI protein. The cassettes further included a nucleic acid encoding a myc epitope which was inserted between the nucleic acid encoding the GR2 coiled coil peptide and the GPI protein. The myc epitope is optional but had been included in the expression cassettes in order to provide an epitope to facilitate detecting the expressed GPI protein attached to the cell surface using a commercially available anti-myc antibody.
(134)
(135) The Pichia pastoris URA6 locus was chosen as an integrating site for the GPI anchoring protein expression cassettes. The URA6 gene was PCR amplified from Pichia pastoris genomic DNA and cloned into pCR2.1 TOPO to produce plasmid pGLY1849. The Bgl2 and EcoR1 sites within the gene were mutated by silent mutation for cloning purposes. The TRP2 targeting nucleic acid of plasmid pGLY2184 was replaced with the Pichia pastoris URA6 gene from pGLY1849. In addition, the Pichia pastoris ARG1 selection marker was replaced with the with Arsenite marker cassette from plasmid pGF18. The final plasmid was named pGF130t and was used to make the plasmids shown in Table 2.
(136) TABLE-US-00003 TABLE 2 Plasmids Containing Cell Surface Anchoring Expression Cassettes Plasmid Description pGLY3015 S. cerevisiae CWP2-GR2 fusion protein pGLY3033 S. cerevisiae SED1-GR2 fusion protein pGLY3034 S. cerevisiae SED1 truncated-GR2 fusion protein pGLY3035 P. pastoris SPI1-GR2 fusion protein pGLY3036 P. pastoris GAS1-GR2 fusion protein pGLY3037 S. cerevisiae GAS1-GR2 fusion protein pGLY3038 S. cerevisiae GAS1 truncated-GR2 fusion protein pGLY3039 H. polymorpha TIP1-GR2 fusion protein PGLY3040 H. polymorpha TIPI truncated-GR2 fusion protein
(137) The antibody and Fab fragment expression cassettes were constructed as follows.
(138) Expression cassette B is capable of producing a full-length antibody fused to a GR1 coiled coil peptide. The first ORF encodes the light chain and the second ORF encodes a fusion protein comprising the heavy chain fused at the C-terminus to the GR1 coiled coil protein. Each ORF is operably linked to an AOX1 promoter. When expression is induced, this expression cassette is capable of producing full-length antibody consisting of the light chain and heavy chain fused at its C-terminus to a GR1 coiled coil peptide. The full-length antibody can be captured by heterodimerization by the GR2 coiled coil peptide fused to the GPI protein, which is on the surface of the cell. Desired antibodies can then be detected by a suitable detection means.
(139) The limitation of expression cassette B is that the full-length antibodies produced will always include the GR1 coiled coil peptide fused to the heavy chain. This limitation may not be desirable for antibodies that are intended for therapeutic purposes. Thus, a new expression cassette must be constructed by isolating from the host cell that produces the desired antibody the nucleic acid that encodes the desired antibody and recloning the nucleic in an expression cassette that does not include the nucleic acid encoding the GR1 coiled coli peptide and which, therefore, produces the full-length antibody without the GR1 coiled coli peptide fused to the C-terminus of the heavy chain. To get around the limitation, expression cassette C was designed.
(140) Expression cassette C under appropriate conditions is capable of producing full-length antibodies that include the GR1 coiled coil peptide fused to the heavy chain for selection of a desired full-length antibody; however, under production conditions, the expression cassette produces the desired antibody in which the heavy chain is not fused to the GR1 coiled coil peptide. Thus, expression cassette C avoids the need to reclone the nucleic acid encoding the desired antibody. In expression cassette C, the second ORF that encodes a fusion protein comprising the heavy chain fused at the C-terminus to the N-terminus of the GR1 coiled coil peptide further includes a single stop codon between the end of the nucleic acid sequence encoding the heavy chain and the nucleic acid encoding the GR1 coiled coil peptide, in which readthrough of the stop codon is inducible. Normally, stop codons signal the ribosome to terminate the decoding of an mRNA template. In yeast, inefficient termination will allow translation to continue; the frequency of read-through varies depending on the yeast strain and stop codon chosen. The cassette is designed with a stop codon in frame with the nucleic acid encoding the full length antibody and separating it from the nucleic acid encoding the coiled coil peptide GR1. therefore, under most conditions, translation of an mRNA transcribed from the expression cassette predominantly terminates at the single stop codon and thus results in production of a full-length antibody that is not fused to the GR1 coiled coil peptide. However, in the presence of the antibiotic G418, translation readthrough through the stop codon is increased, which results in the production of full-length antibodies fused to GR1 coiled coil peptide; however, even in the presence of the antibiotic, expression of full-length antibody not fused to the GR1 coiled coil peptide is the predominant species. This proportional readthrough can reflect the expressability of the full-length antibody; by monitoring both the secreted full-length antibody and the full-length antibody fusion captured at the cell surface, one can screen for high producing host cells. Thus, in the presence of the antibiotic, a population of the full-length antibodies will include the heavy chain-GR1 coiled coil peptide fusion protein. Therefore, when screening a library of antibodies for a desired antibody, the host cells are grown in the presence of the antibiotic. The full-length antibodies comprising the heavy chain GR1 fusion protein are captured at the cell surface by heterodimerization to the GR2 coiled coil peptide fused to the GPI protein on the surface of the cell. Desired antibodies can then be detected by a suitable detection means. However, for production of full-length antibodies in which the heavy chain is not fused to the GR1 coiled coil peptide, host cells that have been identified to produce the desired antibody are grown in the absence of the antibiotic. The premise behind expression cassette C can be adapted to produce Fab fragments that are not fused to the GR1 coiled coil peptide.
(141) TABLE-US-00004 TABLE 3 Plasmid Containing Antibody or Fab Expression Cassettes Cassette Plasmid Type Description pGLY3028 A Anti-Her2 Fab-GR1 fusion protein pGLY3915 A Anti-Her2 Fab-GR1 fusion protein pGLY3026 A Anti-DKK1 Fab-GR1 fusion protein pGLY3916 A Anti-CD20, C2B8 Fab-GR1 fusion protein pGLY3917 A Anti-CD20, Frame grafted Fab-GR1 fusion protein pGLY3918 A Anti-CD20, Frame grafted Fab-GR1 fusion protein pGLY3919 A Anti-CD20, Frame grafted Fab-GR1 fusion protein pGLY3920 A Anti-CD20, Frame grafted Fab-GR1 fusion protein pGLY3939 B Anti-Her2 full-length antibody-GR1 fusion protein pGLY3941 C Anti-her2 full-length antibody-GR1 fusion protein with single stop codon between antibody ORF and GR1 ORF pGLY3942 C Anti CD20 C2B8 full length antibody-GR1 fusion protein single stop codon between antibody ORF and GR1 ORF pGLY3943 C Anti-CD20 Genmab antibody-GR1 fusion protein single stop codon between antibody ORF and GR1 ORF pGLY3944 C Anti-CD20 full length antibody-GR1 fusion protein single stop codon between antibody ORF and GR1 ORF
(142) Plasmids pGLY3028 and pGLY3915. The amino acid sequences for the heavy and light chains of the anti-her2 antibody are shown in SEQ ID NOs:22 and 23, respectively. The nucleic acid sequence encoding the anti-her2 Fab heavy chain fused to GR1 and the ScαMTprepro signal sequence is shown in SEQ ID NO:51. The nucleic acid sequence encoding the anti-her2 light chain fused to the ScαMTprepro signal sequence (SEQ ID NO:49) is shown in SEQ ID NO:52.
(143) Plasmid pGLY3926. The amino acid sequences for the heavy and light chains of the anti-DKK1 antibody are shown in SEQ ID NOs:24 and 25, respectively. The nucleic acid sequence encoding the anti-DKK1 Fab heavy chain fused to GR1 and the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:53. The nucleic acid sequence encoding the anti-DKK1 light chain fused to the Aspergillus niger alpha amylase signal sequence (SEQ ID NO:33) is shown in SEQ ID NO:54.
(144) Plasmid pGLY3916. The amino acid sequences for the heavy and light chains of the anti-CD20 antibody are shown in SEQ ID NOs:26 and 27, respectively. The nucleic acid sequence encoding the anti-CD20, C2B8, Fab heavy chain fused to GR1 and the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:55. The nucleic acid sequence encoding the anti-CD20, C2B8, light chain fused to the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:56.
(145) Plasmids pGLY3917-3920. The amino acid sequences for frame-grafted heavy and light chains of the anti-C20 Fab antibody are shown in SEQ ID NOs:28 and 29, respectively. The nucleic acid sequence encoding the anti-CD20, frame-grafted, Fab heavy chain fused to GR1 and the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:57. The nucleic acid sequence encoding the anti-CD20, frame-grafted, light chain fused to the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:58.
(146) Plasmids pGLY3939 and 41. The amino acid sequences for the heavy and light chains of the anti-her2 antibody are shown in SEQ ID NOs:22 and 23, respectively. The nucleic acid sequence encoding the anti-her2 full length heavy chain fused to GR1 and the ScαMTprepro signal sequence is shown in SEQ ID NO:59 (pGLY3939). The nucleic acid sequence encoding the anti-her2 full length heavy chain with single stop codon between the heavy chain-encoding ORF and GR1 encoding ORF fused to GR1 and the ScαMTprepro signal sequence is shown in SEQ ID NO:60 (pGLY3941). The nucleic acid sequence encoding the anti-her2 light chain fused to the ScαMTprepro signal sequence in both plasmids is shown in SEQ ID NO:52. Plasmid pGLY3942. The amino acid sequences for the heavy and light chains of the anti-CD20 antibody are shown in SEQ ID NOs:26 and 27, respectively. The nucleic acid sequence encoding the anti-CD-20, C2B8, full length heavy chain with single stop codon between the heavy chain-encoding ORF and GR1 encoding ORF fused to GR1 and the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:61. The nucleic acid sequence encoding the anti-CD20, C2B8, light chain fused to the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:56.
(147) Plasmid pGLY3943. The amino acid sequences for Genmab heavy and light chains of the anti-CD20 antibody are shown in SEQ ID NOs:30 and 31, respectively. The nucleic acid sequence encoding the anti-CD-20, Genmab, full length heavy chain with single stop codon, between the heavy chain-encoding ORF and GR1 encoding ORF fused to GR1 and the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:62. The nucleic acid sequence encoding the anti-CD20, Genmab, light chain fused to the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:63.
(148) Plasmid pGLY3944. The nucleic acid sequence encoding the anti-CD-20 full length heavy chain with single stop codon between the heavy chain-encoding ORF and GR1 encoding ORF fused to GR1 and the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:64. The nucleic acid sequence encoding the anti-CD20 light chain fused to the Aspergillus niger alpha amylase signal sequence is shown in SEQ ID NO:65.
(149) Co-Expression of Fab- and Antibody-GR1 Fusion Protein Expression Cassettes and GPI Protein-GR2 Fusion Protein Expression Cassettes in Yeast.
(150) Two different methods were used for transforming the plasmids containing expression cassettes encoding the GPI protein-GR2 fusion proteins and Fab- or antibody-GR1 fusion proteins into glycoengineered yeast.
(151) In the first approach, plasmid vectors containing the GPI protein-GR2 fusion protein expression cassettes and containing a first selection marker is transformed into P. pastoris and plated on medium with the selection means to select for colonies carrying the GPI protein-GR2 expression cassettes. Then, colony PCR is used to screen the positive colonies for the presence of the GPI protein-GR2 fusion proteins. Finally, these cells are transformed with plasmids containing the Fab- or antibody-GR1 fusion expression cassette and containing a gene for conferring a second selection marker and recombinant cells identified by growing the cells in the presence of a second selection means. In the second approach, the plasmids containing the antibody or Fab-GR1 fusion protein expression cassettes are transformed first into the glycoengineered Pichia pastoris followed by transformation with plasmids containing the GPI protein-GR2 fusion protein expression cassettes.
(152)
(153) Table 4 shows a representative number of yeast strains that were made. All the strains were in a GS2.0 background. GS2.0 strains are glycoengineered Pichia pastoris strains that produce glycoproteins having predominantly Man.sub.5GlcNAc.sub.2 N-glycans (strains YGLY638 and YGLY2696. Strains that produce glycoproteins that have predominantly Man.sub.5GlcNAc.sub.2 N-glycans have been described in for example, U.S. Pat. No. 7,029,872 and in Choi et al., Proc. Natl. Acad. Sci. USA 100: 5022-5027 (2003). Strain YGLY2696 is a GS2.0 strain that further has the gene encoding the endogenous chaperone protein PD1 deleted and expresses a nucleic acid encoding a human PD1 chaperone protein and further includes a nucleic acid encoding the human GRP94 protein inserted into the PEP4 locus (See Example 6 below).
(154) TABLE-US-00005 TABLE 4 Yeast Strains Strain Description YGLY638 GS2.0 glycoengineered Pichia pastoris YGLY2696 GS2.0 glycoengineered and humanized chaperones Pichia pastoris YGLY2966 YGLY638/pGLY3026 - expresses anti-DKK1 Fab YGLY4105 YGLY638/pGLY3028 - expresses anti-Her2 Fab YGLY4145 YGLY4102/pGLY3033 - expresses anti Her2 Fab and SED1 anchor YGLY4146 YGLY2966/pGLY3033 - expresses anti-DKK1 Fab and SED1 anchor YGLY5079 YGLY2696/SED1pGLY3033 #1 SED1 anchor YGLY5147 YGLY2696/pGLY3916 Patch #1 - expresses anti-CD20 Fab YGLY5148 YGLY2696/pGLY3917 Patch #4 - expresses anti-CD20 Fab YGLY5149 YGLY5079/pGLY3916 Patch #16 - expresses anti-CD20 Fab and SED1 anchor YGLY5150 YGLY5079/pGLY3916 Patch #18 - expresses anti-CD20 Fab and SED1 anchor YGLY5151 YGLY5079/pGLY3917 Patch #19 - expresses anti-CD20 Fab and SED1 anchor YGLY5152 YGLY5079/pGLY3917 Patch #20 - expresses anti-CD20 Fab and SED1 anchor YGLY5153 YGLY5079/pGLY3918 Patch #22 - expresses anti-CD20 Fab and SED1 anchor YGLY6693 YGLY5079/pGLY3918 Patch #23 - expresses anti-CD20 Fab and SED1 anchor YGLY6694 YGLY5079/pGLY3919 Patch #25 - expresses anti-CD20 Fab and SED1 anchor
The above Pichia pastoris strains are grown in 50 mL BMGY media until OD 600=2. The cells are washed three times with 1 M sorbitol and resuspended in 1 mL 1 M sorbitol. About 1 to 2 μg linearized plasmid are mixed with these competent cells. Transformation is performed with a BioRad electroporation apparatus using the manufacturer's program specific for electroporation of nucleic acids into Pichia pastoris. One mL recovery media is added to the cells, which are then plated out on MG with 300 μg/mL zeocin or YPG with 50 μg/mL arsenite.
Growth and Induction of Fab Displaying Yeast.
(155) Glycoengineered yeast transformed with both Fab-GR1 fusion protein expression cassette and GPI protein-GR2 expression cassette was inoculated using 600 μL BMGY in a 96 deep well plate or 50 mL BMGY in a 250 mL shake flask for two days. The cells were collected by centrifugation and the supernatant was discarded. The cells are induced by incubation in 300 μL or 25 mL BMMY with Pmti-3 inhibitor overnight following the methods taught in WO2007/061631. Pmti-3 is 3-hydroxy-4-(2-phenylethoxyl)benzaldehyde; 3-(1-phenylethoxy)-4-(2-phenylethoxy)-benzaldehyde, which as been described in U.S. Pat. No. 7,105,554 and Published International Application No. WO 2007061631.
(156) Induced cells were labeled with goat anti-human heavy and light chain (H+L) Alexa 488 conjugated antibody and viewed using fluorescence microscopy (as illustrated in
(157) Following the above in which the expression cassette encoded the anti-Her2 Fab-GR1 fusion protein, it was determined that of the nine GPI anchored proteins in the library, cells that expressed the full length Saccharomyces cerevisiae SED1 had the most intense signal followed by S. cerevisiae CWP2 (See
Example 2
Expression Levels of Two Different Fab-GR1 Fusion Proteins Displayed on the Surface of Glycoengineered Pichia pastoris Correlated with the Expression Levels of their Full Length Counterparts
(158) Expression levels of Anti-Her2 full length monoclonal antibodies are generally five times greater than anti-DKK1 full length monoclonal antibodies when both are expressed in glycoengineered Pichia pastoris. Pichia pastoris expressing full-length anti-Her2 antibodies can produce about 1.3 g/L of antibody whereas Pichia pastoris expressing full-length anti-DKK1 antibodies produces about 200 mg/L in 3 L fermentors. In this Example, anti-Her2 Fab-GR1 fusion protein and anti-DKK1-GR1 fusion protein Fab were expressed and displayed on the surface of glycoengineered Pichia pastoris strain 2.0 expressing the SED1-GR2 fusion protein as described in Example 1. The amino acid sequences of the anti-her2 heavy and light chains are shown in SEQ ID NOs:22 and 23, respectively. The amino acid sequences of the anti-DKK1 heavy and light chains are shown in SEQ ID NOs:24 and 25, respectively.
(159) To determine the expression levels of the two Fabs, cells were labeled with goat anti-Human H+L Alexa 488 and photographed according to the method described in the Example 2.
Example 3
(160) Flow cytometry analysis was conducted using the cells expressing anti-Her2 Fab and anti-DKK1 Fab displayed on the surface of the cells. Glycoengineered yeast displaying fluorescently labeled anti-Her2 or anti-DKK1 Fab respectively were prepared as described in Examples 1 and 2. Controls were prepared in which both cell types were not labeled with the detection antibody. Using flow cytometry analysis, anti-Her2 Fab displaying cells were found to have a stronger fluorescence intensity compared to anti-DKK1 Fab displaying cells and both cell types had a stronger signal compared to the signal produced in their corresponding unlabeled controls. In
(161) Fluorescence-activated cell sorting (FACS) profile of a mixture of cells displaying anti-Her2 Fab (strain YGLY4145) and anti-DKK1 Fab (strain YGLY4146) was performed as follows. The cells displaying anti-Her2 Fab and cells displaying anti-DKK1 Fab were mixed together in the following ratios: 1:1, 1:10, 1:100 and 1:1000. Cells were labeled with goat anti-human H+L Alexa 488 prior to mixing.
(162) A second experiment was performed to gain better insight into cell diversity across the observed distribution of high to low levels of fluorescence. Anti-Her2 Fab and anti-DKK1 Fab displaying cells (strains YGLY4145 and YGLY4146, respectively) were mixed at a ratio of 1:1 (See
Example 4
(163) This example illustrates the use of FACS to isolate and enrich for a population of high Fab producing cells from a larger population of low level Fab producing cells.
(164) Fluorescently labeled anti-Her2 Fab and anti-DKK1 Fab displaying cells were labeled, mixed at a ratio of 1:1000, and analyzed by flow cytometry. The cells of highest 1% of fluorescence were isolated (far right of left histogram in
Example 5
(165) This example illustrates surface display of full-length antibodies using the methods disclosed herein.
(166)
(167) Pichia pastoris strain YGLY6724 containing pGLY3941 displays a full length anti-Her2 antibody-GR1 coiled coil fusion protein when the protein is produced under conditions that results in translational readthrough of the stop codon (See SEQ ID 32). Pichia pastoris strain YGLY6722 containing pGLY3939 (no stop codon between the coding sequences for the Her2 antibody and the GR1 peptide) also displays a full length anti-Her2 antibody-GR1 coiled coil fusion. YGLY6724 was grown with increasing amounts of the antibiotic G418 in the medium. G418 inhibits translational termination, thereby increasing stop codon readthrough and increasing fluorescence intensity. To determine the expression levels of the two antibodies, cells were labeled with goat anti-Human H+L Alexa 488 and photographed according to the method described in Example 2.
Example 6
(168) In strain YGLY2696, the gene encoding the endogenous PD1 replaced with a nucleic acid molecule encoding the human PD1 and a nucleic acid molecule encoding the human GRP94 protein inserted into the PEP4 locus. The strain was further engineered to alter the endogenous glycosylation pathway to produce glycoproteins that have predominantly Man.sub.5GlcNAc.sub.2 N-glycans. Strain YGLY2696 has been disclosed in co-pending application Ser. Nos. 61/066,409, filed 20 Feb. 2008, and 61/188,723, filed 12 Aug. 2008, both of which are incorporated herein in their entirety. This strain was shown to be useful for producing immunoglobulins and for producing immunoglobulins that have reduced O-glycosylation. Construction of strain yGLY2696 involved the following steps.
(169) Construction of expression/integration plasmid vector pGLY642 comprising an expression cassette encoding the human PDI protein and nucleic acid molecules to target the plasmid vector to the Pichia pastoris PDI1 locus for replacement of the gene encoding the Pichia pastoris PDI1 with a nucleic acid molecule encoding the human PDI was as follows and is shown in
(170) The nucleotide and amino acid sequences of the Pichia pastoris PDI1 are shown in SEQ ID NOs:39 and 40, respectively. Isolation of nucleic acid molecules comprising the Pichia pastoris PDI1 5′ and 3′ regions was performed by PCR amplification of the regions from Pichia pastoris genomic DNA. The 5′ region was amplified using primers PB248: 5′ ATGAA TTCAG GCCAT ATCGG CCATT GTTTA CTGTG CGCCC ACAGT AG 3′ (SEQ ID NO: 41); PB249: 5′ ATGTT TAAAC GTGAG GATTA CTGGT GATGA AAGAC 3′ (SEQ ID NO: 42). The 3′ region was amplified using primers PB250: 5′ AGACT AGTCT ATTTG GAGAC ATTGA CGGAT CCAC 3′ (SEQ ID NO: 43); PB251:5′ ATCTC GAGAG GCCAT GCAGG CCAAC CACAA GATGA ATCAA ATTTT G-3′ (SEQ ID NO: 44). Pichia pastoris strain NRRL-11430 genomic DNA was used for PCR amplification. The PCR conditions were one cycle of 95° C. for two minutes, 25 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 2.5 minutes, and followed by one cycle of 72° C. for 10 minutes. The resulting PCR fragments, PpPDI1 (5′) and PpPDI1 (3′), were separately cloned into plasmid vector pCR2.1 to make plasmid vectors pGLY620 and pGLY617, respectively. To construct pGLY678, DNA fragments PpARG3-5′ and PpARG-3′ of integration plasmid vector pGLY24, which targets the plasmid vector to Pichia pastoris ARG3 locus, were replaced with DNA fragments PpPD1 (5′) and PpPD1 (3′), respectively, which targets the plasmid vector pGLY678 to the PDI1 locus and disrupts expression of the PDI1 locus.
(171) The nucleic acid molecule encoding the human PDI was then cloned into plasmid vector pGLY678 to produce plasmid vector pGLY642 in which the nucleic acid molecule encoding the human PDI was placed under the control of the Pichia pastoris GAPDH promoter (PpGAPDH). Expression/integration plasmid vector pGLY642 was constructed by ligating a nucleic acid molecule encoding the Saccharomyces cerevisiae alpha mating factor (MF) presequence signal peptide (ScαMFpre-signal peptide) having a NotI restriction enzyme site at the 5′ end and a blunt 3′ end and the expression cassette comprising the nucleic acid molecule encoding the human PDI released from plasmid vector pGLY618 with AfeI and PacI to produce a nucleic acid molecule having a blunt 5′ end and a PacI site at the 3′ end into plasmid vector pGLY678 digested with NotI and PacI. The resulting integration/expression plasmid vector pGLY642 comprises an expression cassette encoding a human PDI1/ScαMFpre-signal peptide fusion protein operably linked to the Pichia pastoris promoter and nucleic acid molecule sequences to target the plasmid vector to the Pichia pastoris PDI1 locus for disruption of the PDI1 locus and integration of the expression cassette into the PDI1 locus.
(172) Construction of expression/integration vector pGLY2233 encoding the human GRP94 protein was as follows and is shown in
(173) The nucleic acid molecule encoding the human GRP94 was released from plasmid vector pGLY2216 with AfeI and FseI. The nucleic acid molecule was then ligated to a nucleic acid molecule encoding the ScαMPpre-signal peptide having NotI and blunt ends as above and plasmid vector pGLY2231 digested with NotI and FseI carrying nucleic acid molecules comprising the Pichia pastoris PEP4 5′ and 3′ regions (PpPEP4-5′ and PpPEP4-3′ regions, respectively) to make plasmid vector pGLY2229. Plasmid vector pGLY2229 was digested with BglII and NotI and a DNA fragment containing the PpPDI1 promoter was removed from plasmid vector pGLY2187 with BglII and NotI and the DNA fragment ligated into pGLY2229 to make plasmid vector pGLY2233. Plasmid vector pGLY2233 encodes the human GRP94 fusion protein under control of the Pichia pastoris PDI promoter and includes the 5′ and 3′ regions of the Pichia pastoris PEP4 gene to target the plasmid vector to the PEP4 locus of genome for disruption of the PEP4 locus and integration of the expression cassette into the PEP4 locus.
(174) Construction of plasmid vectors pGLY1162, pGLY1896, and pGF1207t was as follows. All Trichoderma reesei α-1,2-mannosidase expression plasmid vectors were derived from pGF1165, which encodes the T. reesei α-1,2-mannosidase catalytic domain (See published International Application No. WO2007061631) fused to S. cerevisiae αMATpre signal peptide (ScαMPpre-signal peptide) herein expression is under the control of the Pichia pastoris GAP promoter and wherein integration of the plasmid vectors is targeted to the Pichia pastoris PRO1 locus and selection is using the Pichia pastoris URA5 gene. A map of plasmid vector pGF1165 is shown in
(175) Plasmid vector pGLY1162 was made by replacing the GAP promoter in pGF1165 with the Pichia pastoris AOX1 (PpAOX1) promoter. This was accomplished by isolating the PpAOX1 promoter as an EcoR1 (made blunt)-BglII fragment from pGLY2028, and inserting into pGF1165 that was digested with NotI (made blunt) and BglII. Integration of the plasmid vector is to the Pichia pastoris PRO1 locus and selection is using the Pichia pastoris URA5 gene. A map of plasmid vector pGLY1162 is shown in
(176) Plasmid vector pGLY1896 contains an expression cassette encoding the mouse α-1,2-mannosidase catalytic domain fused to the S. cerevisiae MNN2 membrane insertion leader peptide fusion protein (See Choi et al., Proc. Natl. Acad. Sci. USA 100: 5022 (2003)) inserted into plasmid vector pGF1165 (
(177) Plasmid vector pGF1207t is similar to pGLY1896 except that the URA5 selection marker was replaced with the S. cerevisiae ARR3 (ScARR3) gene, which confers resistance to arsenite. This was accomplished by isolating the ScARR3 gene from pGFI166 digested with AscI and the AscI ends made blunt) and BglII, and inserting the fragment into pGLY1896 that digested with SpeI and the SpeI ends made blunt and BglII. Integration of the plasmid vector is to the Pichia pastoris PRO1 locus and selection is using the Saccharomyces cerevisiae ARR3 gene. A map of plasmid vector pGFI2007t is shown in
(178) Yeast transfections with the above expression/integration vectors were as follows. Pichia pastoris strains were grown in 50 mL YPD media (yeast extract (1%), peptone (2%), dextrose (2%)) overnight to an OD of between about 0.2 to 6. After incubation on ice for 30 minutes, cells were pelleted by centrifugation at 2500-3000 rpm for 5 minutes. Media was removed and the cells washed three times with ice cold sterile 1 M sorbitol before resuspending in 0.5 ml ice cold sterile 1 M sorbitol. Ten μL linearized DNA (5-20 μg) and 100 μL cell suspension was combined in an electroporation cuvette and incubated for 5 minutes on ice. Electroporation was in a Bio-Rad GenePulser Xcell following the preset Pichia pastoris protocol (2 kV, 25 μF, 200Ω), immediately followed by the addition of 1 mL YPDS recovery media (YPD media plus 1 M sorbitol). The transfected cells were allowed to recover for four hours to overnight at room temperature (26° C.) before plating the cells on selective media.
(179) Generation of Cell Lines was as follows and is shown in
(180) Strains yGLY702 and yGLY704 were generated in order to test the effectiveness of the human PD11 expressed in Pichia pastoris cells in the absence of the endogenous Pichia pastoris PDI gene. Strains yGLY702 and yGLY704 (huPDI) were constructed as follows. Strain yGLY702 was generated by transfecting yGLY24-1 with plasmid vector pGLY642 containing the expression cassette encoding the human PDI under control of the constitutive PpGAPDH promoter. Plasmid vector pGLY642 also contained an expression cassette encoding the Pichia pastoris URA5, which rendered strain yGLY702 prototrophic for uracil. The URA5 expression cassette was removed by counterselecting yGLY702 on 5-FOA plates to produce strain yGLY704 in which, so that the Pichia pastoris PDI1 gene has been stably replaced by the human PDI gene and the strain is auxotrophic for uracil.
(181) Strain yGLY733 was generated by transfecting with plasmid vector pGLY1162, which comprises an expression cassette that encodes the Trichoderma Reesei mannosidase (TrMNS1) operably linked to the Pichia pastoris AOX1 promoter (PpAOX1-TrMNS1) and the Saccharomyces cerevisiea αMAT pre signal sequence, into the PRO1 locus of yGLY704. This strain has the gene encoding the Pichia pastoris PDI replaced with the expression cassette encoding the human PDI1, has the PpAOX1-TrMNS1 expression cassette integrated into the PRO1 locus, and is a URA5 auxotroph. The PpAOX1 promoter allows overexpression when the cells are grown in the presence of methanol.
(182) Strain yGLY762 was constructed by integrating expression cassettes encoding TrMNS1 and mouse mannosidase IA (MuMNS1A), each operably linked to the Pichia pastoris GAPDH promoter in plasmid vector pGF1207t into control strain yGLY733 at the 5′ PRO1 locus UTR in Pichia pastoris genome. This strain has the gene encoding the Pichia pastoris PD1 replaced with the expression cassette encoding the human PDI1, has the PpGAPDH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integrated into the PRO1 locus, and is a URA5 auxotroph.
(183) Strain yGLY2677 was generated by counterselecting yGLY762 on 5-FOA plates. This strain has the gene encoding the Pichia pastoris PDI replaced with the expression cassette encoding the human PDI1, has the PpAOX1-TrMNS1 expression cassette integrated into the PRO1 locus, has the PpGAPH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integrated into the PRO1 locus, and is a URA5 prototroph.
(184) Strains yGLY2696 was generated by integrating plasmid vector pGLY2233, which encodes the human GRP94 protein, into the PEP4 locus. This strain has the gene encoding the Pichia pastoris PD1 replaced with the expression cassette encoding the human PDI1, has the PpAOX1-TrMNS1 expression cassette integrated into the PRO1 locus, has the PpGAPDH-TrMNS1 and PpGAPDH-MuMNS1A expression cassettes integrated into the PRO1 locus, has the human GRP64 integrated into the PEP4 locus, and is a URA5 prototroph. The genealogy of this chaperone-humanized strain is shown in
Example 7
(185) Construction of plasmid pGLY5107, pGLY5108 and pGLY5110 encoding various antibody heavy and light chains to make Fab fragments 1H23 and 1D05 (low and high affinity Fab fragments specific to PCSK9, Proprotein convertase subtilisin/kexin type 9) and anti-CD20 Fab fragment Genmab was as follows.
(186) Fab display vector pGLY3958 (
(187) Nucleic acid molecules encoding the variable regions of the heavy and light chains of 1D05, 1H23, and anti-CD20 Genmab were codon optimized, reverse translated, and synthesized by GeneArt based on their amino acid sequences. Nucleic acid molecules encoding the Aspergillus amylase signal sequence (SEQ ID NO:33) was added in-frame to the 5′ end of the open reading frames encoding the 1H23 heavy and light chains (SEQ ID NO:70 and 72, respectively) during the gene synthesis. The open reading frame encoding the heavy chain also included the nucleotide sequence encoding GR1. Nucleic acid molecules encoding the Saccharomyces cerevisiae mating factor pre-signal peptide (alpha-MAT-pro; SEQ ID NO:49) signal sequence was added in-frame to the 5′ end of the open reading frames encoding the 1D05 heavy and light chains (SEQ ID NO:66 and 68, respectively) during the gene synthesis. The open reading frame encoding the heavy chain also included the nucleotide sequence encoding GR1. During synthesis, EcoR1 site was introduced at the 5′ of the nucleic acid molecules encoding the heavy chains and Pst1 sites were introduced at the 5′ ends of the nucleic acid molecules encoding the light chains. Xho1 and Kpn1 sites were created at the 3′ ends of the heavy and light chains, respectively, using nucleic acid molecules encoding heavy chain and light chain constant regions conserved amino acids. The nucleic acid molecules encoding the variable regions of the heavy chain and light chains were cloned into pGLY3958 (
(188) Yeast transformation for making 1D05, 1H23 and anti-CD20 Genmab Fab display strains were as follows. Plasmids pGLY5107, pGLY5108 and pGLY5110 were linearized by Spe1 digestion at 37° C. and linearization was confirmed by gel electrophoresis. DNA was precipitated down using standard procedure using cold ethanol. Grew Pichia host YGLY5079 (expresses ScSED1-GR2 fusion protein in YGLY2696) in 50 mL BMGY media overnight to a cell density of between 1-2 of OD.sub.600. Cells were washed three times with cold sterile water and 1 M sorbitol to render the cells competent for transformation. The linearized DNA was mixed with competent cells and shocked using the Bio-Rad electroporation machine. Then 1 mL recovery media was added to the shocked cells and the cells incubated at room temperature for 1 to 2 hours. Then the cells were plated on YPG plates with appropriate Zeocin concentration to select for transformants. The strains produced are shown in Table 5.
(189) TABLE-US-00006 TABLE 5 Strains Fab displayed Host Plasmid YGLY7761 Anti-CD20 (Genmab) YGLY5079 pGLY5107 YGLY7762 1D05 YGLY5079 PGLY5108 YGLY7764 1H23 YGLY5079 pGLY5110
Example 8
(190) It has been reported for Saccharomyces cerevisiae that assembly of heavy and light chains expressed in yeast can be problematic. Therefore, the ratio of heavy chain to light chain in the Fab fragments displayed on the cell surface was measured to determine the intactness of the Fab fragments displayed on the cell surface.
(191) Strain YGLY7762 (expresses 1D05 Fab fragment heavy and light chains) and strain YGLY7764 (expresses 1H23 Fab fragment heavy and light chains) were grown in 200 mL BMGY and expression induced in a Micro24 bioreactor according to the description of Micro24 cell culture and induction. Then remove about 20-40 uL of induced yeast culture, add 1 mL of blocking solution to the sample, centrifuge at 10,000 rpm for 30 seconds and wash the cell pellet three times with 1 mL blocking solution. Measure OD.sub.600 and calculate the volume needed to get an OD.sub.600 of 1 in desired final volume. (Usually the final volume is about 200 uL). Blocking solution: 60 g BSA from Omni Pur, 200 mL 0.5% Tween 20, 200 mL 10×PBS (from Omni Pur), and dH.sub.20 up to two liters.
(192) Anti-human IgG.sub.2 Fd biotin-conjugated antibody (CALTAG Laboratories, code #MH1522, lot#443408A: anti-heavy chain antibody) coupled with strepavidin Alexa Fluor 488 (2 mg/mL, Invitrogen, lot#53729A) was used for detecting the displayed Fab via the Fd region of the heavy chain and anti-human kappa allophycocyanin-conjugated antibody (CALTAG Laboratories, code# MH10515, lot #358897A: anti-light chain antibody) was used for detecting the light chain. In general, three uL of anti-heavy chain antibody was incubated with the cells at room temperature for 30 minutes on a rotator kept in the dark. Then the cells were washed four times with 3% BSA-0.05% Tween 20-PBS buffer. After this, three uL of Strepavidin Alexa Fluor 488 and 3 uL of anti-light chain antibody Were added and the mixtures incubated at room temperature for 30 minutes on a rotator in the dark. Then, cells were wash three times with 3% BSA-0.05% Tween 20-PBS buffer. The cells were analyzed by FACS. Fluorescent intensity of light chain and heavy chain (Fd) were plotted using FluoJo.
(193) Flow cytometric analysis showed that displayed heavy chains corresponded with displayed light chains. This is shown in
(194) Cell Culture and Induction in Micro 24
(195) Yeast display cells are grown in 200 mL BMGY medium in regular shake flask for two days at room temperature. The yeast culture is centrifuged and the spent supernatant is decanted. The remaining cell pellet is suspended in fresh induction media (see below for recipe) to an OD.sub.600 of between 100 and 200 depending on the experiment. About 4.5 mL of the resulting culture is inoculated into a well of an Applikon Microreactor cassette and a gas-permeable, low evaporation adhesive membrane is used to seal the cassette. The induced cells are run using a constant agitation rate of 800 rpm with a pH set-point of 6.5. Each well is aerated with a continuous flow of 1vvm (4.5 mL/min). Under these conditions the culture will typically consume 2.5% methanol in about 16-20 hours. After 16-20 hours or when a dissolved oxygen spike is observed and additional bolus of 1%-2.5% methanol will be added so the cells remain in an induction start. Once the desired length of induction is achieved the Microreactor is stopped and the culture can be removed from the well for labeling.
(196) TABLE-US-00007 BMGY Medium KH.sub.2PO.sub.4 11.9 g/L K.sub.2HPO.sub.4 2.5 g/L Yeast Nitrogen Base 13.4 g/L Biotin (400 mg/L stock) 10 ml/L Sorbitol 18.2 g/L Soytone 20 g/L Yeast Extract 10 g/L Methanol 25 g/L Sigma 204 8 drops/L
Example 8
(197) This example shows that the method can sort cells that display the antibody or Fab fragments of interest from cells that do not display the antibody of Fab of interest.
(198) In a first experiment, Pichia pastoris cells engineered to display anti-CD20 Fab fragments (YGLY7761) were mixed with Pichia pastoris cells engineered to display anti-PCSK-9 Fab fragments (YGLY7762).
(199) Strains YGLY7762, YGLY7761, and YGLY7764 were incubated at 24° C. for 24 hours and expression induced in Micro24 with BMMY and PMT inhibitor as described previously for 18 hours. Induced cells were harvested and transferred into 50 mL tubes; centrifuged at 2500 rpm for five minutes at 4° C. Supernatant fractions were decanted and the pellets resuspended in 50 mL of blocking solution. The cells were pelleted as before and the cell pellet washed once more in 50 mL of blocking solution and cells pelleted. The pellet was resuspended in blocking solution and the OD.sub.600 was adjusted with blocking solution to give about three OD units. Then the cells were mixed in a 1:1 ratio and then labeled sequentially with fluorophore-conjugated PCSK9 antigen (Alexa 647-conjugated) for one hour at room temperature and fluorophore-conjugated generic H+L antibody (Alexa Fluor488-conjugated) for 30 minutes at room temperature. Afterwards, the cells were washed and the flow cytometric profile was determined.
(200)
(201) In
Example 9
(202) This example shows that the method can sort cells that display the antibody or Fab fragments of interest from a majority of cells that do not display the antibody of Fab of interest.
(203) In a first experiment, Pichia pastoris cells engineered to display anti-PCSK-9 Fab fragments were mixed with Pichia pastoris cells engineered to display anti-CD20 Fab fragments. The cell populations were mixed at ratios of 1:1,000; 1:10,000; and 1:100,000. Each ratio of cells was then labeled sequentially with fluorophore-conjugated PCSK9 antigen (Alexa 647-conjugated) for one hour at room temperature and fluorophore-conjugated generic H+L antibody (Alexa Fluor488-conjugated) for 30 minutes at room temperature. Afterwards, the cells were washed and the flow cytometric profile was determined.
(204) The cells from the area corresponding to the highest 1% fluorescence (area expected for the anti-PCSK-9 Fab fragments) were isolated. The cells were plated out on selection media and incubated three to four days. The cells were then collected by washing the plate with BMGY media and re-induced with BMMY. The re-induced cells were labeled and subsequently sorted. This first round of sorting resulted in two distinct populations of cells (
(205) For the 1:10,000 and 1; 100,000 dilutions, the cells with the highest fluorescence were isolated and, as in the first round of sorting, grown, collected, induced, and labeled again. These cells were again analyzed using flow cytometry (
(206) In a second experiment, Pichia pastoris cells engineered to display high affinity anti-PCSK-9 Fab fragments (1D05) were mixed with Pichia pastoris cells engineered to display low affinity anti-PCSK-9 Fab fragments (1H23). The cell populations were mixed at ratios of 1:10,000 and 1:100,000. The cells were labeled with fluorophore-conjugated PCSK9 antigen (Alexa 647-conjugated) for one hour at room temperature. The cells were washed and the flow cytometric profile was determined.
(207) The cells from the area corresponding to the highest 1% fluorescence (area expected for high affinity 1D05 Fab fragments were isolated). The cells were plated out on selection media and incubated three to four days. The cells were then collected by washing the plate with BMGY media and re-induced with BMMY. The re-induced cells were labeled and subsequently sorted. This first round of sorting resulted in two distinct populations of cells (
(208) For the 1:10,000 and 1; 100,000 dilutions, the cells with the highest fluorescence were isolated and, as in the first round of sorting, grown, collected, induced, and labeled again. These cells were again analyzed using flow cytometry (
(209) These experiments in this example clearly demonstrate the versatility and power of a cell-sorting based approach to isolate and enrich for particular population of antibody or Fab fragments. The methods herein can be used to isolate and enrich for cells expressing particular populations of antibodies or Fab fragments.
BRIEF DESCRIPTION OF THE SEQUENCES
(210) TABLE-US-00008 SEQ ID NO: Name Sequence (5′ to 3′) 1 c-fos zipper LQAETDQLEDEKSALQTEIANLLKEKEKL 2 c-jun zipper LEEKVKTLKAQNSELASTANMLREQVAQL 3 c-fos zipper LTDTLQAETDQLEDEKSALQTEIANLLKEKE KLEFILA 4 c-jun zipper RIARLEEKVKTLKAQNSELASTANMLREQVA QLKQKVMN 5 c-jun zipper LEEKVKTLKAQNSELASTFNMLREQFAQL 6 c-jun zipper LEEKVKTLKAQNSELASTANMLREQVAQF 7 c-jun zipper LEEKVKTFKAQNSELASTANMLREQVAQF 8 c-jun zipper LEEKVKSFKAQNSEHASTANMLREQVAQL 9 S. cerevisiae VDESAAAISQITDGQIQATTTATTEATTTAA CWP2 PSSTVETVSPSSTETISQQTENGAAKAAVGM GAGALAAAAMLL 10 S. cerevisiae VDTTEATTTAAPSSTVETVSPSSTETISQQT CWP2 ENGAAKAAVGMGAGALAAAAMLL truncated version 11 S. cerevisiae VDQFSNSTSASSTDVTSSSSISTSSGSVTIT SED1 SSEAPESDNGTSTAAPTETSTEAPTTAIPTN GTSTEAPTTAIPTNGTSTEAPTDTTTEAPTT ALPTNGTSTEAPTDTTTEAPTTGLPTNGTTS AFPPTTSLPPSNTTTTPPYNPSTDYTTDYTV VTEYTTYCPEPTTFTTNGKTYTVTEPTTLTI TDCPCTIEKPTTTSTTEYTVVTEYTTYCPEP TTFTTNGKTYTVTEPTTLTITDCPCTIEKSE APESSVPVTESKGTTTKETGVTTKQTTANPS LTVSTVVPVSSSASSHSVVINSNGANVVVPG ALGLAGVAMLFL 12 S. cerevisiae VDLTVSTVVPVSSSASSHSVVINSNGANVVV SED1 PGALGLAGVAMLFL truncated version 13 Pichia VDLVSNSSSSVIVVPSSDATIAGNDTATPAP pastoris SPI1 EPSSAAPIFYNSTATATQYEVVSEFTTYCPE PTTFVTNGATFTVTAPTTLTITNCPCTIEKP TSETSVSSTHDVETNSNAANARAIPGALGLA GAVMMLL 14 S. cerevisiae VDDVPAIEVVGNKFFYSNNGSQFYIRGVAYQ GAS1 ADTANETSGSTVNDPLANYESCSRDIPYLKK LNTNVIRVYAINTTLDHSECMKALNDADIYV IADLAAPATSINRDDPTWTVDLFNSYKTVVD TFANYTNVLGFFAGNEVTNNYTNTDASAFVK AAIRDVRQYISDKNYRKIPVGYSSNDDEDTR VKMTDYFACGDDDVKADFYGINMYEWCGKSD FKTSGYADRTAEFKNLSIPVFFSEYGCNEVT PRLFTEVEALYGSNMTDVWSGGIVYMYFEET NKYGLVSIDGNDVKTLDDFNNYSSEINKISP TSANTKSYSATTSDVACPATGKYWSAATELP PTPNGGLCSCMNAANSCVVSDDVDSDDYETL FNWICNEVDCSGISANGTAGKYGAYSFCTPK EQLSFVMNLYYEKSGGSKSDCSFSGSATLQT ATTQASCSSALKEIGSMGTNSASGSVDLGSG TESSTASSNASGSSSKSNSGSSGSSSSSSSS SASSSSSSKKNAATNVKANLAQVVFTSIISL SIAAGVGFALV 15 Pichia VDADFPTIEVTGNKFFYSNNGSQFYIKGVAY pastoris GAS1 QKDTSGLSSDATFVDPLADKSTCERDIPYLE ELGTNVIRVYAVDADADHDDCMQMLQDAGIY VIADLSQPNNSIITTDPEWTVDLYDGYTAVL DNLQKYDNILGFFAGNEVITNKSNTDTAPFV KAAIRDMKTYMEDKGYRSIPVGYSANDDELT RVASADYFACGDSDVKADFYGINMYEWCGKA TFSNSGYKDRTAEFKNLSIPVFFSEYGCNEV QPRLFTEVQSLYGDDMTDVWSGGIVYMYFEE TNNYGLVTIKSDGDVSTLEDFNNLKTELASI SPSIATQSEVSATATEIDCPATGSNWKASTD LPPVPEQAACQCMADALSCVVSEDVDTDDYS DLFSYVCENVSSCDGVSADSESGEYGSYSFC SSKEKLSFLLNLYYSENGAKSSACDFSGSAT LVSGTTASECSSILSAAGTAGTGSITGITGS VEAATQSGSNSGSSKSSSASQSSSSNAGVGG GASGSSWAMTGLVSISVALGMIMSF 16 Pichia VDSILSAAGTAGTGSITGITGSVEAATQSGS pastoris GAS1 NSGSSKSSSASQSSSSNAGVGGGASGSSWAM truncated TGLVSISVALGMIMSF version 17 H. polymorpha VDAAATSSVAAAASEVSSSSAAASSTQAAAA TIP1 ASTSAAASTEATTSAAAAATSSSEAASSSAH VHSHAAESTSAVESTSAAHSHAAESSSAAHS HAVESSSAAHVHSHAAESSSAAHSHAAGSSS AASNSSGHISTFSGAGAKLAVGAGAGIVGLA ALLM 18 H. polymorpha VDSSAAHSHAVESSSAAHVHSHAAESSSAAH TIP1 SHAAGSSSAASNSSGHISTFSGAGAKLAVGA truncated GAGIVGLAALLM version 19 Human GR2, TSRLEGLQSENHRLRMKITELDKDLEEVTMQ coiled coil LQDVGGC peptide sequence 20 SED 1 MVAWWSLFLYGLQVAAPALATSRLEGLQSEN Fusion Leader HRLRMKITELDKDLEEVTMQLQDVGGCEQKL GR2 ISEEDLVDQFSNSTSASSTDVTSSSSISTSS cMyc GSVTITSSEAPESDNGTSTAAPTETSTEAPT SED1 TAIPTNGTSTEAPTTA1PTNGTSTEAPTDTT TEAPTTALPTNGTSTEAPTDTTTEAPTTGLP TNGTTSAFPPTTSLPPSNTTTTPPYNPSTDY TTDYTVVTEYTTYCPEPTTFTTNGKTYTVTE PTTLTITDCPCTIEKPTTTSTTEYTVVTEYT TYCPEPTTFTTNGKTYTVTEPTTLTITDCPC TIEKSEAPESSVPVTESKGTTTKETGVTTKQ TTANPSLTVSTVVPVSSSASSHSVVINSNGA NVVVPGALGLAGVAMLFL 21 Human GR1 EEKSRLLEKENRELEKIIAEKEERVSELRHQ coiled coil LQSVGGC peptide sequence 22 mAb1 (anti- EVQLVESGGGLVQPGGSLRLSCAASGFNIKD her2) Heavy TYIHWVRQAPGKGLEWVARIYPTNGYTRYAD chain SVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 23 mAb1 (anti- DIQMTQSPSSLSASVGDRVTITCRASQDVNT her2) Light AVAWYQQKPGKAPKLLIYSASFLYSGVPSRF chain SGSRSGTDFTLTISSLQPEDFATYYCQQHYT TPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC 24 mAb2 (anti- EVQLVQSGAEVKKPGASVKVSCKASGYTFTD DKK1) YYIHWVRQAPGQGLEWMGWIHSNSGATTYAQ Heavy chain KFQARVTMSRDTSSSTAYMELSRLESDDTAM YFCSREDYWGQGTLVTVSSASTKGPSVFPLA PCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVTSS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTFRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKTKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 25 mAb2 (anti- QSVLTQPPSVSGAPGQRVTISCTGSSSNIGA DKK1) Light GYDVHWYQQLPGTAPKLLIYGYSNRPSGVPD Chain RFSGSKSGASASLAITGLRPDDEADYYCQSY DNSLSSYVFGGGTQLTVLSQPKANPTVTLFP PSSEELQANKATLVCLISDFYPGAVTVAWKA DGSPVKAGVETTKPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTEC 26 mAb3 (anti- QVQLQQPGAELVKPGASVKMSCKASGYTFTS CD20, C2B8) YNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQ Heavy chain KFKGKATLTADKSSSTAYMQLSSLTSEDSAV YYCARSTYYGGDWYFNVWGAGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 27 mAb3 (anti- QIVLSQSPAILSASPGEKVTMTCRASSSVSY CD20, C2B8) IHWFQQKPGSSPKPWIYATSNLASGVPVRFS Light chain GSGSGTSYSLTISRVEAEDAATYYCQQWTSN PPTFGGGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 28 Protein QVQLVQSGAEVKKPGSSVKVSCKASGYTFTS mAb4 (anti- YNMHWVRQAPGQGLEWMGAIYPGNGDTSYNQ CD20, Frame KFKGRVTITADESTSTAYMELSSLRSEDTAV grafted YYCARSTYYGGDWYFNVWGQGTLVTVSSAST Heavy chain KGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 29 mAb4 (anti- EIVLTQSPATLSLSPGERATLSCRASSSVSY CD20, Frame IHWYQQKPGQAPRLLIYATSNLASGIPARFS grafted) GSGSGTDFTLTISSLEPEDFAVYYCQQWTSN Light chain PPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 30 mAb5 (anti- AVQLVESGGGLVQPGRSLRLSCAASGFTFGD CD20, YTMHWVRQAPGKGLEWVSGISWNSGSIGYAD Genmab) SVKGRFTISRDNAKNSLYLQMNSLRAEDTAL Heavy chain YYCTKDNQYGSGSTYGLGVWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 31 mAb5 (anti- EIVLTQSPATLSLSPGERATLSCRASQSVSS CD20, Genmab) YLAWYQQKPGQAPRLLIYDASNRATGIPARF Light chain SGSGSGTDFTLTISSLEPEDFAVYYCQQRSN WPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC 32 Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKD mAb heavy TYIHWVRQAPGKGLEWVARIYPTNGYTRYAD chain SVKGRFTISADTSKNTAYLQMNSLRAEDTAV readthrough YYCSRWGGDGFYAMDYWGQGTLVTVSSASTK coiled coil GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE peptide PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS with one SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK stop codon VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP X - unkown KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN aa incor- WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT porated at VLHQDWLNGKEYKCKVSNKALPAPIEKTISK stop codon AKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKXAAAYPYDVPDYAGG HHHHHHHHHGGEEKSRLLEKENRELEKIIAE KEERVSELRHQLQSVGGC 33 Alpha amylase ATGGTTGCTT GGTGGTCCTT signal GTTCTTGTAC GGATTGCAAG sequence TTGCTGCTCC AGCTTTGGCT (from Aspergillus niger α- amylase) (DNA) 34 Alpha amylase MVAWWSLFLY GLQVAAPALA signal sequence (from Aspergillus niger α- amylase) 35 PCR primer AGCGCTGACGCCCCCGAGGAGGAGGACCAC hPDI/UP1 36 PCR primer CCTTAATTAATTACAGTTCATCATGCACAGC hPDI/LP- TTTCTGATCAT PacI 37 human PDI GACGCCCCCGAGGAGGAGGACCACGTCTTGG Gene (DNA) TGCTGCGGAAAAGCAACTTCGCGGAGGCGCT GGCGGCCCACAAGTACCCGCCGGTGGAGTTC CATGCCCCCTGGTGTGGCCACTGCAAGGCTC TGGCCCCTGAGTATCCAAAGCCGCTGGGAAG CTGAAGGCAGAAGGTTCCGAGATCAGGTTGG CCAAGGTGGACGCCACGGAGGAGTCTGACCT AGCCCAGCAGTACGGCGTGCGCGGCTATCCC ACCATCAAGTTCTTCAGGAATGGAGACACGG CTTCCCCCAAGGAATATACAGCTGGCAGAGA GGCTGATGACATCGTGAACTGGCTGAAGAAG CGCACGGGCCCGGCTGCCACCACCCTGCCTG ACGGCGCAGCTGCAGAGTCCTTGGTGGAGTC CAGCGAGGTGGCCGTCATCGGCTTCTTCAAG GACGTGGAGTCGGACTCTGCCAAGCAGTTTT TGCAGGCAGCAGAGGCCATCGATGACATACC ATTTGGGATCACTTCCAACAGTGACGTGTTC TCCAAATACCAGCTCGACAAAGATGGGGTTG TCCTCTTTAAGAAGTTTGATGAAGGCCGGAA CAACTTTGAAGGGGAGGTCACCAAGGAGAAC CTGCTGGACTTTATCAAACACAACCAGCTGC CCCTTGTCATCGAGTTCACCGAGCAGACAGC CCCGAAGATTTTTGGAGGTGAAATCAAGACT CACATCCTGCTGTTCTTGCCCAAGAGTGTGT CTGACTATGACGGCAAACTGAGCAACTTCAA AACAGCAGCCGAGAGCTTCAAGGGCAAGATC CTGTTCATCTTCATCGACAGCGACCACACCG ACAACCAGCGCATCCTCGAGTTCTTTGGCCT GAAGAAGGAAGAGTGCCCGGCCGTGCGCCTC ATCACCTTGGAGGAGGAGATGACCAAGTACA AGCCCGAATCGGAGGAGCTGACGGCAGAGAG GATCACAGAGTTCTGCCACCGCTTCCTGGAG GGCAAAATCAAGCCCCACCTGATGAGCCAGG AGCTGCCGGAGGACTGGGACAAGCAGCCTGT CAAGGTGCTTGTTGGGAAGAACTTTGAAGAC GTGGCTTTTGATGAGAAAAAAAACGTCTTTG TGGAGTTCTATGCCCCATGGTGTGGTCACTG CAAACAGTTGGCTCCCATTTGGGATAAACTG GGAGAGACGTACAAGGACCATGAGAACATCG TCATCGCCAAGATGGACTCGACTGCCAACGA GGTGGAGGCCGTCAAAGTGCACGGCTTCCCC ACACTCGGGTTCTTTCCTGCCAGTGCCGACA GGACGGTCATTGATTACAACGGGGAACGCAC GCTGGATGGTTTTAAGAAATTCCTAGAGAGC GGTGGCCAAGATGGGGCAGGGGATGTTGACG ACCTCGAGGACCTCGAAGAAGCAGAGGAGCC AGACATGGAGGAAGACGATGACCAGAAAGCT GTGAAAGATGAACTGTAA 38 human PDI DAPEEEDHVLVLRKSNFAEALAAHKYPPVEF Gene HAPWCGHCKALAPEYAKAAGKLKAEGSEIRL (protein) AKVDATEESDLAQQYGVRGYPTIKFFRNGDT ASPKEYTAGREADDIVNWLKKRTGPAATTLP DGAAAESLVESSEVAVIGFFKDVESDSAKQF LQAAEAIDDIPFGITSNSDVFSKYQLDKDGV VLFKKFDEGRNNFEGEVTKENLLDFIKHNQL PLVIEFTEQTAPKIFGGEIKTHILLFLPKSV SDYDGKLSNFKTAAESFKGKILFIFIDSDHT DNQRILEFFGLKKEECPAVRLITLEEEMTKY KPESEELTAERITEFCHRFLEGKIKPHLMSQ ELPEDWDKQPVKVLVGKNFEDVAFDEKKNVF VEFYAPWCGHCKQLAPIWDKLGETYKDHENI VIAKMDSTANEVEAVKVHGFPTLGFFPASAD RTVIDYNGERTLDGFKKFLESGGQDGAGDVD DLEDLEEAEEPDMEEDDDQKAVHDEL 39 Pichia ATGCAATTCAACTGGAATATTAAAACTGTGG pastoris PDI1 CAAGTATTTTGTCCGCTCTCACACTAGCACA Gene (DNA) AGCAAGTGATCAGGAGGCTATTGCTCCAGAG GACTCTCATGTCGTCAAATTGACTGAAGCCA CTTTTGAGTCTTTCATCACCAGTAATCCTCA CGTTTTGGCAGAGTTTTTTGCCCCTTGGTGT GGTCACTGTAAGAAGTTGGGCCCTGAACTTG TTTCTGCTGCCGAGATCTTAAAGGACAATGA GCAGGTTAAGATTGCTCAAATTGATTGTACG GAGGAGAAGGAATTATGTCAAGGCTACGAAA TTAAAGGGTATCCTACTTTGAAGGTGTTCCA TGGTGAGGTTGAGGTCCCAAGTGACTATCAA GGTCAAAGACAGAGCCAAAGCATTGTCAGCT ATATGCTAAAGCAGAGTTTACCCCCTGTCAG TGAAATCAATGCAACCAAAGATTTAGACGAC ACAATCGCCGAGGCAAAAGAGCCCGTGATTG TGCAAGTACTACCGGAAGATGCATCCAACTT GGAATCTAACACCACATTTTACGGAGTTGCC GGTACTCTCAGAGAGAAATTCACTTTTGTCT CCACTAAGTCTACTGATTATGCCAAAAAATA CACTAGCGACTCGACTCCTGCCTATTTGCTT GTCAGACCTGGCGAGGAACCTAGTGTTTACT CTGGTGAGGAGTTAGATGAGACTCATTTGGT GCACTGGATTGATATTGAGTCCAAACCTCTA TTTGGAGACATTGACGGATCCACCTTCAAAT CATATGCTGAAGCTAACATCCCTTTAGCCTA CTATTTCTATGAGAACGAAGAACAACGTGCT GCTGCTGCCGATATTATTAAACCTTTTGCTA AAGAGCAACGTGGCAAAATTAACTTTGTTGG CTTAGATGCCGTTAAATTCGGTAAGCATGCC AAGAACTTAAACATGGATGAAGAGAAACTCC CTCTATTTGTCATTCATGATTTGGTGAGCAA CAAGAAGTTTGGAGTTCCTCAAGACCAAGAA TTGACGAACAAAGATGTGACCGAGCTGATTG AGAAATTCATCGCAGGAGAGGCAGAACCAAT TGTGAAATCAGAGCCAATTCCAGAAATTCAA GAAGAGAAAGTCTTCAAGCTAGTCGGAAAGG CCCACGATGAAGTTGTCTTCGATGAATCTAA AGATGTTCTAGTCAAGTACTACGCCCCTTGG TGTGGTCACTGTAAGAGAATGGCTCCTGCTT ATGAGGAATTGGCTACTCTTTACGCCAATGA TGAGGATGCCTCTTCAAAGGTTGTGATTGCA AAACTTGATCACACTTTGAACGATGTCGACA ACGTTGATATTCAAGGTTATCCTACTTTGAT CCTTTATCCAGCTGGTGATAAATCCAATCCT CAACTGTATGATGGATCTCGTGACCTAGAAT CATTGGCTGAGTTTGTAAAGGAGAGAGGAAC CCACAAAGTGGATGCCCTAGCACTCAGACCA GTCGAGGAAGAAAAGGAAGCTGAAGAAGAAG CTGAAAGTGAGGCAGACGCTCACGACGAGCT TTAA 40 Pichia MQFNWNIKTVASILSALTLAQASDQEAIAPE pastoris PDI1 DSHVVKLTEATFESFITSNPHVLAEFFAPWC Gene GHCKKLGPELVSAAEILKDNEQVKIAQIDCT (protein) EEKELCQGYEIKGYPTLKVFHGEVEVPSDYQ GQRQSQSIVSYMLKQSLPPVSEINATKDLDD TIAEAKEPVIVQVLPEDASNLESNTTFYGVA GTLREKFTFVSTKSTDYAKKYTSDSTPAYLL VRPGEEPSVYSGEELDETHLVHWIDIESKPL FGDIDGSTFKSYAEANIPLAYYFYENEEQRA AAADIIKPFAKEQRGKINFVGLDAVKFGKHA KNLNMDEEKLPLFVIHDLVSNKKFGVPQDQE LTNKDVTELIEKFIAGEAEPIVKSEPIPEIQ EEKVFKLVGKAHDEVVFDESKDVLVKYYAPW CGHCKRMAPAYEELATLYANDEDASSKVVIA KLDHTLNDVDNVDIQGYPTLILYPAGDKSNP QLYDGSRDLESLAEFVKERGTHKVDALALRP VEEEKEAEEEAESEADAHDEL 41 PCR primer ATGAATTCAGGC PB248 CATATCGGCCATTGTTTACTGTGCG CCCACAGTAG 42 PCR primer ATGTTTA PB249 AACGTGAGGATTACTGGTGATGAAAGAC 43 PCR primer AGACTAGTCTATTTGGAG PB250 ACATTGACGGATCCAC 44 PCR primer ATCTCGAGAGGCCATGCAGGCCAACCACAAG PB251 ATGAATCAAATTTTG 45 PCR primer AGCGCTGACGATGAAGTTGATGTGGATGGTA hGRP94/UP1 CAGTAG 46 PCR primer GGCCGGCCTTACAATTCATCATG hGRP94/LP1 TTCAGCTGTAGATTC 47 human GATGATGAAGTTGACGTTGACGGTACTGTTG GRP94 Gene AAGAGGACTTGGGAAAGTCTAGAGAGGGTTC (DNA) CAGAACTGACGACGAAGTTGTTCAGAGAGAG GAAGAGGCTATTCAGTTGGACGGATTGAACG CTTCCCAAATCAGAGAGTTGAGAGAGAAGTC CGAGAAGTTCGCTTTCCAAGCTGAGGTTAAC AGAATGATGAAATTGATTATCAACTCCTTGT ACAAGAACAAAGAGATTTTCTTGAGAGAGTT GATCTCTAACGCTTCTGACGCTTTGGACAAG ATCAGATTGATCTCCTTGACTGACGAAAACG CTTTGTCCGGTAACGAAGAGTTGACTGTTAA GATCAAGTGTGACAAAGAGAAGAACTTGTTG CACGTTACTGACACTGGTGTTGGAATGACTA GAGAAGAGTTGGTTAAGAACTTGGGTACTAT CGCTAAGTCTGGTACTTCCGAGTTCTTGAAC AAGATGACTGAGGCTCAAGAAGATGGTCAAT CCACTTCCGAGTTGATTGGTCAGTTCGGTGT TGGTTTCTACTCCGCTTTCTTGGTTGCTGAC AAGGTTATCGTTACTTCCAAGCACAACAACG ACACTCAACACATTTGGGAATCCGATTCCAA CGAGTTCTCCGTTATTGCTGACCCAAGAGGT AACACTTTGGGTAGAGGTACTACTATCACTT TGGTTTTGAAAGAAGAGGCTTCCGACTACTT GGAGTTGGACACTATCAAGAACTTGGTTAAG AAGTACTCCCAGTTCATCAACTTCCCAATCT ATGTTTGGTCCTCCAAGACTGAGAC TGTTGAGGAACCAATGGAAGAAGAAGAGGCT GCTAAAGAAGAGAAAGAGGAATCTGACGACG AGGCTGCTGTTGAAGAAGAGGAAGAAGAAAA GAAGCCAAAGACTAAGAAGGTTGAAAAGACT GTTTGGGACTGGGAGCTTATGAACGACATCA AGCCAATTTGGCAGAGACCATCCAAAGAGGT TGAGGAGGACGAGTACAAGGCTTTCTACAAG TCCTTCTCCAAAGAATCCGATGACCCAATGG CTTACATCCACTTCACTGCTGAGGGTGAAGT TACTTTCAAGTCCATCTTGTTCGTTCCAACT TCTGCTCCAAGAGGATTGTTCGACGAGTACG GTTCTAAGAAGTCCGACTACATCAAACTTTA TGTTAGAAGAGTTTTCATCACTGACGACTTC CACGATATGATGCCAAAGTACTTGAACTTCG TTAAGGGTGTTGTTGATTCCGATGACTTGCC ATTGAACGTTTCCAGAGAGACTTTGCAGCAG CACAAGTTGTTGAAGGTTATCAGAAAGAAAC TTGTTAGAAAGACTTTGGACATGATCAAGAA GATCGCTGACGACAAGTACAACGACACTTTC TGGAAAGAGTTCGGAACTAACATCAAGTTGG GTGTTATTGAGGACCACTCCAACAGAACTAG ATTGGCTAAGTTGTTGAGATTCCAGTCCTCT CATCACCCAACTGACATCACTTCCTTGGACC AGTACGTTGAGAGAATGAAAGAGAAGCAGGA CAAAATCTACTTCATGGCTGGTTCCTCTAGA AAAGAGGCTGAATCCTCCCCATTCGTTGAGA GATTGTTGAAGAAGGGTTACGAGGTTATCTA CTTGACTGAGCCAGTTGACGAGTACTGTATC CAGGCTTTGCCAGAGTTTGACGGAAAGAGAT TCCAGAACGTTGCTAAAGAGGGTGTTAAGTT CGACGAATCCGAAAAGACTAAAGAATCCAGA GAGGCTGTTGAGAAAGAGTTCGAGCCATTGT TGAACTGGATGAAGGACAAGGCTTTGAAGGA CAAGATCGAGAAGGCTGTTGTTTCCCAGAGA TTGACTGAATCCCCATGTGCTTTGGTTGCTT CCCAATACGGATGGAGTGGTAACATGGAAAG AATCATGAAGGCTCAGGCTTACCAAACTGGA AAGGACATCTCCACTAACTACTACGCTTCCC AGAAGAAAACTTTCGAGATCAACCCAAGACA CCCATTGATCAGAGACATGTTGAGAAGAATC AAAGAGGACGAGGACGACAAGACTGTTTTGG ATTTGGCTGTTGTTTTGTTCGAGACTGCTAC TTTGAGATCCGGTTACTTGTTGCCAGACACT AAGGCTTACGGTGACAGAATCGAGAGAATGT TGAGATTGTCCTTGAACATTGACCCAGACGC TAAGGTTGAAGAAGAACCAGAAGAAGAGCCA GAGGAAACTGCTGAAGATACTACTGAGGACA CTGAACAAGACGAGGACGAAGAGATGGATGT TGGTACTGACGAAGAGGAAGAGACAGCAAAG GAATCCACTGCTGAACACGACGAGTTGTAA 48 human DDEVDVDGTVEEDLGKSREGSRTDDEVVQRE GRP94 Gene EEAIQLDGLNASQIRELREKSEKFAFQAEVN (protein) RMMKLIINSLYKNKEIFLRELISNASDALDK IRLISLTDENALSGNEELTVKIKCDKEKNLL HVTDTGVGMTREELVKNLGTIAKSGTSEFLN KMTEAQEDGQSTSELIGQFGVGFYSAFLVAD KVIVTSKHNNDTQHIWESDSNEFSVIADPRG NTLGRGTTITLVLKEEASDYLELDTIKNLVK KYSQFINFPIYVWSSKTETVEEPMEEEEAAK EEKEESDDEAAVEEEEEEKKPKTKKVEKTVW DWELMNDIKPIWQRPSKEVEEDEYKAFYKSF SKESDDPMAYIHFTAEGEVTFKSILFVPTSA PRGLFDEYGSKKSDYIKLYVRRVFITDDFHD MMPKYLNFVKGVVDSDDLPLNVSRETLQQHK LLKVIRKKLVRKTLDMIKKIADDKYNDTFWK EFGTNIKLGVIEDHSNRTRLAKLLRFQSSHH PTDITSLDQYVERMKEKQDKIYFMAGSSRKE AESSPFVERLLKKGYEVIYLTEPVDEYCIQA LPEFDGKRFQNVAKEGVKFDESEKTKESREA VEKEFEPLLNWMKDKALKDKIEKAVVSQRLT ESPCALVASQYGWSGNMERIMKAQAYQTGKD ISTNYYASQKKTFEINPRHPLIRDMLRRIKE DEDDKTVLDLAVVLFETATLRSGYLLPDTKA YGDRIERMLRLSLNIDPDAKVEEEPEEEPEE TAEDTTEDTEQDEDEEMDVGTDEEEETAKES TAEHDEL 49 Saccharomyces ATG AGA TTC CCA TCC ATC TTC ACT cerevisiae GCT GTT TTG TTC GCT GCT TCT TCT mating factor GCT TTG GCT pre-signal peptide (DNA) 50 Saccharomyces MRFPSIFTAVLFAASSALA cerevisiae mating factor pre-signal peptide (protein) 51 Fab Anti- ATGAGATTCCCATCCATCTTCACTGCTGTTT Her2 HC-GR1 TGTTCGCTGCTTCTTCTGCTTTGGCTGAGGT fusion with TCAGTTGGTTGAATCTGGAGGAGGATTGGTT Pre-pro CAACCTGGTGGTTCTTTGAGATTGTCCTGTG α- mating CTGCTTCCGGTTTCAACATCAAGGACACTTA factor signal CATCCACTGGGTTAGACAAGCTCCAGGAAAG peptide GGATTGGAGTGGGTTGCTAGAATCTACCCAA (ScαMTprepro) CTAACGGTTACACAAGATACGCTGACTCCGT (DNA) TAAGGGAAGATTCACTATCTCTGCTGACACT TCCAAGAACACTGCTTACTTGCAGATGAACT CCTTGAGAGCTGAGGATACTGCTGTTTACTA CTGTTCCAGATGGGGTGGTGATGGTTTCTAC GCTATGGACTACTGGGGTCAAGGAACTTTGG TTACTGTTTCCTCCGCTTCTACTAAGGGACC ATCTGTTTTCCCATTGGCTCCATCTTCTAAG TCTACTTCCGGTGGTACTGCTGCTTTGGGAT GTTTGGTTAAAGACTACTTCCCAGAGCCAGT TACTGTTTCTTGGAACTCCGGTGCTTTGACT TCTGGTGTTCACACTTTCCCAGCTGTTTTGC AATCTTCCGGTTTGTACTCTTTGTCCTCCGT TGTTACTGTTCCATCCTCTTCCTTGGGTACT CAGACTTACATCTGTAACGTTAACCACAAGC CATCCAACACTAAGGTTGACAAGAAGGTTGA GCCAAAGTCCTGTGGTGGTGGTGGTAGTGGA GGTGGTGGAAGTGGTGGCGGTGGTTCTGCGG CCGCTTATCCATATGATGTTCCAGACTACGC TGGAGGTCATCATCATCACCACCATCACCAT CATGGTGGTGAAGAGAAGTCCAGATTGTTGG AGAAAGAGAACAGAGAGTTGGAGAAGATCAT CGCTGAGAAAGAAGAGAGAGTTTCCGAGTTG AGACACCAATTGCAATCCGTTGGTGGTTGTT AATAG 52 Anti-Her2 LC ATGAGATTCCCATCCATCTTCACTGCTGTTT with Pre-pro TGTTCGCTGCTTCTTCTGCTTTGGCTGACAT α- mating CCAAATGACTCAATCCCCATCTTCTTTGTCT factor signal GCTTCCGTTGGTGACAGAGTTACTATCACTT peptide GTAGAGCTTCCCAGGACGTTAATACTGCTGT (ScαMTprepro) TGCTTGGTATCAACAGAAGCCAGGAAAGGCT (DNA) CCAAAGTTGTTGATCTACTCCGCTTCCTTCT TGTACTCTGGTGTTCCATCCAGATTCTCTGG TTCCAGATCCGGTACTGACTTCACTTTGACT ATCTCCTCCTTGCAACCAGAAGATTTCGCTA CTTACTACTGTCAGCAGCACTACACTACTCC ACCAACTTTCGGACAGGGTACTAAGGTTGAG ATCAAGAGAACTGTTGCTGCTCCATCCGTTT TCATTTTCCCACCATCCGACGAACAGTTGAA GTCTGGTACAGCTTCCGTTGTTTGTTTGTTG AACAACTTCTACCCAAGAGAGGCTAAGGTTC AGTGGAAGGTTGACAACGCTTTGCAATCCGG TAACTCCCAAGAATCCGTTACTGAGCAAGAC TCTAAGGACTCCACTTACTCCTTGTCCTCCA CTTTGACTTTGTCCAAGGCTGATTACGAGAA GCACAAGGTTTACGCTTGTGAGGTTACACAT CAGGGTTTGTCCTCCCCAGTTACTAAGTCCT TCAACAGAGGAGAGTGTTAATAG 53 Fab Anti- ATGGTCGCTTGGTGGTCTTTGTTTCTGTACG DKK1 HC-GR1 GTCTTCAGGTCGCTGCACCTGCTTTGGCTGA fusion with GGTTCAGTTGGTTCAATCTGGTGCTGAGGTT Alpha amylase AAGAAACCTGGTGCTTCCGTTAAGGTTTCCT signal GTAAGGCTTCCGGTTACACTTTCACTGACTA peptide CTACATCCACTGGGTTAGACAAGCTCCAGGT (from CAAGGATTGGAATGGATGGGATGGATTCACT Aspergillus CTAACTCCGGTGCTACTACTTACGCTCAGAA niger α- GTTCCAGGCTAGAGTTACTATGTCCAGAGAC amylase) ACTTCTTCTTCCACTGCTTACATGGAATTGT (DNA) CCAGATTGGAATCCGATGACACTGCTATGTA CTTTTGTTCCAGAGAGGACTACTGGGGACAG GGAACTTTGGTTACTGTTTCCTCCGCTTCTA CTAAAGGGCCCTCTGTTTTTCCATTGGCTCC ATGTTCTAGATCCACTTCCGAATCCACTGCT GCTTTGGGATGTTTGGTTAAGGACTACTTCC CAGAGCCAGTTACTGTTTCTTGGAACTCCGG TGCTTTGACTTCTGGTGTTCACACTTTCCCA GCTGTTTTGCAATCTTCCGGTTTGTACTCCT TGTCCTCCGTTGTTACTGTTACTTCCTCCAA CTTCGGTACTCAGACTTACACTTGTAACGTT GACCACAAGCCATCCAACACTAAGGTTGACA AGACTGTTGAGAGAAAGTGTGGTGGTGGTGG TAGTGGAGGTGGTGGAAGTGGTGGCGGTGGT TCTGCGGCCGCTTATCCATATGATGTTCCAG ACTACGCTGGAGGTCATCATCATCACCACCA TCACCATCATGGTGGTGAAGAGAAGTCCAGA TTGTTGGAGAAAGAGAACAGAGAGTTGGAGA AGATCATCGCTGAGAAAGAAGAGAGAGTTTC CGAGTTGAGACACCAATTGCAATCCGTTGGT GGTTGTTAATAGG 54 Anti-DKK1 ATGGTCGCTTGGTGGTCTTTGTTTCTGTACG LC with GTCTTCAGGTCGCTGCACCTGCTTTGGCTCA Alpha amylase GTCCGTTTTGACACAACCACCATCTGTTTCT signal GGTGCTCCAGGACAGAGAGTTACTATCTCCT peptide GTACTGGTTCCTCTTCCAACATTGGTGCTGG (from TTACGATGTTCACTGGTATCAACAGTTGCCA Aspergillus GGTACTGCTCCAAAGTTGTTGATCTACGGTT niger α- ACTCCAACAGACCATCTGGTGTTCCAGACAG amylase) ATTCTCTGGTTCTAAGTCTGGTGCTTCTGCT (DNA) TCCTTGGCTATCACTGGATTGAGACCAGATG ACGAGGCTGACTACTACTGTCAATCCTACGA CAACTCCTTGTCCTCTTACGTTTTCGGTGGT GGTACTCAGTTGACTGTTTTGTCCCAGCCAA AGGCTAATCCAACTGTTACTTTGTTCCCACC ATCTTCCGAAGAACTGCAGGCTAATAAGGCT ACTTTGGTTTGTTTGATCTCCGACTTCTACC CAGGTGCTGTTACTGTTGCTTGGAAGGCTGA TGGTTCTCCAGTTAAGGCTGGTGTTGAGACT ACTAAGCCATCCAAGCAGTCCAATAACAAGT ACGCTGCTAGCTCTTACTTGTCCTTGACACC AGAACAATGGAAGTCCCACAGATCCTACTCT TGTCAGGTTACACACGAGGGTTCTACTGTTG AAAAGACTGTTGCTCCAACTGAGTGTTCCTA ATGAG 55 Fab Anti- ATGGTTGCTTGGTGGTCTTTGTTCTTGTACG CD20, C2B8 GATTGCAAGTTGCTGCTCCAGCTTTGGCTca HC with agttcagctgcaacaaccaggtgctgaattg Alpha amylase gttaagcctggtgcttctgttaagatgtctt signal gtaaggcttctggttacactttcacttccta peptide caacatgcactgggttaagcaaactccaggt (from agaggattggaatggattggtgctatctacc Aspergillus caggtaacggtgacacttcttataaccaaaa niger α- gttcaagggaaaggctactttgactgctgac amylase) aaatcttcttctactgcttacatgcaattgt (DNA) cctccttgacttctgaagattctgctgttta ctactgtgctagatccacttactacggtggt gactggtactttaatgtttggggtgctggta ctactgttactgtctcgagtgcttctactaa gggaccatctgttttcccattggctccatct tctaagtctacttccggtggtacCGCTGCTT TGGGATGTTTGGITAAAGACTACTTCCCAGA GCCAGTTACTGTTTCTTGGAACTCCGGTGCT TTGACTTCTGGTGTTCACACTTTCCCAGCTG TTTTGCAATCTTCCGGTTTGTACTCTTTGTC CTCCGTTGTTACTGTTCCATCCTCTTCCTTG GGTACTCAGACTTACATCTGTAACGTTAACC ACAAGCCATCCAACACTAAGGTTGACAAGAA GGTTGAGCCAAAGTCCTGTGGTGGTGGTGGT AGTGGAGGTGGTGGAAGTGGTGGCGGTGGTT CTGCGGCCGCTTATCCATATGATGTTCCAGA CTACGCTGGAGGTCATCATCATCACCACCAT CACCATCATGGTGGTGAAGAGAAGTCCAGAT TGTTGGAGAAAGAGAACAGAGAGTTGGAGAA GATCATCGCTGAGAAAGAAGAGAGAGTTTCC GAGTTGAGACACCAATTGCAATCCGTTGGTG GTTGTTAATAG 56 Anti-CD20, ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG C28B LC GATTGCAAGTTGCTGCTCCAGCTTTGGCTga with Alpha gatcgttttgacacagtccccagctactttg amylase tctttgtccccaggtgaaagagctacattgt signal cctgtagagcttcctcttccgtttcctacat peptide (from ccactggtatcaacaaaagccaggacaggct Aspergillus ccaagattgttgatctacgctacttccaact niger α- tggcttccggtattccagctagattctctgg amylase) ttctggttccggtactgacttcactttgact (DNA) atctcttccttggaaccagaggacttcgctg tttactactgtcaacagtggacttctaaccc accaactttcggacaaggtactaaggttgag atcaagcgtacggttgctgctccttccgttt tcattttcccaccatccgacgaacaattgaa gtctggtacCGCTTCCGTTGTTTGTTTGTTG AACAACTTCTACCCACGTGAGGCTAAGGTTC AGTGGAAGGTTGACAACGCTTTGCAATCCGG TAACTCCCAAGAATCCGTTACTGAGCAGGAT TCTAAGGATTCCACTTACTCATTGTCCTCCA CTTTGACTTTGTCCAAGGCTGATTACGAGAA GCACAAGGTTTACGCATGCGAGGTTACACAT CAGGGTTTGTCCTCCCCAGTTACTAAGTCCT TCAACAGAGGAGAGTGTTAA 57 Fab Anti- ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG CD20 frame GATTGCAAGTTGCTGCTCCAGCTTTGGCTca grafted HC- agttcagctggttcaatctggtgctgaggtt GR1 with aagaagcctggttcctccgttaaggtttcct Alpha amylase gtaaggcttccggttacactttcacttccta signal caacatgcactgggttagacaagctccaggt peptide caaggattggaatggatgggtgctatctacc (from caggtaacggtgacacttcttacaaccagaa Aspergillus gttcaagggtagagttactatcactgctgac niger α- gaatccacttccactgcttacatggaattgt amylase) cctcattgagatccgaggacactgctgttta (DNA) ctactgtgctagatccacttactacggtggt gactggtactttaatgtttggggacagggaa ctttggttactgtctcgagtgcttctactaa gggaccatccgtttttccattggctccatcc tctaagtctacttccggtggtacCGCTGCTT TGGGATGTTTGGTTAAAGACTACTTCCCAGA GCCAGTTACTGTTTCTTGGAACTCCGGTGCT TTGACTTCTGGTGTTCACACTTTCCCAGCTG TTTTGCAATCTTCCGGTTTGTACTCTTTGTC CTCCGTTGTTACTGTTCCATCCTCTTCCTTG GGTACTCAGACTTACATCTGTAACGTTAACC ACAAGCCATCCAACACTAAGGTTGACAAGAA GGTTGAGCCAAAGTCCTGTGGTGGTGGTGGT AGTGGAGGTGGTGGAAGTGGTGGCGGTGGTT CTGCGGCCGCTTATCCATATGATGTTCCAGA CTACGCTGGAGGTCATCATCATCACCACCAT CACCATCATGGTGGTGAAGAGAAGTCCAGAT TGTTGGAGAAAGAGAACAGAGAGTTGGAGAA GATCATCGCTGAGAAAGAAGAGAGAGTTTCC GAGTTGAGACACCAATTGCAATCCGTTGGTG GTTGTTAATAG 58 Anti-CD20 ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG frame grafted GATTGCAAGTTGCTGCTCCAGCTTTGGCTga LC with gatcgttttgacacagtccccagctactttg Alpha amylase tctttgtccccaggtgaaagagctacattgt signal cctgtagagcttcctcttccgtttcctacat peptide ccactggtatcaacaaaagccaggacaggct (from ccaagattgttgatctacgctacttccaact Aspergillus tggcttccggtattccagctagattctctgg niger α- ttctggttccggtactgacttcactttgact amylase) atctcttccttggaaccagaggacttcgctg (DNA) tttactactgtcaacagtggacttctaaccc accaactttcggacaaggtactaaggttgag atcaagcgtacggttgctgctccttccgttt tcattttcccaccatccgacgaacaattgaa gtctggtacCGCTTCCGTTGTTTGTTTGTTG AACAACTTCTACCCACGTGAGGCTAAGGTTC AGTGGAAGGTTGACAACGCTTTGCAATCCGG TAACTCCCAAGAATCCGTTACTGAGCAGGAT TCTAAGGATTCCACTTACTCATTGTCCTCCA CTTTGACTITGTCCAAGGCTGATTACGAGAA GCACAAGGTTTACGCATGCGAGGTTACACAT CAGGGTTTGTCCTCCCCAGTTACTAAGTCCT TCAACAGAGGAGAGTGTTAA 59 Anti-Her2 ATGAGATTCCCATCCATCTTCACTGCTGTTT full length TGTTCGCTGCTTCTTCTGCTTTGGCTGAGGT HC with GR1 TCAGTTGGTTGAATCTGGAGGAGGATTGGTT ORF and Pre- CAACCTGGTGGTTCTTTGAGATTGTCCTGTG pro α- mating CTGCTTCCGGTTTCAACATCAAGGACACTTA factor signal CATCCACTGGGTTAGACAAGCTCCAGGAAAG peptide GGATTGGAGTGGGTTGCTAGAATCTACCCAA (ScαMTprepro) CTAACGGTTACACAAGATACGCTGACTCCGT (DNA) TAAGGGAAGATTCACTATCTCTGCTGACACT TCCAAGAACACTGCTTACTTGCAGATGAACT CCTTGAGAGCTGAGGATACTGCTGTTTACTA CTGTTCCAGATGGGGTGGTGATGGTTTCTAC GCTATGGACTACTGGGGTCAAGGAACTTTGG TTACTGTTTCCTCCGCTTCTACTAAGGGACC ATCTGTTTTCCCATTGGCTCCATCTTCTAAG TCTACTTCCGGTGGTACTGCTGCTTTGGGAT GTTTGGTTAAAGACTACTTCCCAGAGCCAGT TACTGTTTCTTGGAACTCCGGTGCTTTGACT TCTGGTGTTCACACTTTCCCAGCTGTTTTGC AATCTTCCGGTTTGTACTCTTTGTCCTCCGT TGTTACTGTTCCATCCTCTTCCTTGGGTACT CAGACTTACATCTGTAACGTTAACCACAAGC CATCCAACACTAAGGTTGACAAGAAGGTTGA GCCAAAGTCCTGTGACAAGACTCATACTTGT CCACCATGTCCAGCTCCAGAATTGTTGGGTG GTCCTTCCGTTTTTTTGTTCCCACCAAAGCC AAAGGACACTTTGATGATCTCCAGAACTCCA GAGGTTACATGTGTTGTTGTTGACGTTTCTC ACGAGGACCCAGAGGTTAAGTTCAACTGGTA CGTTGACGGTGTTGAAGTTCACAACGCTAAG ACTAAGCCAAGAGAGGAGCAGTACAACTCCA CTTACAGAGTTGTTTCCGTTTTGACTGTTTT GCACCAGGATTGGTTGAACGGAAAGGAGTAC AAGTGTAAGGTTTCCAACAAGGCTTTGCCAG CTCCAATCGAAAAGACTATCTCCAAGGCTAA GGGTCAACCAAGAGAGCCACAGGTTTACACT TTGCCACCATCCAGAGATGAGTTGACTAAGA ACCAGGTTTCCTTGACTTGTTTGGTTAAGGG ATTCTACCCATCCGACATTGCTGTTGAATGG GAGTCTAACGGTCAACCAGAGAACAACTACA AGACTACTCCACCTGTTTTGGACTCTGACGG TTCCTTTTTCTTGTACTCCAAGTTGACTGTT GACAAGTCCAGATGGCAACAGGGTAACGTTT TCTCCTGTTCCGTTATGCATGAGGCTTTGCA CAACCACTACACTCAAAAGTCCTTGTCTTTG TCCCCTGGTAAGGCGGCCGCTTATCCATATG ATGTTCCAGACTACGCTGGAGGTCATCATCA TCACCACCATCACCATCATGGTGGTGAAGAG AAGTCCAGATTGTTGGAGAAAGAGAACAGAG AGTTGGAGAAGATCATCGCTGAGAAAGAAGA GAGAGTTTCCGAGTTGAGACACCAATTGCAA TCCGTTGGTGGTTGTTAATAG 60 Anti-Her2 ATGAGATTCCCATCCATCTTCACTGCTGTTT full length TGTTCGCTGCTTCTTCTGCTTTGGCTGAGGT HC with TCAGTTGGTTGAATCTGGAGGAGGATTGGTT single stop CAACCTGGTGGTTCTTTGAGATTGTCCTGTG codon between CTGCTTCCGGTTTCAACATCAAGGACACTTA Ab ORF and CATCCACTGGGTTAGACAAGCTCCAGGAAAG GR1 ORF with GGATTGGAGTGGGTTGCTAGAATCTACCCAA Pre-pro α- CTAACGGTTACACAAGATACGCTGACTCCGT mating factor TAAGGGAAGATTCACTATCTCTGCTGACACT signal TCCAAGAACACTGCTTACTTGCAGATGAACT peptide CCTTGAGAGCTGAGGATACTGCTGTTTACTA (ScαMTprepro) CTGTTCCAGATGGGGTGGTGATGGTTTCTAC (DNA) GCTATGGACTACTGGGGTCAAGGAACTTTGG TTACTGTTTCCTCCGCTTCTACTAAGGGACC ATCTGTTTTCCCATTGGCTCCATCTTCTAAG TCTACTTCCGGTGGTACTGCTGCTTTGGGAT GTTTGGTTAAAGACTACTTCCCAGAGCCAGT TACTGTTTCTTGGAACTCCGGTGCTTTGACT TCTGGTGTTCACACTTTCCCAGCTGTTTTGC AATCTTCCGGTTTGTACTCTTTGTCCTCCGT TGTTACTGTTCCATCCTCTTCCTTGGGTACT CAGACTTACATCTGTAACGTTAACCACAAGC CATCCAACACTAAGGTTGACAAGAAGGTTGA GCCAAAGTCCTGTGACAAGACTCATACTTGT CCACCATGTCCAGCTCCAGAATTGTTGGGTG GTCCTTCCGTTTTTTTGTTCCCACCAAAGCC AAAGGACACTTTGATGATCTCCAGAACTCCA GAGGTTACATGTGTTGTTGTTGACGTTTCTC ACGAGGACCCAGAGGTTAAGTTCAACTGGTA CGTTGACGGTGTTGAAGTTCACAACGCTAAG ACTAAGCCAAGAGAGGAGCAGTACAACTCCA CTTACAGAGTTGTTTCCGTTTTGACTGTTTT GCACCAGGATTGGTTGAACGGAAAGGAGTAC AAGTGTAAGGTTTCCAACAAGGCTTTGCCAG CTCCAATCGAAAAGACTATCTCCAAGGCTAA GGGTCAACCAAGAGAGCCACAGGTTTACACT TTGCCACCATCCAGAGATGAGTTGACTAAGA ACCAGGTTTCCTTGACTTGTTTGGTTAAGGG ATTCTACCCATCCGACATTGCTGTTGAATGG GAGTCTAACGGTCAACCAGAGAACAACTACA AGACTACTCCACCTGTTTTGGACTCTGACGG TTCCTTTTTCTTGTACTCCAAGTTGACTGTT GACAAGTCCAGATGGCAACAGGGTAACGTTT TCTCCTGTTCCGTTATGCATGAGGCTTTGCA CAACCACTACACTCAAAAGTCCTTGTCTTTG TCCCCTGGTAAGTAGGCGGCCGCTTATCCAT ATGATGTTCCAGACTACGCTGGAGGTCATCA TCATCACCACCATCACCATCATGGTGGTGAA GAGAAGTCCAGATTGTTGGAGAAAGAGAACA GAGAGTTGGAGAAGATCATCGCTGAGAAAGA AGAGAGAGTTTCCGAGTTGAGACACCAATTG CAATCCGTTGGTGGTTGTTAATAGGGCCGGC CATTTAA 61 Anti-CD20 ATGGTTGCTTGGTGGTCTTTGTTCTTGTACG C2B8 full GATTGCAAGTTGCTGCTCCAGCTTTGGCTca length HC agttcagctgcaacaaccaggtgctgaattg with stop gttaagcctggtgcttctgttaagatgtctt codon gtaaggcttctggttacactttcacttccta between Ab caacatgcactgggttaagcaaactccaggt ORF and GR1 agaggattggaatggattggtgctatctacc ORF with caggtaacggtgacacttcttataaccaaaa Alpha amylase gttcaagggaaaggctactttgactgctgac signal aaatcttcttctactgcttacatgcaattgt peptide cctccttgacttctgaagattctgctgttta (from ctactgtgctagatccacttactacggtggt Aspergillus gactggtactttaatgtttggggtgctggta niger α- ctactgttactgtctcgagtgcttctactaa amylase) gggaccatctgttttcccattggctccatct (DNA) tctaagtctacttccggtggtacCGCTGCTT TGGGATGTTTGGTTAAAGACTACTTCCCAGA GCCAGTTACTGTTTCTTGGAACTCCGGTGCT TTGACTTCTGGTGTTCACACTTTCCCAGCTG TTTTGCAATCTTCCGGTTTGTACTCTTTGTC CTCCGTTGTTACTGTTCCATCCTCTTCCTTG GGTACTCAGACTTACATCTGTAACGTTAACC ACAAGCCATCCAACACTAAGGTTGACAAGAA GGTTGAGCCAAAGTCCTGTGACAAGACTCAT ACTTGTCCACCATGTCCAGCTCCAGAATTGT TGGGTGGTCCTTCCGTTTTTTTGTTCCCACC AAAGCCAAAGGACACTTTGATGATCTCCAGA ACTCCAGAGGTTACATGTGTTGTTGTTGACG TTTCTCACGAGGACCCAGAGGTTAAGTTCAA CTGGTACGTTGACGGTGTTGAAGTTCACAAC GCTAAGACTAAGCCAAGAGAGGAGCAGTACA ACTCCACTTACAGAGTTGTTTCCGTTTTGAC TGTTTTGCACCAGGATTGGTTGAACGGAAAG GAGTACAAGTGTAAGGTTTCCAACAAGGCTT TGCCAGCTCCAATCGAAAAGACTATCTCCAA GGCTAAGGGTCAACCAAGAGAGCCACAGGTT TACACTTTGCCACCATCCAGAGATGAGTTGA CTAAGAACCAGGTTTCCTTGACTTGTTTGGT TAAGGGATTCTACCCATCCGACATTGCTGTT GAATGGGAGTCTAACGGTCAACCAGAGAACA ACTACAAGACTACTCCACCTGTTTTGGACTC TGACGGTTCCTTTTTCTTGTACTCCAAGTTG ACTGTTGACAAGTCCAGATGGCAACAGGGTA ACGTTTTCTCCTGTTCCGTTATGCATGAGGC TTTGCACAACCACTACACTCAAAAGTCCTTG TCTTTGTCCCCTGGTAAGTAGGCGGCCGCTT ATCCATATGATGTTCCAGACTACGCTGGAGG TCATCATCATCACCACCATCACCATCATGGT GGTGAAGAGAAGTCCAGATTGTTGGAGAAAG AGAACAGAGAGTTGGAGAAGATCATCGCTGA GAAAGAAGAGAGAGTTTCCGAGTTGAGACAC CAATTGCAATCCGTTGGTGGTTGTTAATAG 62 Anti-CD20 ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG Genmab full GATTGCAAGTTGCTGCTCCAGCTTTGGCTgc length HC tgttcagctggttgaatctggtggtggattg with single gttcaacctggtagatccttgagattgtcct stop codon gtgctgcttccggattactttcggtgactac between Ab actatgcactgggttagacaagctccaggaa ORF and GR1 agggattggaatgggtttccggtatttcttg ORF with gaactccggttccattggttacgctgattcc Alpha amylase gttaagggaagattcactatctccagagaca signal acgctaagaactccttgtacttgcagatgaa peptide ctccttgagagctgaggatactgctttgtac (from tactgtactaaggacaaccaatacggttctg Aspergillus gttccacttacggattgggagtttggggaca niger α- gggaactttggttactgtctcgagtgcttct amylase) actaagggaccatccgtttttccattggctc (DNA) catcctctaagtctacttccggtggtacCGC TGCTTTGGGATGTTTGGTTAAAGACTACTTC CCAGAGCCAGTTACTGTTTCTTGGAACTCCG GTGCTTTGACTTCTGGTGTTCACACTTTCCC AGCTGTTTTGCAATCTTCCGGTTTGTACTCT TTGTCCTCCGTTGTTACTGTTCCATCCTCTT CCTTGGGTACTCAGACTTACATCTGTAACGT TAACCACAAGCCATCCAACACTAAGGTTGAC AAGAAGGTTGAGCCAAAGTCCTGTGACAAGA CTCATACTTGTCCACCATGTCCAGCTCCAGA ATTGTTGGGTGGTCCTTCCGTTTTTTTGTTC CCACCAAAGCCAAAGGACACTTTGATGATCT CCAGAACTCCAGAGGTTACATGTGTTGTTGT TGACGTTTCTCACGAGGACCCAGAGGTTAAG TTCAACTGGTACGTTGACGGTGTTGAAGTTC ACAACGCTAAGACTAAGCCAAGAGAGGAGCA GTACAACTCCACTTACAGAGTTGTTTCCGTT TTGACTGTTTTGCACCAGGATTGGTTGAACG GAAAGGAGTACAAGTGTAAGGTTTCCAACAA GGCTTTGCCAGCTCCAATCGAAAAGACTATC TCCAAGGCTAAGGGTCAACCAAGAGAGCCAC AGGTTTACACTTTGCCACCATCCAGAGATGA GTTGACTAAGAACCAGGTTTCCTTGACTTGT TTGGTTAAGGGATTCTACCCATCCGACATTG CTGTTGAATGGGAGTCTAACGGTCAACCAGA GAACAACTACAAGACTACTCCACCTGTTTTG GACTCTGACGGTTCCTTTTTCTTGTACTCCA AGTTGACTGTTGACAAGTCCAGATGGCAACA GGGTAACGTTTTCTCCTGTTCCGTTATGCAT GAGGCTTTGCACAACCACTACACTCAAAAGT CCTTGTCTTTGTCCCCTGGTAAGTAGGCGGC CGCTTATCCATATGATGTTCCAGACTACGCT GGAGGTCATCATCATCACCACCATCACCATC ATGGTGGTGAAGAGAAGTCCAGATTGTTGGA GAAAGAGAACAGAGAGTTGGAGAAGATCATC GCTGAGAAAGAAGAGAGAGTTTCCGAGTTGA GACACCAATTGCAATCCGTTGGTGGTTGTTA ATAG 63 Anti-CD20 ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG Genmab LC GATTGCAAGTTGCTGCTCCAGCTTTGGCTga Alpha amylase gatcgttttgacacagtccccagctactttg signal tctttgtccccaggtgaaagagctacattgt peptide cctgtagagcttcccaatctgtttcctccta (from cttggcttggtatcaacaaaagccaggacag Aspergillus gctccaagattgttgatctacgacgcttcca niger α- atagagctactggtatcccagctagattctc amylase) tggttctggttccggtactgacttcactttg (DNA) actatctcttccttggaaccagaggacttcg ctgtttactactgtcagcagagatccaattg gccattgactttcggtggtggtactaaggtt gagatcaagcgtacggttgctgctccttccg ttttcattttcccaccatccgacgaacaatt gaagtctggtacCGCTTCCGTTGTTTGTTTG TTGAACAACTTCTACCCACGTGAGGCTAAGG TTCAGTGGAAGGTTGACAACGCTTTGCAATC CGGTAACTCCCAAGAATCCGTTACTGAGCAG GATTCTAAGGATTCCACTTACTCATTGTCCT CCACTTTGACTTTGTCCAAGGCTGATTACGA GAAGCACAAGGTTTACGCATGCGAGGTTACA CATCAGGGTTTGTCCTCCCCAGTTACTAAGT CCTTCAACAGAGGAGAGTGTTAA 64 Anti-CD20 ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG full length GATTGCAAGTTGCTGCTCCAGCTTTGGCTca HC with stop agttcagctggttcaatctggtgctgaggtt codon aagaagcctggttcctccgttaaggtttcct between Ab gtaaggcttccggttacactttcacttccta ORF and GR1 caacatgcactgggttagacaagctccaggt ORF with caaggattggaatggatgggtgctatctacc Alpha amylase caggtaacggtgacacttcttacaaccagaa signal gttcaagggtagagttactatcactgctgac peptide gaatccacttccactgcttacatggaattgt (from cctcattgagatccgaggacactgctgttta Aspergillus ctactgtgctagatccacttactacggtggt niger α- gactggtactttaatgtttggggacagggaa amylase) ctttggttactgtctcgagtgcttctactaa (DNA) gggaccatccgtttttccattggctccatcc tctaagtctacttccggtggtacCGCTGCTT TGGGATGTTTGGTTAAAGACTACTTCCCAGA GCCAGTTACTGTTTCTTGGAACTCCGGTGCT TTGACTTCTGGTGTTCACACTTTCCCAGCTG TTTTGCAATCTTCCGGTTTGTACTCTTTGTC CTCCGTTGTTACTGTTCCATCCTCTTCCTTG GGTACTCAGACTTACATCTGTAACGTTAACC ACAAGCCATCCAACACTAAGGTTGACAAGAA GGTTGAGCCAAAGTCCTGTGACAAGACTCAT ACTTGTCCACCATGTCCAGCTCCAGAATTGT TGGGTGGTCCTTCCGTTTTITTGTTCCACCA AAGCCAAAGGACACTTTGATGATCTCCAGAA CTCCAGAGGTTACATGTGTTGTTGTTGACGT TTCTCACGAGGACCCAGAGGTTAAGTTCAAC TGGTACGTTGACGGTGTTGAAGTTCACAACG CTAAGACTAAGCCAAGAGAGGAGCAGTACAA CTCCACTTACAGAGTTGTTTCCGTTTTGACT GTTTTGCACCAGGATTGGTTGAACGGAAAGG AGTACAAGTGTAAGGTTTCCAACAAGGCTTT GCCAGCTCCAATCGAAAAGACTATCTCCAAG GCTAAGGGTCAACCAAGAGAGCCACAGGTTT ACACTTTGCCACCATCCAGAGATGAGTTGAC TAAGAACCAGGTTTCCTTGACTTGTTTGGTT AAGGGATTCTACCCATCCGACATTGCTGTTG AATGGGAGTCTAACGGTCAACCAGAGAACAA CTACAAGACTACTCCACCTGTTTTGGACTCT GACGGTTCCTTTTTCTTGTACTCCAAGTTGA CTGTTGACAAGTCCAGATGGCAACAGGGTAA CGTTTTCTCCTGTTCCGTTATGCATGAGGCT TTGCACAACCACTACACTCAAAAGTCCTTGT CTTTGTCCCCTGGTAAGTAGGCGGCCGCTTA TCCATATGATGTTCCAGACTACGCTGGAGGT CATCATCATCACCACCATCACCATCATGGTG GTGAAGAGAAGTCCAGATTGTTGGAGAAAGA GAACAGAGAGTTGGAGAAGATCATCGCTGAG AAAGAAGAGAGAGTTTCCGAGTTGAGACACC AATTGCAATCCGTTGGTGGTTGTTAATAG 65 Anti-CD20 ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG LC with GATTGCAAGTTGCTGCTCCAGCTTTGGCTga Alpha amylase gatcgttttgacacagtccccagctactttg signal tctttgtccccaggtgaaagagctacattgt peptide cctgtagagcttcctcttccgtttcctacat (from ccactggtatcaacaaaagccaggacaggct Aspergillus ccaagattgttgatctacgctacttccaact niger α- tggcttccggtattccagctagattctctgg amylase) ttctggttccggtactgacttcactttgact (DNA) atctcttccttggaaccagaggacttcgctg tttactactgtcaacagtggacttctaaccc accaactttcggacaaggtactaaggttgag atcaagcgtacggttgctgctccttccgttt tcattttcccaccatccgacgaacaattgaa gtctggtacCGCTTCCGTTGTTTGTTTGTTG AACAACTTCTACCCACGTGAGGCTAAGGTTC AGTGGAAGGTTGACAACGCTTTGCAATCCGG TAACTCCCAAGAATCCGTTACTGAGCAGGAT TCTAAGGATTCCACTTAUCATTGTCCTCCAC TTTGACTTTGTCCAAGGCTGATTACGAGAAG CACAAGGTTTACGCATGCGAGGTTACACATC AGGGTTTGTCCTCCCCAGTTACTAAGTCCTT CAACAGAGGAGAGTGTTAA 66 DNA ATGAGATTCCCATCCATCTTCACTGCTGTTT sequence of TGTTCGCTGCTTCTTCCGCTTTGGCTCAGGT 1D05 Heavy TCAATTGGTTCAATCCGGTGCTGAAGTTAAG chain with AAGCCTGGTTCCTCCGTTAAGGTTTCCTGTA Saccharomyces AGGCTTCTGGTGGTACTTTTAACTCCCACGC cerevisiae TATCTCTTGGGTTAGACAAGCTCCAGGTCAA mating factor GGATTGGAATGGATGGGTGGTATCAACCCAA pre-signal TTTTGGGTATCGCTAACTACGCTCAAAAGTT peptide and CCAGGGTAGAGTTACTATTACTGCTGACGAA GR1 TCCACTTCCACTGCTTACATGGAATTGTCCT CATTGAGATCCGAGGACACTGCTGTTTACTA CTGTGCTAGACACTACGAGATCCAGATCGGT AGATACGGAATGAACGTTTACTACTTGATGT ACAGATTCGCTTCTTGGGGACAGGGAACTTT GGTTACTGTCTCGAGTGCTTCTACTAAGGGG CCCTCTGTTTTTCCATTGGCTCCATGTTCTA GATCCACTTCCGAATCCACTGCTGCTTTGGG ATGTTTGGTTAAGGACTACTTCCCAGAGCCA GTTACTGTTTCTTGGAACTCCGGTGCTTTGA CTTCTGGTGTTCACACTTTCCCAGCTGTTTT GCAATCTTCCGGTTTGTACTCCTTGTCCTCC GTTGTTACTGTTACTTCCTCCAACTTCGGTA CTCAGACTTACACTTGTAACGTTGACCACAA GCCATCCAACACTAAGGTTGACAAGACTGTT GAGAGAAAGGGTGGTGGTGGTAGTGGAGGTG GTGGAAGTGGTGGCGGTGGTTCTGCGGCCGC TTATCCATATGATGTTCCAGACTACGCTGGA GGTCATCATCATCACCACCATCACCATCATG GTGGTGAAGAGAAGTCCAGATTGTTGGAGAA AGAGAACAGAGAGTTGGAGAAGATCATCGCT GAGAAAGAAGAGAGAGTTTCCGAGTTGAGAC ACCAATTGCAATCCGTTGGTGGTTGTTAATA G 67 Amino acid MRFPSIFTAVLFAASSALAQVQLVQSGAEVK sequence of KPGSSVKVSCKASGGTFNSHAISWVRQAPGQ 1D05 HC GLEWMGGINPILGIANYAQKFQGRVTITADE with STSTAYMELSSLRSEDTAVYYCARHYEIQIG Saccharomyces RYGMNVYYLMYRFASWGQGTLVTVSSASTKG cerevisiae PSVFPLAPCSRSTSESTAALGCLVKDYFPEP mating factor VTVSWNSGALTSGVHTFPAVLQSSGLYSLSS pre-signal VVTVTSSNFGTQTYTCNVDHKPSNTKVDKTV peptide ERK 68 DNA ATGAGATTCCCATCCATCTTCACTGCTGTTT sequence of TGTTCGCTGCTTCTTCTGCTTTGGCTGACAT 1D05 light CCAAATGACACAATCCCCATCTTCCTTGTCT chain with GCTTCCGTTGGTGACAGAGTTACTATCACTT Saccharomyces GTAGAGCTTCCCAAGGTATCAGATCCGCTTT cerevisiae GAACTGGTATCAACAGAAGCCAGGAAAGGCT mating factor CCAAAGTTGTTGATCTACAACGGTTCCACTT pre-signal TGCAATCTGGTGTTCCATCTAGATTCTCTGG peptide TTCCGGTTCTGGTACTGACTTCACTTTGACT ATCTCTTCCTTGCAACCAGAGGACTTCGCTG TTTACTACTGTCAACAGTTCGATGGTGACCC AACTTTTGGACAGGGTACTAAGGTTGAGATC AAGAGAACTGTTGCTGCTCCATCCGTTTTCA TTTTCCCACCATCCGACGAACAATTGAAGTC TGGTACCGCTTCCGTTGTTTGTTTGTTGAAC AACTTCTACCCACGTGAGGCTAAGGTTCAGT GGAAGGTTGACAACGCTTTGCAATCCGGTAA CTCCCAAGAATCCGTTACTGAGCAGGATTCT AAGGATTCCACTTACTCATTGTCCTCCACTT TGACTTTGTCCAAGGCTGATTACGAGAAGCA CAAGGTTTACGCTTGCGAGGTTACACATCAG GGTTTGTCCTCCCCAGTTACTAAGTCCTTCA ACAGAGGAGAGTGTTAATAG 69 Amino acid MRFPSIFTAVLFAASSALADIQMTQSPSSLS sequence of ASVGDRVTITCRASQGIRSALNWYQQKPGKA 1D05 LC with PKLLIYNGSTLQSGVPSRFSGSGSGTDFTLT Saccharomyces ISSLQPEDFAVYYCQQFDGDPTFGQGTKVEI cerevisiae KRTVAAPSVFIFPPSDEQLKSGTASVVCLLN mating factor NFYPREAKVQWKVDNALQSGNSQESVTEQDS pre-signal KDSTYSLSSTLTLSKADYEKHKVYACEVTHQ peptide GLSSPVTKSFNRGEC 70 DNA ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG sequence of GATTGCAAGTTGCTGCTCCAGCTTTGGCTCA 1H23 heavy AGTTCAGTTGGTTGAATCCGGTGGTGGATTG chain with GTTCAACCTGGTGGTTCTTTGAGATTGTCCT Aspergillus GTGCTGCTTCCGGTTTTACTTTCTCCGACTA amylase CTACATGCACTGGGTTAGACAAGCACCTGGA signal AAGGGATTGGAATGGGTTTCCAACATTTCTG sequence, GTTCCGGTTCCACTACTTACTACGCTGATTC linker and CGTTAAGGGAAGATTCACTATCTCCAGAGAC GR1 AACTCCAAGAACACTTTGTACTTGCAGATGA ACTCCTTGAGAGCTGAGGATACTGCTGTTTA CTACTGTGCTAGAGGAATGTTTGACTTCTGG GGACAGGGAACTTTGGTTACTGTCTCGAGTG CTTCTACTAAGGGGCCCTCTGTTTTTCCATT GGCTCCATGTTCTAGATCCACTTCCGAATCC ACTGCTGCTTTGGGATGTTTGGTTAAGGACT ACTTCCCAGAGCCAGTTACTGTTTCTTGGAA CTCCGGTGCTTTGACTTCTGGTGTTCACACT TTCCCAGCTGTTTTGCAATCTTCCGGTTTGT ACTCCTTGTCCTCCGTTGTTACTGTTACTTC CTCCAACTTCGGTACTCAGACTTACACTTGT AACGTTGACCACAAGCCATCCAACACTAAGG TTGACAAGACTGTTGAGAGAAAGGGTGGTGG TGGTAGTGGAGGTGGTGGAAGTGGTGGCGGT GGTTCTGCGGCCGCTTATCCATATGATGTTC CAGACTACGCTGGAGGTCATCATCATCACCA CCATCACCATCATGGTGGTGAAGAGAAGTCC AGATTGTTGGAGAAAGAGAACAGAGAGTTGG AGAAGATCATCGCTGAGAAAGAAGAGAGAGT TTCCGAGTTGAGACACCAATTGCAATCCGTT GGTGGTTGTTAATAG 71 Amino acid MVAWWSLFLYGLQVAAPALAQVQLVESGGGL sequence of VQPGGSLRLSCAASGFTFSDYYMHWVRQAPG 1H23 HC KGLEWVSNISGSGSTTYYADSVKGRFTISRD with NSKNTLYLQMNSLRAEDTAVYYCARGMFDFW Aspergillus GQGTLVTVSSASTKGPSVFPLAPCSRSTSES amylase TAALGCLVKDYFPEPVTVSWNSGALTSGVHT signal FPAVLQSSGLYSLSSVVTVTSSNFGTQTYTC sequence NVDHKPSNTKVDKTVERK 72 DNA ATGGTTGCTTGGTGGTCCTTGTTCTTGTACG sequence of GATTGCAAGTTGCTGCTCCAGCTTTGGCTGA 1H23 light CATCGTTTTGACACAGTCCCCAGCTACTTTG chain with TCTTTGTCCCCAGGTGAAAGAGCTACATTGT Aspergillus CCTGTAGAGCTTCCCAATCCGTTAACTCCAA amylase CTACTTGGCTTGGTATCAACAAAAGCCAGGA signal CAGGCTCCAAGATTGTTGATCTACGGTGCTT sequence CTTCTAGAGCTACTGGTGTTCCAGCTAGATT CTCTGGTTCTGGTTCCGGTACTGACTTCACT TTGACTATCTCTTCCTTGGAACCAGAGGACT TCGCTGTTTACTACTGTCAACAGTGGGGTGA CGTTCCAATTACTTTCGGACAGGGTACTAAG GTTGAGATCAAGAGAACTGTTGCTGCTCCTT CCGTTTTCATTTTCCCACCATCCGACGAACA ATTGAAGTCTGGTACCGGTACCGCTTCCGTT GTTTGTTTGTTGAACAACTTCTACCCACGTG AGGCTAAGGTTCAGTGGAAGGTTGACAACGC TTTGCAATCCGGTAACTCCCAAGAATCCGTT ACTGAGCAGGATTCTAAGGATTCCACTTACT CATTGTCCTCCACTTTGACTTTGTCCAAGGC TGATTACGAGAAGCACAAGGTTTACGCTTGC GAGGTTACACATCAGGGTTTGTCCTCCCCAG TTACTAAGTCCTTCAACAGAGGAGAGTGTTA ATAG 73 Amino acid MVAWWSLFLYGLQVAAPALADIVLTQSPATL sequence of SLSPGERATLSCRASQSVNSNYLAWYQQKPG 1H23 light QAPRLLIYGASSRATGVPARFSGSGSGTDFT chain with LTISSLEPEDFAVYYCQQWGDVPITFGQGTK Aspergillus VEIKRTVAAPSVFIFPPSDEQLKSGTGTASV amylase VCLLNNFYPREAKVQWKVDNALQSGNSQESV signal TEQDSKDSTYSLSSTLTLSKADYEKHKVYAC sequence EVTHQGLSSPVTKSFNRGEC 74 amino acid QEDEDGDYEELVLALRSEEDGLAEAPEHGTT sequence of ATFHRCAKDPWRLPGTYVVVLKEETHLSQSE PCSK9 RTARRLQAQAARRGYLTKILHVFHGLLPGFL without 30 VKMSGDLLELALKLPHVDYIEEDSSVFAQSI amino acid PWNLERITPPRYRADEYQPPDGGSLVEVYLL signal DTSIQSDHREIEGRVMVTDFENVPEEDGTRF peptide HRQASKCDSHGTHLAGVVSGRDAGVAKGASM RSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQ PVGPLVVLLPLAGGYSRVLNAACQRLARAGV VLVTAAGNFRDDACLYSPASAPEVITVGATN AQDQPVTLGTLGTNFGRCVDLFAPGEDIIGA SSDCSTCFVSQSGTSQAAAHVAGIAAMMLSA EPELTLAELRQRLIHFSAKDVINEAWFPEDQ RVLTPNLVAALPPSTHGAGWQLFCRTVWSAH SGPTRMATAIARCAPDEELLSCSSFSRSGKR RGERMEAQGGKLVCRAHNAFGGEGVYAIARC CLLPQANCSVHTAPPAEASMGTRVHCHQQGH VLTGCSSHWEVEDLGTHKPPVLRPRGQPNQC VGHREASIHASCCHAPGLECKVKEHGIPAPQ EQVTVACEEGWTLTGCSALPGTSHVLGAYAV DNTCVVRSRDVSTTGSTSEEAVTAVAICCRS RHLAQASQELQ
(211) While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.