Expression and secretion system

Abstract

To provide an expression and secretion system, and methods of using the same, for the expression and secretion of one fusion protein in prokaryotic cells and a second fusion protein in eukaryotic cells.SOLUTION: Provided herein are nucleic acid molecules, vectors and host cells comprising such vectors and nucleic acid molecules. The nucleic acid molecule encodes a first polypeptide comprising VH-HVR1, VH-HVR2 and VH-HVR3 of a variable heavy chain domain (VH) and / or a second polypeptide comprising VL-HVR1, VL-HVR2 and VL-HVR3 of a variable light chain domain (VL). The nucleic acid molecule further encodes a signal sequence which is functional in both prokaryotic and eukaryotic cells and is encoded by a nucleic acid sequence that is operably linked to the first and / or second polypeptide sequence. A full-length antibody is expressed from the first and / or second polypeptide of the nucleic acid molecule.SELECTED DRAWING: Figure 14

Description

DISCLOSURE OF THEINVENTION

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 61 / 668,397, filed July 5, 2012, 61 / 852,483, filed March 15, 2013, and 61 / 819,063, filed May 3, 2013, all of which are incorporated by reference in their entireties. [Technical field]

[0002] FIELD OF THEINVENTION The present invention relates to expression and secretion systems and methods of use thereof for the expression and secretion of one Fab fusion protein when the nucleic acid is transformed into a prokaryotic cell for phage display, and separate or identical Fab fusion proteins when the nucleic acid is transfected into a eukaryotic cell for expression and purification. Also provided herein are nucleic acid molecules, vectors and host cells containing such vectors and nucleic acid molecules. [Background technology]

[0003] background Phage display of peptides or proteins on filamentous phage particles is an in vitro technique that allows the selection of peptides or proteins with desired properties from a large pool of variant peptides or proteins (McCafferty et al., Nature, 348: 552-554 (1990); Sidhu et al., Current Opinion in Biotechnology, 11: 610-616 (2000); Smith et al., Science, 228: 1315-1317 (1985)). Phage display can be used in the context of antibody detection to display diverse libraries of peptides or proteins, including antibody fragments such as Fab, on the surface of filamentous phage particles that are then selected for binding to a specific antigen of interest. Antibody fragments can be displayed on the surface of filamentous phage particles by fusing the genes for the antibody fragment to the genes for a phage coat protein, resulting in phage particles that display the encoded antibody fragments on their surface. This technique allows the isolation of antibody fragments with desired affinities for multiple antigens from large phage libraries.

[0004] For phage-based antibody discovery, evaluation of the properties of selected antibody fragments and their cognate IgG in functional assays (target binding, cell-based activity assays, in vivo half-life, etc.) requires reformatting of the Fab heavy chain (HC) and light chain (LC) sequences into full-length IgG by subcloning DNA sequences encoding the HC and LC from the vector used for phage display into a mammalian expression vector for IgG expression. The laborious process of subcloning tens or hundreds of selected HC / LC pairs represents a major bottleneck in the phage-based antibody discovery process. Moreover, a significant proportion of selected Fabs, once reformatted, are unable to function satisfactorily in initial screening assays, so increasing the number of clones carried through this reformatting / screening step significantly increases the probability of final success.

[0005] Here we describe the generation of an expression and secretion system to drive expression of Fab-phage fusions when transformed into E. coli, and to drive expression of full-length IgG with the same Fab fragment when transfected into mammalian cells. We have shown that the mammalian signal sequence from mouse binding immunoglobulin protein (mBiP) (Haas et al., Immunoglobulin heavy chain binding protein, Nature, 306: 387-389 (1983); Munro et al., An Hsp70-like protein in the ER: identify with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein, Cell, 4:291-300 (1986)) can drive efficient protein expression in both prokaryotic and eukaryotic cells. Human IgG 1 To remove a synthetic intron containing a phage fusion peptide inserted within the hinge region of the HC, we used mammalian mRNA splicing to generate two distinct proteins in a host cell-dependent manner: a Fab fragment fused to a phage-displayed adapter peptide in E. coli and native human IgG in mammalian cells. 1 This technique allows the selection of Fab fragments that bind to an antigen of interest from a phage display library, with subsequent expression in mammalian cells and purification of the cognate full-length IgG, without the need for subcloning. Summary of the Invention

[0006] overview In one aspect, the invention is based in part on experimental findings demonstrating that (1) a functional signal sequence of non-prokaryotic origin in a prokaryotic cell and (2) different Fab fusion proteins are expressed from the same nucleic acid molecule in a host cell-dependent manner (Fab phage fusion protein in prokaryotic cells and Fab-Fc fusion protein in eukaryotic cells) when mRNA processing occurs in eukaryotic cells but not in prokaryotic cells. Thus, described herein are nucleic acid molecules and methods of use for the expression and secretion of Fab fragments fused to phage particle proteins, coat proteins or adapter proteins for phage display in bacteria when the nucleic acid is transformed into a prokaryotic host cell (e.g., E. coli) without the need for subcloning, and Fab fragments fused to Fc when the nucleic acid is transformed into a eukaryotic cell (e.g., mammalian cell).

[0007] In one embodiment, the invention provides a nucleic acid molecule encoding a first polypeptide comprising variable heavy domains (VH) VH-HVR1, VH-HVR2 and HVR3, and / or a second polypeptide comprising variable light domains VL-HVR1, VL-HVR2 and VL-HVR3, wherein the nucleic acid molecule is functional in both prokaryotic and eukaryotic cells and further encodes a signal sequence encoded by a nucleic acid sequence operably linked to the first and / or second polypeptide sequence, and a full-length antibody is expressed from the first and / or second polypeptide of the nucleic acid molecule. In another embodiment, the first and / or second polypeptide further comprises a variable heavy (VH) domain and a variable light (VL) domain. In a further embodiment, the VH domain is linked to CH1 and the VL domain is linked to CL.

[0008] In one aspect, the invention provides a nucleic acid molecule comprising a prokaryotic promoter and a eukaryotic promoter encoding VH-HVR1, VH-HVR2 and VH-HVR3 of a variable heavy domain (VH) and VL-HVR1, VL-HVR2 and VL-HVR3 of a variable light domain (VL), operably linked to the HVR of VH and / or the HVR of VL, enabling expression of the HVR of VH and the HVR of VL in prokaryotic and eukaryotic cells, wherein the HVR of VH and / or the HVR of VL are linked to a utility peptide when expressed by a eukaryotic cell, and wherein the nucleic acid further encodes a signal sequence that is functional in both prokaryotic and eukaryotic cells.

[0009] In one aspect, the invention provides a nucleic acid molecule comprising a prokaryotic promoter and a eukaryotic promoter encoding a variable heavy (VH) domain and a variable light (VL) domain, operably linked to the VH domain and / or the VL domain, and enabling expression of the VH domain and / or the VL domain in prokaryotic and eukaryotic cells, wherein the VH domain and / or the VL domain are linked to a utility peptide when expressed by a eukaryotic cell, and wherein the nucleic acid further encodes a signal sequence that is functional in both prokaryotic and eukaryotic cells.

[0010] In one embodiment, the VL and VH are linked to a utility peptide. In a further embodiment, the VH is further linked to CH1 and the VL is linked to CL. The utility peptide is selected from the group consisting of Fc, a tag, a label and a control protein. In one embodiment, the VL is linked to a control protein and the VH is linked to Fc. For example, the control protein is a gD protein, or a fragment thereof.

[0011] In yet another embodiment, the first and / or second polypeptide of the invention is a coat protein (e.g., pI, pII, pIII, pIV, pV, pVI, pVII, pVIII, pIX and pX of bacteriophage M13, f1 or fd, or fragments thereof, such as amino acids 267-421 or 262-418 of the pIII protein ("pI", "PII", "pIII", "pIV", "pV", "pVI", "pVII", "pVIII", "pIX" and "pX" as used herein refer to full-length proteins or fragments thereof unless otherwise specified)) or an adaptor protein. TIFF2024170391000002.tif33169 or a variant thereof (amino acids of SEQ ID NO:12 and SEQ ID NO:13 that may be modified include, but are not limited to, those underlined and bolded), where the variant has an amino acid modification that is fused to an adaptor protein, another adaptor protein, or SEQ ID NO:6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO:7 (ASLDELEAEIEQLEEENYALEKEIE 15 (Hinge:CPPCPG)。 The nucleic acid molecule encoding the coat protein or adaptor protein is contained within a synthetic intron. The synthetic intron is located between the nucleic acid encoding the VH domain and the nucleic acid encoding the Fc. The synthetic intron further comprises a nucleic acid encoding a naturally occurring intron from IgG1, where the naturally occurring intron may be selected from the group consisting of intron 1, intron 2 or intron 3 from IgG1.

[0012] In one embodiment, the invention provides a nucleic acid molecule in which a first fusion protein is expressed in a prokaryotic cell and a second fusion protein is expressed in a eukaryotic cell. The first and second fusion proteins may be the same or different. In a further embodiment, the first fusion protein may be a Fab-phage fusion protein (e.g., a Fab-phage fusion protein comprising a VH / CH1 fused to pIII) and the second fusion may be a Fab-Fc or Fab-hinge-Fc fusion protein (e.g., a Fab-Fc or Fab-hinge-Fc fusion protein comprising a VH / CH1 fused to Fc).

[0013] In one embodiment, the invention provides nucleic acid molecules in which a signal sequence directs secretion of a protein into the endoplasmic reticulum or outside of a cell in a eukaryotic cell and / or a signal sequence directs secretion of a protein into the periplasm or outside of a cell in a prokaryotic cell. Furthermore, the signal sequence can be encoded by a nucleic acid sequence encoding an amino acid sequence comprising the amino acid sequence of SEQ ID NO:10 (XMKFTVVAAALLLLGAVRA, where X=0 amino acids or 1 or 2 amino acids (e.g., X=M (SEQ ID NO:3; MMKFTVVAAALLLLGAVRA; wild type mBIP) or X=MT (SEQ ID NO:19; MTMKFTVVAAALLLLGAVRA) or X is absent (SEQ ID NO:20; MKFTVVAAALLLLGAVRA), or by a nucleic acid sequence encoding mBIP (SEQ ID NO:4; ATG ATG AAA TTT ACC GTG GTG GCG GCG GCG CTG CTG CTG CTG GGC GCG GTC CGC GCG) and variants thereof, or by a nucleic acid sequence encoding an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence selected from SEQ ID NO:3 (mBIP amino acid sequence), and wherein the signal sequence functions in both prokaryotic and eukaryotic cells, or by a nucleic acid sequence encoding an amino acid sequence comprising the amino acid sequence of SEQ ID NO:11 (consensus mBIP sequence, X ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN, where N=A, T, C or G, where X=ATG (SEQ ID NO:5; ATG ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN), X=ATG ACC (SEQ ID NO:21; ATG ACC ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN) or X=not present (SEQ ID NO:22; ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN), or by the nucleic acid sequence of SEQ ID NO:16 (mBIP.It may be encoded by a nucleic acid sequence selected from the group consisting of: Opt1: ATG ATG AAA TTT ACC GTT GTT GCT GCT GCT CTG CTA CTT CTT GGA GCG GTC CGC GCA), SEQ ID NO: 17 (mBIP.Opt2: ATG ATG AAA TTT ACT GTT GTT GCG GCT GCT CTT CTC CTT CTT GGA GCG GTC CGC GCA), and SEQ ID NO: 18 (mBIP.Opt3: ATG ATG AAA TTT ACT GTT GTC GCT GCT GCT CTT CTA CTT CTT GGA GCG GTC CGC GCA).

[0014] In a further embodiment, the synthetic intron in the nucleic acid molecule is flanked at its 5' end by a nucleic acid encoding CH1 and at its 3' end by a nucleic acid encoding Fc. Furthermore, the nucleic acid encoding the CH1 domain comprises a portion of a natural splice donor sequence, and the nucleic acid encoding Fc comprises a portion of a natural splice acceptor sequence. Alternatively, the nucleic acid encoding the CH1 domain comprises a portion of a modified splice donor sequence, where the modified splice donor sequence comprises a modification of at least one nucleic acid residue, and where the modification increases splicing.

[0015] In one embodiment, the prokaryotic promoter is a phoA, Tac, Tphac or Lac promoter, and / or the eukaryotic promoter is a CMV or SV40 or Moloney murine leukemia virus U3 region or a caprine arthritis encephalitis virus U3 region or a Visna virus U3 region or a retroviral U3 region sequence. Expression by a prokaryotic promoter occurs in a bacterial cell, and expression by a eukaryotic promoter occurs in a mammalian cell. In a further embodiment, the bacterial cell is an E. coli cell, and the eukaryotic cell is a yeast cell, a CHO cell, a 293 cell or an NSO cell.

[0016] In another embodiment, the invention provides a vector comprising a nucleic acid molecule described herein, and / or a host cell transformed with the vector. The host cell may be a bacterial cell (e.g., an E. coli cell) or a eukaryotic cell (e.g., a yeast cell, a CHO cell, a 293 cell, or an NSO cell).

[0017] In another embodiment, the invention provides a method for producing an antibody comprising culturing a host cell as described herein such that a nucleic acid is expressed, the method further comprising recovering the antibody expressed by the host cell, and the antibody is recovered from the host culture medium.

[0018] In one aspect, the invention provides an adaptor protein comprising an alteration of at least one residue of the amino acid sequence of SEQ ID NO: 8, 9, 12, 13, 14 or 15. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NO: 6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO: 7 (ASLDELEAEIEQLEEENYALEKEIEDLEKELEKLGGC). In one embodiment, the invention provides a nucleic acid encoding such an adaptor protein.

[0019] In one aspect, the invention provides a nucleic acid molecule encoding a mBIP polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof, or a polypeptide having an amino acid sequence with 85% homology to the amino acid sequence of SEQ ID NO:3, which is functional in both prokaryotic and eukaryotic cells. In one embodiment, the invention provides a method of expressing a mBIP polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof in both prokaryotic and eukaryotic cells. In one embodiment, the invention provides a bacterial cell expressing a mBIP sequence comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof.

[0020] In one aspect, the invention provides that a synthetic intron is located between the nucleic acid encoding the VH domain of the antibody and the nucleic acid encoding the Fc or hinge, between the nucleic acid encoding the CH2 and CH3 domains of the antibody, between the nucleic acid encoding the hinge region and the CH2 domain of the antibody.

[0021] In one aspect, the present invention relates to a signal sequence comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof, a polypeptide comprising a variable heavy domain (VH) and a variable light domain (VL) in which the VH domain is linked to the N-terminus of the VL domain, or a signal sequence comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof, a polypeptide comprising a variable heavy domain (VH) and a variable light domain (VL) in which the VH domain is linked to the C ... or a polypeptide comprising a signal sequence comprising the amino acid sequence of SEQ ID NO: 3, and a variable light domain (VL) comprising VL-HVR1, VL-HVR2, and VL-HVR3, or a signal sequence comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof, a variable heavy domain (VH) comprising VH-HVR1, VH-HVR2, and VH-HVR3, and a variable light domain (VL) comprising VL-HVR1, VL-HVR2, and VL-HVR3. In one embodiment, the polypeptide of the present invention is an antibody or an antibody fragment. The antibody or antibody fragment of the present invention may be selected from the group consisting of F(ab')2 and Fv fragments, diabodies, and single chain antibody molecules.

[0022] In one aspect, the invention includes a mutant helper phage for enhancing phage display of a protein. In one embodiment, the nucleotide sequence of the helper phage includes an amber mutation in pill, and the helper phage includes the amber mutation to enhance the display of a protein fused to pill on the phage. In a further embodiment, the nucleotide sequence of claim 70, wherein the amber mutation is a mutation at nucleotides 2613, 2614, and 2616 of the nucleic acid of M13KO7. In yet another embodiment, the nucleotide sequence of claim 71, wherein the mutation at nucleotides 2613, 2614, and 2616 of the nucleic acid of M13KO7 introduces an amber stop codon. [Brief description of the drawings]

[0023] [Figure 1] (A) Her2 phage ELISA of purified phages showing anti-Her2 Fabs under the control of four different eukaryotic signal sequences (mBiP, Gaussia princeps, yBGL2, HGH). The heat-stable enterotoxin II (STII) prokaryotic signal sequence commonly used in phagemids serves as a benchmark. (B) Anti-Her2 Fab phage display fused to the wild-type eukaryotic mBiP signal sequence (mBiP.wt) and codon-optimized versions (mBiP.Opt1, mBiP.Opt2 and mBip.Opt3 (SEQ ID NO: 16-18)) obtained by phage library panning. [Diagram 2] (A) Expression yields from 30 mL 293 cell suspension cultures of individual clones and (B) total statistics of hIgG1 clones expressed as fusions to either the eukaryotic mBiP or the prokaryotic native IgG HC (VHS) signal sequence. [Diagram 3](A) Genomic structure of human IgG1 HC containing three naturally occurring introns. Intron 1 is immediately preceding the hinge region. (B) HC construct containing a synthetic intron from intron 1 or 3 and containing a phage adaptor fusion peptide. The synthetic intron is flanked by a natural intron splice donor (D) and an acceptor (A) from intron 1 or 3. (C) HC construct containing a synthetic intron from intron 1 or 3 and containing a phage coat fusion protein. The synthetic intron is flanked by a natural intron splice donor (D) and an acceptor (A) from intron 1 or 3. Both constructs (B) and (C) contain a stop codon at the 3' end of the adaptor peptide or phage coat protein sequence. [Figure 4] (A) Expression levels of h4D5 IgG from constructs containing either no intron, a synthetic intron with a phage adaptor peptide (see FIG. 3B), or a synthetic intron with a phage coat protein (gene III, see FIG. 3C). (B) RT-PCR of hIgG1 HC from transfected cells. The expected size for a properly spliced ​​mRNA HC is 1650 nt. The band in the adaptor+intron 1 construct represents unspliced ​​precursor mRNA. The lower band in the adaptor and gene-III containing construct is misspliced ​​by a cryptic splice donor in the VH. [Diagram 5] (A) Point mutations generated in the natural intron 1 splice donor improve its compatibility with the consensus splice donor of mammalian mRNAs. (B) Optimization of the intron splice donor eliminates the accumulation of unspliced ​​and misspliced ​​HC mRNAs and (C) increases expression in mammalian cells to levels observed in the absence of the intron. [Figure 6](A) Modulation of display using pDV.5.0 and either wild type KO7 (monovalent display) or adaptor KO7 (multivalent display). (B) Expression of four different mAbs from pDV.5.0 in three different mammalian cell lines. [Figure 7] Schematic diagram of vectors for expression and secretion of polypeptides in prokaryotic and eukaryotic cells. The synthetic intron may contain either an adapter sequence or a phage coat protein sequence along with any of the naturally occurring intron sequences from hIgG1. Both the HC and LC can have either 1) a mammalian promoter and a bacterial promoter upstream of the ORF, 2) only a bacterial promoter upstream of the ORF (see also FIG. 14), or 3) only a mammalian promoter upstream of the ORF. Constructs are shown in which both the HC and LC have both promoter types. The cassette containing gene-III with adapter peptide fusion (pDV5.0, shown) is present only if the synthetic intron contains the adapter peptide fusion, but not if the phage coat protein fusion is not present in the synthetic intron. [Figure 8] Nucleotide sequence of pIII of mutant helper phage amber KO7 (nucleotides 1579 to 2853 (SEQ ID NO:24)), which enhances display of proteins fused on the pIII of M13 phage. Amber KO7 has an amber codon introduced into the M13KO7 helper phage genome by site-directed mutagenesis. The underlined residues are mutations at nucleotides 2613, 2614, and 2616 (T2613C, C2614T, and A2616G) that introduce an amber stop (TAG) at codon 346 and silent mutations for the AvrII restriction site at codon 345 of M13KO7 gene III. Nucleotide 1 of M13KO7 is the third residue of a unique HpaI restriction site. [Figure 9]Enhanced display of Fab fragments on pIII of M13 phage by use of amber KO7 helper phage. A conventional high display phagemid containing wild type M13KO7 (open diamonds) increases the level of Fab display significantly higher than that achieved by a low display phagemid vector when wild type M13KO7 is used for phage production (closed squares). Use of modified M13KO7 with an amber mutation in pIII (amber KO7) increases the display level of the low display phagemid (closed triangles) to that of the high display phagemid of wild type M13KO7 (open diamonds). [Figure 10] Figure 10 is a bar graph showing binding (as measured by phage ELISA) to immobilized VEGF of clones selected from phage library selection of a naive double vector Fab phage library described in Example 5. After four rounds of selection, individual clones were picked and phage supernatants were tested for binding to immobilized antigen (VEGF) and an unrelated protein (Her2) to assess binding specificity. [Figure 11] Figure 11 shows screening of phage clones selected in IgG format by BIAcore for antigen binding to VEGF as measured by Fc capture assay on a BIAcore T100 instrument. 96 clones picked for sequence analysis and phage ELISA were transfected into 293S cells (1 mL) and cultured for 7 days for expression of IgG. Supernatants were 0.2 μm filtered and used to assess VEGF antigen binding by Fc-capture assay on a Biacore T100 instrument. [Figure 12]Figure 12 shows the sequences of positive binders from the VEGF panning experiment in Example 5. The heavy chain CDR sequences of eight clones (VEGF50 (respectively in order of appearance, SEQ ID NOs: 25-27), VEGF51 (respectively in order of appearance, SEQ ID NOs: 28-30), VEGF52 (respectively in order of appearance, SEQ ID NOs: 31-33), VEGF59 (respectively in order of appearance, SEQ ID NOs: 34-36), VEGF55 (respectively in order of appearance, SEQ ID NOs: 37-39), VEGF60 (respectively in order of appearance, SEQ ID NOs: 40-42), VEGF61 (respectively in order of appearance, SEQ ID NOs: 43-45) and VEGF64 (respectively in order of appearance, SEQ ID NOs: 46-48)) are shown. All clones share the same light chain CDR sequences. [Figure 13] Figure 13 shows the ability of selected anti-VEGF IgGs selected from phage sorting against VEGF to inhibit binding of VEGF to one of its natural receptors, VEGF-R1. Antibodies selected from sorting against VEGF were expressed in CHO cells and purified IgG was used to measure the ability of selected clones to inhibit VEGF binding to VEGF-R1. One clone (VEGF55) inhibited VEGF-R1 binding with an IC50 that was within 3.5-fold of that of bevacizumab (Avastin). [Figure 14] Figure 14 shows a schematic of a vector for expression and secretion of polypeptides in prokaryotic and eukaryotic cells, where a synthetic intron contains pIII with either a naturally occurring intron sequence from hIgG1, and the LC has a bacterial promoter upstream of the ORF, and the HC has both a mammalian and bacterial promoter upstream of the ORF. Unlike the vector shown in Figure 7, this vector (pDV6.5) does not require an additional gIII cassette for fusion to the phage particle. The proteins resulting from expression in E. coli and mammalian cells are shown below the vector schematic. The dashed line indicates the intron in the spliced ​​heavy chain transcript in mammalian cells. Note that part of the sequence encoding the hinge of IgG1 is repeated in the vector to allow for inclusion of the protein expressed in both E. coli and mammalian cells. [Figure 15] Figure 15 shows the properties of full-length anti-VEGF IgG expressed from pDV6.5. IgG was expressed in 100 mL transfected CHO cell cultures and purified by Protein A chromatography. Final yields of purified IgG are shown along with scores in a baculovirus ELISA used to measure non-specific binding. Positive or negative binding of each clone in phage format (phage ELISA) or IgG format (Biacore) is also shown.

[0024] DETAILED DESCRIPTION OF EMBODIMENTS OF THE PRESENTINVENTION I. Definition The term "synthetic intron" is used herein to define a segment of nucleic acid located between the nucleic acid encoding CH1 and the nucleic acid encoding hinge-Fc or Fc. A "synthetic intron" may be any nucleic acid that does not code for protein synthesis, such as a protein or coat protein of a phage particle (e.g., PI, PII, pIII, pIV, PV, PVI, pVII, pVIII, pIX, pX), or an adaptor protein (e.g., leucine zipper, etc.), or any combination thereof. In one embodiment, a "synthetic intron" includes a portion of a splice donor sequence and a splice acceptor sequence that allow a splice event. The splice donor sequence and the splice acceptor sequence allow a splice event and may include natural or synthetic nucleic acid sequences.

[0025] The term "utility polypeptide" is used herein to refer to a polypeptide that is useful for a number of activities, including protein purification, protein tagging, protein labeling (e.g., labeling with a detectable compound or composition, such as a radioactive, fluorescent, or enzymatic label). The label can be indirectly conjugated to an amino acid side chain, an activated amino acid side chain, a cysteine ​​engineered antibody, or the like. For example, an antibody can be conjugated to biotin, and any of the three broad categories of labels described above can be conjugated to avidin or streptavidin, or vice versa. Biotin selectively binds to streptavidin, and thus the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the polypeptide variant with the label, the polypeptide variant is conjugated to a small hapten (e.g., digoxin), and one of the different types of labels described above is conjugated to the anti-hapten polypeptide variant (e.g., an anti-digoxin antibody). In this manner, indirect conjugation of the label to the polypeptide variant can be achieved (Hermanson, G. (1996) in Bioconjugate Techniques Academic Press, San Diego).

[0026] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are present in a nucleic acid molecule such that they have a functional relationship to each other as nucleic acids or as the protein expressed by them. They may or may not be contiguous. In the case of secretory leaders, they are often contiguous and in reading phase. Enhancers, however, need not be contiguous. Linkage is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used.

[0027] A VH or VL domain is "linked" to a phage when nucleic acid encoding a heterologous protein sequence (e.g., the VH or VL domain) is inserted directly into nucleic acid encoding a phage coat protein (e.g., pII, pVI, pVII, pVIII, or pIX). When introduced into a prokaryotic cell, phage are generated whose coat protein is capable of displaying the VH or VL domain. In one embodiment, the resulting phage particle displays an antibody fragment fused to the amino- or carboxy-terminus of the phage coat protein.

[0028] As used herein, the terms "linked" or "linked" or "linkage" refer to the covalent linking together of two amino acid sequences or two nucleic acid sequences via a peptide bond or a phosphodiester bond, respectively, and such a linkage can include any number of additional amino acid sequences or nucleic acid sequences between the two amino acid sequences or nucleic acid sequences being linked, for example, a direct peptide bond linkage between the first and second amino acid sequences or a linkage that includes one or more amino acid sequences between the first and second amino acid sequences.

[0029] As used herein, "linker" refers to an amino acid sequence that is two or more amino acids long. The linker may be composed of naturally occurring polar or non-polar amino acids. The linker may be, for example, 2 to 100 amino acids long, for example, between 2 and 50 amino acids long, for example, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The linker may be "cleavable", for example, by self-cleavage or enzymatic or chemical cleavage. Cleavage sites within an amino acid sequence, as well as enzymes and chemicals that cleave at such sites, are well known in the art and are described herein.

[0030] The term "function of a signal sequence" refers to the biological activity of a signal sequence that directs a secreted protein to the ER (in eukaryotes) or periplasm (in prokaryotes) or outside the cell.

[0031] As used herein, a "control protein" refers to a protein sequence whose expression is measured to quantify the level of display of the protein sequence. For example, the protein sequence can be an "epitope tag" that allows the VH or VL to be easily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the epitope tag. Tag polypeptides and their suitable respective antibodies include poly-histidine (poly-His) or poly-histidine-glycine (poly-his-gly) tags; influenza HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; c-myc tag and its 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and herpes simplex virus glycoprotein D (gD) tag and its antibodies [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

[0032] As used herein, "coat protein" refers to any of the five capsid proteins that are components of phage particles, including pIII, pVI, pVII, pVIII, and pIX. In one embodiment, a "coat protein" can be used to display proteins or peptides (see Phage Display, A Practical Approach, Oxford University Press, edited by Clackson and Lowman, 2004, p. 1-26). In one embodiment, the coat protein can be the pIII protein or a variant, part, and / or derivative thereof. For example, the C-terminal portion of the pIII coat protein of M13 bacteriophage (cP3) can be used, such as the sequence encoding the C-terminal residues 267 to 421 of protein III of M13 phage. In one embodiment, the pIII sequence is SEQ ID NO:1 (AEDIEFASGGGSGAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAF The amino acid sequence of HSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES).In one embodiment, the pill fragment comprises the amino acid sequence of SEQ ID NO:2 (SGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES).

[0033] As used herein, "adapter protein" refers to a protein sequence that specifically interacts with other adaptor protein sequences in solution. In one embodiment, an "adapter protein" comprises a heteromultimerization domain. In one embodiment, the adaptor protein is a cJUN protein or a Fos protein. In another embodiment, the adaptor protein comprises the sequence of SEQ ID NO: 6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO: 7 (ASLDELEAEIEQLEEENYALEKEIEDLEKELEKLGGC).

[0034] As described herein, a "heteromultimerization domain" refers to a modification or addition to a biomolecule that promotes heteromultimer formation and inhibits homomultimer formation. Any heterodimerization domain that has a strong preference for heterodimer formation over homodimer formation is within the scope of the present invention. Illustrative examples include, but are not limited to, U.S. Patent Publication No. 20030078385 (Arathoon et al. Genentech; describing knobs-into-holes); WO 2007147901 (Kjaergaard et al. Novo Nordisk: describing ionic interactions); WO 2009089004 (Kannan et al. Amgen: describing electrostatic steering effects); and WO 2011 / 034605 (Christensen et al. - Genentech; describing coiled coils). See also, e.g., Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992), which describes leucine zippers, or Pack et al., Bio / Technology 11, 1271-1277 (1993), which describes helix-turn-helix motifs. The terms "heteromultimerization domain" and "heterodimerization domain" are used interchangeably herein.

[0035] The term "Fab fusion protein" as used herein refers to a Fab phage fusion protein in prokaryotic cells and / or a Fab-Fc fusion protein in eukaryotic cells. The Fab-Fc fusion can also be a Fab-hinge-Fc fusion.

[0036] The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0037] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0038] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further subdivided (isotypes), e.g., IgG 1 , IgG 2 IgG 3 , IgG 4 , I.G.A. 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0039] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0040] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0041] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0042] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0043] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Typically, a naturally occurring four-chain antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), the latter being of greatest sequence variability and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). With the exception of CDR1 of VH, CDRs generally contain amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs", which are residues that contact the antigen. SDRs are contained within a region of CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).Unless otherwise specified, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0044] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0045] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0046] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0047] An "isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and including such nucleic acid molecules present in one or more locations in a host cell.

[0048] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies, e.g., including naturally occurring mutations or variants that arise during the manufacture of monoclonal antibody preparations and are generally present in minor amounts. In contrast to polyclonal antibody preparations, which generally contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0049] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0050] "Native antibodies" refer to immunoglobulin molecules that occur in nature and assume a variety of structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0051] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, including information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings concerning the use of such therapeutic products.

[0052] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated by using a VH or VL domain from an antibody that specifically binds to the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0053] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0054] II. Detailed Description The detection process for phage-based antibodies involves the identification of individual phage clones. 1-3 The phage display technology is used to select Fab fragments with the desired binding specificity from a large pool of Fab fragments. In this approach, phage libraries consisting of Fab fragments fused directly or indirectly to M13 filamentous phage particles via one of the major coat proteins and containing diverse complementarity determining regions (CDRs) are generated using established molecular biology techniques and specialized phage display vectors (Tohidkia et al., Journal of drug targeting, 20: 195-208 (2012); Bradbury et al., Nature biotechnology, 29: 245-254 (2011); Qi et al., Journal of molecular biology, 417: 129-143 (2012)). The theoretical diversity of such libraries could reach 10 25 Although the number of unique sequences in a given library can easily exceed 10, practical limitations in constructing phage pools limit the actual diversity to ≦10 in a given library. 11 (Sidhu et al., Methods in enzymology, 328: 333-363 (2000)).

[0055] Considering the substantial number of unique sequences that can be included in the starting library, the throughput of screening of selected clones is crucial. For phage-based antibody discovery, full characterization of selected Fabs and their cognate full-length IgGs in functional assays (target binding, cell-based activity assays, in vivo half-life, etc.) requires reformatting of the Fab heavy chain (HC) and light chain (LC) sequences into full-length IgGs by subcloning DNA sequences encoding the HC and LC from the phagemid vector used for display into a mammalian expression vector for IgG expression. The laborious process of subcloning tens or hundreds of selected HC / LC pairs represents a major bottleneck in the phage-based antibody discovery process. Moreover, a significant proportion of selected Fabs, once reformatted, are unable to function satisfactorily in the initial screening assays, so increasing the number of clones carried through this reformatting / screening step significantly increases the probability of final success.

[0056] We describe herein the creation of an expression and secretion system for the expression and secretion of one Fab fusion protein in prokaryotic cells, and different (or identical) Fab fusions in eukaryotic cells. For example, the expression and secretion system drives the expression of a Fab phage fusion when transformed into E. coli, and drives the expression of a full-length IgG with the same Fab fragment when transfected into mammalian cells. We have used mouse binding immunoglobulin protein (mBiP) 8,9 We demonstrate that mammalian signal sequences from human IgG can drive efficient protein expression in both prokaryotic and eukaryotic cells. 1 To remove a synthetic intron containing a phage fusion peptide inserted within the hinge region of the HC, we used mammalian mRNA splicing to generate two distinct proteins in a host cell-dependent manner: a Fab fragment fused to a phage-displayed adapter peptide in E. coli and native human IgG in mammalian cells. 1This technique allows the selection of Fab fragments that bind to an antigen of interest from a phage display library, with subsequent expression in mammalian cells and purification of the cognate full-length IgG, without the need for subcloning.

[0057] The present invention is based in part on experimental discoveries demonstrating that (1) signal sequences of non-bacterial origin function in prokaryotic cells at levels sufficient for selection of phage libraries without compromising expression of IgG in eukaryotic cells, and (2) different Fab fusion proteins are expressed from the same nucleic acid molecule in a host cell-dependent manner (Fab phage fusion proteins in prokaryotic cells and Fab-Fc fusion proteins in eukaryotic cells) when mRNA processing occurs in eukaryotic cells but not in prokaryotic cells. Thus, described herein are expression and secretion systems for the expression and secretion of Fab fragments fused to phage particle proteins, coat proteins or adapter proteins for phage display in prokaryotic host cells (e.g., E. coli) and Fab fragments fused to Fc in eukaryotic cells (e.g., mammalian cells) without the need for subcloning, and methods for the construction and use of the expression and secretion systems. In particular, described herein are vectors for the expression and secretion of Fab-phage fusion proteins in prokaryotic cells and Fab-Fc fusion proteins in eukaryotic cells, nucleic acid molecules for the expression and secretion of proteins or peptides in prokaryotic and eukaryotic cells, and host cells containing such vectors.Further described herein are methods of using the expression and secretion systems, including methods of using the expression and secretion systems for screening and selection of novel antibodies against a protein of interest.

[0058] Modes for carrying out the invention The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology, 4th ed. (DM Weir & CC Blackwell, eds., Blackwell Science Inc., 1987); Gene Transfer Vectors for Mammalian Cells (JM Miller & MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JE Coligan et al., eds., 1991).

[0059] Expression and secretion systems for prokaryotic and eukaryotic cells Expression and secretion systems for prokaryotic and eukaryotic cells include vectors containing regulatory and coding sequences of a protein of interest (e.g., heavy or light chain of an IgG molecule) where a prokaryotic promoter and a eukaryotic promoter (e.g., CMV (eukaryotic) and PhoA (prokaryotic)) are placed in tandem upstream of the gene of interest and a single signal sequence drives expression of the protein of interest in prokaryotic and eukaryotic cells. The present invention provides a means for this vector to generate two different fusion forms of the protein of interest by using a synthetic intron located between the VH / CH1 and hinge-Fc regions of IgG1, which is spliced ​​out during mRNA processing in eukaryotic cells, in a host cell dependent manner.

[0060] A. Signal Sequences that Function in Both Prokaryotic and Eukaryotic Cells One problem in constructing vectors capable of expressing a protein of interest in prokaryotic (E. coli) and eukaryotic (mammalian) cells arises from the differences in signal sequences found in these cell types. While certain features of signal sequences are generally conserved in both prokaryotic and eukaryotic cells (e.g., a patch of hydrophobic residues located in the middle of the sequence and polar / charged residues adjacent to the cleavage site at the N-terminus of the mature polypeptide), other features are more characteristic of one cell type than the other. It is also known in the art that different signal sequences can greatly affect expression levels in mammalian cells, even if the sequences are all of mammalian origin (Hall et al., J of Biological Chemistry, 265: 19996-19999 (1990); Humphreys et al., Protein Expression and Purification, 20: 252-264 (2000)). For example, bacterial signal sequences typically have a positively charged residue (most commonly a lysine) immediately following the initiating methionine, whereas mammalian signal sequences do not necessarily have one. Although signal sequences capable of directing secretion in both cell types are known, such signal sequences typically direct high levels of protein secretion only in one cell type or the other.

[0061] Although bacterial signal sequences have only rarely been shown to exhibit any functionality in mammalian cells, signal sequences of mammalian origin capable of driving translocation into the bacterial periplasm have been reported (Humphreys et al., The Protein Expression and Purification, 20: 252-264 (2000)). However, the mere functionality of a signal sequence is not suitable for the robust dual expression system used for phage display and IgG expression. Rather, the selected signal sequence must function sufficiently in both expression systems for phage display, where low levels of display would impair the ability of the system to perform phage panning experiments.

[0062] The present invention is based in part on the discovery that signal sequences of non-bacterial origin function in prokaryotic cells at levels sufficient for selection of phage libraries without compromising expression of IgG in eukaryotic cells.

[0063] The present invention provides any signal sequence (including consensus signal sequences) that can be used to target a polypeptide of interest to the periplasm in prokaryotes and to the endoplasmic / plasmic reticulum in eukaryotes. Signal sequences that can be used include, but are not limited to, mouse binding immunoglobulin protein (mBiP) signal sequence (UniProtKB: Accession P20029), human growth hormone derived signal sequence (hGH) (UniProtKB: Accession BIA4G6), Gaussia princeps luciferase (UniProtKB: Accession Q9BLZ2), yeast endo-1,3-glucanase (yBGL2) (UniProtKB: Accession P15703). In one embodiment, the signal sequence is a natural or synthetic signal sequence. In a further embodiment, the synthetic signal sequence is an optimized signal secretion sequence that drives the level of display at an optimized level compared to a non-optimized natural signal sequence.

[0064] Suitable assays for determining the ability of a signal sequence to drive display of a polypeptide of interest in a prokaryotic cell include, for example, the phage ELISA described herein.

[0065] A suitable assay for determining the ability of a signal sequence to drive the display of a polypeptide of interest in eukaryotic cells includes, for example, transfection of cultured mammalian cells with a mammalian expression vector encoding the polypeptide of interest with a signal of interest, growing the cells for a period of time, harvesting supernatant from the cultured cells, and purifying IgG from the supernatant by affinity chromatography, as described herein.

[0066] B. Synthetic introns that result in host-dependent fusion protein expression from the same nucleic acid The present invention is based, in part, on the discovery that different Fab fusion proteins can be expressed from the same nucleic acid molecule in a host cell-dependent manner by exploiting the natural process of intron splicing that occurs during mRNA processing in eukaryotic cells but not in prokaryotic cells.

[0067] The genomic sequence of the HC constant region of hIgG1 contains three naturally occurring introns (Figure 3A), intron 1, intron 2, and intron 3. Intron 1 is a 391 base pair intron located between the HC variable domain / CH1 (VH / CH1) and the hinge region. Intron 2 is a 118 base pair intron located between the hinge region and CH2. Intron 3 is a 97 base pair intron located between CH2 and CH3.

[0068] The invention provides vectors that include intron 1 located between VH / CH1 and the hinge region. Other examples include intron 2 or intron 3 located between VH / CH1 and the hinge region. In some vectors, a nucleic acid encoding a coat protein or adaptor protein is inserted into an intron located between VH / CH1 and the hinge region, with the natural splice donor for the intron at its 5' end and the natural splice acceptor at its 3' end. Other examples include mutant splice donors with substitutions at positions 1 and 5 of the eight positions in the splice donor.

[0069] For example, phage ELISA can be used to analyze expression and secretion systems in prokaryotic cells.

[0070] For example, purification of IgG from culture supernatants using protein A and gel filtration chromatography can be used to analyze expression and secretion systems in eukaryotic cells. Furthermore, RT-PCR can be used to analyze splicing of HC cassettes containing synthetic introns in eukaryotic cells.

[0071] C. Vectors for the expression and secretion of polypeptides in prokaryotic and eukaryotic cells. Expression and secretion systems for the expression and secretion of Fab fusion proteins in prokaryotic and eukaryotic cells can be constructed using a variety of techniques that are within the skill of the art.

[0072] In one embodiment, the expression and secretion system comprises a vector comprising: (1) a mammalian promoter; (2) an LC cassette comprising (in 5' to 3' order): a bacterial promoter, a signal sequence, an antibody light chain sequence, and a control protein (gD); (3) a synthetic cassette comprising (in 5' to 3' order): a mammalian polyadenylation / transcription termination signal, a transcription terminator sequence for terminating transcription in prokaryotic cells, a mammalian promoter and a bacterial promoter for driving expression of the HC; (4) an HC cassette comprising a signal sequence and an antibody heavy chain sequence; (5) a second synthetic cassette comprising a mammalian polyadenylation / transcription termination signal and a transcription terminator sequence for terminating transcription in prokaryotic cells. The secretion signal sequence preceding the LC and HC may be the same signal sequence (e.g., a mammalian mBiP signal sequence) that functions in both prokaryotic and eukaryotic cells. In one embodiment, the antibody heavy chain sequence comprises a synthetic intron. The synthetic intron is located in the VH / CH1 domain (at its 5' end) and in the hinge region (at its 3' end). In one embodiment, the synthetic intron is flanked by an optimized splice donor sequence at the 5' end and the splice acceptor sequence of the native intron 1 at the 3' end. In one embodiment, the synthetic intron comprises a nucleotide sequence encoding a phage coat protein (e.g., pill) for direct fusion display (see FIG. 14) or an adapter protein fused at the nucleotide level to intron 1 for indirect fusion display (see FIG. 7). For indirect fusion display, the vector further comprises a separate bacterial expression cassette comprising (in 5' to 3' order) a bacterial promoter, a bacterial signal sequence, a phage coat protein (e.g., pill) with a partner adapter peptide fused at the nucleotide level to the N-terminus of the coat protein, and a transcription terminator sequence (see FIG. 7).Additionally, different embodiments of the above constructs are possible in which both the HC and LC are controlled by tandem mammalian and bacterial promoters (see FIG. 7), or in which only one (e.g., the HC) cassette is controlled by tandem mammalian and bacterial promoters while the other (e.g., the LC) cassette is controlled only by a bacterial promoter (see FIG. 14).

[0073] Additionally, the vector can include a bacterial origin of replication, a mammalian origin of replication, a nucleic acid encoding a polypeptide useful as a control (e.g., gD protein) or useful for such activities as protein purification, protein tagging, protein labeling (labeling with a detectable compound or composition, such as a label (e.g., a radioactive label, a fluorescent label or an enzyme label)).

[0074] In one embodiment, a mammalian promoter and a bacterial promoter and signal sequence are operably linked to the antibody light chain sequence, and a mammalian promoter and a bacterial promoter and signal sequence are operably linked to the antibody heavy chain sequence.

[0075] D. Selecting and Screening Antibodies Against the Antigen of Interest The present invention provides a method for screening and selecting antibodies against a protein of interest by phage or bacterial display of ab-based libraries, or optimizing existing antibodies by similar methods. The use of the dual vectors described above can be used for screening and selection of Fab fragments in prokaryotic cells, and for selection of Fabs that can be easily expressed as full-length IgG molecules for further testing without the need for subcloning.

[0076] Antibodies of the invention In a further aspect of the invention, the antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2In other embodiments, the antibody is a full length antibody, such as an intact IgG1, IgG2, IgG3 or IgG4 antibody, or other antibody class or isotype as defined herein.

[0077] In further aspects, the antibodies according to any of the above embodiments can incorporate any of the features, either alone or in combination, as described in Sections 1-7 below:

[0078] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10- 8 M or less, e.g., 10- 8 M to 10- 13 M, e.g., 10- 9 M to 10- 13 M).

[0079] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of the antibody of interest and its antigen, as illustrated by the following assay: 125 Solution binding affinity of Fab to antigen is measured by equilibrating Fab with I)-labeled antigen and capturing bound antigen with anti-Fab antibody-coated plates (see, for example, Chen, et al., J. Mol. Biol. 293:865-881 (1999)). To determine assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23° C.) for 2-5 hours. Non-adsorbent plates (Nunc #269620) are coated with 100 pM or 26 pM [ 125I] Antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al. (1997) Cancer Res. 57: 4593-4599). The Fab of interest is then incubated overnight; however, the incubation may take longer (e.g., approximately 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with PBS containing 0.1% polysorbate 20 (Tween 20®). Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20 TM Packard) and the plate was TOPCOUNT TM The mixture is counted for 10 minutes in a gamma counter (Packard). Concentrations of Fab that give 20% or less of maximal binding are selected for use in competitive binding assays.

[0080] According to another embodiment, Kd is measured using a surface plasmon resonance assay with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25° C. using an immobilized antigen CM5 chip with ∼10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (∼0.2 μM) in 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block non-reactive populations. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were incubated at 25° C. with a flow rate of approximately 25 μl / min in 0.05% polysorbate 20 (TWEEN 20 TM) in PBS containing a surfactant (PBST). Association rates (kon) and dissociation rates (koff) were calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. Equilibrium dissociation constants (Kd) were calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). When the binding rate by the surface plasmon resonance assay described above was 10 6 M- 1 s- 1 If the measurement exceeds 100 nm, a spectrometer, e.g., a stop-flow equipped spectrophometer (Aviv Instruments) or an 8000 series SLM-AMINCO equipped with a stirred cuvette, may be used. TM The binding rate can be measured using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass = 16 nm) of 20 nM anti-antigen antibody (Fab type) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured with a spectrophotometer (ThermoSpectronic).

[0081] 2. Antibody fragment In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. Fab and F(ab') fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives. 2 For a discussion of fragments, see US Pat. No. 5,869,046.

[0082] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0083] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516 B1).

[0084] Antibody fragments can be produced by a variety of techniques, including, but not limited to, digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phages), as described herein.

[0085] 3. Chimeric and humanized antibodies In some embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. The chimeric antibody includes an antigen-binding fragment thereof.

[0086] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0087] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al. al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0088] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from FR library screening (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Immunol., 151:2623 (1993)). et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0089] 4. Human antibodies In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0090] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or human variable regions or intact antibodies in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., XENOMOUSE, J. Med. 2003, 143:1117-1125 (2005). TM See also U.S. Patent Nos. 6,075,181 and 6,150,584, which describe HuMab® technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies generated in such animals can be further modified, for example, by combining with different human constant regions.

[0091] Human antibodies can be produced by hybridoma-based methods. Human myeloma and mouse-human heterologous cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0092] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences may then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0093] 5. Library-derived antibodies Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0094] In a given phage display method, repertoires of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol. 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0095] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0096] 6. Multispecific antibodies In some embodiments, the antibody provided herein is a multispecific antibody, e.g., a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In some embodiments, one of the binding specificities is for a first antigen, and the other is for any other antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of a first antigen. Bispecific antibodies can also be used to localize cytotoxic drugs to cells expressing the first antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0097] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1993)); (1994)); and by preparing trispecific antibodies, for example as described in Tutt et al. J. Immunol. 147: 60 (1991).

[0098] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576A1).

[0099] The antibodies or fragments herein also include "Dual Acting FAbs" or "DAFs" that contain antigen binding sites that bind to a first antigen as well as other, different antigens (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0100] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired properties, e.g., antigen binding.

[0101] a) Substitution, insertion, and deletion mutants In some embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Conservative Substitutions". More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are further described below with reference to classes of amino acid side chains. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0102] TIFF2024170391000003.tif165170

[0103] Amino acids can be divided into groups based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0104] Non-conservative substitutions would entail exchanging a member of one of these classes for another class.

[0105] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (such as a humanized or human antibody). Typically, the resulting variant selected for further study will have an altered (e.g., improved) certain biological property compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a certain biological activity (e.g., binding affinity).

[0106] Modifications (e.g., substitutions) can be made in the HVRs, e.g., to improve antibody affinity. Such changes can be made in "hot spots" of the HVRs, i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting from a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted.

[0107] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made within an HVR. Such modifications may be outside of HVR "hot spots" or SDRs. In certain embodiments of the variant VH or VL sequences provided above, each HVR is either unchanged or contains only one, two, or three amino acid substitutions.

[0108] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antigen with the antibody is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex identifies contact points between the antibody and the antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. The mutants can be screened to determine whether they contain the desired properties.

[0109] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to a polypeptide containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionine residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., in the case of ADEPT) or to a polypeptide which increases the serum half-life of the antibody.

[0110] b) Glycosylation Mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0111] If the antibody contains an Fc region, the sugars attached thereto can be varied. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can contain a variety of carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, as well as fucose, attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be made to generate antibody variants with certain improved properties.

[0112] In one embodiment, an antibody variant is provided with a glycostructure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the fucose content of such an antibody can be between 1% and 80%, between 1% and 65%, between 5% and 65%, or between 20% and 40%.

[0113] The amount of fucose is determined by calculating the average amount of fucose in the glycan of Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues), however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations of the antibody. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "non-fucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, U.S. Patent Application Publication No. 2003 / 0115614, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, and U.S. Patent Application Publication No. 2005 / 0132141. No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO 2005 / 053742, WO 2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing nonfucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Publication No. 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0114] The antibody variants further comprise bisected oligosaccharides, e.g., where a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairett et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0115] c) Fc region mutants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The variant Fc region may comprise the sequence of a human Fc region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0116] In certain embodiments, the present invention contemplates antibody variants that retain some, but not all, effector functions that make them desirable candidates for applications where in vivo antibody half-life is important, but where certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay techniques can be used (e.g., ACTI for flow cytometry). TMSee non-radioactive cytotoxicity assays (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., (USA) 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind body C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004). FcRn binding and in vivo clearance / half-life measurements can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0117] Antibodies with reduced effector function include those with substitutions in one or more of residues 238, 265, 269, 270, 297, 327, 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0118] Certain antibody variants with improved or diminished binding to FcRs have been described (e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0119] In certain embodiments, the antibody variant comprises one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU residue numbering) of the Fc region.

[0120] In some embodiments, modifications are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0121] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those having one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434 of the Fc region, e.g., a substitution at residue 434 of the Fc region (U.S. Patent No. 7,371,826).

[0122] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0123] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0124] e) Antibody derivative In certain embodiments, the antibodies provided herein can be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0125] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005). The radiation can be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells, but heat the non-protein moiety to a temperature that kills cells proximal to the antibody-non-protein moiety.

[0126] Recombinant methods and compositions Antibodies can be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided encoding an antibody described herein. Such a nucleic acid can encode an amino acid sequence comprising the VL of the antibody, and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In further embodiments, a host cell comprising such a nucleic acid is provided. In one embodiment, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese hamster ovary (CHO) cell, or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as described above, and, optionally, recovering the antibody from the host cell (or host cell culture medium).

[0127] For recombinant production of an antibody, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell, e.g., as described above. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the antibody heavy and light chains).

[0128] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibodies in E. coli.) After expression, the antibody is isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0129] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal or yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0130] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0131] Plant cell cultures can be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR ANTIBODY PRODUCTION IN TRANSGENIC PLANTS).TM See description of technology.

[0132] Vertebrate cells can also be used as hosts, for example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney lines (293 or 293T cells described in Graham et al., J. Gen Virol. 36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For an evaluation of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0133] Assay The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0134] 1. Binding and other assays In one embodiment, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, etc.

[0135] In another embodiment, a competitive assay can be used to identify antibodies that compete with the antibodies of the present invention for binding to an antigen of interest. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by the antibodies of the present invention. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0136] In a typical competitive assay, an immobilized antigen of interest is incubated in a solution containing a first labeled antibody (e.g., an antibody of the present invention) that binds to the antigen of interest, and a second unlabeled antibody that is to be tested for its ability to compete with the first antibody for binding to the antigen of interest. The second antibody may be present in a hybridoma supernatant. As a control, the immobilized antigen of interest is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to the antigen of interest, excess unbound antibody is removed and the amount of label bound to the immobilized antigen of interest is measured. If the amount of label bound to the immobilized antigen of interest is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to the antigen of interest. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0137] 2. Activity Assay In one embodiment, assays are provided to identify antibodies that have biological activity. Antibodies that have such biological activity in vivo and / or in vitro are also provided.

[0138] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0139] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0140] All patents and literature references cited herein are expressly incorporated herein by reference in their entirety. EXAMPLES

[0141] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that other various embodiments may be practiced.

[0142] M13KO7 helper phage was from New England Biolabs. Bovine serum albumin (BSA) and Tween 20 were from Sigma. Casein was obtained from Pierce. Anti-M13 conjugated horseradish peroxidase (HRP) was from Amersham Pharmacia. Maxisorp immunoplates were from NUNC. Tetramethylbenzidine (TMB) substrate was from Kirkegaard and Perry Laboratories. All other protein antigens were generated by research groups at Genentech.

[0143] Example 1: Selection of signal sequences for expression in prokaryotic and eukaryotic cells To address whether the vectors could express the protein of interest in both E. coli and eukaryotic (mammalian) cells, four signal sequences of non-bacterial origin were selected for which there is anecdotal evidence supporting the notion that they could function in mammalian cells. We tested these signal sequences for their ability to drive anti-Her2 (h4D5) Fab display on M13 phage using phage ELISA (Figure 1A). The level of display was assessed in comparison to the signal sequence of bacterial heat-stable enterotoxin II (STII). The ability of signal sequences to drive Fab phage display varied greatly, with one signal sequence, from mouse binding immunoglobulin protein (mBiP), driving a level of display that likely allowed efficient levels of Fab display.

[0144] To further improve the performance of the mBiP signal sequence, we utilized a phage-based codon optimization approach, since it has previously been demonstrated that the function of eukaryotic signal sequences in bacteria is strongly influenced by codon usage (Humphreys et al., The Protein Expression and Purification, 20: 252-264 (2000)). The mBiP signal sequence was expressed as h4D5 Fab in a standard phagemid vector. We constructed a phage library in which the mBiP signal peptide was fused to the N-terminus of HC. The DNA sequence of the mBiP signal peptide was diversified at the third base of each codon following the first two methionines, which allows only silent mutations. After four rounds of solid-phase panning on immobilized Her2, individual clones were picked and sequenced. We found that the consensus sequence of the selected clones strongly preferred adenine or thymine at the randomized positions, rather than guanine or cytosine. This result is highlighted by the fact that while 15 of the 17 codons in the sequence of wild-type mBiP contain a guanine or cytosine at the third base position, each of the 17 codons in the screened library contained an adenine or thymine at these positions 60-90% of the time. When tested in ELISA phage, the optimized mBiP signal sequence was able to bind h4D5 at a level comparable to the prokaryotic STII signal sequence. This suggests that the mBiP signal sequence can be utilized in place of the prokaryotic STII signal sequence for phage display and panning experiments, driving the display of Fabs without any apparent loss of capacity (Figure 1B).

[0145] The ability of the mBiP signal sequence to support the expression and secretion of IgG in mammalian cells was then assessed. h4D5 hIgG, each with the mBiP signal sequence fused to its N-terminus 1Mammalian expression vectors encoding the HC and LC of mBiP were cotransfected into 293S cells in suspension and grown for 5 days, after which the supernatant was harvested and IgG purified by affinity chromatography. Yields of IgG from 30 mL cultures were routinely around 2.0 mg, comparable to yields obtained using native HC signals (VHS) for both chains (data not shown). Interestingly, use of wild-type versus codon-optimized forms of the mBiP signal sequence had no discernible effect on IgG expression levels (data not shown). Gel filtration chromatography and mass spectrometry confirmed that the purified protein was >90% monomeric in solution and that the mBiP signal sequence was completely cleaved at the appropriate positions for both the HC and LC (data not shown). Since h4D5 is known to be a good expresser, we tested the performance of mBiP relative to VHS on a pool of unidentified clones randomly selected from phage panning experiments. The average yield from these clones was approximately 1.0 mg from 30 mL of suspension culture, and no significant difference was observed between the two signal sequences (FIGS. 2A and B).

[0146] In summary, the mBiP mammalian secretory signal sequence was capable of expressing IgG in mammalian cells at sufficient levels for screening, and also, once codon-optimized, was capable of driving robust Fab display on phage without compromising IgG expression levels.

[0147] Example 2: Expression of alternative Fab fusions in prokaryotic and eukaryotic cells To generate different Fab fusion proteins in a host cell-dependent manner, we sought to exploit the natural process of intron splicing that occurs during mRNA processing in eukaryotes but not in prokaryotes. 1 The genomic sequence of the HC constant region of V contains three naturally occurring introns (Figure 3A). The first of these (intron 1) is located in the HC variable domain (V H) and the hinge region. As assessed by RT-PCR and sequencing of the transcripts, the HC expression vector containing intron 1 and an optimized splice donor sequence expressed fully spliced ​​mRNA and, when co-transfected with the LC vector, produced IgG1 at levels comparable to the intron-free vector. 1 was expressed (Figure 5).

[0148] To determine whether a Fab fragment to a phage adaptor peptide embedded within the sequence of intron 1 would allow both display on phage and expression of IgG in bacterial and mammalian cells, respectively, an adaptor peptide (Figure 3B) or a phage coat protein gene III (Figure 3C) was inserted into the h4D5 HC.intron 1 construct at the 5' end of intron 1 or intron 3, whereas the natural splice donor from intron 1 or 3 was moved immediately upstream of the adaptor peptide. When the HC-adaptor.intron 1 construct was coexpressed with h4D5 LC in mammalian cells, expression of h4D5 IgG was approximately 40% (for the adaptor-containing intron) or approximately 5% (for the gene-III-containing intron) of that of the intron-free control construct (Figure 4A). RT-PCR demonstrated that a portion of the HC-adaptor mRNA was properly spliced ​​(Figure 4B, middle band) while the V H We demonstrated that a significant amount of mRNA was either not spliced ​​(Fig. 4B, upper band) or incorrectly spliced ​​(Fig. 4B, lower band) from the cryptic splice donor in the region. The mRNA of HC-gene-III was almost entirely spliced ​​from the cryptic splice donor. Silent mutations in the cryptic splice donor sequence only resulted in the accumulation of unspliced ​​mRNA (not shown).

[0149] In light of the failure of efficient splicing of the intron when adapter sequences were inserted, we compared the sequence of the natural splice donor to known consensus sequences of splice donors for mammalian mRNAs (Stephens et al., J of Molecular Biology, 228: 1124-1136 (1992)). As shown in FIG. 5, 1 The native splice donor from intron 1 of differs from the consensus donor sequence at three out of eight positions. Substitutions at positions 1 and 5 were analyzed further as these positions are more conserved than position 8. A mutant splice donor (donor 1) in which the bases at positions 1 and 5 were altered to match the consensus sequence (Figure 5A) was generated and the ability of these modified donors to mediate splicing of the synthetic intron in HC was tested. This optimized splice donor fully restored splicing of the synthetic intron (Figure 5B), with a concomitant increase in expression of h4D5 IgG to levels consistent with those of the intron-free control construct (Figure 5C). Improved splicing and IgG expression was observed whether the synthetic intron contained an adaptor peptide or gene III and also whether the synthetic intron was based on intron 1 or intron 3 of hIgG1.

[0150] Example 3: Generation of expression and secretion systems for prokaryotic and eukaryotic cells For the generation of the dual vector plasmid, we used pRS, a phagemid vector derived from pBR322 currently used for phage display. This bicistronic vector consists of a bacterial PhoA promoter driving the expression of an antibody light chain cassette with its associated STII signal sequence, followed by an antibody heavy chain cassette with its associated STII signal sequence. At the end of the light chain sequence is a gD epitope tag for detection of Fab display on the phage particle. In conventional phagemids, the heavy chain sequence is the V of hIgG H and C HIt consists of only one domain and is fused at the nucleotide level to a utility peptide such as gene III, which codes for a phage fusion protein, most often the phage coat protein pIII or an adapter peptide. The 3' ends of the light and heavy chain cassettes contain lambda transcription terminator sequences to stop transcription in E. coli. Since this vector produces light and heavy chain pIII from a single mRNA transcript, there are no transcriptional regulatory elements between the LC and HC sequences. The vector contains the β-lactamase (BLA) gene conferring ampicillin resistance, a pMB1 origin for replication in E. coli, and an f1 origin for expression of pili on the bacterial surface allowing infection by M13 phage. Another form of this vector also contains the SV40 origin of replication for episomal replication of the plasmid in an appropriate strain of mammalian cells.

[0151] For construction of the initial dual vector (referred to here as "pDV.6.0"), we first inserted the mammalian CMV promoter from pRK (a mammalian expression vector used for expression of IgG and other proteins) upstream of the phoA promoter driving the LC-HC cistron. At the end of the LC antibody coding sequence, we inserted an amber stop codon followed by a gD epitope tag, which allows detection of the tagged LC on the phage when displayed in an amber suppressor E. coli strain. The epitope tag is not present when the vector is expressed in mammalian cells. Thus, the LC cassette contains (in 5' to 3' order): the eukaryotic promoter, the bacterial promoter, the signal sequence, the antibody light chain (LC) coding sequence, and the epitope tag (gD).

[0152] Next, between the HC and LC cassettes, we inserted a synthetic cassette containing (from 5′ to 3′) the SV40 mammalian polyadenylation / transcription termination signal, a lambda terminator sequence for transcription termination in E. coli, the CMV promoter, and the PhoA promoter.

[0153] The SV40 mammalian polyadenylation / transcription termination signal and lambda terminator sequence were then inserted after the HC cassette, which contains the signal sequence and the antibody heavy chain (HC) coding sequence.

[0154] To allow for secretion of the fusion protein of interest in both prokaryotic and eukaryotic cells, we replaced the STII signal sequence in front of the LC and HC with the eukaryotic mouse binding immunoglobulin protein (mBiP) signal sequence. Screening of several candidate signal sequences led us to the discovery that this signal sequence could function in applications requiring prokaryotic expression (i.e., phage display) and / or eukaryotic expression (i.e., expression of IgG in mammalian cells), and that mBiP performed as well in both of these settings as each of the signal sequences used prior to this work had performed.

[0155] To enable expression of Fab phages in E. coli and IgG in mammalian cells, we generated a synthetic intron in the HC cassette. We modified the natural intron from intron 1 or intron 3 of human IgG1 to create a synthetic intron containing fusion protein (gene III) for display on phage particles. The genomic sequence of intron 1 (or intron 3) from human IgG1 was inserted immediately after the gene III sequence separated by a stop codon to generate Fab HC-p3 fusions in E. coli. The placement of the natural splice donor octanucleotide in the 5' flanking region of the synthetic intron required two amino acid mutations in the hinge region (E212G and P213K, Kabat numbering) when expressed in E. coli, and the mutations to create the optimized splice donor resulted in both of these residues being mutated to lysines. These mutations do not affect the level of display on the phage (not shown) and will not be present in full-length IgG expressed in mammalian cells, as the phage hinge region is removed during the splicing process.

[0156] For use in adapter phage display, we also generated a vector similar to the pDV6.0 vector described above, but with a different synthetic intron (called pDV5.0, shown in FIG. 7). The gene III sequence was replaced with one of the two members of a leucine zipper pair (herein called "adapter"). In this synthetic intron, the adapter peptide sequence is followed by a stop codon and the genomic sequence of intron 1 or 3. In this construct, we also inserted a separate bacterial expression cassette consisting of gene III fused to its cognate member of the leucine zipper pair. This separate bacterial expression cassette, introduced upstream of the LC CMV promoter and controlled by the PhoA promoter, contains the STII signal sequence that restricts expression of the adapter gene III to E. coli, and immediately downstream contains the lambda terminator. When expressed in E. coli, the heavy and light chains assemble in the periplasm to form Fab, and the adapter fused to the heavy chain stably binds to its cognate adapter on the pIII-adapter protein. Packaging of this assembled Fab-adapter pIII complex into a phage particle will yield a phage that displays the Fab of interest. In addition, we have generated a custom variant of the KO7 helper phage in which a partner adapter is fused to the N-terminus of gene III (adapter-KO7). Infection of E. coli harboring adapter-KO7 and pDV.5.0 will result in all copies of pIII present on the mature phagemid fused to the adapter. As a result, all copies of pIII that bind to the Fab adapter are available, rather than only those copies of pIII that originate from pDV5. However, sometimes a low level of display may be desirable when rare high affinity clones are sought (e.g., in affinity maturation applications). In this case, infection of E. coli harboring pDV.5.0 with the conventional KO7 helper phage will result in a mixture of adapter-pIII (from pDV.5.0) and wild-type pIII (from the KO7 helper phage) displayed on the phage particle.In this scenario, the resulting display levels will be lower than with the KO7 adaptor, since only a subset of the total pIII pool will be able to bind the adaptor-Fab.The ability to modulate the display levels simply by selecting the appropriate helper phage is a unique advantage of the present invention.

[0157] We evaluated the ability of pDV5.0 to express different IgGs in mammalian cells. The HC and LC from four different human IgGs were subcloned into pDV5.0 and expressed in 293 cells. Somewhat surprisingly, the total yield from pDV was consistently about 10-fold lower than from the two-plasmid system. However, the yield is still on the order of about 0.1-0.4 mg per 30 mL culture (Figure 6B). This amount of material is more appropriate for routine screening assays and can be easily scaled up to 0.1-1 L or more if larger amounts of material are required. The IgGs were shown to be >90% monomeric in solution by gel filtration chromatography.

[0158] Example 4: Construction of a mutant helper phage, M13KO7, carrying an amber mutation in gene-III (AmberKO7) To enhance the display of proteins fused to pIII on M13 phage, we used site-directed mutagenesis to generate a mutant helper phage, AmberKO7, which has an amber codon introduced into the M13KO7 helper phage genome by site-directed mutagenesis. The nucleotide sequence of pIII (nucleotides 1579 to 2853 of mutant helper phage AmberKO7) is shown in Figure 8.

[0159] To generate amber KO7, helper phage M13KO7 was transformed with E. coli strain CJ236 (genotype dut - / ung -) and progeny virions were harvested to purify ssDNA using a ssDNA purification kit (QIAGEN). A synthetic oligonucleotide (sequence 5'-GTGAATTATCACCGTCACCGACCTAGGCCATTTGGGAATTAGAGCCA-3') (SEQ ID NO: 23) was used to mutate gene-III of M13KO7 by oligonucleotide-directed site-directed mutagenesis. The mutagenized DNA was used to transform E. coli XL1-Blue cells (Agilent Technologies) and plated onto a lawn of uninfected XL1-Blue cells on soft agar plates. Plaques were individually picked and cells were grown in LB medium containing 50 μg / ml kanamycin. Double-stranded replicative form (RF) DNA was extracted using a DNA miniprep kit and sequenced to confirm the presence of amber stop mutations. The homogeneity of the population was confirmed by AvrII restriction endonuclease digestion of RF DNA and agar gel electrophoresis. All recombinant DNA manipulation steps were performed as described (Sambrook, J. et al., A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0160] The level of Fab display on phage particles generated with Amber KO7 was measured by phage ELISA. Antigen (Her2) was immobilized on immunoplates and phages bearing anti-Her2 Fab were generated in XL1-Blue cells using either wild type KO7 (WT KO7) or modified M13KO7 with amber mutation in pIII helper phage (Amber KO7). Binding was detected by incubation with mouse anti-M13-HRP conjugate followed by TMB substrate OD measurement at 450 nm. Use of Amber KO7 resulted in higher display levels from low display phagemids (closed triangles) compared to the levels achieved by the same phagemids when wild type KO7 was used for phage generation (closed squares) (Figure 9). The level of Fab display by low display phagemids using Amber KO7 (closed triangles) was also similar to the level of Fab display observed when using high display phagemids with wild type KO7 (open diamonds) (Figure 9).

[0161] Example 5: Creation of prokaryotic and eukaryotic expression and secretion systems for the generation of naive HC-only libraries and use of the systems for phage panning In addition to the direct and indirect fusion vectors featuring prokaryotic and eukaryotic promoters for both the HC and LC described in Example 3 (pDV5.0 and pDV6.0), we generated a modified direct fusion dual vector construct (pDV.6.5 shown in Figure 14) in which the Fab LC is fused to the STII signal sequence and driven solely by the bacterial PhoA promoter, while the Fab HC (containing gene III-synthetic intron sequences and hIgG Fc sequences for expression of full-length hIgG1 HC in mammalian cells) is driven by both the eukaryotic CMV promoter and the prokaryotic PhoA promoter. This construct was used to recreate a synthetic human Fab library previously described in which diversity was introduced only in the HC (Lee, et al., Journal of Molecular Biology, 340. 1073-1093 (2004)). Expression of full-length IgG from this vector requires the co-introduction of a mammalian expression vector encoding the LC.

[0162] A phage display library was generated using oligonucleotide-directed (Kunkel) mutagenesis and a "stop template" version of pDV.6.5 in which stop codons (TAA) were placed in all three heavy chain CDRs. These stops were repaired during a mutagenesis reaction with a mixture of oligonucleotides that annealed across the regions encoding CDRH1, H2 and H3 and replaced the codons at the positions selected for randomization with degenerate codons. The mutagenesis reaction was electroporated into XL1-Blue cells and the cultures were grown using a temperature shift protocol (4 h at 37°C, followed by 36 h at 30°C) in 2YT broth supplemented with Amber KO7 helper phage, 50 μg / ml carbenicillin and 25 μg / ml kanamycin. Phage were recovered from the cultures by precipitation with PEG / NaCl. Each electroporation reaction used approximately 5 μg of DNA and contained 1 × 10 8 -7×10 8 Transformants were obtained.

[0163] Panning of the naive phage library generated in this vector was performed against human vascular endothelial growth factor (VEGF). For selection of the phage library, the protein antigen was immobilized on a maxisorp immunoplate and the library was subjected to 4 to 5 rounds of binding selection. Wells were alternately blocked with BSA or casein in alternating rounds. Random clones selected through rounds 3 to 5 were assayed using phage ELISA to compare binding to the target antigen (VEGF) and an irrelevant protein (Her2) to check for non-specific binding. Briefly, phage clones were grown overnight in 1.6 mL of 2YT broth supplemented with amber.KO7 helper phage (Example 4). Supernatants were allowed to bind to plates coated with immobilized antigen or irrelevant protein for 1 h at room temperature. After washing, bound phages were detected using HRP-conjugated anti-M13 antibody (20 min at room temperature) followed by detection with TMB substrate. We isolated multiple clones that were ELISA positive for VEGF but not for an unrelated control protein (Her2) (Figure 10--bar graph).

[0164] DNA from those clones that demonstrated specificity for VEGF was then used to express full-length IgG by co-transfection of a mammalian expression vector encoding the consensus LC in 293 cells in small-scale suspension cultures for expression of full-length hIgG1. 1 mL cultures were transfected using Expifectamine or JetPEI according to the manufacturer's instructions and incubated at 37°C / 8% CO. 2 The cells were incubated at RT for 5–7 days. Scaled-up transfections were performed in 30 mL of 293 cells.

[0165] Culture supernatants were then used to screen IgG for VEGF binding in an Fc capture assay on a Biacore T100 instrument (Figure 11). IgG supernatants from 1 mL of culture were screened for antigen binding. Anti-human Fc capture antibody was immobilized on a series S CM5 sensor chip (10000RU). To allow capture of IgG from the supernatants (50-150RU), the supernatants were sequentially flowed over flow cells 2, 3 and 4 (5 μL / min for 4 min), after which antigen (100-1000 nM) was flowed over the immobilized IgG (30 μL / min for 2 min) and the binding response was measured.

[0166] Sequencing of the positive binders revealed eight unique sequences with positive binding properties (heavy chain CDR sequences are shown in Figure 12). The sequencing data (Figure 12) combined with phage ELISA (Figure 10) and Biacore data (Figure 11) were used to select a pool of eight anti-VEGF clones for further analysis.

[0167] Expression of these eight clones was scaled up to 100 mL Chinese Hamster Ovary (CHO) cell cultures (see Figure 15), and purified material was used to assess the ability of anti-VEGF clones to block VEGF binding to one of its cognate receptors (VEGFR1) via receptor blocking ELISA. Biotinylated hVEGF165 (2 nM) was incubated with anti-VEGF antibodies (upper limit concentration of 200 nM) serially diluted 3-fold in PBS / 0.5% BSA / 0.05% Tween-20. After 1-2 hours of incubation at room temperature, the mixture was transferred to a VEGFR1-immobilized plate and incubated for 15 minutes. VEGF bound to VEGFR-1 was then detected by streptavidin-HRP for 30 minutes, followed by development with TMB substrate, and IC50 values ​​were measured.

[0168] We identified one clone (VEGF55) with an IC50 comparable to that of the commercially available anti-VEGF antibody, bevacizumab (Figure 13). In this way, we were able to go directly from phage panning to IgG expression and triage the pool of clones down to a single candidate, all without the need to subclone.

[0169] In summary, this modified direct fusion double vector (pDV.6.5) could be used to construct a phage display library with randomized heavy and constant light chains in E. coli, and could also be used to subsequently express selected clones as native IgG1 in mammalian cells without subcloning when complemented with a light chain expression vector. Because the mammalian CMV promoter is upstream of only the HC, PDV expressed both Fab LC and Fab HC-pIII in E. coli, but only hIgG1HC in mammalian cells. This vector was used to select Fab fragments from a naive synthetic Fab library that bind multiple antigens, and then express full-length native hIgG1 from selected clones in mammalian 293 and CHO cells by co-introducing the modified direct fusion double vector clones with a mammalian expression vector encoding the common LC. Native IgG1 was derived from these expression experiments to run several assays, e.g., from a pool of 8 unique anti-VEGF clones showing binding activity by ELISA and Biacore, allowing us to triage the order down to a single candidate bore to evaluate the solution behavior, nonspecific binding, and biological activity of the IgG form of the candidates without the need to subclone the HC sequence from the original phage vector clone.

[0170] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are incorporated by reference in their entireties. SEQUENCE LISTING <110> GENENTECH, INC. ET AL. <120> EXPRESSION AND SECRETION SYSTEM <130> P4825R1-WO <140> <141> <150> 61 / 819,063 <151> 2013-05-03 <150> 61 / 852,483 <151> 2013-03-15 <150> 61 / 668,397 <151> 2012-07-05 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 418 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 1 Ala Glu Asp Ile Glu Phe Ala Ser Gly Gly Gly Ser Gly Ala Glu Thr 1 5 10 15 Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe Thr Asn 20 25 30 Val Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly 35 40 45 Cys Leu Trp Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr 50 55 60 Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn 65 70 75 80 Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu 85 90 95 Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile Pro Gly 100 105 110 Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly Thr Glu 115 120 125 Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu 130 135 140 Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln Gly Ala 145 150 155 160 Leu Thr Val Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pro Val Lys 165 170 175 Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Tyr Asp Ala 180 185 190 Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly Phe Asn 195 200 205 Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro 210 215 220 Gln Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 225 230 235 240 Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 245 250 255 Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp 260 265 270 Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala 275 280 285 Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val 290 295 300 Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser 305 310 315 320 Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn 325 330 335 Ser Gln Met Ala Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn 340 345 350 Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro 355 360 365 Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp 370 375 380 Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala 385 390 395 400 Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys 405 410 415 Glu Ser <210> 2 <211> 158 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala 1 5 10 15 Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu 20 25 30 Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly 35 40 45 Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly 50 55 60 Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln 65 70 75 80 Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr 85 90 95 Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe Gly 100 105 110 Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu 115 120 125 Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr 130 135 140 Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 145 150 155 <210> 3 <211> 19 <212> PRT <213> Mus sp. <400> 3 Met Met Lys Phe Thr Val Val Ala Ala Ala Leu Leu Leu Leu Gly Ala 1 5 10 15 Val Arg Ala <210> 4 <211> 57 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 4 atgatgaaat ttaccgtggt ggcggcggcg ctgctgctgc tgggcgcggt ccgcgcg 57 <210> 5 <211> 57 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> modified_base <222> (9)..(9) <223> a, c, t or g <220> <221> modified_base <222> (12)..(12) <223> a, c, t or g <220> <221> modified_base <222> (15)..(15) <223> a, c, t or g <220> <221> modified_base <222> (18)..(18) <223> a, c, t or g <220> <221> modified_base <222> (21)..(21) <223> a, c, t or g <220> <221> modified_base <222> (24)..(24) <223> a, c, t or g <220> <221> modified_base <222> (27)..(27) <223> a, c, t or g <220> <221> modified_base <222> (30)..(30) <223> a, c, t or g <220> <221> modified_base <222> (33)..(33) <223> a, c, t or g <220> <221> modified_base <222> (36)..(36) <223> a, c, t or g <220> <221> modified_base <222> (39)..(39) <223> a, c, t or g <220> <221> modified_base <222> (42)..(42) <223> a, c, t or g <220> <221> modified_base <222> (45)..(45) <223> a, c, t or g <220> <221> modified_base <222> (48)..(48) <223> a, c, t or g <220> <221> modified_base <222> (51)..(51) <223> a, c, t or g <220> <221> modified_base <222> (54)..(54) <223> a, c, t or g <220> <221> modified_base <222> (57)..(57) <223> a, c, t or g <400> 5 atgatgaant tnacngtngt ngcngcngcn ctnctnctnc tnggngcngt ncgngcn 57 <210> 6 <211> 37 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 6 Ala Ser Ile Ala Arg Leu Arg Glu Arg Val Lys Thr Leu Arg Ala Arg 1 5 10 15 Asn Tyr Glu Leu Arg Ser Arg Ala Asn Met Leu Arg Glu Arg Val Ala 20 25 30 Gln Leu Gly Gly Cys 35 <210> 7 <211> 37 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 7 Ala Ser Leu Asp Glu Leu Glu Ala Glu Ile Glu Gln Leu Glu Glu Glu 1 5 10 15 Asn Tyr Ala Leu Glu Lys Glu Ile Glu Asp Leu Glu Lys Glu Leu Glu 20 25 30 Lys Leu Gly Gly Cys 35 <210> 8 <211> 38 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 8 Glu Glu Lys Ser Arg Leu Leu Glu Lys Glu Asn Arg Glu Leu Glu Lys 1 5 10 15 Ile Ile Ala Glu Lys Glu Glu Arg Val Ser Glu Leu Arg His Gln Leu 20 25 30 Gln Ser Val Gly Gly Cys 35 <210> 9 <211> 38 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 9 Thr Ser Arg Leu Glu Gly Leu Gln Ser Glu Asn His Arg Leu Arg Met 1 5 10 15 Lys Ile Thr Glu Leu Asp Lys Asp Leu Glu Glu Val Thr Met Gln Leu 20 25 30 Gln Asp Val Gly Gly Cys 35 <210> 10 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> VARIANT <222> (1)..(2) <223> / replace=" " <220> <221> misc_feature <222> (1)..(2) <223> / note="This region may encompass 'Met' or 'Met-Thr' or be absent" <220> <221> misc_feature <222> (1)..(2) <223> / note="Variant residues given in the sequence have no preference with respect to those in the annotation for the variant position" <400> 10 Met Thr Met Lys Phe Thr Val Val Ala Ala Ala Leu Leu Leu Leu Gly 1 5 10 15 Ala Val Arg Ala 20 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> variation <222> (1)..(6) <223> / replace=" " <220> <221> misc_feature <222> (1)..(6) <223> / note="This region may encompass 'ATG' or 'ATGACC' or be absent" <220> <221> misc_feature <222> (1)..(6) <223> / note="Variant nucleotides given in the sequence have no preference with respect to those in the annotation for the variation position" <220> <221> modified_base <222> (12)..(12) <223> a, c, t or g <220> <221> modified_base <222> (15)..(15) <223> a, c, t or g <220> <221> modified_base <222> (18)..(18) <223> a, c, t or g <220> <221> modified_base <222> (21)..(21) <223> a, c, t or g <220> <221> modified_base <222> (24)..(24) <223> a, c, t or g <220> <221> modified_base <222> (27)..(27) <223> a, c, t or g <220> <221> modified_base <222> (30)..(30) <223> a, c, t or g <220> <221> modified_base <222> (33)..(33) <223> a, c, t or g <220> <221> modified_base <222> (36)..(36) <223> a, c, t or g <220> <221> modified_base <222> (39)..(39) <223> a, c, t or g <220> <221> modified_base <222> (42)..(42) <223> a, c, t or g <220> <221> modified_base <222> (45)..(45) <223> a, c, t or g <220> <221> modified_base <222> (48)..(48) <223> a, c, t or g <220> <221> modified_base <222> (51)..(51) <223> a, c, t or g <220> <221> modified_base <222> (54)..(54) <223> a, c, t or g <220> <221> modified_base <222> (57)..(57) <223> a, c, t or g <220> <221> modified_base <222> (60)..(60) <223> a, c, t or g <400> 11 atgaccatga anttnacngt ngtngcngcn gcnctnctnc tnctnggngc ngtncgngcn 60 <210> 12 <211> 37 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 12 Ala Ser Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln 1 5 10 15 Asn Tyr Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala 20 25 30 Gln Leu Gly Gly Cys 35 <210> 13 <211> 37 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 13 Ala Ser Ile Asp Glu Leu Gln Ala Glu Val Glu Gln Leu Glu Glu Arg 1 5 10 15 Asn Tyr Ala Leu Arg Lys Glu Val Glu Asp Leu Gln Lys Gln Ala Glu 20 25 30 Lys Leu Gly Gly Cys 35 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 14 Ala Gly Ser Cys 1 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 15 Cys Pro Pro Cys Pro Gly 1 5 <210> 16 <211> 57 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 16 atgatgaaat ttaccgttgt tgctgctgct ctgctacttc ttggagcggt ccgcgca 57 <210> 17 <211> 57 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 17 atgatgaaat ttactgttgt tgcggctgct cttctccttc ttggagcggt ccgcgca 57 <210> 18 <211> 57 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 18 atgatgaaat ttactgttgt cgctgctgct cttctacttc ttggagcggt ccgcgca 57 <210> 19 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 19 Met Thr Met Lys Phe Thr Val Val Ala Ala Ala Leu Leu Leu Leu Gly 1 5 10 15 Ala Val Arg Ala 20 <210> 20 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 20 Met Lys Phe Thr Val Val Ala Ala Ala Leu Leu Leu Leu Gly Ala Val 1 5 10 15 Arg Ala <210> 21 <211> 60 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> modified_base <222> (12)..(12) <223> a, c, t or g <220> <221> modified_base <222> (15)..(15) <223> a, c, t or g <220> <221> modified_base <222> (18)..(18) <223> a, c, t or g <220> <221> modified_base <222> (21)..(21) <223> a, c, t or g <220> <221> modified_base <222> (24)..(24) <223> a, c, t or g <220> <221> modified_base <222> (27)..(27) <223> a, c, t or g <220> <221> modified_base <222> (30)..(30) <223> a, c, t or g <220> <221> modified_base <222> (33)..(33) <223> a, c, t or g <220> <221> modified_base <222> (36)..(36) <223> a, c, t or g <220> <221> modified_base <222> (39)..(39) <223> a, c, t or g <220> <221> modified_base <222> (42)..(42) <223> a, c, t or g <220> <221> modified_base <222> (45)..(45) <223> a, c, t or g <220> <221> modified_base <222> (48)..(48) <223> a, c, t or g <220> <221> modified_base <222> (51)..(51) <223> a, c, t or g <220> <221> modified_base <222> (54)..(54) <223> a, c, t or g <220> <221> modified_base <222> (57)..(57) <223> a, c, t or g <220> <221> modified_base <222> (60)..(60) <223> a, c, t or g <400> 21 atgaccatga anttnacngt ngtngcngcn gcnctnctnc tnctnggngc ngtncgngcn 60 <210> 22 <211> 54 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> modified_base <222> (6)..(6) <223> a, c, t or g <220> <221> modified_base <222> (9)..(9) <223> a, c, t or g <220> <221> modified_base <222> (12)..(12) <223> a, c, t or g <220> <221> modified_base <222> (15)..(15) <223> a, c, t or g <220> <221> modified_base <222> (18)..(18) <223> a, c, t or g <220> <221> modified_base <222> (21)..(21) <223> a, c, t or g <220> <221> modified_base <222> (24)..(24) <223> a, c, t or g <220> <221> modified_base <222> (27)..(27) <223> a, c, t or g <220> <221> modified_base <222> (30)..(30) <223> a, c, t or g <220> <221> modified_base <222> (33)..(33) <223> a, c, t or g <220> <221> modified_base <222> (36)..(36) <223> a, c, t or g <220> <221> modified_base <222> (39)..(39) <223> a, c, t or g <220> <221> modified_base <222> (42)..(42) <223> a, c, t or g <220> <221> modified_base <222> (45)..(45) <223> a, c, t or g <220> <221> modified_base <222> (48)..(48) <223> a, c, t or g <220> <221> modified_base <222> (51)..(51) <223> a, c, t or g <220> <221> modified_base <222> (54)..(54) <223> a, c, t or g <400> 22 atgaanttna cngtngtngc ngcngcnctn ctnctnctng gngcngtncg ngcn 54 <210> 23 <211> 47 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 23 gtgaattatc accgtcaccg acctaggcca tttgggaatt agagcca 47 <210> 24 <211> 1275 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 24 gtgaaaaaat tattattcgc agttccttta gttgttcctt tctattctca ctcagctgag 60 actgttgaaa gttgtttagc aaaaccccat acagaaaatt catttactaa cgtctggaaa 120 gacgacaaaa ctttagatcg ttacgctaac tatgagggtt gtctgtggaa tgctacaggc 180 gttgtagttt gtactggtga cgaaactcag tgttacggta catgggttcc tattgggctt 240 gctatccctg aaaatgaggg tggtggctct gagggtggcg gttctgaggg tggcggttct 300 gagggtggcg gtactaaacc tcctgagtac ggtgatacac ctattccggg ctatacttat 360 atcaaccctc tcgacggcac ttatccgcct ggtactgagc aaaaccccgc taatcctaat 420 ccttctcttg aggagtctca gcctcttaat actttcatgt ttcagaataa taggttccga 480 aataggcagg gggcattaac tgtttatacg ggcactgtta ctcaaggcac tgaccccgtt 540 aaaacttatt accagtacac tcctgtatca tcaaaagcca tgtatgacgc ttactggaac 600 ggtaaattca gagactgcgc tttccattct ggctttaatg aggatccatt cgtttgtgaa 660 tatcaaggcc aatcgtctga cctgcctcaa cctcctgtca atgctggcgg cggctctggt 720 ggtggttctg gtggcggctc tgagggtggt ggctctgagg gtggcggttc tgagggtggc 780 ggctctgagg gaggcggttc cggtggtggc tctggttccg gtgattttga ttatgaaaag 840 atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc gctacagtct 900 gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat cgatggtttc 960 attggtgacg tttccggcct tgctaatggt aatggtgcta ctggtgattt tgctggctct 1020 aattcccaaa tggcctaggt cggtgacggt gataattcac ctttaatgaa taatttccgt 1080 caatatttac cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt tagcgctggt 1140 aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg tgtctttgcg 1200 tttcttttat atgttgccac ctttatgtat gtattttcta cgtttgctaa catactgcgt 1260 aataaggagt cttaa 1275 <210> 25 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 25 Thr Ser Tyr Ala 1 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 26 Gly Gly Ile Ser Pro Tyr Gly Gly Asn Thr Tyr 1 5 10 <210> 27 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 27 Ala Arg Pro Gly Pro Gly Gly Gly Phe Asp Ser Tyr Tyr Tyr Gly Met 1 5 10 15 Asp Tyr <210> 28 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 28 Thr Asp Tyr Ala 1 <210> 29 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 29 Gly Phe Ile Tyr Pro Tyr Ser Gly Asp Thr Tyr 1 5 10 <210> 30 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 30 Ala Arg Glu Val His Phe Trp Tyr Tyr Ser Val Met Asp Tyr 1 5 10 <210> 31 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 31 Ser Ser Tyr Gly 1 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 32 Gly Trp Ile Tyr Pro Asn Ser Gly Asn Thr Tyr 1 5 10 <210> 33 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 33 Ala Arg Phe Gly Tyr Asp Val Leu Arg Tyr Trp Asp Tyr Tyr Tyr Gly 1 5 10 15 Met Ala Tyr <210> 34 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 34 Ser Asn Thr Ser 1 <210> 35 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 35 Gly Trp Ile Tyr Pro Tyr Gly Gly Ser Thr Asn 1 5 10 <210> 36 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 36 Ala Arg Phe Gly Tyr Gln His Glu Val Gln Phe Ser Asp His Tyr Tyr 1 5 10 15 Ala Met Asp Tyr 20 <210> 37 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 37 Ser Gly Thr Tyr 1 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 38 Gly Phe Ile Ser Pro Tyr Asp Gly Tyr Thr Asp 1 5 10 <210> 39 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Ala Arg Leu Gln Phe Asn Thr Met Trp Val Met Asp Tyr 1 5 10 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Ser Ser Tyr Ala 1 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Gly Ser Ile Asn Pro Asn Ser Gly Tyr Thr Asn 1 5 10 <210> 42 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 42 Ala Arg Ile Gly Phe Gly Ser Leu Cys Phe Asp Cys Asn Leu Tyr Tyr 1 5 10 15 Gly Met Asp Tyr 20 <210> 43 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Ser Ser Thr Ala 1 <210> 44 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 44 Ala Gly Ile Thr Pro Tyr Ser Gly Asn Thr Tyr 1 5 10 <210> 45 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 45 Ala Arg Ile Gly Ser Gly Ser His Trp Ser Ala Phe Asp His Tyr Tyr 1 5 10 15 Ala Met Asp Tyr 20 <210> 46 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 46 Ser Ser Tyr Ala 1 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 47 Gly Ser Ile Asn Pro Asn Ser Gly Tyr Thr Asn 1 5 10 <210> 48 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 48 Ala Arg Thr Gly Phe Gly Gly Ile Val Val Asp Trp Ser Leu Tyr Tyr 1 5 10 15 Gly Met Asp Tyr 20

Claims

1. A nucleic acid molecule encoding a first polypeptide comprising a variable heavy domain (VH) VH-HVR1, VH-HVR2 and VH-HVR3 and / or a second polypeptide comprising a variable light domain (VL) VL-HVR1, VL-HVR2 and VL-HVR3, which nucleic acid molecule is functional in both prokaryotic and eukaryotic cells and further encodes a signal sequence encoded by a nucleic acid sequence operably linked to the first and / or second polypeptide sequence, and a full length antibody is expressed from the first and / or second polypeptide of the nucleic acid molecule.

2. A nucleic acid molecule encoding a variable heavy (VH) domain and a variable light (VL) domain, operably linked to the VH domain and / or the VL domain, comprising a prokaryotic and / or eukaryotic promoter enabling expression of the VH domain and / or the VL domain in prokaryotic and eukaryotic cells, wherein the VH domain and / or the VL is linked to a utility peptide when expressed by a eukaryotic cell, and the nucleic acid further encodes a signal sequence that is functional in both prokaryotic and eukaryotic cells.

3. The nucleic acid molecule of claim 1 , wherein the VL and VH are linked to a utility peptide.

4. The nucleic acid molecule of claim 3, wherein the VH is further linked to CH1 and the VL domain is linked to CL.

5. The nucleic acid molecule of claim 4 , wherein the utility peptide is selected from the group consisting of Fc, a tag, a label and a control protein.

6. The nucleic acid molecule of claim 5 , wherein the VL is linked to a control protein and the VH is linked to Fc.

7. The nucleic acid molecule of claim 6 , wherein the control protein is a gD protein or a fragment thereof.

8. The nucleic acid molecule of claim 1 , wherein the first and / or second polypeptide is fused to a coat protein or an adaptor protein.

9. The nucleic acid molecule of claim 8, wherein the nucleic acid molecule encoding the coat protein or adaptor protein is contained within a synthetic intron.

10. 10. The nucleic acid molecule of claim 9, wherein the synthetic intron is located between the nucleic acid encoding the VH domain and the nucleic acid encoding the Fc or hinge.

11. The nucleic acid molecule of claim 10 , wherein the synthetic intron comprises a nucleic acid encoding a naturally occurring intron from IgG1.

12. 12. The nucleic acid molecule of claim 11, wherein the naturally occurring intron is selected from the group comprising intron 1, intron 2 or intron 3 from IgG1.

13. 13. The nucleic acid molecule of claim 12, wherein the naturally occurring intron is intron 1 from IgG1.

14. The nucleic acid molecule of claim 8 , wherein the adapter protein is a leucine zipper.

15. The nucleic acid molecule of claim 8, wherein the adapter protein comprises the amino acid sequence of SEQ ID NO: 8, 9, 12, 13, 14 or 15.

16. 9. The nucleic acid molecule of claim 8, wherein the adaptor protein comprises an amino acid sequence having at least one amino acid sequence modification of SEQ ID NO: 8, 9, 12, 13, 14 or 15, and wherein the adaptor protein comprises a modification at at least one residue of SEQ ID NO: 8, 9, 12, 13, 14 or 15, and wherein the modification maintains or increases affinity of the adaptor protein to another adaptor protein.

17. 9. The nucleic acid molecule of claim 8, wherein the coat protein is selected from the group consisting of pI, pII, pIII, pIV, pV, pVI, pVII, pVIII, pIX and pX of bacteriophage M13, f1 or fd.

18. 18. The nucleic acid molecule of claim 17, wherein the coat protein is a pill protein or a fragment thereof.

19. 19. The nucleic acid molecule of claim 18, wherein the pill fragment is amino acids 267 to 421 or 262 to 418 of the pill protein.

20. 18. The nucleic acid molecule of claim 17, wherein a first fusion protein is expressed in a prokaryotic cell and a second fusion protein is expressed in a eukaryotic cell.

21. 21. The nucleic acid molecule of claim 20, wherein the first fusion protein and the second fusion protein are the same.

22. The nucleic acid molecule of claim 20 , wherein the first fusion protein and the second fusion protein are different.

23. The nucleic acid molecule of claim 22, wherein the first fusion protein is a Fab-phage fusion protein.

24. The nucleic acid molecule of claim 23, wherein the Fab-phage fusion protein comprises VH / CH1 fused to pill.

25. The nucleic acid molecule of claim 24, wherein the second fusion is a Fab-Fc or a Fab-hinge-Fc fusion protein.

26. The nucleic acid molecule of claim 25, wherein the Fab-Fc or Fab-hinge-Fc fusion protein comprises a VH / CH1 fused to an Fc.

27. The nucleic acid molecule of claim 1 , wherein the signal sequence directs secretion of the protein into the endoplasmic reticulum or outside the cell in a eukaryotic cell.

28. 2. The nucleic acid molecule of claim 1, wherein the signal sequence directs secretion of the protein into the periplasm or outside the cell in a prokaryotic cell.

29. 29. The nucleic acid molecule of claim 28, wherein the nucleic acid encoding the CH1 domain comprises a portion of a natural splice donor sequence.

30. The nucleic acid molecule of claim 28 , wherein the nucleic acid encoding the Fc comprises a portion of a natural splice acceptor sequence.

31. 30. The nucleic acid molecule of claim 28, wherein the nucleic acid encoding the CH1 domain comprises a portion of a modified splice donor sequence.

32. 32. The nucleic acid molecule of claim 31 , wherein the modified splice donor sequence comprises a modification of at least one nucleic acid residue, which modification increases splicing.

33. 9. The nucleic acid molecule of claim 8, wherein the adapter protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO: 7 (ASLDELEAEIEQLEEENYALEKEIEDLEKELEKLGGC).

34. The nucleic acid molecule of claim 2 , wherein the prokaryotic promoter is a phoA, Tac, Tphac or Lac promoter.

35. The nucleic acid molecule of claim 2 , wherein the eukaryotic promoter is CMV or SV40.

36. 29. The nucleic acid molecule of claim 27 or 28, wherein the signal sequence is encoded by a nucleic acid sequence that encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10 (XMKFTVVAAALLLLGAVRA, where X=0 amino acids or 1 or 2 amino acids (e.g., X=M or X=MT or X is not present).

37. 29. The nucleic acid molecule of claim 27 or 28, wherein the signal sequence is encoded by a nucleic acid sequence encoding mBIP and variants thereof.

38. 29. The nucleic acid molecule of claim 27 or 28, wherein the signal sequence is encoded by a nucleic acid sequence that encodes an amino acid sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from SEQ ID NO: 3 (mBIP amino acid sequence), and the signal sequence functions in both prokaryotic and eukaryotic cells.

39. 29. The nucleic acid molecule of claim 27 or 28, wherein the signal sequence is encoded by the nucleic acid sequence of SEQ ID NO: 11 (consensus mBIP sequence, X ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN, where N=A, T, C or G, where X=ATG, X=ATG ACC or X is not present).

40. 28. The nucleic acid molecule of claim 27, wherein the signal sequence is encoded by a nucleic acid sequence selected from the group of SEQ ID NO: 16 (Opt1), SEQ ID NO: 17 (Opt2) and SEQ ID NO: 18 (Opt3).

41. The nucleic acid molecule of claim 2 , wherein expression from a prokaryotic promoter occurs in a bacterial cell.

42. The nucleic acid molecule of claim 2, wherein expression by a eukaryotic promoter occurs in a mammalian cell.

43. 42. The nucleic acid molecule of claim 41, wherein the bacterial cell is an E. coli cell.

44. 43. The nucleic acid molecule of claim 42, wherein the mammalian cell is a yeast cell, a CHO cell, a 293 cell or an NSO cell.

45. 45. A vector comprising a nucleic acid molecule according to any one of claims 1 to 44.

46. A host cell transformed with the vector of claim 45.

47. The host cell of claim 46 , wherein the host cell is a bacterial cell.

48. 48. The host cell of claim 47, wherein the bacterial cell is an E. coli cell.

49. The host cell of claim 46 , wherein the host cell is a eukaryotic cell.

50. 50. The host cell of claim 49, wherein the eukaryotic cell is a yeast cell, a CHO cell, a 293 cell, or an NSO cell.

51. 47. A method for producing an antibody comprising culturing the host cell of claim 46 so that the nucleic acid is expressed.

52. 52. The method of claim 51, further comprising recovering the antibody expressed by the host cell.

53. 53. The method of claim 52, wherein the antibody is recovered from the host cell culture medium.

54. An adapter protein comprising an alteration of at least one residue in the amino acid sequence of SEQ ID NO: 8, 9, 12, 13, 14 or 15.

55. 55. The adapter protein of claim 54, wherein the adapter protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO: 7 (ASLDELEAEIEQLEEENYALEKEIEDLEKELEKLGGC).

56. 56. A nucleic acid molecule encoding an adapter protein according to claim 54 or 55.

57. An isolated nucleic acid molecule encoding an mBIP polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof, which nucleic acid molecule functions in both prokaryotic and eukaryotic cells.

58. An isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from SEQ ID NO:

3.

59. A bacterial cell expressing a mBIP polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof.

60. A method for expressing a mBIP polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a variant thereof in both prokaryotic and eukaryotic cells.

61. The nucleic acid molecule of claim 9 , wherein the synthetic intron is located between the nucleic acids encoding the CH2 and CH3 domains of the antibody.

62. The nucleic acid molecule of claim 9 , wherein the synthetic intron is located between the nucleic acid encoding the hinge region and the CH2 domain of the antibody.

63. A signal sequence comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof; a polypeptide comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH domain is linked to the N-terminus of the VL domain.

64. A signal sequence comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof; a polypeptide comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH domain is linked to the C-terminus of the VL domain.

65. A polypeptide comprising a signal sequence comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof, and variable heavy domains (VH) VH-HVR1, VH-HVR2, and VH-HVR3.

66. A polypeptide comprising a signal sequence comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof, and variable light domains (VL) VL-HVR1, VL-HVR2, and VL-HVR3.

67. A polypeptide comprising a signal sequence comprising the amino acid sequence of SEQ ID NO:3, or a variant thereof, variable heavy domains (VH) VH-HVR1, VH-HVR2 and VH-HVR3, and variable light domains (VL) VL-HVR1, VL-HVR2 and VL-HVR3.

68. 68. The polypeptide of any one of claims 63 to 67, wherein the polypeptide is an antibody or an antibody fragment.

69. 69. The antibody or antibody fragment of claim 1 or 68, wherein the antibody is selected from the group consisting of F(ab')2 and Fv fragments, diabodies, and single chain antibody molecules.

70. Nucleotide sequence of a helper phage containing an amber mutation in pill, which enhances the display of a protein fused to pill on the phage.

71. 71. The nucleotide sequence of claim 70, wherein the amber mutations are mutations at nucleotides 2613, 2614 and 2616 of the M13KO7 nucleic acid.

72. 72. The nucleotide sequence of claim 71, wherein the mutations at nucleotides 2613, 2614 and 2616 of the M13KO7 nucleic acid introduce an amber stop codon.

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