HK1204479B - Expression and secretion system - Google Patents

Description

Expression and secretion system

Cross-references to related applications

This application claims the benefit of priority to U.S. Provisional Application Nos. 61/668,397, filed on July 5, 2012, 61/852,483, filed on March 15, 2013, and 61/819,063, filed on May 3, 2013, all of which are incorporated herein by reference in their entirety.

Field of the Invention

The present invention relates to expression and secretion systems and methods for using the same for expressing and secreting a Fab fusion protein when nucleic acids are transformed into prokaryotic cells for phage display, and for expressing and secreting different or identical Fab fusion proteins when nucleic acids are transfected into eukaryotic cells for expression and purification. Also provided herein are nucleic acid molecules, vectors, and host cells comprising such vectors and nucleic acid molecules.

Background of the Invention

Phage display of polypeptides 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 library of multiple 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 to display different libraries of peptides or proteins (including antibody fragments such as Fab in the field of antibody research and development) on the surface of filamentous phage particles, and then select those that bind to a specific antigen of interest. Antibody fragments can be displayed on the surface of filamentous phage particles by fusing the gene for the antibody fragment to the gene for the phage coat protein, producing phage particles that display the encoded antibody fragment on their surface. This technology allows the isolation of antibody fragments with desired affinity for a variety of antigens from large phage libraries.

For phage-based antibody development, evaluating the properties of selected antibody fragments and their cognate IgG in functional assays (e.g., target binding, cell-based activity assays, in vivo half-life, etc.) requires reformatting the Fab heavy chain (HC) and light chain (LC) sequences into full-length IgG by isolating the DNA sequences encoding the HC and LC from the vector used for phage display and subcloning them 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 phage-based antibody development methods. Furthermore, once reformatted, since a significant proportion of selected Fabs do not perform satisfactorily in the initial screening assays, increasing the number of clones subjected to the reformatting/screening process will greatly increase the ultimate probability of success.

Herein, we describe the generation of an expression and secretion system for driving the expression of Fab-phage fusions when transformed into E. coli and for driving the expression of full-length IgG comprising the same Fab fragments when transfected into mammalian cells. We demonstrate that mammalian signal sequences from mouse binding immunoglobulin (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 78kd 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. Using mammalian mRNA splicing to remove a synthetic intron containing a phage fusion peptide inserted within the hinge region of human IgG ₁ HC, we can produce two different proteins in a host cell-dependent manner: Fab fragments fused to a linker peptide for phage display in E. coli and native human IgG ₁ in mammalian cells. This technology allows the selection of Fab fragments that bind to the antigen of interest from a phage display library and the subsequent expression and purification of homologous full-length IgG in mammalian cells without the need for subcloning.

SUMMARY OF THE INVENTION

In one aspect, the present invention is based in part on the experimental findings that (1) signal sequences of non-prokaryotic origin can function in prokaryotes and (2) different Fab fusion proteins (Fab-phage fusion proteins in prokaryotes and Fab-Fc fusion proteins in eukaryotic cells) can be expressed from the same nucleic acid molecule in a host cell-dependent manner when mRNA processing occurs in eukaryotic cells rather than prokaryotes. Thus, described herein are nucleic acid molecules and methods of use for bacterial expression and secretion of Fab fragments fused to phage particle proteins, coat proteins, or linker proteins for phage display when the nucleic acid is transformed into a prokaryotic host cell (e.g., E. coli), and Fab fragments fused to Fc when the nucleic acid is transformed into a eukaryotic cell (e.g., a mammalian cell), without the need for subcloning.

In one embodiment, the invention provides such a nucleic acid molecule, which encodes a first polypeptide comprising VH-HVR1, VH-HVR2 and 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, and wherein the nucleic acid molecule further encodes a signal sequence that functions in both prokaryotes and eukaryotic cells, and which is encoded by a nucleic acid sequence operatively linked to the first and/or second polypeptide sequence, and wherein 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 chain (VH) domain and a variable light chain (VL) domain. In another embodiment, the VH domain is connected to CH1, and the VL domain is connected to CL.

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

In another aspect, the present invention provides a nucleic acid molecule encoding a variable heavy chain (VH) domain and a variable light chain (VL) domain, and comprising a prokaryotic promoter and a eukaryotic promoter, said promoter being operably linked to the VH domain and/or VL domain to allow expression of the VH domain and/or VL domain in prokaryotic and eukaryotic cells, and wherein when expressed by eukaryotic cells, the VH domain and/or VL is linked to a utility peptide, and wherein the nucleic acid further encodes a signal sequence that functions in both prokaryotic and eukaryotic cells.

In one embodiment, VL and VH are linked to a utility peptide. In another embodiment, VH is also linked to CH1, and VL is linked to CL. The utility peptide is selected from Fc, a tag, a marker, and a control protein. In one embodiment, VL is linked to a control protein, and VH is linked to Fc. For example, the control protein is gD protein or a fragment thereof.

In even further embodiments, the first and/or second polypeptide of the invention is fused to a coat protein (e.g., pi, pII, pill, pIV, pV, pVI, pVII, pVIII, pIX, and pX of phage M13, f1, or fd, or a fragment thereof, such as amino acids 267-421 or 262-418 of the pill protein (unless otherwise indicated, as used herein, "pi,""pII,""pIII,""pIV,""pV,""pVI,""pVII,""pVIII,""pIX," and "pX" refer to the full-length protein or a fragment thereof)), or a linker protein (e.g., a leucine zipper protein or a polypeptide comprising SEQ ID NO: 12 (cJUN(R): ASIARL E E K VKTL K A Q NYEL A S T ANMLRE Q VAQLGGC) or SEQ ID NO: 13 (FosW(E): AS I DEL Q AE V EQLEE R NYAL R KE V EDL Q K Q A EKLGGC) or a variant thereof (modified amino acids in SEQ ID NO: 12 and SEQ ID NO: 13, including but not limited to those indicated by underline and bold), wherein the variant has an amino acid modification, wherein the modification retains or increases the affinity of the adaptor protein for another adaptor protein or a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO: 7 (ASLDELEAEIEQLEEENYALEKEIEDLEKELEKLGGC)), or a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 (GABA-R1: EEKSRLLEKE NRELEKIIAE KEERVSELRH QLQSVGGC) or SEQ ID NO: 9 (GABA-R2: TSRLEGLQSE NHRLRMKITE LDKDLEEV™ QLQDVGGC) or SEQ ID NO: 14 (Cys: AGSC) or SEQ ID NO: 15 (hinge: CPPCPG). The nucleic acid molecule encoding the coat protein or the 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 also includes a nucleic acid encoding a naturally occurring intron from IgG1, wherein the naturally occurring intron is selected from intron 1, intron 2, or intron 3 of IgG1.

In one embodiment, the invention provides such nucleic acid molecules, wherein in prokaryotic cell, the first fusion protein is to be expressed, and in eukaryotic cell, the second fusion protein is to be expressed.The first fusion protein and the second fusion protein can be identical or different.In other embodiments, the first fusion protein can be Fab-phage fusion protein (Fab-phage fusion protein that for example comprises the VH/CH1 that merges with pill), and the second fusion protein can be Fab-Fc or Fab-hinge-Fc fusion protein (Fab-Fc or Fab-hinge-Fc fusion protein that for example comprises the VH/CH1 that merges with Fc).

In one embodiment, the present invention provides nucleic acid molecules wherein the signal sequence directs protein secretion to the endoplasmic reticulum or extracellularly in eukaryotic cells and/or wherein the signal sequence directs protein secretion to the periplasm or extracellularly in prokaryotic cells. In addition, nucleic acid sequences that can encode a signal sequence include nucleic acid sequences encoding an amino acid sequence comprising SEQ ID NO: 10 (XMKFTVVAAALLLLGAVRA), wherein 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 deleted (SEQ ID NO: 20; MKFTVVAAALLLLGAVRA), or nucleic acid sequences encoding mBIP (SEQ ID NO: 4; ATG ATG AAA TTT ACC GTG GTG GCG GCG GCG CTG CTG CTG CTG GGC GCGGTC CGC GCG) and variants thereof, or nucleic acid sequences 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 is functional in both prokaryotes and eukaryotes, or nucleic acid sequences encoding mBIP (SEQ ID NO: 4; ATG ATG AAA TTT ACC GTG GTG GCG GCG GCG CTG CTG CTG CTG GGC GCGGTC CGC GCG) and variants thereof. NO: 11 (consensus mBIP sequence, X ATG AAN TTNACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN, wherein N＝A, T, C or G, wherein X＝ATG (SEQ ID NO: 5; ATG ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTNCTN GGN GCN GTN CGN GCN), X＝ATG ACC (SEQ ID NO: 21; ATG ACC ATG AAN TTN ACN GTNGTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN) or X＝is deleted (SEQ ID NO: 22; ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN), or a nucleic acid sequence selected from SEQ ID NO: 16 (mBIP. Opt 1: ATG ATG AAA TTT ACC GTT GTT GCT GCTGCT CTG CTA CTT CTT GGA GCG GTC CGC GCA), SEQ ID NO: 17 (mBIP.Opt2: ATG ATG AAATTT 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 GCGGTC CGC GCA) nucleic acid sequence.

In another embodiment, the synthetic intron in the nucleic acid molecule is flanked by nucleic acid encoding CH1 at its 5' end and nucleic acid encoding Fc at its 3' end. In addition, the nucleic acid encoding the CH1 domain comprises a portion of a native splice donor sequence, and the nucleic acid encoding Fc comprises a portion of a native splice acceptor sequence. Alternatively, the nucleic acid encoding the CH1 domain comprises a portion of a modified splice donor sequence, wherein the modified splice donor sequence comprises a modification of at least one nucleic acid residue, and wherein the modification increases splicing.

In one embodiment, the prokaryotic promoter is phoA, Tac, Tphac or Lac promoter and/or the eukaryotic promoter is CMV or SV40 or Moloney murine leukemia virus U3 region or caprine arthritis encephalitis virus U3 region or ovine demyelinating leukoencephalitis virus U3 region or a retroviral U3 region sequence. Expression from the prokaryotic promoter occurs in bacterial cells, and expression from the eukaryotic promoter occurs in mammalian cells. In another embodiment, the bacterial cell is Escherichia coli, and the eukaryotic cell is a yeast cell, a CHO cell, a 293 cell or a NSO cell.

In another embodiment, the present invention provides a vector comprising a nucleic acid molecule as described herein and/or a host cell transformed with such a vector. The host cell can 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 a NSO cell).

In another embodiment, the present invention provides a method for producing an antibody, comprising culturing a host cell as described herein to allow expression of the nucleic acid. The method further comprises recovering the antibody expressed by the host cell, and wherein the antibody is recovered from the host cell culture medium.

In one aspect, the present invention provides an adaptor protein comprising at least one modified 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 SEQ ID NO: 6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO: 7 (ASLDELEAEIEQLEEENYALEKEIEDLEKELEKLGGC). In one embodiment, the present invention provides a nucleic acid encoding such an adaptor protein.

In one aspect, the present invention provides nucleic acid molecules that function in both prokaryotic and eukaryotic cells, encoding an mBIP polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and variants thereof, or a polypeptide having an amino acid sequence with 85% homology to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the present invention provides methods for expressing an mBIP polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and variants thereof in both prokaryotic and eukaryotic cells. In one embodiment, the present invention provides bacterial cells that can express an mBIP sequence comprising the amino acid sequence of SEQ ID NO: 3 and variants thereof.

In one aspect, the invention provides synthetic introns located between nucleic acid encoding a VH domain and nucleic acid encoding an Fc or hinge of an antibody, between nucleic acid encoding the CH2 and CH3 domains of an antibody, or between nucleic acid encoding the hinge region and CH2 domain of an antibody.

In one aspect, the present invention comprises a polypeptide comprising a signal sequence comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof, a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH domain is linked to the amino terminus of the VL domain, or the polypeptide comprises a signal sequence comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof, a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH domain is linked to the carboxyl terminus of the VL domain, or the polypeptide comprises a signal sequence comprising the amino acid sequence of SEQ ID NO: 3 and VH-HVR1, VH-HVR2 and VH-HVR3 of the variable heavy chain domain (VH), or the polypeptide comprises a signal sequence comprising the amino acid sequence of SEQ ID NO: 3 and VL-HVR1, VL-HVR2 and VL-HVR3 of the variable light chain domain (VL), or the polypeptide comprises a signal sequence comprising the amino acid sequence of SEQ ID NO: 3 and VL-HVR1, VL-HVR2 and VL-HVR3 of the variable light chain domain (VL), or the polypeptide comprises a signal sequence comprising the amino acid sequence of SEQ ID NO: 3 The polypeptide of the present invention is an antibody or antibody fragment. The antibody or antibody fragment of the present invention can be selected from F(ab')2 and Fv fragments, diabodies and single-chain antibody molecules.

In one aspect, the present invention includes mutated helper phage for enhancing phage display of proteins. In one embodiment, the nucleotide sequence of the helper phage comprises an amber mutation in pill, wherein the helper phage comprising the amber mutation enhances phage display of proteins fused to pill. In another embodiment, in the nucleotide sequence of claim 70, the amber mutation is a mutation in nucleotides 2613, 2614, and 2616 of the M13KO7 nucleic acid. In an even further embodiment, the nucleotide sequence of claim 71, wherein the mutation in nucleotides 2613, 2614, and 2616 of the M13KO7 nucleic acid introduces an amber stop codon.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. (A) Her2 phage ELISA of purified phage displaying anti-Her2 Fabs under the control of four different eukaryotic signal sequences (mBiP, Gaussia princeps, yBGL2, hGH). The prokaryotic signal sequence of heat-stable enterotoxin II (STII), commonly used in phagemids, was used as a benchmark. (B) Phage display of anti-Her2 Fabs fused to the wild-type eukaryotic mBiP signal sequence (mBiP.wt) and codon-optimized versions obtained by phage library panning (mBiP.Opt1, mBiP.Opt2, and mBiP.Opt3 (SEQ ID NOs: 16-18)).

Figure 2. (A) Expression yields from 30 mL 293 cell suspension cultures of individual clones and (B) aggregate statistics analysis of hIgG1 clones expressed as fusions with either the eukaryotic mBiP or the prokaryotic native IgG HC (VHS) signal sequence.

Figure 3. (A) Genomic organization of human IgG1 HC containing three natural introns. Intron 1 occurs immediately before the hinge region. (B) HC construct containing a synthetic intron derived from intron 1 or 3 and containing a phage linker fusion peptide. The synthetic intron is flanked by a natural intron splice donor (D) and acceptor (A) from intron 1 or 3. (C) HC construct containing a synthetic intron derived 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 acceptor (A) from intron 1 or 3. Both constructs (B) and (C) contain a STOP codon at the 3' end of the linker peptide or phage coat protein sequence.

FIG4. (A) Expression levels of h4D5 IgG from constructs without introns, containing a synthetic intron with a phage linker peptide (see FIG3B ), or containing a synthetic intron with a phage coat protein (gene-III, see FIG3C ). (B) RT-PCR of hIgG1 HC from transfected cells. The predicted size of correctly spliced HC mRNA is 1,650 nt. The upper band in the linker + intron 1 construct represents unspliced pre-mRNA. The lower band in the linker- and gene-III-containing construct represents incorrect splicing in VH via a cryptic splice donor.

Figure 5. (A) Point mutations were generated in the native intron 1 splice donor to increase consensus splice donor identity for mammalian mRNAs. (B) Optimization of the intronic splice donor eliminated the accumulation of unspliced and incorrectly spliced HC mRNA and (C) increased expression in mammalian cells to levels observed in the absence of the intron.

Figure 6. (A) Modulated display using pDV.5.0 and wild-type KO7 (monovalent display) or linker KO7 (multivalent display). (B) Expression of four different mAbs from pDV.5.0 in three different mammalian cell lines.

Figure 7. Schematic diagram of a vector for expressing and secreting polypeptides in prokaryotic and eukaryotic cells. Synthetic introns can contain linker sequences or phage coat proteins, as well as any naturally occurring intron sequence from hIgG1. Both the HC and LC can have: 1) mammalian and bacterial promoters upstream of the ORF, 2) only bacterial promoters upstream of the ORF (see also Figure 14), or 3) only mammalian promoters upstream of the ORF. Constructs are shown in which both the HC and LC have two promoter types. The construct containing gene-III with a linker peptide fusion (shown in pDV5.0) is present only when the synthetic intron contains a linker peptide fusion, but not when the phage coat protein fusion is present in the synthetic intron.

Figure 8. The nucleotide sequence of pill of the helper phage Amber KO7 (nucleotides 1579 to 2853 (SEQ ID NO: 24)) was mutated to enhance display of proteins fused to pill on M13 phage. Amber KO7 has an amber codon introduced into the M13KO7 helper phage genome by site-directed mutagenesis. The underlined residues are mutations in nucleotides 2613, 2614, and 2616 (T2613C, C2614T, and A2616G) that introduce an amber stop (TAG) in codon 346 of gene III of M13KO7, and a silent mutation that introduces an AvrII restriction site in codon 345. Nucleotide 1 of M13KO7 is the third residue of a unique HpaI restriction site.

Figure 9. Enhanced display of Fab fragments on pill of M13 phage using the Amber KO7 helper phage. When wild-type M13KO7 was used for phage production, conventional high-display phagemids with wild-type M13KO7 (open diamonds) drove significantly higher Fab display levels than those achieved by low-display phagemid vectors (closed squares). The use of modified M13KO7 carrying an amber mutation in pill (Amber KO7) increased the display levels of the low-display phagemid (closed triangles) to those of the high-display phagemid with wild-type M13KO7 (open diamonds).

Figure 10 is a bar graph showing binding (measured by phage ELISA) to immobilized VEGF of clones selected from the naive dual-vector Fab-phage library of phage library sorting Example 5. After four rounds of selection, individual clones were picked, and phage supernatants were tested for binding to immobilized antigen (VEGF) and to an unrelated protein (Her2) to evaluate binding specificity.

Figure 11 shows selected phage clones screened in IgG format for antigen binding to VEGF as measured by Fc capture assay on a BIAcore T100 instrument. 96 clones selected for sequencing analysis and phage ELISA were transfected into 293S cells (1 mL) and cultured for 7 days for IgG expression. The supernatant was filtered through 0.2 μm and used to evaluate VEGF antigen binding by Fc capture assay on a BIAcore T100 instrument.

Figure 12 shows the sequences of positive binders from the VEGF panning experiment in Example 5. The heavy chain CDR sequences of eight clones are shown: VEGF50 (SEQ ID NOS 25-27, in order of appearance), VEGF51 (SEQ ID NOS 28-30, in order of appearance), VEGF52 (SEQ ID NOS 31-33, in order of appearance), VEGF59 (SEQ ID NOS 34-36, in order of appearance), VEGF55 (SEQ ID NOS 37-39, in order of appearance), VEGF60 (SEQ ID NOS 40-42, in order of appearance), VEGF61 (SEQ ID NOS 43-45, in order of appearance), and VEGF64 (SEQ ID NOS 46-48, in order of appearance). All clones shared the same light chain CDR sequences.

Figure 13 shows the ability of selected anti-VEGF IgGs from phage sorting against VEGF to inhibit the binding of VEGF to one of its natural receptors, VEGF-R1. Selected antibodies from the VEGF sort were expressed in CHO cells and purified IgG was used to measure the ability of selected clones to inhibit the binding of VEGF to VEGF-R1. One clone (VEGF55) inhibited VEGF-R1 binding with an IC50 3.5 times that of bevacizumab (Avastin).

Figure 14 shows the schematic diagram of the vector for expressing and secreting polypeptide in prokaryotic and eukaryotic cells, wherein the synthetic intron contains pill, and any naturally occurring intron sequence from hIgG1, and wherein LC has a bacterial promoter upstream of the ORF, and HC is both mammalian and bacterial promoters upstream of the ORF. Unlike the vector shown in Figure 7, this vector (pDV6.5) does not need an extra gIII box for fusion with phage particles. Proteins produced from expression in Escherichia coli and mammalian cells are shown in the vector schematic diagram below. The dashed line represents the intron in the heavy chain transcript spliced in mammalian cells. It should be noted that the sequence portion encoding the IgG1 hinge in this vector is repeated to allow it to be included in proteins expressed in Escherichia coli and mammalian cells.

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. The final yield of purified IgG is shown, as well as the score in the baculovirus ELISA for measuring nonspecific binding. Positive or negative binding of each clone in phage format (phage ELISA) or IgG format (BIAcore) is also shown.

Detailed description of embodiments of the present invention

I. Definition

As used herein, the term "synthetic intron" is used to define a nucleic acid segment between a nucleic acid encoding CH1 and a nucleic acid encoding hinge-Fc or Fc. A "synthetic intron" can be any nucleic acid that does not encode protein synthesis, any nucleic acid that encodes protein synthesis (e.g., phage particle proteins or coat proteins (e.g., pI, pII, pIII, pIV, pV, pVI, pVII, pVIII, pIX, pX) or linker proteins (e.g., leucine zippers, etc.)), or a combination thereof. In one embodiment, a "synthetic intron" comprises a portion of a splicing donor sequence and a splicing acceptor sequence that allow a splicing event. The splicing donor and splicing acceptor sequences allow a splicing event and can comprise a natural or synthetic nucleic acid sequence.

As used herein, the term "utility polypeptide" refers to a polypeptide that is used for several activities, such as protein purification, protein tagging, protein labeling (e.g., labeling with a detectable compound or composition (e.g., radiolabeled, fluorescently labeled, or enzymatically labeled)). The label can be indirectly conjugated to an amino acid side chain, an activated amino acid side chain, an antibody with a modified cysteine residue, or the like. For example, an antibody can be conjugated to biotin, and one of the three major types 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 label to the polypeptide variant, the polypeptide variant is conjugated to a small hapten (e.g., digoxigenin), and one of the different types of labels described above is conjugated to an anti-hapten polypeptide variant (e.g., an anti-digoxigenin antibody). Thus, indirect conjugation of the label to the polypeptide variant is achieved (Hermanson, G. (1996) in Bioconjugate Techniques Academic Press, San Diego).

When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is "operably linked". For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is effectively linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of the sequence, it is effectively linked to the coding sequence; if a ribosome binding site is located in a position that can assist translation, it is effectively linked to the coding sequence. In short, "operably linked" means that the DNA sequences that are linked are present in a nucleic acid molecule so that they can function as nucleic acids or in a functional relationship with each other as proteins expressed by them. They can be continuous or discontinuous. In the case of a secretory leader, they are generally continuous and in the reading phase. However, enhancers do not have to be continuous. Connection can be achieved by ligation at suitable restriction sites. If there is no such site, a synthetic oligonucleotide linker or connector can be used.

When a nucleic acid encoding a heterologous protein sequence (e.g., a VH or VL region) is directly inserted into a nucleic acid encoding a phage coat protein (e.g., pII, pVI, pVII, pVIII, or pIX), the VH or VL domain is "linked" to the phage. When introduced into a prokaryotic cell, phage can be produced in which the coat protein can display the VH or VL domain. In one embodiment, the resulting phage particle displays an antibody fragment fused to the amino or carboxyl terminus of the phage coat protein.

As used herein, the term "linked" or "linked" is intended to refer to two amino acid sequences or two nucleic acid sequences being covalently bound together via a peptide bond or a phosphodiester bond, respectively. The binding may include any number of additional amino acid or nucleic acid sequences between the two bound amino acid sequences or nucleic acid sequences. For example, the first and second amino acid sequences may be directly linked by a peptide bond, or one or more amino acid sequences may be linked between the first and second amino acid sequences.

As used herein, "linker" means an amino acid sequence of two or more amino acids in length. The linker can be composed of neutral polar or non-polar amino acids. The linker can be, for example, 2-100 amino acids in length, for example, a length between 2 and 50 amino acids, for example, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length. The linker can be "cleavable", for example, by self-cleavage or enzymatic or chemical cleavage. Cleavage sites in amino acid sequences and enzymes or chemicals that cut at these sites are well known in the art and are also described herein.

The term "signal sequence function" refers to the biological activity of a signal sequence directing the secretion of a protein into the ER (in eukaryotes) or the periplasm (in prokaryotes) or outside the cell.

As used herein, a "control protein" refers to a protein sequence whose expression is measured to quantify the display level of the protein sequence. For example, the protein sequence can be an "epitope tag" that can be readily purified by affinity purification of the VH or VL using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Examples of tag polypeptides and suitable respective antibodies thereof 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 antibody [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 T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87 : 6393-6397 (1990)].

As used herein, "coat protein" refers to one of the five capsid proteins that make up the phage particle, including pill, 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 pill protein or some variant, part, and/or derivative thereof. For example, the carboxyl terminal portion (cP3) of the M13 phage pill coat protein can be used, such as a sequence encoding the carboxyl terminal residues 267-421 of protein III of the M13 phage. In one embodiment, the pill sequence comprises SEQ ID NO: 1. NO: 1: (AEDIEFASGGGSGAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKP PEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNED PFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGD The amino acid sequence of VSGLANGNGATGDFAGSNSQMAVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES). In one embodiment, the pill fragment comprises the amino acid sequence of SEQ ID NO: 2 (SGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES).

As used herein, "adapter protein" refers to a protein sequence that specifically interacts with another adaptor protein sequence in solution. In one embodiment, the "adapter protein" comprises a heterotypic multimerization 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).

As used herein, a "heteromeric multimerization domain" refers to a modification or addition to a biomolecule that promotes heteromeric formation and discourages homomeric formation. Any heteromeric multimerization domain that has a strong preference for forming heterodimers relative to homodimers is within the scope of the present invention. Illustrative examples include, but are not limited to, U.S. Patent Application 20030078385 (Arathoon et al. - Genentech; describing knob into holes); WO2007147901 (et al. - Novo Nordisk: describing ionic interactions); WO 2009089004 (Kannan et al. - Amgen: describing electrostatic steering effects); WO2011/034605 (Christensen et al. - Genentech; describing coiled coils). See also, Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992) describing leucine zippers or Pack et al., Bio/Technology 11, 1271-1277 (1993) describing helix-turn-helix motifs. The phrases "heterotypic multimerization domain" and "heterotypic dimerization domain" are used interchangeably herein.

As used herein, the term "Fab-fusion protein" refers to a Fab-phage fusion protein in prokaryotes and/or a Fab-Fc fusion protein in eukaryotes. The Fab-Fc fusion can also be a Fab-hinge-Fc fusion.

Herein, the term "antibody" is used in its broad sense and includes various 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.

An "antibody fragment" refers to a molecule comprising a portion of an intact antibody, other than an intact antibody, that binds to 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 ; diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

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

"Framework" or "FR" refers to variable region residues other than the hypervariable region (HVR) residues. The variable region FR typically consists of four FR regions: FR1, FR2, FR3, and FR4. Thus, in a VH (or VL), the HVR and FR sequences typically appear in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies that have a structure substantially similar to a native antibody structure or have heavy chains that comprise an Fc region as defined herein.

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," which include the initially transformed cell and its derived progeny (regardless of the number of passages). Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as the screened or selected initially transformed cell are included herein.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable region that is highly variable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), which have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops are present in 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) are found at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35 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 in VH, CDRs typically include the amino acid residues that form the hypervariable loops. CDRs also include "specificity determining residues" or "SDRs," which are residues that contact the antigen. The SDRs are contained within the region of the CDRs and are referred to as "abbreviated-CDRs" or a-CDRs. Exemplary α-CDRs (α-CDR-L1, α-CDR-L2, α-CDR-L3, α-CDR-H1, α-CDR-H2, and α-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 indicated, HVR residues, as well as other variable region residues (e.g., FR residues), are numbered herein according to Kabat et al., supra.

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

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

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated antibody-encoding nucleic acid" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

As used herein, the term "monoclonal antibody" refers to the antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variant antibodies (such as variant antibodies occurring during the generation of naturally occurring mutations or monoclonal antibody preparations, such variant antibodies are generally present in less amounts), the individual antibodies included in the population are identical and/or in conjunction with the same epi-position. Contrary to polyclonal antibody preparations (which generally include different antibodies directed against different determinants (epi-positions)), each monoclonal antibody of a monoclonal antibody preparation is directly directed against a single determinant on the antigen. Therefore, the modifier "monoclonal" refers to the feature of the antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody used in accordance with the present invention can be prepared by various techniques, including but not limited to hybridoma method, recombinant DNA method, phage-display method, and the method using all or part of a transgenic animal containing human immunoglobulin locus, and other exemplary methods of such methods and for the preparation of polyclonal antibodies are described herein.

A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

"Natural antibodies" refer to naturally occurring immunoglobulin molecules with variable structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From amino to carboxyl terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from amino to carboxyl terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be designated as one of two types (called κ and λ).

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

The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al. Kuby Immunology, 6 ^ed ., WH Freeman and Co., page 91 (2007). A single VH or VL domain is sufficient to confer antigen binding specificity. In addition, VH and VL domains from antibodies that bind to an antigen can be used to screen libraries of complementary VL or VH domains, respectively, to isolate antibodies that bind to a specific antigen. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been 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."

II. Detailed Description

The phage-based antibody development method uses phage display technology to select Fab ^fragments with desired binding specificity from a large library of individual phage particles. In this method, established molecular biology techniques and specific phage display vectors are used to generate phage libraries consisting of Fab fragments fused directly or indirectly through one of the major coat proteins to M13 filamentous phage particles and containing varying complementarity determining regions (CDRs) (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)). Although the theoretical diversity of this library can easily exceed 10 ²⁵ unique sequences, practical limitations of phage library construction typically restrict the actual diversity to ≤ 10 ¹¹ clones for a given library (Sidhu et al., Methods in enzymology , 328:333-363 (2000)).

Assuming that the starting library can contain a basic number of unique sequences, the screening throughput of the selected clones is very important. For phage-based antibody research and development, the characteristics of the selected Fab and its cognate full-length IgG are thoroughly evaluated in functional assays (target binding, cell-based activity assays, in vivo half-life, etc.). It is necessary to reformat the Fab heavy chain (HC) and light chain (LC) sequences into full-length IgG by isolating the DNA sequences encoding HC and LC from the phagemid vector used for display and subcloning them into a mammalian expression vector for IgG expression. The laborious method of subcloning dozens or hundreds of selected HC/LC pairs represents the main bottleneck of the phage-based antibody research and development method. In addition, once reformatted, since a considerable proportion of the selected Fabs do not perform satisfactorily in the initial screening assay, increasing the number of clones subjected to the reformatting/screening method will greatly increase the ultimate probability of success.

Here, we describe the generation of an expression and secretion system for expressing and secreting one Fab fusion protein in prokaryotes and a different (or identical) Fab fusion protein in eukaryotic cells. For example, this 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 containing the same Fab fragment when transfected into mammalian cells. We demonstrate that a mammalian signal sequence from murine binding immunoglobulin (mBiP) ^8,9 can drive efficient protein expression in both prokaryotes and eukaryotic cells. Using mammalian mRNA splicing to remove a synthetic intron containing a phage fusion peptide inserted within the hinge region of human IgG ₁ HC, we can produce two different proteins in a host cell-dependent manner: Fab fragments fused to a linker peptide for phage display in E. coli and native human IgG ₁ in mammalian cells. This technology allows the selection of Fab fragments that bind to the antigen of interest from a phage display library and allows the subsequent expression and purification of homologous full-length IgG in mammalian cells without the need for subcloning.

The present invention is based in part on the following experimental findings: (1) signal sequences of non-bacterial origin can function in prokaryotes at levels sufficient for sorting phage libraries without impairing IgG expression in eukaryotic cells and (2) different Fab fusion proteins (Fab-phage fusion proteins in prokaryotes and Fab-Fc fusion proteins in eukaryotic cells) can be expressed from the same nucleic acid molecule in a host cell-dependent manner when mRNA processing occurs in eukaryotic cells rather than prokaryotes. Thus, described herein are expression and secretion systems and methods related to the construction and use of expression and secretion systems for expressing and secreting Fab fragments fused to phage particle proteins, coat proteins, or linker 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. In particular, described herein are vectors for expressing and secreting Fab-phage fusion proteins in prokaryotic cells and Fab-Fc fusion proteins in eukaryotic cells, nucleic acid molecules for expressing and secreting proteins or peptides in prokaryotic and eukaryotic cells, and host cells comprising such vectors. In addition, described herein are methods for using the expression and secretion systems, including methods for using the expression and secretion systems for screening and selecting new antibodies against a protein of interest.

Mode for Carrying Out the Invention

The practice of the present invention may 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 fully described in the literature, for example, in "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology", 4th edition (D.M. Weir & C.C. Blackwell, eds., Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", 4th edition (D.M. Weir & C.C. Blackwell, eds., Blackwell Science Inc., 1987); Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991).

Expression and secretion systems for prokaryotic and eukaryotic cells

The expression and secretion system for prokaryotic and eukaryotic cells involves a vector containing regulatory and coding sequences for a protein of interest (e.g., the heavy or light chain of an IgG molecule), wherein prokaryotic and eukaryotic promoters (e.g., CMV (eukaryotic) and PhoA (prokaryotic)) are arranged in tandem upstream of the gene of interest, and a single signal sequence drives expression of the protein of interest in both prokaryotic and eukaryotic cells. The present invention provides a means for this vector to produce two different fusion forms of the protein of interest in a host cell-dependent manner by using a synthetic intron located between the VH/CH1 and hinge-Fc regions of IgG1.

A. Signal sequences that function in prokaryotes and eukaryotes

One challenge in constructing vectors that can express target proteins in both prokaryotic (E. coli) and eukaryotic (mammalian) cells arises from the differences in signal sequences found in these cell types. Although some features of signal sequences are widely conserved in prokaryotic and eukaryotic cells (e.g., a small portion of hydrophobic residues is located in the middle of the sequence, and polar/charged residues are adjacent to the cleavage site at the amino terminus of the mature polypeptide), other features are different in most cases between different cell types. In addition, it is known in the art that different signal sequences have a significant effect on expression levels in mammalian cells, even if these sequences are 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 cell sequences are usually followed by a positively charged residue (most commonly lysine) after the initiator methionine, which is not typically found in mammalian signal sequences. Although there are known signal sequences that can direct secretion in both cell types, such signal sequences typically direct high-level secretion of a protein in only one cell type or the other.

Although bacterial signal sequences have rarely been shown to exhibit any function in mammalian cells, mammalian-derived signal sequences have been reported to drive transport into the bacterial periplasm (Humphreys et al., The Protein Expression and Purification, 20: 252-264 (2000)). However, simply having a functional signal sequence is not sufficient for a robust dual expression system for phage display and IgG expression. Instead, the selected signal sequence must be functional in both expression systems, particularly for phage display, where low levels of display may impair the system's ability to perform phage panning experiments.

The present invention is based in part on the discovery that signal sequences of non-bacterial origin can function in prokaryotes at levels sufficient for phage library sorting without impairing IgG expression in eukaryotic cells.

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 reticulum in eukaryotic cells. Signal sequences that can be used include, but are not limited to, the mouse binding immunoglobulin (mBiP) signal sequence (UniProtKB: accession P20029), the signal sequence from human growth hormone (hGH) (UniProtKB: accession BIA4G6), Gaussia princeps luciferase (UniProtKB: accession Q9BLZ2), and yeast endo-1,3-glucanase (yBGL2) (UniProtKB: accession P15703). In one embodiment, the signal sequence is a natural or synthetic signal sequence. In another embodiment, the synthetic signal sequence is an optimized signal secretion sequence that drives expression at an optimized level compared to its non-optimized natural signal sequence.

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

As described herein, suitable assays for determining the ability of a signal sequence to drive expression of a polypeptide of interest in eukaryotic cells include, for example, transfecting a mammalian expression vector encoding the polypeptide of interest with a signal of interest into cultured mammalian cells, culturing the cells for a period of time, collecting the supernatant from the cultured cells, and purifying IgG from the supernatant by affinity chromatography.

B. Synthetic introns leading to expression of host-dependent fusion proteins 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 rather than prokaryotes.

The genomic sequence of the hIgG1 HC constant region contains three native 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.

The present invention provides vectors comprising 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. For some vectors, nucleic acid encoding a coat protein or an adaptor protein is inserted into an intron located between VH/CH1 and the hinge region, with a natural splice donor for the intron at its 5' end and a natural splice acceptor at its 3' end.

Other examples include splice donors with mutations having substitutions at positions 1 and 5 in addition to position 8 of the splice donor.

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

For example, IgG purified from culture supernatants using protein A and gel filtration chromatography can be used to analyze expression and secretion systems in eukaryotic cells.

C. Vectors for 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 various techniques within the skill in the art.

In one aspect, the expression and secretion system comprises a vector comprising: (1) a mammalian promoter; (2) an LC cassette comprising (in order from 5' to 3') a bacterial promoter, a signal sequence, an antibody light chain sequence, a control protein (gD); (3) a synthetic cassette comprising (in order from 5' to 3') a mammalian polyadenylation/transcription termination signal, a transcription terminator sequence for terminating transcription in prokaryotes, a mammalian promoter and a bacterial promoter for driving HC expression; (4) an HC cassette comprising a signal sequence and an antibody heavy chain sequence; and (5) a second synthetic cassette comprising a mammalian polyadenylation/transcription termination signal and a transcription terminator sequence for terminating transcription in prokaryotes. The secretory signal sequence preceding the LC and HC can be the same signal sequence that functions in both prokaryotes and eukaryotic cells (e.g., a mammalian mBiP signal sequence). In one embodiment, the antibody heavy chain sequence comprises a synthetic intron. The synthetic intron is located between the VH/CH1 domain (at its 5' end) and 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 a native intron 1 splice acceptor sequence 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 Figure 14), or encoding an adaptor protein fused to intron 1 at the nucleotide level for indirect fusion display (see Figure 7). For indirect fusion display, the vector further comprises an isolated bacterial expression cassette comprising (in order from 5' to 3') a bacterial promoter, a bacterial signal sequence, a phage coat protein (e.g., pill) having a partner adaptor peptide fused to the amino terminus of the coat protein at the nucleotide level, and a transcription terminator sequence (see Figure 7). In addition, various embodiments of the above constructs are possible, wherein the HC and LC are controlled by mammalian and bacterial promoters in series (see Figure 7) or only one (e.g., HC) cassette is controlled by mammalian and bacterial promoters in series, while the other (e.g., LC) cassette is controlled only by bacterial promoters (see Figure 14).

In addition, the vector includes a bacterial origin of replication, a mammalian origin of replication, a nucleic acid encoding a protein used as a control (e.g., gD protein), or a protein for activities such as protein purification, protein tagging, protein labeling, for example, labeling with a detectable compound or composition (e.g., radiolabeling, fluorescent labeling, or enzymatic labeling).

In one embodiment, mammalian and bacterial promoters and signal sequences are operably linked to the antibody light chain sequence, and mammalian and bacterial promoters and signal sequences are operably linked to the antibody heavy chain sequence.

D. Selection and screening of antibodies against target antigens

The present invention provides a method for screening and selecting antibodies against a target protein by displaying Fab-based libraries on phage or bacteria, or by optimizing existing antibodies using similar methods. The dual vectors can be used to screen and select Fab fragments in prokaryotic cells, and selected Fabs can be readily expressed as full-length IgG molecules for further testing without the need for subcloning.

Antibodies of the present invention

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

In further aspects, the antibody according to any of the above embodiments may comprise any of the properties described in Sections 1-7 below, alone or in combination.

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).

In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) using a Fab version of the antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fab for antigen is measured by equilibrating Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen and then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-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., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to the capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 in PBS. When the plate is dry, 150 μl/well of scintillant (MICROSCINT-20 ^™ ; Packard) is added and counted for 10 minutes in a TOPCOUNT ^™ gamma counter (Packard). Concentrations of each Fab that produce less than or equal to 20% of maximal binding are selected for use in competitive binding assays.

According to another embodiment, immobilized antigen CM5 chip is used, with～10 response units (RU), uses or (BIAcore, Inc., Piscataway, NJ) at 25 ℃, uses surface plasmon resonance determination method to measure Kd.In brief, according to supplier's instructions, use N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and: N-hydroxysuccinimide (NHS) activation carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.).Before injecting with the flow rate of 5 μ l/ minute, with 10mM sodium acetate, pH 4.8, antigen is diluted to 5 μ g/ml (～0.2 μ M), to realize about 10 response units (RU) of coupling protein.After injecting antigen, 1M ethanolamine is injected, to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 μl/min in PBS with 0.05% polysorbate 20 (TWEEN-20 ^™ ) surfactant at 25°C. Association rates (k _on ) and dissociation rates (k off ) were calculated using a simple one-to-one Langmuir binding model (Evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K d ) was calculated as the ratio k _off /k _on . See, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the on-rate determined by the surface plasmon resonance assay described above exceeds 10 ⁶ M ^-1 s ^-1 , the on-rate can be determined using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen at 25°C, as measured using a stirred cuvette in a spectrophotometer, such as a flow-through equipped spectrophotometer (Aviv Instruments) or an 8000-series SLM-AMINCO ^™ spectrophotometer (ThermoSpectronic).

2. Antibody fragments

In certain 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, for example, Pluckthün, 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. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, 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).

Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable region or all or a portion of the light chain variable region of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

As described herein, antibody fragments can be prepared by a variety of techniques, including, but not limited to, hydrolytic digestion of intact antibodies, and production by recombinant host cells (eg, E. coli or phage).

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described in, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class switched" antibody, in which the class or subclass is changed from the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Generally, humanized non-human antibodies are used to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable regions, wherein HVR, such as CDR (or a portion thereof) are derived from non-human antibodies, and FR (or a portion thereof) are derived from human antibody sequences. Humanized antibodies optionally also include at least a portion of human constant regions. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from non-human antibodies (e.g., antibodies derived from HVR residues), to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for making them are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, e.g., 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., 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 the “guided selection” method of FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of the light or heavy chain variable regions of a particular subgroup of human antibodies (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 screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996))

4. Human Antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of 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).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that is modified to produce complete human antibodies or complete antibodies with human variable regions when responding to an antigen attack. Such animals typically contain all or part of a human immunoglobulin locus, all or part of which replaces an endogenous immunoglobulin locus, or all or part of which exists outside the chromosome or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patents 6,075,181 and 6,150,584 describing XENOMOUSE ^™ technology; U.S. Patent 5,770,429 describing technology; U.S. Patent 7,041,870 describing KM technology, and U.S. Patent Application Publication No. US 2007/0061900 describing technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, 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 produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing the 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).

Can also be by being selected from the Fv clone variable region sequence of the separation of people-derived phage display library, produce people's antibody.Then, can be by this type of variable region sequence and desired people's constant region combination.The technology for selecting people's antibody from antibody library is described below.

5. Antibodies derived from the library

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, by 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, by 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).

In some phage display methods, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994), the full repertoire of VH and VL genes is cloned respectively by polymerase chain reaction (PCR) and randomly combined in a phage library, which can then be screened for antigen binding. Phages typically display antibody fragments, single-chain Fv (scFv) fragments or Fab fragments. In the absence of the need to construct hybridomas, libraries from immune sources provide high-affinity antibodies to the immunogen. Alternatively, as described by Griffiths et al., EMBO J, 12: 725-734 (1993), a natural full repertoire (e.g., from humans) can be cloned to provide a wide range of non-self and autoantibodies to a single source of antibodies without any immunization. Finally, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), natural libraries can also be prepared by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and perform in vitro rearrangement. Patent literature describing human antibody phage libraries includes, for example: U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites. In certain embodiments, one binding specificity is for a first antigen, and the other binding specificity is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of a first antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing the first antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression 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). The electrostatic steering effect for preparing antibody Fc-heterodimer molecules can also be modified (WO2009/089004A1); cross-linking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using antibodies for preparing bispecific antibodies. Multispecific antibodies can be prepared using the "diabody" technology of synthesizing specific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "Octopus antibodies," see, for example, US 2006/0025576A1.

As used herein, antibodies or fragments also include "Dual Acting FAbs" or "DAFs," which comprise an antigen binding site that binds to a first antigen as well as a second, different antigen (see, eg, US 2008/0069820).

7. Antibody variants

In certain embodiments, it is contemplated that amino acid sequence variants of antibodies are provided herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or replacement residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and replacements may be performed to obtain the final construct, as long as the final construct possesses desired characteristics such as antigen binding.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVRs and FRs. In Table 1, conservative substitutions are shown under the heading "conservative substitutions." In Table 1, more substitution changes are provided under the heading "exemplary substitutions," and are further described below with reference to amino acid side chain types. Amino acid substitutions can be introduced into the target antibody and the product can be screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 1

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect chain direction: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

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

One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the variants selected for further study have modifications (e.g., improvements) (e.g., increased affinity, reduced immunogenicity) and/or certain biological properties of the parent antibody that are substantially retained relative to the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conventionally produced using, for example, affinity maturation techniques based on phage display as described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

Changes (e.g., replacements) can be made in HVR to improve antibody affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that undergo high-frequency mutations during the somatic maturation process) (see, e.g., Chowdhury, Methods Mol.Biol.207:179-196 (2008)) and/or SDR (a-CDR), and the resulting variant VH or VL binding affinity is tested. For example, Hoogenboom et al. have described affinity maturation by constructing and reselecting from a second library 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-guided mutagenesis). The second library is then produced. The library is then screened to identify any antibody variant with the desired affinity. Another approach to introducing diversity involves an HVR-guided approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues that participate in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

In certain embodiments, substitutions, insertions or deletions may occur in one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in the HVRs. Such changes may be outside the HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

As described in Cunningham and Wells (1989) Science, 244: 1081-1085, a useful method for identifying residues or regions of antibodies that can be targets for mutagenesis is called "alanine scanning mutagenesis". In this method, residues or target residue groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen will be affected. Additional replacements can be introduced at amino acid positions that initially exhibit functional sensitivity to replacement. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted and removed as candidates for replacement. Variants can be screened to determine whether they contain desired properties.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging from one residue to polypeptides of several hundred or more residues in length, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an amino-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the amino- or carboxyl-terminus of an antibody with an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of antibody glycosylation sites can be routinely achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When antibody comprises Fc district, the sugar that it is connected can be changed.Natural antibodies produced by mammalian cells usually comprise branched, bibranched oligosaccharides, which are usually connected to the Asn297 of the CH2 domain in the Fc district by N-connection.See, for example, Wright et al. TIBTECH15:26-32 (1997).Oligosaccharides can include multiple sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose that is connected with GlcNAc in the " stem " of bibranched oligosaccharide structure.In some embodiments, can carry out the modification of oligosaccharides in antibody of the present invention, to produce antibody variants with some improved characteristics.

In one embodiment, the antibody variants provided have a sugar structure lacking fucose connected (directly or indirectly) to the Fc region. For example, the amount of fucose in the antibody can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. As described in WO 2008/077546, the amount of fucose can be determined, for example, by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high mannose structures) connected to Asn 297 as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues); however, due to less sequence variation in antibodies, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants can improve ADCC function. See, for example, US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of literature related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; 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 defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. US 2003/0157108A1, Presta, L; and WO 2004/056312A1, Adams et al., particularly in Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (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).

Also provided are antibody variants having bisected oligosaccharides, for example, bibranched oligosaccharides connected to the Fc region of the antibody by bisecting with GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide connected to the Fc region. Such antibody variants have improved CDC function. Such antibody variants are described in, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc region variants

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 Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., replacement) at one or more amino acid positions.

In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions that make the antibody variant a desirable candidate for applications where the in vivo half-life of the antibody is important and certain effector functions (e.g., complement and ADCC) are unnecessary or disadvantageous. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/loss of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that regulate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression 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 for assessing ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 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 assays can be used (see, e.g., ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activity, 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 assays 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)).

Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including an Fc mutant designated "DANA" having residues 265 and 297 substituted with alanine ( U.S. Patent No. 7,332,581 ).

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

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) 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).

US2005/0014934A1 (Hinton et al.) describes 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)). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., an Fc variant having substitutions at Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

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 for other examples of Fc region variants.

d) Cysteine engineered antibody variants

In certain embodiments, it is desirable to produce cysteine-engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced with cysteine residues. In a specific embodiment, the replaced residues are present at accessible sites of the antibody. As further described herein, by replacing these residues with cysteine, active thiol groups are placed at accessible sites of the antibody and can be used for conjugation of the antibody with other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates. 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) in the heavy chain Fc region. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatizing 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly (N-vinyl pyrrolidone) polyethylene glycol, propropylene glycol copolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde has advantages in production. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can be varied, and if 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 specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used therapeutically under defined conditions, etc.

In another embodiment, a polymer of an antibody and a non-protein portion is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein portion 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 do not harm normal cells, but which can heat the non-protein portion to a temperature that can kill cells adjacent to the antibody-non-protein portion.

Recombinant methods and compositions

Antibodies can be produced using, for example, recombinant methods and compositions as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody as described herein is provided. Such nucleic acid can encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH (e.g., a light chain and/or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been 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 a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of producing an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of antibodies, nucleic acids encoding the antibodies, such as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. The nucleic acids can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibodies).

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

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

Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrates include plant and insect cells. Numerous baculovirus strains have been identified that can be used for conjugation with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also 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 (describing PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cells suitable for cultivation in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell lines (COS-7) transformed by SV40; human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A)); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT) 060562); TRI cells, e.g., as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 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 a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Assay

The antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.

1. Binding and other assays

In one aspect, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blot, and the like.

On the other hand, competition assays can be used to identify antibodies that compete with the antibodies of the present invention for binding to the antigen of interest. In certain embodiments, the competing antibodies bind to the same epitope (e.g., linear or conformational epitope) that the antibodies of the present invention bind to. Detailed exemplary methods for mapping epitopes that are bound to antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competitive assay, an immobilized antigen of interest is incubated in a solution comprising a first labeled antibody (e.g., antibody of the present invention) that is bound to the antigen of interest and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to the antigen of interest. The second antibody can be present in a hybridoma supernatant. As a control, the immobilized antigen of interest is incubated in a solution comprising the first labeled antibody but not comprising the second unlabeled antibody. After incubation under conditions allowing the first antibody to bind to the antigen of interest, excess unbound antibody is removed, and the amount of the labeling associated with the immobilized antigen of interest is measured. If the amount of the labeling associated with the immobilized antigen of interest is significantly reduced in the test sample relative to the control sample, it is represented that the second antibody competes with the first antibody for binding to the antigen of interest. Referring to Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity Assay

In one aspect, assays are provided for identifying antibodies having biological activity. Antibodies having the biological activity in vivo and/or in vitro are also provided.

In certain embodiments, the antibodies of the invention are tested for this biological activity.

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

All patent and literature references cited in this specification are hereby incorporated by reference in their entirety.

III. Examples

The following are examples of methods and compositions of the present invention.It is understood that various other embodiments may be practiced, given the general description provided above.

M13KO7 helper phage was from New England Biolabs. Bovine serum albumin (BSA) and Tween 20 were from Sigma. Casein was from Pierce. Anti-M13 conjugated to 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 the research group at Genentech, Inc.

Example 1: Selection of signal sequences for expression in prokaryotic and eukaryotic cells

In order to illustrate whether the vector can express the target protein in both Escherichia coli and eukaryotic (mammalian) cells, 4 signal sequences of non-bacterial origin were selected. For these 4 signal sequences of non-bacterial origin, there is no control evidence to support the view that they can function in mammalian cells. We used phage ELISA to test the ability of these signal sequences to drive anti-Her2 (h4D5) Fab to be displayed on M13 phage (Figure 1A). Relative to the bacterial heat-stable enterotoxin II (STII) signal sequence, the level of display was evaluated. The ability of signal sequences to drive Fab-phage display varies greatly, and a signal sequence from mouse binding immunoglobulin (mBiP) has driven a display level that can allow effective level Fab display.

In order to further improve the performance of the mBiP signal sequence, we used a phage-based codon optimization method because it has been shown that the function of eukaryotic signal sequences in bacteria can be greatly affected by codon selection (Humphreys et al., The Protein Expression and Purification, 20: 252-264 (2000)). A phage library was constructed in which the mBiP signal sequence was fused to the amino terminus of h4D5Fab HC in a standard phagemid vector. The DNA sequence of the mBiP signal sequence was diversified in the third base of each codon after the first two methionines that only allow silent mutations. After 4 rounds of solid phase panning for immobilized Her2, each clone was selected and sequenced. We found that the consensus sequence of the selected clones strongly favored adenine or thymine at the randomized position, rather than guanine or cytosine. This result is underscored by the fact that 15 of the 17 codons in the wild-type mBiP sequence contain a guanine or cytosine at the third base position, but in the sorted libraries, each of the 17 codons contained an adenine or thymine at these positions 60-90% of the time. When tested in a phage ELISA, the optimized mBiP signal sequence drove h4D5 Fab display at levels comparable to the prokaryotic STII signal sequence, indicating that the mBiP signal sequence can replace the prokaryotic STII signal sequence for phage display and panning experiments without significantly reducing performance.

Next, the ability of the mBiP signal sequence to support IgG expression and secretion in mammalian cells was evaluated. Mammalian expression vectors encoding the HC and LC of _h4D5hIgG1 (each with the mBiP signal sequence fused to the amino terminus) were co-transfected into suspended 293S cells and cultured for 5 days. The supernatant was then collected and the IgG purified by affinity chromatography. The IgG yield in a 30 mL culture was routinely ~2.0 mg, comparable to the yield obtained using the native HC signal (VHS) for both chains (data not shown). Interestingly, the use of wild-type versus codon-optimized versions 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 in solution was >90% monomeric and that the mBiP signal sequence was completely cleaved at the appropriate positions on both the HC and LC (data not shown). Since h4D5 is known to be a good expressor, we tested the performance of mBiP against VHS on a library of uncharacterized clones randomly selected from phage panning experiments. The average yield of these clones was -1.0 mg in 30 mL suspension culture, and no significant differences were observed between the two signal sequences (Fig. 2A and B).

In summary, the mBiP mammalian secretion signal sequence can express IgG in mammalian cells at levels sufficient for selection and, once codons are optimized, can also drive strong Fab display on phage without compromising IgG expression levels.

Example 2: Expression of altered Fab fusions in prokaryotic and eukaryotic cells

To produce different Fab-fusion proteins in a host cell-dependent manner, we sought to exploit a natural process for intron splicing that occurs during mRNA processing in eukaryotic cells rather than prokaryotes. The genomic sequence of the hIgG ₁ HC constant region contains three natural introns ( FIG3A ). The first of these introns (intron 1) is a 384-base pair sequence located between the HC variable domain ( _VH ) and the hinge region. HC expression vectors containing intron 1 and an optimized splice donor sequence expressing fully spliced mRNA were evaluated by RT-PCR and sequencing transcripts, and when co-transfected with LC vectors, IgG ₁ was expressed at levels comparable to vectors without introns ( FIG5 ).

To determine whether Fab fragments fused to a phage linker peptide embedded within the intron 1 sequence allow both phage display in bacteria and IgG expression in mammalian cells, respectively, a linker peptide ( FIG3B ) or phage coat protein gene III ( FIG3C ) was inserted into the h4D5HC intron 1 construct at the 5′ end of intron 1 or intron 3. The natural splice donor from intron 1 or 3 was removed immediately upstream of the linker peptide. When the HC-linker.intron 1 construct was co-expressed with h4D5LC in mammalian cells, h4D5 IgG expression was approximately 40% (for linker-containing introns) or 5% (for gene-III-containing introns) of the h4D5 IgG expression from a control construct without an intron ( FIG4A ). RT-PCR showed that although the HC-linker mRNA portion was correctly spliced (Figure 4B, middle band), a significant amount of mRNA was either not spliced (Figure 4B, upper band) or incorrectly spliced from the cryptic splice donor in the _VH region (Figure 4B, lower band). The HC-gene-III mRNA was almost completely spliced from the cryptic splice donor. Silent mutations in the cryptic splice donor sequence resulted in only the accumulation of unspliced mRNA (not shown).

Given the failure to efficiently splice introns when linker sequences were inserted, we compared the sequences of the natural splice donors with the known consensus sequences of mammalian mRNA splice donors (Stephens et al., J of Molecular Biology, 228: 1124-1136 (1992)). As shown in FIG5 , the natural splice donor from hIgG ₁ intron 1 differs from the consensus donor sequence at 3 of 8 positions. Substitutions at positions 1 and 5 were further analyzed because these positions are more conserved than position 8. A mutant splice donor (donor 1) was generated in which the bases at positions 1 and 5 were changed to match the consensus sequence ( FIG5A ), and the ability of these modified donors to mediate splicing of synthetic introns in HC was tested. This optimized splice donor fully restored splicing of the synthetic intron ( FIG5B ), accompanied by an increase in h4D5IgG expression to a level matching that of the control construct without the intron ( FIG5C ). Improvements in splicing and IgG expression were observed regardless of whether the synthetic intron contained a linker peptide or gene-III, and regardless of whether the synthetic intron was based on hIgG1 intron 1 or intron 3.

Example 3: Generation of expression and secretion systems for prokaryotic and eukaryotic cells

For the production of dual-vector plasmids, we used a pBR322-derived phagemid vector, pRS, currently used for phage display. This bicistronic vector consists of the bacterial PhoA promoter driving expression of the antibody light chain cassette and its associated STII signal sequence, as well as the antibody heavy chain and its associated STII sequence. At the terminus of the light chain sequence is a gD epitope tag for detection of Fabs displayed on the phage particle. In conventional phagemids, the heavy chain sequence consists solely of the _VH and _CHI domains of hIgG and is fused at the nucleotide level to a utility peptide, such as a phage fusion protein, most commonly gene encoding the phage coat protein pill, or a linker peptide. The 3' ends of the light and heavy chain cassettes contain a lambda transcriptional terminator sequence for transcription termination in E. coli. Because this vector produces the light and heavy chain pills from a single mRNA transcript, no transcriptional regulatory elements are present between the LC and HC sequences. The vector also contains a β-lactamase (bla) gene to confer ampicillin resistance, the pMB1 origin of replication in E. coli, and the f1 origin for bacterial surface expression of pillus, allowing infection by M13 phage. Another form of the vector also includes the SV40 origin of replication for episomal replication of the plasmid in a suitable mammalian cell line.

To construct the initial binary vector (designated "pDV.6.0"), we first inserted the mammalian CMV promoter from pRK (a mammalian expression vector for IgG and other proteins) upstream of the PhoA promoter driving the LC-HC cistron. At the end of the sequence encoding the LC antibody, we inserted an amber stop codon before the gD epitope tag, allowing detection of the tagged LC on phage when displayed in an E. coli strain with an amber suppressor. When this vector is expressed in mammalian cells, the epitope tag is absent. Thus, the LC cassette contains (in 5' to 3' order) a eukaryotic promoter, a bacterial promoter, a signal sequence, a sequence encoding the antibody light chain (LC), and an epitope tag (gD).

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

Next, the SV40 mammalian polyadenylation/transcription termination signal and lambda terminator sequence are inserted after the HC cassette. The HC cassette contains a signal sequence and a sequence encoding the antibody heavy chain (HC).

To allow secretion of the fusion protein of interest in both prokaryotic and eukaryotic cells, we replaced the STII signal sequence preceding the LC and HC with the eukaryotic murine binding immunoglobulin (mBiP) signal sequence. Screening of several candidate signal sequences led us to the discovery that this signal sequence is functional in applications requiring prokaryotic expression (i.e., phage display) and/or eukaryotic expression (i.e., expression of IgG in mammalian cells), and that mBiP in these settings performed as well as the respective signal sequences used prior to this work.

To allow for expression of Fab-phage in E. coli and IgG in mammalian cells, we created a synthetic intron within the HC cassette. We modified the native introns from either intron 1 or intron 3 of human IgG1 to create synthetic introns containing fusion proteins (gene-III) for display on phage particles. Genomic sequence from intron 1 (or intron 3) of human IgG1 was inserted immediately following the gene-III sequence, separated by a stop codon, to produce Fab HC-p3 fusions in E. coli. When expressed in E. coli, placement of the native splice donor octanucleotide at the 5' flanking region of the synthetic intron necessitated two amino acid mutations in the hinge region (E212G and P213K, Kabat numbering), and mutations that generated optimized splice donors resulted in both of these residues being mutated to lysine. These mutations did not affect the levels of display on phage (not shown), and since the phage hinge region is removed during the splicing process, it is absent in full-length IgG expressed in mammalian cells.

Alternatively, for the use of linker phage display, we have produced a vector (referred to as pDV5.0 in this article, shown in Figure 7) with different synthetic introns similar to the above-mentioned pDV6.0 vector. The gene-III sequence is replaced by one of the two members of the leucine zipper pair (referred to as " linker " in this article). In this synthetic intron, the linker peptide sequence is followed by the genomic sequence of stop codon and intron 1 or 3. In this construct, we have also inserted an isolated bacterial expression cassette consisting of the gene-III fused to the homologous members of the leucine zipper pair. The isolated bacterial expression cassette is introduced into the upstream of the LC CMV promoter and is controlled by the PhoA promoter. The isolated bacterial expression cassette contains the STII signal sequence to limit the linker-gene-III expression of Escherichia coli, and immediately contains the lambda terminator in the downstream. When expressed in Escherichia coli, the heavy chain and light chain are assembled in the periplasm to form Fab, and the linker fused to the heavy chain stably binds to the homologous linker on the pIII-linker protein. The Fab-joint-pIII complex of this assembling is packaged into the phage particle and obtained the phage that displays purpose Fab.In addition, we have produced the KO7 helper phage of conventional mutation, wherein the aminoterminal fusion (joint-KO7) of partner joint and gene-III.Infect the Escherichia coli carrying pDV.5.0 with joint-KO7 and cause the pill of all copies to be presented on the mature phagemid that merges with joint.As a result, the pill of all copies can be associated with Fab-joint, and not just those pill copies that derive from pDV5.However, in some cases, when seeking extremely high affinity clone (for example, in affinity maturation application), expect lower level display.In this case, the mixture of joint-pIII (from pDV.5.0) and the wild-type pill (from KO7 helper phage) displayed on phage particle was produced with conventional KO7 helper phage infection of the Escherichia coli carrying pDV.5.0. In this case, since only a subset of the entire pill repertoire can associate with linker-Fab, the resulting display level is lower than when linker-KO7 is used.This ability to modulate display levels by selecting appropriate helper phage is a unique advantage of the present invention.

We evaluated the ability of pDV5.0 to express different IgGs in mammalian cells. HC and LC from 4 different human IgGs were subcloned into pDV5.0 and expressed in 293 cells. Somewhat surprisingly, the overall yield of pDV was consistently ~10 times lower than that of the dual-plasmid system. However, the yield was still on the order of ~0.1-0.4 mg per 30 mL culture (Figure 6B). This amount of material is sufficient for routine screening assays, and if larger amounts of material are needed, it can be easily increased to 0.1-1 L or more. By gel filtration chromatography, it was shown that >90% of the IgG in the solution was monomeric.

Example 4: Construction of a mutant helper phage M13KO7 with an amber mutation in gene III (AMBER KO7)

To enhance display of proteins fused to pill on M13 phage, we used site-directed mutagenesis to generate a mutant helper phage, Amber KO7. Amber KO7 has an amber codon introduced into the M13KO7 helper phage by site-directed mutagenesis. The nucleotide sequence of pill is shown in Figure 8 (nucleotides 1579 to 2853 of the mutant helper phage, Amber KO7).

To generate Amber KO7, helper phage M13KO7 was used to infect E. coli CJ236 strain (genotype ^dut- / ^ung- ), and progeny viral particles were harvested to purify ssDNA using an ssDNA purification kit (QIAGEN). A synthetic oligonucleotide (sequence 5'-GTGAATTATCACCGTCACCGACCTAGGCCATTTGGGAATTAGAGCCA-3') (SEQ ID NO: 23) was used to mutate gene-III in M13KO7 by oligonucleotide-directed mutagenesis. The mutagenized DNA was used to transform E. coli XL1-Blue cells (Agilent Technologies) and inoculated onto a lawn of non-infected XL1-Blue cells on soft agar plates. Individual plaques were picked and the cells were cultured in LB medium containing 50 μg/ml kanamycin. Double-stranded replicative form (RF) DNA was extracted using a DNA microprep kit and sequenced to confirm the presence of the amber stop mutation. The homogeneity of the population was confirmed by AvrII restriction endonuclease digestion and agarose gel electrophoresis of RF DNA. All recombinant DNA procedures were performed as described in Sambrook, J. et al., A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratroy Press, Cold Spring Harbor, NY, 2001.

The level of Fab displayed on phage particles using Amber KO7 to produce was measured by phage ELISA. Antigen (Her2) was immobilized on immunoplate, and modified M13KO7, carrying amber mutations, was used to produce phages carrying anti-Her2 Fab in XL1-Blue cells. Incubation with mouse anti-M13-HRP conjugates was followed by measuring TMB substrate OD at 450 nm to detect binding. Compared to the level (closed squares) achieved by identical phagemids when WT KO7 was used for phage production, the use of Amber KO7 resulted in a higher display level (closed triangles) (Fig. 9) of low display phagemid display. The Fab level (closed triangles) displayed using the low display phagemid of Amber KO7 was also similar to the Fab display level (open diamonds) observed when the high display phagemid of WT KO7 was used (Fig. 9).

Example 5: Generation of expression and secretion systems for prokaryotic and eukaryotic cells to produce only unprocessed HC libraries And use of the system for phage panning

In addition to the direct and indirect fusion vectors (pDV5.0 and pDV6.0) described in Example 3, which feature prokaryotic and eukaryotic promoters on both the HC and LC, we also 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 the gene-III-synthetic intron and the hIgG Fc sequence 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 recapitulate the previously described synthetic human Fab library (Lee, et al., Journal of Molecular Biology, 340, 1073-1093 (2004)), in which diversity was introduced only into the HC. Expression of full-length IgG from this vector requires co-transfection of a mammalian expression vector encoding the LC.

Using oligonucleotide site-directed mutagenesis and the "stop template" version of pDV.6.5, a phage display library was generated in which a stop codon (TAA) was placed in all three heavy chain CDRs. A mixture of oligonucleotides annealed in the regions encoding CDRH1, H2, and H3 were used to repair these stops during the mutagenesis reaction and replace the codons with degenerate codons at the positions selected by randomization. The mutagenesis reaction was electroporated into XL1-Blue cells and cultured using a temperature conversion method (37°C for 4 hours, then 30°C for 36 hours) in 2YT culture medium supplemented with Amber.KO7 helper phage, 50 μg/ml carbenicillin, and 25 μg/ml kanamycin. Phage was harvested from the culture medium by precipitation with PEG/NaCl. Each electroporation reaction used ~5 μg of DNA and produced 1 × 10 ⁸ –7 × 10 ⁸ transformants.

Panning of the untreated phage library produced in this vector was performed for human vascular endothelial growth factor (VEGF). For phage library sorting, the protein antigen was immobilized on a Maxisorp immunoplate and the library was subjected to 4 to 5 rounds of binding selection. In alternating rounds, BSA or casein was used to seal the plate wells. Random clones selected from the 3rd to 5th rounds were assayed using phage ELISA to compare binding with the target antigen (VEGF) and an unrelated protein (Her2) for checking nonspecific binding. In brief, phage clones were cultured overnight in 1.6 mL of 2YT culture medium supplemented with Amber.KO7 helper phage (Example 4). The supernatant was combined with an immobilized antigen or an unrelated protein-coated plate at room temperature for 1 hour. After washing, the bound phage was detected using an HRP-conjugated anti-M13 antibody (room temperature 20 minutes) followed by TMB substrate detection. We isolated several clones that were ELISA positive for VEGF but not for an irrelevant control protein (Her2) (Figure 10 - bar graph).

DNA from clones that showed specificity for VEGF was then used to express full-length hIgG in small-scale 293 cell suspension cultures expressing full-length hIgG1 by co-transfection with a mammalian expression vector encoding a consensus LC. One mL of culture was transfected using Expifectamine or JetPEI according to the manufacturer's instructions and incubated at 37°C/8% _CO₂ for 5-7 days. Scaled-up transfections were performed in 30 mL of 293 cells.

The culture supernatant was then used to screen for IgG binding to VEGF in an Fc capture assay on a BIAcore T100 instrument (Figure 11). IgG supernatants from 1 mL of culture were used to screen for antigen binding. Anti-human Fc capture antibodies were immobilized on a series of S CM5 sensor chips (~10,000 RU). Supernatants were sequentially flowed through flow cells 2, 3, and 4 (5 μL/min for 4 minutes) to allow capture of IgG (50-150 RU) from the supernatant, followed by flow of antigen (100-1000 nM) through the immobilized IgG (30 μL/min for 2 minutes) to measure binding responses.

Sequencing of the positive binders revealed that eight unique sequences (heavy chain CDR sequences are shown in FIG12 ) had positive binding properties ( FIG12 ). The sequencing data ( FIG12 ) combined with the phage ELISA ( FIG10 ) and BiaCore data ( FIG11 ) were used to select a pool of eight anti-VEGF clones for further analysis.

The expression of these eight clones was scaled up to 100 mL Chinese hamster ovary (CHO) cell cultures (see Figure 15), and the purified material was used to evaluate the ability of the anti-VEGF clones to block the binding of VEGF to one of its cognate receptors (VEGFR1) by receptor-blocking ELISA. Biotinylated hVEGF165 (2 nM) was incubated with a 3-fold serial dilution of anti-VEGF antibody (200 nM highest concentration) in PBS/0.5% BSA/0.05% Tween-20. After incubation at room temperature for 1-2 hours, the mixture was transferred to a plate immobilized with VEGFR1 and incubated for 15 minutes. VEGFR-1 bound VEGF was then detected by incubation with streptavidin-HRP for 30 minutes, followed by development with TMB substrate, and IC50 values were measured.

We identified a clone ( _VEGF55 ) with an _IC50 comparable to that of bevacizumab (a commercially available anti-VEGF antibody) (Figure 13). Thus, we were able to proceed directly from phage panning to IgG expression without the need for subcloning and to reduce the clone library to a single candidate.

In summary, this modified direct fusion binary vector (pDV.6.5) can be used in E. coli to construct phage display libraries with randomized heavy chains and constant light chains, and when supplemented with a light chain expression vector, can also be used in mammalian cells for subsequent expression of selected clones as native IgG1 without subcloning. Because the mammalian CMV promoter is only present upstream of the HC, pDV expresses both Fab LC and Fab HC-pIII in E. coli, but only expresses hIgG1 HC in mammalian cells. This vector can be used to select Fab fragments from a library of unprocessed synthetic Fabs that bind to multiple antigens, and then full-length native hIgG1 can be expressed from selected clones in mammalian 293 and CHO cells by co-transfecting the modified direct fusion binary vector clone with a mammalian expression vector encoding the common LC. Native IgG1s obtained from expression experiments were subjected to several assays, such as those obtained from a pool of eight unique anti-VEGF clones that showed binding activity by ELISA and BIAcore. We were able to sort individual candidates without the need to subclone the HC sequence from the original phage vector clone to evaluate the candidates' in-solution properties, nonspecific binding, and biological activity in an IgG format.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the illustration and example should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (15)

1. A nucleic acid molecule encoding a first polypeptide, a second polypeptide, and a signal sequence encoding mBiP, wherein: (a) The first polypeptide contains a variable heavy chain domain (VH) comprising VH-HVR1, VH-HVR2 and VH-HVR3; (b) The second polypeptide contains a variable light chain domain (VL) comprising VL-HVR1, VL-HVR2 and VL-HVR3; (c) The signal sequence is encoded by a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:3; (d) The first and second polypeptides form an antibody, antibody fragment, or Fab fragment, and (e) wherein the first and/or second polypeptide is fused with a capsid protein or a adaptor protein, wherein the capsid protein is selected from pI, pII, pIII, pIV, pV, pVI, pVII, pVIII, pIX, and pX of phage M13, f1, or fd, and the adaptor protein is selected from SEQ ID NO:6 and SEQ ID NO:7, wherein the nucleic acid encoding the capsid protein is contained in a synthetic intron, wherein the synthetic intron is a nucleic acid located between a nucleic acid encoding CH1 and a nucleic acid encoding the hinge region -Fc or Fc. 2. The nucleic acid molecule of claim 1, wherein the VL and VH are linked to Fc, a tag, and/or a label. 3. The nucleic acid molecule of claim 1 or 2, wherein the VL is linked to a tag and the VH is linked to an Fc. 4. The nucleic acid molecule of claim 1 or 2, wherein the synthesized intron is located between the nucleic acid encoding the CH2 and CH3 domains of the antibody, or wherein the synthesized intron is located between the nucleic acid encoding the hinge region and the CH2 domain of the antibody. 5. The nucleic acid molecule of claim 1, wherein the outer shell protein is a pIII protein or a fragment thereof. 6. The nucleic acid molecule of claim 5, wherein the pIII fragment is amino acid 267-421 or 262-418 of the pIII protein. 7. A vector comprising the nucleic acid molecule of any one of claims 1-5. 8. Host cells transformed using the vector of claim 7. 9. The host cell of claim 8, wherein the host cell is a bacterial cell. 10. The host cell of claim 9, wherein the bacterial cell is an Escherichia coli cell. 11. The host cell of claim 8, wherein the host cell is a eukaryotic cell. 12. The host cell of claim 11, wherein the eukaryotic cell is a yeast cell, a CHO cell, a 293 cell, or an NSO cell. 13. A method for generating antibodies, comprising culturing the host cell of claim 8 to express the nucleic acid. 14. The method of claim 13, wherein the method further comprises recovering the antibody expressed by the host cell. 15. The method of claim 14, wherein the antibody is recovered from the host cell culture medium.