Bacterial host strains

专利摘要:
The present invention lacks the chromosome degP and prc encoding proteases DegP and Prc, respectively, and has a mutant spr gene encoding a protein that inhibits the growth phenotype represented by the strain carrying the prc mutation. E. coli strains. Preferably, said strain comprises a nucleic acid encoding a polypeptide which is heterologous to and can be produced by this strain.
公开号:KR20030057579A
申请号:KR10-2003-7007918
申请日:2001-12-07
公开日:2003-07-04
发明作者:크리스티나 유-칭 첸
申请人:제넨테크, 인크.；
IPC主号:

专利说明:

Bacterial Host Strains {BACTERIAL HOST STRAINS}
[2] E. lacking a protease, or a gene that controls protease regulation. E. coli strains are known. See, eg, Beckwith and Strauch, WO 88/05821 published August 11, 1988; Chaudhury and Smith, J. Bacteriol., 160: 788-791 (1984); Elish et al., J. Gen. Microbiol., 134: 1355-1364 (1988); Baneyx and Georgiou, "Expression of proteolytically sensitive polypeptides in Escherichia coli," In: Stability of Protein Pharmaceuticals, Vol. 3: Chemical and Physical Pathways of Protein Degradation, Ahern and Manning, eds. ( Plenum Press, New York, 1992), p. 69-108).
[3] In the past, attempts have been made to effectively produce proteins that are sensitive to proteolysis, particularly those that are medically potent or commercially important, using some of these strains. US Pat. No. 5,508,192 to Georgiou et al. Describes the production of a number of protease-deficient and / or heat shock protein-deficient bacterial strains. Such hosts include one-, two-, three-, or four-protease deficient bacteria and one-protease bacteria that also carry mutations in the rpo H gene. Examples of the disclosed protease-deficient strains are strains without deg P, omp T, ptr3 and / or prc (tsp) , and these. The reported to produce large titers of recombinant proteins in E. coli include a deg P rpoH strain. Another document (Park et al., Biotechnol. Prog., 15: 164-167 (1999)) also found that a strain (HM114) lacking two cell-envelope proteases ( deg P , prc ) was more protease. It is reported to grow slightly faster than other strains that lacked and produced more fusion proteins. These documents reveal that the strain grew to a cell dry weight of 47.86 g / L after 29 hours by pH-stat, fed-batch culture. The protein produced was a protein A-β-lactamase fusion protein, which showed 30% higher β-lactamase activity than the protein obtained from its parent strain KS272.
[4] According to Hara et al., J. Bacteriol., 173: 4799-4813 (1991), the Prc protein is a periplasmic protease that cleaves the carboxyl-terminus of penicillin binding protein 3 (PBP3) in the periplasm. Was first isolated. The Prc protein was then also identified as a protease that selectively degrades proteins with non-polar C-terminus and was renamed Tsp (Silber et al., Proc. Natl. Acad. Sci. USA, 89: 295). -299 (1992). The prc gene is known to encode a 75-kDa protein required to protect cells from stress combinations of heat and osmotic pressure (Hara et al., supra). Its C-terminal sequence was found to determine substrate selectivity (Keiler et al., Protein Sci., 4 ,: 1507-1515 (1995)). The amount of cleavage is sensitive in identifying residues or functional groups at the C-terminus of the substrate protein. The presence of free ∂-carboxyl groups is important in determining whether closely related peptides with non-polar C-terminal sequences are effectively cleaved by Prc.
[5] Prc homologs include several cyanobacteria (Brand et al., Plant Mol. Bio., 20: 481-491 (1992); Shestakov et al., J. Biol. Chem., 269: 19354-19359 (1994)), Neisseria gonorrhoeae (Black et al., J. Bacteriol., 177: 1952-1958 (1995)), Haemophilus influenzae (Fleischmann et al., Science, 269: 496) -512 (1995)), and other prokaryotic groups including Bartonella bacilliformis (GenBank Accession No. L37094). The domains of the Prc family of proteins are similar to those of retinol-binding proteins, which represent a common folding domain that can form binding pockets for hydrophobic substrates in these proteins (Silber et al., Supra; Shestakov et. al., supra).
[6] Literature reveals that (Hara et al., Supra) in the heat resistance △ reverse mutation of prc mutants (revertant) the exogenous suppressor (spr) mutations contained. They also confirm that the wild-type spr gene product is the lipoprotein of the envelope portion. They thought that the wild type spr gene could be a gene encoding peptidoglycan-hydrolase (Hara et al., Microbial Drug Resistance, 2: 63-72 (1996)). If spr was not functional in the prc -plus background, the inhibitor for spr mutations was found to be another penicillin-binding protein, PBP7 (Hara et al., 1996, supra). there (not decomposed by the Spr is a protease), produced in the cloning and △ prc mutant of the spr is also described in literature (Hara et al., Abstract for Table Ronde Roussel Uclat no. 86, Versailles, May 1997), the disclosures In, the authors conclude that prc and spr are mutual inhibitors.
[7] Also this. Three multicopy prc inhibitors were isolated using the conditional lethal phenotype of the prc (tsp) null strain of E. coli (Bass et al., J. Bacteriol., 178: 1154-1161 (1996)). None of these were related to the spr gene. One set of these inhibitors is two putative protease genes that are in line, mapping at 72.5 minutes on the chromosome. These two genes are htr A homologues, which encode proteins that show 58% and 35% identity to HtrA (DegP) serine protease, respectively. The check the different type of inhibition and (I dnak inhibition) dks A gene, which is also the number of copies inhibitor for a defect in the thermal shock genes dank, which are, dnaj and grp E. The dks A gene was also isolated independently as a multicopy inhibitor of muk B mutations, which is required for chromosome division. The third type is the truncated lipoprotein A ( rlp A) gene.
[8] Gene deg P appears to regulate the synthesis of cell-envelope protease DegP (HtrA). According to Beckwith and Strauch, supra, the first deg P deficient mutation was generated. Recombination into E. coli chromosome. HtrA has a high molecular mass of about 500 kDa, for example at high temperatures in excess of 42 ° C. It is a heat shock protein with proteolytic activity essential for the survival of E. coli (Skorko-Glonek et al., Gene, 163: 47-52 (1995)). deg P mutations can stabilize many cell-envelope proteins that are generally unstable (Strauch and Beckwith, Proc. Natl. Acad. Sci. USA, 85: 1676-1580 (1988)). Recently, by electron microscopy and chemical crosslinking analysis, HtrA protein has been reported to behave as a duplex consisting of two hexameric rings (Kim et al., J. Mol. Biol., 294: 1363-1374 ( 1999). For example, if the protein substrate is unfolded by exposure to high temperatures or reduction of disulfide bonds, it is possible to access the internal chamber of the bicyclic HtrA where cleavage of peptide bonds can occur (Kim et al., supra).
[9] Many heterologous polypeptides are produced in various strains that lack proteases. However, most of these strains exhibit relatively low production titers and / or poor growth. There is a need to provide protease deficient bacterial strains that provide high product titers without showing a decrease in product.
[10] <Overview of invention>
[11] Accordingly, the invention is as claimed. In one aspect, the invention lacks a chromosomal degP and prc, each encoding a protease DegP and Prc, and holds a mutant spr gene, to inhibit the growth phenotype indicating the strain in which the mutant spr gene the product holding prc mutant Being this. Provides E. coli strains. Preferably, the strain lacks chromosome ptr3 encoding protease III and chromosome omp T encoding protease OmpT. Preferably, the cell density is high. By introducing the mutant spr gene into the deg P Δ prcΔ strain for survival in the stagnant phase of the E. coli fermentation process. Manipulate E. coli strains.
[12] In other embodiments, the strain comprises a nucleic acid encoding a polypeptide heterologous thereto, preferably a polypeptide sensitive to proteolysis, more preferably a eukaryotic polypeptide.
[13] In a further embodiment, the invention provides a method of making a heterologous polypeptide, ie, a polypeptide that is heterologous to a strain. The method of this is to inhibit the growth phenotype is first strain chromosomal prc encoding protease lacking Prc and holds or contains a mutant spr gene, the product of the mutant spr gene have the prc mutants shown. Culturing E. coli strains. This strain also includes nucleic acids encoding heterologous polypeptides. The culture is for allowing the nucleic acid to be expressed. In a second step of the method, the polypeptide is recovered from the strain, whether from the cytoplasm or the plasma membrane space of the strain, from the culture medium, preferably from the plasma membrane space or the culture medium, most preferably from the whole fermentation broth. Preferably, the polypeptide is an Apo2 ligand or an antibody including antibody fragments.
[1] The present invention relates to the use of bacterial host strains lacking protease. More specifically, the present invention relates to such host strains which exclude degradation of heterologous polypeptides and improve the yield of such polypeptides.
[14] 1A-1E show the complete nucleotide and encoded amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of the expression cassette for generation of pY0317, the production plasmid for anti-VEGF Fab. Bold residues represent CDR residues from the original murine A.4.6.1 antibody. Italic and underlined residues represent murine framework residues required for antigen binding.
[15] 2A and 2B show the plasmid scheme for pY0317 (FIG. 2A) and the plasmid construction of pY0317tet20 (FIGS. 2A and 2B).
[16] 3 shows a plasmid schematic for pAPApo2-P2RU.
[17] 4 shows the nucleotide sequence of human Apo-2 ligand cDNA (SEQ ID NO: 3) and the amino acid sequence derived therefrom (SEQ ID NO: 4). "N" at nucleotide position 447 (in SEQ ID NO: 3) is used to indicate that the nucleotide base may be "T" or "G".
[18] 5 is this. A scheme for inducing E. coli strains 59A7, 49A5 and 43H1 is shown.
[19] 6 shows the 2-D gel results of fermented cell pellets derived from strain 49A5 ( prc -plus strain) expressing the rhuFab'2 anti-CD18-LZ fusion as a heterologous polypeptide. All LC-related spots are circled.
[20] 7 shows the 2-D gel results of fermented cell pellets derived from strain 43H1 ( prc -minus strain) expressing the rhuFab'2 anti-CD18-LZ fusion as a heterologous polypeptide. In this gel, no LC-cleaving product appeared.
[21] FIG. 8 shows five peaks resolved by analysis using AME5® / reverse phase column, thus providing a split comparison of the rhuFab'2 LZ (xCD18) antibody fragment thus resolved. The y-axis is the specific peak area of peaks 1-5. The x-axis represents three rhuFab'2 LZ (xCD18) producing strains 43H1 ( prc −), 49A5 ( prc +) and 58H2 ( prc − recovered 43H1). The gray bars edged by thick lines are LC-115, the black bars are LC, the white bars are LC dimers, the gray bars edged by thin lines are Fab-like molecules, and the brick-like bars Fab'2-LZ. It can be seen that the peak 1 (LC-115) did not appear in the prc -deleted strain.
[22] 9 shows prc -minus without a mutant spr gene (58B3 transformed with pS1130) (square) and prc -minus with mutant spr gene (59A7 transformed with pS1130), expressed as OD550 as a function of fermentation time ( Diamond) strains show high growth density of standard fermentation.
[23] FIG. 10 shows the humanized anti-CD18 kappa LC sequence (SEQ ID NO: 5) with the calculated pi value of the putative LC degradation product. Cut sites highlighted with a slash (/) were identified by mass spectrometry. See Table 3.
[24] FIG. 11 is shown using different hosts and three types of proteins in a gel with seven lanes. This gel shows that a 20-kD LC clip (LC 182) is not present in 43H1 ( prc −) cells expressing the anti-VEGF Fab and anti-tissue factor Fab′2-LZ fusion molecules. Lane 1 is anti-tissue factor F (ab ') 2 LZ 6xHis, host strain 33B6, lane 2 is anti-tissue factor F (ab') 2 LZ 6xHis, host strain 43H1, and lane 3 is anti-CD18 F ( ab ') 2 LZ 6xHis, host strain 49A5, lane 4 is anti-CD18 F (ab') 2 LZ 6xHis, host strain 41H1, lane 5 is pBR322, host strain 49A5, lane 6 is anti-VEGF Fab, Host strain 43H1, lane 7 is anti-VEGF Fab, host strain 43E7. The markings of HC and H represent heavy chains and the LC and L marks represent light chains.
[25] 12 shows 2-D gel results of shake flask cell pellets derived from strain 59A7 ( prc -minus strain) expressing anti-VEGF Fab (pY0317tet20) as a heterologous polypeptide. In this gel, the LC-cleavage product and two HC-cleavage fragments found in prc -plus cells did not appear. Two separate HC clips detected only in 59A7 are also shown, which are OmpT- or Ptr3-cut products.
[26] FIG. 13 shows 2-D gel results of shake flask cell pellets derived from strain 60C1 ( prc -plus strain) expressing anti-VEGF Fab (pY0317tet20) as a heterologous polypeptide. In this gel, several LC-cleaved fragments and two HC-cleaved fragments were detected.
[27] <Detailed Description of the Preferred Embodiments>
[28] Justice
[29] As used herein, "polypeptide" generally refers to peptides and proteins having about 10 or more amino acids. A “heterologous” polypeptide is a polypeptide that is heterologous to the host cell used, such as E. coli. It is a human protein produced by E. coli. Such polypeptides may be of prokaryotes or eukaryotes, but are preferably of eukaryotes, more preferably of mammals.
[30] Examples of mammalian polypeptides include molecules such as lenin; Growth hormones including human growth hormone and bovine growth hormone; Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipoprotein; 1-antitrypsin; Insulin A-chain; Insulin B-chain; Proinsulin; Thrombopoietin; Follicle stimulating hormone; Calcitonin; Progesterone; Glucagon; Coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; Anti-coagulation factors such as protein C; Atrial sodium excretion factor; Waste surfactants; Plasminogen activators such as urokinase or human urine or tissue-type plasminogen activator (t-PA); Bombesin; Thrombin; Hematopoietic growth factor; Tumor necrosis factor-alpha and -beta; ErbB2, such as 2C4 (WO 01/00245; hybridoma ATCC HB-12697) that binds to a region within the extracellular domain of ErbB2 (eg, any one or more residues within the region of about 22 to about 584 residues of ErbB2 included) Antibodies to domain (s), enkephalinases; Serum albumin, eg human serum albumin; Muellerian-inhibiting substance; Relaxin A-chain; Relaxin B-chain; Prolylacin; Mouse gonadotropin-associated peptide; Microbial proteins such as beta-lactamase; DNase; Inhibin; Activin; Vascular endothelial growth factor (VEGF); Receptors for hormones or growth factors; Integrin; Protein A or D; Rheumatoid factor; Neurotrophic factors such as bone-induced neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or Nerve growth factors such as NGF; Cardiotropin (heart hypertrophy factor), for example cardiotropin-1 (CT-1); Platelet-induced growth factor (PDGF); Fibroblast growth factors such as aFGF and bFGF; Epidermal growth factor (EGF); Transforming growth factors (TGF), for example TGF-alpha and TGF-beta (including TGF-1, TGF-2, TGF-3, TGF-4 or TGF-5); Insulin-like growth factor-I and -II (IGF-I and IGF-II); Des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8 and CD19; Erythropoietin; Osteoinduction factors; Immunotoxins; Bone morphogenic protein (BMP); Interferons such as interferon-alpha, -beta and -gamma; Serum albumin such as human serum albumin (HSA) or bovine serum albumin (BSA); Colony stimulating factors (CSFs) such as M-CSF, GM-CSF and G-CSF; Interleukin (IL), for example IL-1 to IL-10; Anti-HER-2 antibodies; Apo2 ligand; Superoxide dismutase; T-cell receptor; Surface membrane proteins; Decline facilitating factor; Viral antigens such as portions of AIDS envelopes; Carrier protein; Homing receptors; Addressin; Regulatory proteins; Antibodies; And fragments of any of the polypeptides listed above.
[31] Preferred target polypeptides include, for example, HSA, BSA, anti-IgE, anti-CD20, anti-IgG, t-PA, gp120, anti-CD11a, anti-CD18, 2C4, anti-VEGF, VEGF, TGF-beta, Activin, Inhibin, Anti-HER-2, DNase, IGF-I, IGF-II, Brain IGF-I, Growth Hormone, Relaxin Chain, Growth Hormone Release Factor, Insulin Chain or Pro-Insulin, NGF, NT- 3, BDNF, Apo2 ligand and polypeptides such as urokinase. Particularly preferred mammalian polypeptides are antibodies, and antibodies include full length antibodies, antibody fragments and Apo2 ligands. More preferably, these antibodies are human antibodies or humanized antibodies. These include, for example, anti-IgE, anti-IgG, anti-Her-2, anti-CD11a, anti-CD18, anti-CD20 and anti-VEGF, 2C4, BSA or HSA. Even more preferably, the antibody is an anti-CD18, anti-VEGF, anti-tissue factor, 2C4, anti-Her-2, anti-CD20, anti-CD40 or anti-CD11a antibody. Antibody fragments included in the polypeptide definition include, for example, Fab, Fab ', Fab'2, or Fab'2-leucine zipper (LZ), most preferably anti-CD18 Fab'2-LZ, Anti-tissue factor Fab'2 LZ-6xhis, anti-VEGF Fab, anti-CD8 his-tagged Fab'2 LZ, and anti-CD18 lys-tagged Fab'2LZ.
[32] As used herein, the expression “protease sensitive” for a polypeptide is prone to cleavage, susceptibility to cleavage, or one or more E. coli in the natural state or during secretion. A polypeptide that is cleaved by E. coli protease.
[33] "Cell-dense" fermentations or cultures usually add some nutrients to the batch first to allow cell growth, and dissolved oxygen, which is easy to measure using the relationship between O ₂ consumption and glucose consumption By means of means to control the addition of glucose. To reach high cell density, ammonia can be added continuously and additional minor nutrients (e.g., P, K, S and Mg) can be added at various stages of fermentation so as to be described in more detail in the examples below. Can support growth
[34] "A mutant spr gene (the product of this gene inhibits the growth phenotype exhibited by the strain carrying the prc mutation)" E. coli with the sequence reported in Hara et al., 1996, supra. E. coli prc inhibitor ( spr ) (coding Prc ^sup ) or mutated, provided that this gene product acts to inhibit the growth phenotype of strains containing prc mutations. Preferably, the mutation consists of one point mutation. Most preferred is the point mutation W148R, where the TGG codon is changed to CGG to change tryptophan to arginine at amino acid 148.
[35] The term "antibody" is used herein in its broadest sense and specifically includes circular monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) formed from two or more circular antibodies, and Antibody fragments, provided that they exhibit the desired biological activity.
[36] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, ie the individual antibodies that make up this population are identical except for possible naturally occurring mutations that may be present in small amounts. Do. Monoclonal antibodies are highly specific for a single antigenic site. In addition, unlike polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to these specificities, monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier “monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of antibodies in any particular manner. For example, the monoclonal antibodies used in the present invention can be prepared by the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (US Patent 4,816,567). "Monoclonal antibodies" are also described, eg, in Clackson et al., Nature, 352: 624-628 (1991); and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Isolation from phage antibody libraries can be performed using the techniques described in)).
[37] In particular the monoclonal antibodies herein have the same or homologous to the corresponding sequences of the heavy and / or light chains as derived from a particular species or an antibody belonging to a particular antibody class or subclass, but the rest of the chain As well as antibodies derived from another species or antibodies belonging to another antibody class or subclass, as well as "chimeric" antibodies which are identical or homologous to the corresponding sequences of said antibody fragments so long as they exhibit the desired biological activity, (US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include primatized antibodies comprising variable domain antigen-binding sequences and human constant region sequences derived from non-human “primates” (eg, Old World Monkey, Ape, etc.).
[38] An “antibody fragment” includes a portion of a circular antibody, preferably comprising an antigen binding region or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; Diabodies; Linear antibodies; Single chain antibody molecules; And multispecific antibodies formed from antibody fragment (s).
[39] "Circular" antibodies are antibodies comprising a light chain constant domain (C _L ) and a heavy chain constant domains C _H 1, C _H 2 and C _H 3 as well as an antigen binding variable region. Such constant domains may be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the circular antibody has one or more effector functions.
[40] "Effector function" of an antibody means a biological activity attributable to the Fc region (an Fc region with an amino acid sequence variation or a native sequence Fc region) of an antibody. Examples of antibody effector functions include C1q binding; Complement dependent cytotoxicity; Fc receptor binding; Antibody-dependent cell mediated cytotoxicity (ADCC); Phagocytosis; Down regulation of cell surface receptors (eg B cell receptor; BCR).
[41] Circular antibodies can be assigned to different "classes" depending on the amino acid sequence of their heavy chain constant domains. The five major classes of circular antibodies are IgA, IgD, IgE, IgG and IgM, some of which may be further classified as "subclass (isotype)," eg, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. Can be. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional arrangements of other classes of immunoglobulins are well known.
[42] "Antibody-dependent cell mediated cytotoxicity" and "ADCC" are antibodies in which nonspecific cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages) that express Fc receptors (FcRs) are bound on target cells. After recognition, it refers to a cell mediated response that causes lysis of the target cell. NK cells, the major cells that mediate 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: 457-92 (1991). To assess ADCC activity of a subject molecule, in vitro ADCC assays can be performed as described in US Pat. No. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the subject molecule can be assessed in vivo in an animal model as described, for example, in Clynes et al., PNAS (USA) 95: 652-656 (1998).
[43] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably such cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, but PBMCs and NK cells are preferred. The effector cells can be isolated from natural sources, such as blood or PBMCs as described herein.
[44] Generally “natural antibodies” are heterotetrameric glycoproteins of about 150,000 Daltons that are composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one disulfide covalent bond, but the number of disulfide bonds varies for the heavy chains of other immunoglobulin isotypes. Each heavy and light chain also has regularly positioned intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) and a number of constant domains following this domain. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the light and heavy chain variable domains.
[45] The term "variable" refers to the fact that certain portions of the variable domains exhibit broad sequence differences between antibodies and are used for the binding and specificity of specific antibodies with their specific antigens. However, this variability is not evenly distributed throughout the variable domains of antibodies. The variability is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains predominantly in the form of beta-sheets and comprise four FRs linked to three hypervariable regions, which form loop linkages and in some cases β-sheet structures Form a part. The hypervariable regions in each chain are closely held together by the FRs, and the hypervariable regions from the other chain help to form the antigen-binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD. (1991). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
[46] As used herein, the term "hypervariable region" refers to an amino acid residue of an antibody that is involved in antigen-binding. Such hypervariable regions are generally amino acid residues from the "complementarity determining regions" or "CDRs" (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) and heavy chains in the light chain variable domain). Residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda , MD. (1991)) and / or amino acid residues from “hypervariable loops” (eg, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) and heavy chains in the light chain variable domain). Residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
[47] Cleavage of the antibody with papain yields two identical antigen-binding fragments, each with a single antigen-binding site, called "Fab" fragments, and the remaining "Fc" fragments (these names reflect the ability to readily crystallize). do. Pepsin treatment of the antibody produces a F (ab ′) ₂ fragment that has a bivalent antigen-binding site and is still able to crosslink with the antigen.
[48] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition site and an antigen-binding site. This region consists of dimers in which one heavy chain variable region domain and one light chain variable region domain are tightly linked to each other by non-covalent bonds. This form is where three hypervariable regions of each variable domain interact to form an antigen-binding site on the surface of the V _H -V _L dimer. In total, the six hypervariable regions provide antigen binding sites for antibodies. However, even one variable domain (or half of the Fv comprising only three hypervariable regions specific for a particular antigen), although less affinity than the entire antigenic site, has the ability to recognize and bind antigens. Have
[49] Fab fragments also comprise the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain comprising one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab 'in which the cysteine residue (s) of the constant domains bear one or more free thiol groups. F (ab ') ₂ antibody fragments originally were produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
[50] The “light chains” of antibodies from any vertebrate species can be assigned to one of two distinctly different types called kappa (κ) and lambda (λ) based on the amino acid sequences of their constant domains.
[51] "Single-chain Fv" or "scFv" antibody fragments comprise the V _H and V _L antibody domains present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V _H domain and the V _L domain, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994). Anti-ErbB2 antibody scFv fragments are described in WO 93/16185; US 5,571,894 and US 5,587,458.
[52] The term "diabody" refers to a small antibody with two antigen binding sites, comprising a variable heavy domain (V _H ) linked to a variable light domain (V _L ) within the same polypeptide chain (V _H -V _L ). Means a fragment. By using a linker that is too short to pair between these two domains on the same chain, the domains are paired with the complementary domains of the other chain to form two antigen binding sites. Diabodies are described, for example, in European Patent No. 404,097; WO 93/11161; And Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
[53] A “humanized” form of a non-human (eg rodent) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are those of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates whose residues in the hypervariable region of the recipient have the desired antibody specificity, affinity and ability. Human immunoglobulin (receptor antibody) replaced with a residue in the region. In some cases, the framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Moreover, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications are made to further enhance antibody performance. In general, humanized antibodies will comprise substantially all of one or more, and typically two, variable domains, where all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all Or substantially all FRs are FRs of a human immunoglobulin sequence. In addition, the humanized antibody will optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct Biol. 2: 593-596 (1992)).
[54] An "isolated antibody" is an antibody identified, isolated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that prevent antibodies from being used for diagnosis or treatment, and examples include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody comprises (1) greater than 95% by weight, most preferably greater than 99% by weight, as measured by the Lowry method, and (2) using a spinning cup sequencer. Sufficient to obtain an N-terminal or internal amino acid sequence consisting of 15 or more residues, or (3) uniformity by performing SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Purify. Isolated antibodies include antibodies in situ in recombinant cells, since at least one antibody natural environment component will not be present. Ordinarily, isolated antibodies will be prepared in one or more purification steps.
[55] The expression "regulatory sequence" means in particular the DNA sequence necessary for the expression of a coding sequence operably linked in a host organism. Suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites.
[56] Nucleic acids are “operably linked” when placed in a functional relationship with other nucleic acid sequences. For example, the DNA of a presequence or secretion leader is operably linked to the DNA for that polypeptide when expressed as a pro-protein involved in secretion of that polypeptide, and the promoter affects transcription of the sequence. Operably linked to a coding sequence, and a ribosomal binding site is operably linked to a coding sequence when placed to facilitate translation. In general, "operably linked" means that the DNA sequences to be linked are located adjacent, and in the case of a secretory leader, not only are located adjacent, but also exist at the same leading face. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used according to conventional methods.
[57] As used herein, the expressions "cell," "cell line," and "cell culture" are interchangeable, all of which include their progeny. Thus, the terms “transformer” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to intentional or accidental mutations. Mutant progeny that have the same function or biological activity as screened for the originally transformed cell are included. Where otherwise indicated, this is clearly indicated herein.
[58] Practice of the Invention
[59] The present invention is directed to a method in which the chromosomal degP and prc encoding the proteases DegP and Prc, respectively, lack a mutant spr gene and wherein the mutant spr gene product inhibits the growth phenotype exhibited by a strain carrying a prc mutation. Provides E. coli strains. The strain may optionally further lack chromosome ptr3 encoding protease III and / or chromosome ompT encoding protease OmpT.
[60] In other embodiments, the strain comprises a nucleic acid encoding a polypeptide that is heterologous to this strain. This strain is preferably transformed with a nucleic acid, more preferably DNA (cDNA or genomic DNA) as by the use of recombinant expression vectors.
[61] In a further aspect, the present invention provides a method for preparing the heterologous polypeptide. In this method, the E. coli also comprises a nucleic acid encoding the polypeptide. E. coli strains are cultured to express nucleic acids. The polypeptide is then recovered from the strain. Recovery may be performed from the plasma membrane periphery or culture medium. Preferably, the culturing is carried out under fermentation conditions, more preferably under high cell density fermentation conditions.
[62] Culture parameters are used and polypeptide preparation is carried out in conventional manner, for example by the method described below.
[63] A. Selection of Nucleic Acids and Modifications thereof
[64] The nucleic acid encoding the subject polypeptide is suitably RNA, cDNA or genomic DNA from any source so long as it encodes the subject polypeptide (s). this. Methods of selecting appropriate nucleic acids for expression of heterologous polypeptides (including variants thereof) in E. coli are well known.
[65] When producing monoclonal antibodies, DNA encoding the monoclonal antibodies can be conventionally employed (e.g., using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of a murine antibody). Easily isolate and sequence using Hybridoma cells serve as a preferred source of the DNA. If isolated, the DNA can be located in an expression vector, which is then transformed with the bacterial host cell of the present disclosure to synthesize monoclonal antibodies in the recombinant host cell. Review literature on recombinant expression of DNA encoding antibodies in bacteria includes Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-. 188 (1992).
[66] Methods of humanizing non-human antibodies are described in the prior art. Preferably, the humanized antibody has one or more amino acid residues introduced into it from a non-human source. Such non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization is in essence a method of Winter and its collaborators (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al. , Science, 239: 1534-1536 (1988)), by replacing the corresponding sequences of human antibodies with hypervariable region sequences. Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) and the circular human variable domains are substantially not replaced at all by the corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.
[67] The selection of human variable domains of the light and heavy chains used to prepare humanized antibodies is of great importance in reducing antigenicity. According to the so-called "most-suitable" method, the sequence of the variable domains of rodent antibodies is screened against the entire library of known human variable domain sequences. The human sequence closest to that of the rodent is then accepted as the human framework region (FR) for humanized antibodies (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies in a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 ( 1993).
[68] It is more important that the antibody be humanized with high affinity for the antigen and other beneficial biological properties. To achieve this goal, humanized antibodies are prepared by methods of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences, in accordance with preferred methods. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and represent possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Investigation of such displays allows analysis of the possible role of residues in the function of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of a candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined with receptor and import sequences to achieve the desired antibody properties, eg, increased affinity for the target antigen (s). In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.
[69] Various forms of humanized antibodies or affinity-matured antibodies are contemplated. For example, the humanized antibody or affinity-matured antibody can be a Fab that can optionally bind an antibody fragment, eg, one or more target substance (s), to form an immunoconjugate. Alternatively, the humanized antibody or affinity-matured antibody may be a circular antibody, eg, a circular IgG1 antibody.
[70] Fab'-SH fragments to E. coli. Direct recovery from E. coli and chemical coupling can form F (ab ') ₂ fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). According to another method, F (ab ') ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the preparation of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (WO 93/16185; US Pat. Nos. 5,571,894 and 5,587,458). The antibody fragment may also be a "linear antibody" as described, for example, in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.
[71] Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the Dkk-1 protein. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab ') ₂ bispecific antibodies).
[72] According to another method, an antibody variable domain exhibiting the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant domain sequence. This fusion is preferably a fusion with an immunoglobulin heavy chain constant domain comprising at least a portion of a hinge, CH2 and CH3 region. It is preferred to have a first heavy chain constant region (CH1) containing a site necessary for light chain binding, present in one or more of these fusions. The DNA encoding the immunoglobulin heavy chain fusions and optionally the immunoglobulin light chain are inserted into a separate expression vector and cotransfected into suitable bacterial host cells. This provides even greater flexibility in controlling the mutual ratios of these three polypeptide fragments in embodiments in which unequal proportions of three polypeptide chains used in the construction provide optimal yields. However, if expression of two or more polypeptide chains in equal proportions results in high yield, or if such proportions are not particularly important, the coding sequences for two or all of the three polypeptide chains are inserted into one expression vector. It is possible to do
[73] In a preferred embodiment of this method, the bispecific antibody comprises a hybrid immunoglobulin heavy chain that exhibits first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain pair (second binding specificity) on another arm. Provided). This asymmetric structure has been found to facilitate separation of the desired bispecific compounds from undesirable immunoglobulin chain combinations, which is easy because the immunoglobulin light chain is present in only half of these bispecific molecules. This is because a separation method is provided. This method is described in WO 94/04690. For further details on bispecific antibody production, see, eg, Suresh et al., Methods in Enzymology, 121: 210 (1986).
[74] According to another method described in US Pat. No. 5,731,168, the interface between antibody molecule pairs can be engineered to maximize the percentage of heterodimer recovered from recombinant cell culture. Preferred interfaces include at least a portion of the C _H 3 domain of the antibody constant domains. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Replacing large amino acid side chains with smaller amino acids (such as alanine or threonine) produces “cavities” of the same or similar size as the large side chain (s) on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other undesirable end products (eg homodimers).
[75] Bispecific antibodies include crosslinked antibodies or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other can be coupled to biotin. Such antibodies are for example used to target immune system cells to unwanted cells (US Pat. No. 4,676,980) and to treat HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be prepared using conventional crosslinking methods. Suitable crosslinkers are well known in the art with many crosslinking techniques and are described in US Pat. No. 4,676,980.
[76] Techniques for generating bispecific antibodies from antibody fragments have also been reported in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., Science, 229: 81 (1985) describe the process of cleaving protoplasts with proteases to produce F (ab ') ₂ fragments. These fragments are reduced in the presence of dithiol binder former, sodium arsenite, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab 'fragment thus generated is then converted to thionitrobenzoate (TNB) derivatives. Subsequently, one of the Fab'-TNB derivatives is converted back to Fab'-thiol by reduction with mercaptoethylamine, which is mixed with an equimolar amount of other Fab'-TNB derivatives to form a bispecific antibody. The bispecific antibody thus produced can be used as a substance for selective immobilization of enzymes.
[77] In addition, Fab'-SH fragments were purified from E. coli. Direct recovery from E. coli and chemical coupling can form bispecific antibodies (Shalaby et al., J. Exp. Med., 175: 217-225 (1992)).
[78] Various techniques have also been reported for preparing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies were generated using leucine zippers (Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)). The leucine zipper peptides derived from Fos and Jun proteins were linked to Fab 'portions of two different antibodies by gene fusion. This antibody homodimer was reduced in the hinge region to form a monomer and then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The "diabody" technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provided another mechanism for preparing bispecific antibody fragments. Such fragments comprise a heavy chain variable domain (V _H ) linked to the light chain variable domain (V _L ) by a linker that is too short to pair between two domains on the same chain. Thus, the V _H and V _L domains of one fragment are paired with the complementary V _L and V _H domains of another fragment, thereby forming two antigen binding sites. Another method for preparing bispecific antibody fragments by using single chain Fv (sFv) dimers is also described in Grubber et al., J. Immunol., 152: 5368 (1994).
[79] Bivalent or higher antibodies are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60 (1991)).
[80] Nucleic acid molecules encoding polypeptide variants are prepared by a variety of methods known in the art. Such methods include isolation from natural sources (for naturally occurring amino acid sequence variants), or oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, or prefabricated polypeptide variants or non- Preparation by cassette mutagenesis of polypeptides in variant forms is included, but is not limited thereto.
[81] For example, it may be desirable to modify the antibodies of the invention to effector function in order to increase Fc receptor binding. This can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or additionally, the introduction of cysteine residue (s) into the Fc region can allow for interchain disulfide bond formation in this region.
[82] To increase the serum half-life of the antibody, salvage receptor binding epitopes can be introduced into the antibody (especially antibody fragments), as described, for example, in US Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to the Fc region of an IgG molecule (eg, IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ) which serves to increase serum half-life in vivo of the IgG molecule. Means epitope.
[83] Other modifications to the antibodies are contemplated herein. For example, the antibody can be linked to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol.
[84] B. Insertion of Nucleic Acids into Replicable Vectors
[85] Suitably the heterologous nucleic acid (eg cDNA or genomic DNA) is inserted into a replicable vector for expression in bacteria under the control of a suitable promoter. Many vectors are available for this purpose, and the choice of the appropriate vector will depend primarily on the size of the nucleic acid inserted into the vector and the particular host cell transformed with the vector. Include them. Depending on the particular type of host, the vector component generally includes, but is not limited to, one or more of signal sequences, origins of replication, one or more marker genes, promoters and transcription termination sequences.
[86] In general, plasmid vectors comprising regulatory sequences derived from species suitable for replicon and host cells are described in E. coli. Use with E. coli host. This vector typically carries not only a replication site, but also a marking sequence that can provide phenotypic selection in the transformed cell. For example, this. E. coli using pBR322, a plasmid derived from E. coli species. E. coli is transformed (see, e.g., Bolivar et al., Gene, 2: 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance, thus providing an easy means to identify transformed cells. The pBR322 plasmid or other bacterial plasmids or phages are also commonly used for the expression of selectable marker genes. It includes or is modified to include a promoter that can be used by the E. coli host.
[87] (i) signal sequence components
[88] The DNA encoding the polypeptide of interest of the present invention can not only be expressed directly, but also with other polypeptides, preferably with a fusion with a signal sequence or with another polypeptide having a specific cleavage site at the N-terminus of the mature polypeptide. It can be expressed as water. In general, the signal sequence may be a component of the vector or part of a polypeptide DNA inserted into the vector. The heterologous signal sequence selected should be one that is recognized and processed (ie, cleaved by signal peptidase) by the host cell.
[89] For prokaryotic host cells that do not recognize and process native or eukaryotic polypeptide signal sequences, the signal sequences are for example prokaryotic selected from the group consisting of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leader. Substituted by a biosignal sequence.
[90] (ii) origin of replication components
[91] Expression vectors include nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are well known for various bacteria. The origin of replication from plasmid pBR322 is E. coli. It is suitable for most Gram-negative bacteria such as E. coli.
[92] (iii) selection gene components
[93] Expression vectors generally contain a selection gene, also called a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells growing in selective culture medium. Host cells not transformed with the vector comprising the selection gene will not survive in the culture medium. Such selectable markers are independent of the genetic markers used and defined in the present invention. Typical selection genes are nutrients other than (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, or (b) caused by the presence of genetic marker (s). There is a gene that encodes a protein that compensates for a deficiency in demand, or (c) supplies important nutrients that are not available from the complex medium, for example Bacillus.
[94] One example of a screening scheme is to use drugs that stop the growth of host cells. In this case, cells successfully transformed with the nucleic acid of interest produce a polypeptide that confers drug resistance to withstand the screening scheme. Examples of such dominant selection include neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327 (1982)), mycophenolic acid (Mulligan et al., Science, 209: 1422 (1980)) as a drug. ) Or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 (1985)). The three examples given above use bacterial genes under prokaryotic control to confer resistance to suitable drugs G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
[95] (iv) promoter components
[96] Expression vectors for the production of a subject polypeptide include suitable promoters recognized by the host organism and operably linked to the nucleic acid encoding the subject polypeptide. Suitable promoters for use in prokaryotic hosts include the beta-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)), Arabies North promoter system (Guzman et al., J. Bacteriol., 174: 7716-7728 (1992)), alkaline phosphatase, tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980) and European patents 36,776) and hybrid promoters, for example the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)). However, other known bacterial promoters are also suitable. Their nucleotide sequences have been published, and therefore one skilled in the art will be able to operatively ligate these sequences to the DNA encoding the polypeptide of interest using a linker or an adapter that provides any necessary restriction sites (Siebenlist et al., Cell, 20: 269 (1980)).
[97] Generally promoters used in bacterial systems also include a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of interest. This promoter can be removed from the bacterial source DNA by restriction enzyme digestion and inserted into the vector containing the target DNA.
[98] (v) Construction and analysis of vectors
[99] Construction of suitable vectors comprising one or more of the components listed above utilizes standard ligation techniques. The isolated plasmid or DNA fragment is cut, trimmed and ligated back into the desired form to produce the required plasmid.
[100] For analysis to identify the correct sequence in the constructed plasmid, ligation mixtures were used. E. coli K12 strain 294 (ATCC 31,446) or other strains were transformed and successful transformants were optionally selected by ampicillin or tetracycline resistance. Plasmids from the transformants were prepared, analyzed and / or sequenced by restriction endonuclease cleavage (Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977). ) Or by the method of Messing et al., Nucleic Acids Res., 9: 309 (1981), or by the method of Maxam et al., Methods in Enzymology, 65: 499 (1980).
[101] C. Selection and Transformation of Host Cells
[102] E. suitable as parent host for expression plasmids herein. E. coli host. E. coli W3110 (ATCC 27,325). E. coli 294 (ATCC 31,446). E. coli B, and Lee. E. coli X1776 (ATCC 31,537). These examples are intended to be illustrative rather than restrictive. Mutant cells for any of the above mentioned strains can also be used as starting hosts, and then further mutated to at least contain the minimum genotype required herein. this. E. coli strain W3110 is the preferred parent host because it is a common host strain for recombinant DNA producing fermentations. Departures used as parent host E. Examples of E. coli hosts, along with their genotypes, are included in the table below:
[103]
[104] Also suitable are intermediates in making strain 36F8, namely 27B4 (US Pat. No. 5,304,472) and 35E7 (spontaneous temperature-resistant colony isolates that grow better than 27B4). Further suitable strains are E. coli with the mutant plasma membrane periphery protease (s) disclosed in US Pat. No. 4,946,783, issued August 7, 1990. E. coli strains.
[105] Strains of the invention can be produced by chromosomal integration or other techniques of the parent strain, including the techniques described in the Examples below.
[106] A nucleic acid encoding a polypeptide is inserted into a host cell. Preferably, this process is accomplished by transforming the host cells with the expression vectors described above and culturing in conventional nutrient media modified to be suitable for inducing various promoters.
[107] Transformation means introducing DNA into an organism so that the DNA can be replicated as an extrachromosomal element or as a chromosomal integration.
[108] Depending on the host cell used, transformation is carried out using standard techniques suitable for such cells. As described in section 1.82 of Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), calcium treatment with calcium chloride generally results in prokaryotic or substantial cell wall barriers. It is used against other cells, including. Another transformation method uses polyethyleneglycol / DMSO as described in Chung and Miller, Nucleic Acids Res., 16: 3580 (1988). Another method uses a technique called electroporation.
[109] D. Host Cell Culture
[110] Prokaryotic cells used to produce the polypeptide of interest are cultured in a suitable medium as generally described in Sambrook et al., Supra. Culture conditions such as temperature and pH are those already used in the host cell selected for expression and will be apparent to those skilled in the art.
[111] When using an alkaline phosphatase promoter, the E. coli used to produce the subject polypeptides of the invention. E. coli cells are cultured in a suitable medium, wherein the alkaline phosphatase promoter can generally be partially or fully induced, as described, for example, in Sambrook et al., Supra. The culturing need not be carried out in the absence of inorganic phosphate or in the absence of phosphate. First, the medium contains inorganic phosphate in an amount above the induction level of protein synthesis and sufficient for bacterial growth. As cells grow and use phosphate, they reduce phosphate levels in the medium, thus inducing the synthesis of polypeptides.
[112] In addition to carbon, nitrogen and inorganic phosphate sources, any other necessary media components may be included at suitable concentrations, either alone or as a mixture with other components or media, such as a complex nitrogen source. The pH of the medium may be any pH from about 5-9, depending mainly on the host organism.
[113] If the promoter is an inducible promoter, in order for the induction to occur, the cells are usually cultured until a specific optical density is reached, for example, A ₅₅₀ is reached to about 200 using a high cell density method. At this point, induction is initiated (eg, by addition of an initiator, by depletion of media components, etc.) to induce the expression of the gene encoding the polypeptide of interest.
[114] E. Expression Detection
[115] Conventional Southern blotting, Northern blotting to quantify transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)), based on the sequence of the polypeptide, for example Gene expression in samples can be measured directly by loting (DNA analysis), or in situ hybridization using appropriately labeled probes. Various labels can be used, most commonly radioisotopes, in particular ³² P. However, other techniques may be used, such as using biotin-modified nucleotides for introduction into polynucleotides. Biotin then serves as a site that binds to avidin or an antibody that can be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, and the like. Alternatively, proteins can be detected using assays or gels.
[116] For secretion of the expressed gene product, the host cells are cultured under conditions sufficient for the secretion of the gene product. Such conditions include, for example, temperature, nutrients, and cell density conditions that allow secretion by the cell. These conditions are also conditions under which a cell can perform basic cellular functions such as transcription, translation, and delivery of proteins from one compartment of the cell to another, as is known to those skilled in the art.
[117] F. Polypeptide Purification
[118] Each of the following methods, or a combination thereof, is an example of a suitable purification method, and the particular method (s) used depend on the type of polypeptide: fractionation on an immunoaffinity or ion exchange column; Ethanol precipitation; Reverse phase HPLC; Hydrophobic-interaction chromatography; Chromatography on silica; Ion exchange resins such as S-SEPHAROSE® and DEAE phase chromatography; Chromatofocusing; SDS-PAGE; Ammonium-sulfate precipitation; And gel filtration using, for example, SEPHADEX® G-75.
[119] Monoclonal antibodies may suitably be separated from the culture medium by conventional antibody purification methods such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. have.
[120] The present invention will be understood in more detail with reference to the following examples. However, these should not be considered as limiting the scope of the present invention. All publications and patent citations herein are incorporated by reference.
[121] <Example 1>
[122] Materials and methods
[123] A. Expression Plasmid
[124] 1. Plasmids for expression of rhuFab'2 LZ (xCD18) and tagged derivatives
[125] pS1130
[126] Plasmid pS1130 is a pBR322-based plasmid described in US Pat. Nos. 6,180,367 and 6,258,560. Synthesis of rhuFab'2 LZ (xCD18) Controlled by an E. coli alkaline phosphatase (AP) promoter. If the AP promoter is induced by phosphate depletion, the promoter may comprise a STII signal-kappa light chain coding sequence; Di-cistronic messenger RNA is formed in the order of the STII signal-heavy chain coding sequence and the leucine zipper sequence. The lambda warrior terminator is located near the translation termination codon.
[127] pcyc34
[128] Plasmid pcyc34 is the tac II promoter counterpart of pS1130.
[129] pxCD18-7T3
[130] PxCD18-7T3, a double-promoter plasmid containing two separate translation units, allows the transcription of the light chain to be temporarily separated from the transcription of the heavy chain. As in pS1130, the light chain is under the control of the pho A promoter. However, in pxCD18-7T3, the λ t ₀ transcription terminator is present after the light chain coding sequence. Downstream of this terminator, a tac II promoter was added to regulate transcription of the heavy chain fragment / C-terminal leucine zipper (DeBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)). . The second λt ₀ transcription terminator is next to this coding sequence. Silent codon variants of the STII signal sequence were used to direct secretion of both chains (Simmons and Yansura, Nature Biotechnology, 14: 629-634 (1996)). Specifically, the nucleotides of the STII signal sequence are modified so that the light chain has a TIR relative intensity of 7, the heavy chain has a TIR relative intensity of 3, and the last three nucleotides of the signal sequences present in front of these light and heavy chains are GCT. It was. In this two-promoter system, the pho A promoter sequence and DNA for the light and heavy chains of the antibody are the same as in pS1130.
[131] pAB3
[132] Plasmid pAB3 is E. coli. It was designed to express anti-CD18 F (ab ') 2 under the control of an alkaline phosphatase promoter in the plasma membrane periplasm (Kikuchi et al., Nucleic Acids Res., 9 (21): 5671-5678 (1981)). , Has a leucine zipper and is His-tagged. Heat-stable enterotoxin II signal sequence (Picken et al., Infect. Immun., 42: 269-275 (1983)) is present in front of the light and heavy chains, followed by six histidine residues on the C-terminus of the heavy chain. Fusion with the following yeast GCN4 leucine zippers. The coding sequences of the light and heavy chains exist in a polycistronic arrangement with the λ ₀ transcription terminator behind the heavy chain gene (Scholtissek and Grosse, Nucleic Acids Res., 15: 3185 (1987)).
[133] Three DNA was ligated with the fragment constructed plasmid pAB3, the first one of the fragment was the vector pS1130 the small Kpn I- Sph I fragment removed. The second part of the ligation was the approximately 645 base-pair Kpn I- Hind III fragment from pS1130. The last part of the ligation was a synthetic DNA duplex with the following sequence:
[134] 5'-AGCTTGTCGGGGAGCGCCATCACCATCACCATCACTAAGCATG (SEQ ID NO: 6)
[135] ACAGCCCCTCGCGGTAGTGGTAGTGGTAGTGATTC-5 '(SEQ ID NO: 7)
[136] pAB21
[137] Plasmid pAB21 is a pAB3 derivative in which six histidine residues on the C-terminus of the heavy chain have been replaced with six lysine residues. This plasmid was constructed in the same way as pAB3 except that the synthetic DNA used for ligation was as follows:
[138] 5'-AGCTTGTCGGGGAGCGCAAAAAGAAAAAGAAAAAGTAAGCATG (SEQ ID NO: 8)
[139] ACAGCCCCTCGCGTTTTTCTTTTTCTTTTTCATTC-5 '(SEQ ID NO: 9)
[140] 2. Plasmids for Expression of Anti-TF Fab'2 LZ-6xhis
[141] The plasmid D3H44-F (ab ') 2 (also known as pD3h44f2), constructed to direct the production of the anti-tissue factor Fab'2 leucine zipper-6xhis, showed that the variable regions of HC and LC were xTF VL from xCD18 VL / VH. It has exactly the same backbone DNA sequence as pAB3 except that it is changed to / VH. The construction of this plasmid is described in WO 01/70984 published September 27, 2001.
[142] Specifically, first, plasmids for the expression of anti-TF Fab (D3H44-F (ab)) were prepared as follows: Plasmid pEMX1 used for mutagenesis and expression of F (ab) s in E. coli is described in Werther et al., J. Immunol., 157: 4986-4995 (1996). In sum, this plasmid comprises a DNA fragment and an alkaline phosphatase promoter encoding consensus human κ subgroup I light chain (VLκI-CL) and consensus human subgroup III heavy chain (VHIII-CH1). The use of consensus sequences for VL and VH is described in Carter et al., Bio / Technology, 10: 163-167 (1992); Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992).
[143] Site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488-492 (1985)) was performed on the deoxyuridine-containing template of pEMX1. Six CDRs were changed to the murine D3 sequence, and residues included in each CDR were CDR-H1 from Kabat et al, supra, and Chothia et al., Nature, 342: 877-833 (1989). CDR-based sequences (Kabat et al., Sequences of proteins of immunological interest), except for CDR-H1 (ie CDR-H1 is defined as extending from residues H26-H35 in the heavy chain) defined using a combination of definitions , Ed. 5, Public Health Service (National Institutes of Health, Bethesda, MD, (1991)) Thus, D3H44-F (ab) is a complete human framework (VLκ) with six complete murine CDR sequences. F (ab) consisting of subgroup I and VH subgroup III) was coded.
[144] A heavy chain hinge (CPPCPAPELLGG; SEQ ID NO: 10) was added to the C-terminus of D3H44-F (ab), followed by the addition of a GCN4 leucine zipper and (his) 6 tag to generate D3H44-F (ab ') 2 (See the description of pAB3 above for leucine zippers and hisx6 tags).
[145] 3. Plasmids for Expression of Anti-VEGF Fab
[146] pY0317
[147] Affinity-matured anti-VEGF Fab protein Y0317 is described in Chen et al., J. Mol. Biol., 293: 865-881 (1999). In order to construct a plasmid, pY0317, to generate it, in short, an expression cassette was used. Cloned to Eco RI site in framework of E. coli plasmid pBR322 (Sutcliffe, Cold Spring Harbor Symp. Quant. Biol., 43: 77-90 (1978)). The expression cassette contained at least the following basic components: (1) pho A promoter for regulation of transcription; (2) λ t ₀ terminator for termination of transcription; And (3) to facilitate translation; Shine-Dalgarno sequence from E. coli trp or heat stable enterotoxin II (STII) gene, or a combination of both. The basic components of the bacterial expression cassettes are known in the art and are described, for example, in Kikuchi et al., Nucleic Acids Res., 9 (21): 5671-5678 (1981) (for the pho A promoter); Scholtissek and Grosse, Nucleic Acids Res., 15: 3185 (1987) (for λt ₀ terminators); Yanofsky et al., Nucleic Acids Res., 9: 6647-6668 (1981) (for trp ); Picken et al., Infect. Immun., 42: 269-275 (1983) (for STII); and Chang et al., Gene, 55: 189-196 (1987) (when using a combination of trp and STII Shine-Dalgano sequences). In addition, the STII signal sequence, or silent codon variant thereof, is ahead of the coding sequence for both the light and heavy chains at pY0317 for the production of anti-VEGF Fabs, indicating that the protein is secreted into the periplasm. (Picken et al., Infect. Immun., 42: 269-275 (1983); Simmons and Yansura, Nature Biotechnology, 14: 629-634 (1996)). For recombinant protein production Nucleotide and amino acid sequences for the expression cassette of the 1952-base pair inserted at the Eco RI site are shown in FIG. 1 (SEQ ID NOS: 1 and 2, respectively).
[148] For other antibodies, see Carter et al., Proc. Natl. Acad. Sci. USA. 89: 4285-4289 (1992); Presta et al., J. Immunol., 151: 2623-2632 (1993); A.4.6.1 of mice (Presta et al., Cancer Res., 57: 4593-4599 (1997) using the method previously described in Werther et al., J. Immunol. 157: 4986-4995 (1996)). )) Monoclonal antibodies were humanized to generate RhuFab V2 Y0317. In sum, cDNA encoding muMAb A.4.6.1 variable light and variable heavy chains was isolated from mouse monoclonal antibody producing hybridoma cells using RT-PCR. These cDNAs were cloned and fused to human CL and human CH1 domains (Werther et al., J. Immunol., 157: 4986-4995 (1996)) to generate mouse-human chimeric Fabs. Six complementarity-determining regions (CDRs) (shown in bold in FIG. 1) were implanted into already humanized antibody vectors encoding consensus human K subgroup I light chain and consensus human subgroup III heavy chain (Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992). Binding to the VEGF antigen was reduced 1,000-fold by only delivering CDR residues to the human framework. Several framework residues near the CDRs (represented in italics and underline in FIG. 1) were also altered to enhance binding to the target (Presta et al., Cancer Res., 57: 4593-4599 (1997)). In all, seven heavy chain residues and one light chain residue were altered outside of the CDRs. The heavy and light chains were then transferred into phage-display vectors (Baca et al., J. Biol. Chem., 272: 10678-10684 (1997)) to replace the hGH gene of phGHam-g3 (Bass et al. , Proteins, 8: 309-314 (1990)). VL Met4Leu was altered by site-directed mutagenesis to interfere with methionine oxidation, and VH Thr231Leu was altered to facilitate cloning into gene III fusions. This vector was named Y0101 and was used as a starting point for the optimization of CDRs in binding to VEGF antigens (Muller et al., Structure, 6: 1153-1167 (1998)). Only mutations in CDRs H1 and H3 have been shown to enhance binding and incorporated into the final form of pY0317. The change from pY0101 plasmid to pY0317 plasmid is as follows: Thr28Asp, Asn31His, His101Tyr, Ser105Thr. These changes are all within the variable heavy chain region. The pY0317 plasmid is a Fab phage display vector. The plasmid schematic of this plasmid appears to be as in FIG. 2A.
[149] pY0317tet20
[150] this. Plasmid pY0317tet20 was constructed to direct the production of rhuFab V-2 in E. coli. 2A and 2B show flow charts for the plasmid constructs beginning with pY0317. Plasmid pY0317tet20 is a modified form of the well characterized pBR322 plasmid. A 639-base pair of Ava I- Pvu II fragments were removed from the pBR322 site of the plasmid. This deletion eliminates the rop gene involved in copy number regulation (Cesareni et al., Proc. Natl. Acad. Sci., USA, 79: 6313-6317 (1982)). Thus, the plasmid has a slightly increased copy number compared to pBR322. An expression cassette of a 1952-base pair (FIG. 1) was inserted into the Eco RI site for recombinant protein production. Plasmid pY0317tet20 is resistant to both tetracycline and β-lactam antibiotics. The expression cassette comprises a single copy of the light and heavy chains connected in line. Each gene is transcribed into a single di-cistronic mRNA. Directed by the E. coli pho A promoter (Chang et al., Gene, 44: 121-125 (1986)). The translation-initiation signal for each strand is two. E. coli STII (heat-stable enterotoxin) is provided by the shine-dalgano sequence. Translation of each chain begins with 23 residues of the STII signal peptide that direct the peptides to cross the cytoplasmic membrane to the periplasm (Picken et al., Infection and Immunity, 42: 269-275 (1983)). Thereafter, the STII signal peptide was identified as E. coli. Eliminated by E. coli leader peptidase. The light and heavy chains are folded into their natural form after secretion into the periplasm, and are covalently linked by intermolecular disulfide bonds.
[151] Tetracycline resistance is located on the final vector via modification of pY0317 (see FIGS. 2A and 2B). The 3642-base pair Sap I / Apa I fragment of pY0317, containing half of the replication origin of pBR322, the β-lactamase gene, the pho A promoter, the entire light chain and the heavy (VH) amino terminus, was converted to the 2738-base pair SapI of p6G4V11N35A.PEG. / ApaI fragment was ligated. This second fragment comprises the CH1 region of the heavy chain and the tetracycline-resistant gene from pBR322. This fragment also contains four particular amino acids at the carboxyl terminus of the heavy chain for site-specific modification of the protein. The region comprising these four special residues and the CH1 region were removed by Bss HII / Hpa I cleavage and replaced with the Bss HII / Xba I fragment of pY0317 to recover the original heavy chain sequence and delete the site-specific modification region. . Xba I cleavage was performed first, and the overhang portion was filled with Klenow and deoxynucleotides. Then, the Bss HII cutting water gel purification of the 433- bp fragment is carried out. Nhe I- in a pBR322 - by replacing the Nde I fragment, a Nhe I / Nde I fragment of pBR322 that contains the 639- bp Ava I- Pvu II deletion was carried out the last operation of the plasmid. The final plasmid pY0317tet20 is resistant to tetracycline and β-lactam antibiotics and includes the pho A promoter and genes encoding the light and heavy chains of anti-VEGF.
[152] 4. Plasmids for Expression of Apo2L
[153] pAPApo2-P2RU is described in WO 01/00832, published January 4, 2001. In short, the plasmid, the construct shown in FIG. 3, encodes the coexpression of Apo-2L (amino acid residues 114-281 ) and tRNA encoded by pro2 and argU , which coexpression is regulated by an alkaline phosphatase promoter . pBR322-based plasmid (Sutcliffe, Cold Spring Harbor Symp. Quant. Biol., 43: 77-90 (1978)) using pAPApo2-P2RU. E. coli produced Apo-2L. Transcription and translation sequences required for expression of Apo-2L are provided by the alkaline phosphatase promoter and trp shine-dalgano as described for plasmid phGH1 (Chang et al., Gene, 55: 189-196 (1987)). . The coding sequences (114-281) for Apo-2L are located downstream of the promoter and shine-dalgano sequences and behind the starting methionine. This coding sequence is Apo-2L (Fig. 4 (SEQ ID NO: 3 for the nucleotide and amino acid sequences, respectively) except that the codon encoding residue Pro119 has been replaced with "CCG" instead of "CCT" to eliminate potential secondary structure. And nucleotides encoding residues 114 to 281 of 4) (shown in FIG. 4). The sequence encoding the lambda t ₀ transcription terminator (Scholtissek et al., Nucleic Acids Res., 15: 3185 (1987)) is behind the Apo-2L coding sequence.
[154] In addition, the plasmids include pro2 (Komine et al., J. Mol. Biol., 212: 579-598 (1990)) and argU / dnaY (Garcia et al., Cell, 45: 453-459 (1986)). Sequences for expression). The gene was purified by PCR. Cloned from E. coli W3110, this gene was located downstream of the lambda relative to the lambda t ₀ transcription-termination sequence. The plasmid confers both tetracycline and ampicillin resistance to the production host.
[155] B. Cell Transformation
[156] Competent cells of the relevant strains were prepared and transformed into appropriate plasmids by standard methods, and successful transformants were selected and cultured. For plasmids resistant to tetracycline, transformants were picked out from LB plates (LB + Tet20) containing 20 μg / mL tetracycline, streaked-purified, and 30 ° C. shaker / incubator. In an LB broth containing 20 μg / mL tetracycline and then stored at −80 ° C. in DMSO.
[157] For plasmid pxCD18-7T3 and pcyc34, additional plasmid pMS421 was co-transformed with pxCD18-7T3 or pcyc34. pMS421 is pSC101- substrate is IPTG is added The plasmid is inhibited as to release the suppression of the tac II promoter and inhibit the induction of the promoter, plasmids for overexpression cut Iac Iq suppressed to suppress the induction of the tac II promoter was also Confers spectinomycin and streptomycin resistance. The plasmid provides in multiple copies the lac Iq chromosomal gene from the lac Iq strain under the control of its own promoter, which is inserted into the affinity plasmid pSC101.
[158] C. Antibody Extraction
[159] 20 OD-mL pellets were suspended in 500 μl of 200 mM TRIS-HCl (pH 8.0) containing 20 μl of 0.1 M EDTA (pH 8.0) and 10 μl of lysozyme (6 mg / mL). Soluble fractions of E. coli cells were prepared. The mixture was vortexed, sonicated with 7-10 pulses, and then centrifuged at 15,000 rpm for 15 minutes at 4 ° C. The supernatant fraction after centrifugation is called high-salt extract (HSE). The remaining pellets were used for insoluble fraction analysis.
[160] D. Protein Identification
[161] One-dimensional SDS-PAGE gel electrophoresis was performed in a 4-12% linear acrylamide gradient from Novex. Specifically, the system used was the NOVEX® NuPage® system consisting of NuPAGE Bis-TRIS Pre-Cast gel (for low to medium molecular weight proteins).
[162] Fixed pH gradient in the first dimension, purchased from Amersham Pharmacia Biotech, as described in Champion et al., Electrophoresis, 20 (4-5): 994-1000 (1999) Two-dimensional gel electrophoresis was performed with (pH 3-10) and linear acrylamide gradient (9-18% T) in the second dimension. Identification of the protein was determined using a combination of silver / Coomassie staining, NH ₂ -terminal sequencing and mass spectrometry analysis. For analytical gels, E. coli lysates (about 40 μg of protein) were combined with the rehydration solution as described in Champion et al., Supra. A non-linear fixed pH gradient (IPG) gel strip (Amersham Pharmacia Biotech) at 18 cm, pH 3-10 was used for a total of 50,000 Vh isoelectric focusing.
[163] The preloaded gel was blotted onto polyvinylidene difluoride (PVDF) membrane (ProBlott; Applied Biosystems) as described by the manufacturer. NH ₂ -terminal sequencing was performed using a 20-minute Edman cycle and multiple sample horizontal flow reactors for sequencing of PVDF-electroblotted proteins (Henzel et al., Analytical Biochemistry, 267: 148-160 ( 1999). Samples eluted from the gels were analyzed by MALDI-TOF mass spectrometry and capillary LC-MS to evaluate low molecular weight light chain-specific spots (Champion et al., Supra).
[164] E. Measurement of Target Protein Species
[165] As described below, anti-CD18 F (ab ′) 2 LZ titers were determined using AME5® reversed phase double-column analysis (AME5® / RP double-column analysis).
[166] F. AME5® / RP Double-Column Analysis
[167] 1. Tools and Equipment
[168] The INTEGRAL® workstation (PerSeptive Biosytems) was set up in a double-column gradient batch. Target proteins were captured using an affinity column containing an anti-light chain (kappa) Fab antibody (AME5®) immobilized on controlled-pore glass (CPG). The captured antibody species was further developed using a reversed phase column controlled at 60 ° C. Activated aldehyde immunoaffinity resin (AL-20), reverse phase POROS resin (R220) and column-packing device were purchased from Perceptive Biosystems (Cambridge, Mass., USA). CPG Empty PEEK columns (30 × 2.1 mm (100 μl)) were purchased from Upchurch Scientific (Oak Harbor, WA, USA). Using an ACRODISC® PF syringe 5-micron filter (manufactured by Gelman Sciences), E. coli. Coli samples were filtered.
[169] 2.AME5® anti-human kappa FAb (his-gly) ₄purification of his- (lys)
[170] Phosphate-buffered saline (pH 7.2) (PBS) comprising 9.4 mM sodium phosphate, 136.9 mM sodium chloride and 2.7 mM potassium chloride is referred to herein as a loading buffer. Monoclonal antibodies are E. coli. Obtained from AME5® anti-human kappa FAb (his-gly) ₄ his- (lys) ₃ , a mouse FAb herein referred to as AME5® FAb hgk, purified from E. coli paste. this. E. coli paste was obtained from 10-liter fermentation of 27C7 cells. Microfluidizer was used to homogenize the cells after suspension in 20 mM sodium phosphate, 0.25 M sodium chloride, 10 mM magnesium chloride and 2 mM imidazole, pH 7.0. 0.2% polyethyleneimine (PEI) was added and centrifuged to give E. coli. E. coli extract was cleared. The clarified extract was purified using a combination of ion exchange and immobilized metal-ion-chelating (IMAC) chromatography steps. Chelating SEPHAROSE FAST FLOW® and SP SEPHAROSE FAST FLOW® resins were purchased from Amersham Pharmacia.
[171] 3. Immobilization of AME5® FAb hgk with Activated Glyceryl-Coated CPG
[172] Purified Fab was immobilized on periodate-activated glyceryl-coated controlled pore glass (CPG) to prepare an affinity resin. The method of Roy et al., J. Chromatography, 303: 225-228 (1984) was modified to immobilize AME5® FAb hgk antibody on activated glyceryl-coated CPG.
[173] Dry CPG was wet with purified water, packed on a chromatography column, and activated for 30 minutes by recycling 1% sodium metaperiodate (Sigma S-1878®) through the column. The activated resin was then washed in 20 mM sodium phosphate, 0.15 M sodium chloride, pH 7.2, coupling buffer.
[174] AME5® FAb hgk antibody at a concentration of approximately 5 mg / mL in coupling buffer with 1 μg / mL reducing agent sodium cyanoborohydride (Sigma S8628) was recycled through the activated resin layer. The coupling of the antibody to the resin was monitored by a decrease in absorbance at 280 nm. When the absorbance was not further reduced, any remaining antibody was washed and recovered using coupling buffer. Coupling density was determined by the difference between the starting amount and recovery after the reaction was terminated and reported in mg FAb per mL of resin.
[175] Thereafter, 1 M ethanolamine (pH 8.0) (ICN, Cat. No. 151078) was recycled for 2 hours in the presence of 1 μg / mL sodium cyanoborohydride to react with the active site remaining on the resin. The resin was then washed in coupling buffer containing 0.01% thimerosal (GDL International) for storage. The resin was precirculated three times between the equilibration buffer and the elution buffer to be used, then any protein was added.
[176] 4. Reagents and Methods of Analysis
[177] Solvent stocks were as follows: solvent 1A, affinity loading buffer; Solvent 1B, reversed phase aqueous buffer and affinity elution buffer, 0.1% TFA in water; Solvent 2A, water; Solvent 2B, reverse phase organic elution buffer, 0.09% TFA / 80% acetonitrile. this. E. coli HSE (diluted 1: 2), or 50 μl of supernatant of fermentation broth in loading buffer was injected. All forms of anti-CD18 present in fermented cell extracts were determined by comparing 2-D-gels of affinity-captured (AME5®) material from blank development, production development, and production development. Captured by the AME5® antibody. Non-specific adsorption was reduced (by washing with PBS), then the affinity column was placed in line with the reverse phase column and eluted with dilute acid to deliver the captured components. The reverse phase column was then eluted with a small acetonitrile gradient to analyze the components. Detection was performed by measuring absorbance at 280 nm, and the circular antibodies were quantified by comparing the peak areas of similarly treated standards.
[178] G. Identifying Peaks in the Chromatogram
[179] This assay resolved the anti-CD18 fragment into five antibody-related peaks, which peaks represent the following antibody fragments:
[180] Peak 1: LC-115 (115 amino acid degradation product of kappa light chain)
[181] Peak 2: Unassembled Free Light and Glutathiated Light Chains
[182] Peak 3: Light Chain Dimers
[183] Peak 4: Fab-Like Fragments
[184] Peak 5: Fab'2-LZ or Fab'2 Fragment
[185] Purified bulk anti-CD18 F (ab) '2 release material (5 mg / mL) was used as standard. E. coli derived from high cell density fermentation of 49A5 / pS1130. E. coli extract was frozen at -70 ° C and used as a positive control. Equivalent cell mass was loaded for all samples to be compared.
[186] H. Total HC / LC POROS (R) Reverse Phase Analysis
[187] To assess the total amount of light and heavy chain fragments produced in the fermentation, selective reverse phase HPLC analysis (RP-HPLC) was used. For total antibody expression, 100 μl of total broth was added to 100 μl of 0.2 M TRIS 8.0. After sonication with 10 pulses, 650 μl guanidine-HCl / 50 mM TRIS (pH 9) and 50 μl 2 M DTT were added and incubated for 15 minutes at room temperature. After loading into the column, 200 μl of acetonitrile was added and filtered through a size-exclusion spin column (Pharmacia). 5 μl of this suspension was analyzed by POROS® reversed phase analysis.
[188] For the reverse phase method, a perceptive POROS® R-1 reverse phase column was used for HEWLETT-PACKARD® 1100 HPLC. The analysis was performed on a column heated to 60 ° C. and the UV absorbance at 278 nm was monitored. The column was equilibrated in a 28% acetonitrile solution in water with 0.1% trifluoroacetic acid. 25 μl of sample was then loaded onto the column, eluted over 20 minutes using acetonitrile linear gradient of 28% to 38%, then regenerated with 95% acetonitrile for 17 minutes, 28% aceto Re-equilibrate with nitrile. For identification, peaks for light and heavy chain related species were identified by comparing the standard and analyte using a HEWLETT-PACKARD® mass screening detector. Fermentation samples from blank development using the same host were similarly prepared and analyzed with the exception of plasmids that did not contain sequences for the heavy and light chains to determine the appropriate base level for analysis. Integration of peak areas was performed using the HEWLETT-PACKARD® 1100 software, and a calibration curve was generated by spiking a standard into a blank developing sample to determine the relative amounts of the various species in the sample.
[189] For soluble samples, lysates were prepared for ion-exchange analysis. Typically, 100 μl of sample was diluted with 650 μl of 6 M guanidine-HCl, 50 mM TRIS-HCl, pH 9. Then 50 μl of 2 M dithiothreitol (freshly thawed) was added, 200 μl of acetonitrile was added, filtered through a 0.2 μm filter and then loaded onto HPLC.
[190] In addition, PBS-washed insoluble pellets obtained after cell extraction were resuspended in 100 μl of 0.2 M TRIS 8.0 and mixed well to analyze insoluble lysate samples similarly. Then 650 μl 6 M guanidine-HCl / 50 mM TRIS-HCl (pH 9), 50 μl 2 M DTT and 200 μl acetonitrile were added. The sample was then filtered and the filtered sample was analyzed using the same method as for the soluble lysate sample.
[191] I. CSX Analysis
[192] Cleavage of anti-CD18 Fab'2 LZ was analyzed by HPLC cation-exchange chromatography. Specifically, the BAKERBONDJ® Carboxy-Sulfone (CsX) 50 x 4.6-mm column (JT Baker, Phillipsburg, NJ) was diluted at least 1: 1 and kept at 55 ° C on a Hewlett-Packard 1090 HPLC system. )). Samples were eluted using approximately 5-50 mM sodium phosphate (pH 7.0) gradient over 14 minutes, and the peaks were monitored using absorbance at 278 nm. Peaks containing anti-CD18 Fab'2-leucine zippers were identified and quantified in comparison to purified standards.
[193] J. Cell Line Generation
[194] The host used for the rhuFab'2 LZ (xCD18) fermentation was E. coli. Derivatives of E. coli W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219), 49A5, 58B3, 59A7, 43H1, 58H2, 45F8, 41H1 and Represented by 33D3. 5 is this. A scheme for inducing E. coli strains 59A7, 49A5 and 43H1 is shown.
[195] 1.Strain 49A5
[196] The complete genotype of 49A5 is ΔfhuA phoA ΔE15Δ (argF-lac) 169 deoC2 degP41 (Δpst1-Kan ^r ) IN (rrD-rrE) 1 ilvG2096 (Val ^r ) ΔfucP ΔmalE . Lee, the starting strain. E. coli W3110 is F'- and lambda minus. Derivative of E. coli K-12. It is known to retain the inversion of chromosomes between rrnD and rrnE (Bachmann., Supra; Hill and Harnish, Proc. Natl. Acad. Sci. USA, 78: 7069-7072 (1981)). The fhu A gene ( formerly known as ton A) was deleted from W3110 by incorrect cleavage of Tn 10 followed by insertion into its fhu A gene. The resulting strain 1A2 is resistant to bacteriophage T1, T5 and ø80.
[197] Two deletion mutations, phoA ΔE15 (Sarthy A. et al., J. Bacteriol., 145: 288-292 (1981)) and arg-lac 169 (Schweizer et al., Mol. Gen. Genet) , 192: 293-294 (1983)) were simultaneously introduced into strain 1A2 by P1 cotransduction with Tn5 inserts linked in the pro C gene. Accurate cleavage of the transposon restored the pro C gene. The phoA ΔE15 mutation eliminates alkaline phosphatase expression, and the Δ (argF-lac) 169 mutation represents the lac ⁻ phenotype of this strain, denoted 7C1.
[198] Deo C2 mutations with deoxyribose phosphate aldolase expression were introduced by P1 cotransduction . deo C locus is genetically associated with threonine biosynthetic locus. Threonine nutrients were generated by Tn 10 insertion and incorrect cleavage. Subsequently, threonine nutrients were transduced with threonine proteome using P1 phage grown on deo C3 mutants. The presence of the deo C2 mutation was confirmed by the resultant strain 16C9 could not grow on 0.2% thymidine as a carbon source.
[199] The degP41 (ΔPst1-Kan ^r ) mutation, which is a mutation in the gene for the plasma membrane protease, was introduced by transduction. This mutation was made by replacing the deg P gene portion with the kanamycin-resistant gene in vitro (Strauch and Beckwith, J. Bacteriol., 171: 2689-2696 (1989)). This is not a transposon, and deletions can be selected using kanamycin resistance. The resulting strain is represented by 23E3.
[200] ilvG2096 (Val ^r ) mutation (Lawther et al., Proc. Natl. Acad. Sci. USA, 78: 922-925 (1981)) was introduced by homogenotization. This mutation is a wild type. Restores the frameshift that makes the Coli K-12 sensitive to valine. Strain 23E3 was transformed using the plasmid pAH29 (Lawther et al., supra) comprising the ilvG2096 (Val ^r ) marker and the ampicillin-resistant gene. The strain represented by 33B6, which naturally lost the plasmid and obtained the desired allele, was identified by screening for ampicillin-sensitive clones for valine resistance.
[201] Finally, two mutations in the carbohydrate-use pathway were introduced to allow the host to be distinguished from other recombinant hosts by a simple carbohydrate utilization test. Deletion mutations in fuc P and mal E were made by PCR and introduced separately into plasmid vectors containing beta-lactamase and levan sucrase (Bass et al., supra). Each whole plasmid was recombined into chromosomes of W3110 derivatives that did not support independent replication of the plasmid vector (Bass et al., Supra). Strain 33B6 was then transduced with carbenicillin resistance using P1 phage grown on W3110 derivatives carrying the fuc P deletion plasmid integrated into the chromosome. Levan sucrase, and therefore derivatives that no longer express sucrose resistance, were selected and screened for reduced carbenicillin resistance and loss of fucose availability. PCR confirmed that the resulting strain, 49B2, retained the planned fuc P deletion.
[202] The above steps were repeated to introduce mal E deletions. Strain 49B2 was transduced with carbenicillin resistance using P1 phage and grown on strains with mal E deletion plasmids integrated into the chromosome. Subsequently, sucrose-resistant derivatives were selected and screened for loss of carbenicillin resistance and loss of fucose availability, and the presence of mal E deletion was confirmed by PCR.
[203] Important features of strain 49A5 include the following:
[204] ㆍ Resistant to T1 phage.
[205] • When phosphate is depleted (conditions used to induce product synthesis), do not overproduce alkaline phosphatase.
[206] • Lack of protease.
[207] ㆍ Not sensitive to valine toxicity.
[208] Can be distinguished from other hosts by carbohydrate-use tests.
[209] 2. Strain 58B3
[210] Strain 58B3 was also derived from strain 33B6. Δprc :: pS1080 genotype (Bass et al., Supra ; Metcalf et al., Gene, 138: 1-720 (1994)) was introduced into the kan ^s derivative (56G4) of strain 33B6 by P1 transduction, 42 Screened for poorly growing colonies on half concentration LB containing less salt at &lt; RTI ID = 0.0 &gt; The kan ^s strain had a deg P deletion derived from pKS16 (Strauch and Beckwith, 1989, supra) and exhibited a kanamycin-sensitive phenotype. Thus, the 58B3 strain is a kan ^s strain bearing both deg P and prc deletions.
[211] The complete genotype of the 58B3 strain is W3110 ΔfhuA phoAΔE15 Δ (argF-lac) 169 deoC degP41 IN (rrD-rrE) 1 Kan ^s ilvG2096 (Val ^r ) Δprc .
[212] 3. Strain 59A7
[213] This strain was generated by introducing a Prc inhibitor (Spr mutant) into the 58B3 strain. P1 phage lysate of 51B9 strain ( tonA prc prc sup zeg722 :: Tn10 ) was transduced into 58B3 strain, screened for tet-resistant colonies and at half concentration of Prc inhibitor phenotype (42 ° C. with less salt) Screened well on LB). The new strain is called 58F1. Δprc mutations cannot survive at 42 ° C. Tetracycline-resistant genes were plated on Malloy plates to remove from 58F1, resulting in tet ^s -sensitive strains, denoted 59A7. The complete genotype of the 59A7 strain is W3110 ΔfhuA phoAΔE15 Δ (argF-lac) 169 deoC degP41 IN (rrD-rrE) 1 Kan ^s ilvG2096 (Val ^r ) Δprc spr W148R.
[214] The original 51B9 strain has a Prc inhibitor Spr with point mutation W148R which is the same Spr as in the 43H1 and 59A7 strains.
[215] 4. Strain 43H1
[216] The complete genotype of the 43H1 strain is very similar to that of 49A5: W3110 ΔfhuA phoAΔE15 Δ (argF-lac) 169 degP41 (Δpst1-Kan ^r ) IN (rrD-rrE) 1 ilvG2096 (Val ^r ) ptr3ΔompT prc :: kan ^r spr W148R. This strain carries three more protease markers Ptr3 OmpT and Prc than 49A5. This strain has a point mutation (W148R) in Spr. The strain is Kan ^r .
[217] 5. Strain 58H2
[218] 43H1 strain was transduced with tet ^r using P1 phage grown on strain 42E3. This strain (58F9) was recovered for the prc :: kan ^r mutation and thus became kan ^s . This strain was then plated on minimal glucuronic acid medium to remove eda :: Tn 10. The newly generated strain 58H2 was kan ^s and became a three-protease mutation with wild type prc . The complete genotype of the 58H2 strain is W3110 ΔfhuA phoAΔE15 Δ (argF-lac) 169 degP41 (Δpst1-Kan ^r ) IN (rrD-rrE) 1 ilvG2096 (Val ^r ) ptr3 ΔompT spr W148R.
[219] 6. Strain 45F8
[220] The complete genotype of the 45F8 strain is W3110 ΔfhuA Δ (argF-lac) 169 degP41 Kan ^s ΔompT ptr3 ilvG2096 (Val ^r ) phoS * (T104) . This strain is a pho S strain with three protease markers.
[221] 7. Strain 41H1
[222] The complete genotype of the 41H1 strain is W3110 ΔfhuA phoS * (T104) Δ (argF-lac) 169 degP41 (Δpst1-Kan ^r ) ptr3 ilvG2096 (Val ^r ) T adapted at 37 ° C. This strain is a pho S strain with a two-protease marker.
[223] 8. Strain 33D3
[224] The complete genotype of the 33D3 strain is W3110 ΔfhuA ptr3 lacIq lacL8 ΔompT degP41 (ΔpstI-kan ^R ) . A description of strain generation can be found, for example, in US Pat. No. 5,789,199.
[225] K. Shake Flasks and Fermentation Cultures
[226] In shake-flask experiments, Luria-Bertani (LB) broth and CRAP Minimal Medium containing 5 μg / mL AMPICILLINE® antibiotic were used. CRAP minimal media was prepared as follows: (NH ₄ ) ₂ SO ₄ 3.57 g, NaCitrate-2H ₂ O 0.71 g, KCl 1.07 g, yeast extract 5.36 g and HYCASE SF-SHEFFIELD® 5.36 g were mixed and The pH was adjusted to 7.3 with KOH, and the volume was adjusted to 872 mL with deionized water. The mixture was then autoclaved and cooled to 55 ° C. 110 mL of 1 M MOPS buffer (pH 7.3), 11 mL of 50% glucose and 7.0 mL of 1 M MgSO ₄ were added.
[227] As used herein. E. coli fermentation method was a high cell density method as defined above. In order to reach higher cell density, ammonia was added continuously and additional micronutrients (P, K, S and Mg) were added at various stages of the fermentation process to support cell growth. Reducing the amount of nutrients constituted another method of lower final optical density of broth containing products of equivalent quality, which is referred to herein as a low cell density method.
[228] Single vial containing 1.5 mL of culture in 10-15% DMSO, 1-liter shake containing 500 mL of LB medium supplemented with 0.5 mL of tetracycline solution (5 mg / mL) and 2.5 mL of 1 M sodium phosphate solution Thaw in the flask. These seed cultures were incubated at 30 ° C. for approximately 16 hours and then used to inoculate 10-liter fermenters.
[229] Initially, the fermentor is about 4.4 g of glucose, 100 mL of 1 M magnesium sulfate, trace element solution (100 mL of hydrochloric acid in 1 liter of final volume, 27 g of iron chloride hexahydrate, 8 g of zinc sulfate heptahydrate, 7 g of cobalt chloride hexahydrate, 7 g of sodium molybdate dihydrate, 8 g of copper sulfate pentahydrate, 2 g of boric acid, and 5 g of manganese sulfate monohydrate, 10 mL of tetracycline solution (in 5 mg / mL ethanol), FERMAX ADJUVANT 27 (registered trademark) ( Or 10 mL of some equivalent anti-foam), HCD salt (37.5 g ammonium sulfate, 19.5 g potassium phosphate dibasic, 9.75 g sodium phosphate monobasic dihydrate, 7.5 g sodium citrate dihydrate, and 11.3 g potassium phosphate monobasic) and about 6.5 L of medium containing 200 g of NZ amine A (protein hydrolyzate). Fermentation was carried out with an air stream of 10 slpm at 30 ° C. and adjusted at pH 7.0 ± 0.2 (in some cases it may sometimes be outside this range). The rate of oxygen delivery in the fermenter was controlled by varying the back pressure and the stirring rate of the fermentor, and consequently, the cellular respiration rate.
[230] The fermentor was inoculated with the cell containing medium from the shake flask and then cultured at high cell density in the fermentor using a computer based algorithm to feed the concentrated glucose solution into the fermentor. In addition, when it was necessary to adjust the pH, ammonium hydroxide (58% solution) and sulfuric acid (24% solution) were fed to the fermentor. In some cases, the addition of anti-foam was added to control foaming. When the culture reached a cell density of about 40 OD550, 100 mL of 1 M magnesium sulfate was further added to the fermentor. In addition, with a concentrated salt (about 10 g of ammonium sulfate in 1 L of water, 26 g of potassium dibasic phosphate, 13 g of monobasic sodium phosphate dihydrate, 2 g of sodium citrate dihydrate and 15 g of monobasic potassium phosphate) fed to the fermentor Feed), starting at a rate of 2.5 mL / min when the OD550 of the culture reached about 20 and continuing until about 1,250 mL was added to the fermentation. Fermentation typically continued for 72 to 80 hours.
[231] During fermentation, when dissolved oxygen reached a set point for fermentation, a concentrated glucose solution was supplied based on the dissolved oxygen irradiation signal that controls the dissolved oxygen concentration at that set point. Thus, in this controlled scheme, the adjustment of fermentor operating parameters such as stirring speed or back pressure affects the oxygen transfer capacity in the fermentation, thus controlling the rate of oxygen uptake or metabolism of the cells.
[232] The mass spectrometer was used to monitor the composition of the offgas from the fermentation and the oxygen uptake and carbon dioxide emission rates could be calculated in the fermentation.
[233] When the OD550 of the culture reached a cell density of about 220, the stirring speed was slowed from the initial 1,000 rpm to about 725 rpm over about 12 hours.
[234] Fermentation of cells transformed with pMS421 and pcyc34 where the expression of both heavy and light chains was controlled using the tac II promoter, or fermentation of cells transformed with pMS421 and double-promoter plasmid pxCD18-7T3 heavy chain expression was controlled using the tac II promoter), approximately 12 hours after the OD550 of the culture reached a cell density of 200, heavy and light chain synthesis for pcyc34 by adding 50 mL of 200 mM IPTG, and pxCD18- Heavy chain synthesis for 7T3 was induced.
[235] result
[236] A. Discovery and Identification of Kappa Light Chain Cleavage Products
[237] Soluble E. suspended in SDS sample buffer (commercially available product commonly used for the development of SDS gels). E. coli extract (see HSE in Materials and Methods) and residual pellets were analyzed by SDS-PAGE. E. coli in a 49A5 strain carrying the pS1130 plasmid for the production of rhuF (ab) '2LZ (xCD18). Samples were derived from 20 OD-mL pellets collected during high cell density (HCD) fermentation of E. coli. In the soluble fraction, a 115 amino acid long kappa LC cleavage fragment was identified. In the insoluble fraction, a 182 amino acid long kappa LC cleavage fragment was identified. All fragments were sequenced by delivery to PVDF membrane. Both had the correct N-terminus as the processed form of kappa-LC. Masses measured by mass spectrometry were 12488.5 and 19857.2 Da, respectively. Proteolytic cleavage sites were between residues Val 115 and Phe 116 for LC-115 and between residues Ser182 and Lys183 for LC-182. Only one site appears to be a typical Prc clipping site.
[238] At the end of said fermentation, E. coli 20 OD-mL pellets were analyzed by two-dimensional gel electrophoresis. Teeth of pellets. E. coli lysates (about 40 μg of protein) were combined with the rehydration solution as described in Champion et al., Supra. On a 2-D gel pattern of cells derived from 49A5 / pS1130 fermentation, the resulting gel was compared at a similar time point (49A5 / pBR322) to a kappa-light chain-specific spot compared to the blank 2-D gel derived from the cell pellet of the fermentation. Confirmed. The pellets were selected from the same time point for both fermentations and assume that the cells should be in a similar metabolic state. All kappa LC spots were identified by immunoblot using alkaline phosphatase-linked anti-human kappa LC antibodies.
[239] In addition to the two main clips identified by the 1-D gel analysis, the 2-D gel showed circular LC, iso-form of circular LC, and at least 5 minor LC clips (see FIG. 6). Corresponding spots were eluted and sequenced. All LC-specific peptides had the correct N-terminus, indicating that they were all processed sufficiently to cleave the STII signal. All these peptides were analyzed by mass spectrometry to determine the approximate mass. Because minority clips are present in traces, the exact mass to determine the clipping site of the fragment could not be obtained.
[240] Three minor clips were clustered with kappa LC-115 clips at pI values of about 9. The fourth clip had a pi value of about 6.5 and the fifth clip had the same pi value as the LC-182 clip of pi about 6. To determine the solubility of this LC fragment, HSE of the same pellet was loaded on a 2-D gel. Only LC182 fragments were present in the insoluble fraction.
[241] B. Prc is the only protease that performs cleavage of kappa-light chains
[242] E. coli expressing anti-CD18 Fab'2 LZ molecules. 1-D SDS-PAGE gels were loaded that were loaded with insoluble fractions of cells derived from four different fermentations of the E. coli protease mutants 49A5, 45F8, 41H1 and 43H1. LC-182 proteolytic cleavage was present in three of the four samples (not present in the prc -deleting strain 43H1), suggesting that Prc protease may be involved in kappa-LC cleavage. Peak 1, corresponding to the LC-115 clip, present in the sample derived from strain 49A5 ( prc -plus), was also 43H1-derived as compared to chromatograms resolved by AME5® / RP double-column analysis. No sample was taken. This assay selectively absorbed kappa-LC-containing antibody species and then resolved them into five peaks as described in the Materials and Methods section above.
[243] Analysis of the 2-D gel of the 43H1-derived cell pellet confirmed that not only the LC-115 and LC-182 fragments appeared in the gel, but also all other LC-related minor species (see FIG. 7). These results strongly suggest that Prc is the only enzyme responsible for cleaving kappa-LC. These 43H1 cell pellets were derived from low cell density fermentation.
[244] C. Strain Formation Confirms that Prc is the Only Enzyme Involved in Kappa-Light Chain Cleavage
[245] One. prcPlus prcDeletion strain
[246] By recovering the 43H1 strain ( prc -minus host with four protease markers) into the prc -plus, three-protease strain (58H2), evidence was obtained that Prc was the only enzyme involved in kappa-LC cleavage. Strain 42E3 has eda-51 :: Tn10 cotransduced with prc. 43H1 strain was transduced with tet ^r using P1 phage grown on 42E3. The resulting strain (58F9) was repaired for the prc :: kan ^r mutation, so the strain became kan ^s . This strain was then plated on minimal glucuronic acid medium to remove eda :: Tn10 . The newly generated strain 58H2 became three protease mutations with wild type prc . This isolate is a transconductor or natural Eda ⁺ isolate. The prc -plus genotype was confirmed by PCR. This 58H2 strain still retains a prc inhibitor ( spr ^W148R ) derived from ^43H1 and is kan ^s . LC-clips reappeared in the 58H2 strain were detected by AME5® / RP double-column analysis (see FIG. 8).
[247] 2. prcNatural strains that lack genes prc-It became negative
[248] Strain 49A5 was a prc wild type strain as described above. When the prc deletion was introduced into the strain background to generate a 58B3 strain and the cell extract was analyzed by the AME5® / RP double-column method, no LC-115 clip (Peak 1) appeared. Strain 58B3 was derived from the 33B6 strain carrying only DegP, a protease marker. Δprc :: pS1080 (Bass et al., Supra ; Metcalf et al., Supra) was introduced into the kan ^s derivative (56G4) of 33B6 by P1 transduction to generate the degP Δprc two-protease strain 59A7. .
[249] A summary of the cleavage results of all seven strains is shown in Table 1 below.
[250] E. coli expressing anti-CD18 F (ab) '2 leucine zipper. E. coli host strain this. E. coli hostProtease markersLC decomposition 49A5DegP+ 45F8DegP Ptr3+ 41H1DegP Ptr3 OmpT+ 43H1DegP Ptr3 OmpT △ Prc Spr ^W148R - 58H2DegP Ptr3 OmpT Spr ^W148R + 58B3DegP △ Prc- 59A7DegP △ Prc Spr ^W148R -
[251] D. prcImproved Yield of rhuFab'2 LZ (xCD18) in Negative Hosts
[252] 1. Shake Flask Results
[253] Three strains expressing rhuFab'2 LZ (xCD18) (49A5, 43H1 and 58H2) were first incubated overnight at 30 ° C. in LB broth + Amp. All cultures were then subjected to C.R.A.P. Equivalently inoculated into shake flasks containing a minimum of medium plus 25 mL of Amp and shaking continued at 30 ° C. overnight. 20 OD-mL pellets were collected to make soluble lysate (HSE). 25 μl in 530 μl were loaded on an AME5® / reverse phase column.
[254] 8 shows a bar graph showing five peaks resolved by this analysis. The Y-axis is the specific peak area of peaks 1-5 (see Materials and Methods). X-axis represents the rhuFab'2 LZ (xCD18) producing strain. prc + strains, which are 49A5 and 58H2 both were nearly produce the same amount of the product, all of which are almost the same amount of LC-115 fragment (peak 1), compared with that do not appear at all the peak 1 to peak 5 product in the △ prc strain (43H1) Indicated. This graph shows the cleavage of antibody fragments. Higher amounts of soluble, circular LC and LC dimers were observed in 43H1 hosts than in 49A5 and 58H2 hosts. In shake flasks, was prc- host produced almost 5-fold more than the number of rhuFab'2 LZ (xCD18) product than a native prc strain.
[255] 2. Fermentation Results
[256] The average rhuFab'2 LZ (xCD18) titer obtained by standard high cell density (HCD) fermentation was 893 mg in wild-type prc host (49A5, n = 6) based on AME5® / RP double-column analysis. / L. Nearly twice the potency improvement was observed from 43H1 / pS1130 fermentation. While not wishing to be bound by any one theory, there was a large difference that appears to be due to differences in product secretion efficiency between shake flask (5x) and fermentation (less than 2) titers for 43H1 and 49A5 hosts, respectively. When analyzing the total lysates of shake flask pellets, only 50% of the antibody fragments were correctly processed on the prc -plus background and derived from 43H1 shake-flask cells or cells from all fermentations ( prc -plus and minus). The lysates showed 100% processing. Processing of the Prc protein was found to be secY, secA dependent (Hara et al., 1991, supra). While not wishing to be bound by any one theory, it is believed that shake flasks indicate that Prc proteins competed with antibody fragments in translocation.
[257] 3. Total expression of antibody fragments was measured
[258] POROS® column analysis of the whole broth fermentation sample was performed as described in the Materials and Methods section above to assess the efficiency of antibody folding and assembly. When comparing equivalent infusions of whole broth samples derived from three anti-CD18 HCD fermentations in different hosts, 43H1 fermentation expressed similar amounts of HC as 49A5 fermentation, but circular kappa-LC produced more. It was shown to express the amount (see Table 2). The rhuFab'2 LZ (xCD18) titer was 1830 mg / L for 43H1 compared to 887.8 mg / L for 49A5. Not only did 59A7 ferment produce a special antibody fragment, the titer of rhuFab'2 LZ (xCD18) was highest at 2,403 mg / L.
[259] Total expression of antibody fragments and Fab'2-LZ titers of other strains expressing rhuFab'2LZ (xCD18) by standard HCD fermentation methods Fermentation samplehostTotal LC (g / L)Total HC (g / L)Total HC + LC (g / L)Fab'2-LZ (mg / L)Hours (h) One49A52.232.274.5 40 2〃4.753.968.71887.872 343H16.57410.57 62 4〃7.384.1811.56183072 559A712.496.8719.36 68 6〃13.767.4621.22240372
[260] E. Prc Inhibitors Are Required for Stagnation Survival
[261] 58B3 strains carrying deg P and prc deletions are known to show dissolution during prolonged stagnation growth of HCD fermentation expressing anti-CD18 Fab'2 LZ molecules. Cell lysis began 50 hours after inoculation. The strain produced only 320 mg / L of rhuFab'2LZ (xCD18), but 59A7 / pS1130 fermentation showed good growth in plateau during 72 hours when HCD fermentation reached high cell density (OD ₅₅₀ -mL of about 300). Maintained. 9 shows a growth comparison of these two fermentations. In addition, both HC and LC fragments showed particularly high expression in this strain background, which increased the yield of rhuFab'2 LZ (xCD18) molecules to 2,403 mg / L. In addition, no kappa-LC clips were present in the samples derived from both 58B3 and 59A7 prc -deleting strains.
[262] The prc suppressor ( spr ) (coding Prc ^sup ) was first isolated from strain 40A6 ( prc :: kan spr ) as a heat resistant back mutation of a natural mutation, ie a prc deletion mutant. After sequencing this gene and mapping the conjugation, it was found that the gene was located about 48 minutes on the E. coli chromosome. The nucleotide sequence of its PCR product is described in Hara et al., 1996, except for one point mutation at amino acid 148 (wherein the TGG codon is converted to CGG such that the tryptophan residue is changed to arginine (W148R)). reported on supra). Matched the sequence of E. coli spr gene. When introduced into 59A7 strain, the prc inhibitor exhibited a W148R mutation. The wild type spr gene has been reported to encode lipoproteins in the envelope portion, which is thought to be a peptidoglycan-hydrolase (Hara et al., 1996, supra).
[263] Prc inhibitors were introduced into the 59A7 strain by Tn 10 linked to these inhibitors, and cotransductors were selected that were resistant to tetracycline and could grow on half concentrations of LB low-salt plates at 42 ° C. New point mutations occurred when Tn 10 was removed by the Maloy plate.
[264] Based on the anti-CD18 Fab'2 fermentation results of the 58B3 vs. 59A7 strain, the Prc inhibitors exhibited successful growth of ΔPrc mutations, particularly those with high cell density. It was shown to be necessary for growth in coli fermentation. The strain designated 58B3 had exactly the same genotype as 59A7 except for spr (W148R) and could not survive 50 h in standard HCD fermentation.
[265] F. Prc deletion mutations can increase various antibody production levels due to the location of the Prc clipping site
[266] 10 shows a humanized kappa LC sequence (SEQ ID NO: 5). The calculated pI values of potential Prc clips are shown in Table 3 below.
[267] Calculated pI value of potential Prc clip Calculated pI ValueCutting siteProtease typesLC-clip 5.97S / KSerine specificLC-182 9.14V / F PrcLC-115 9.14S / VSerine specificLC-114 9.14V / A PrcLC-110
[268] While not wishing to be bound by any one theory, based on FIG. 10 and Table 3, the Prc protease begins to clip kappa-LC from the 9 or 18 amino acids at its C-terminus into the LC sequence, which is then gradually increased to N-. It is believed to break in the terminal direction to open the S / K site where the serine-specific protease acts. Other kappa LC species (possibly in other folding states) could be cleaved with up to 115 amino acids. Many potential cleavage products have molecular weights and calculated pi values that match fairly well with kappa LC spots found from 2-D gels.
[269] Figure 11 shows that the prc -deleting strain (43H1) expresses anti-VEGF Fab, anti-CD18 Fab'2 LZ, anti-CD18 Fab'2-LZ-6xHis molecule and anti-tissue factor Fab'2-LZ-6xHis molecule. The LC-182 clip was removed from the cells. Fermentation samples derived from cab2826 (33B6 / D3H44-F (ab ') 2) and cab2847 (43H1 / D3H44-F (ab') 2) were intended to express the anti-tissue factor Fab'2 LZ-6xhis molecule. It was a high cell density fermentation. Fermentation method was the same as the standard HCD method described above for the anti-CD18 Fab'2 LZ fermentation. Cab2793 was 49A5 / pAB3 fermentation intended to express anti-CD18 Fab'2 LZ-6xHis molecule. Cab 2846 was 41H1 / pS1130 fermentation intended to express anti-CD18 Fab'2 LZ molecules. JJ81 (43H1 / pY0317) and JJ67 (43E7 / pY0317) fermentations were intended to produce anti-VEGF Fabs. Cab 2814 was a blank fermentation (49A5 / pBR322) containing a similar plasmid backbone, which did not contain antibody-expressing genes.
[270] 20-OD fermentation pellets were extracted with TRIS / EDTA / lysozyme to remove soluble HSE. The remaining pellet was suspended in 400 μl of 1 × SDS sample buffer + 20 μl of beta-mercaptoethanol and then heated at 95 ° C. for 5 minutes on a heating block. 5 μl was then loaded onto a 4-12% NUPAGE® gel. The 33B6, 41H1, 49A5 and 43E7 strains were prc -plus strains. The 43H1 strain was a prc -minus strain. All natural prc -strain-derived samples had 19.8-kD LC degradation products. Cab2829 (33B6 / pD3H44TB) fermentation samples expressing anti-TF Fab were also able to detect LC-lysed fragments of the same size. All of these fragments were amino acid sequenced and found to have their exact N-terminal LC sequence.
[271] G. strain 59A7 shows good expression for anti-CD18 His- and Lys-tagged Fab'2 LZ and Apo2L cytoplasmic proteins in shake flasks
[272] Additional shake flask data shown in Table 4 below shows that strain 59A7 expressed pAB3 (anti-CD18 His-tagged Fab'2 LZ) better than strains 43H1 and 49A5. Strain 59A7 expressed 2.4-fold more pAB21 (Lys-tagged Fab'2 LZ) than 33B6 strain. Strains 59A7 and 43H1 expressed 2.9-fold more pS1130 (Tag'less Fab'2 LZ) than the 49A5 strain. However, the fermentation results showed that strain 59A7 was always superior to strain 43H1 in pS31130 expression.
[273] For Apo2L, a non-antibody cytoplasmic protein, the specific activity is about 20-30% higher when expressed in strain 59A7 than in strain 43E7 (in shake-flask). Since strain 43E7 showed higher and higher OD550 values, the total expression was similar. The 43E7 strain is an ompT ptr3 degP strain without prc and spr .
[274] High specific potency of several proteins expressed in 59A7 and other strains of shake flask culture StrainProtease Marker (s)pS1130pAB3PAB21Apo2L mg / L / OD-mLmg / L / OD-mLmg / L / OD-mLmg / L / OD-mL 33B6DegPN.A.N.A.0.33N.A. 49A5DegP0.380.46N.A.N.A. 43H1DegP Ptr3 OmpT Prc Spr ^W148R 1.10.3N.A.N.A. 59A7DegP Spr ^W148R 1.10.70.814.415.2 43E7DegP Ptr3 OmpTN.A.N.A.N.A.12.211.4
[275] H. strain 59A7 shows good expression of anti-CD18 Fab'2 LZ by fermentation
[276] Table 5 below shows that strain 59A7 was superior to 33D3 in expressing anti-CD18 Fab'2 LZ from the dual-promoter plasmid pxCD18-7T3 and superior to 49A5 in expressing anti-CD18 Fab'2 LZ from plasmid pcyc34. Indicates.
[277] High specific titers of anti-CD18 Fab'2 LZ expressed at 59A7 compared to 33D3 and 49A5 by fermentation using two different plasmids StrainPlasmidAnti-CD18 Fab'2 LZ Titers (mg / L) (Average) by CSX Analysis 33D3pxCD18-7T3 / pMS4212,500 59A7pxCD18-7T3 / pMS4214,000 49A5pcyc34 / pMS421341.3 59A7pcyc34 / pMS4212,067.1
[278] discussion
[279] In this study, E. coli expressing an anti-CD18 Fab'2-LZ molecule. Kappa-LC degradation in E. coli cells was investigated. Prior studies have identified many potential Prc substrates, but as best be identi fi ed, have not been reported for the discovery of antibody fragments as substrates of these proteases. In this study, Prc was used. It was found to be the only protease involved in kappa LC cleavage within E. coli cells. The Prc protein appeared to selectively cleave kappa-LC at separate sites, with two major clips (LC-115 and LC-182) and five special minor cleavage products observed from the 2-D gel results. Was formed. One of the main clips was more fully investigated because it was an S / K cleavage product that did not fit the characteristics of the Prc clipping site (Keiler et al., Supra). In the present invention. Degradation of kappa light chains in E. coli cells. It has been found to be associated with E. coli plasma membrane protease (Prc / Tsp). Kappa light chain cleavage products were derived from several protease deficient strains expressing anti-CD18 F (ab) '2 leucine zipper molecules. Confirmation method (1-D / 2-D SDS PAGE, mass spectrometry and N-terminal sequencing analysis) for E. coli extract confirmed that Prc / Tsp is the only enzyme that acts on kappa light chain cleavage.
[280] Special combinations of degP prc deletions and prc inhibitors ( spr mutations) result in a very large amount of recombinant protein or the only E. coli that can produce high specific activity of this protein, as exemplified by the Apo2 ligand and active antibody herein. It was found to be an E. coli strain.
[281] Fermentation using the degP prc spr strains herein yields high yields of rhuxCD18 Fab'2 leucine zipper products compared to high cell density growth (OD 300 or higher), and expression of antibodies in wild type strains or other protease deficient strains. To produce.
[282] Cell fermentation weights of 100-200 g / L can be produced after 72 hours by the fermentation method of the present application, increasing the active antibody produced in 59A7, one preferred strain with degP prc spr combination, by over 200%. The complete genotype of the 59A7 strain is W3110 ΔfhuA phoA ΔE15 Δ (argF-lac) 169 deoC degP41 IN (rrD-rrE) 1 kan ^s ilvG2096 (Val ^r ) ^Δprc spr ^W148R . Its parent strain is 58B3 with the same genetic markers as the 59A7 strain except for the absence of the prc inhibitor spr . 58B3 strain is E. coli. Growth could not be maintained in the plateau of the high density of the coli fermentation process. This strain produced less antibodies than the native prc strain (49A5), was holding the degP deletion marker, 49A5 is a kanamycin-resistant prc native strain but 59A7 is a kanamycin-except that the sensitivity △ prc strain others 59A7 strain and They had the same genotype.
[283] The present invention thus reveals that prc inhibitors ( spr ) are essential for good growth and high levels of antibody production not only in degP prc deletion strains, especially in high cell density fermentation processes, but also in low cell density fermentation processes. lost.
[284] DegP △ single-protease mutant and other multiple-containing degP △ - protease deficient strains did not produce a particularly high level of recombinant product. The two strains mentioned above, degP rpoH and degP prc , expressed more product than many other strains compared to them, but not as many as the 59A7 strain. More specifically, strain 58B3 with degP prc combination without spr inhibitor showed no advantage in producing antibody fragments as exemplified by the anti-CD18 Fab'2 LZ molecule.
[285] E. expressing a humanized anti-CD18 F (ab) '2-leucine zipper molecule. Assays are provided demonstrating that cleavage of kappa LCs in E. coli cells is associated with the C-terminal processing protein (Prc). Prc protein is the only protease that serves to cleave kappa light chains, which has been demonstrated by two-dimensional gel electrophoresis and genetic manipulation of antibody producing strains. The kappa LC- cleavage product was again in time to confirm that the Prc protease is the only enzyme involved in kappa LC cleavage actually △ The prc strain was recovered from natural prc strains. Similarly, when the prc gene was deleted from the native prc strain, no LC-cleavage product appeared. Formation of the two strains was performed by P1 transduction.
[286] Also, with or without prc deletion. A titer comparison of anti-CD18 F (ab) '2-leucine zipper molecules derived from E. coli protease mutations is provided. This data demonstrates that the 59A7 strain is a producer of high levels of antibody. Wherein -CD18 F (ab) '2- Louis has been a variety of nucleic acid configured in order to express a new zipper molecule technology, any expression plasmid was transformed to strain 59A7 degP prc mutant △ than degP no single protease mutant or spr Antibody fragments were produced in higher amounts. 43H1, another strain with genotype degP prc spr in addition to ompP and ptr3 mutations, had the same spr mutation as 59A7 in that the amino acid W changed from position 148 to R, including the change from T to C at position 520 Nonetheless, they did not grow as sufficiently as the 59A7 strain. The strain produced anti-CD18 Fab'2 LZ with higher titer than that produced by degP strain (49A5), but was not as high as produced by 59A7 strain in the fermentor.
[287] Prc proteases cleave their substrates at many isolated sites but have been reported to have broad sequence specificity (Keiler et al., Supra). The Prc cleavage site in the kappa-LC fragment was found to be located in the constant region, which is the backbone sequence commonly used to construct other humanized antibody expression plasmids. Based on the results herein, the Prc deletion mutant is E. coli. It is thought to enhance the titers of several antibody fragments, such as Fab, Fab ', Fab'2 (with or without leucine zipper), including full-length antibodies expressed in E. coli cells. It is also contemplated that antibody fragments flanked with His tag or Lys tag sequences at the C-terminus of HC would be advantageous.
[288] Strain 59A7 is superior to strain 49A5 in expressing pAB3, better than strain 43E7 in specific expression of Apo2L cytoplasmic protein in shake flasks, and in expressing pS1130 and pcyc34 ( tac II promoter equivalent of pS1130) by fermentation It was shown to be superior to 43H1 and 49A5 strains. The strain was also superior to strain 33D3 in expressing the dual-promoter plasmid pxCD18-7T3.
[289] <Example 2>
[290] Materials and methods
[291] A. Expression Plasmid
[292] Plasmid D3H44-F (ab ') 2 is described in Example 1.
[293] Plasmid pY0317tet20 is described in Example 1.
[294] B. strain
[295] The strain used for xVEGF Fab expression is similar to the other strains described in Example 1. This strain is Lee. Is a derivative of E. coli W3110 and is represented by 60C1. The complete genotype of the 60C1 strain is W3110 ΔfhuA Δ (argF-lac) 169 ptr3 degP41 Kan ^s ompT ilvG2096 (Val ^r ) Δ (nmpc-fepE) ΔssrA . The strain carries three protease markers without prc, similar to the 45F8 strain.
[296] Strains 43H1, 59A7 and 33B6 are all described in Example 1.
[297] C. Culture Method
[298] Incubation in shake flasks was performed as described in Example 1. Growth of shake-flask cultures expressing xTF Fab'2 LZ-6x his molecules was extended at 30 ° C. to 42 hours and two sets of samples were taken at different growth stages and compared. In comparison of xVEGF Fab expression, two identical cultures were grown and taken only at the 24 hour time point.
[299] D. Protein Identification
[300] 2-D gel electrophoresis was performed as described in Example 1.
[301] result
[302] Data for shake flask cultures are shown in Table 6 below. As evident in Example 1 for the production of rhuFab'2LZ (xCD18), the Prc- strains 43H1 and 59A7 were produced in terms of the amount of product produced (anti-VEGF Fab 'and anti-tissue factor Fab'2 LZ-6xhis). It was superior to the Prc + strains 60C1 and 33B6.
[303] Figure 12 shows prc deletion (strain 59A7, prc -minus strain) expressing anti-VEGF Fab (pY0317tet20) removes all digested anti-VEGF LCs and two digested xVEGF HC fragments (found in prc -plus strains). However, it is a 2-D gel showing that two separate HC clips were found in 59A7, an OmpT- or Ptr3-cut product. FIG. 13 is a 2-D gel showing that strain 60C1 ( prc -plus strain) expressing anti-VEGF Fab (pY0317tet20) as a heterologous polypeptide included multiple digested anti-VEGF LC and two digested HC fragments.
[304] prc +/- shake for comparing xVEGF Fab and prc +/- xTF Fab'2 LZ-6x his Expression in a host in a host data flask Strain24-hour incubation42-hour incubation pst statusAnti-VEGF Fab (mg / L / OD)59A7 / pY0317tet202.44 - 59A7 / pY0317tet202.53 - 60C1 / pY0317tet200.82 + 60C1 / pY0317tet201.03 +Anti-TF Fab'2 LZ-6xhis (mg / L / OD)33B6 / pd3h44f20.680.54+ 43H1 / pd3h44f21.601.88- 59A7 / pd3h44f22.183.98-

权利要求:
Claims (24)
[1" claim-type="Currently amended] The protease of the chromosomal degP and prc encoding a DegP and Prc, respectively, and to the lack holds a mutant spr gene, inhibiting the growth phenotype represented by the strain that is the product of the mutant spr gene have the prc mutants. E. coli strains.
[2" claim-type="Currently amended] The strain of claim 1, wherein the strain lacks chromosome ptr3 encoding protease III or chromosome ompT encoding protease OmpT.
[3" claim-type="Currently amended] The strain of claim 1 comprising a nucleic acid encoding a polypeptide that is heterologous to said strain.
[4" claim-type="Currently amended] The strain of claim 3, wherein said polypeptide is sensitive to proteolysis.
[5" claim-type="Currently amended] The strain of claim 3, wherein said polypeptide is a eukaryotic polypeptide.
[6" claim-type="Currently amended] 6. The strain of claim 5, wherein said polypeptide is a mammalian polypeptide.
[7" claim-type="Currently amended] 4. The strain of claim 3, wherein said strain is transformed with said nucleic acid.
[8" claim-type="Currently amended] (a) is a protease and is lacking the chromosomal prc encoding Prc mutant spr gene, and holding, to prevent the growth phenotype represented by the strain that is the product of the mutant spr gene have the prc mutants. Culturing the E. coli strain, the strain comprising an expression nucleic acid encoding a polypeptide heterologous thereto; And
(b) recovering the heterologous polypeptide from the strain
Comprising a method for producing a polypeptide.
[9" claim-type="Currently amended] The method of claim 8, wherein the heterologous polypeptide is sensitive to proteolysis.
[10" claim-type="Currently amended] The method of claim 8, wherein said culturing is carried out in a fermentor.
[11" claim-type="Currently amended] The method of claim 10, wherein said culturing is performed under fermentation conditions with high cell density.
[12" claim-type="Currently amended] The method of claim 10, wherein said culturing is performed under fermentation conditions of low cell density.
[13" claim-type="Currently amended] The method of claim 8, wherein the polypeptide is recovered from the periplasm or culture medium of the strain.
[14" claim-type="Currently amended] The method of claim 8, wherein said polypeptide is an antibody or Apo2 ligand.
[15" claim-type="Currently amended] The method of claim 14, wherein said polypeptide is an antibody.
[16" claim-type="Currently amended] The method of claim 15, wherein said antibody is a humanized antibody.
[17" claim-type="Currently amended] The method of claim 15, wherein said antibody is a full length antibody.
[18" claim-type="Currently amended] The method of claim 15, wherein the antibody is an anti-CD18, anti-VEGF, anti-tissue factor, 2C4, anti-Her-2, anti-CD20, anti-CD40 or anti-CD11a antibody.
[19" claim-type="Currently amended] The method of claim 15, wherein said antibody is an antibody fragment.
[20" claim-type="Currently amended] The method of claim 19, wherein said antibody fragment has a light chain.
[21" claim-type="Currently amended] The method of claim 20, wherein the light chain is a kappa light chain.
[22" claim-type="Currently amended] The method of claim 19, wherein said antibody fragment is a Fab, Fab ', Fab'2, or Fab'2-leucine zipper fusion.
[23" claim-type="Currently amended] The antibody fragment of claim 22, wherein said antibody fragment is an anti-CD18 Fab'2-leucine zipper fusion, an anti-tissue factor Fab'2-leucine zipper fusion, or an anti-VEGF Fab with or without a histidine or lysine tag. Way.
[24" claim-type="Currently amended] The antibody fragment of claim 22, wherein said antibody fragment is an anti-CD18 Fab'2-leucine zipper fusion, an anti-tissue factor Fab'2-leucine zipper fusion with a 6-histidine tag, anti-VEGF Fab, 6-. Anti-CD18 Fab'2-leucine zipper fusions with histidine tags, and anti-CD18 Fab'2-leucine zipper fusions with 6-lysine tags.

类似技术:

---

公开号 | 公开日 | 专利标题

---

JP2014239697A|2014-12-25|METHOD FOR HIGH-LEVEL EXPRESSION OF ACTIVE LYMPHOTOXIN-β RECEPTOR IMMUNOGLOBULIN CHIMERIC PROTEINS AND THEIR PURIFICATION

---

AU2009213095B2|2012-01-19|Methods of synthesizing heteromultimeric polypeptides in yeast using a haploid mating strategy

---

DE69233008T2|2004-01-22|Fusion of peptides and proteins with thioredoxin and thioredoxin-like molecules

---

US7575893B2|2009-08-18|Methods for producing humanized antibodies and improving yield of antibodies or antigen binding fragments in cell culture

---

EP2240595B2|2019-07-24|Glycosylated protein expression in prokaryotes

---

US6627438B2|2003-09-30|Direct expression of peptides into culture media

---

Wong1995|Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins

---

ES2533964T3|2015-04-16|Use of human myeloid leukemic cells for antibody expression

---

AU2003301318B2|2008-09-04|E.coli host cells with modified PhoS/psts periplasmic phosphate-binding proteins, and method of manufacturing recombinant fabs

---

JP4889185B2|2012-03-07|Improved in vitro method for the synthesis of active proteins containing disulfide bonds

---

JP3749257B2|2006-02-22|Bacterial production method of heterologous polypeptide having disulfide bond

---

Huang et al.2012|Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements

---

DE69635076T3|2009-01-15|Method for controlling sialylation of protein made by mammalian cell culture

---

US6528286B1|2003-03-04|Mammalian cell culture process for producing glycoproteins

---

AU2017202434B2|2019-05-16|Bacterial host strain expressing recombinant DsbC and having reduced Tsp activity

---

KR100452572B1|2005-02-07|Production of tryptophan by Escherichia coli

---

KR101299417B1|2013-08-28|Method for producing carboxy-terminal amidified peptides

---

JP2930984B2|1999-08-09|E. FIG. Excretion of heterologous proteins from Coli

---

JP4664684B2|2011-04-06|Production of IL-21 in prokaryotic hosts

---

US20140051124A1|2014-02-20|Methods for reducing accumulation of lactate during culturing and method for producing polypeptide

---

US5508192A|1996-04-16|Bacterial host strains for producing proteolytically sensitive polypeptides

---

EP0866876B1|2002-02-13|Process for the preparation of recombinant proteins in e.coli by high cell density fermentation

---

JP2016005483A|2016-01-14|Method for obtaining antibodies

---

JP3503705B2|2004-03-08|Methods for regulating polypeptide production in bacteria

---

Jonda et al.1999|Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm

同族专利:

---

公开号 | 公开日

---

EP1341899B1|2006-03-01|

---

NO328987B1|2010-07-12|

---

ZA200304101B|2005-07-27|

---

US20020142388A1|2002-10-03|

---

HU0303892A2|2004-03-01|

---

NZ526134A|2005-07-29|

---

JP4056880B2|2008-03-05|

---

RU2003121232A|2005-01-20|

---

WO2002048376A3|2003-06-19|

---

CN1233820C|2005-12-28|

---

IL156111D0|2003-12-23|

---

DK1341899T3|2006-04-10|

---

CA2430249A1|2002-06-20|

---

US6828121B2|2004-12-07|

---

NO20032694D0|2003-06-13|

---

KR100841486B1|2008-06-25|

---

PL366194A1|2005-01-24|

---

MXPA03005271A|2003-09-25|

---

CN1526010A|2004-09-01|

---

AU3957602A|2002-06-24|

---

HU0303892A3|2011-03-28|

---

WO2002048376A2|2002-06-20|

---

NO20032694L|2003-08-14|

---

CZ20031523A3|2003-10-15|

---

HU230065B1|2015-06-29|

---

BR0116680A|2005-04-05|

---

AT318890T|2006-03-15|

---

AU2002239576B2|2007-03-29|

---

RU2287574C2|2006-11-20|

---

JP2004537262A|2004-12-16|

---

EP1341899A2|2003-09-10|

---

DE60117601T2|2006-11-16|

---

HK1058686A1|2006-08-25|

---

ES2259338T3|2006-10-01|

---

IL156111A|2010-05-31|

---

CZ304484B6|2014-05-21|

---

EP1341899B9|2007-03-21|

---

DE60117601D1|2006-04-27|

---

PL205897B1|2010-06-30|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
2000-12-14|Priority to US25616200P

---

2000-12-14|Priority to US60/256,162

---

2001-12-07|Application filed by 제넨테크, 인크.

---

2001-12-07|Priority to PCT/US2001/047581

---

2003-07-04|Publication of KR20030057579A

---

2008-06-25|Application granted

---

2008-06-25|Publication of KR100841486B1

优先权:

---

申请号 | 申请日 | 专利标题

---

US25616200P| true| 2000-12-14|2000-12-14|

---

US60/256,162|2000-12-14|

---

PCT/US2001/047581|WO2002048376A2|2000-12-14|2001-12-07|Bacterial host strains|