WO2005047335A1 - Method for themass production of immunoglobulin constant region - Google Patents
Method for themass production of immunoglobulin constant region Download PDFInfo
- Publication number
- WO2005047335A1 WO2005047335A1 PCT/KR2004/002943 KR2004002943W WO2005047335A1 WO 2005047335 A1 WO2005047335 A1 WO 2005047335A1 KR 2004002943 W KR2004002943 W KR 2004002943W WO 2005047335 A1 WO2005047335 A1 WO 2005047335A1
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- WIPO (PCT)
- Prior art keywords
- constant region
- immunoglobulin constant
- coli
- protein
- immunoglobulin
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- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Definitions
- the present invention relates to a recombinant expression vector comprising a nucleotide sequence encoding an E. coli-derived signal sequence and a nucleotide sequence encoding an immunoglobulin constant region, a transformant transformed with the expression vector, and a method of mass-producing an immunoglobulin constant region by culturing the transformant and expressing the immunoglobulin constant region in a water-soluble form.
- protein drugs are susceptible to denaturator or proteolytic degradation in the body, and it is difficult to sustain in vivo concentrations and titers for a long period of time.
- Developing a technique to maintain the in vivo concentrations of protein drugs at suitable levels by enhancing protein stability is important to promote the efficacy of therapy, to help the patients who need to take their protein drug in frequent injections and to reduce the cost of treatment.
- Many attempts have been made to enhance the in vivo stability of protein drugs for a long time, and such attempts include the changing a protein formulation, fusing a protein to another protein, or chemically or biologically attaching a suitable polymer to the surface of a protein.
- the Fc fragment mediates effector functions such as complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) , as well as antigen binding capacity that is the unique function of immunoglobulins .
- CDC complement-dependent cytotoxicity
- ADCC antibody-dependent cell-mediated cytotoxicity
- the Fc fragment contains a sequence participating in the binding to the neonatal Fc receptor (FcRn) , which plays a role in regulating serum IgG levels by increasing the transport of maternal IgG to neonates and the half-life of the IgG (Ghetie and Ward, Immunology Today 18: 592-598, 1997), and the sequence regulates the interaction between protein A and protein G.
- FcRn neonatal Fc receptor
- Korean Pat. No. 249572 discloses a fusion protein which is prepared by linking an IgGl heavy chain constant region (Fc) at an amino terminal end thereof to a carboxyl terminal end of a protein, such as IL4 receptor, IL7 receptor, G-CSF receptor or EPO receptor, and producing the resulting fusion protein in mammalian cells.
- a protein such as IL4 receptor, IL7 receptor, G-CSF receptor or EPO receptor
- U.S. Pat. No. 5,605,690 describes a fusion protein comprising tumor necrosis factor receptor fused at its carboxyl terminal end to a human IgGl Fc derivative, the fusion protein being produced in animal cells.
- Tanox Inc. reported in U.S.
- 20030082679 discloses a fusion protein with an extended serum half-life, which comprises human G-CSF linked at its carboxyl terminal end to the amino terminal end of IgGl Fc and is produced in animal cells.
- Fc fusion protein with a longer serum half-life than a native protein, which comprises an IgGl Fc or Fc derivative linked at its amino terminal end through a peptide linker or without a peptide linker to the carboxyl terminal end of human EPO, TPO, human growth hormone or human interferon beta, the Fc fusion protein being produced in animal cells.
- Fc fusion proteins as described above, increase the serum half-life of a target protein, but are problematic in terms of mediating effector functions by the Fc fragment
- Fc may have increased immunogenicity or antigenicity compared to the native human Fc fragment due to the presence of unsuitable amino acids and may lose preferable Fc functions.
- an aglycosylated antibody derivative as an anti-CD3 antibody can be prepared by replacing a glycosylated residue of antibodies, the asparagine residue at position 297 of the CH2 domain, with another amino acid.
- This aglycosylated antibody derivative exhibits reduced effector functions, but still retains its binding affinity to FcRn receptor with no change of serum half-life.
- U.S. Pat. Publication No. 20030073164 discloses a method of producing an Fc derivative using E. coli devoid of glycosylation ability so as to prepare a therapeutic antibody deficient in effector functions.
- the American company Amgen Inc. described, in U.S. Pat. No. 6,660,843 and U.S. Pat. Publication Nos.
- a methionine residue is added to the N-terminus of the expression product due to the feature of the protein expression system of E. coli .
- the aforementioned expression products of Amgen Inc. have an N- terminal methionine residue, which may induce immune responses upon repeated or excessive administration to the body.
- these fusion molecules are expressed in a fusion protein form in E. coli through linking a gene encoding a therapeutic protein to an Fc gene, they are difficult to express in E. coli, or a therapeutic protein is difficult to produce in E. coli if its expression in E. coli in a fused form results in a significant decrease or loss or activity.
- the present inventors previously prepared an Fc fragment and a protein drug as separate polypeptides not using a fusion method based on genetic recombination but using the best expression systems and covalently linking the two polypeptides together to use the Fc fragment as a drug carrier.
- it is possible to prepare a conjugate of a glycosylated polypeptide drug and an aglycosylated Fc which does not induce undesirable immune responses but has satisfactory properties of physiological drug activity, in vivo duration and stability.
- a prokaryotic expression system such as E. coli is used.
- Protein production methods using an E. coli expression system have several advantages compared to methods using animal cells, as follows.
- An E. coli expression vector can be easily constructed, thus allowing rapid evaluation for protein expression. Due to its rapid growth rate, E. coli allows mass production of a protein of interest at low cost. Also, a relatively simple expression process can be used. Thus, E. coli is more useful for commercial production than other host cells.
- the intensive and through research into a method capable of mass- producing immunoglobulin constant regions such as an immunoglobulin Fc fragment resulted in the finding that, when a nucleotide sequence encoding an immunoglobulin constant region such as an immunoglobulin Fc fragment is expressed in E. coli in a form fused to an E. coli signal sequence, the immunoglobulin constant region is expressed not as inclusion bodies but in a water-soluble form in E. coli .
- an object of the present invention to provide a method of mass-producing an immunoglobulin constant region, comprising transforming a prokaryotic cell with a recombinant expression vector including a nucleotide sequence encoding an E. coli-derived signal sequence and a nucleotide sequence encoding an immunoglobulin constant region; culturing a resulting transformant; and isolating and purifying the immunoglobulin constant region expressed in a water-soluble form by the transformant. It is another object of the present invention to provide an immunoglobulin constant region prepared by the above method.
- FIG. 1 shows the results of Western blotting of immunoglobulin Fc fragments expressed in E. coli transformants
- FIG. 2 shows the results of non-reduced and reduced SDS-PAGE of dimeric immunoglobulin Fc fragments expressed in E. coli transformants
- FIG. 3 shows the results of non-reduced and reduced SDS-PAGE of mono eric immunoglobulin Fc fragments expressed in E. coli transformants
- FIG. 4 shows the results of ELISA for the Clq binding capacity of immunoglobulin Fc fragments expressed in E. coli transformants and a native immunoglobulin
- FIG. 5 is a graph showing the results of pharmacokinetic analysis of a native EPO, Aranesp and an EPO-PEG-.E. coli-derived Fc conjugate.
- the present invention relates to a method of mass-producing an immunoglobulin constant region, comprising transforming a prokaryotic cell with a recombinant expression vector including a nucleotide sequence encoding an E. coli-derived signal sequence and a nucleotide sequence encoding an immunoglobulin constant region; culturing a resulting transformant; and isolating and purifying the immunoglobulin constant region expressed in a water-soluble form by the transformant.
- Immunoglobulins are divided into variable regions that perform antibody functions of specifically binding to antigens and exhibit many variations on sequences, and constant regions that have fixed sequences and effector functions, including activating the complement system, conferring an ability to pass across the placenta and acting as ligands for receptors on various immune cells .
- the present invention relates to a method of mass- producing an immunoglobulin constant region useful as a carrier for protein drugs .
- the immunoglobulin constant region capable of being produced by the present invention may be a native form isolated from humans and other animals including goats, swine, mice, rabbits, hamsters, rats and guinea pigs, or may be a recombinant or a derivative thereof, obtained from transformed animal cells or microorganisms.
- Preferred may be a constant region of IgG, IgA, IgM, IgE and IgD from humans, or a combination or hybrid thereof.
- the term "combination”, as used herein, means that polypeptides encoding single-chain immunoglobulin Fc fragments of the same origin is linked to a single-chain polypeptide of a different origin to form a dimer or multimer.
- the term "hybrid”, as used . herein, means that sequences encoding two or more immunoglobulin Fc fragments of different origins are present in a single- chain immunoglobulin Fc fragment.
- the immunoglobulin may preferably be a constant region of IgGl, IgG2, IgG3 and lgG4, or a combination or hybrid thereof.
- heavy chains there are five distinct types of heavy chains based on differences in the amino acid sequences of their constant regions: gamma ( ⁇ ) , mu ( ⁇ ) , alpha ( ⁇ ) , delta ( ⁇ ) and epsilon ( ⁇ ) , and the heavy chains include the following subclasses : gamma 1 ( ⁇ l) , gamma 2 ( ⁇ 2 ) , gamma 3 ( ⁇ 3) , gamma 4 ( ⁇ 4 ) , alpha 1 ( ⁇ l) and alpha 2 ( ⁇ 2) .
- Nucleotide sequences encoding human immunoglobulin constant regions and amino acid sequencing limiting the same, useful in the present invention may be those encoded by nucleotide sequences from GenBank and/or
- the heavy chain constant region of IgG, IgA, IgM, IgE consists of three domains: C H 1, C H 2 and C H 3, and the heavy chain of IgM further includes C H 4.
- the light chain has one constant domain, C L .
- the immunoglobulin constant region of the present invention is one or more selected from the group consisting of C H 1, C H 2, C H 3, C H 4 and C L domains.
- the selected domain may be the whole or a fragment thereof (form having 30 or more amino acid deletions) .
- the immunoglobulin constant region of the present invention includes an amino acid sequence derivative.
- the amino acid sequence derivative means to have a sequence in which one or more amino acid residues differ from a wild- type amino acid sequence, and may naturally occur or be artificially generated.
- the immunoglobulin constant region includes derivatives resulting from a deletion, an insertion, a non-conservative or conservative substitution or combinations thereof.
- An insertion is typically made by the addition of a consecutive amino acid sequence of about 1 to 20 amino acids, or may be made with a longer sequence.
- a deletion is typically in the range of about 1 to 30 amino acid residues .
- Amino acid exchanges in proteins and peptides, which do not generally alter the activity of the proteins, or peptides are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979) .
- the immunoglobulin constant region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
- the present invention may include a hinge region that allow a dimer formation.
- the hinge region may include, in addition to a native form, derivatives by a deletion, an insertion, a non-conservative or conservative substitution or combinations thereof.
- the immunoglobulin derivative of the present invention is preferably a functional equivalent to its natural form having a similar biological activity, or, if desired, could be made by altering the property of the natural form.
- the derivatives of the immunoglobulin constant region are proteins that have increased structural stability against heat, pH, etc., or solubility, or that have improved characteristics in terms of disulfide bond formation, compatibility with an expression host, complement binding, Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) , so long as the derivatives produced do not induce unwanted immune responses in humans and animals .
- Preferred derivatives are as follows .
- a cysteine residue at a hinge region, responsible for disulfide bond formation, may be removed.
- twelve residues among fifteen amino acids of the hinge region of IgGl, from the 1st amino acid, Glu, to the 12th amino acid, Pro may be deleted to remove two cysteine residues.
- an IgGl constant region may be altered in a specific residue to have reduced affinity to Fc receptors mediating antibody-dependent cell-mediated cytotoxicity (ADCC) .
- a derivative produced may contain a deletion or a replacement with another amino acid in the leucine residue at position 234 of an IgGl CH2 sequence (see, the sequence from Kobat database for the numbering of the amino acid residues) .
- Leu234 is replaced by phenylalanine, an amino acid residue at a corresponding position in IgG4.
- a derivative of an IgGl hinge region may contain a substitution at the first amino acid, proline.
- a proline residue unlike other amino acid residues forms a closed ring structure. If proline is present at an amino terminal end, it may negatively affect the protein expression and elimination of a signal sequence.
- the proline residue may be replaced by another amino acid, preferably serine, an amino acid residue at a corresponding position in IgG4.
- the immunoglobulin constant region of the present invention includes a heavy chain constant region.
- a heavy chain constant region containing a hinge region to allow the formation of an IgGl dimer has the amino acid sequence of SEQ ID NO. 25, 21, 22 or 23.
- a heavy chain constant region not containing a hinge region to allow the formation of an IgGl monomer has the amino acid sequence of SEQ ID NO. 27.
- a heavy chain constant region containing a hinge region to allow the formation of an IgG2 dimer has the amino acid sequence of SEQ ID NO. 35.
- a heavy chain constant region containing a hinge region to allow the formation of an IgG4 dimer has the amino acid sequence of SEQ ID NO. 29 or 24.
- a heavy chain constant region not containing a hinge region to allow the formation of an IgG4 monomer has the amino acid sequence of SEQ ID NO. 30.
- the immunoglobulin constant region of the present invention includes a light chain constant region.
- the immunoglobulin light chain constant region has the amino acid sequence of SEQ ID NO. 34.
- the immunoglobulin constant region of the present invention includes both heavy chain and light chain constant regions. Preferred is a dimer or tetramer that contains a heavy chain constant region having the amino acid sequence of SEQ ID NO.
- an immunoglobulin heavy chain region is overexpressed in a prokaryotic cell such as E. coli so as to be produced in an aglycosylated form, it is not completely folded and forms insoluble, inactive inclusion bodies by various mechanisms . Since inclusion bodies do not have activity as proteins, they need an additional denaturation and refolding process, which is conjugate and time-consuming, in order to be converted to a soluble form having biological activity. Thus, there is a need for the establishment of a mass production system useful in prokaryotic cells for the expression of an immunoglobulin heavy chain region as inclusion bodies . E.
- signal sequence refers to a specific amino acid sequence that allows transport and secretion of a protein to the outside of the cytosol.
- the signal sequence of the present invention is an E. coli- derived signal sequence, which an E. coli secretory protein possesses.
- the E. coli-derived signal sequence is composed of 18 to 30 amino acids, and has several common features, as follows.
- the typical signal sequence possesses a very short amino-terminal N-domain of one to several positively charged amino acids, followed by a relatively long hydrophobic region, H-domain. At the amino terminal end, many polar positively charged amino acids, such as Lys and Arg, are present.
- the H-domain mainly contains hydrophobic residues such as Ala and Leu.
- a C-domain is located between the H-domain and a protein to be practically secreted, and has a sequence recognized by a signal peptidase.
- a protein having such a signal sequence arrives at the membrane by interaction with several proteins and is then cleaved at a specific site by a signal peptidase, releasing the mature protein.
- Non-limiting examples of the E Non-limiting examples of the E.
- coli-derived signal sequence include alkaline phosphatase, penicillinase, Ipp, heat-stable enterotoxin II, LamB, PhoE, PelB, OmpA and maltose binding protein.
- the signal sequence of the present invention includes derivatives resulting from a deletion, an insertion, a non-conservative or conservative substitution or combinations thereof, so long as a protein expressed along with the signal sequence is allowed to be secreted to the outside of the cytosol. In particular, derivatives improving secretion efficacy of a fusion protein may be desirable.
- the . signal sequence is preferably heat-stable enterotoxin II.
- the heat-stable enterotoxin II includes its native form having the amino acid sequence of SEQ ID NO.
- An enterotoxin II derivative preferably has a replacement of an amino acid sequence of one or more amino acid residues at positions 2, 4, 5, 12, 20 and 22 by another amino acid sequence, and more preferably by the amino acid sequence of SEQ ID NO. 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.
- an enterotoxin II having the amino acid sequence of SEQ ID NO. 38 was used, which is encoded by the nucleotide sequence of SEQ ID NO.
- a nucleic acid encoding the native enterotoxin II may be naturally isolated or synthesized by a gene synthesis method.
- An enterotoxin II derivative may be prepared by performing site-directed mutagenesis for a naturally isolated native form, or may be synthesized by a gene synthesis method.
- the term "fusion protein", as used herein, means two or more different polypeptides that are linked through a peptide bond to become a single-chain polypeptide, and may be easily prepared by a genetic recombination method allowing translation into a single polypeptide. With the objects of the present invention, the fusion protein refers to comprise an immunoglobulin constant region fused to an E. coli-derived signal sequence.
- the signal sequence and the immunoglobulin constant region are in frame and translated into a single chain under the control of a single promoter, but the signal sequence is finally removed by cleavage from the translated polypeptide.
- a method of preparing the fusion protein is not specifically limited.
- the fusion protein is prepared by digesting a nucleic acid sequence encoding a signal sequence and another nucleic acid sequence encoding an immunoglobulin constant region with general restriction enzymes and ligating them with each other using an enzyme such as ligase.
- a nucleic acid sequence encoding the fusion protein is expressed .in the form of being inserted into a recombinant expression vector.
- recombinant expression vector which describes a vector capable of expressing a target protein in a suitable host cell, refers to a genetic construct that comprises essential regulatory elements to which a gene insert is operably linked in such a manner as to be expressed in a host cell.
- operably linked refers to a functional linkage between a nucleic acid expression control sequence and a second nucleic acid sequence coding for a target protein in such a manner as to allow general functions.
- the operable linkage to a recombinant vector may be prepared using a genetic recombinant technique well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes generally known in the art.
- a suitable expression vector includes expression regulatory elements, such as a promoter, an initiation codon, a stop codon, a polyadenylation signal and an enhancer.
- a promoter such as E. coli is used as a host, the following promoters may be used: trp promoter, lac promoter, recA promoter, ⁇ P L promoter, lpp promoter and T7 promoter.
- the following promoters may be used: SPOl promoter, SP02 promoter and penP promoter.
- the initiation and stop codons are necessary to be functional in an individual to whom a genetic construct has been administered, and must be in frame with the coding sequence.
- the promoter of the vector may be constitutive or inducible.
- expression vectors include a selectable marker that allows selection of host cells containing the vector, and replicable expression vectors include a replication origin.
- the expression vector of the present invention is a vector essentially containing an E. coli signal sequence, and preferably containing an E. coli heat-stable enterotoxin II signal sequence.
- a vector, which includes an enterotoxin II Shine-Dalgarno (SD) sequence along with an enterotoxin II signal sequence, is more preferable because it further enhances expression levels of a target protein.
- SD enterotoxin II Shine-Dalgarno
- a nucleotide sequence encoding an enterotoxin II Shine-Dalgarno (SD) sequence and an enterotoxin II signal sequence is linked to another nucleotide sequence encoding an immunoglobulin heavy chain region by a genetic recombination method in such a way as to be expressed in frame under the control of a promoter.
- SD enterotoxin II Shine-Dalgarno
- the following recombinant expression vectors are prepared: pSTIIGlCHl_3 expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO.
- pSTIIdCGlFc expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 21
- pSTIIdCGlSFc expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 22
- pSTIIdCGlSFFc expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 23
- pSTIIGlMo expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO.
- pSTIIdCG2Fc expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO.. 35
- pSTIldCG4Fc expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 29
- pSTIIG4CHl_3 expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 24
- pSTIIG4Mo expressing an immunoglobulin heavy chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 30.
- a recombinant expression vector expresses an immunoglobulin'' heavy chain constant region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 24, and an immunoglobulin light chain region including an enterotoxin II signal sequence and the amino acid sequence of SEQ ID NO. 34, under the control of independent promoters.
- the recombinant expression vectors expressing the fusion proteins are transformed into host cells.
- the host cells are prokaryotic cells where glycosylation does not occur.
- Examples of these prokaryotic cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus .
- Preferred is E. coli .
- Non-limiting examples of E. coli strains include BL21 (DE3) , JM109, DH series, TOP10 and HB101, and the BL21 (DE3) strain is more preferable.
- E. coli is used as a host cell, since E. coli does not have a system for protein glycosylation, an immunoglobulin constant region is produced in the form of being devoid of sugar moieties that are present in a CH2 domain of a native immunoglobulin.
- sugar moieties of the immunoglobulin CH2 domain do not affect the structural stability of immunoglobulins, but cause immunoglobulins to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) upon association with cells expressing Fc receptors and immune cells to secrete cytokines to induce inflammation. Also, the sugar moieties bind to the Clq part of the first complement component Cl, leading to complement fixation. Thus, when an immunoglobulin constant region is produced in an aglycosylated form and linked to a therapeutic protein, the therapeutic protein is present in the serum for a prolonged period of time without the undesirable effector functions of immunoglobulins .
- ADCC antibody-dependent cell-mediated cytotoxicity
- the transformation of the recombinant expression vectors into prokaryotic cells includes any methods by which nucleic acids can be introduced into cells, and, as known in the art, may be performed by selecting suitable standard techniques according to host cells. These methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaP0 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation with silicon carbide fiber, and PEG-, dextran sulfate- and lipofectamine-mediated transformation.
- the recombinant expression vectors are individually introduced into E.
- coli thus generating the following transformants: BL21/pSTIIGlCHl_3 (HM10935) transformed with pSTIIGlCHl_3; BL21/pSTIIdCGlFc (HM10927) transformed with pSTIIdCGlFc; BL21/pSTIIdCGlSFc (HM10928) transformed with pSTIIdCGlSFc; BL21/pSTIIdCGlSFFc (HM10929) transformed with pSTIIdCGlSFFc; BL21/pSTIIGlMo (HM10930) transformed with pSTIIGlMo; BL21/pSTIIdCG2Fc (HM10936) transformed with pSTIIdCG2Fc; BL21/pSTIIdCG4Fc (HM10932) transformed with pSTIIdCG4Fc; BL21/pSTIIG4CHl_3 (HM10931) transformed with pSTIIG4CHl_3; B 21/pSTIIG4Mo (HM10933)
- the transformants transformed with the recombinant expression vectors are cultured by a general method. Culture conditions may be easily adjusted by those skilled in the art to be suitable for selected bacterial strains .
- a medium used in the culturing should contain all nutrients essential for the growth and survival of cells .
- the medium should contain a variety of carbon sources, nitrogen sources and trace elements. Examples of available carbon sources include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid.
- nitrogen sources may be used singly or in combination of two or more.
- available nitrogen sources include organic nitrogen sources, such as peptone, yeast extract, meat extract, malt extract, corn steep liquor (CSL) and soybean whey, and inorganic nitrogen sources, such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used singly or in combinations of two or more .
- a phosphorus source may be contained in the medium, which includes potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium-containing salts.
- the medium may contain a metal salt, such as magnesium sulfate or iron sulfate.
- the medium may further include amino acids, vitamins, suitable precursors, and the like.
- the pH of the culture may be adjusted by adding a compound, such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid, to the culture by a suitable method.
- antifoaming agents such as polyglycol fatty acid esters, may be used to prevent bubble formation.
- oxygen or an oxygen-containing gas e.g., air
- the temperature of the culture is generally 20°C to 45°C, and preferably 25°C to 45°C. After protein expression is confirmed in a small volume of the medium, large-scale expression may be carried out using a fermentor.
- Protein production using a fermentor should be carried out taking into consideration several factors, including the growth rate of host cells and protein expression levels . Protein expression may be induced through adding, for example, IPTG to the medium under suitable culture conditions .
- the immunoglobulin constant region fused to a signal sequence according to the present invention, expressed in a prokaryotic cell according to the above method, was surprisingly not expressed in the periplasmic space but was overexpressed in a water-soluble form in the cytoplasm, and the signal sequence Was accurately processed. In the detailed practice of the present invention, the amount of the fusion protein secreted into the medium or periplasmic space was negligible.
- the lipase protein aggregated by overexpression should undergo an additional complex process including dissolution and denaturation in a -suitable solution and refolding using a refolding agent such as urea, guanidine or arginine (Kohno, Meth. Enzym. 185:187-195, 1990).
- a refolding agent such as urea, guanidine or arginine
- the protein refolding efficiency is very low, and refolded proteins have lower activities than soluble proteins.
- this refolding method has no industrial benefits.
- Second there are large differences between proteins to be expressed as recombinant forms in the degree of secretion into the periplasmic space or medium, aggregation degree after secretion, expression efficiency, and the like.
- an immunoglobulin constant region overexpressed according to the present method may be purified by a general technique.
- the immunoglobulin constant regions of the present invention may be isolated and purified by disrupting cells using a French press, an ultrasonicator, etc., and subjecting a water- soluble fraction containing the immunoglobulin constant region to, for example, salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation using acetone, ethanol, etc.), dialysis, various chromatographies, such as gel filtration, ion exchange and reverse phase column chromatography, and ultrafiltration.
- salting out e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.
- solvent precipitation e.g., protein fraction precipitation using acetone, ethanol, etc.
- dialysis various chromatographies, such as gel filtration, ion exchange and reverse phase column chromatography, and ultrafiltration.
- the present invention relates to an immunoglobulin constant region prepared according to the above method.
- the immunoglobulin constant region produced in prokaryotic cells such as E.
- coli according to the present method does not specifically have limited industrial applications .
- One exemplary application is use as a carrier for the formation of a conjugate with a certain drug.
- Construction of the conjugate comprising the immunoglobulin constant region linked to a drug is not specifically limited.
- the immunoglobulin constant region and the drug may be linked together at various ratios, and the linkage may be mediated, for example, through a linker.
- the drug includes polypeptides, compounds, extracts and nucleic acids.
- Preferred is a polypeptide drug (used with meaning identical to the word "protein") .
- the linker includes peptide and non-peptide linkers. Preferred is a non-peptide linker, and more preferred is a non-peptide polymer.
- any physiologically active polypeptide may be used without specific limitation as a protein partner of the immunoglobulin constant region prepared according to the present method to form a conjugate.
- physiologically active polypeptides include those used for treating or preventing human diseases, which include cytokines, interleukins, interleukin binding protein, enzymes, antibodies, growth factors, transcription regulatory factors, coagulation factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists, and derivatives and analogues thereof.
- physiologically active polypeptide examples include human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons and interferon receptors (e.g., interferon- ⁇ , - ⁇ and - ⁇ , water-soluble type I interferon receptor, etc.), colony stimulating factors, interleukins (e.g., interleukin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, - 18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, etc.) and interleukin receptors (e.g., IL-1 receptor, IL-4 receptor, etc.), enzymes (e.g., glucocerebrosidase, iduronate-2-sulfatase, alpha-
- interleukin and cytokine binding proteins e.g., IL-18bp, TNF-binding protein, etc.
- acrophage activating factor macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, factor VII, factor Vila, factor VIII, factor IX, factor XIII, plasminogen activating factor, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C,
- the physiologically active polypeptide useful in the present invention may be a native form, may be produced by genetic recombination using prokaryotic cells such as E. coli or eukaryotic cells such as yeast cells, insect cells and animal cells, or may be a derivative having one or more amino acid mutations but having the biological activity identical to the native form.
- an immunoglobulin constant region fragment produced by the BL21/pSTIIdCGlSFFc (HM10929) transformant was linked to human erythropoietin (EPO) using a water-soluble polymer, polyethylene glycol, thus providing an EPO-PEG- immunoglobulin constant region protein conjugate.
- the immunoglobulin constant region produced in a water- soluble form using an E. coli signal sequence according to the present invention in the form of being linked to a physiologically active polypeptide, may be useful for enhancing the serum half-life and physiological activity of the physiologically active polypeptide with no risk of inducing immune responses .
- a better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.
- EXAMPLE 1 Construction of human immunoglobulin IgGl Fc constant region expression vector
- RNA isolated from human blood cells was isolated from about 6 ml of blood using a Qiamp RNA blood kit (Qiagen) , and gene amplification was performed using the total RNA as a template and a One-Step RT-PCR kit (Qiagen) .
- Qiagen Qiamp RNA blood kit
- a pair of primers represented by SEQ ID Nos SEQ ID Nos .
- DNA sequencing analysis revealed that two genes cloned into the cloning vector, IgGl heavy chain CH1- hinge-CH2-CH3 gene and light chain constant region gene, have nucleotide sequences of SEQ ID Nos. 5 and 6, respectively.
- PCR was carried out using the plasmid as prepared above as a template to amplify genes encoding the following constant region fragments .
- a pair of primers represented by SEQ ID Nos. 7 and 2 was designed to amplify the CHI, hinge region, CH2 and CH3 of IgGl.
- An Fc fragment lacking cysteine residues not participating in disulfide bond formation in the hinge region was amplified using three primer pairs of SEQ ID Nos.
- a gene amplified using a primer pair of SEQ ID Nos. 7 and 2 and the pBGlCHl-3 plasmid as a template contained the CH1- hinge-CH2-CH3 of IgGl and encoded a protein having the amino acid sequence represented by SEQ ID No. 25.
- SEQ ID No. 8 is a nucleotide sequence starting from the 13th residue, proline, of 15 amino acid residues of the hinge region (Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro; SEQ ID No. 26) .
- a gene amplified using a primer pair of SEQ ID Nos is a nucleotide sequence starting from the 13th residue, proline, of 15 amino acid residues of the hinge region (Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro; SEQ ID No. 26) .
- SEQ ID No. 21 an amino acid sequence that encodes an amino terminal end starting from the Pro-Cys-Pro sequence of the hinge region and CH2 and CH3 domains, among the whole IgGl constant region.
- SEQ ID No. 9 was prepared by replacing the 13th proline residue of the hinge region with serine
- SEQ ID No. 10 was prepared by replacing the 13th proline residue of the hinge region with serine and the 4th leucine residue of the CH2 with phenylalanine .
- an expression vector pT14SlSH-4T20V22Q (Korean Pat. No. 38061), previously developed by the present inventors, was used as a starting vector.
- This expression vector contains an E. coli heat-stable enterotoxin signal sequence derivative having the nucleotide sequence represented by SEQ ID No. 12.
- a Stul recognition site was inserted into an end of the E. coli heat-stable enterotoxin signal sequence derivative of the pTl4SlSH-4T20V22Q plasmid through site-directed mutagenesis using a pair of primers represented by SEQ ID Nos. 13 and 14 to induce mutagenesis to introduce the Stul site at a nucleotide sequence coding for the last amino acid residue of the signal sequence.
- This insertion of the Stul site was identified to be successful by DNA sequencing.
- the resulting pT14SlSH-4T20V22Q plasmid containing a Stul site was designated as "pmSTII".
- the pmSTII plasmid was treated with Stul and BamHI and subjected to agarose gel electrophoresis, and a large fragment (4.7 kb) was purified, which contained the E. coli heat-stable enterotoxin signal sequence derivative. Then, the amplified genes were digested with BamHI and ligated with the linearized expression vector, thus providing pSTIIGlCHl_3 (PCR product obtained using the primers of SEQ ID Nos. 7 and 2), and pSTIIdCGlFc (PCR product obtained using the ⁇ primers of SEQ ID Nos. 8 and 2), pSTIIdCGlSFc (PCR product obtained using the primers of SEQ ID Nos .
- ⁇ l-2> Construction of monomeric IgGl constant region expression vector
- PCR was carried out using a pair of primers represented by SEQ ID Nos. 11 and 2 and the same template as in the above ⁇ 1-1>.
- the PCR product was cloned into an expression vector, pmSTII, according to the same procedure as in the above ⁇ 1- 1>, thus providing pSTIIGlMo (PCR product obtained using the primers of SEQ ID NOS. 11 and 2) .
- This expression vector was transformed into E.
- BL21/pSTIIGlMo HM10930
- KCCM Korean Culture Center of Microorganisms
- EXAMPLE 2 Construction of human immunoglobulin IgG2 Fc expression vector To clone an IgG2 Fc gene, RT-PCR was carried out according to the same method as in the ⁇ 1-1> of Example 1 using RNA isolated from human blood cells as a template and a One-Step RT-PCR kit (Qiagen) . To obtain a desired gene sequence, a pair of primers represented by SEQ ID Nos. 31 and 32 was used. SEQ ID No. 31 is a nucleotide sequence starting from the 10th residue, proline, of 12 amino acid residues of the hinge region (Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro; SEQ ID NO. 33) .
- the gene amplified using the pair of primers represented by SEQ ID Nos. 31 and 32 was identified to contain an amino terminal end starting with the Pro-Cys-Pro sequence of the hinge region and CH2 and CH3 domains, among a full-length IgG2 Fc gene sequence, and has the nucleotide sequence of SEQ ID No. 35.
- the amplified IgG2 Fc gene into an expression vector containing an E. coli signal sequence the aforementioned pmSTII vector was used.
- the pmSTII plasmid was treated with Stul and BamHI and subjected to agarose gel electrophoresis, and a large fragment (4.7 kb) was purified, which contained the E. coli heat-stable enterotoxin signal sequence derivative. Then, the amplified IgG2 Fc gene was digested with BamHI and ligated with the linearized expression vector, thus providing pSTIIdCG2Fc.
- This vector expresses in a host cell a protein that has the amino acid sequence of SEQ ID No. 35 and is present in a dimeric form by disulfide bonds between cysteine residues in the hinge region.
- the final expression vector was transformed into E. coli BL21 (DE3) , and the resulting transformant was designated as "BL21/pSTIIdCG2Fc (HM10936)".
- SEQ ID No. 16 is a nucleotide sequence starting from the 10th residue, _ serine, of 12 amino acid residues of the IgG4 hinge region (Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro: SEQ ID No. 28) .
- SEQ ID No. 16 is a nucleotide sequence starting from the 10th residue, _ serine, of 12 amino acid residues of the IgG4 hinge region (Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro: SEQ ID No. 28) .
- a gene amplified using the above primer pair encoded an amino terminal end starting from the Ser-Cys-Pro sequence of the hinge region and CH2 and CH3 domains, among the whole IgG4 constant region.
- the amplified gene was treated with BamHI and inserted into a StuI/BamHI-digested pmSTII plasmid, thus providing pSTIIG4Fc.
- a protein expressed by this expression vector has the amino acid sequence of SEQ ID No. 29 and is present in a dimeric form through disulfide bonds between cysteine residues of the hinge region.
- an expression vector for a constant region (CH1-Hinge-CH2-CH3) containing a CHI domain PCR was carried out using the pBG4CHl-3 as a template and a pair of primers represented by SEQ ID Nos. 19 and 17, and the PCR product was cloned according to the same method as described above, thus providing pSTIIG4CHl_3.
- a protein expressed by this expression vector has the amino acid sequence of SEQ ID No. 24.
- a vector expressing both heavy chain and light chain constant regions of an immunoglobulin was constructed as follows.
- a desired gene was amplified using the pBK plasmid prepared in the ⁇ 1-1> of Example 1 as a template and a set of primers represented by SEQ ID Nos . 20 and 4, and cloned into a pmSTII vector containing an E. coli heat-stable enterotoxin II signal sequence derivative, thus generating a pSTIIK plasmid.
- the pSTIIK plasmid included a gene fragment comprising a kappa light chain constant region linked to an E. coli heat-stable enterotoxin II signal sequence derivative.
- the pSTIIK plasmid was digested with Hindi11 and Sail to obtain an about 1.2-kb gene fragment containing sequences required for protein expression, including a promoter and a signal sequence.
- the 1.2-kb gene fragment was inserted into an Nrul site of the pSTIIG4CHl_3 vector, thus providing a pSTIIG4H_K vector.
- This expression vector expresses a heavy chain constant region and a light chain constant region under the control of different promoters, and the expressed products form a dimer or tetramer linked through free cysteine residues in each chain.
- the above expression vectors were individually transformed into E.
- coli BL21 (DE3) , and the resulting transformants were designated as "BL21/pSTIIG4CHl_3(HM10931)", “BL21/pSTIIdCG4Fc (HM10932)” and “BL21/pSTIIG4H_K (HM10934)", and deposited at KCCM (accession numbers: KCCM-10596, KCCM-10597 and KCCM-10599) .
- a protein expressed by the expression vector has the amino acid sequence of SEQ ID No. 30, is expressed from the CH2 domain, and is present in a monomeric form because it lacks the hinge region.
- the recombinant expression vectors, capable of expressing an immunoglobulin constant region in a water- soluble form in E. coli using an E. coli heat-stable enterotoxin II signal sequence derivative, and microorganisms transformed with the expression vectors are summarized in Table 1, below.
- Bacterial transformants prepared in Examples 1, 2 were individually inoculated in a fermentor (Marubishi Company) and allowed to ferment, and were evaluated to determine whether they express immunoglobulin constant region fragments .
- each transformant was grown in 100 ml of LB medium with agitation overnight and inoculated in the fermentor for large-scale culture. The fermentor was maintained at 30°C or 35°C. To prevent conversion from an aerobic to an anaerobic environment, the cultures were aerated with 20-wm air and stirred at 500 rpm. To compensate for the insufficient nutrients for bacterial growth during fermentation, the cultures were supplemented with glucose and yeast extracts according to the fermented states of bacteria.
- an inducer IPTG
- the cultures were further cultured for 40 to 45 hrs until the OD value at 600 nm increased to 100 to 120.
- the expression of immunoglobulin Fc in the E. coli transformants and the expressed sites, water solubility and dimer formation of the expressed lg Fc were examined as follows. To determine whether an expressed product is secreted to the fermentation fluid or the periplasmic space of E. coli by the signal sequence fused to the expression vector, the fermentation fluid was centrifuged to obtain a cell-free fermentation fluid and collect cells.
- the cell- free fermentation fluid and a periplasmic space solution obtained by osmotic shock of the collected cells were subjected to Western blot analysis. As a result, a very small amount of immunoglobulin Fc was detected.
- cells were disrupted using an ultrasonicator (Misonix Company) . The resulting cell lysate was centrifuged to separate water- soluble substances from water-insoluble substances, and the water-soluble substances were subjected to Western blot analysis, as follows.
- the water-soluble substances were mixed with a protein sample buffer not containing a reducing agent such as DTT or ⁇ -mercaptoethanol, and separated on a 15% SDS-PAGE gel (Criterion Gel, Bio-Rad) . Then, proteins were transferred onto a nitrocellulose membrane and detected with an HRP-conjugated anti-human Fc antibody (Sigma) . As shown in FIG. 1, immunoglobulin Fc was overexpressed in a water-soluble form and located in the cytosol of E. coli . Also, products, expressed by transformants transformed with expression vectors expressing lg Fc having a portion of a hinge region, were expressed as dimers . In FIG.
- lanes 1, 2 and 3 show products expressed in HM10927, HM10932 and HM10936, respectively, and lane 4 shows Fc generated by papain treatment of immunoglobulins produced in animal cells, which showed a slightly larger size due to its sugar moieties on the SDS-PAGE gel than that produced in E. coli .
- ⁇ 4-2> N-terminal sequence analysis The water-soluble dimeric lg Fc fragments, which were located in the cytosol of E. coli as demonstrated in the above ⁇ 4-l>, were designed to be translated in a fused form to a signal sequence. Thus, to determine whether the lg Fc fragments are located in E.
- N-terminal amino acid sequences of the lg Fc fragments were determined by the Basic Science Research Institute, Seoul, Korea. Samples used in the N- terminal amino acid sequence analysis were prepared as follows . First, a PVDF membrane (Bio-Rad) was immersed in methanol for about 2-3 sec to be activated, and was sufficiently wet with a blocking buffer (170 mM glycine, 25 mM Tris-HCl (pH 8.0), 20% methanol).
- the protein samples separated on a non-reduced SDS-PAGE gel, prepared in the above ⁇ 4-l>, were blotted onto a PVDF membrane for about one hour using a blotting kit (Hoefer Semi-Dry Transfer unit, Amersham) . Proteins transferred onto the PVDF membrane were stained with a protein dye, Coomassie Blue R- 250 (Amnesco) , for 3-4 sec, and washed with a destaining solution (water: acetic acid: methanol 5: 1: 4). Then, fragments containing proteins from the membrane were cut out with scissors and subjected to N-terminal sequence analysis .
- a protein dye Coomassie Blue R- 250 (Amnesco)
- the IgGl Fc protein was found to have an N-terminal sequence of Pro-Cys-Pro-Ala-Pro-Glu-Leu-Leu-Gly- Gly, the lgG4 Fc protein had an N-terminal sequence of Ser- Cys-Pro-Ala-Pro-Glu-Phe-Leu-Gly-Gly, and the IgG2 Fc protein had an N-terminal sequence of Pro-Cys-Pro-Ala-Pro- Pro-Val-Ala-Gly-Pro .
- the Fc fragments expressed by the E. coli transformants of the present invention were found to have an accurate N-terminal sequence.
- the cell lysates were subjected to two- step column chromatography to purify recombinant immunoglobulin constant regions present in the cytosol.
- 5 ml of a protein-A affinity column (Pharmacia) was equilibrated with PBS, and the cell lysates were loaded onto the column at a flow rate of 5 ml/min. Unbound proteins were washed out with PBS, and bound proteins were eluted with 100 mM citrate (pH 3.0).
- the collected fractions were desalted using a HiPrep 26/10 desalting column (Pharmacia) with 10 mM Tris buffer (pH 8.0).
- FIGS. 2 and 3 show the results of SDS-PAGE analysis of the purified immunoglobulin constant regions in a dimeric or monomeric form under non-reduced and reduced conditions using a criterion gel (Bio-Rad) , wherein the constant regions were evaluated for differential migration on reduced versus non-reduced gels.
- a criterion gel Bio-Rad
- the A region shows proteins separated on a non-reduced SDS-PAGE gel
- the B region shows proteins on a reduced SDS-PAGE gel.
- Lane M indicates a prestained low-range standard protein marker (Bio-Rad)
- lanes 1 to 4 indicate protein samples for immunoglobulin constant regions produced by E. coli transformants, HM10927, HM10928, HM10929 and HM10932 respectively.
- the lg Fc fragments were present in a monomeric form because disulfide bonds formed between cysteine residues of the hinge region were reduced, and were thus migrated the monomer distance.
- the lg Fc fragments were present in a dimeric form by disulfide bonds and thus had a migration distance of about 42 kDa.
- the A region shows proteins separated on a non-reduced SDS-PAGE gel
- the B region shows proteins on a reduced SDS-PAGE gel.
- Lane M indicates the standard protein marker
- lanes 1 and 2 indicate protein samples for immunoglobulin constant regions produced by E. coli transformants, HM10930 and HM10934, respectively.
- the proteins did not show a large difference in migration on reduced versus non-reduced .gels, and only displayed a slightly different migration due to the reduction of intramolecular disulfide bonds.
- an enzyme linked immunosorbent assay was carried out as follows.
- test groups immunoglobulin constant regions produced by the ' HM10929, HM10930, HM10932 and HM10933 transformants prepared in the above Examples were used.
- standards a glycosylated immunoglobulin (IVIGG-globulin S, Green Cross PBM) was used.
- the test and standard samples were prepared in 10 mM carbonate buffer (pH 9.6) at a concentration of 1 ⁇ g/ml .
- the samples were aliquotted into a 96-well plate (Nunc) in an amount of 200 ng per well, and the plate was coated overnight at 4°C. Then, each well was washed with PBS-T (137 mM NaCI, 2 mM KCl, 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , 0.05% Tween 20) three times, • blocked with 250 ⁇ l of a blocking buffer (1% bovine serum albumin in PBS-T) at room temperature for 1 hr, and washed again with the same PBS-T three times .
- PBS-T 137 mM NaCI, 2 mM KCl, 10 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , 0.05% Tween 20
- the standard and test samples were diluted in PBS-T to a predetermined concentration and added to antibody-coated wells, and the plate was incubated at room temperature for 1 hr and washed with PBS-T three times. Thereafter, 2 ⁇ g/ml Clq (R&D Systems) was added to the plate and reacted at room temperature for 2 hrs, and the plate was washed with PBS-T six times. 200 ⁇ l of a 1:1000 dilution of a human anti-human Clq antibody- peroxidase conjugate (Biogenesis, USA) in the blocking buffer was added to each well and reacted at room temperature for 1 hr.
- the immunoglobulin constant region proteins of the present invention rarely have the risk of inducing immune responses such as cytotoxicity and inflammation in the body when used as a carrier for physiologically active polypeptides in a conjugate form.
- ⁇ 6-l> Preparation of human EPO
- an EPO gene was amplified by RT-PCR using total RNA isolated from blood cells and cloned into a pBluscript II (Stratagen) vector, thus generating a pBlueEP vector.
- the pBlueEP was digested with HindiII and BamHI, and an EPO gene-containing fragment was inserted into the animal cell expression vector treated with the same restriction enzymes, thus providing pcmvEP.
- This expression vector carrying an EPO gene was transfected into CHO cells allowing protein expression using a Lipofectamine reagent (Gibco) .
- the cells were treated with MTX, and MTX concentrations gradually increased to 120 nM to elevate expression levels. EPO was expressed at high levels, higher than 100 mg per liter.
- the reaction mixture was subjected to size exclusion chromatography using a Superdex R column (Pharmacia) .
- the EPO-PEG complex was eluted from the column using 10 mM potassium phosphate buffer (pH 6.0) as an elution buffer, and EPO not linked to PEG, unreacted PEG and dimer byproducts where PEG was linked to two EPO molecules were removed.
- the purified EPO- PEG complex was concentrated to 5 mg/ml.
- the immunoglobulin constant region fragment (about 53 kDa) prepared in the ⁇ 4-3> was dissolved in 10 mM phosphate buffer and mixed with the EPO-PEG complex at an EPO-PEG complex: constant region molar ratio of 1:1, 1:2, 1:4 and 1:8.
- a reducing agent, NaCNBH 3 was added to the reaction solution at a final concentration of 20 mM and was allowed to react at 4°C for 20 hrs with gentle agitation.
- the optimal reaction molar ratio for EPO-PEG complex to constant region fragment providing the highest reactivity and generating the fewest byproducts such as dimers, was found to be 1:2.
- the reaction mixture was subjected to high-pressure liquid chromatography so as to eliminate unreacted substances and byproducts.
- the coupling reaction solution was desalted using a HiPrep 26/10 desalting column (Pharmacia) with 10 mM Tris buffer (pH 8.0). Then, the reaction solution was then loaded onto 50 ml of a Q HP 26/10 column (Pharmacia) at a flow rate of 8 ml/min, and this column was eluted with a linear NaCI gradient of 0-0.2 M to obtain desired fractions.
- Aranesp (Amgen) having greater sialic acid content to increase the half-life
- test group the EPO-PEG-Fc conjugate (test group) prepared in the ⁇ 5-3> of Example 5 were subcutaneously injected at a dose of 100 ⁇ g/kg to five SD rats per group. After the subcutaneous injection, blood samples were collected at 0.5, 1, 2, 4, 6, 12, 24 and 48 hrs in the control groups, and, in the test groups, at 1, 12, 24, 30,
- the blood samples were collected in 1.5 ml tubes, coagulated, and centrifuged for 10 min using an Eppendorf high-speed micro centrifugator to remove blood cells. Serum protein levels were measured by ELISA using an antibody specific to EPO. Table 3, below, and FIG. 5 show serum- half-lives of the native protein and the protein conjugate, respectively.
- EPO-PEG-Fc (E. coli) protein conjugate prepared using the immunoglobulin constant region produced according to the present invention as a carrier, exhibited a longer serum half-life than the native EPO. This extended half- life was found to be higher than that of Aranesp, a hyperglycosylated EPO having a ' long serum half-life.
- the present method allows mass production of an immunoglobulin constant region in a water-soluble form using an E. coli signal sequence.
- the produced immunoglobulin constant region is useful when linked to a physiologically active protein for enhancing the serum half-life and physiological activity of the physiologically active protein with no risk of inducing immune responses .
- INTERNATIONAL DEPOSITARY AUTHORITY Name Korean Culture Center of Microorganisms Signatured) of person(s) having the power to represent the International Depositary Address ' ⁇ 361-221, Yurim B D Authority or of autl Ho ⁇ gje-1-dong, Seodaemun-gu SEOUL 120-091 Date: Sep. 21.
- INTERNATIONAL DEPOSITARY AUTHORITY Name Korean Culture Center of Microorganisms Signature(s) of person(s) having the power to represent the International Depositary Address : 361-221. Yurim B D Authority or of mt o ⁇ acti atfi ⁇ LsX, Hongje-1-dong, Seodaemun-gu SEOUL 120-091 Date: Sep. 21.
Abstract
Description
Claims
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EP04800090A EP1682582B1 (en) | 2003-11-13 | 2004-11-13 | Method for the mass production of immunoglobulin constant region |
JP2006539397A JP4762904B2 (en) | 2003-11-13 | 2004-11-13 | Mass production method of immunoglobulin constant region |
US10/535,312 US8029789B2 (en) | 2003-11-13 | 2004-11-13 | Method for the mass production of immunoglobulin constant region |
AT04800090T ATE522548T1 (en) | 2003-11-13 | 2004-11-13 | METHOD FOR MASS PRODUCTION OF CONSTANT REGION OF IMMUNOLOBULIN |
US13/087,593 US8822650B2 (en) | 2003-11-13 | 2011-04-15 | Method for the mass production of immunoglobulin constant region |
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US13/087,593 Continuation US8822650B2 (en) | 2003-11-13 | 2011-04-15 | Method for the mass production of immunoglobulin constant region |
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PCT/KR2004/002944 WO2005047336A1 (en) | 2001-11-13 | 2004-11-13 | Protein complex using immunoglobulin fragment andmethod for the preparation thereof |
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AR056142A1 (en) * | 2005-10-21 | 2007-09-19 | Amgen Inc | METHODS TO GENERATE THE MONOVALENT IGG ANTIBODY |
US8067562B2 (en) | 2005-11-01 | 2011-11-29 | Amgen Inc. | Isolated nucleic acid molecule comprising the amino acid sequence of SEQ ID NO:1 |
EP1968621A2 (en) * | 2005-12-06 | 2008-09-17 | Amgen Inc. | Uses of myostatin antagonists |
CN100475270C (en) | 2006-01-20 | 2009-04-08 | 清华大学 | Medicine for treating tumor, and application thereof |
CN101002945B (en) | 2006-01-20 | 2012-09-05 | 清华大学 | Novel complex used for treating tumor |
CA3149553C (en) * | 2006-06-12 | 2023-11-21 | Aptevo Research And Development Llc | Single-chain multivalent binding proteins with effector function |
UA99262C2 (en) * | 2006-08-28 | 2012-08-10 | Арес Трейдінг С.А. | Process for reducing the concentration of free fc-moieties in a fluid comprising an fc-containing protein |
US20140147441A1 (en) * | 2006-09-12 | 2014-05-29 | The General Hospital Corporation | Compositions containing alpha-1-antitrypsin and methods for use |
AU2007343796A1 (en) | 2006-10-25 | 2008-07-24 | Amgen Inc. | Toxin peptide therapeutic agents |
US20080181903A1 (en) * | 2006-12-21 | 2008-07-31 | Pdl Biopharma, Inc. | Conjugate of natriuretic peptide and antibody constant region |
JP2008169195A (en) * | 2007-01-05 | 2008-07-24 | Hanmi Pharmaceutical Co Ltd | Insulinotopic peptide drug combo using carrier material |
CN101219219B (en) * | 2007-01-10 | 2013-02-13 | 北京普罗吉生物科技发展有限公司 | Complex containing vascellum chalone or fragment, preparation method and application thereof |
WO2008127780A2 (en) * | 2007-02-21 | 2008-10-23 | Nantero, Inc. | Symmetric touch screen system with carbon nanotube-based transparent conductive electrode pairs |
US8501678B2 (en) | 2007-03-06 | 2013-08-06 | Atara Biotherapeutics, Inc. | Variant activin receptor polypeptides and uses thereof |
TWI454479B (en) | 2007-03-06 | 2014-10-01 | Amgen Inc | Variant activin receptor polypeptides and uses thereof |
WO2008153745A2 (en) | 2007-05-22 | 2008-12-18 | Amgen Inc. | Compositions and methods for producing bioactive fusion proteins |
KR101580938B1 (en) | 2007-06-01 | 2015-12-30 | 유니버시티 오브 매릴랜드, 발티모어 | Immunoglobulin constant region Fc receptor binding agents |
CA2689492A1 (en) * | 2007-06-06 | 2008-12-18 | Boehringer Ingelheim International Gmbh | Natriuretic fusion proteins |
EP2164871A2 (en) * | 2007-06-12 | 2010-03-24 | Wyeth a Corporation of the State of Delaware | Anti-cd20 therapeutic compositions and methods |
CN101990439A (en) * | 2007-07-06 | 2011-03-23 | 特鲁比昂药品公司 | Binding peptides having a c-terminally disposed specific binding domain |
WO2009052415A1 (en) * | 2007-10-17 | 2009-04-23 | The Regents Of The University Of California | Peptide for the induction of immune tolerance as treatment for systemic lupus erythematosus |
WO2009059278A1 (en) * | 2007-11-02 | 2009-05-07 | Centocor, Inc. | Semi-synthetic glp-1 peptide-fc fusion constructs, methods and uses |
MX340204B (en) * | 2008-04-11 | 2016-06-30 | Emergent Product Dev Seattle | Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof. |
MY151184A (en) * | 2008-07-23 | 2014-04-30 | Hanmi Science Co Ltd | A polypeptide complex comprising non-peptidyl polymer having three functional ends |
RU2457856C2 (en) * | 2008-08-05 | 2012-08-10 | Виктор Владимирович Чалов | Antiviral and antimicrobial oral composition |
MX339137B (en) * | 2008-08-07 | 2016-05-13 | Ipsen Pharma Sas | Analogues of glucose-dependent insulinotropic polypeptide (gip) modified at n-terminal. |
ES2574835T3 (en) | 2008-08-07 | 2016-06-22 | Ipsen Pharma S.A.S. | Glucose-dependent insulinotropic polypeptide analogs |
KR20110043686A (en) * | 2008-08-07 | 2011-04-27 | 입센 파마 에스.에이.에스 | Analogues of glucose-dependent insulinotropic polypeptide |
EP2334334A1 (en) * | 2008-09-19 | 2011-06-22 | Nektar Therapeutics | Polymer conjugates of nesiritide peptides |
US20110166063A1 (en) | 2008-09-19 | 2011-07-07 | Nektar Therapeutics | Polymer conjugates of therapeutic peptides |
EP2344527A4 (en) * | 2008-10-31 | 2012-04-04 | Amgen Inc | Materials and methods relating to stem cell mobilization by multi-pegylated granulocyte colony stimulating factor |
EA201100832A1 (en) | 2008-11-26 | 2011-12-30 | Амген Инк. | OPTIONS OF POLYPEPTIDES OF ACTIVIN IIB RECEPTOR AND THEIR APPLICATION |
WO2010065578A2 (en) * | 2008-12-04 | 2010-06-10 | Leukosight Inc. | POLYPEPTIDES COMPRISING Fc FRAGMENTS OF IMMUNOGLOBULIN G (IgG) AND METHODS OF USING THE SAME |
US20100158893A1 (en) * | 2008-12-19 | 2010-06-24 | Baxter International Inc. | Systems and methods for obtaining immunoglobulin from blood |
KR20100084996A (en) * | 2009-01-19 | 2010-07-28 | 한미홀딩스 주식회사 | Method for producing physiologically active protein or peptide using immunoglobulin fragment |
WO2010096394A2 (en) | 2009-02-17 | 2010-08-26 | Redwood Biosciences, Inc. | Aldehyde-tagged protein-based drug carriers and methods of use |
CN102333872B (en) | 2009-02-25 | 2014-07-09 | 默沙东公司 | Metabolic engineering of a galactose assimilation pathway in the glycoengineered yeast pichia pastoris |
US20120142593A1 (en) * | 2009-03-24 | 2012-06-07 | Bayer Healthcare Llc | Factor VIII Variants and Methods of Use |
RU2503688C2 (en) * | 2009-04-22 | 2014-01-10 | Алтеоген, Инк | Fused protein or peptide with increased half lifetime in vivo, which is maintained due to slow release in vivo, and method for increasing half lifetime in vivo using it |
EP2496262A2 (en) * | 2009-11-02 | 2012-09-12 | Université de Genève | Stabilized protein formulations and use thereof |
JP2013515004A (en) | 2009-12-18 | 2013-05-02 | エクソドス ライフ サイエンシーズ リミテッド パートナーシップ | Methods and compositions for stable liquid medicaments |
WO2011075185A1 (en) | 2009-12-18 | 2011-06-23 | Oligasis | Targeted drug phosphorylcholine polymer conjugates |
ES2654102T3 (en) * | 2010-01-19 | 2018-02-12 | Hanmi Science Co., Ltd. | Liquid formulations for long-acting C-CSF conjugate |
JP5638628B2 (en) * | 2010-01-19 | 2014-12-10 | ハンミ サイエンス カンパニー リミテッドHanmi Scienceco.,Ltd. | Long-acting erythropoietin conjugate solution |
WO2011093470A1 (en) * | 2010-01-28 | 2011-08-04 | 協和発酵キリン株式会社 | Pharmaceutical composition for treatment of bone diseases, which contains protein comprising bone morphogenetic protein receptor 1b (bmpr1b) extracellular domain or mutant thereof |
BR112012020882A2 (en) | 2010-02-24 | 2015-11-03 | Merck Sharp & Dohme | methods for producing a heterologous glycoprotein and for producing a glycoprotein composition, host cell, glycoprotein composition, and use of recombinant pichia pastoris. |
DK2977088T3 (en) * | 2010-03-08 | 2017-10-09 | Ge Healthcare Bioprocess R&D Ab | IMMUNOGLOBULIN G FC REGION BINDING POLYPEPTIDE |
AR081755A1 (en) * | 2010-04-02 | 2012-10-17 | Hanmi Holdings Co Ltd | FORMULATION OF PROLONGED ACTION OF THE FOLICULES STIMULATING HORMONE WHERE AN IMMUNOGLOBULIN FRAGMENT, PREPARATION METHOD AND METHOD TO TREAT A SUBJECT SUFFERING A REPRODUCTIVE DISORDER ARE USED |
AR081066A1 (en) | 2010-04-02 | 2012-06-06 | Hanmi Holdings Co Ltd | INSULIN CONJUGATE WHERE AN IMMUNOGLOBULIN FRAGMENT IS USED |
US9981017B2 (en) | 2010-04-02 | 2018-05-29 | Hanmi Science Co., Ltd. | Insulin conjugate using an immunoglobulin fragment |
AR080993A1 (en) * | 2010-04-02 | 2012-05-30 | Hanmi Holdings Co Ltd | FORMULATION OF PROLONGED BETA INTERFERTION ACTION WHERE AN IMMUNOGLOBULIN FRAGMENT IS USED |
SG185079A1 (en) | 2010-04-30 | 2012-12-28 | Alexion Pharma Internat Sarl | Methods, compositions, and kits for the treatment of matrix mineralization disorders |
EP2571992B1 (en) * | 2010-05-21 | 2018-04-25 | Merrimack Pharmaceuticals, Inc. | Bi-specific fusion proteins |
KR101330868B1 (en) * | 2010-06-08 | 2013-11-18 | 한미사이언스 주식회사 | Insulin derivative drug conjugate using an immunoglobulin fragment |
KR20120002129A (en) * | 2010-06-30 | 2012-01-05 | 한미홀딩스 주식회사 | A FACTOR VIIa COMPLEX USING AN IMMUNOGLOBULIN FRAGMENT |
KR101337797B1 (en) * | 2010-07-14 | 2013-12-06 | 한미사이언스 주식회사 | A liquid formulation of long acting human growth hormone conjugate |
KR101382593B1 (en) * | 2010-07-21 | 2014-04-10 | 한미사이언스 주식회사 | Novel long-acting glucagon conjugate and pharmaceutical composition comprising the same for the prevention and treatment of obesity |
DK2598533T3 (en) | 2010-07-28 | 2019-04-23 | Gliknik Inc | Fusion proteins of natural human protein fragments to create in an orderly fashion multimerized immunoglobulin FC compositions |
WO2012053828A2 (en) * | 2010-10-20 | 2012-04-26 | 주식회사 한독약품 | Human interleukin-1 receptor antagonist - hybrid fc fusion protein |
KR101333958B1 (en) * | 2010-10-20 | 2013-11-27 | 주식회사 한독 | Human Interleukin-1 receptor antagonist-hybrid Fc fusion protein |
KR101303388B1 (en) * | 2010-10-26 | 2013-09-03 | 한미사이언스 주식회사 | Liquid formulations of long acting interferon alpha conjugate |
EP2465536A1 (en) | 2010-12-14 | 2012-06-20 | CSL Behring AG | CD89 activation in therapy |
JP5766296B2 (en) | 2010-12-23 | 2015-08-19 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Polypeptide-polynucleotide complexes and their use in targeted delivery of effector components |
US9266939B2 (en) | 2010-12-27 | 2016-02-23 | Alexion Pharmaceuticals, Inc. | Compositions comprising natriuretic peptides and methods of use thereof |
CA2824143C (en) | 2011-01-14 | 2018-12-18 | Redwood Bioscience, Inc. | Aldehyde-tagged immunoglobulin polypeptides and method of use thereof |
CN102309765B (en) * | 2011-02-28 | 2013-10-16 | 北京韩美药品有限公司 | Long-acting anticoagulant polypeptide including immunoglobulin Fc segments as carriers and preparation method of same |
CN103533951B (en) | 2011-04-08 | 2017-04-19 | 安姆根有限公司 | Method of treating or ameliorating metabolic disorders using growth differentiation factor 15 (GDF-15) |
UA113626C2 (en) * | 2011-06-02 | 2017-02-27 | A COMPOSITION FOR THE TREATMENT OF DIABETES CONTAINING THE DURABLE INSULIN CON conjugate AND THE DUAL ACTION INSULINOTROPIC PIPIDE | |
CN102807619B (en) * | 2011-06-03 | 2016-08-03 | 北京韩美药品有限公司 | Containing immunoglobulin Fc segments and the complex of granulocyte-macrophage colony stimutaing factor and pharmaceutical composition thereof |
EP2718318B1 (en) | 2011-06-10 | 2018-07-25 | Hanmi Science Co., Ltd. | Novel oxyntomodulin derivatives and pharmaceutical composition for treating obesity comprising the same |
UA114709C2 (en) * | 2011-06-17 | 2017-07-25 | Ханмі Сайенс Ко., Лтд. | CONJUGATE CONTAINING OXYNTOMODULIN DERIVATIVES, THE FACILITY OF THE IMMUNOGLOBULIN AND THE NON-REPTIDYL POLYMER, AND ITS APPLICATION |
KR102103476B1 (en) * | 2011-06-24 | 2020-04-23 | 더 리젠츠 오브 더 유니버시티 오브 콜로라도, 어 바디 코포레이트 | Compositions, methods and uses for alpha-1 antitrypsin fusion molecules |
US10400029B2 (en) | 2011-06-28 | 2019-09-03 | Inhibrx, Lp | Serpin fusion polypeptides and methods of use thereof |
EP2726093A4 (en) * | 2011-06-28 | 2015-08-19 | Inhibrx Llc | Wap domain fusion polypeptides and methods of use thereof |
US8980266B2 (en) | 2011-06-28 | 2015-03-17 | Inhibrx, Llc | Serpin fusion polypeptides and methods of use thereof |
CA2847837A1 (en) * | 2011-09-05 | 2013-03-14 | Hanmi Science Co., Ltd. | A pharmaceutical composition for treating cancer, comprising interferon alpha conjugate |
US9458214B2 (en) * | 2011-09-26 | 2016-10-04 | Novartis Ag | Dual function fibroblast growth factor 21 proteins |
US9597378B2 (en) * | 2011-10-06 | 2017-03-21 | Hanmi Science Co., Ltd. | Blood coagulation factor VII and VIIa derivatives, conjugates and complexes comprising the same, and use thereof |
EP2784080B1 (en) * | 2011-10-31 | 2019-12-18 | Shimadzu Corporation | Peptide-hinge-free flexible antibody-like molecule |
KR20130049671A (en) * | 2011-11-04 | 2013-05-14 | 한미사이언스 주식회사 | Method for preparation of biological active polypeptide conjugate |
MX358358B (en) | 2011-12-19 | 2018-08-15 | Amgen Inc | Variant activin receptor polypeptides, alone or in combination with chemotherapy, and uses thereof. |
CN103172745A (en) * | 2011-12-21 | 2013-06-26 | 北京韩美药品有限公司 | Long-acting human endothelium chalone containing immune globulin Fc segment |
KR102041412B1 (en) * | 2011-12-30 | 2019-11-11 | 한미사이언스 주식회사 | Derivatives of Immunglobulin Fc fragment |
KR101895047B1 (en) * | 2011-12-30 | 2018-09-06 | 한미사이언스 주식회사 | A site-specific GLP-2 conjugate using an immunoglobulin fragment |
JP6684490B2 (en) | 2012-01-09 | 2020-04-22 | ザ・スクリップス・リサーチ・インスティテュート | Ultralong complementarity determining regions and uses thereof |
AU2013208007A1 (en) | 2012-01-09 | 2014-07-31 | The Scripps Research Institute | Humanized antibodies with ultralong CDR3 |
EP2802653A4 (en) | 2012-01-10 | 2015-09-02 | Univ Colorado Regents | Compositions, methods and uses for alpha-1 antitrypsin fusion molecules |
MX2014009129A (en) | 2012-01-26 | 2014-11-21 | Amgen Inc | Growth differentiation factor 15 (gdf-15) polypeptides. |
AR090281A1 (en) | 2012-03-08 | 2014-10-29 | Hanmi Science Co Ltd | IMPROVED PROCESS FOR THE PREPARATION OF A PHYSIOLOGICALLY ACTIVE POLYPEPTIDE COMPLEX |
BR112014022240A2 (en) | 2012-03-09 | 2017-08-01 | Csl Behring Ag | compositions comprising secretory-like immunoglobulins |
EP2636681A1 (en) | 2012-03-09 | 2013-09-11 | CSL Behring AG | Process for enriching IgA |
EP2636684A1 (en) | 2012-03-09 | 2013-09-11 | CSL Behring AG | Prevention of infection |
US10064951B2 (en) | 2012-03-30 | 2018-09-04 | Hanmi Science Co., Ltd. | Liquid formulation of highly concentrated long-acting human growth hormone conjugate |
CA2871468C (en) * | 2012-04-23 | 2021-09-21 | Nrl Pharma, Inc. | Lactoferrin fusion protein and method for preparation thereof |
US10052366B2 (en) | 2012-05-21 | 2018-08-21 | Alexion Pharmaceuticsl, Inc. | Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof |
CA2875989A1 (en) | 2012-06-08 | 2013-12-12 | Sutro Biopharma, Inc. | Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use |
DK2863955T3 (en) | 2012-06-26 | 2017-01-23 | Sutro Biopharma Inc | MODIFIED FC PROTEINS, INCLUDING LOCATION-SPECIFIC NON-NATURAL AMINO ACID RESIDUES, CONJUGATES THEREOF, METHODS OF PRODUCING ITS AND PROCEDURES FOR USE THEREOF |
BR112014032193A2 (en) | 2012-06-27 | 2017-06-27 | Hoffmann La Roche | bispecific antibody production and combination determination methods, bispecific antibody, formulation and use of bispecific antibody |
AR091902A1 (en) | 2012-07-25 | 2015-03-11 | Hanmi Pharm Ind Co Ltd | LIQUID FORMULATION OF A PROLONGED INSULIN CONJUGATE |
US10441665B2 (en) | 2012-07-25 | 2019-10-15 | Hanmi Pharm. Co., Ltd. | Liquid formulation of long acting insulinotropic peptide conjugate |
KR101968344B1 (en) | 2012-07-25 | 2019-04-12 | 한미약품 주식회사 | A composition for treating hyperlipidemia comprising oxyntomodulin analog |
AR094821A1 (en) * | 2012-07-25 | 2015-09-02 | Hanmi Pharm Ind Co Ltd | LIQUID FORMULATION OF AN INSULINOTROPIC PEPTIDE CONJUGATE OF PROLONGED ACTION |
AR092862A1 (en) | 2012-07-25 | 2015-05-06 | Hanmi Pharm Ind Co Ltd | LIQUID FORMULATION OF PROLONGED ACTION INSULIN AND AN INSULINOTROPIC PEPTIDE AND PREPARATION METHOD |
AU2013305885B2 (en) | 2012-08-20 | 2017-12-21 | Gliknik Inc. | Molecules with antigen binding and polyvalent Fc gamma receptor binding activity |
HUE045227T2 (en) | 2012-08-31 | 2019-12-30 | Sutro Biopharma Inc | Modified amino acids comprising an azido group |
US9561275B2 (en) | 2012-09-14 | 2017-02-07 | The Johns Hopkins University | Compositions and methods for rendering tumor cells susceptible to CD8+ T cell-mediated killing |
KR101993393B1 (en) * | 2012-11-06 | 2019-10-01 | 한미약품 주식회사 | A composition for treating diabetes or diabesity comprising oxyntomodulin analog |
MY197849A (en) | 2012-11-06 | 2023-07-20 | Hanmi Pharm Ind Co Ltd | Liquid formulation of protein conjugate comprising the oxyntomodulin and an immunoglobulin fragment |
US9644021B2 (en) | 2013-01-11 | 2017-05-09 | The California Institute For Biomedical Research | Bovine fusion antibodies |
CN103217489B (en) * | 2013-01-15 | 2016-03-09 | 珠海市丽珠单抗生物技术有限公司 | A kind ofly measure the glycosylation of sample in protein purification technological process and the method for end modified situation |
KR102073748B1 (en) * | 2013-01-31 | 2020-02-05 | 한미약품 주식회사 | Recombinant yeast transformant and process for preparing immunoglobulin Fc fragment employing the same |
CA2899889A1 (en) | 2013-02-01 | 2014-08-07 | Santa Maria Biotherapeutics, Inc. | Administration of an anti-activin-a compound to a subject |
WO2014130529A1 (en) * | 2013-02-22 | 2014-08-28 | Zoetis Llc | In ovo administration of growth factors for improving poultry performance |
TWI647240B (en) * | 2013-03-05 | 2019-01-11 | 韓美藥品股份有限公司 | Improved process for preparing physiologically active polypeptide complexes in high yield |
US20140271641A1 (en) * | 2013-03-14 | 2014-09-18 | University Of Guelph | Thrombospondin-1 polypeptides and methods of using same |
JP6397479B2 (en) | 2013-03-15 | 2018-09-26 | エータイアー ファーマ, インコーポレイテッド | Histidyl-tRNA synthetase Fc conjugate |
SG10201913874TA (en) | 2013-03-15 | 2020-03-30 | Biogen Ma Inc | Factor ix polypeptide formulations |
KR101895634B1 (en) * | 2013-05-31 | 2018-09-05 | 한미약품 주식회사 | IgG4 Fc fragment comprising modified hinge region |
US9764039B2 (en) | 2013-07-10 | 2017-09-19 | Sutro Biopharma, Inc. | Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use |
AR096890A1 (en) * | 2013-07-12 | 2016-02-03 | Hanmi Pharm Ind Co Ltd | CONJUGATING FC OF IMMUNOGLOBULINA, THAT MAINTAINS THE UNION AFFINITY OF THE FC FRAGMENT OF THE IMMUNOGLOBULIN TO FCRN |
AR096891A1 (en) * | 2013-07-12 | 2016-02-03 | Hanmi Pharm Ind Co Ltd | CONJUGATE OF BIOMOLOGICALLY ACTIVE POLYPEPTIDE MONOMER AND CONJUGATE OF FRAGMENTO FC OF IMMUNOGLOBULINA, THAT SHOWS CLEARING THROUGH REDUCED RECEPTOR, AND THE METHOD FOR PREPARING THE SAME |
WO2015010100A2 (en) | 2013-07-18 | 2015-01-22 | Fabrus, Inc. | Humanized antibodies with ultralong complementarity determining regions |
JP6509852B2 (en) * | 2013-07-31 | 2019-05-08 | アムジエン・インコーポレーテツド | Constructs of growth differentiation factor 15 (GDF-15) |
AU2014312190A1 (en) | 2013-08-28 | 2016-02-18 | Bioasis Technologies Inc. | CNS-targeted conjugates of antibodies |
PL3041513T3 (en) | 2013-09-08 | 2021-01-25 | Kodiak Sciences Inc. | Factor viii zwitterionic polymer conjugates |
US9657059B2 (en) | 2013-09-09 | 2017-05-23 | Canimguide Therapeutics Ab | Immune system modulators |
KR102276304B1 (en) | 2013-09-27 | 2021-07-14 | 한미약품 주식회사 | A formulation of long-acting human growth hormone conjugate |
WO2015047890A1 (en) * | 2013-09-30 | 2015-04-02 | 3M Innovative Properties Company | Fibers and wipes with epoxidized fatty ester disposed thereon, and methods |
PL3068797T3 (en) | 2013-11-11 | 2020-06-29 | Wake Forest University Health Sciences | Constructs for multi-valent targeting of tumors |
EA201691374A1 (en) | 2014-01-20 | 2016-12-30 | Ханми Фарм. Ко., Лтд. | INSULIN LONG ACTION AND ITS APPLICATION |
US10421796B2 (en) * | 2014-01-21 | 2019-09-24 | Gray D Shaw | Variants of IgG Fc with limited amine groups |
WO2015152618A1 (en) * | 2014-03-31 | 2015-10-08 | 한미약품 주식회사 | Method for improving solubility of protein and peptide by using immunoglobulin fc fragment linkage |
CN106459205B (en) * | 2014-04-11 | 2021-04-09 | 免疫医疗有限责任公司 | Conjugated compounds comprising cysteine engineered antibodies |
CA2947255A1 (en) * | 2014-04-28 | 2015-11-05 | The National Institute for Biotechnology in the Negev Ltd. | Variants of dr3 and use thereof |
KR20150133576A (en) * | 2014-05-20 | 2015-11-30 | 삼성전자주식회사 | Chemically modified targeting protein and use thereof |
AR100639A1 (en) | 2014-05-29 | 2016-10-19 | Hanmi Pharm Ind Co Ltd | COMPOSITION TO TREAT DIABETES THAT INCLUDES CONJUGATES OF PROLONGED INSULIN ANALOGS AND CONJUGATES OF PROLONGED INSULINOTROPIC PEPTIDES |
TWI684458B (en) | 2014-05-30 | 2020-02-11 | 南韓商韓美藥品股份有限公司 | Composition for treating diabetes mellitus comprising insulin and a glp-1/glucagon dual agonist |
WO2015197598A2 (en) * | 2014-06-27 | 2015-12-30 | Innate Pharma | Multispecific antigen binding proteins |
US10519234B2 (en) | 2014-06-27 | 2019-12-31 | Innate Pharma | NKp46 binding proteins |
US9840553B2 (en) | 2014-06-28 | 2017-12-12 | Kodiak Sciences Inc. | Dual PDGF/VEGF antagonists |
WO2016007873A1 (en) | 2014-07-11 | 2016-01-14 | The Regents Of The University Of Michigan | Compositions and methods for treating craniosynostosis |
BR112017001403A2 (en) * | 2014-07-24 | 2017-11-21 | Genentech Inc | methods for conjugating an agent with a thiol moiety to a protein containing at least one trisulfide bond |
TWI772252B (en) | 2014-09-16 | 2022-08-01 | 南韓商韓美藥品股份有限公司 | Use of a long acting glp-1/glucagon receptor dual agonist for the treatment of non-alcoholic fatty liver disease |
US9616114B1 (en) | 2014-09-18 | 2017-04-11 | David Gordon Bermudes | Modified bacteria having improved pharmacokinetics and tumor colonization enhancing antitumor activity |
EP3207128B1 (en) * | 2014-10-17 | 2022-07-27 | Kodiak Sciences Inc. | Butyrylcholinesterase zwitterionic polymer conjugates |
WO2016090251A1 (en) | 2014-12-05 | 2016-06-09 | Alexion Pharmaceuticals, Inc. | Treating seizure with recombinant alkaline phosphatase |
KR102418477B1 (en) | 2014-12-30 | 2022-07-08 | 한미약품 주식회사 | Gluagon Derivatives |
AU2015372818A1 (en) | 2014-12-30 | 2017-07-27 | Hanmi Pharm. Co., Ltd. | Glucagon derivatives with improved stability |
CA2973883A1 (en) | 2015-01-28 | 2016-08-04 | Alexion Pharmaceuticals, Inc. | Methods of treating a subject with an alkaline phosphatase deficiency |
ES2853582T3 (en) | 2015-03-06 | 2021-09-16 | Canimguide Therapeutics Ab | Immune system modulators and compositions |
EP3280727B1 (en) | 2015-04-06 | 2021-02-17 | Acceleron Pharma Inc. | Single-arm type i and type ii receptor fusion proteins and uses thereof |
BR112017022658A2 (en) | 2015-04-22 | 2018-07-17 | Alivegen Usa Inc | isolated protein, isolated nucleic acid molecule, recombinant vector, host cell, method of producing a hybrid actriib protein, pharmaceutical composition, methods of treating myostatin or activin-related disorders, muscle wasting disease, disease cardiovascular disease, metabolic disorders, cancer cells, kidney disease, inflammatory / autoimmune disease, fibrosis disease, anemia, pain, aging condition, bone disorders, muscle wasting or metabolic disorder or fibrotic or inflammatory or activin-related in individuals, and a method of inducing stem cell growth for tissue repair or organ regeneration in an individual |
KR20170138569A (en) * | 2015-04-29 | 2017-12-15 | 메디오라늄 파마세우티시 에스.피.에이. | Soluble chimeric interleukin-10 receptor and its therapeutic use |
CN107835693B (en) * | 2015-06-12 | 2021-11-02 | 联亚药业股份有限公司 | Immunoglobulin fusion proteins and uses thereof |
US11820807B2 (en) * | 2015-06-12 | 2023-11-21 | Ubi Pharma Inc | Immunoglobulin fusion proteins and uses thereof |
AU2016284871B2 (en) | 2015-06-23 | 2022-09-29 | Innate Pharma | Multispecific NK engager proteins |
EP4324519A2 (en) * | 2015-06-30 | 2024-02-21 | Hanmi Pharm. Co., Ltd. | Glucagon derivative and a composition comprising a long acting conjugate of the same |
KR20170012145A (en) | 2015-07-24 | 2017-02-02 | 한미약품 주식회사 | Method for producing physiologically active polypeptide conjugate |
RU2745528C2 (en) | 2015-08-17 | 2021-03-26 | Алексион Фармасьютикалз, Инк. | Production of alkaline phosphatases |
UY36870A (en) | 2015-08-28 | 2017-03-31 | Hanmi Pharm Ind Co Ltd | NEW INSULIN ANALOGS |
WO2017053469A2 (en) | 2015-09-21 | 2017-03-30 | Aptevo Research And Development Llc | Cd3 binding polypeptides |
WO2017052329A1 (en) * | 2015-09-24 | 2017-03-30 | Hanmi Pharm. Co., Ltd. | Protein complex by use of a specific site of an immunoglobulin fragment for linkage |
TW201718629A (en) * | 2015-09-25 | 2017-06-01 | 韓美藥品股份有限公司 | A protein conjugate comprising multiple physiological polypeptides and an immunoglobulin Fc region |
US11229686B2 (en) | 2015-09-28 | 2022-01-25 | Alexion Pharmaceuticals, Inc. | Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia |
WO2017059168A1 (en) | 2015-10-01 | 2017-04-06 | Heat Biologics, Inc. | Compositions and methods for adjoining type i and type ii extracellular domains as heterologous chimeric proteins |
CN108367048B (en) * | 2015-10-02 | 2022-08-12 | 银溪制药股份有限公司 | Bispecific therapeutic proteins for tissue repair |
US20170095577A1 (en) * | 2015-10-06 | 2017-04-06 | Washington University | Noninvasive imaging of focal atherosclerotic lesions using fluorescence molecular tomography |
WO2017074466A1 (en) | 2015-10-30 | 2017-05-04 | Alexion Pharmaceuticals, Inc. | Methods for treating craniosynostosis in a patient |
US20190002516A1 (en) * | 2015-12-21 | 2019-01-03 | Hefei Lifeon Pharmaceutical Co., Ltd. | Drug Design Method, Obtained Drug and Application Thereof |
KR20170079409A (en) * | 2015-12-30 | 2017-07-10 | 한미약품 주식회사 | A novel liquid formulation of long acting human growth hormone conjugate |
RU2744860C2 (en) | 2015-12-30 | 2021-03-16 | Кодиак Сайенсиз Инк. | Antibodies and their conjugates |
TWI731015B (en) | 2015-12-31 | 2021-06-21 | 南韓商韓美藥品股份有限公司 | Long-acting conjugate of triple glucagon/glp-1/gip receptor agonist |
US11708413B2 (en) | 2016-01-27 | 2023-07-25 | Sutro Biopharma, Inc. | Anti-CD74 antibody conjugates, compositions comprising anti-CD74 antibody conjugates and methods of using anti-CD74 antibody conjugates |
AR107483A1 (en) | 2016-01-29 | 2018-05-02 | Hanmi Pharm Ind Co Ltd | CONJUGATE OF THERAPEUTIC ENZYMES |
EP3426286A4 (en) | 2016-03-08 | 2019-12-04 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in children |
WO2017173395A1 (en) | 2016-04-01 | 2017-10-05 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in adolescents and adults |
CA3019726A1 (en) | 2016-04-01 | 2017-10-05 | Alexion Pharmaceuticals, Inc. | Treating muscle weakness with alkaline phosphatases |
US11208632B2 (en) | 2016-04-26 | 2021-12-28 | R.P. Scherer Technologies, Llc | Antibody conjugates and methods of making and using the same |
WO2017191817A1 (en) | 2016-05-02 | 2017-11-09 | 味の素株式会社 | Azide group-containing fc protein |
CN106008722B (en) * | 2016-05-13 | 2019-10-15 | 未名生物医药有限公司 | A kind of recombinant beta-hNGF-Fc fusion protein, Preparation method and use |
WO2017214130A1 (en) | 2016-06-06 | 2017-12-14 | Alexion Pharmaceuticals, Inc. | Metal impact on manufacturing of alkaline phosphatases |
CA3026420A1 (en) | 2016-06-07 | 2017-12-14 | Gliknik Inc. | Cysteine-optimized stradomers |
IL263934B2 (en) | 2016-06-29 | 2023-10-01 | Hanmi Pharm Ind Co Ltd | Glucagon derivative, conjugate thereof, composition comprising same and therapeutic use thereof |
JP7018933B2 (en) | 2016-08-18 | 2022-02-14 | アレクシオン ファーマシューティカルズ, インコーポレイテッド | How to treat bronchomalacia |
JOP20190019A1 (en) * | 2016-08-30 | 2019-02-12 | Genexine Inc | PHARMACEUTICAL COMPOSITION FOR TREATING GROWTH HORMONE DEFICIENCY CONTAINING hGH FUSION PROTEIN |
WO2018056764A1 (en) | 2016-09-23 | 2018-03-29 | 한미약품 주식회사 | Insulin analog having reduced binding force to insulin receptor, and use thereof |
EP3549950A4 (en) * | 2016-12-05 | 2020-08-19 | Hanmi Pharm. Co., Ltd. | Conjugate having attenuated immune response |
US11180535B1 (en) | 2016-12-07 | 2021-11-23 | David Gordon Bermudes | Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria |
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
EP3551647A4 (en) | 2016-12-09 | 2021-01-13 | Gliknik Inc. | Manufacturing optimization of gl-2045, a multimerizing stradomer |
EP3578204A4 (en) * | 2017-02-03 | 2021-01-06 | Hanmi Pharm. Co., Ltd. | Conjugate of bioactive material having enhanced sustainability and use thereof |
CA3054132A1 (en) | 2017-02-27 | 2018-08-30 | Shattuck Labs, Inc. | Csf1r-based chimeric proteins |
MX2019009812A (en) | 2017-02-27 | 2019-10-14 | Shattuck Labs Inc | Tigit- and light-based chimeric proteins. |
WO2018174668A2 (en) | 2017-03-23 | 2018-09-27 | 한미약품 주식회사 | Insulin analog complex with reduced affinity for insulin receptor and use thereof |
AU2018243320A1 (en) | 2017-03-31 | 2019-10-10 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia (HPP) in adults and adolescents |
US11767520B2 (en) | 2017-04-20 | 2023-09-26 | Atyr Pharma, Inc. | Compositions and methods for treating lung inflammation |
US20200131225A1 (en) * | 2017-04-20 | 2020-04-30 | Novo Nordisk A/S | Methods of purification of albumin fusion proteins |
CN109206522B (en) * | 2017-07-07 | 2021-11-09 | 北京三有利和泽生物科技有限公司 | Long-acting anticoagulant fusion protein and application thereof |
JP6566324B2 (en) * | 2017-09-29 | 2019-08-28 | サイデン化学株式会社 | Adhesive sheet |
KR20190038456A (en) * | 2017-09-29 | 2019-04-08 | 한미약품 주식회사 | A long acting protein complex having an enhanced efficiency |
EP3692070A1 (en) | 2017-10-02 | 2020-08-12 | Denali Therapeutics Inc. | Fusion proteins comprising enzyme replacement therapy enzymes |
CN108218998A (en) * | 2017-12-31 | 2018-06-29 | 武汉班科生物技术股份有限公司 | A kind of Fc segments of saltant type humanized IgG and preparation method and application |
KR101974305B1 (en) * | 2018-02-14 | 2019-04-30 | 한미사이언스 주식회사 | Method for preparation of biological active polypeptide conjugate |
EP3772926A1 (en) * | 2018-03-26 | 2021-02-17 | Regeneron Pharmaceuticals, Inc. | Humanized rodents for testing therapeutic agents |
KR102209108B1 (en) | 2018-03-27 | 2021-01-28 | 국립암센터 | A composition for inhibiting stemness comprising peptides for blocking the function of OCT4 |
US11913039B2 (en) | 2018-03-30 | 2024-02-27 | Alexion Pharmaceuticals, Inc. | Method for producing recombinant alkaline phosphatase |
AU2019253462A1 (en) | 2018-04-09 | 2020-11-26 | Amgen Inc. | Growth differentiation factor 15 fusion proteins |
US10780121B2 (en) | 2018-08-29 | 2020-09-22 | Shattuck Labs, Inc. | FLT3L-based chimeric proteins |
JP2022530216A (en) * | 2019-04-23 | 2022-06-28 | エルジー・ケム・リミテッド | Fusion polypeptide containing Fc region of immunoglobulin and GDF15 |
US11267858B2 (en) * | 2019-05-31 | 2022-03-08 | Spectrum Pharmaceuticals, Inc. | Methods of treatment using G-CSF protein complex |
CN112142850A (en) * | 2019-06-27 | 2020-12-29 | 深圳市卫光生物制品股份有限公司 | Human nerve growth factor-lactoferrin recombinant protein and application |
CA3157509A1 (en) | 2019-10-10 | 2021-04-15 | Kodiak Sciences Inc. | Methods of treating an eye disorder |
CN110669134A (en) * | 2019-10-15 | 2020-01-10 | 广东菲鹏生物有限公司 | IgM-FC fragment, IgM-FC antibody, preparation method and application |
CN114867739A (en) * | 2019-12-11 | 2022-08-05 | 株式会社Lg化学 | Fusion polypeptide comprising GDF15 and a polypeptide region capable of O-glycosylation |
CN111153996B (en) * | 2020-01-10 | 2021-12-14 | 苏州睿瀛生物技术有限公司 | Antibody of G protein coupled receptor, preparation method thereof and G protein coupled receptor kit |
CN111505291B (en) * | 2020-04-14 | 2023-04-25 | 山东省千佛山医院 | Method for eliminating interference of macroenzyme molecules on serum enzyme concentration detection |
CA3179707A1 (en) | 2020-05-22 | 2021-11-25 | Ji Hye Moon | Liquid formulation |
CN112098639B (en) * | 2020-09-21 | 2024-01-02 | 天津医科大学 | Synthesis and application of secondary antibody with graphene oxide as carrier |
WO2023147489A2 (en) * | 2022-01-28 | 2023-08-03 | argenx BV | Anti-musk antibodies for use in treating neuromuscular disorders |
WO2023168403A2 (en) * | 2022-03-03 | 2023-09-07 | The Trustees Of The University Of Pennsylvania | Viral vectors encoding parathyroid hormone fusions and uses thereof in treating hypoparathyroidism |
CN114588315A (en) * | 2022-03-14 | 2022-06-07 | 东莞市人民医院 | Preparation method of anti-inflammatory protein coating, bioengineering functional material and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001003737A1 (en) * | 1999-07-13 | 2001-01-18 | Bolder Biotechnology Inc. | Immunoglobulin fusion proteins |
US20020081664A1 (en) * | 1999-05-19 | 2002-06-27 | Kin-Ming Lo | Expression and export of interferon-alpha proteins as Fc fusion proteins |
Family Cites Families (118)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4399216A (en) | 1980-02-25 | 1983-08-16 | The Trustees Of Columbia University | Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials |
GB8504099D0 (en) | 1985-02-18 | 1985-03-20 | Wellcome Found | Physiologically active substances |
JPS62153300A (en) * | 1985-12-26 | 1987-07-08 | Teijin Ltd | Fc region protein of human immunoglobulin g and production thereof |
JPH0728746B2 (en) | 1986-02-28 | 1995-04-05 | 理化学研究所 | Novel plasmid, microbial cell, and method for producing human immunoglobulin G Fc region protein |
US5643758A (en) * | 1987-03-10 | 1997-07-01 | New England Biolabs, Inc. | Production and purification of a protein fused to a binding protein |
JPS63245691A (en) | 1987-03-31 | 1988-10-12 | Teijin Ltd | Human immunoglobulin gfc region protein and production thereof |
JPS63290899A (en) | 1987-05-22 | 1988-11-28 | Takeda Chem Ind Ltd | Fragment of human igefc protein and production thereof |
US6710169B2 (en) * | 1987-10-02 | 2004-03-23 | Genentech, Inc. | Adheson variants |
DE3889546T2 (en) | 1987-12-21 | 1994-09-08 | Univ Toledo | TRANSFORMATION OF Germinating PLANT SEEDS WITH THE HELP OF AGROBACTERIUM. |
US5567584A (en) | 1988-01-22 | 1996-10-22 | Zymogenetics, Inc. | Methods of using biologically active dimerized polypeptide fusions to detect PDGF |
US6004781A (en) | 1988-01-22 | 1999-12-21 | The General Hospital Corporation | Nucleic acid encoding Ig-CD4 fusion proteins |
US6018026A (en) | 1988-01-22 | 2000-01-25 | Zymogenetics, Inc. | Biologically active dimerized and multimerized polypeptide fusions |
GB8824869D0 (en) | 1988-10-24 | 1988-11-30 | Stevenson G T | Synthetic antibody |
US5225538A (en) | 1989-02-23 | 1993-07-06 | Genentech, Inc. | Lymphocyte homing receptor/immunoglobulin fusion proteins |
US6406697B1 (en) | 1989-02-23 | 2002-06-18 | Genentech, Inc. | Hybrid immunoglobulins |
US5116964A (en) | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
US6307025B1 (en) | 1989-04-28 | 2001-10-23 | Biogen, Inc. | VCAM fusion proteins and DNA coding therefor |
FR2650598B1 (en) | 1989-08-03 | 1994-06-03 | Rhone Poulenc Sante | DERIVATIVES OF ALBUMIN WITH THERAPEUTIC FUNCTION |
US6541610B1 (en) | 1989-09-05 | 2003-04-01 | Immunex Corporation | Fusion proteins comprising tumor necrosis factor receptor |
US5605690A (en) | 1989-09-05 | 1997-02-25 | Immunex Corporation | Methods of lowering active TNF-α levels in mammals using tumor necrosis factor receptor |
GB9009106D0 (en) | 1990-04-23 | 1990-06-20 | 3I Res Expl Ltd | Processes and intermediates for synthetic antibody derivatives |
US5349053A (en) | 1990-06-01 | 1994-09-20 | Protein Design Labs, Inc. | Chimeric ligand/immunoglobulin molecules and their uses |
EP0835939B8 (en) | 1990-06-28 | 2006-01-11 | Sanofi-Aventis Deutschland GmbH | Fusion proteins with parts of immunoglobulins, their production and use |
US7253264B1 (en) | 1990-06-28 | 2007-08-07 | Sanofi-Arentideutschland GmbH | Immunoglobulin fusion proteins, their production and use |
US5650150A (en) | 1990-11-09 | 1997-07-22 | Gillies; Stephen D. | Recombinant antibody cytokine fusion proteins |
US5191066A (en) | 1990-12-07 | 1993-03-02 | Abbott Laboratories | Site-specific conjugation of immunoglobulins and detectable labels |
CA2080226C (en) * | 1991-02-08 | 2004-05-04 | Gary A. Beaudry | Cd4-gamma2 and cd4-igg2 chimeras |
ES2199935T3 (en) | 1991-03-15 | 2004-03-01 | Amgen Inc. | PEGILATION OF POLYPEPTIDES. |
WO1992018626A1 (en) * | 1991-04-17 | 1992-10-29 | Laboratoire Europeen De Biotechnologie | WATER-SOLUBLE POLYPEPTIDES HAVING HIGH AFFINITY FOR INTERFERONS α AND $g(b) |
EP0533006A1 (en) | 1991-09-18 | 1993-03-24 | F.Hoffmann-La Roche & Co. Aktiengesellschaft | Chimaeric interleukin 5-receptor/immunoglobulin polypeptides |
US20020037558A1 (en) | 1991-10-23 | 2002-03-28 | Kin-Ming Lo | E.coli produced immunoglobulin constructs |
FR2686899B1 (en) | 1992-01-31 | 1995-09-01 | Rhone Poulenc Rorer Sa | NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
FR2686901A1 (en) | 1992-01-31 | 1993-08-06 | Rhone Poulenc Rorer Sa | NOVEL ANTITHROMBOTIC POLYPEPTIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
GB9206422D0 (en) | 1992-03-24 | 1992-05-06 | Bolt Sarah L | Antibody preparation |
US5447851B1 (en) | 1992-04-02 | 1999-07-06 | Univ Texas System Board Of | Dna encoding a chimeric polypeptide comprising the extracellular domain of tnf receptor fused to igg vectors and host cells |
JPH0640945A (en) | 1992-07-23 | 1994-02-15 | Kureha Chem Ind Co Ltd | Fc fragment binding antitumor agent |
EP0664710A4 (en) * | 1992-08-07 | 1998-09-30 | Progenics Pharm Inc | NON-PEPTIDYL MOIETY-CONJUGATED CD4-GAMMA2 AND CD4-IgG2 IMMUNOCONJUGATES, AND USES THEREOF. |
IT1263831B (en) | 1993-01-29 | 1996-09-04 | Paolo Chiesi | MULTI-COMPONENT INCLUSION COMPLEXES WITH HIGH SOLUBILITY CONSTITUTED BY A BASIC-TYPE DRUG, AN ACID AND A CYCLODEXTRINE |
US6482919B2 (en) | 1993-02-01 | 2002-11-19 | Bristol-Myers Squibb Company | Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell |
US5470952A (en) | 1993-10-20 | 1995-11-28 | Regeneron Pharmaceuticals, Inc. | CNTF and IL-6 antagonists |
US5738846A (en) | 1994-11-10 | 1998-04-14 | Enzon, Inc. | Interferon polymer conjugates and process for preparing the same |
US6410008B1 (en) | 1994-12-12 | 2002-06-25 | Beth Israel Hospital Association | Chimeric IL-10 proteins and uses thereof |
US6030613A (en) | 1995-01-17 | 2000-02-29 | The Brigham And Women's Hospital, Inc. | Receptor specific transepithelial transport of therapeutics |
EP0816381B1 (en) | 1995-03-10 | 2004-01-14 | NAKAMURA, Toshikazu | Polyethylene glycol modified hepatocyte growth factor (hgf) |
US6096871A (en) | 1995-04-14 | 2000-08-01 | Genentech, Inc. | Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life |
GB9511935D0 (en) | 1995-06-13 | 1995-08-09 | Smithkline Beecham Plc | Novel compound |
BR9610492A (en) * | 1995-09-21 | 1999-03-30 | Andaris Ltd | Transcytosis vehicles and intensifiers for drug release |
US6936439B2 (en) | 1995-11-22 | 2005-08-30 | Amgen Inc. | OB fusion protein compositions and methods |
US6620413B1 (en) | 1995-12-27 | 2003-09-16 | Genentech, Inc. | OB protein-polymer chimeras |
US5723125A (en) | 1995-12-28 | 1998-03-03 | Tanox Biosystems, Inc. | Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide |
US6750334B1 (en) * | 1996-02-02 | 2004-06-15 | Repligen Corporation | CTLA4-immunoglobulin fusion proteins having modified effector functions and uses therefor |
DE69731289D1 (en) | 1996-03-18 | 2004-11-25 | Univ Texas | IMMUNGLOBULIN-LIKE DOMAIN WITH INCREASED HALF-VALUE TIMES |
KR19980038061A (en) | 1996-11-23 | 1998-08-05 | 유우준 | Food Waste Water Separation Treatment Device |
US7122636B1 (en) * | 1997-02-21 | 2006-10-17 | Genentech, Inc. | Antibody fragment-polymer conjugates and uses of same |
US6277375B1 (en) | 1997-03-03 | 2001-08-21 | Board Of Regents, The University Of Texas System | Immunoglobulin-like domains with increased half-lives |
TW577759B (en) * | 1997-04-18 | 2004-03-01 | Ipsen Pharma Biotech | Sustained release compositions in the form of microcapsules or implants and the process for their preparation |
US5990237A (en) * | 1997-05-21 | 1999-11-23 | Shearwater Polymers, Inc. | Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines |
AU8182298A (en) | 1997-07-10 | 1999-02-08 | Beth Israel Deaconess Medical Center | Recombinant erythropoietin / immunoglobulin fusion proteins |
US6165476A (en) | 1997-07-10 | 2000-12-26 | Beth Israel Deaconess Medical Center | Fusion proteins with an immunoglobulin hinge region linker |
US6451986B1 (en) * | 1998-06-22 | 2002-09-17 | Immunex Corporation | Site specific protein modification |
US6703381B1 (en) * | 1998-08-14 | 2004-03-09 | Nobex Corporation | Methods for delivery therapeutic compounds across the blood-brain barrier |
KR100316347B1 (en) * | 1998-09-15 | 2002-08-27 | 한미약품(주) | Recombinant microorganisms expressing a fusion protein of Escherichia coli enterotoxin II signal peptide and fusion protein of human growth hormone and a method of producing human growth hormone using the same |
PT1121382E (en) | 1998-10-16 | 2006-10-31 | Biogen Idec Inc | BETA INTERFERENCE FUSE PROTEINS AND THEIR RESPECTIVE USES |
US6660843B1 (en) | 1998-10-23 | 2003-12-09 | Amgen Inc. | Modified peptides as therapeutic agents |
KR20020007287A (en) | 1999-01-07 | 2002-01-26 | 추후보정 | EXPRESSION AND EXPORT OF ANTI-OBESITY PROTEINS AS Fc FUSION PROTEINS |
US6737056B1 (en) * | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
AU3224700A (en) | 1999-02-08 | 2000-08-25 | Chiron Corporation | Fibroblast growth factor receptor-immunoglobulin fusion |
US20040266673A1 (en) * | 2002-07-31 | 2004-12-30 | Peter Bakis | Long lasting natriuretic peptide derivatives |
US6833349B2 (en) * | 1999-06-08 | 2004-12-21 | Regeneron Pharmaceuticals, Inc. | Methods of treating inflammatory skin diseases |
JO2291B1 (en) | 1999-07-02 | 2005-09-12 | اف . هوفمان لاروش ايه جي | Erythopintin derivatives |
EP1194451A1 (en) * | 1999-07-02 | 2002-04-10 | Genentech, Inc. | Fusion peptides comprising a peptide ligand domain and a multimerization domain |
KR100360594B1 (en) * | 2000-01-19 | 2002-11-13 | 한미약품공업 주식회사 | Expression and secretion vector for human interferon alpha and process for producing human interferon alpha by employing same |
WO2001081376A2 (en) * | 2000-04-21 | 2001-11-01 | Amgen Inc. | Apo-ai/aii peptide derivatives |
US6756480B2 (en) * | 2000-04-27 | 2004-06-29 | Amgen Inc. | Modulators of receptors for parathyroid hormone and parathyroid hormone-related protein |
US20020168367A1 (en) * | 2000-04-28 | 2002-11-14 | Planet Biotechnology Incorporated | Novel immunoadhesins for treating and preventing viral and bacterial diseases |
US6677136B2 (en) * | 2000-05-03 | 2004-01-13 | Amgen Inc. | Glucagon antagonists |
DE10021731B4 (en) | 2000-05-04 | 2005-12-08 | Aventis Pharma Deutschland Gmbh | Cyclipostins, process for their preparation and pharmaceutical preparation thereof |
US6417237B1 (en) | 2000-06-08 | 2002-07-09 | The Board Of Trustees Of The University Of Illinois | Macromolecular drug complexes and compositions containing the same |
PT1355942E (en) | 2000-12-07 | 2008-11-21 | Lilly Co Eli | Glp-1 fusion proteins |
US6979556B2 (en) | 2000-12-14 | 2005-12-27 | Genentech, Inc. | Separate-cistron contructs for secretion of aglycosylated antibodies from prokaryotes |
US6828121B2 (en) | 2000-12-14 | 2004-12-07 | Genentech, Inc. | Bacterial host strains |
US7754208B2 (en) | 2001-01-17 | 2010-07-13 | Trubion Pharmaceuticals, Inc. | Binding domain-immunoglobulin fusion proteins |
US7829084B2 (en) | 2001-01-17 | 2010-11-09 | Trubion Pharmaceuticals, Inc. | Binding constructs and methods for use thereof |
JP2004525621A (en) | 2001-01-18 | 2004-08-26 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | Bifunctional fusion protein having glucocerebrosidase activity |
JP2004532620A (en) * | 2001-02-19 | 2004-10-28 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | Artificial proteins with reduced immunogenicity |
KR100566911B1 (en) | 2001-06-25 | 2006-04-03 | 주식회사 삼양사 | Negatively charged amphiphilic block copolymer as drug carrier and complex thereof with positively charged drug |
EP1411983A4 (en) * | 2001-06-26 | 2006-06-21 | Imclone Systems Inc | Bispecific antibodies that bind to vegf receptors |
JP2005501052A (en) * | 2001-07-31 | 2005-01-13 | イムノメディクス, インコーポレイテッド | Polymer delivery system |
US6900292B2 (en) * | 2001-08-17 | 2005-05-31 | Lee-Hwei K. Sun | Fc fusion proteins of human erythropoietin with increased biological activities |
US6797493B2 (en) * | 2001-10-01 | 2004-09-28 | Lee-Hwei K. Sun | Fc fusion proteins of human granulocyte colony-stimulating factor with increased biological activities |
US7125843B2 (en) * | 2001-10-19 | 2006-10-24 | Neose Technologies, Inc. | Glycoconjugates including more than one peptide |
US7138370B2 (en) | 2001-10-11 | 2006-11-21 | Amgen Inc. | Specific binding agents of human angiopoietin-2 |
KR100811138B1 (en) | 2001-11-13 | 2008-03-07 | 오리온피디피주식회사 | method of manufacturing a multilayer circuit board using low temperature cofired ceramic on metal, and a multilayer circuit board manufactured thereby |
WO2003049684A2 (en) * | 2001-12-07 | 2003-06-19 | Centocor, Inc. | Pseudo-antibody constructs |
AU2003217912A1 (en) * | 2002-03-01 | 2003-09-16 | Xencor | Antibody optimization |
EP1572936A2 (en) | 2002-03-05 | 2005-09-14 | Eli Lilly And Company | Heterologous g-csf fusion proteins |
CN101143221A (en) | 2002-03-15 | 2008-03-19 | 布赖汉姆妇女医院 | Central airway administration for systemic delivery of therapeutics |
US20030191056A1 (en) * | 2002-04-04 | 2003-10-09 | Kenneth Walker | Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins |
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US20050281829A1 (en) | 2003-05-06 | 2005-12-22 | Hehir Cristina A T | Fc chimeric proteins with anti-HIV drugs |
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CA2581505A1 (en) | 2004-09-27 | 2006-04-06 | Centocor, Inc. | Srage mimetibody, compositions, methods and uses |
KR100594607B1 (en) * | 2004-11-03 | 2006-06-30 | 재단법인서울대학교산학협력재단 | Probiotic microorganisms producing human growth hormone fused with Fc fragment of human IgG for oral delivery system and a method for producing the same |
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JP2010106019A (en) | 2008-10-02 | 2010-05-13 | Kanazawa Univ | Agent of prophylaxis, therapy, and or symptom alleviation for peripheral neuropathy resulting from cancer chemotherapy comprising limaprost |
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KR20120002129A (en) * | 2010-06-30 | 2012-01-05 | 한미홀딩스 주식회사 | A FACTOR VIIa COMPLEX USING AN IMMUNOGLOBULIN FRAGMENT |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020081664A1 (en) * | 1999-05-19 | 2002-06-27 | Kin-Ming Lo | Expression and export of interferon-alpha proteins as Fc fusion proteins |
WO2001003737A1 (en) * | 1999-07-13 | 2001-01-18 | Bolder Biotechnology Inc. | Immunoglobulin fusion proteins |
Non-Patent Citations (1)
Title |
---|
VAN DER POLL T. ET AL.: "Effects of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans", BLOOD, vol. 89, no. 10, May 1997 (1997-05-01), pages 3727 - 3734, XP008110247 * |
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