WO2002079232A2 - Reducing the immunogenicity of fusion proteins - Google Patents
Reducing the immunogenicity of fusion proteins Download PDFInfo
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- WO2002079232A2 WO2002079232A2 PCT/US2002/009815 US0209815W WO02079232A2 WO 2002079232 A2 WO2002079232 A2 WO 2002079232A2 US 0209815 W US0209815 W US 0209815W WO 02079232 A2 WO02079232 A2 WO 02079232A2
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- protein
- fusion protein
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Classifications
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- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/475—Growth factors; Growth regulators
- C07K14/505—Erythropoietin [EPO]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/52—Cytokines; Lymphokines; Interferons
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C07K—PEPTIDES
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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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
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- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S424/00—Drug, bio-affecting and body treating compositions
- Y10S424/809—Drug, bio-affecting and body treating compositions involving immunoglobulin or antibody fragment, e.g. fab', fv, fc, heavy chain or light chain
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/868—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving autoimmunity, allergy, immediate hypersensitivity, delayed hypersensitivity, immunosuppression, or immunotolerance
Definitions
- the present invention relates generally to methods and compositions for making and using modified fusion proteins with reduced or no immunogenicity as therapeutic agents. More specifically, the invention relates to fusion proteins, made less immunogenic by identifying candidate T-cell epitopes and modifying the amino acid sequence to eliminate such epitopes.
- Many therapeutic proteins are normal human proteins.
- interleukin-2, erythropoietin, and growth hormone are all human proteins that are given to humans who already usually make endogenous levels of these proteins.
- immune responses against completely normal human proteins are rare when these proteins are used as therapeutics.
- Enbrel is a fusion of the extracellular domain of a TNF receptor with an IgGl Fc region. Enbrel is used to treat rheumatoid arthritis, and is thought to function by titrating TNF and preventing TNF action. However, a significant incidence of anti-Enbrel antibodies have been noted in patients treated with Enbrel.
- immunocytokines Another example of a therapeutically useful class of fusion proteins is the immunocytokines. These proteins include an antibody moiety and a cytokine moiety, and are useful for targeting cytokines to diseased cells, such as cancer cells. However, the therapeutic use of many of these fusion proteins is curtailed due to their immunogenicity in mammals, especially humans.
- the present invention features methods and compositions useful for producing fusion proteins with reduced immunogenicity for use in therapy.
- the invention features immunocytokines, immunofusins, immunoligands, other antibody and Fc fusion proteins, cytokine-cytokine fusion proteins, and albumin fusion proteins with decreased immunogenicity.
- the invention relates in part to the insight that fusion proteins contain sequences that are "non-self."
- the region surrounding the fusion junction comprises a peptide sequence that is not normally present in the human body.
- a protein drug such as Enbrel is derived from two normal human proteins: TNF receptor and IgGl.
- the junction between TNF receptor and IgGl is a peptide sequence that is not normally found in the human body.
- Preferred methods of the invention involve reducing the immunogenicity of a fusion protein by reducing the ability of a junctional epitope (junctional peptide) to interact with a T-cell receptor by reducing its ability to bind (its binding affinity) to MHC molecules.
- the junctional epitope or peptide is preferably "non-self.”
- proteins, including therapeutic proteins are immunogenic, in part because proteins are endocytosed by antigen-presenting cells and proteolyzed, and the resulting peptides bind to molecules called major histocompatibility complex (MHC) that present the peptides to T cells.
- MHC major histocompatibility complex
- the antigenic peptide - MHC complex on the surface of an antigen presenting cell activates T-cells to proliferate, differentiate and release cytokines.
- APC antigen presenting cell
- B-cell differentiation and antibody production is induced which may further limit the therapeutic protein's effectiveness due to clearance.
- the antigenic peptide if derived from a therapeutic protein, is capable of inducing a series of undesired immune responses.
- the therapeutic protein's effectiveness is limited due to titration by antibodies, and the induction of T-cell and B-cell responses is often deleterious due to inflammatory and allergic reactions in the patient.
- the invention provides (1) the identification of novel amino acid sequences in the region of the immunoglobulin - target protein junction with one or more candidate T-cell epitopes; and (2) the modification of these amino acid sequences to reduce or eliminate the presence of peptides, derived from the junction sequence, that function as T-cell epitopes.
- the invention provides two general classes of compositions and methods relating to the reduction of immunogenicity. According to one embodiment of the invention, potential non-self T-cell epitopes are identified in sequences that span a fusion junction. For example, potential non-self T-cell epitopes are identified by computational methods based on modeling peptide binding to MHC Class II molecules.
- one or more glycosylation sites is introduced at a fusion junction.
- An N-linked glycosylation site is preferably used, although an O-linked glycosylation site may also be used.
- amino acids in a junction region surrounding a fusion junction of wild-type sequence are mutated such that the last amino acid of the N-terminal fusion partner is mutated to an asparagine, and the first two amino acids of the second fusion partner are mutated to a glycine followed by a serine or a threonine.
- removal of MHC Class II binding is preferred in situations where a protein is to be produced in bacteria or in an organism that does not generate a mammalian glycosylation pattern, such as yeast or insect cells.
- a component of the fusion protein is a cytokine.
- cytokine is used herein to describe naturally occurring or recombinant proteins, analogs thereof, and fragments thereof that elicit a specific response in a cell that has a receptor for that cytokine.
- cytokines are proteins that may be produced and excreted by a cell.
- cytokines include interleukins such as interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, LL-10, IL-12, IL-13, IL-14, IL-15, IL-16 and IL-18, hematopoietic factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), G-CSF and erythropoietin, tumor necrosis factors (TNF) such as TNF ⁇ , lymphokines such as lymphotoxin, regulators of metabolic processes such as leptin, and interferons such as interferon ⁇ , interferon ⁇ , and interferon ⁇ and chemokines.
- interleukins such as interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, LL-10, IL-12, IL-13, IL-14, IL-15, IL-16 and IL-18
- the antibody-cytokine fusion protein of the present invention displays a cytokine specific biological activity.
- a component of the fusion protein is an anti- obesity cytokine.
- a component is leptin, CNTF, or a portion of Acrp30.
- a component of the fusion protein is a hormone.
- a component may be insulin, growth hormone, or glucagon- like peptide l(GLP-l).
- a component of the fusion protein is a ligand-binding protein with biological activity.
- an extracellular domain of TNF receptor is used.
- a fusion protein of the invention comprises the N-terminus of a non-antibody moiety fused to the C-terminus of an antibody moiety.
- a fusion protein of the invention comprises the C-terminus of a non-antibody moiety fused to the N-terminus of an antibody moiety.
- an antibody moiety can be an intact immunoglobulin or a portion of an intact immunoglobulin.
- a portion of an immunoglobulin can include a variable region or a constant region or both.
- Preferred immunoglobulins include Fc regions or portions thereof.
- a preferred embodiment of the invention includes an IgGl immunoglobulin isotype, or a portion thereof, modified to be less immunogenic and/or to have a longer serum half-life.
- an IgGl with modification of amino acid residues near the CH3 - cytokine junction is preferred.
- antibody moieties from IgG2 or IgG4 isotypes are preferred.
- Immunocytokines are only one example of a tumor-targeted fusion protein therapy. Other tumor-toxic molecules can also be targeted to tumors by fusion to tumor-specific antibodies.
- antibody fusion proteins can attack other types of diseased cells, such as virus-infected cells.
- Fc-X and X-Fc technology where X is a polypeptide.
- Fc-X fusion proteins a signal peptide, followed by the Fc fragment of an immunoglobulin gene is the N-terminal fusion partner to the target protein.
- Fc-Fc orientation a fusion protein in the X-Fc orientation.
- lymphocyte cell surface glycoprotein (LHR) (US patent 5,428,130), and glucagon-like peptide (GLP-1).
- LHR lymphocyte cell surface glycoprotein
- GLP-1 glucagon-like peptide
- methods and compositions of the invention provide forms of Fc- X and X-Fc fusion proteins with reduced-immunogenicity.
- the immunogenicity of a fusion protein can be assayed according to a method known in the art or disclosed herein.
- Methods and compositions of the invention also provide albumin fusion proteins with reduced immunogenicity.
- Human serum albumin (HSA), due to its remarkably long half-life, its.
- albumin fusion proteins such as HSA-CD4 have a novel junction which generally contains one or more T-cell epitopes capable of being presented on MHC class II molecules.
- useful albumin proteins include species, allelic, and mutant variants of albumin, including fragments thereof.
- Preferred albumin proteins retain the structural and functional properties of a wild-type albumin protein such as HSA.
- the invention provides de-immunized antibody fusion proteins with normal, mutant, or hybrid isotypes that comprise useful mutations. These mutations may be near the junction or at positions distinct from the region of the junction.
- the invention provides a de-immunized immunocytokine, modified at the junction, with a point mutation at the junction between the IgG and non-IgG moieties.
- the cytokine moiety includes any cytokine but preferably IL-2 or IL-12.
- the amino acid changes involve changing the C-terminal lysine of the antibody moiety to a hydrophobic amino acid such as alanine or leucine.
- the invention provides immunocytokines comprising a hybrid antibody moiety that includes domains from different Ig isotypes, preferably from both IgGl and IgG2 isotypes, and a de-immunizing modification at the fusion junction.
- the invention provides a. de-immunized, junction-modified immunocytokine using an IgG2 and an IgG2h hybrid (IgG2 modified in the hinge region to IgGl).
- the hybrid fusion protein consists of a de-immunized immunoglobulin moiety composed of an IgG ( ⁇ l:CHl-H)( ⁇ 2: CH2- CH3) and a cytokine moiety.
- nucleic acid sequences that encode fusion proteins with reduced immunogenicity or facilitate the expression, production, and secretion of fusion proteins with reduced immunogenicity.
- nucleic acids are generated according to standard recombinant DNA techniques.
- a nucleic acid molecule encodes an immunocytokine fusion protein.
- a preferred immunocytokine includes a cytokine, for example, Interleukin 2, and a tumor specific monoclonal antibody such as an antibody to human epithelial cell adhesion molecule KSA (EP-CAM)(huKS).
- nucleic acid molecules encode Fc fusion proteins in various configurations.
- the nucleic acid molecule encodes serially in a 5' to 3' direction, (i) a signal sequence, an immunoglobulin Fc region and a target protein sequence or (ii) a signal sequence, a target protein, and an immunoglobulin Fc region, or (iii) a signal sequence, a first target protein, an immunoglobulin Fc region, and a second target protein.
- the resulting nucleic acid molecule thereby encodes an Fc-X, X-Fc, or X-Fc-Y structure where X and Y are a target protein, hi an alternative embodiment, a nucleic acid encodes an Fc-X, X-Fc, or X-Fc-Y protein without a signal sequence.
- a nucleic acid of the invention encodes an Ig fusion protein with mutant or hybrid isotypes.
- the nucleic acid provides antibody moieties with hybrid isotypes, or alternatively with altered hinge regions.
- the fusion protein consists of an IgG2, modified to contain fewer disulfide bonds in the hinge region, or an IgG2 CH2 and CH3 region in which the hinge region derives from another antibody, preferably a normal or mutant IgGl hinge region.
- a nucleic acid of the invention is preferably incorporated in operative association into a replicable expression vector which is then introduced into a mammalian host cell competent to produce the fusion protein.
- the resultant fusion protein is produced efficiently and secreted from the mammalian host cell.
- the secreted fusion protein is subsequently collected from the culture media without lysing the mammalian host cell.
- the protein product is assayed for activity and/or purified using common reagents as desired, and/or cleaved from the fusion partner, all using conventional techniques.
- the invention also provides methods for producing fusion proteins with reduced immunogenicity.
- compositions of the invention are also useful to provide therapeutic treatment using a fusion protein that has been rendered less immunogenic.
- An overall object of the invention is to provide processes that are both efficient and inexpensive as well as proteins that are less immunogenic.
- Preferred therapeutic compositions of the invention include a therapeutically effective amount of de- immunized fusion protein.
- the de-immunized fusion protein is administered along with a pharmaceutically acceptable carrier.
- T-cells T-lymphocytes
- B-cells B- lymphocytes
- Antibody production against the therapeutic agent is detrimental since it leads to more rapid elimination of the therapeutic agent and may induce an allergic response.
- the present invention provides methods of reducing the immunogenicity of fusion proteins. According to one method of this invention, potential T-cell epitopes are identified in the junction region of a fusion junction in a fusion protein. T-cell epitopes are identified by a variety of computer and non-computer methods, including prediction based on structure-based computer modeling or by synthesis of peptides and testing for binding to specific MHC Class II molecules or in an immunogenicity assay.
- a fusion junction is defined as the position between the last (C-terminal) amino acid of a first protein or peptide and the first (N- terminal) amino acid of a second protein or peptide in a fusion protein. Accordingly, a fusion junction includes any amino acids between the last amino acid of one protein and the first amino acid of a second protein, hi one embodiment, the fusion junction includes a linker.
- a junction region is the region of a fusion protein surrounding or spanning the fusion junction between two proteins.
- a junction region preferably includes between 1 and about 100 amino acids, more preferably between 1 and about 50 amino acids, or between 1 and about 25 amino acids, and even more preferably between 1 and about 15 amino acids, or between 1 and 9 amino acids.
- a junction region comprises a spacer or linker peptide inserted at the junction point between the two proteins.
- a junction region including a spacer or linker peptide can also be de-immunized to minimize the response of a patient to a fusion protein including the spacer or linker.
- ajunctional T-cell epitope is defined as a peptide sequence capable of binding an MHC Class II containing at least one amino acid derived from each of at least two different fusion partner proteins.
- Paul (Fundamental Immunology, Chapter 8, Table 8, p. 276 [2000] 4 th ed.) illustrates segments of 10 amino acids that can bind to an MHC Class II molecule.
- these 10 amino acid peptides are derived from different fusion partners.
- a potential or candidate T-cell epitope that spans a fusion junction preferably includes
- I to 8 amino acids from either side of the junction and more preferably 1 to 10 or 1 to
- ajunctional T-cell epitope of the invention includes at least one amino acid from each side of the junction, preferred candidate T-cell epitopes are junctional epitopes that include 1-8 (or 1-10, or 11) amino acids from one side of the junction and also include a complementary number of amino acids from the other side of the junction to result in an epitope having 9-12 amino acids, and most preferably 9 amino acids.
- anchor residues within ajunctional T-cell epitope are then mutated to prevent binding to an MHC Class II molecule.
- a fusion of wild-type sequences is a fusion in which the sequences at the N-terminal and C-terminal sides of the fusion junction are derived directly from naturally occurring sequences.
- a de-immunized fusion junction is a junction sequence in which one or more substitution mutations have been introduced relative to a junction of wild-type sequences.
- deimmunization of a fusion junction does not involve introduction of a linker, such as a 'non-immunogenic' Gly-Ser linker, and the spatial relationship between the fusion partners is not altered in a de-immunized fusion protein.
- one or more amino acids can be substituted or changed in the junction region either N-terminally to the fusion junction, C-terminally to the fusion junction, or both N- terminally and C-terminally to the fusion junction.
- a potential T-cell epitope is a sequence that, when considered as an isolated peptide, is predicted to bind to an MHC Class II molecule or an equivalent in a non-human species.
- a potential T-cell epitope is defined without consideration of other aspects of antigen processing, such as the efficiency of protein uptake into antigen-presenting cells, the efficiency of cleavage at sites in an intact protein to yield a peptide that can bind to MHC Class II, and so on.
- the set of T-cell epitopes that are actually presented on MHC Class II after administration of a protein to an animal is a subset of the potential T-cell epitopes.
- a T-cell epitope is an epitope on a protein that interacts with an MHC class II molecule.
- a T-cell epitope is an amino acid sequence in a protein or a fusion protein, that failed to undergo the negative T-cell selection process during T- cell development and therefore will be expected to be presented by an MHC Class II molecule and recognized by a T-cell receptor.
- the non-self T-cell epitopes are present in the junction region at the fusion junction of two proteins that form a fusion protein.
- the invention provides non-computer methods for reducing or eliminating the number of T-cell epitopes in a fusion protein junction without requiring elaborate computer simulations or protein three-dimensional structures.
- a method of the invention takes advantage of the fact that a core segment of nine amino acids interacts with both the MHC class II molecule as well as the T-cell receptor during antigen presentation.
- the N-terminal most amino acid is called an "anchor" position residue that binds to a deep pocket within the MHC class II molecule.
- One of the following amino acids is typically present at the anchor position which is important for binding to an MHC class II molecule: Leucine, Naline, Isoleucine, Methionine, Phenylalanine, Tyrosine and Tryptophan.
- an additional 2 to 3 amino acids adjacent to the core 9 amino acids also affect the interaction with MHC molecules.
- the C-terminal most amino acid in the first protein of the fusion protein can generally be substituted. This is useful especially when the ⁇ -terminal fusion partner or first protein is known to be active when fused to the C-terminal fusion partner or second protein at the C-terminus of the first protein.
- a general method of the invention includes mutating any Leucines, Valines, Isoleucines, Methionines, Phenylalanines, Tyrosines or Tryptophans that occur in the C-terminal most eight amino acids of an ⁇ -terminal fusion partner in a fusion protein.
- one or more of these amino acids in a candidate junctional T-cell epitope amino acids is preferentially mutated to a Threonine, an Alanine or a Proline. This retains some of the hydrophobic nature of the amino acid that is replaced, hi further embodiments of the invention, one more more of the above-mentioned amino acids is deleted from a candidate or potential junctional T-cell epitope, or replaced with an appropriate amino acid analog.
- an amino acid is deleted to destroy a potential T-cell epitope, care is taken not to generate a new T- cell epitope that includes amino acids near the deletion.
- it is often useful to construct a generalized expression plasmid construction intermediate comprising the coding sequence for an ⁇ -terminal fusion partner containing a mutation of one or more hydrophobic residues in the last eight amino acids.
- a plasmid has one or more convenient restriction enzyme sites at or near the D ⁇ A encoding the C-terminus of the ⁇ - terminal fusion partner.
- a plasmid construction intermediate is to construct expression plasmids encoding a fusion protein in which one or more N-terminal fusion partners has one or more substitutions of a Leucine, Valine, Isoleucine, Methionine, Phenylalanine, Tyrosine, or Tryptophan to another amino acid in the eight C-terminal amino acids.
- the construction of such final expression plasmids may be accomplished by a variety of other methods well known in the art, such as generation of PCR fragments or synthetic nucleic acids, followed by ligation of the fragment into an appropriated vector or attachment with other sequences through well-known PCR techniques.
- Specific preferred embodiments include Fc-X fusion plasmids, albumin-X fusion plasmids, scFv-X fusion plasmids, and Fab-X fusion plasmids.
- Fc(gamma)-X it is useful to introduce mutations into the coding sequence to bring about amino acid substitutions of the Leucine-Serine-Leucme-Serine segment near C-terminus the Fc region of an IgGl, IgG2, IgG3, or IgG4 molecule, as diagrammed here for IgGl : Amino acid sequences of human Fc regions derived from IgGl, IgG2, IgG3 and IgG4 are depicted in SEQ ID NOs: 1, 2, 3 and 4 respectively.
- KSLSLSPGK (SEQ ID NO: 5) is changed to KSATATPGK (SEQ ID NO: 6). This mutation is designed to eliminate potential junctional T-cell epitopes and also remove a T-cell epitope in which the upstream Phenylalanine or Tyrosine serves as a position 1 anchor residue.
- KSLSLSPGK SEQ ID NO: 5
- KSATATPGA SEQ ID NO: 7
- Other embodiments include substitutions in the LSLS segment to other amino acids such as Glycine or Proline.
- QKTIDRLAGKPTH SEQ ID NO: 8 changed to QKTADRTAGKPTH (SEQ ID NO: 9)
- Additional de-immunized sequences in an IgA-X fusion protein are, QKTPTRTAGKPTH (SEQ ID NO: 10) QKTPTRPAGKPTH (SEQ ID NO: 11) QKTATRPAGKPTH (SEQ ID NO: 12).
- albumin-X In the case of an albumin-X fusion, it is useful to introduce the following changes in an albumin-X expression plasmid such that the C-terminus of albumin is modified as follows:
- KKLNAASQAALGL (SEQ ID NO: 13) changed to KKLNAASQAATTA (SEQ ID NO: 14).
- the invention provides nucleic acid sequences and proteins that are useful in construction of less immunogenic fusion proteins. Specifically, the invention provides proteins with mutations of any Leucines, Nalines, Isoleucines, Methionines, Phenylalanines, Tyrosines, or Tryptophans in the last eight amino acids.
- the proteins are preferably human proteins with sequences that generally correspond to sequences found in the human body.
- the invention also provides nucleic acid sequences encoding such proteins.
- the nucleic acid sequences for this aspect of the invention may exist as plasmids, PCR-generated fragments, or nucleic acids produced by chemical synthesis.
- the invention also provides expression plasmids encoding a fusion protein in which one or more ⁇ -terminal fusion partners has one or more mutations of a Leucine, Naline, Isoleucine, Methionine, Phenylalanine, Tyrosine, or Tryptophan to another amino acid in the eight C-terminal amino acids.
- plasmids encoding an Fc-IL2 or whole-antibody-IL2 fusion protein in which the Fc region is mutated as described above are provided by the invention.
- the invention also provides a method for reducing immunogenicity of a fusion protein junction by introducing an ⁇ -linked or O-linked glycosylation site near, or preferably, at a fusion junction.
- the amino acids Asparagine, Serine or Threonine, and a third residue are introduced as follows. Consider a sequence in which X's represent amino acids of an N-terminal fusion partner, and Z's represent amino acids of a C-terminal fusion partner.
- binding of a junction peptide is not necessarily blocked by introduction of the glycosylation site.
- any peptide that is bound in the MHC Class II groove and has the glycosylated asparagine C-terminal to the N- terminal-most anchor residue will not function as a T-cell epitope.
- the presence of the large glycosylation moiety will sterically hinder recognition of the MHC Class II/pe ⁇ tide complex.
- a preferred glycosylation site includes the sequence Asn-X-Ser or Asn-X-Thr wherein X is preferably Gly, but can be any amino acid.
- the introduction of mutations introducing Glycine and Serine residues does not create new T-cell epitopes.
- a fusion protein in principle, is cleaved between the glycosylated Asparagine and the Glycine or between the Glycine and the Serine. In either case, the resulting peptides have the mutant Glycine and or Serine residues N-terminal to an anchor residue, and thus the mutant Glycine and/or Serine residues are not recognized by a T cell receptor, since residues N-terminal to an anchor residue are outside the region recognized by the TCR.
- a fusion junction region already contains a Serine or Threonine preceded by an amino acid residues such as Glycine, Serine, Alanine, etc.
- the second method is preferably used when a junction region is flexible and displaced from the hydrophobic core of each fusion partner, so that the novel N- linked glycosylation does not interfere with the folding or function of either fusion partner.
- both the N-terminal and the C- terminal partner of the fusion protein are human proteins. Potential T-cell epitopes in such fusion proteins are created from the final 8 amino acids of the N-terminal partner
- first protein combined with the first 8 amino acids of the C-terminal partner (second protein).
- second protein This provides a series of 8 hybrid 9-mers created from the first and second proteins. Any aliphatic or aromatic residue (Leucine, Valine, Isoleucine, Methionine,
- Phenylalanine, Tryptophan or Tyrosine) in the last 8 amino acids of the first protein presents a high risk of creating an MHC binding peptide with the amino acid in the first position (anchor position) that binds the pocket of the MHC molecule.
- Proline or Threonine
- T-cell epitope will remove a candidate T-cell epitope.
- Fc fusion protein containing the sequence: HNHYTQKSLSLSPGKGGGGSGGGGSGGGGS (SEQ ID NO: 15)
- the leucine residues create two potential epitopes. Therefore, the sequence can be de-immunized as;
- HNHYTQKSATATPGKGGGGSGGGGSGGGGS SEQ ID NO: 16
- L to A and S to T These changes remove epitopes with Leucine as the first amino acid in the MHC binding pocket and Tyrosine as the first amino acid in the
- Methods of the invention are generally applicable in all vertebrate organisms, preferably in mammals and most preferably in humans.
- the invention is illustrated further by the following non-limiting examples .
- HuKS-IL2 consists of humanized N ⁇ and N L regions combined with human H and L chain constant regions. The H chain was fused at its carboxyl terminus to the mature sequence of human IL-2 as described previously. This H chain is of the ⁇ l isotype and has high affinity for Fc receptors. Because of this high affinity HuKS-IL2 was cleared quickly from the circulation. Without wishing to be bound by theory, the clearance of HuKS-IL2 presumably occurs via FcR-bearing cells in the liver (Kupffer cells) and spleen (antigen presenting cells).
- NH1 a de-immunized form of huKS-IL2 with no T-cell epitopes in the VH and NL regions, derived from mouse N regions with surface-exposed mouse B-cell epitopes veneered to human residues.
- NH2 a de-immunized form of huKS-IL2 with one remaining T-cell epitope in CDR3, derived from mouse N regions with surface-exposed mouse B-cell epitopes veneered to human residues, in which the NH contains one T-cell epitope.
- huKS-mIL2 constructed with either KOL or EU C ⁇ l regions (rather than KS) (to compare allotypic reactivity); (5) huKS-mIL2 - a molecule with the huKS N regions fused to mouse C regions and mouse IL-2;
- Immunoglobulin fusion proteins and fragments were purified by protein A Sepharose chromatography and were coated on 96-well plates in bicarbonate buffer and then blocked with 1% goat serum containing 1% BSA. Dilutions of patient sera were incubated and then unbound material was removed by three washes with PBS- Tween. Bound human antibodies from the patient sera were detected with various HRP-conjugated antibodies depending on the bound protein. Generally, goat anti- human ⁇ chain HRP conjugate was used because most of the plate-bound proteins consisted of human Fc and human chains. [0070] Certain patient sera showed a clear reactivity to huKS-IL2 that was not detectable in pre-injection sera from the same patients.
- Preimmune antisera was used to establish a baseline non-immunized control. Reactivity seen in patient sera can be attributed to (1) anti-IL2 reactivity, (2) anti Fc (allotypic) reactivity, (3) reactivity to the novel junction sequence or (4) anti-idiotypic reactivity with the KS idiotype, or a combination of reactivities .
- Example 2 Modification of amino acid residues at the junction of an antibodv- cytokine fusion protein to reduce immunogenicity by elimination of MHC Class II binding motifs
- Peptide threading analysis identified two overlapping peptide segments with strong MHC binding potential at the junction between the Fc and LL2 portion of the immunocytokine.
- the peptide threading and identification of potential T-cell epitopes was performed as disclosed in Carr (WO00/34317). Amino acid changes were introduced such that the existing potential MHC Class II binding epitopes were eliminated, but new potential MHC Class II epitopes were not introduced.
- junction-derived peptides are not effective as T-cell epitopes, because when the junction-derived peptides are presented to T cells by MHC Class II, the large N-glycosylation moiety prevents specific docking between a T cell-receptor and MHC Class II.
- Example 4 Characterization of the immune reactivity of antigen presenting cells to immunocytokine huKs-DL2 in comparison to a de-immunized huKS-IL2 immunocytokine.
- the hyphen is the KS-IL2 junction
- the hyphen is the KS-IL2 junction
- the ability of those T cells to proliferate in response to a subsequent challenge with the peptide antigen serves as a measure of immunogenicity of that peptide.
- peripheral blood mononuclear cells are isolated from leukopacks by standard density gradient techniques. Mononuclear cells are resuspended in serum-free Aim V culture media and allowed to adhere. After 2 h at 37 °C nonadherent cells are removed. Adherent cells are cultured for 7 days in media containing human GM-CSF (50 ng/ml) and IL-4 (20ng/ml) to derive immature dendritic cells (DC). After 7 days, the cells are harvested and phenotypically characterized by flow cytometry with appropriate FITC-labeled Abs for MHC class I, MHC class II, CD80 and CD40 to confirm the immature DC phenotype.
- GM-CSF 50 ng/ml
- IL-4 20ng/ml
- Non-adherent cells are cultured with IL2 and IL7 to obtain autologous effector cells (T-cells) to be used in subsequent functional studies.
- T-cells are added to immature dendritic cells (10:1 ratio) and co-cultured with huKS, de- immunized huKS, peptide junction 13 mer (KSLSLSP GK -APTS) (SEQ ID NO: 20) and the modified, de-immunized 13 mer peptide (KSATATPGK-APTS) (SEQ ID NO : 21).
- Example 5 Deduction of immunogenic reactive epitopes found in albumin fusion proteins and modification of amino acid residues at a fusion junction to reduce immunogenicity.
- HSA Human serum albumin
- a genetically engineered HSA-CD4 hybrid has been shown to block the entry of the human immunodeficiency virus into CD4+ cells while exhibiting antiviral in vitro properties similar to those of soluble CD4 (Yeh et al, PNAS 89:1904-1908, 1992).
- the genetic fusion of bioactive peptides to HSA is useful for designing and recovering secreted therapeutic HSA derivatives.
- HSA-CD4 has a novel junction which can be immunogenic and contains T-cell epitopes capable of being presented on MHC class II molecules. Analysis of the junction between HSA and CD4 using the methods of Examples 1, 2, 3, and 4 identifies peptides with MHC binding potential. The potentially immunogenic sequences are modified to decrease or eliminate potential T and B-cell epitopes in order to reduce immunogenicity.
- the HS A-IFNalpha fusion protein junction region contains three candidate T-cell epitopes,
- LGLCDLPQTHSLG (SEQ ID NO: 24).
- the peptide threading score can be reduced significantly by changing
- T-cell epitope in SEQ ID NO: 24 can be de-immunized by changing the second L (in bold ) to an A.
- X can be any protein, deimmunization of the fusion junction is achieved by changing the amino acid sequence AALGL(SEQ ID NO: 25) to TATTA (SEQ ID NO: 26).
- Example 6 X-Fc fusion proteins and modification of amino acid residues at a fusion junction to reduce immuno enicity.
- a target protein is a N-terminal fusion protein and a Fc fragment follows.
- GLP-1 glucagon-like peptide
- a GLP-1 -Fc fusion protein is constructed according to standard techniques described in the art. This fusion protein has the C-terminus of GLP-1 joined to the hinge of the ⁇ l heavy chain.
- the ⁇ l hinge sequence containing a Cys to Ser mutation (residue 5) which eliminates the Cys residue that forms a disulphide bond with the light chain in IgGl is used.
- the non-mutant Fc sequence is EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 28) with the hinge region being underlined, followed by the start of the CH2 domain sequence.
- the fusion junction between GLP-1 (7-37) and mutant Fc is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG - EPKSSDKTHTCPPCPAPELLG (SEQ ID NO: 29).
- GLP-1 (7-37) and normal Fc are: SYLEGQAAKEFIAWLVKGRG - EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 30)
- AWLVKGRGEPKSS (SEQ ID NO: 33).
- peptide shown in SEQ ID NO: 31 is de-immunized by changing the Lysine (shown in bold) to a Threonine and the Arginine(shown in bold) to a Threonine.
- the peptide shown in SEQ ID NO: 32 is de-immunized by replacing the Isoleucine (shown in bold) with an Alanine or a Proline and the peptide in SEQ TD NO: 33 is de- immunized by replacing the Leucine with an Alanine or a Proline.
- the resulting de- immunized junction is:
- HAEGTFTSDVSSYLEGQAAKEFAAWAVTGTG - EPKSSDKTHTCPPCPAPELLG SEQ ID NO: 34.
- Example 7 Deduction of immunogenic reactive epitopes of Enbrel, a TNFR-Fc fusion protein and modification of amino acid residues at a fusion junction to reduce immunogenicity.
- ENBREL or etanercept a X-Fc fusion protein approved by the FDA, is a tumor necrosis factor (TNF) inhibitor used to treat rheumatoid arthritis.
- ENBREL is a dimeric fusion protein consisting of an extracellular-ligand-binding domain of TNF receptor linked to an Fc protein of human IgGl .
- TNFR-Fc competitively inhibits binding of TNF to its receptor and renders the bound TNF biologically inactive, resulting in significant reduction in inflammatory activity.
- GLP-1 - Fc TNFR-Fc has a novel junction which contains potential T-cell epitopes.
- STSFLLPMGPSPPAEGSTGN - GSKSCDKTHTCPPCPAPELLG (SEQ ID NO: 37).
- Example 8 Deduction of immunogenic reactive epitopes for Fc-X-Y fusion proteins such as Fc-IL12-IL2 and modification of amino acid residues at the fusion junction to reduce immunogenicity.
- Fusion proteins of a Fc-X-Y orientation such as Fc-TL12-IL2 have multiple novel fusion junctions which are potentially immunogenic.
- Fc-IL12 has a fusion junction similar to other Fc-X fusion proteins or immunocytokines (Example 1) but is novel due to the usage of the cytokine IL12.
- the fusion junction is analyzed for immunogenic binding sites and modified accordingly.
- there is an X-Y fusion junction comparable to that described in Example 5, with two different cytokines constituting a fusion protein. Peptide thread analysis is used for each of the fusion junctions.
- sequence (2) above is modified to introduce a glycosylation site by introducing an Asparagine and Glycine at the first two positions within IL-2.
- This strategy uses the naturally occurring Threonine at position 3 of mature IL-2.
- it is important to not disrupt the formation of a disulfide bond in the p40 moiety, so it is useful to separate the glycosylation site by at least one or two amino acids from the Cysteine in p40.
- RAQDRYYSSSWSEWASVPCS - NGTSSSTKKTQLQLEHLLLD SEQ ID NO:
- ASVPCSNGTSSST (SEQ ID NO: 43)
- glycosylation of the T-cell epitope prevents MHC Class II binding thus resulting in reduced immunogenicity.
- Example 9 Deduction of immunogenic reactive epitopes in junction of an X-Fc-Y fusion protein and modification of amino acid residues at a fusion junction to reduce MHC Class ⁇ binding.
- Fusion proteins of the X-Fc-Y configuration such as IL4-Fc-GMCSF, have multiple novel fusion junctions that contain potential T-cell epitopes.
- the IL4-Fc is a junction analogous to other X-Fc fusion proteins (Examples 6 and 7) but is novel due to the use of the cytokine IL4.
- a form of Fc using a hinge region, CH2, and CH3 domain from human ⁇ l is used.
- a ⁇ l hinge sequence in pdCs- huFc ⁇ l may contain a Cys to Ser mutation (underlined) that eliminates the Cys residue that forms a disulphide bond with a light chain in IgGl (Lo et al., (1998) Protein Engineering 11 :495-500), thereby creating a third potentially immunogenic fusion junction for analysis.
- the fusion junction is analyzed for potential T-cell epitopes and modified according to the methods of Examples 1-4.
- MHEALHNHYTQKSLSLSPGK -parspspstqpwehvnaiqe (SEQ ID NO: 45) between the C-terminus of Fc (bold text) and the N-terminus of GMCSF (plain text) by peptide threading identifies peptides with MHC binding potential.
- the potential T-cell epitopes are modified to decrease or eliminate potential T epitopes in order to reduce immunogenicity.
- a candidate T-cell epitope at the junction of IL4-Fc fusion protein is, EKYSKCSSEPKSC (SEQ ID NO: 46), where changing E (in bold) to T reduces the peptide threading score or the MHC Class II binding potential significantly.
- the sequence of the modified IL4-Fc fusion is as follows:
- the Fc-GMCSF fusion junction is de-immunized by changing the sequence LSLS to AT AT as shown below.
- MHEALHNHYTQKSATATPGK-parspspstqpwehvnaiqe SEQ ID NO: 48.
- Example 10 Modification of amino acid residues at a fusion junction of immunocytokines and immunofusins prepared with a hybrid isotype to remove T-cell epitopes.
- Fusion proteins with hybrid isotypes may be modified according to the invention to reduce immunogenicity.
- An antibody fusion protein with the following components is constructed by standard recombinant DNA techniques: a light chain and a heavy chain, the V regions recognizing a tumor-specific antigen, the light chain being a typical light chain, and the heavy chain comprising CHI, CH2, and CH3 domains from IgG2 and a hinge region > from IgGl, with a cytokine fused to the C-terminus of the heavy chain involving a fusion junction as described above.
- This protein contains novel junctions between CHlg2 and hinge-gl, and hinge-gl and CH2g2. The identification and modification of potential T-cell epitopes in these junctions is performed as follows.
- TKVDKTVEPKSCD SEQ ID NO: 51
- KTVEPKSCDKTHT SEQ ID NO: 52
- the IgG2CHl-IgGl hinge fusion junction is de-immunized by changing the V (in bold) to an A, a T or a P.
- the sequence of the modified fusion junction is depicted in SEQ ID NO: 53.
- the ⁇ l hinge sequence in pdCs-huFc ⁇ l may contain a Cys to Ser mutation (underlined) that eliminates the Cys residue that forms a disulphide bond with the light chain in IgGl (Lo et al., (1998) Protein Engineering 11:495-500), thereby creating two additional potentially immunogenic fusion junctions for analysis and modification:
- Example 11 Generation of Fc-EPO fusion protein using hybrid isotype Fc components of IgGl and !gG4.
- This Xmal-Xhol DNA fragment was inserted into a plasmid vector that encodes a hinge region from IgGl and a CH2 and CH3 region from IgG2, except that there were two sets of mutations that resulted in amino acid substitutions in the region of the CH3 C- terminus, such that the sequence at the junction of the CH3 C-terminus and the Epo N- terminus is as follows: . . . . TQKSATATPGA-APPRLI . . . .(SEQ ID NO: 57)
- the first set of mutations which change the sequence KSLSLSPG (SEQ TD NO: 58) of the IgG2 CH3 region to KSATATPG (SEQ ID NO: 59), is disclosed in U.S. Patent Application Serial No. 60/280,625.
- the effect of the substitution of Leu-Ser-Leu-Ser (position 3 to position 6 of SEQ ID NO: 58) with Ala-Thr-Ala-Thr (position 3 to position 6 of SEQ ID NO: 59) is to remove potential human non-self T-cell epitopes that may arise because the junction between human Fc and human erythropoietin contains non-self peptide sequences.
- the second set consisting of the single amino acid substitution K to A at the C-terminal amino acid of the CH3 region, is disclosed in U.S. Patent Application Serial No. 09/780,668.
- the resulting plasmid was transfected into NS/0 cells and the Fc-Epo fusion protein was expressed and purified according to the procedures known in the art. After purification based on binding to protein A, the huFcy2h-huEpo protein containing the IgG2 CH3 and erythropoietin substitutions described above was characterized by size exclusion chromatography and found to consist of 97% monomer and 90% monomer in two independent preparations.
- the huFc ⁇ 2h-huEpo protein containing the IgG2 CH3 and erythropoietin substitutions described above was found to be about as active, on a molar basis, as human erythropoietin in a cell-based assay that measured the ability of an erythropoietin protein to stimulate TF-1 cell division.
- the assay was performed as described in WO01/36489.
- NS/0 cells were transfected with the Fc ⁇ l- Epo, Fcy2-Epo, and Fcy2h-Epo expression plasmids, and stable clones were isolated after screening an approximately equal number of clones for each plasmid.
- the best- producing clones yielded 50 ⁇ g/ml for Fc ⁇ l-Epo, 20 ⁇ g/ml for Fc ⁇ 2-Epo, and 120 ⁇ g/ml for Fc ⁇ 2h-Epo.
- the following example describes in detail a preferred method for identification of immunogenic sequence regions (T-cell epitopes) within the sequences of the fusion proteins as disclosed in this invention. However, it should be pointed out, that said . molecules can be obtained by other known methods.
- Example 12 Identification of T-cell epitopes by computational methods [0119]
- epitopes in a junction region of a fusion protein can be modified using methods for introducing mutations into proteins to modulate their interaction with the immune system.
- known methods in the art that can be adapted according to the invention include those described in the prior art (WO 92/10755 and WO 96/40792 (Novo Nordisk), EP 0519 596 (Merck & Co.), EP 0699 755(Centro de hnmunologia Moelcular), WO 98/52976 and WO 98/59244 (Biovation Ltd.) or related methods.
- the peptide bond i.e., that bond which joins the amino acids in the chain together, is a covalent bond.
- This bond is planar in structure, essentially a substituted amide.
- An "amide" is any of a group of organic compounds containing the grouping -CONH-.
- planar peptide bond linking C ⁇ of adjacent amino acids may be represented as depicted below:
- a second factor that plays an important role in defining the total structure or conformation of a polypeptide or protein is the angle of rotation of each amide plane about the common G ⁇ linkage.
- the terms "angle of rotation” and “torsion angle” are hereinafter regarded as equivalent terms. Assuming that the O, C, N, and H atoms remain in the amide plane (which is usually a valid assumption, although there may be some slight deviations from planarity of these atoms for some conformations), these angles of rotation define the N and R polypeptide's backbone conformation, i.e., the structure as it exists between adjacent residues. These two angles are known as ⁇ and ⁇ .
- a set of the angles ⁇ i, ⁇ ls where the subscript i represents a particular residue of a polypeptide chain thus effectively defines the polypeptide secondary structure.
- the conventions used in defining the ⁇ , ⁇ angles i.e., the reference points at which the amide planes form a zero degree angle, and the definition of which angle is ⁇ , and which angle is ⁇ , for a given polypeptide, are defined in the literature. See, e.g Berry Ramachandran et al. Adv. Prot. Chem. 23:283-437 (1968), at pages 285-94, which pages are incorporated herein by reference.
- the present method can be applied to any protein, and is based in part upon the discovery that in humans the primary Pocket 1 anchor position of MHC Class II molecule binding grooves has a well designed specificity for particular amino acid side chains.
- the specificity of this pocket is determined by the identity of the amino acid at position 86 of the beta chain of the MHC Class II molecule. This site is located at the bottom of Pocket 1 and determines the size of the side chain that can be accommodated by this pocket. Marshall, K.W., J Immunol, 152:4946-4956 (1994).
- this residue is a glycine
- all hydrophobic aliphatic and aromatic amino acids hydrophobic aliphatics being: valine, leucine, isoleucine, methionine and aromatics being: phenylalanine, tyrosine and tryptophan
- this pocket residue is a valine
- the side chain of this amino acid protrudes into the pocket and restricts the size of peptide side chains that can be accommodated such that only hydrophobic aliphatic side chains can be accommodated.
- a computational method embodying the present invention profiles the likelihood of peptide regions to contain T-cell epitopes as follows: (1) The primary sequence of a peptide segment of predetermined length is scanned, and all hydrophobic aliphatic and aromatic side chains present are identified. (2) The hydrophobic aliphatic side chains are assigned a value greater than that for the aromatic side chains; preferably about twice the value assigned to the aromatic side chains, e.g., a value of 2 for a hydrophobic aliphatic side chain and a value of 1 for an aromatic side chain.
- each amino acid residue of the peptide is assigned a value that relates to the likelihood of a T-cell epitope being present in that particular segment (window).
- the values calculated and assigned as described in Step 3, above, can be plotted against the amino acid coordinates of the entire amino acid residue sequence being assessed. (5) All portions of the sequence which have a score of a predetermined value, e.g., a value of 1, are deemed likely to contain a T-cell epitope and can be modified, if desired.
- This particular aspect of the present invention provides a general method by which the regions of peptides likely to contain T-cell epitopes can be described. Modifications to the peptide in these regions have the potential to modify the MHC Class II binding characteristics.
- T-cell epitopes can be predicted with greater accuracy by the use of a more sophisticated computational method which takes into account the interactions of peptides with models of MHC Class II alleles.
- the computational prediction of T-cell epitopes present within a peptide contemplates the construction of models of at least 42 MHC Class ⁇ alleles based upon the structures of all known MHC Class II molecules and a method for the use of these models in the computational identification of T-cell epitopes, the construction of libraries of peptide backbones for each model in order to allow for the known variability in relative peptide backbone alpha carbon (C ⁇ ) positions, the construction of libraries of amino-acid side chain conformations for each backbone dock with each model for each of the 20 amino-acid alternatives at positions critical for the interaction between peptide and MHC Class II molecule, and the use of these libraries of backbones and side-chain conformations in conjunction with a scoring function to select the optimum backbone and side-chain conformation for a particular peptide docked with a particular MHC Class II molecule and the derivation of a binding score from this interaction.
- Models of MHC Class II molecules can be derived via homology modeling from a number of similar structures found in the Brookhaven Protein Data Bank ("PDB"). These may be made by the use of semi-automatic homology modeling software (Modeller, Sali A. & Blundell TL., 1993. J MolBiol 234:779-815) which incorporates a simulated annealing function, in conjunction with the CHARMm force-field for energy minimization (available from Molecular Simulations Inc., San Diego, Ca.). Alternative modeling methods can be utilized as well.
- PDB Brookhaven Protein Data Bank
- the present method differs significantly from other computational methods which use libraries of experimentally derived binding data of each amino-acid alternative at each position in the binding groove for a small set of MHC Class II molecules (Marshall, K.W., et al, Biomed. Pept. Proteins Nucleic Acids, 1(3):157-162) (1995) or yet other computational methods which use similar experimental binding data in order to define the binding characteristics of particular types of binding pockets within the groove, again using a relatively small subset of MHC Class II molecules, and then 'mixing and matching' pocket types from this pocket library to artificially create further 'virtual' MHC Class II molecules (Stumiolo T., et al., Nat. Biotech, 17(6): 555-561 (1999).
- Both prior methods suffer the major disadvantage that, due to the complexity of the assays and the need to synthesize large numbers of peptide variants, only a small number of MHC Class ⁇ molecules can be experimentally scanned. Therefore the first prior method can only make predictions for a small number of MHC Class II molecules.
- the second prior method also makes the assumption that a pocket lined with similar amino-acids in one molecule will have the same binding characteristics when in the context of a different Class ⁇ allele and suffers further disadvantages in that only those MHC Class II molecules can be 'virtually' created which contain pockets contained within the pocket library.
- the structure of any number and type of MHC Class II molecules can be deduced, therefore alleles can be specifically selected to be representative of the global population.
- the number of MHC Class ⁇ molecules scanned can be increased by making further models further than having to generate additional data via complex experimentation [0132]
- the use of a backbone library allows for variation in the positions of the C ⁇ atoms of the various peptides being scanned when docked with particular MHC Class II molecules. This is again in contrast to the alternative prior computational methods described above which rely on the use of simplified peptide backbones for scanning amino-acid binding in particular pockets.
- the present backbone library is created by superposing the backbones of all peptides bound to MHC Class II molecules found within the Protein Data Bank and noting the root mean square (RMS) deviation between the C ⁇ atoms of each of the eleven amino-acids located within the binding groove. While this library can be derived from a small number of suitable available mouse and human structures (currently 13), in order to allow for the possibility of even greater variability, the RMS figure for each C"- ⁇ position is increased by 50%. The average C ⁇ position of each amino-acid is then determined and a sphere drawn around this point whose radius equals the RMS deviation at that position plus 50%. This sphere represents all allowed C ⁇ positions.
- RMS root mean square
- the sphere is three-dimensionally gridded, and each vertex within the grid is then used as a possible location for a C ⁇ of that amino-acid.
- the subsequent amide plane, corresponding to the peptide bond to the subsequent amino-acid is grafted onto each of these C ⁇ s and the ⁇ and ⁇ angles are rotated step-wise at set intervals in order to position the subsequent C ⁇ . If the subsequent C ⁇ falls within the 'sphere of allowed positions' for this C ⁇ than the orientation of the dipeptide is accepted, whereas if it falls outside the sphere then the dipeptide is rejected.
- This process is then repeated for each of the subsequent C ⁇ positions, such that the peptide grows from the Pocket 1 C ⁇ 'seed', until all nine subsequent C ⁇ s have been positioned from all possible permutations of the preceding C ⁇ s.
- the process is then repeated once more for the single C ⁇ preceding pocket 1 to create a library of backbone C ⁇ positions located within the binding groove.
- the number of backbones generated is dependent upon several factors: The size of the 'spheres of allowed positions'; the fineness of the gridding of the 'primary sphere' at the Pocket 1 position; the fineness of the step-wise rotation of the ⁇ and ⁇ angles used to position subsequent C ⁇ s. Using this process, a large library of backbones can be created.
- backbone library The larger the backbone library, the more likely it will be that the optimum fit will be found for a particular peptide within the binding groove of an MHC Class II molecule. Inasmuch as all backbones will not be suitable for docking with all the models of MHC Class II molecules due to clashes with amino-acids of the binding domains, for each allele a subset of the library is created comprising backbones which can be accommodated by that allele.
- the use of the backbone library, in conjunction with the models of MHC Class ⁇ molecules creates an exhaustive database consisting of allowed side chain conformations for each amino-acid in each position of the binding groove for each MHC Class II molecule docked with each allowed backbone.
- This data set is generated using a simple steric overlap function where a MHC Class II molecule is docked with a backbone and an amino-acid side chain is grafted onto the backbone at the desired position.
- Each of the rotatable bonds of the side chain is rotated step-wise at set intervals and the resultant positions of the atoms dependent upon that bond noted.
- the interaction of the atom with atoms of side-chains of the binding groove is noted and positions are either accepted or rejected according to the following criteria:
- the sum total of the overlap of all atoms so far positioned must not exceed a pre-determined value.
- the stringency of the conformational search is a function of the interval used in the step-wise rotation of the bond and the pre-determined limit for the total overlap.
- a protein is scanned for potential T-cell epitopes by subjecting each possible peptide of length varying between 9 and 20 amino-acids (although the length is kept constant for each scan) to the following computations:
- An MHC Class II molecule is selected together with a peptide backbone allowed for that molecule and the side-chains corresponding to the desired peptide sequence are grafted on.
- Atom identity and interatomic distance data relating to a particular side-chain at a particular position on the backbone are collected for each allowed conformation of that amino-acid (obtained from the database described above). This is repeated for each side-chain along the backbone and peptide scores derived using a scoring function.
- the best score for that backbone is retained and the process repeated for each allowed backbone for the selected model.
- the scores from all allowed backbones are compared and the highest score is deemed to be the peptide score for the desired peptide in that MHC Class II model. This process is then repeated for each model with every possible peptide derived from the protein being scanned, and the scores for peptides versus models are displayed.
- each ligand presented for the binding affinity calculation is an amino-acid segment selected from a peptide or protein as discussed above.
- the ligand is a selected stretch of amino acids about 9 to 20 amino acids in length derived from a peptide, polypeptide or protein of known sequence.
- amino acids and “residues” are hereinafter regarded as equivalent terms.
- the ligand in the form of the consecutive amino acids of the peptide to be examined grafted onto a backbone from the backbone library, is positioned in the binding cleft of an MHC Class ⁇ molecule from the MHC Class II molecule model library via the coordinates of the C"- ⁇ atoms of the peptide backbone and an allowed conformation for each side-chain is selected from the database of allowed conformations.
- the relevant atom identities and interatomic distances are also retrieved from this database and used to calculate the peptide binding score.
- Ligands with a high binding affinity for the MHC Class II binding pocket are flagged as candidates for site-directed mutagenesis.
- Amino-acid substitutions are made in the flagged ligand (and hence in the protein of interest) which is then retested using the scoring function in order to deten ine changes which reduce the binding affinity below a predetermined threshold value. These changes can then be incorporated into the protein of interest to remove T-cell epitopes.
- Binding between the peptide ligand and the binding groove of MHC Class II molecules involves non-covalent interactions including, but not limited to: hydrogen bonds, electrostatic interactions, hydrophobic (lipophilic) interactions and Van der Waal's interactions. These are included in the peptide scoring function as described in detail below.
- a hydrogen bond is a non-covalent bond which can be formed between polar or charged groups and consists of a hydrogen atom shared by two other atoms.
- the hydrogen of the hydrogen donor has a positive charge where the hydrogen acceptor has a partial negative charge.
- hydrogen bond donors may be either nitrogens with hydrogen attached or hydrogens attached to oxygen or nitrogen.
- electrostatic bonds may be formed between arginine, histidine or lysine and aspartate or glutamate.
- the strength of the bond will depend upon the pKa of the ionizing group and the dielectric constant of the medium although they are approximately similar in strength to hydrogen bonds.
- Lipophilic interactions are favorable hydrophobic-hydrophobic contacts that occur between he protein and peptide ligand. Usually, these will occur between hydrophobic amino acid side chains of the peptide buried within the pockets of the binding groove such that they are not exposed to solvent.
- Nan der Waal's bonds are non-specific forces found between atoms which are 3- 4A apart. They are weaker and less specific than hydrogen and electrostatic bonds. The distribution of electronic charge around an atom changes with time and, at any instant, the charge distribution is not symmetric. This transient asymmetry in electronic charge induces a similar asymmetry in neighboring atoms.
- the resultant attractive forces between atoms reaches a maximum at the Nan der Waal's contact distance but diminishes very rapidly at about 1 A to about 2A. Conversely, as atoms become separated by less than the contact distance, increasingly strong repulsive forces become dominant as the outer electron clouds of the atoms overlap. Although the attractive forces are relatively weak compared to electrostatic and hydrogen bonds (about 0.6 Kcal mol), the repulsive forces in particular may be very important in determining whether a peptide ligand may bind successfully to a protein.
- the B ⁇ hm scoring function (SCORE1 approach) is used to estimate the binding constant. (B ⁇ hm, H J., J. Comput Aided Mol. Des., 8(3):243-256 (1994) which is hereby incorporated in its entirety).
- the scoring function (SCORE2 approach) is used to estimate the binding affinities as an indicator of a ligand containing a T-cell epitope (B ⁇ hm, H.J., J. Comput Aided Mol. Des., 12(4):309- 323 (1998) which is hereby incorporated in its entirety).
- the B ⁇ hm scoring functions as described in the above references are used to estimate the binding affinity of a ligand to a protein where it is already known that the ligand successfully binds to the protein and the protein ligand complex has had its structure solved, the solved structure being present in the Protein Data Bank ("PDB"). Therefore, the scoring function has been developed with the benefit of known positive binding data. In order to allow for discrimination between positive and negative binders, a repulsion term must be added to the equation. In addition, a more satisfactory estimate of binding energy is achieved by computing the lipophilic interactions in a pairwise manner rather than using the area based energy term of the above B ⁇ hm functions. Therefore, in a preferred embodiment, the binding energy is estimated using a modified B ⁇ hm scoring function.
- the binding energy between protein and ligand is estimated considering the following parameters: The reduction of binding energy due to the overall loss of translational and rotational entropy of the ligand ( ⁇ G 0 ); contributions from ideal hydrogen bonds ( ⁇ G hb ) where at least one partner is neutral; contributions from unperturbed ionic interactions ( ⁇ Gi on ic); lipophilic interactions between lipophilic ligand atoms and lipophilic acceptor atoms ( ⁇ GH PO ); the loss of binding energy due to the freezing of internal degrees of freedom in the ligand, i.e., the freedom of rotation about each C-C bond is reduced ( ⁇ G rot ); the energy of the interaction between the protein and ligand (Evaw)- Consideration of these terms gives equation 1 :
- ( ⁇ G bind ) ( ⁇ G 0 ) + ( ⁇ G hb xN hb ) + ( ⁇ G ion i c Nionic) + ( ⁇ G ⁇ ipo xNiipo) + (
- N is the number of qualifying interactions for a specific term and, in one embodiment, ⁇ G 0 , ⁇ G b , Gi 0n ic 5 ⁇ G]jp 0 and ⁇ G ro t are constants which are given the values: 5.4, -4.7, -4.7, -0.17, and 1.4, respectively.
- Nhb is calculated according to equation 2:
- ⁇ is the deviation of the hydrogen bond angle Z N /O- H ..
- Nneighb is the number of non-hydrogen protein atoms that are closer than 5A to any given protein atom.
- N HB is the number of hydrogen bonds
- Nij p0 is calculated according to equation 5 below:
- ⁇ rot is the number of rotable bonds of the amino acid side chain and is taken to be the number of acyclic sp 3 - sp 3 and sp 3 - sp 2 bonds. Rotations of terminal -CH 3 or -NH 3 are not taken into account.
- Si and ⁇ 2 are constants dependent upon atom identity n vdw +r vdw are the Van der Waal's atomic radii r is the distance between a pair of atoms.
- the constants ⁇ and ⁇ are given the atom values: C: 0.245, N: 0.283, O: 0.316, S: 0.316, respectively (i.e. for atoms of Carbon, Nitrogen, Oxygen and Sulphur, respectively).
- the Van der Waal's radii are given the atom values C: 1.85, N: 1.75, O: 1.60, S: 2.00A.
- the present prediction method can be calibrated against a data set comprising a large number of peptides whose affinity for various MHC Class II molecules has previously been experimentally determined. By comparison of calculated versus experimental data, a cut of value can be determined above which it is known that all experimentally determined T-cell epitopes are correctly predicted. [0145] It should be understood that, although the above scoring function is relatively simple compared to some sophisticated methodologies that are available, the calculations are performed extremely rapidly. It should also be understood that the objective is not to calculate the true binding energy per se for each peptide docked in the binding groove of a selected MHC Class II protein.
- the underlying objective is to obtain comparative binding energy data as an aid to predicting the location of T-cell epitopes based on the primary structure (i.e. amino acid sequence) of a selected protein.
- a relatively high binding energy or a binding energy above a selected threshold value would suggest the presence of a T-cell epitope in the ligand.
- the ligand may then be subjected to at least one round of amino-acid substitution and the binding energy recalculated. Due to the rapid nature of the calculations, these manipulations of the peptide sequence can be performed interactively within the program's user interface on cost-effectively available computer hardware. Major investment in computer hardware is thus not required. [0146] It would be apparent to one skilled in the art that other available software could be used for the same purposes.
- more sophisticated software which is capable of docking ligands into protein binding-sites may be used in conjunction with energy minimization.
- docking software are: DOCK (Kuntz et al, J. Mol. BioL, 161:269-288 (1982)), LUDI (B ⁇ hm, H.J., J. Comput Aided Mol. Des., 8:623-632 (1994)) and FLEXX (Rarey M., et al., ISMB, 3:300-308 (1995)).
- Examples of molecular modeling and manipulation software include: AMBER (Tripos) and CHARMm (Molecular Simulations Inc.).
Abstract
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DK02757870.7T DK1373301T3 (en) | 2001-03-30 | 2002-03-30 | Reduction of the immunogenicity of fusion proteins |
BR0208207-1A BR0208207A (en) | 2001-03-30 | 2002-03-30 | Reduction of fusion protein immunogenicity |
MXPA03008715A MXPA03008715A (en) | 2001-03-30 | 2002-03-30 | Reducing the immunogenicity of fusion proteins. |
DE60238472T DE60238472D1 (en) | 2001-03-30 | 2002-03-30 | WEAKNESS OF IMMUNOGENICITY OF FUSION PROTEINS |
KR10-2003-7012819A KR20030087040A (en) | 2001-03-30 | 2002-03-30 | Reducing the immunogenicity of fusion proteins |
HU0303753A HUP0303753A2 (en) | 2001-03-30 | 2002-03-30 | Reducing the immunogenicity of fusion proteins |
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US8926973B2 (en) | 2001-03-30 | 2015-01-06 | Merck Patent Gmbh | Reducing the immunogenicity of fusion proteins |
US8937158B2 (en) | 2003-03-03 | 2015-01-20 | Xencor, Inc. | Fc variants with increased affinity for FcγRIIc |
EP2423222B1 (en) | 2007-01-30 | 2015-04-29 | Epivax, Inc. | Regulatory t cell epitopes, compositions and uses thereof |
US9029330B2 (en) | 2005-12-30 | 2015-05-12 | Merck Patent Gmbh | Methods of treating cancer using interleukin-12p40 variants having improved stability |
US9040041B2 (en) | 2005-10-03 | 2015-05-26 | Xencor, Inc. | Modified FC molecules |
WO2016054603A2 (en) | 2014-10-02 | 2016-04-07 | City Of Hope | Multivalent meditopes, meditope-binding antibodies and uses thereof |
US9371369B2 (en) | 2009-02-03 | 2016-06-21 | Amunix Operating Inc. | Extended recombinant polypeptides and compositions comprising same |
US9376672B2 (en) | 2009-08-24 | 2016-06-28 | Amunix Operating Inc. | Coagulation factor IX compositions and methods of making and using same |
US9416171B2 (en) | 2011-12-23 | 2016-08-16 | Nicholas B. Lydon | Immunoglobulins and variants directed against pathogenic microbes |
US9475881B2 (en) | 2010-01-19 | 2016-10-25 | Xencor, Inc. | Antibody variants with enhanced complement activity |
US9617349B2 (en) | 2004-01-22 | 2017-04-11 | Merck Patent Gmbh | Anti-cancer antibodies with reduced complement fixation |
US9714282B2 (en) | 2003-09-26 | 2017-07-25 | Xencor, Inc. | Optimized Fc variants and methods for their generation |
EP3278813A1 (en) | 2009-06-08 | 2018-02-07 | Amunix Operating Inc. | Growth hormone polypeptides and methods of making and using same |
US9938331B2 (en) | 2005-09-27 | 2018-04-10 | Amunix Operating Inc. | Biologically active proteins having increased in vivo and/or in vitro stability |
US9988439B2 (en) | 2011-12-23 | 2018-06-05 | Nicholas B. Lydon | Immunoglobulins and variants directed against pathogenic microbes |
US10072092B2 (en) | 2005-12-30 | 2018-09-11 | Merck Patent Gmbh | Methods of use of anti-CD19 antibodies with reduced immunogenicity |
EP3372617A2 (en) | 2010-04-02 | 2018-09-12 | Amunix Operating Inc. | Binding fusion proteins, binding fusion protein-drug conjugates, xten-drug conjugates and methods of making and using same |
EP3406347A2 (en) | 2012-02-27 | 2018-11-28 | Amunix Operating Inc. | Xten conjugate compositions and methods of making same |
US10246501B2 (en) | 2014-01-08 | 2019-04-02 | Prosit Sole Biotechnology (Beijing) Co, Ltd | Fusion polypeptides and methods of use |
US10370430B2 (en) | 2012-02-15 | 2019-08-06 | Bioverativ Therapeutics Inc. | Recombinant factor VIII proteins |
US10421798B2 (en) | 2012-02-15 | 2019-09-24 | Bioverativ Therapeutics Inc. | Factor VIII compositions and methods of making and using same |
US10548953B2 (en) | 2013-08-14 | 2020-02-04 | Bioverativ Therapeutics Inc. | Factor VIII-XTEN fusions and uses thereof |
US10604576B2 (en) | 2016-06-20 | 2020-03-31 | Kymab Limited | Antibodies and immunocytokines |
US10745680B2 (en) | 2015-08-03 | 2020-08-18 | Bioverativ Therapeutics Inc. | Factor IX fusion proteins and methods of making and using same |
WO2021097186A1 (en) | 2019-11-13 | 2021-05-20 | Amunix Pharmaceuticals, Inc. | Barcoded xten polypeptides and compositions thereof, and methods for making and using the same |
US11020454B2 (en) | 2015-07-15 | 2021-06-01 | Prosit Sole Biotechnology (Beijing) Co. Ltd | Fusion polypeptides and methods of use |
US11401348B2 (en) | 2009-09-02 | 2022-08-02 | Xencor, Inc. | Heterodimeric Fc variants |
US11753479B2 (en) | 2014-03-04 | 2023-09-12 | Kymab Limited | Nucleic acids encoding anti-OX40L antibodies |
US11779604B2 (en) | 2016-11-03 | 2023-10-10 | Kymab Limited | Antibodies, combinations comprising antibodies, biomarkers, uses and methods |
Families Citing this family (69)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2312188C (en) | 1997-12-08 | 2010-06-29 | Lexigen Pharmaceuticals Corp. | Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation |
US20030105294A1 (en) * | 1998-02-25 | 2003-06-05 | Stephen Gillies | Enhancing the circulating half life of antibody-based fusion proteins |
BR9909583A (en) * | 1998-04-15 | 2002-01-15 | Lexigen Pharm Corp | Increased immune response mediated by an antibody-cytokine fusion protein by coadministration with angiogenesis inhibitor |
US6617135B1 (en) * | 1999-08-09 | 2003-09-09 | Emd Lexigen Research Center Corp. | Multiple cytokine protein complexes |
US20050202538A1 (en) * | 1999-11-12 | 2005-09-15 | Merck Patent Gmbh | Fc-erythropoietin fusion protein with improved pharmacokinetics |
BR0112111A (en) * | 2000-06-29 | 2003-05-06 | Merck Patent Gmbh | Enhancement of antibody-cytokine fusion protein-mediated immune responses by combined treatment with immunocytocin uptake enhancers |
US20120148585A1 (en) * | 2001-05-01 | 2012-06-14 | Andrew Saxon | Fusion molecules and methods for treatment of immune diseases |
DK1383785T3 (en) | 2001-05-03 | 2011-05-23 | Merck Patent Gmbh | Recombinant tumor-specific antibody and its use |
DE10122140A1 (en) * | 2001-05-08 | 2002-11-28 | Apotech Res & Dev Ltd | Recombinant fusion proteins and their trimers |
AU2002357784B2 (en) | 2001-12-04 | 2008-07-31 | Merck Patent Gmbh | Immunocytokines with modulated selectivity |
WO2005056606A2 (en) * | 2003-12-03 | 2005-06-23 | Xencor, Inc | Optimized antibodies that target the epidermal growth factor receptor |
US7611700B2 (en) * | 2002-09-09 | 2009-11-03 | Hanall Pharmaceuticals, Co., Ltd. | Protease resistant modified interferon alpha polypeptides |
AU2003280761B2 (en) * | 2002-11-15 | 2009-10-01 | Nipro Corporation | Liposome |
JP4494977B2 (en) | 2002-12-17 | 2010-06-30 | 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 ハフツング | Humanized antibody (H14.18) of mouse 14.18 antibody that binds to GD2 and its IL-2 fusion protein |
US20070161087A1 (en) * | 2003-05-29 | 2007-07-12 | Wolfgang Glaesner | Glp-1 fusion proteins |
AU2004251145C1 (en) * | 2003-06-12 | 2011-04-14 | Eli Lilly And Company | GLP-1 analog fusion proteins |
US20050069521A1 (en) * | 2003-08-28 | 2005-03-31 | Emd Lexigen Research Center Corp. | Enhancing the circulating half-life of interleukin-2 proteins |
EP1673398B1 (en) | 2003-10-16 | 2010-12-29 | Micromet AG | Multispecific deimmunized cd3-binders |
CN100427599C (en) * | 2003-12-26 | 2008-10-22 | 吉林大学第一医院 | ALBSIN/IL-18MAT hybridization cytokine |
PT1737961E (en) | 2004-03-19 | 2013-08-26 | Merck Patent Gmbh | Modified bouganin proteins, cytotoxins and methods and uses thereof |
EP1732613A2 (en) * | 2004-04-06 | 2006-12-20 | Affibody AB | Use of serum albumin binding peptides conjugates for the preparation of a medicament |
US7670595B2 (en) * | 2004-06-28 | 2010-03-02 | Merck Patent Gmbh | Fc-interferon-beta fusion proteins |
KR101151957B1 (en) | 2004-07-22 | 2012-06-01 | 로저 킹돈 크레이그 | binding molecules |
US20060204512A1 (en) | 2004-09-23 | 2006-09-14 | Vasgene Therapeutics, Inc. | Polypeptide compounds for inhibiting angiogenesis and tumor growth |
KR20070085886A (en) * | 2004-12-09 | 2007-08-27 | 메르크 파텐트 게엠베하 | Il-7 variants with reduced immunogenicity |
SI1831252T1 (en) | 2004-12-22 | 2009-12-31 | Lilly Co Eli | Glp-1 analog fusion protein formulations |
CA2658673A1 (en) * | 2005-07-27 | 2008-02-01 | Gerald J. Prud'homme | Composition and method for prevention and treatment of type i and type ii diabetes |
US20070104689A1 (en) * | 2005-09-27 | 2007-05-10 | Merck Patent Gmbh | Compositions and methods for treating tumors presenting survivin antigens |
ES2856451T3 (en) | 2005-10-11 | 2021-09-27 | Amgen Res Munich Gmbh | Compositions comprising specific antibodies for different species, and uses thereof |
ES2595091T3 (en) | 2005-12-21 | 2016-12-27 | Amgen Research (Munich) Gmbh | Pharmaceutical compositions with soluble CEA resistance |
US7625564B2 (en) * | 2006-01-27 | 2009-12-01 | Novagen Holding Corporation | Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo |
EP2038417A2 (en) * | 2006-07-06 | 2009-03-25 | Merck Patent GmbH | Compositions and methods for enhancing the efficacy of il-2 mediated immune responses |
WO2008045380A2 (en) * | 2006-10-04 | 2008-04-17 | Codon Devices, Inc. | Nucleic acid libraries and their design and assembly |
EP2120998B1 (en) | 2006-11-28 | 2013-08-07 | HanAll Biopharma Co., Ltd. | Modified erythropoietin polypeptides and uses thereof for treatment |
WO2009014835A2 (en) * | 2007-06-21 | 2009-01-29 | Angelica Therapeutics, Inc. | Modified toxins |
US8137979B2 (en) * | 2007-06-25 | 2012-03-20 | Qinetiq Limited | Preconcentrator device |
US8067548B2 (en) | 2007-07-26 | 2011-11-29 | Novagen Holding Corporation | Fusion proteins having mutated immunoglobulin hinge region |
JP6035009B2 (en) | 2007-08-22 | 2016-11-30 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Activable binding polypeptides and methods for identification and use thereof |
WO2009110944A1 (en) * | 2008-02-29 | 2009-09-11 | Angelica Therapeutics, Inc. | Modified toxins |
WO2010080124A2 (en) | 2008-12-18 | 2010-07-15 | Dana-Farber Cancer Institute, Inc. | Nkg2d-fc for immunotherapy |
JP5851842B2 (en) | 2009-01-12 | 2016-02-03 | サイトムエックス セラピューティクス, インク.CytomX Therapeutics, Inc. | Modified antibody composition and methods of making and using the same |
WO2010096838A2 (en) | 2009-02-23 | 2010-08-26 | Cytomx Therapeutics, Llc | Proproteins and methods of use thereof |
AU2010238858A1 (en) | 2009-04-22 | 2011-12-08 | Merck Patent Gmbh | Antibody fusion proteins with modified FcRn binding sites |
BRPI1011236A2 (en) | 2009-06-15 | 2019-07-09 | 4S3 Bioscience Inc | methods and compositions for treating myotubular myopathy using chimeric polypeptides comprising miotubularih1 (mtm1) polypeptides |
JP5822841B2 (en) * | 2009-11-05 | 2015-11-24 | テバ・ファーマシューティカルズ・オーストラリア・ピーティワイ・リミテッド | Treatment of cancer containing mutant KRAS or BRAF gene |
CN104610454A (en) | 2010-02-16 | 2015-05-13 | 米迪缪尼有限公司 | HSA-related compositions and methods of use |
WO2012119989A2 (en) | 2011-03-04 | 2012-09-13 | Oryzon Genomics, S.A. | Methods and antibodies for the diagnosis and treatment of cancer |
PT2697257T (en) * | 2011-04-13 | 2016-12-28 | Bristol Myers Squibb Co | Fc fusion proteins comprising novel linkers or arrangements |
CN110272484A (en) * | 2011-05-05 | 2019-09-24 | 阿尔布梅迪克斯医疗有限公司 | Albumin variants |
CN102222177A (en) * | 2011-07-08 | 2011-10-19 | 上海生物信息技术研究中心 | Auxiliary prediction method for molecular modification of antibody protein |
US9714291B2 (en) * | 2012-10-05 | 2017-07-25 | Kyowa Hakko Kirin Co., Ltd | Heterodimer protein composition |
US10017581B2 (en) | 2013-02-20 | 2018-07-10 | Valerion Therapeutics, Llc | Methods and compositions for treatment of Pompe disease |
US10993420B2 (en) | 2013-03-15 | 2021-05-04 | Erasmus University Medical Center | Production of heavy chain only antibodies in transgenic mammals |
WO2014150600A2 (en) | 2013-03-15 | 2014-09-25 | Angelica Therapeutics, Inc. | Modified toxins |
WO2015085311A1 (en) | 2013-12-07 | 2015-06-11 | Case Western Reserve University | Compositions and methods of treating thrombosis |
AU2015204446A1 (en) | 2014-01-13 | 2016-07-14 | Valerion Therapeutics, Llc | Internalizing moieties |
US9676863B2 (en) | 2014-02-10 | 2017-06-13 | Merck Patent Gmbh | Targeted TGFβ inhibitors |
CA2950178A1 (en) * | 2014-06-06 | 2015-12-10 | The California Institute For Biomedical Research | Methods of constructing amino terminal immunoglobulin fusion proteins and compositions thereof |
EP3677285B1 (en) | 2014-08-04 | 2023-12-13 | Case Western Reserve University | Targeting peptides for detecting prostate cancer |
WO2016087514A1 (en) | 2014-12-02 | 2016-06-09 | Cemm - Forschungszentrum Für Molekulare Medizin Gmbh | Anti-mutant calreticulin antibodies and their use in the diagnosis and therapy of myeloid malignancies |
EP3270958A4 (en) * | 2015-03-18 | 2018-11-14 | Omnicyte | Fusion proteins comprising modified alpha virus surface glycoproteins and tumor associated antigen and methods thereof |
WO2017083612A1 (en) | 2015-11-13 | 2017-05-18 | Dana-Farber Cancer Institute, Inc. | An nkg2d-ig fusion protein for cancer immunotherapy |
DK3387019T3 (en) | 2015-12-09 | 2022-01-10 | Scripps Research Inst | RELAXIN-IMMUNOGLOBULIN INFUSION PROTEINS AND METHODS OF USE |
KR20190039200A (en) | 2016-08-12 | 2019-04-10 | 메르크 파텐트 게엠베하 | Combination therapy for cancer |
WO2018102777A1 (en) * | 2016-12-01 | 2018-06-07 | University Of South Florida | Peptibodies, compositions thereof, and methods of treating atrial fibrillation |
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
EP3873612A2 (en) | 2018-11-01 | 2021-09-08 | Merck Patent GmbH | Methods of administering anti-tim-3 antibodies |
CN113272017A (en) | 2018-11-01 | 2021-08-17 | 默克专利有限公司 | anti-TIM-3 antibodies |
JP2022543669A (en) * | 2019-08-08 | 2022-10-13 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Novel antigen-binding molecule format |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992010755A1 (en) | 1990-12-05 | 1992-06-25 | Novo Nordisk A/S | Proteins with changed epitopes and methods for the production thereof |
EP0519596A1 (en) | 1991-05-17 | 1992-12-23 | Merck & Co. Inc. | A method for reducing the immunogenicity of antibody variable domains |
EP0699755A2 (en) | 1994-06-30 | 1996-03-06 | Centro de Inmunologia Molecular | Method for obtaining modified immunoglobulins with reduced immunogenicity of murine antibody variable domains, compositions containing them |
WO1996040792A1 (en) | 1995-06-07 | 1996-12-19 | Novo Nordisk A/S | Modification of polypeptides |
WO1998052976A1 (en) | 1997-05-21 | 1998-11-26 | Biovation Limited | Method for the production of non-immunogenic proteins |
WO1998059244A1 (en) | 1997-06-20 | 1998-12-30 | Eclagen Limited | Identification of mhc binding peptides |
WO2000034317A2 (en) | 1998-12-08 | 2000-06-15 | Biovation Limited | Method for reducing immunogenicity of proteins |
WO2001036489A2 (en) | 1999-11-12 | 2001-05-25 | Merck Patent Gmbh | Erythropoietin forms with improved properties |
WO2002066514A2 (en) | 2001-02-19 | 2002-08-29 | Merck Patent Gmbh | Artificial fusion proteins with reduced immunogenicity |
Family Cites Families (234)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US44423A (en) * | 1864-09-27 | Improved screw for music-stools | ||
US147311A (en) * | 1874-02-10 | Improvement in electric telegraphs | ||
US192222A (en) * | 1877-06-19 | Improvement in watches | ||
US105294A (en) * | 1870-07-12 | Erasmus w | ||
US166877A (en) * | 1875-08-17 | Improvement in electric lights | ||
US53539A (en) * | 1866-03-27 | Improvement in revolving fire-arms | ||
US348237A (en) * | 1886-08-31 | richards | ||
US3529A (en) * | 1844-04-10 | yvolfe | ||
US142374A (en) * | 1873-09-02 | Improvement in stopping mechanisms for doubling and twisting machines | ||
US12789A (en) * | 1855-05-01 | Office | ||
US49227A (en) * | 1865-08-08 | Improvement in construction of railway trains and cars | ||
US193570A (en) * | 1877-07-24 | Improvement in washing-boards | ||
US81664A (en) * | 1868-09-01 | Island | ||
US166163A (en) * | 1875-07-27 | Improvement in bee-hives | ||
US1803686A (en) * | 1923-11-23 | 1931-05-05 | Herman A Brassert | Gas production |
US4196265A (en) * | 1977-06-15 | 1980-04-01 | The Wistar Institute | Method of producing antibodies |
US4522811A (en) * | 1982-07-08 | 1985-06-11 | Syntex (U.S.A.) Inc. | Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides |
US4469797A (en) * | 1982-09-23 | 1984-09-04 | Miles Laboratories, Inc. | Digoxigenin immunogens, antibodies, labeled conjugates, and related derivatives |
US4737462A (en) * | 1982-10-19 | 1988-04-12 | Cetus Corporation | Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β |
US4966843A (en) * | 1982-11-01 | 1990-10-30 | Cetus Corporation | Expression of interferon genes in Chinese hamster ovary cells |
US4816567A (en) * | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
NZ210501A (en) * | 1983-12-13 | 1991-08-27 | Kirin Amgen Inc | Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence |
US4703008A (en) | 1983-12-13 | 1987-10-27 | Kiren-Amgen, Inc. | DNA sequences encoding erythropoietin |
KR850004274A (en) * | 1983-12-13 | 1985-07-11 | 원본미기재 | Method for preparing erythropoietin |
US5082658A (en) * | 1984-01-16 | 1992-01-21 | Genentech, Inc. | Gamma interferon-interleukin-2 synergism |
EP0158198A1 (en) | 1984-03-29 | 1985-10-16 | Takeda Chemical Industries, Ltd. | DNA and use thereof |
US5807715A (en) | 1984-08-27 | 1998-09-15 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin |
GB8422238D0 (en) | 1984-09-03 | 1984-10-10 | Neuberger M S | Chimeric proteins |
US4667016A (en) * | 1985-06-20 | 1987-05-19 | Kirin-Amgen, Inc. | Erythropoietin purification |
US4690915A (en) | 1985-08-08 | 1987-09-01 | The United States Of America As Represented By The Department Of Health And Human Services | Adoptive immunotherapy as a treatment modality in humans |
US5679543A (en) | 1985-08-29 | 1997-10-21 | Genencor International, Inc. | DNA sequences, vectors and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi |
US5643565A (en) | 1985-09-20 | 1997-07-01 | Chiron Corporation | Human IL-2 as a vaccine adjuvant |
US4676980A (en) * | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
US4935233A (en) * | 1985-12-02 | 1990-06-19 | G. D. Searle And Company | Covalently linked polypeptide cell modulators |
DE3712985A1 (en) | 1987-04-16 | 1988-11-03 | Hoechst Ag | BIFUNCTIONAL PROTEINS |
US5359035A (en) | 1985-12-21 | 1994-10-25 | Hoechst Aktiengesellschaft | Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF) |
JPS63267278A (en) | 1986-03-14 | 1988-11-04 | Toray Ind Inc | Base sequence coding bonded interferon |
EP0237019A3 (en) | 1986-03-14 | 1988-03-09 | Toray Industries, Inc. | Interferon conjugate and production thereof using recombinant gene |
US5225539A (en) * | 1986-03-27 | 1993-07-06 | Medical Research Council | Recombinant altered antibodies and methods of making altered antibodies |
GB8607679D0 (en) | 1986-03-27 | 1986-04-30 | Winter G P | Recombinant dna product |
DK173067B1 (en) | 1986-06-27 | 1999-12-13 | Univ Washington | Human erythropoietin gene, method of expression thereof in transfected cell lines, the transfected cell lines |
US4954617A (en) | 1986-07-07 | 1990-09-04 | Trustees Of Dartmouth College | Monoclonal antibodies to FC receptors for immunoglobulin G on human mononuclear phagocytes |
US4894227A (en) | 1986-08-01 | 1990-01-16 | Cetus Corporation | Composition of immunotoxins with interleukin-2 |
US4946778A (en) * | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
US5508031A (en) | 1986-11-21 | 1996-04-16 | Cetus Oncology Corporation | Method for treating biological damage using a free-radial scavenger and interleukin-2 |
US4732683A (en) * | 1986-12-02 | 1988-03-22 | Biospectrum, Inc. | Purification method for alpha interferon |
US5019368A (en) * | 1989-02-23 | 1991-05-28 | Cancer Biologics, Inc. | Detection of necrotic malignant tissue and associated therapy |
WO1988007089A1 (en) * | 1987-03-18 | 1988-09-22 | Medical Research Council | Altered antibodies |
US5258498A (en) | 1987-05-21 | 1993-11-02 | Creative Biomolecules, Inc. | Polypeptide linkers for production of biosynthetic proteins |
EP0623679B1 (en) | 1987-05-21 | 2003-06-25 | Micromet AG | Targeted multifunctional proteins |
US5091513A (en) * | 1987-05-21 | 1992-02-25 | Creative Biomolecules, Inc. | Biosynthetic antibody binding sites |
ES2073394T3 (en) | 1987-06-10 | 1995-08-16 | Dana Farber Cancer Inst Inc | BIFUNCTIONAL ANTIBODY CONSTRUCTS AND THEIR USE TO SELECTIVELY DESTROY CELLULAR POPULATIONS. |
US5064646A (en) | 1988-08-02 | 1991-11-12 | The University Of Maryland | Novel infectious bursal disease virus |
EP0305967B1 (en) | 1987-09-02 | 1993-05-05 | Ciba-Geigy Ag | Conjugates of interferon alpha with immunoglobulins |
PT88641B (en) | 1987-10-02 | 1993-04-30 | Genentech Inc | METHOD FOR PREPARING A VARIETY OF ADHESION |
PT89121A (en) | 1987-12-04 | 1989-12-29 | Du Pont | PROCESS FOR THE PREPARATION OF INTERLEUQUIN-2 FIXED AND INTERLEUKIN-2 CONTAINING AN EXTENSION IN THE TERMINAL-CARBOXYL WITH ACTIVITY OF INTERLEUQUIN-2 NATURAL |
WO1989006692A1 (en) * | 1988-01-12 | 1989-07-27 | Genentech, Inc. | Method of treating tumor cells by inhibiting growth factor receptor function |
CA1341588C (en) | 1988-01-26 | 2009-01-06 | Michel Revel | Human ifn-beta2/i1-6, its purification and use |
US5120525A (en) * | 1988-03-29 | 1992-06-09 | Immunomedics, Inc. | Radiolabeled antibody cytotoxic therapy of cancer |
DE3812605A1 (en) | 1988-04-15 | 1990-06-07 | Leskovar Peter Dipl Ing Dr Hab | IMMUNEGULATIVE SUBSTANCES AND SUBSTANCE MIXTURES FOR ACTIVE INFLUENCING OF THE DISEASE PROCESS |
US4975369A (en) | 1988-04-21 | 1990-12-04 | Eli Lilly And Company | Recombinant and chimeric KS1/4 antibodies directed against a human adenocarcinoma antigen |
IE62463B1 (en) | 1988-07-07 | 1995-02-08 | Res Dev Foundation | Immunoconjugates for cancer diagnosis and therapy |
US5601819A (en) * | 1988-08-11 | 1997-02-11 | The General Hospital Corporation | Bispecific antibodies for selective immune regulation and for selective immune cell binding |
IL91933A (en) | 1988-10-11 | 1994-12-29 | Univ Southern California | Vasoactive immunoconjugates useful for increasing the vascular permeability or the blood supply to neoplastic or otherwise diseased tissues |
US5457038A (en) | 1988-11-10 | 1995-10-10 | Genetics Institute, Inc. | Natural killer stimulatory factor |
US5242824A (en) | 1988-12-22 | 1993-09-07 | Oncogen | Monoclonal antibody to human carcinomas |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5225538A (en) * | 1989-02-23 | 1993-07-06 | Genentech, Inc. | Lymphocyte homing receptor/immunoglobulin fusion proteins |
US5116964A (en) | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
US5166322A (en) | 1989-04-21 | 1992-11-24 | Genetics Institute | Cysteine added variants of interleukin-3 and chemical modifications thereof |
US6750329B1 (en) * | 1989-05-05 | 2004-06-15 | Research Development Foundation | Antibody delivery system for biological response modifiers |
IE63847B1 (en) | 1989-05-05 | 1995-06-14 | Res Dev Foundation | A novel antibody delivery system for biological response modifiers |
US6291158B1 (en) | 1989-05-16 | 2001-09-18 | Scripps Research Institute | Method for tapping the immunological repertoire |
US5399346A (en) * | 1989-06-14 | 1995-03-21 | The United States Of America As Represented By The Department Of Health And Human Services | Gene therapy |
EP0739904A1 (en) | 1989-06-29 | 1996-10-30 | Medarex, Inc. | Bispecific reagents for aids therapy |
ATE123065T1 (en) * | 1989-07-07 | 1995-06-15 | Takeda Chemical Industries Ltd | PROTEINS AND THEIR PRODUCTION. |
US5073627A (en) | 1989-08-22 | 1991-12-17 | Immunex Corporation | Fusion proteins comprising GM-CSF and IL-3 |
ES2103274T3 (en) | 1989-09-20 | 1997-09-16 | Abbott Lab | PRODUCTION METHOD OF FUSION PROTEINS. |
US5856298A (en) * | 1989-10-13 | 1999-01-05 | Amgen Inc. | Erythropoietin isoforms |
AU646822B2 (en) | 1989-10-13 | 1994-03-10 | Kirin-Amgen Inc. | Erythropoietin isoforms |
DE69034247T2 (en) | 1989-12-22 | 2008-04-17 | F. Hoffmann-La Roche Ag | Cytotoxic lymphocyte maturation factor 40kD subunit and monoclonal antibodies specific for it |
US5314995A (en) | 1990-01-22 | 1994-05-24 | Oncogen | Therapeutic interleukin-2-antibody based fusion proteins |
WO1991013166A1 (en) | 1990-03-02 | 1991-09-05 | Abbott Biotech, Inc. | Antibody constructs with enhanced binding affinity |
AU654811B2 (en) | 1990-03-20 | 1994-11-24 | Trustees Of Columbia University In The City Of New York, The | Chimeric antibodies with receptor binding ligands in place of their constant region |
US5349053A (en) | 1990-06-01 | 1994-09-20 | Protein Design Labs, Inc. | Chimeric ligand/immunoglobulin molecules and their uses |
US7253264B1 (en) | 1990-06-28 | 2007-08-07 | Sanofi-Arentideutschland GmbH | Immunoglobulin fusion proteins, their production and use |
JPH06504262A (en) | 1990-07-27 | 1994-05-19 | レブリゲン コーポレーション | Novel methods and compositions for the treatment of angiogenic diseases |
ATE218889T1 (en) | 1990-11-09 | 2002-06-15 | Stephen D Gillies | CYTOKINE IMMUNOCONJUGATES |
WO1992008801A1 (en) | 1990-11-09 | 1992-05-29 | Abbott Laboratories | Bridging antibody fusion constructs |
US5650150A (en) * | 1990-11-09 | 1997-07-22 | Gillies; Stephen D. | Recombinant antibody cytokine fusion proteins |
US5709859A (en) * | 1991-01-24 | 1998-01-20 | Bristol-Myers Squibb Company | Mixed specificity fusion proteins |
GB9105245D0 (en) | 1991-03-12 | 1991-04-24 | Lynxvale Ltd | Binding molecules |
US6072039A (en) | 1991-04-19 | 2000-06-06 | Rohm And Haas Company | Hybrid polypeptide comparing a biotinylated avidin binding polypeptide fused to a polypeptide of interest |
US5199942A (en) * | 1991-06-07 | 1993-04-06 | Immunex Corporation | Method for improving autologous transplantation |
WO1993003157A1 (en) | 1991-07-29 | 1993-02-18 | Dana Farber Cancer Institute | Plasmids for the rapid preparation of modified proteins |
US20020037558A1 (en) * | 1991-10-23 | 2002-03-28 | Kin-Ming Lo | E.coli produced immunoglobulin constructs |
US5376367A (en) | 1991-11-22 | 1994-12-27 | Immunex Corporation | Fusion proteins comprising MGF and IL-3 |
US6627615B1 (en) | 1991-12-17 | 2003-09-30 | The Regents Of The University Of California | Methods and compositions for in vivo gene therapy |
ATE260971T1 (en) | 1992-04-01 | 2004-03-15 | Univ Rockefeller | METHOD FOR THE IN VITRO CULTIVATION OF DENDRITIC PRECURSOR CELLS AND THEIR USE FOR IMMUNOGENIC PRODUCTION |
AU687733B2 (en) | 1992-04-01 | 1998-03-05 | Rockefeller University, The | Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens |
ATE311895T1 (en) * | 1992-05-26 | 2005-12-15 | Immunex Corp | NEW CYTOKINE THAT BINDS CD30 |
EP0646178A1 (en) | 1992-06-04 | 1995-04-05 | The Regents Of The University Of California | expression cassette with regularoty regions functional in the mammmlian host |
US5614184A (en) * | 1992-07-28 | 1997-03-25 | New England Deaconess Hospital | Recombinant human erythropoietin mutants and therapeutic methods employing them |
EP0671926B1 (en) | 1992-08-11 | 2002-11-13 | President And Fellows Of Harvard College | Immunomodulatory peptides |
US5429199A (en) * | 1992-08-26 | 1995-07-04 | Kennametal Inc. | Cutting bit and cutting insert |
DE4228839A1 (en) | 1992-08-29 | 1994-03-03 | Behringwerke Ag | Methods for the detection and determination of mediators |
EP0627932B1 (en) | 1992-11-04 | 2002-05-08 | City Of Hope | Antibody construct |
ES2276390T3 (en) * | 1992-11-05 | 2007-06-16 | Sloan-Kettering Institute For Cancer Research | SPECIFIC MEMBRANE ANTIGEN OF THE PROSTATE. |
US5738849A (en) * | 1992-11-24 | 1998-04-14 | G. D. Searle & Co. | Interleukin-3 (IL-3) variant fusion proteins, their recombinant production, and therapeutic compositions comprising them |
US5543297A (en) * | 1992-12-22 | 1996-08-06 | Merck Frosst Canada, Inc. | Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity |
US6096331A (en) * | 1993-02-22 | 2000-08-01 | Vivorx Pharmaceuticals, Inc. | Methods and compositions useful for administration of chemotherapeutic agents |
WO1994024267A1 (en) * | 1993-04-20 | 1994-10-27 | Robinson, William, S. | Methods and materials for treatment of individuals infected with intracellular infectious agents |
WO1994024160A2 (en) | 1993-04-21 | 1994-10-27 | Brigham And Women's Hospital | Erythropoietin muteins with enhanced activity |
US5759551A (en) * | 1993-04-27 | 1998-06-02 | United Biomedical, Inc. | Immunogenic LHRH peptide constructs and synthetic universal immune stimulators for vaccines |
AU6776194A (en) | 1993-04-28 | 1994-11-21 | Hybritech Incorporated | Method for creating optimized regulatory regions affecting protein expression and protein trafficking |
CN1125401A (en) * | 1993-04-29 | 1996-06-26 | 艾博特公司 | Erythropoietin analog compositions and methods |
US5554512A (en) | 1993-05-24 | 1996-09-10 | Immunex Corporation | Ligands for flt3 receptors |
CA2125763C (en) * | 1993-07-02 | 2007-08-28 | Maurice Kent Gately | P40 homodimer of interleukin-12 |
GB9316989D0 (en) | 1993-08-16 | 1993-09-29 | Lynxvale Ltd | Binding molecules |
CN1057534C (en) | 1993-08-17 | 2000-10-18 | 柯瑞英-艾格公司 | Erythropoietin analogs |
EP0742830B1 (en) | 1994-02-01 | 2001-07-18 | THE UNITED STATES OF AMERICA, as represented by the Secretary of the Department of Health and Human Services | Fusion proteins that include antibody and nonantibody portions |
WO1995028427A1 (en) | 1994-04-15 | 1995-10-26 | Imclone Systems Incorporated | Chimeric interleukin-3/mutein interleukin-6 lymphokine |
US5837682A (en) | 1996-03-08 | 1998-11-17 | The Children's Medical Center Corporation | Angiostatin fragments and method of use |
US5639725A (en) * | 1994-04-26 | 1997-06-17 | Children's Hospital Medical Center Corp. | Angiostatin protein |
NZ285501A (en) * | 1994-04-26 | 1998-02-26 | Childrens Medical Center | Plasminogen analogs (angiostatin), endothelial inhibitors, assays and kits |
WO1995031483A1 (en) | 1994-05-13 | 1995-11-23 | Eclagen Limited | Improvements in or relating to peptide delivery |
US6429199B1 (en) | 1994-07-15 | 2002-08-06 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules for activating dendritic cells |
AU3382595A (en) | 1994-07-29 | 1996-03-04 | Smithkline Beecham Corporation | Novel compounds |
US6309853B1 (en) | 1994-08-17 | 2001-10-30 | The Rockfeller University | Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof |
US5888773A (en) * | 1994-08-17 | 1999-03-30 | The United States Of America As Represented By The Department Of Health And Human Services | Method of producing single-chain Fv molecules |
US5541087A (en) * | 1994-09-14 | 1996-07-30 | Fuji Immunopharmaceuticals Corporation | Expression and export technology of proteins as immunofusins |
ES2167391T3 (en) | 1994-09-16 | 2002-05-16 | Merck Patent Gmbh | IMMUNOCONJUGADOS II. |
WO1996018412A1 (en) | 1994-12-12 | 1996-06-20 | Beth Israel Hospital Association | Chimeric cytokines and uses thereof |
US6485726B1 (en) | 1995-01-17 | 2002-11-26 | The Brigham And Women's Hospital, Inc. | Receptor specific transepithelial transport of therapeutics |
US6086875A (en) * | 1995-01-17 | 2000-07-11 | The Brigham And Women's Hospital, Inc. | Receptor specific transepithelial transport of immunogens |
US5691309A (en) | 1995-01-31 | 1997-11-25 | Eli Lilly And Company | Anti-obesity proteins |
US5552524A (en) | 1995-01-31 | 1996-09-03 | Eli Lilly And Company | Anti-obesity proteins |
US5891680A (en) * | 1995-02-08 | 1999-04-06 | Whitehead Institute For Biomedical Research | Bioactive fusion proteins comprising the p35 and p40 subunits of IL-12 |
AU712585B2 (en) * | 1995-03-10 | 1999-11-11 | Genentech Inc. | Receptor activation by gas6 |
US5719266A (en) * | 1995-03-17 | 1998-02-17 | Eli Lilly And Company | Anti-obesity proteins |
WO1996031526A1 (en) | 1995-04-06 | 1996-10-10 | Amylin Pharmaceuticals, Inc. | Anti-obesity agents |
US6281010B1 (en) * | 1995-06-05 | 2001-08-28 | The Trustees Of The University Of Pennsylvania | Adenovirus gene therapy vehicle and cell line |
WO1997000317A1 (en) | 1995-06-07 | 1997-01-03 | Osteosa Inc. | Osteoclast growth regulatory factor |
GB9511935D0 (en) | 1995-06-13 | 1995-08-09 | Smithkline Beecham Plc | Novel compound |
NL1000614C2 (en) * | 1995-06-20 | 1996-12-23 | Leer Koninklijke Emballage | Method for manufacturing a laminated product. |
US5736852A (en) * | 1995-06-21 | 1998-04-07 | Alliedsignal Truck Brake Systems Co. | Circuit and method for conditioning a wheel speed sensor signal |
CZ416797A3 (en) * | 1995-06-30 | 1998-06-17 | Eli Lilly And Company | Use of leptin or lepton mimetics for preparing medicaments for treating or prevention of diabetes mellitus and composition containing thereof |
US6406689B1 (en) * | 1995-10-03 | 2002-06-18 | Frank W. Falkenberg | Compositions and methods for treatment of tumors and metastatic diseases |
KR19990066981A (en) | 1995-10-23 | 1999-08-16 | 윌리엄 뉴우 | Therapeutic angiogenesis composition and method |
US5854205A (en) | 1995-10-23 | 1998-12-29 | The Children's Medical Center Corporation | Therapeutic antiangiogenic compositions and methods |
AU1407997A (en) | 1995-12-01 | 1997-06-19 | Beth Israel Hospital | Il-12 p40 subunit fusion polypeptides and uses thereof |
RU2178307C2 (en) | 1995-12-27 | 2002-01-20 | Джинентех Инк. | Derivatives of protein ob |
US6080409A (en) * | 1995-12-28 | 2000-06-27 | Dendreon Corporation | Immunostimulatory method |
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 |
US7063958B1 (en) | 1996-01-16 | 2006-06-20 | The Rockefeller University | Nucleic acids db, the receptor for leptin |
US6750334B1 (en) | 1996-02-02 | 2004-06-15 | Repligen Corporation | CTLA4-immunoglobulin fusion proteins having modified effector functions and uses therefor |
WO1997030089A1 (en) | 1996-02-13 | 1997-08-21 | Regents Of The University Of California | Novel antibody-cytokine fusion protein, and methods of making and using the same |
US5756541A (en) | 1996-03-11 | 1998-05-26 | Qlt Phototherapeutics Inc | Vision through photodynamic therapy of the eye |
AU2527397A (en) | 1996-03-13 | 1997-10-01 | Protein Design Labs, Inc. | Fas ligand fusion proteins and their uses |
CA2249195A1 (en) | 1996-03-18 | 1997-09-25 | Board Of Regents, The University Of Texas System | Immunoglobin-like domains with increased half lives |
WO1997043316A1 (en) | 1996-05-10 | 1997-11-20 | Beth Israel Deaconess Medical Center, Inc. | Physiologically active molecules with extended half-lives and methods of using same |
CA2205757C (en) * | 1996-05-30 | 2006-01-24 | F. Hoffmann-La Roche Ag | Pyridazinone derivatives and their use as inhibitors of prostaglandin g/h synthase i and ii(cox i and ii) |
US5922685A (en) * | 1996-06-05 | 1999-07-13 | Powderject Vaccines, Inc. | IL-12 gene therapy of tumors |
CA2258593A1 (en) | 1996-07-02 | 1998-01-08 | Abraham Nudelman | Retinoyloxy(substituted)alkylene butyrates useful for the treatment of cancer and other proliferative diseases |
WO1998006752A1 (en) | 1996-08-16 | 1998-02-19 | The Rockefeller University | A leptin binding protein and its use in methods for diagnosing and treating abnormalities of the endogenous leptin pathway |
ES2176574T3 (en) * | 1996-09-03 | 2002-12-01 | Gsf Forschungszentrum Umwelt | USE OF BI AND TRIESPECIFIC ANTIBODIES FOR INDUCTION OF TUMOR IMMUNITY. |
US5994104A (en) | 1996-11-08 | 1999-11-30 | Royal Free Hospital School Of Medicine | Interleukin-12 fusion protein |
KR100937550B1 (en) | 1996-12-20 | 2010-01-19 | 암젠 인코포레이티드 | Ob fusion protein compositions and process for producing such protein |
US6737057B1 (en) | 1997-01-07 | 2004-05-18 | The University Of Tennessee Research Corporation | Compounds, compositions and methods for the endocytic presentation of immunosuppressive factors |
US6100387A (en) * | 1997-02-28 | 2000-08-08 | Genetics Institute, Inc. | Chimeric polypeptides containing chemokine domains |
US6277375B1 (en) * | 1997-03-03 | 2001-08-21 | Board Of Regents, The University Of Texas System | Immunoglobulin-like domains with increased half-lives |
AU6954398A (en) * | 1997-04-11 | 1998-11-11 | G.D. Searle & Co. | Antagonistic anti-avb3 integrin antibodies |
ATE223229T1 (en) | 1997-04-17 | 2002-09-15 | Amgen Inc | COMPOSITIONS OF CONJUGATES OF THE STABLE, ACTIVE, HUMAN OB PROTEIN WITH THE FC CHAIN OF IMMUNOGLOBULINS AND RELATED METHODS |
AU8182298A (en) * | 1997-07-10 | 1999-02-08 | Beth Israel Deaconess Medical Center | Recombinant erythropoietin / immunoglobulin fusion proteins |
BR9812267B1 (en) * | 1997-07-14 | 2013-11-26 | Recombinant Growth Hormone. | |
CA2312188C (en) * | 1997-12-08 | 2010-06-29 | Lexigen Pharmaceuticals Corp. | Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation |
US20030105294A1 (en) | 1998-02-25 | 2003-06-05 | Stephen Gillies | Enhancing the circulating half life of antibody-based fusion proteins |
CA2320403A1 (en) | 1998-02-25 | 1999-09-02 | Lexigen Pharmaceuticals Corporation | Enhancing the circulating half-life of antibody-based fusion proteins |
BR9909583A (en) | 1998-04-15 | 2002-01-15 | Lexigen Pharm Corp | Increased immune response mediated by an antibody-cytokine fusion protein by coadministration with angiogenesis inhibitor |
CZ20003817A3 (en) * | 1998-04-17 | 2002-08-14 | Lexigen Pharmaceuticals Corporation | Medicament for inducing cytokine immune response |
US6284536B1 (en) | 1998-04-20 | 2001-09-04 | The Regents Of The University Of California | Modified immunoglobin molecules and methods for use thereof |
AU751823B2 (en) | 1998-05-14 | 2002-08-29 | Merck Patent Gmbh | Fused protein |
DZ2788A1 (en) | 1998-05-15 | 2003-12-01 | Bayer Ag | Selective IL-2 agonists and antagonists. |
US6620382B1 (en) * | 1998-05-22 | 2003-09-16 | Biopheresis Technologies, Llc. | Method and compositions for treatment of cancers |
WO1999062944A2 (en) | 1998-06-03 | 1999-12-09 | The Children's Medical Center Corporation | Protein oligomer compositions comprising endostatin protein and methods of using the same |
US6548653B1 (en) | 1998-06-15 | 2003-04-15 | Genzyme Transgenics Corporation | Erythropoietin analog-human serum albumin fusion |
GB9814383D0 (en) | 1998-07-02 | 1998-09-02 | Cambridge Antibody Tech | Improvements relating to antibodies |
US20020142374A1 (en) | 1998-08-17 | 2002-10-03 | Michael Gallo | Generation of modified molecules with increased serum half-lives |
AU761027B2 (en) * | 1998-08-25 | 2003-05-29 | Merck Patent Gmbh | Expression and export of angiostatin and endostatin as immunofusis |
US6646113B1 (en) | 1998-09-17 | 2003-11-11 | The Trustees Of The University Of Pennsylvania | Nucleic acid molecule encoding human survival of motor neuron-interacting protein 1 (SIP1) deletion mutants |
US6335176B1 (en) * | 1998-10-16 | 2002-01-01 | Pharmacopeia, Inc. | Incorporation of phosphorylation sites |
ES2283139T5 (en) | 1998-10-23 | 2018-03-05 | Amgen Inc. | Methods and compositions for the prevention and treatment of anemia |
BR0007414A (en) * | 1999-01-07 | 2001-10-16 | Lexigen Pharm Corp | Expression and export of anti-obesity proteins as fc fusion proteins |
PT1151102E (en) * | 1999-02-12 | 2006-07-31 | Amgen Inc | GLYCOSILATED LEPTINE COMPOSITIONS AND RELATED METHODS |
KR100773109B1 (en) | 1999-05-06 | 2007-11-02 | 웨이크 포리스트 유니버시티 | Compositions And Methods For Identifying Antigens Which Elicit An Immune Response |
IL145785A0 (en) | 1999-05-07 | 2002-07-25 | Genentech Inc | Novel chimpanzee erythropoietin (chepo) polypeptides and nucleic acids encoding the same |
US6348192B1 (en) * | 1999-05-11 | 2002-02-19 | Bayer Corporation | Interleukin-2 mutein expressed from mammalian cells |
CZ20014123A3 (en) | 1999-05-19 | 2002-06-12 | Lexigen Pharmaceuticals Corp. | Expression and export of alpha interferons as Fc fusion proteins |
WO2000078334A1 (en) | 1999-06-17 | 2000-12-28 | University Of Maryland Biotechnology Institute | Chimeric chemokine-antigen polypeptides and uses therefor |
CZ299516B6 (en) * | 1999-07-02 | 2008-08-20 | F. Hoffmann-La Roche Ag | Erythropoietin glycoprotein conjugate, process for its preparation and use and pharmaceutical composition containing thereof |
JO2291B1 (en) * | 1999-07-02 | 2005-09-12 | اف . هوفمان لاروش ايه جي | Erythopintin derivatives |
SK782002A3 (en) | 1999-07-21 | 2003-08-05 | Lexigen Pharm Corp | FC fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
US7067110B1 (en) * | 1999-07-21 | 2006-06-27 | Emd Lexigen Research Center Corp. | Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens |
US6617135B1 (en) * | 1999-08-09 | 2003-09-09 | Emd Lexigen Research Center Corp. | Multiple cytokine protein complexes |
US20050202538A1 (en) | 1999-11-12 | 2005-09-15 | Merck Patent Gmbh | Fc-erythropoietin fusion protein with improved pharmacokinetics |
DK1252192T3 (en) | 2000-02-11 | 2006-11-20 | Merck Patent Gmbh | Enhancement of the serum half-life of antibody-based fusion proteins |
ATE333900T1 (en) * | 2000-02-24 | 2006-08-15 | Philogen Spa | COMPOSITIONS AND METHODS FOR TREATING ANGIOGENESIS IN PATHOLOGICAL DEFECTS |
US6586398B1 (en) * | 2000-04-07 | 2003-07-01 | Amgen, Inc. | Chemically modified novel erythropoietin stimulating protein compositions and methods |
US6946134B1 (en) * | 2000-04-12 | 2005-09-20 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20020019342A1 (en) * | 2000-05-12 | 2002-02-14 | Robert Bayer | In vitro modification of glycosylation patterns of recombinant glycopeptides |
BR0112111A (en) * | 2000-06-29 | 2003-05-06 | Merck Patent Gmbh | Enhancement of antibody-cytokine fusion protein-mediated immune responses by combined treatment with immunocytocin uptake enhancers |
MXPA03006294A (en) * | 2001-01-18 | 2003-09-16 | Merck Patent Gmbh | Bifunctional fusion proteins with glucocerebrosidase activity. |
EP1366455B1 (en) | 2001-02-19 | 2008-07-02 | MERCK PATENT GmbH | Method for identification of t-cell epitopes and use for preparing molecules with reduced immunogenicity |
MXPA03008031A (en) * | 2001-03-07 | 2003-12-04 | Merck Patent Gmbh | Expression technology for proteins containing a hybrid isotype antibody moiety. |
WO2002079415A2 (en) | 2001-03-30 | 2002-10-10 | Lexigen Pharmaceuticals Corp. | Reducing the immunogenicity of fusion proteins |
DK1383785T3 (en) | 2001-05-03 | 2011-05-23 | Merck Patent Gmbh | Recombinant tumor-specific antibody and its use |
CA2456470A1 (en) | 2001-08-13 | 2003-02-27 | University Of Southern California | Interleukin-2 mutants with reduced toxicity |
AU2002357784B2 (en) | 2001-12-04 | 2008-07-31 | Merck Patent Gmbh | Immunocytokines with modulated selectivity |
CN101143221A (en) | 2002-03-15 | 2008-03-19 | 布赖汉姆妇女医院 | Central airway administration for systemic delivery of therapeutics |
US6996009B2 (en) * | 2002-06-21 | 2006-02-07 | Micron Technology, Inc. | NOR flash memory cell with high storage density |
JP4494977B2 (en) * | 2002-12-17 | 2010-06-30 | 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 ハフツング | Humanized antibody (H14.18) of mouse 14.18 antibody that binds to GD2 and its IL-2 fusion protein |
US20050069521A1 (en) * | 2003-08-28 | 2005-03-31 | Emd Lexigen Research Center Corp. | Enhancing the circulating half-life of interleukin-2 proteins |
BRPI0418286A (en) * | 2003-12-30 | 2007-05-02 | Merck Patent Gmbh | il-7 fusion proteins |
KR20060124656A (en) | 2003-12-31 | 2006-12-05 | 메르크 파텐트 게엠베하 | Fc-erythropoietin fusion protein with improved pharmacokinetics |
EP1706428B1 (en) | 2004-01-22 | 2009-09-23 | MERCK PATENT GmbH | Anti-cancer antibodies with reduced complement fixation |
US7670595B2 (en) | 2004-06-28 | 2010-03-02 | Merck Patent Gmbh | Fc-interferon-beta fusion proteins |
KR20070085886A (en) * | 2004-12-09 | 2007-08-27 | 메르크 파텐트 게엠베하 | Il-7 variants with reduced immunogenicity |
US20070104689A1 (en) * | 2005-09-27 | 2007-05-10 | Merck Patent Gmbh | Compositions and methods for treating tumors presenting survivin antigens |
ATE555125T1 (en) * | 2005-12-30 | 2012-05-15 | Merck Patent Gmbh | INTERLEUKIN 12P40 VARIANTS WITH IMPROVED STABILITY |
ES2382164T3 (en) | 2005-12-30 | 2012-06-05 | Merck Patent Gmbh | Anti-IL-6 antibodies that prevent the binding of IL-6 in complex with IL-6R () to GP130 |
JP2009521912A (en) | 2005-12-30 | 2009-06-11 | 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 ハフツング | Anti-CD19 antibody with reduced immunogenicity |
PL1999154T3 (en) | 2006-03-24 | 2013-03-29 | Merck Patent Gmbh | Engineered heterodimeric protein domains |
EP2038417A2 (en) * | 2006-07-06 | 2009-03-25 | Merck Patent GmbH | Compositions and methods for enhancing the efficacy of il-2 mediated immune responses |
-
2002
- 2002-03-29 WO PCT/US2002/009650 patent/WO2002079415A2/en not_active Application Discontinuation
- 2002-03-29 US US10/112,582 patent/US6992174B2/en not_active Expired - Lifetime
- 2002-03-30 DE DE60238472T patent/DE60238472D1/en not_active Expired - Lifetime
- 2002-03-30 EP EP02757870A patent/EP1373301B1/en not_active Expired - Lifetime
- 2002-03-30 CA CA2442363A patent/CA2442363C/en not_active Expired - Lifetime
- 2002-03-30 AT AT02757870T patent/ATE490262T1/en active
- 2002-03-30 PL PL366826A patent/PL209110B1/en unknown
- 2002-03-30 RU RU2003129523/13A patent/RU2333919C2/en not_active IP Right Cessation
- 2002-03-30 MX MXPA03008715A patent/MXPA03008715A/en active IP Right Grant
- 2002-03-30 ES ES02757870T patent/ES2356620T3/en not_active Expired - Lifetime
- 2002-03-30 CN CNB028075056A patent/CN100488984C/en not_active Expired - Lifetime
- 2002-03-30 DK DK02757870.7T patent/DK1373301T3/en active
- 2002-03-30 AU AU2002306976A patent/AU2002306976B2/en not_active Expired
- 2002-03-30 KR KR10-2003-7012819A patent/KR20030087040A/en not_active Application Discontinuation
- 2002-03-30 JP JP2002577856A patent/JP4638129B2/en not_active Expired - Fee Related
- 2002-03-30 PT PT02757870T patent/PT1373301E/en unknown
- 2002-03-30 WO PCT/US2002/009815 patent/WO2002079232A2/en active Application Filing
- 2002-03-30 HU HU0303753A patent/HUP0303753A2/en unknown
- 2002-03-30 BR BR0208207-1A patent/BR0208207A/en not_active IP Right Cessation
- 2002-03-30 EP EP10010015A patent/EP2311882A1/en not_active Withdrawn
-
2003
- 2003-10-29 ZA ZA200308430A patent/ZA200308430B/en unknown
-
2005
- 2005-09-23 US US11/233,683 patent/US7601814B2/en not_active Expired - Fee Related
-
2009
- 2009-09-01 US US12/552,076 patent/US7973150B2/en not_active Expired - Fee Related
-
2010
- 2010-07-02 JP JP2010151743A patent/JP5291671B2/en not_active Expired - Fee Related
-
2011
- 2011-05-26 US US13/116,830 patent/US8926973B2/en not_active Expired - Fee Related
-
2015
- 2015-01-02 US US14/588,702 patent/US20150141626A1/en not_active Abandoned
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992010755A1 (en) | 1990-12-05 | 1992-06-25 | Novo Nordisk A/S | Proteins with changed epitopes and methods for the production thereof |
EP0519596A1 (en) | 1991-05-17 | 1992-12-23 | Merck & Co. Inc. | A method for reducing the immunogenicity of antibody variable domains |
EP0699755A2 (en) | 1994-06-30 | 1996-03-06 | Centro de Inmunologia Molecular | Method for obtaining modified immunoglobulins with reduced immunogenicity of murine antibody variable domains, compositions containing them |
WO1996040792A1 (en) | 1995-06-07 | 1996-12-19 | Novo Nordisk A/S | Modification of polypeptides |
WO1998052976A1 (en) | 1997-05-21 | 1998-11-26 | Biovation Limited | Method for the production of non-immunogenic proteins |
WO1998059244A1 (en) | 1997-06-20 | 1998-12-30 | Eclagen Limited | Identification of mhc binding peptides |
WO2000034317A2 (en) | 1998-12-08 | 2000-06-15 | Biovation Limited | Method for reducing immunogenicity of proteins |
WO2001036489A2 (en) | 1999-11-12 | 2001-05-25 | Merck Patent Gmbh | Erythropoietin forms with improved properties |
WO2002066514A2 (en) | 2001-02-19 | 2002-08-29 | Merck Patent Gmbh | Artificial fusion proteins with reduced immunogenicity |
Non-Patent Citations (1)
Title |
---|
LO ET AL., PROTEIN ENGINEERING, vol. 11, 1998, pages 495 - 500 |
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