Detailed Description
The present invention relates to antibodies, peptides and aptamers that antagonize extracellular PCSK9 function, including the interaction of PCSK9 with LDLR. More particularly, the invention relates to methods of making antagonist PCSK9 antibodies, peptides and aptamers, compositions comprising the antibodies, peptides and/or aptamers, and methods of using the antibodies, peptides and/or aptamers as pharmaceuticals. The antagonist PCSK9 antibodies and peptides can be used to lower LDL-cholesterol levels in the blood and can be used to prevent and/or treat abnormal cholesterol and lipoprotein metabolism, including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis and more generally CVD.
General techniques
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are fully described in the literature, such as molecular cloning: ALaboratoryManual, secondedition (Sambrook et al, 1989), ColdSpringHarbor Press, OligonuclotideYnesis (M.J.Gate, ed.,1984), MethodsinMolecular biology, HumanaPress; cell biology: ALaboratoryNotebook (J.E.cell, ed.,1998) Acidenecpress, animal cell culture (R.I.Freene, ed.,1987), inner cell culture (J.P.origin, Rober. E.Rober, 1998) Plumbum Press, cell culture, 1987, molecular culture, et al, molecular culture, filtration, 1987, molecular culture, et al (C.7, molecular culture, 1987, moisture, 1987), molecular culture, et al, filtration, 1987, et al, filtration, 1987, protein, plant, 19835, moisture, et al, plant culture, plant, 1987, plant, research, et al, plant, research, plant, research, 1987, research, plant, research, 2.
Definition of
An "antibody" is an immunoglobulin molecule that specifically binds a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The term as used herein encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab ', F (ab')2Fv, single chain (ScFv), and domain antibodies), and fusion proteins comprising an antibody site, and any other immunoglobulin molecule containing a modified configuration of an antigen recognition site. Antibodies include any type of antibody, such as IgG, IgA, or IgM (or subtypes thereof), and the antibodies need not be of any particular type. Ammonia from the heavy chain constant domain of an antibodyThere are five major immunoglobulin classes, IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, the heavy chain constant domains corresponding to the different classes of immunoglobulins are known as α, gamma and mu, respectively.
As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies comprise a consistent population except for the possible naturally occurring mutations that are present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, unlike polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the present invention can be prepared by the hybridoma method described at the earliest by Kohlerand Milstein,1975, Nature256:495, or can be prepared by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated, for example, using the techniques described in McCaffertytal, 1990, Nature348: 552-.
"humanized" antibody, as used herein, refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab ', F (ab')2Or other antigen binding subsequence of an antibody). Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a recipient-derived Complementarity Determining Region (CDR) are replaced with residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some examples, the Fv framework of a human immunoglobulinThe region (FR) residues are substituted with the corresponding non-human residues. In addition, the humanized antibody may contain residues not found in the recipient antibody or introduced into the CDR or framework sequences which are included to further improve and optimize the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the corresponding CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also desirably comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Preferred are antibodies having a modified Fc region as described in WO 99/58572. Other forms of humanized antibodies have one or more CDR classes (CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3) that are altered relative to the original antibody, also referred to as one or more CDR classes "derived" from one or more CDR classes from the original antibody.
As used herein, "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody which can be produced by a human and/or prepared using any of the techniques for making human antibodies known to those skilled in the art or disclosed herein. A human antibody as defined herein includes an antibody comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be made using a variety of techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library represents a human antibody (Vaughanetal, 1996, Nature Biotechnology,14: 309-. Human antibodies can also be prepared by immunizing an animal in which the endogenous locus (loci) has been replaced by a human immunoglobulin locus introduced by gene transfer, e.g., a mouse in which the endogenous immunoglobulin gene has been partially or completely inactivated. Such methods are described in U.S. Pat. nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016. Alternatively, the human antibody can be prepared by immortalizing human B lymphocytes that produce antibodies to the target antigen (the B lymphocytes can be collected from the subject or have been immunized in vitro). See, for example, Coleatal. Monoclonal antibodies and C.ancer therapy, AlanR. Liss, p.77,1985, Boerneretal, 1991, J.Immunol.,147(1), 86-95 and U.S. Pat. No. 5,750,373.
The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs) containing the hypervariable region. The CDRs in each chain are drawn tightly together by the FRs and with the CDRs from the other chain result in the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) a manner based on cross-species sequence variability (i.e., Kabat et al, sequence of proteins of immunologicalcalemtest, (5)thed.,1991, national institutes of health, BethesdaMD)); and (2) a mode based on a crystallographic test of an antigen-antibody complex (Al-lazikanetal, 1997, J.Molec.biol.273: 927-948). As used herein, a CDR may refer to a CDR defined in either way or a combination of both ways.
By "constant region" of an antibody, as used in the art, is meant the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination.
The term "PCSK 9" as used herein refers to any form of PCSK9 and variants thereof that retain at least a portion of PCSK9 activity. Unless otherwise indicated such as specifically for human PCSK9, PCSK9 includes the native sequence PCSK9 of all mammalian species, e.g., human, canine, feline, equine, and bovine. An exemplary human PCSK9 is found in Uniprot accession No. Q8NBP 7.
As used herein, a "PCSK 9 antagonist" refers to an antibody, peptide or aptamer that inhibits PCSK9 biological activity and/or downstream pathways mediated by PCSK9 signaling, including PCSK 9-mediated down-regulation of LDLR and PCSK 9-mediated reduction of LDL blood clearance. PCSK9 antagonist antibodies include antibodies that block, antagonize, inhibit, or reduce (to any extent including significantly) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as LDLR interactions and/or induce cellular responses to PCSK 9. For the purposes of the present invention, it is to be expressly understood that the term "PCSK 9 antagonist antibody" encompasses all terms, titles and functional states and features previously identified whereby PCSK9 itself, PCSK9 biological activity (including but not limited to its ability to mediate interaction with LDLR, down-regulate LDLR, and any aspect of reduced blood LDL clearance) or the outcome of such biological activity is substantially abrogated, reduced, or neutralized to any meaningful degree. In some embodiments, the PCSK9 antagonist antibody binds to PCSK9 and prevents interaction with the LDLR. The present invention provides examples of PCSK9 antagonist antibodies.
As used herein, "complete antagonist" refers to an antagonist that substantially completely blocks the measurable effect of PCSK9 at an effective concentration. A partial antagonist is an antagonist that partially blocks a measurable effect, but is not a complete antagonist even at the highest concentration. By substantially completely is meant that at least about 80%, preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% or 99% of the measurable effect is blocked. The relevant "measurable effects" are LDLR down-regulation, in vivo reduction of total cholesterol levels in blood (or in plasma), and in vivo reduction of LDL levels in blood (or in plasma) of PCSK9 antagonists as described herein and including as determined in vitro in Huh7 cells.
The term "clinically significant" as used herein means that LDL-cholesterol levels in human blood are reduced by at least 15% or that total murine blood cholesterol is reduced by at least 15%. It is clear that plasma or serum measurements can be used as a surrogate for blood volume measurements.
The term "PCSK 9 antagonist peptide" or "PCSK 9 antagonist aptamer" as used herein includes any common peptide or polypeptide or aptamer that blocks, antagonizes, inhibits or reduces (to any extent including significantly) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as LDLR interactions and/or elicitation of cellular responses to PCSK 9. PCSK9 antagonist peptides or polypeptides include Fc fusions comprising LDLR and soluble portions of said LDLR, or mutations thereof having higher affinity for PCSK 9.
The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to any chain of amino acids, preferably of any length, which is relatively short (e.g., 10 to 100 amino acids). The chain may be linear or branched, it may comprise modified amino acids and/or may be interrupted by non-amino acids. The term also encompasses amino acid chains that have been modified by natural or human intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling element. The definition also includes, for example, polypeptides that include one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications well known in the art. It is understood that the polypeptide may exist as a single chain or as a bound chain.
"Polynucleotide" or "nucleic acid" as are well known in the art are used interchangeably herein to refer to a strand of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the strand by DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if any, may be made before or after the strand is combined. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as conjugation with a labeling component. Other types of modifications include, for example, "cap end (cap)", substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those having uncharged linkages (links) (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), and charged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)Phosphorothioate, phosphorodithioate, etc.), those containing a pendant group such as, for example, a protein (e.g., nuclease, toxin, antibody, signal peptide, polylysine, etc.), those with an intercalator (e.g., acridine, psoralen, etc.), those with a chelator (e.g., metal, radioactive metal, boron, oxidative metal, etc.), those containing an alkylating agent, those with a modified linker (e.g., α trans-nucleic acid, etc.), and unmodified polynucleotide forms, additionally, any hydroxyl group typically present in a saccharide may be substituted with, for example, a phosphonate, a phosphate, a standard protecting group or activated to make other linkages to additional nucleotides, or may be conjugated to a solid support, the 5 'and 3' OH groups may be phosphorylated or substituted with amines or organic end-capping groups from 1 to 20 carbon atoms, other hydroxyl groups may also be derivatized as standard protecting groups, polynucleotides may also contain similar forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2 '-O-methyl-ribose, 2' -O-allyl-2 '-ribose, 2' -azaribose, or phosphorothioated ribose, phosphorosomes, phosphorothioated ribose, optionally substituted with, phosphorothioated ribose, phosphorothioates, phosphodiester, optionally substituted with, phosphorothioated riboside, phosphorothioated ribose, phosphorothioated riboside analogs, or non-substituted with a non-riboside analogs, S-riboside ribo2("amidates"), P (O) R, P (O) OR', CO OR CH2("formacetal") wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1 to 20 carbons) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. All linkages in a polynucleotide need not be identical. The foregoing applies to all polynucleotides referred to herein, including RNA and DNA.
"PCSK 9 antagonist aptamers" comprising a nucleic acid or protein sequence are selected from, for example, a large number of random sequences and specifically bind to PCSK 9. The nucleic acid of the aptamer is double-stranded DNA or single-stranded RNA. Nucleic acid aptamers may include modified bases or functional groups including, but not limited to, 2 '-fluoro nucleotides and 2' -O-methyl nucleotides. Aptamers may include hydrophilic polymers, such as polyethylene glycol. Aptamers can be made by methods known in the art and PCSK9 antagonist activity can be selected by routine modification of the methods disclosed in the examples.
As used herein, an antibody, peptide or aptamer "interacts" with PCSK9 when the equilibrium dissociation constant, as measured by the method disclosed herein in example 2, is equal to or less than 20nM, preferably less than about 6nM, more preferably less than about 1nM, and most preferably less than about 0.2 nM.
The terms "preferential binding" or "specific binding" (used interchangeably herein) of an epitope to an antibody or polypeptide are well known in the art, as are methods for determining such specific or preferential binding. A molecule is said to exhibit "specific binding" or "preferential binding" if it interacts or binds more frequently, more rapidly, for a longer period of action, and/or with a higher affinity (affinity) with a particular cell or substance than it does with other cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with a higher affinity, avidity, more rapidly, and/or for a longer period of time than it binds to other substances. For example, an antibody that specifically or preferentially binds to a PCSK9 epitope refers to an antibody that binds to this epitope with greater affinity, avidity, more rapidly, and/or for a longer period of time than it binds to other PCSK9 epitopes or non-PCSK 9 epitopes. It will also be appreciated from reading this definition that, for example, an antibody (or group or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, the term "binding" refers to preferential binding.
As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.
"host cell" includes individual cells or cell cultures that may be or have been the recipient of a vector for introducing a polynucleotide insert. Host cells include progeny of a single host cell, and such progeny are not necessarily identical (in morphology or genetic DNA complementarity) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.
The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, as known in the art. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of immunoglobulin heavy chains may differ, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to the carboxy terminus of each other. The numbering of residues in the Fc region is the EU index as in Kabat. Kabat et al, sequence of proteins of immunologica interest,5thPublished healthcare service, national institutes of health, Bethesda, Md.,1991 the Fc region of an immunoglobulin typically comprises two constant domains, CH2 and CH 3.
"Fc receptor" and "FcR" as used in the art describe receptors that bind to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is a receptor that binds IgG antibodies (gamma receptor) and includes Fc γ RI, Fc γ RII, and Fc γ RIII subtype receptors, including paired variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetchand Kinet,1991, Ann.Rev.Immunol.,9:457-92, Capeletal, 1994, Immunomethods,4:25-34 and deHaasetal, 1995, J.Lab.Clin.Med.,126: 330-41. "FcR" also includes the neonatal receptor FcRn, which is responsible for transporting maternal IgG to the fetus (Guyeret al, 1976, J.Immunol.,117:587; and Kimetal, 1994, J.Immunol.,24: 249).
The term "competes" as used herein with respect to an antibody means that a first antibody or antigen-binding site thereof binds to an epitope in a manner sufficiently similar to the binding of a second antibody or antigen-binding site thereof such that the result of binding of the first antibody to its cognate epitope in the presence of the second antibody is detectably reduced as compared to the binding of the first antibody in the absence of the second antibody. Alternatively, it may be, but need not be, that the binding of the second antibody to its epitope is also detectably reduced when the first antibody is present. That is, a first antibody may inhibit the binding of a second antibody to its epitope, but a second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits, whether to the same, a higher or lower degree, the binding of another antibody to its cognate epitope or ligand, the antibodies are said to "cross-compete" with each other for binding to their respective epitope. The invention encompasses competitive and cross-competitive antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, morphological changes, or binding to a common epitope or portion thereof), one of skill in the art will appreciate that such competing and/or cross-competing antibodies comprise and may be used in the methods disclosed herein in accordance with the teachings provided herein.
Antibodies have an epitope that "overlaps" with other (second) epitopes or with the surface of PCSK9 that interacts with the EGF-like domain of LDLR, meaning that they share space with respect to the interacting PCSK9 residues. To calculate the percentage of overlap, for example, the percentage of overlap of the PCSK9 epitope of the claimed antibody and the PCSK9 surface interacting with the EGF-like domain of LDLR, i.e. the surface area of the PCSK9 that is embedded when forming a complex with LDLR, is calculated on a per residue basis. The buried area was also calculated from these residues in the PCSK9: antibody complex. To avoid more than 100% possible overlap, the residue surface area in the PCSK9: antibody complex with higher buried surface area compared to the LDLR: PCSK9 complex was set at the value starting from the LDLR: PCSK9 complex (100%). The area overlap percentage was calculated by summing all the LDLR: PCSK9 interacting residues and weighted by the area of interaction.
A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody dependent cell mediated cytotoxicity, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art for assessing such antibody effector functions.
A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of a naturally found Fc region. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution, e.g., from about 1 to about 10 amino acid substitutions, as compared to the native sequence Fc region or the Fc region of the parent polypeptide, and preferably from about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein will preferably have at least about 80% sequence identity with the native sequence Fc region and/or with the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity therewith, and more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.
As used herein, "treatment" refers to a method of achieving a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: increasing LDL clearance and reducing the incidence of or improving aberrant cholesterol and/or lipoprotein levels caused by metabolic and/or eating disorders or including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis and more generally cardiovascular disease (CVD).
By "reduced incidence" is meant any reduction in severity (which may include reducing the need and/or amount (e.g., exposure) to other drugs and/or treatments commonly used to treat the condition). As will be appreciated by those skilled in the art, individuals may differ with respect to their response to treatment, and thus, for example, "methods of reducing incidence" indicate that administration of PCSK9 antagonist antibodies, peptides or aptamers may result in a reduction in the incidence in that particular individual, as reasonably expected.
By "improving" is meant reducing or ameliorating one or more symptoms as compared to when a PCSK9 antagonist antibody, peptide, or aptamer is not administered. "improving" also includes shortening or reducing the duration of symptoms.
As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition refers to an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in the onset of the disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological manifestations manifested during the course of disease progression. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms of hypercholesterolemia or dyslipidemia, atherosclerosis, CVD or coronary heart disease, reducing the dosage of other drugs required to treat the disease, enhancing the effects of other drugs, and/or delaying disease progression in a patient. An effective dose may be administered in one or more divided doses. For the purposes of the present invention, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. As understood in a clinical sense, an effective dose of a drug, compound or pharmaceutical composition may be achieved with or without combination with another drug, compound or pharmaceutical composition. Thus, an "effective dose" may be considered where one or more therapeutic agents are administered, and a single agent may be considered to be administered in an effective amount if combined with one or more other agents that may or may not achieve the desired result.
An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.
As used herein, "vector" refers to a construct that is capable of being delivered in a host cell and preferably expresses one or more genes or sequences of interest. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plastids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and specific eukaryotic cells such as producer cells.
As used herein, "expression control sequence" refers to a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence may be a promoter, such as a persistent or inducible promoter or promoter. Expression control sequences are operably linked to the nucleic acid sequence to be transcribed.
As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance that, when combined with an active ingredient, enables the ingredient to retain biological activity and not react with the immune system of the subject. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. Preferred diluents for spray or parenteral administration are Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington's pharmaceutical sciences, 18)thedition,A.Gennaro,ed.,MackPublishingCo.,Easton,PA,1990;andRemington,TheScienceandPracticeofPharmacy,20thEd.,MackPublishing,2000)。
The term "K" as used hereinon"refers to the binding rate constant of an antibody to an antigen. In particular, the first and second (c) substrates,rate constant (K)onAnd Koff) The equilibrium dissociation constant was measured using Fab antibody fragments (i.e., monovalent) and PCSK 9.
The term "K" as used hereinoff"refers to the rate constant at which an antibody dissociates from an antibody/antigen complex.
The term "K" as used hereinD"refers to the equilibrium dissociation constant of an antibody-antigen interaction.
A. Methods for preventing or treating disorders associated with hypercholesterolemia
In one aspect, the present invention provides a method for treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease in an individual, the method comprising administering to the individual an effective amount of a PCSK9 antagonist antibody or peptide or aptamer that antagonizes circulating PCSK 9.
In another aspect, the present invention provides an effective amount of a PCSK9 antagonist antibody, peptide or aptamer that antagonizes circulating PCSK9 for use in treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD, or coronary heart disease in an individual. The invention further provides the use of an effective amount of a PCSK9 antagonist antibody, peptide or aptamer that antagonizes extracellular or circulating PCSK9 in the manufacture of a medicament for treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease in an individual.
Advantageously, therapeutic administration of the antibody, peptide or aptamer results in lower cholesterol in the blood and/or lower LDL in the blood. Preferably, the cholesterol and/or LDL in the blood is reduced by at least about 10% or 15% compared to prior to administration. More preferably, the cholesterol and/or LDL in the blood is reduced by at least about 20% compared to before administration of the antibody. Even more preferably, the cholesterol and/or LDL in the blood is reduced by at least 30% compared to before administration of the antibody. Advantageously, the cholesterol and/or LDL in the blood is reduced by at least 40% compared to before administration of the antibody. More advantageously, the cholesterol and/or LDL in the blood is reduced by at least 50% compared to before administration of the antibody. Very preferably, the cholesterol and/or LDL in the blood is reduced by at least 60% compared to before administration of the antibody. Most preferably, the cholesterol and/or LDL in the blood is reduced by at least 70% compared to before administration of the antibody.
For all methods described herein, reference to PCSK9 antagonist antibodies, peptides, and aptamers also includes compositions comprising one or more additional agents. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers well known in the art. The present invention may be used alone or in combination with other conventional methods of treatment.
The PCSK9 antagonist antibody, peptide, or aptamer may be administered to an individual via any suitable route. It will be apparent to those skilled in the art that the examples described herein are not intended to be limiting but rather to illustrate the technology that is available. Thus, in some embodiments, the PCSK9 antagonist antibody, peptide, or aptamer is administered to an individual according to known methods, such as intravenous administration, e.g., bolus injection or continuous infusion over a period of time, intramuscular, intraperitoneal, intracerobrospinal, transdermal, subcutaneous, intra-articular, sublingual, intrasynovial, insufflating, intrathecal, oral, inhalation, or topical routes. Administration can be systemic, e.g., intravenous, or topical. Commercially available nebulizers for liquid preparations including jet nebulizers and ultrasonic nebulizers can be used for administration. The liquid formulation may be applied by direct spray, and the freeze-dried powder may be applied by spray after reconstitution with water. Alternatively, PCSK9 antagonist antibodies, peptides or aptamers can be nebulized using fluorocarbon formulations and metered dose spray inhalers or inhaled as a freeze-dried and ground powder.
In one embodiment, the PCSK9 antagonist antibody, peptide, or aptamer is administered via site-specific or targeted local delivery techniques. Examples of location-specific or targeted local delivery techniques include implantable storage sources or local delivery catheters of various PCSK9 antagonist antibodies, peptides, or aptamers, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial coatings, shunts and stents or other implantable devices, location-specific carriers, direct injection, or direct administration. See, e.g., PCT publication No. WO00/53211 and U.S. Pat. No. 5,981,568.
Various formulations of PCSK9 antagonist antibodies, peptides, or aptamers may be used for administration. In some embodiments, the PCSK9 antagonist antibody, peptide, or aptamer may be administered directly. In some embodiments, the PCSK9 antagonist antibody, peptide, or aptamer can be formulated with pharmaceutically acceptable excipients into various formulations. Pharmaceutically acceptable excipients are relatively inert substances which are known in the art and which aid in the administration of pharmacologically effective substances. For example, excipients may impart a shape or consistency, or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for altering permeability, encapsulating agents, buffering agents, and skin penetration enhancers. Excipients and formulations for parenteral and enteral drug delivery are set forth in Remington, the science and practice of pharmacy,20thEd.,MackPublishing(2000)。
These agents may be combined with pharmaceutically acceptable carriers such as saline, ringer's solution, dextrose solution, and the like. The particular regimen, i.e., dosage, time, and reproducibility will depend upon the particular subject and the medical history of that subject.
The PCSK9 antibody may also be administered by inhalation, as described herein. In general, the initial candidate dose may be about 2mg/kg when the PCSK9 antibody is administered. For the purposes of the present invention, typical daily dosages may range anywhere from about 3 μ g/kg to 30 μ g/kg to 300 μ g/kg to 3mg/kg, to 30mg/kg to 100mg/kg or higher, depending on the factors mentioned above. For example, dosages of about 1mg/kg, about 2.5mg/kg, about 5mg/kg, about 10mg/kg and about 25mg/kg may be used. The treatment is continued for several days or more depending on the condition, until desired suppression of symptoms occurs or until a sufficient therapeutic amount is achieved, e.g., a reduction in the amount of LDL in the blood. An exemplary dosing regimen comprises administration of an initial dose of about 2mg/kg of the PCSK9 antibody, followed by a weekly maintenance dose of about 1mg/kg, or followed by a maintenance dose of about 1mg/kg every two weeks. However, other dosing regimens may be used depending on the pharmacokinetic decay pattern desired by the physician. For example, in some embodiments, one to four times per week administration is contemplated. In other embodiments, once per month administration or once every two months or once every three months may be contemplated. The progress of this treatment can be readily monitored by conventional techniques and assays. The dosing regimen, including the PCSK9 antagonist used, may vary over time.
For purposes of the present invention, the appropriate dosage of a PCSK9 antagonist antibody, peptide, or aptamer will depend on the PCSK9 antagonist antibody, peptide, or aptamer (or combination thereof) employed, the type and severity of the condition being treated, the agent being administered for prophylactic or therapeutic purposes, prior treatments, the patient's clinical history and response to the agent, the amount of PCSK9 in the patient's blood, the rate at which the patient synthesizes and clears PCSK9, the rate at which the patient clears the administered agent, and the discretion of the attending physician. Typically, a physician will administer a PCSK9 antagonist antibody, peptide, or aptamer until a dose is reached that will achieve the desired result. The dosage and/or frequency may vary depending on the course of treatment. Empirical considerations such as half-life will generally influence the decision of the dose. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted according to the course of treatment, typically but not necessarily according to the treatment and/or inhibition and/or amelioration and/or delay of symptoms, such as hypercholesterolemia. Alternatively, a sustained continuous release formulation of a PCSK9 antagonist antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.
In one embodiment, the dosage of the antagonist antibody, peptide or aptamer may be determined empirically in an individual to whom one or more administrations of the antagonist antibody, peptide or aptamer have been administered. The individual lines are administered incremental doses of PCSK9 antagonist antibodies, peptides, or aptamers. To assess efficacy, disease indices can be followed.
Administration of PCSK9 antagonist antibodies, peptides, or aptamers according to the methods of the invention may be continuous or intermittent depending, for example, on the physiological condition of the recipient, the intended therapeutic or prophylactic administration, and other factors known to those skilled in the art. The PCSK9 antagonist antibody, peptide, or aptamer may be administered substantially continuously for a predetermined period of time or in a series of spaced doses.
In some embodiments, more than one antagonist antibody, peptide, or aptamer may be present. At least one, at least two, at least three, at least four, at least five different, or more antagonist antibodies and/or peptides may be present. In general, these PCSK9 antagonist antibodies or peptides may have complementary activities that do not adversely affect each other. PCSK9 antagonist antibodies, peptides or aptamers may also be used in combination with other PCSK9 antagonists or PCSK9 receptor antagonists. For example, one or more of the following PCSK9 antagonists may be used: antisense molecules targeting PCSK9 (including antisense molecules targeting PCSK 9-encoding nucleic acids), PCSK9 inhibitory compounds, and PCSK9 structural analogs. PCSK9 antagonist antibodies, peptides, or aptamers may also be used in combination with other agents that are used to enhance and/or supplement the effectiveness of these agents.
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants include ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol alcohols, butanol or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysineAmino acids, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counter ions such as sodium, metal complexes (e.g. zinc protein complexes) and/or non-ionic surfactants such as TWEENTM、PLURONICSTMOr polyethylene glycol (PEG).
Liposomes containing PCSK9 antagonist antibodies, peptides or aptamers are prepared by methods known in the art, such as described in Epstein, et, 1985, proc.natl.acad.sci.usa82:3688, Hwang, et, 1980, proc.natl.acad.sci.usa77:4030 and U.S. patent nos. 4,485,045 and 4,544,545. Liposomes that enhance circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation methods with lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a screen of defined pore size to produce liposomes of the desired diameter.
The active ingredient may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate-microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington, the science and practice of pharmacy,20thEd.,MackPublishing(2000)。
Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid-phase hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or polyvinyl alcohol), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOTTM(from lactic acid-glycolic acid copolymerAnd leuprolide (leuprolide) injection microsphere, sucrose acetate isobutyrate and poly-D- (-) -3-hydroxybutyric acid.
Formulations to be used for in vivo administration must be sterile. This can be easily achieved by filtration, for example, through sterile filtration membranes. The therapeutic PCSK9 antagonist antibody, peptide, or aptamer composition is typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM、LiposynTM、InfonutrolTM、LipofundinTMAnd LipiphysanTM. The active ingredient may be dissolved in a pre-mixed emulsion composition or it may be dissolved in an oil (e.g., soybean oil, safflower seed oil, cottonseed oil, sesame oil, corn oil or almond oil) to form an emulsion when mixed with a phospholipid (e.g., lecithin, soybean phospholipid or soybean lecithin) and water. It will be appreciated that other ingredients such as glycerol or glucose may be added to adjust the tonicity of the emulsion. Suitable emulsions will generally contain up to 20%, for example between 5 and 20% oil. The fat emulsion may comprise fat droplets of between 0.1 and 1.0 μm, in particular 0.1 to 0.5 μm, and have a pH in the range of between 5.5 and 8.0.
The emulsion composition may be prepared by mixing a PCSK9 antagonist antibody, peptide or aptamer with an IntralipidTMOr its ingredients (soybean oil, lecithin, glycerin and water).
Compositions for inhalation or insufflation include solutions and suspensions and powders in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof. The fluid or solid composition may contain suitable pharmaceutically acceptable excipients as described previously. In some embodiments, the composition is administered in the oral or nasal respiratory route for local or systemic effects. The composition in the preferred sterile pharmaceutically acceptable solvent can be nebulized by gas. The nebulized solution can be inhaled directly from the nebulizing device or the nebulizing device can be connected to a face mask, tent, or intermittent positive pressure respirator. The solution, suspension or powder composition may be administered, preferably orally or nasally, from a device that delivers the formulation in a suitable manner.
PCSK9 antagonists
The methods of the invention use PCSK9 antagonist antibodies, peptides or aptamers that refer to any peptide or nucleic acid molecule that blocks, inhibits or reduces (including significantly reduces) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as inducing a cellular response to PCSK 9.
The PCSK9 antagonist antibody, peptide, or aptamer should have any one or more of the following characteristics: (a) binding to PCSK9, (b) blocking PCSK9 interaction with LDLR, (c) blocking or reducing PCSK 9-mediated down-regulation of LDLR, (d) inhibiting PCSK 9-mediated reduction in LDL blood clearance, (e) increasing LDL clearance by cultured hepatocytes in culture, (f) increasing blood LDL clearance by the liver in vivo, (g) sensitizing to statins, and (h) blocking PCSK9 interaction with other factors still to be recognized.
For the purposes of the present invention, the antibody, peptide or aptamer preferably reacts with PCSK9 in a manner that inhibits PCSK9 signaling function and LDLR interaction. In some embodiments, the PCSK9 antagonist antibody specifically recognizes PCSK9 of a primate. In some embodiments, the PCSK9 antagonist antibody binds to PCSK9 in primates and rodents.
Antibodies used in the invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab ', F (ab')2Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, single chain (ScFv), and mutations thereof, fusion proteins comprising an antibody site (e.g., domain antibodies), human antibodies, humanized antibodies, and any other immunoglobulin molecule containing a modified configuration of an antigen recognition site of a desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may beMouse, rat, human, or any other source (including chimeric or humanized antibodies).
In some embodiments, the PCSK9 antagonist antibody is a monoclonal antibody. The PCSK9 antagonist antibody can also be a humanized antibody. In other embodiments, the antibody is a human antibody.
In some embodiments, the antibody comprises a modified constant region, such as an immunologically inert constant region, i.e., a reduced ability to elicit an immune response. In some embodiments, the constant region is as defined in eur.j.immunol.,1999,29: 2613-2624; modifications described in PCT publication No. WO99/58572 and/or British patent application No. 9809951.8. The Fc may be human IgG2Or human IgG4. The Fc may be a Fc comprising the mutations A330P331 to S330S331 (IgG)2Δa) Human IgG of (1)2Wherein the amino acid residues are numbered with reference to wild-type IgG2 sequence. Eur.J.Immunol.,1999,29: 2613-2624. In some embodiments, the antibody comprises IgG4The constant region of (a), which contains the following mutations (aromatic, 2003, molecular immunology 40585-593): E233F234L235 to P233V234A235 (IgG)4Δc) Wherein the numbering is with reference to wild-type IgG 4. In another embodiment, the Fc is human IgG4E233F234L235 to P233V234A235 and deletion G236 (IgG)4Δb). In another embodiment, the Fc is any human IgG comprising the hinge stabilizing mutations S228 to P2284Fc(IgG4、IgG4ΔbOr IgG4Δc) (Aalberseetal, 2002, Immunology105, 9-19). In another embodiment, the Fc may be a non-glycosylated Fc.
In some embodiments, the constant region is non-glycosylated by mutating the oligosaccharide linking residue (such as Asn297) and/or by flanking the residue in the constant region that is part of the glycosylation recognition sequence. In some embodiments, the N-linked glycosylation of the constant region is enzymatically non-glycosylated. The N-linked glycosylation of the constant region can be non-glycosylated either enzymatically or by expression in a glycosylation deficient host cell.
The binding affinity (KD) of a PCSK9 antagonist antibody to PCSK9 (such as human PCSK9) may be about 0.002 to about 200 nM. In some embodiments, the binding affinity is any of about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, about 60pM, about 50pM, about 20pM, about 15pM, about 10pM, about 5pM, or about 2 pM. In some embodiments, the binding affinity is any of less than about 250nM, about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, about 50pM, about 20pM, about 10pM, about 5pM, or about 2 pM.
One method of determining the binding affinity of an antibody to PCSK9 is to measure the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, the antibody (e.g., IgG) can be cleaved by papain or expressed recombinantly. The affinity of PCSK9Fab fragment of antibody can be determined by surface plasmon resonance (Biacore 3000) equipped with a pre-immobilized streptavidin-sensing chip (SA)TMSurface Plasmon Resonance (SPR) system, Piscataway Biacore, INC, N.J.) was determined using HBS-EP electrophoresis buffer (0.01MHEPES, pH7.4, 0.15NaCl, 3mM EDTA, 0.005% v/v surfactant P20). Biotinylated human PCSK9 (or any other PCSK9) can be diluted in HBS-EP buffer to a concentration of less than 0.5 μ g/ml and injected through individual chip channels using different contact times to achieve either a 50 to 200 Reaction Units (RU) for detailed kinetic experiments or two antigen density ranges of 800 to 1000RU for screening assays. Regeneration experiments have shown that 25% v/v ethanol containing 25mMNaOH effectively removes the bound Fab while maintaining PCSK9 activity on the chip for more than 200 injections. In general, serial dilutions (0.1 to 10 fold predicted K) of purified Fab samplesDCross concentration) was injected at 100 μ l/min for 1 minute and allowed for dissociation times of up to 2 hours. The concentration of the Fab protein is determined by ELISA and/or SDS-PAGE electrophoresis using known concentrations (determined by amino acid analysis) of Fab as a standard. Data were put in their entirety into the 1:1 Lanmuir (Langmuir) binding model (Karlsson, R.Roos, H.Fagerstam, L.Petersson, B.1994. methods enzymology6.99-110) using BIAevaluation software to simultaneously obtain kinetic binding ratesRate (k)on) And dissociation rate (k)off). Equilibrium dissociation constant (K)D) The value of koff/konThis procedure is suitable for determining the binding affinity of an antibody to any PCSK9, including human PCSK9, other mammalian PCSK9 (such as mouse PCSK9, rat PCSK9, primate PCSK9), and various forms of PCSK9 (such as forms α and β).
PCSK9 antagonist antibodies can be prepared by any method known in the art, including the methods provided in example 1. The route and course of immunization of a host animal is generally consistent with established and conventional antibody stimulation and production techniques, as further described herein. General techniques for generating human and mouse antibodies are known in the art and/or described herein. Presently preferred methods of making antibodies comprise immunizing PCSK 9-gene knockout (PCSK9-/-) animals as disclosed herein.
It is contemplated that any mammalian subject, including humans, or antibody producing cells derived from such animals, can be manipulated to serve as a basis for the generation of mammalian (including human) hybridoma cell lines. Generally, the host cell is inoculated with an amount of immunogen, including as described herein, intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally.
Hybridomas can be prepared from lymphocytes and immortal myeloma cells using conventional somatic cell hybridization techniques of Kohler, B.andMilstein, C.,1975, Nature256:495-497 or modified by Buck, D.W., et., 1982, InVitro,18: 377-381. Available myeloma cells include, but are not limited to, X63-Ag8.653 and those from the cell distribution center of the san Diego Saker, Calif. (SalkInstituteCell distribution center) can be used for hybridization. In general, the technique involves fusing myeloma cells with lymphoid cells using a fusion promoting agent such as polyethylene glycol or by electrical methods well known to those skilled in the art. After fusion, the cells are isolated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to remove unhybridized parental cells. Any of the media described herein, with or without supplemental serum, can be used to culture monoclonal antibody-secreting hybridomas. In another alternative to cell fusion techniques, EBV immortalized B cells can be used to produce the PCSK9 monoclonal antibodies of the invention. If desired, these hybridomas are expanded and subcloned, and the supernatants are assayed for anti-immunogen activity by conventional immunoassay methods, such as radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay.
Hybridomas that may be used as antibody sources include progeny cells of all derivatives, parent hybridomas that produce monoclonal antibodies specific for PCSK9 or portions thereof.
Hybridomas producing these antibodies can be grown in vitro or in vivo using known methods. The monoclonal antibodies can be isolated from the culture medium or body fluid by conventional immunoglobulin purification methods, such as ammonium sulfate precipitation, colloidal electrophoresis, dialysis, chromatography, and, if desired, ultrafiltration. If undesirable activity is present, it can be removed, for example, by passing the preparation through an adsorbent consisting of an immunogen linked solid phase and eluting or releasing the desired antibody from the immunogen. Immunization of a host animal with human PCSK9 conjugated with a protein that is immunogenic to the species desired for immunization, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, or a fragment containing the amino acid sequence of the target, produces a population of antibodies (e.g., monoclonal antibodies) that are conjugated using bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimidyl ester (conjugated via cysteine residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2Or R1N = C = NR (where R and R1Are different alkyl groups).
If desired, the PCSK9 antagonist antibody of interest (monoclonal or polyclonal) can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequences encoding the antibody of interest may be maintained in a vector for the host cell and the host cell may then be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be performed by cloning antibody genes from B cells by methods known in the art. See, e.g., tilleret, 2008, j.immunol.methods329, 112; U.S. patent No. 7,314,622.
In alternative embodiments, the polynucleotide sequences may be used in genetic manipulation to "humanize" the antibody or to improve the affinity or other characteristics of the antibody. For example, the constant region may be engineered to more closely resemble a human constant region to avoid an immune response if the antibody is used in clinical trials and treatments for humans. It is desirable to genetically manipulate the antibody sequences to obtain higher affinity for PCSK9 and higher inhibition utility for PCSK 9. It will be apparent to those skilled in the art that one or more polynucleotide changes may be made in a PCSK9 antagonist antibody and still maintain its binding ability to PCSK 9.
There are four general procedures for humanizing monoclonal antibodies. The steps are as follows: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains; (2) designing the humanized antibody, i.e., determining which antibody framework region will be used during the humanization step; (3) actual humanization methods/techniques; and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Pat. nos. 4,816,567; 5,807,715, respectively; 5,866,692, respectively; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.
Some "humanized" antibody molecules comprising antigen-binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated CDRs fused to human constant domains. See, for example, Winteretal, 1991, Nature349: 293-. Other references describe the implantation of rodent CDRs into human supporting Framework Regions (FRs) prior to fusion with appropriate human antibody constant domains. See, for example, Riechmann et al, 1988, Nature332: 323-. Another reference describes rodent CDRs supported by genetically engineered recombinantly engineered rodent framework regions. See, for example, european patent publication No. 0519596. These "humanized" molecules are designed to minimize unwanted immune responses to rodent anti-human antibody molecules that limit the duration and effectiveness of these moieties in therapeutic applications in human recipients. For example, the antibody constant region may be engineered to be immunologically inert (e.g., not to induce complement lysis). See, e.g., PCT publication Nos. WO 99/58572; british patent application No. 9809951.8. Other methods of humanizing antibodies that may also be utilized are described by daughertyetal, 1991, nuclear.acids sres.19:2471-2476 and U.S. patent No. 6,180,377; 6,054,297; 5,997,867, respectively; 5,866,692, respectively; 6,210,671 and 6,350,861 and PCT publication No. WO 01/27160.
In another alternative embodiment, fully human antibodies can be obtained by using commercially available mice that have been genetically engineered to express specific human immunoglobulins. Transgenic animals designed to produce a more desirable or robust immune response may also be used to produce humanized or human antibodies. An example of this technology is Xenomose from Abgenix, Inc. (Ferimen, Calif.)TMOf Medrex, Inc. (Princeton, N.J.), IncAnd TCMouseTMAnd Regeneron pharmaceuticals, Inc. (Talliendon, N.Y.)A mouse.
In alternative embodiments, the antibody may be recombinantly produced and expressed using any method known in the art. In another alternative embodiment, the antibody may be recombinantly produced by phage display technology. See, e.g., U.S. Pat. nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150 and Winteret, 1994, Annu. Rev. Immunol.12: 433-455. Alternatively, the phage display technology (McCaffertytal, 1990, Nature348:552-553) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region (V) domain gene populations of non-immunized donors. According to this technique, antibody V domain genes are cloned in-frame into a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle comprises a single-stranded DNA copy of the phage genome, screening for functional properties of the antibody also results in screening for genes encoding antibodies exhibiting these properties. Thus, the phage mimics several characteristics of B cells. Phage display can be performed in various formats; see, e.g., Johnson, KevinS.andChoswell, DavidJ.,1993, CurrentOpinionStrectural biology3: 564-571. Different sources of V gene segments can be used for phage display. Clacksononet, 1991, Nature352: 624-. V gene populations from human donors not immunized can be constructed and antibodies against various classes of antigens, including self-antigens, can be isolated substantially in accordance with the techniques described in Marketal, 1991, J.mol.biol.222:581-597 or Griffithal, 1993, EMBO J.12: 725-734. In a natural immune response, antibody genes are mutated cumulatively at a high frequency (somatic hypermutation). Several introduced changes will give higher affinity and B cells display high affinity surface immunoglobulins that are preferentially replicated and differentiated during subsequent antigen challenges. This natural process can be simulated by using a technique called "chainshuffling" (Marksetal.,1992, Bio/technol.10: 779-. In this method, the affinity of "primary" human antibodies resulting from phage display can be improved by successive replacement of the heavy and light chain V region genes with a population of naturally occurring variants (populations) of V domain genes from non-immunized donors. This technique allows the generation of antibodies and antibody fragments with affinities ranging from pM to nM. The strategy for generating very large phage antibody populations (also known as "the mother of all libraries") has been described by Waterhouseeet al, 1993, Nucl. acids sRes.21: 2265-. Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodies have affinity and specificity similar to the original rodent antibody. According to this method, also known as "epitope imprinting", the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced by a population of human V domain genes, resulting in rodent-human chimeras. Screening for antigens results in the isolation of human variable regions that restore functional antigen binding sites, i.e., selected epitopes of the control (imprinting) partner. When this method is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT publication No. WO 93/06213). Unlike conventional CDR grafting with humanized rodent antibodies, this technique provides fully human antibodies that do not have rodent-derived framework or CDR residues.
It will be apparent that although the above discussion has been with respect to humanized antibodies, the general principles discussed are applicable to the customization of antibodies for use in, for example, dogs, cats, primates, horses or cattle. It will also be apparent that one or more aspects of the humanized antibodies described herein may be combined, e.g., CDR grafting, framework mutations and CDR mutations.
Antibodies can be recombinantly produced by first isolating the antibody and antibody-producing cells from a host animal, obtaining a gene sequence, and using the gene sequence to recombinantly express the antibody in a host cell (e.g., a CHO cell). Another method that may be employed is the expression of the antibody sequence in plants (e.g., tobacco) or transgenic milk. Methods for recombinant expression of antibodies in plants or milk have been disclosed. See, e.g., Peeters,2001, et al, vaccine19: 2756; lonberg, n.andd.huskzar, 1995, int.rev.immunol13:65 and Pollock, et al, 1999, jimmunol methods231: 147. Methods for making derivatives of antibodies, e.g., humanized, single chain, etc., are known in the art.
Immunoassays and flow cytometric sorting techniques such as Fluorescence Activated Cell Sorting (FACS) can also be used to isolate antibodies specific for PCSK 9.
Antibodies can be bound to many different carriers. The carrier may be active and/or inert. Examples of well known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agar essence and magnetite. The nature of the carrier may be soluble or insoluble for the purposes of the present invention. Those skilled in the art will know or will be able to ascertain using routine experimentation other suitable carriers for binding the antibody. In some embodiments, the vector comprises a moiety that targets myocardium.
DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of this DNA. Once isolated, the DNA may be placed into an expression vector (such as that disclosed in PCT publication No. WO 87/04462), which is then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, to obtain synthesis of monoclonal antibodies in the recombinant host cells. See, for example, PCT publication No. WO 87/04462. The DNA may also be modified, for example, by substituting the coding sequence for the human heavy and light chain constant domains in place of the homologous murine sequences (Morrisonetal, 1984, Proc. Nat. Acad. Sci.81:6851) or by covalently binding all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, "chimeric" or "hybrid" antibodies are prepared to have the binding specificity of the PCSK9 monoclonal antibodies herein.
PCSK9 antagonist antibodies and antibody-derived polypeptides can be identified or characterized using methods known in the art, thereby detecting and/or measuring a reduction, improvement, or neutralization of PCSK9 biological activity. In some embodiments, a PCSK9 antagonist antibody or polypeptide is identified by culturing a candidate agent with PCSK9 and monitoring binding and/or concomitant reduction or neutralization of PCSK9 biological activity. The binding assay may be performed with a purified PCSK9 polypeptide or with cells that naturally express or are transfected to express a PCSK9 polypeptide. In one embodiment, the binding assay is a competitive binding assay, wherein the ability of a candidate antibody to compete for PCSK9 binding with a known PCSK9 antagonist is assessed. The assay can be performed in various formats, including ELISA formats. In other embodiments, the PCSK9 antagonist antibody is identified by culturing the candidate agent with PCSK9 and monitoring binding and consequent inhibition of LDLR expression and/or blood cholesterol clearance.
Following initial identification, the activity of candidate PCSK9 antagonist antibodies can be further confirmed and refined by bioassays known for testing biological activity of the target. Alternatively, the bioassay may be used directly to screen candidates. Several methods for identifying and characterizing PCSK9 antagonist antibodies, peptides, or aptamers are described in detail in the examples.
PCSK9 antagonist antibodies can be characterized using methods well known in the art. For example, one approach is for identifying the epitope or "epitope mapping" to which it binds. There are many methods known in the art for locating and characterizing the position of epitopes on proteins, including disruption of the crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, such as those described in chapter 11 of harlowand lane, using antibodies, a laboratory manual (cold spring harbor laboratory press, cold spring harbor, new york, 1999). In another example, epitope mapping can be used to determine the sequence to which a PCSK9 antagonist antibody binds. Epitope mapping is available from different commercial sources, such as the Pepscan system (8219 PH leirstataidenhawy 15, the netherlands). The epitope may be a linear epitope, i.e., a conformational epitope comprised in a single stretch of amino acids, or formed by the three-dimensional interaction of amino acids not necessarily comprised in a single stretch. Peptides of various lengths (e.g., at least 4 to 6 amino acids in length) can be isolated or synthesized (e.g., recombinant) and used in binding assays for PCSK9 antagonist antibodies. In another example, the epitope to which a PCSK9 antagonist antibody binds can be determined in a systematic screen by using an overlapping peptide derived from the PCSK9 sequence and determining the binding of the PCSK9 antagonist antibody. The open reading frame encoding PCSK9 is segmented randomly or by specific genetic structure according to gene fragment expression assays, and the reactivity of the expression fragment of PCSK9 with the antibody to be tested has been determined. The gene fragment may be generated, for example, by PCR, and then transcribed and translated into protein in the presence of radioactive amino acids in vitro. The binding of the antibody to the radiolabeled PCSK9 fragment was then determined by immunoprecipitation and colloidal electrophoresis. Specific epitopes can also be recognized by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments may be tested for binding to the test antibody in a simple binding assay. In another embodiment, mutagenesis of the antigen binding domain, domain replacement experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain replacement experiments can be performed using mutant PCSK9 in which various fragments of the PCSK9 polypeptide have been replaced (replaced) by PCSK9 sequences from other species or closely related but antigen-unique proteins such as other members of the proprotein convertase family. By detecting binding of the antibody to the mutant PCSK9, the importance of a particular PCSK9 fragment for antibody binding can be assessed.
Another method that may be used to characterize a PCSK9 antagonist antibody is a competition assay using other antibodies that are known to bind to the same antigen, i.e., a different fragment of PCSK9, to determine whether the PCSK9 antagonist antibody binds to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art.
The crystal structure of antibodies and antibody-antigen complexes can also be used to characterize antibodies. Residues were identified by calculating the difference between the accessible surface areas of the L1L3: PCSK9 crystal structure and PCSK9 alone structure. PCSK9 residues that exhibit buried surface area when complexed with L1L3 antibody are included as part of the epitope. The solvent accessible surface of a protein is defined as the central position of the probe microsphere (representing solvent molecules with a radius of 1.4 angstroms) as it rolls over the van der Waals surface of the protein. The solvent accessible surface area is calculated using the software AREAIMOL (Briggs, P.J.,2000, CCP4Newsletter No.38, CCLRC, Daresbury) by creating surface points on an expanded sphere of about each atom (from the center of the atom corresponding to the sum of the atom and the probe radius) and deleting those located within the equivalent sphere associated with the adjacent atom.
The expression vector may be used to directly express a PCSK9 antagonist antibody. One skilled in the art is familiar with the administration of expression vectors to obtain expression of endogenous proteins in vivo and in vitro. See, e.g., U.S. patent nos. 6,436,908; 6,413,942, and 6,376,471. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or transcatheter administration, and topical administration. In another embodiment, the expression vector is administered directly into the sensory nerve trunk or ganglia, or into the coronary arteries, atria, ventricles, or pericardium.
Targeted delivery of therapeutic compositions or subgenomic polynucleotides containing expression vectors may also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeiet, 1993, trends Biotechnol.11:202, Chiouet, 1994, Gene therapeutics: methods and applications of direct Gene transfer (J.A.Wolff, ed.); Wuetal, 1988, J.biol.Chem.263:621, Wuetal.1994, J.biol.Chem.269:542, Zenkeet, 1990, Proc.Natl.Acad.Sci.USA87:3655, Wuetal.1991, J.biol.Chem.266: 338. Therapeutic compositions containing polynucleotides are administered in the range of about 100ng to about 200mg of DNA for topical administration during gene therapy. DNA concentration ranges of about 500ng to about 50mg, about 1 μ g to about 2mg, about 5 μ g to about 500 μ g, and about 20 μ g to about 100 μ g can also be used during gene therapy sessions. The therapeutic polynucleotides and polypeptides may be delivered using a gene delivery vehicle. The gene delivery vehicle may be of viral or non-viral origin (see generally Jolly,1994, cancer Gene therapy1:51; Kimura,1994, HumanGene therapy5:845; Connelly,1995, HumanGene therapy1:185 and Kaplitt,1994, Nature genetics6: 148). Expression of these coding sequences can be induced using endogenous mammalian or heterologous promoters. The appearance of a coding sequence may be continuous or modulated.
Viral-based vectors for delivering desired polynucleotides and expression in desired cells are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB patent No. 2,200,651 and EP patent No. 0345242), alphaviral-based vectors (e.g., Sindbis (Sindbis) viral vectors, Sengliki) forest viruses (ATCCVR-67; ATCCVR-1247), Roche virus (ATCCVR-373; ATCCVR-1246) and Venezuelan equine encephalitis virus (ATCCRR-923; ATCCVR-1250; CVR-1249; CVATCCTR-532)), and adenovirus-related virus (AAV) vectors (see, e.g., WO94/12649, WO93/03769, WO 93/462891, WO 392842/00684, WO 3946/4655 and WO 3911955/11955). DNA ligated to non-living adenovirus may also be administered as described in Curiel,1992, hum.
Non-viral delivery vehicles and methods can also be employed, including but not limited to multivalent cation condensed DNA linked or unlinked to non-living adenovirus alone (see, e.g., Curiel,1992, hum. gene ther.3: 147); DNA linked to a ligand (see, e.g., Wu, j.,1989, biol. chem.264: 16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Pat. No. 5,814,482; PCT publication No. WO 95/07994; WO 96/17072; WO95/30763 and WO97/42338) and nuclear charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary naked DNA introduction methods are described in PCT publication No. WO90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Pat. nos. 5,422,120; PCT publication Nos. WO 95/13796; WO 94/23697; WO91/14445 and EP 0524968. Other methods are described in Philip,1994, mol.Cellbiol.,14:2411 and Wuffendin, 1994Proc.Natl.Acad.Sci.91: 1581.
The invention encompasses compositions (including pharmaceutical compositions) comprising an antibody described herein or produced by the method and having the characteristics described herein. As used herein, a composition comprises one or more antibodies, peptides or aptamers that antagonize the interaction of PCSK9 with LDLR, and/or one or more polynucleotides comprising sequences that encode one or more of these antibodies or peptides. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
The PCSK9 antagonist antibodies and peptides of the invention have any (one or more) of the following characteristics: (a) binds to PCSK 9; (b) blocking the interaction of PCSK9 with LDLR; (c) reducing PCSK 9-mediated down-regulation of LDLR; and (d) inhibits PCSK 9-mediated reduction in LDL blood clearance. Preferably, the PCSK9 antibody has two or more of these characteristics. More preferably, these antibodies have three or more characteristics. Most preferably, these antibodies have all four characteristics.
Accordingly, the invention provides any of the following sequences, or compositions (including pharmaceutical compositions) comprising any antibody having a partial light chain sequence and a partial heavy chain sequence as set forth in table 1. The underlined sequence is the CDR sequence according to Kabat (Kabat) and the bold part is the CDR sequence according to Coxiya (Chothia).
TABLE 1
The invention also provides CDR portions (including CDR sequences of cauchynia and kappa) of the antibody anti-PCSK 9. Determination of CDR regions is well known in the art. It is understood that in some embodiments, the CDRs may be a combination of kaba and cauchy CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDR is a kappa CDR. In other embodiments, the CDR is a cauchynia CDR. In other words, in embodiments having more than one CDR, the CDR can be any kaba, cauchynia, combined CDR, or a combination thereof.
The invention also provides methods of making any of these antibodies or polypeptides. Antibodies of the invention can be prepared using methods known in the art. The polypeptides may be prepared by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Polypeptides of antibodies, particularly shorter polypeptides of up to about 50 amino acids, can be conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be produced by automated polypeptide synthesizers using solid phase methods. See also U.S. patent nos. 5,807,715; 4,816,567 and 6,331,415.
In another alternative embodiment, antibodies and peptides may be recombinantly produced using methods well known in the art. In one embodiment, the polynucleotide comprises a sequence encoding the heavy and/or light chain variable region of antibody 4a5, 5a10, 6F6, 7D4, or L1L 3. The sequences encoding the antibody of interest may be maintained in a vector for the host cell, and the host cell may then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are described in detail herein.
The invention also encompasses scfvs of the antibodies of the invention. Single chain variable region fragments are prepared by linking light and/or heavy chain variable regions using short linking peptides (Birdetal, 1988, Science242: 423-426). An example of a linker peptide is (GGGGS)3(SEQ ID NO:24) bridging the carboxyl terminus of one variable region to the amino terminus of the other variable region by about 3.5 nm. Linkers for other sequences have been designed and used (birdetal, 1988, supra). The linker should be a short and flexible polypeptide, preferably comprising less than about 20 amino acid residues. The linker may also be modified to obtain additional functions, such as attachment to a drug or attachment to a solid support. Single-stranded variants can be produced recombinantly or synthetically. For synthetic generation of scFv, an automated synthesizer can be used. For recombinant production of an scFv, an appropriate plasmid comprising a polynucleotide encoding the scFv may be introduced as appropriateWhether eukaryotic such as yeast, plant, insect or mammalian cells, or prokaryotic such as E.coli. Polynucleotides encoding the scFv of interest can be prepared by routine manipulation, such as ligation of polynucleotides. The formed scFv can be isolated using standard protein purification techniques known in the art.
Other forms of single chain antibodies such as diabodies (diabodies) are also encompassed. Bivalent antibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker is used that is too short to pair the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger, P., et al, 1993, Proc. Natl. Acad. Sci. USA90: 6444-.
For example, bispecific antibodies (monoclonal antibodies having binding specificity for at least two different antigens) can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, e.g., Sureshetal, 1986, Methodsin enzymology121: 210). Traditionally, recombinant production of bispecific antibodies was based on the co-expression of two immunoglobulin heavy chain-light chain pairs, the two heavy chains having different specificities (millstein and ceullo, 1983, nature305, 537-539).
According to one method of making bispecific antibodies, an antibody variable domain (antibody-antigen binding site) having the desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion preferably has an immunoglobulin heavy chain constant domain comprising at least portions of the hinge, CH2, and CH3 regions. Preferably, there is a first heavy chain constant region (CH1) in which at least one fusion exists, the region comprising the position necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into an appropriate host organism. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides a high degree of flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, when at least two polypeptide chains are represented in the same ratio, resulting in high yields, or when the ratio is of no particular importance, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.
In one approach, the bispecific antibody consists of one arm of a hybrid immunoglobulin heavy chain having a first binding specificity and the other arm of a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity). Such immunoglobulin light chains are located in the asymmetric structure of only half of the bispecific molecule, facilitating the isolation of the desired bispecific compound from the undesired immunoglobulin chain combinations. This method is described in PCT publication No. WO 94/04690.
Heteroconjugate antibodies comprising two covalently linked antibodies are also within the scope of the invention. These antibodies have been used to target cells of the immune system to undesired cells (U.S. Pat. No. 4,676,980) and to treat HIV infection (PCT publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents and techniques are well known in the art and are described in U.S. Pat. No. 4,676,980.
Chimeric or hybrid antibodies can also be prepared in vitro using known synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate (iminothiolate) and methyl-4-mercaptobutylimidate (methyl-4-mercaptoimidate).
Humanized antibodies comprising one or more CDRs of antibody 5a10 or 7D4 or derived from one or more CDRs of antibody 5a10 or 7D4 can be prepared using, for example, any method known in the art. For example, four conventional steps can be used to humanize a monoclonal antibody. The steps are as follows: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains; (2) designing the humanized antibody, i.e., determining which antibody framework region will be used during the humanization step; (3) using actual humanization methods/techniques; and (4) transfecting and expressing the humanized antibody. See, e.g., U.S. Pat. nos. 4,816,567; 5,807,715, respectively; 5,866,692, respectively; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.
In recombinant humanized antibodies, the Fc portion may be modified to avoid interaction with the Fc γ receptor and complement and immune system. Techniques for making these antibodies are described in WO 99/58572. For example, the constant region may be engineered to more closely resemble a human constant region to avoid an immune response if the antibody is used in clinical trials and treatments for humans. See, e.g., U.S. patent nos. 5,997,867 and 5,866,692.
Humanized antibodies comprising the light or heavy chain variable regions or one or more CDRs of an antibody or variant thereof set forth in table 1 or derived from one or more CDRs of an antibody or variant thereof set forth in table 2 can be prepared using any method known in the art.
Humanized antibodies can be prepared using any method known in the art.
The present invention encompasses modifications to antibodies and variant polypeptides of the invention as shown in table 1, including functionally equivalent antibodies that do not significantly affect their properties and variants with increased or decreased activity and/or affinity. For example, the amino acid sequence may be mutated to yield an antibody having the desired binding affinity for PCSK 9. Modification of polypeptides is routine in the art and need not be described in detail herein. Modifications of the polypeptides are exemplified in the examples. Examples of modified polypeptides include polypeptides having amino acid residues with conservative substitutions, one or more deletions or additions of amino acids that do not significantly detrimentally alter the functional activity or mature (enhance) the affinity of the polypeptide for its ligand, or the use of chemical analogs.
Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions of single or multiple amino acid residues within a sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertional variants of the antibody molecule include an enzyme or polypeptide to which the N-or C-terminus of the antibody is fused to increase the half-life of the antibody in blood circulation.
Substitution variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted at that position. The positions of most interest for substitution mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Retained substitutions are shown in table 2 under the heading "conservative substitutions". If the substitution results in an alteration in biological activity, then more substantial alterations, referred to in Table 2 as "exemplary substitutions" or in addition to the reference amino acid classes described below, may be introduced and the product screened.
TABLE 2
Substantial modification of the biological properties of antibodies is accomplished by selecting substitutions that differ significantly in their maintenance of the following effects: (a) the structure of the polypeptide backbone of the substitution region, e.g., a sheet or helix configuration, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. Naturally occurring residues are grouped into the following groups according to common side chain properties:
(1) non-polar: norleucine, Met, Ala, Val, Leu, Ile;
(2) polar uncharged: cys, Ser, Thr, Asn, Gln;
(3) acidic (negative charge): asp and Glu;
(4) basic (positive charge): lys, Arg;
(5) residues that affect the directionality of the side chains: gly, Pro; and
(6) aromaticity: trp, Tyr, Phe, His.
Non-retentive substitutions are achieved by swapping members of one of these classes with another.
Any cysteine residues not involved in maintaining the proper configuration of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.
Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region such as a variable region. Changes in the variable region can alter binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions occur within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions occur within a CDR domain. In other embodiments, the CDR domain is CDRH3 and/or CDRL 3.
Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation. The antibody is glycosylated at the conserved positions in its constant regions (JeffersiandLund, 1997, chem. Immunol.65:111-128; WrightandMorrison,1997, TibTECH15: 26-32). The oligosaccharide side chains of immunoglobulins influence the function of the protein (Boydetal.,1996, mol. Immunol.32:1311-1318; WittweandHoward,1990, biochem.29:4175-4180) and the intramolecular interactions between parts of the glycoprotein which influence the configuration and the three-dimensional surface presented by the glycoprotein (JeffersandLund, supra; WyssandWagner,1996, CurrentOpin. Biotech.7: 409-416). Oligosaccharides can also be used to target a given glycoprotein to a particular molecule based on a specific recognition structure. It is also indicated that glycosylation of antibodies affects antibody-dependent cell-mediated cytotoxicity (ADCC). Specifically, it has been shown that CHO cells expressing β (1,4) -N-acetylglucosaminyltransferase III (GnTIII) which is regulated by tetracycline have enhanced ADCC activity (Umanaeal., 1999, Nature Biotech.17: 176-.
Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate group to the side chain of an aspartic acid residue. The tripeptide sequences of aspartyl-X-serine, aspartyl-X-threonine and aspartyl-X-cysteine, where X is any amino acid other than proline, are recognition sequences for the enzymatic attachment of a carbohydrate group to the side chain of aspartyl acid. Thus, the presence of any of these tripeptide sequences in a polypeptide results in a possible glycosylation site. O-linked glycosylation refers to linking one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be accomplished by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (O-linked glycosylation site).
The glycosylation pattern of an antibody can also be altered without altering the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell types used to express recombinant glycoproteins, such as antibodies, as potential therapeutic agents are rarely native cells, differences in antibody glycosylation patterns can be expected (see, e.g., Hseatal, 1997, J.biol.chem.272: 9062-.
In addition to the choice of host cell, factors that influence glycosylation during recombinant production of antibodies include growth pattern, medium formulation, culture density, oxidation, pH, purification mode, and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of specific enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation or a particular kind of glycosylation can be enzymatically removed from the glycoprotein, for example using endoglycosidase h (endoh), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, the recombinant host cell may be genetically engineered to be deficient in the treatment of a particular species of polysaccharide. These and similar techniques are well known in the art.
Other methods of modification include the use of coupling techniques known in the art, including but not limited to enzymatic methods, oxidative substitution, and chelation. Modifications can be used, for example, to attach labels for immunoassays. Modified polypeptides are prepared using methods established in the art, and can be screened using standard assays known in the art, some of which are described in the examples below.
In some embodiments of the invention, the antibody comprises a modified constant region, such as an immunologically or partially inert constant region, e.g., does not cause complement-mediated lysis, does not stimulate ADCC, or does not activate microglia; or reduced activity (as compared to an unmodified antibody) in any one or more of the following: causing complement-mediated lysis, stimulating ADCC, or activating microglia. Different modifications of the constant region can be used to achieve the optimal degree and/or combination of effector functions. See, e.g., Morganet, 1995, Immunology86: 319-. In some embodiments, the constant region is modified as described in EurJ.Immunol.,1999,29:2613-2624, PCT publication No. WO99/58572 and/or British patent application No. 9809951.8. In other embodiments, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering system referenced to wild type IgG2 sequence) (Eur. J. Immunol.,1999,29: 2613-2624). In other embodiments, the N-linked glycosylation of the constant region is non-glycosylated. In some embodiments, the N-linked glycosylation of the constant region is non-glycosylated by mutating the glycosylated amino acid residue or flanking the residue in the constant region that is part of the N-glycosylation recognition sequence. For example, N297 can be mutated to A, Q, K or H at N-glycosylation site. See Taoetal, 1989, J.Immunoglogy143: 2595-. In some embodiments, the N-linked glycosylation of the constant region is non-glycosylated. The N-linked glycosylation of the constant region may be unglycosylated by enzymes (such as by removal of carbohydrates by the enzyme PNGase) or by expression in glycosylation deficient host cells.
Other antibody modifications include antibodies that have been modified as described in PCT publication No. WO 99/58572. These antibodies, in addition to being directed against the binding domain of the target molecule, also comprise an effector domain having an amino acid sequence that is substantially homologous to all or part of the constant domain of a human immunoglobulin heavy chain. These antibodies are capable of binding to the target molecule without causing significant complement-dependent lysis or cell-mediated damage to the target. In some embodiments, the effector domain is capable of specifically binding FcRn and/or fcyriib. These effects are generally based on the derivation from two or more human immunoglobulin heavy chains CH2 domain. Antibodies modified in this manner are particularly useful in chronic antibody therapy to avoid inflammation and other adverse effects of conventional antibody therapy.
The present invention includes affinity maturation embodiments. For example, affinity matured antibodies can be produced using methods known in the art (Marksetal.,1992, Bio/Technology,10:779- & 783; Barbasel., 1994, Proc Nat. Acad. Sci., USA91:3809- & 3813; Schiereal., 1995, Gene,169:147- & 155; Yeltonet., 1995, J.Immunol.,155:1994- & 2004; Jacksonetal.,1995, J.Immunol.,154(7):3310-9; Hawkintal et., 1992, J.Mol.biol.,226:889- & 896 and PCT publication No. WO 2004/058184).
The following methods can be used to adjust the affinity and characterize the CDRs of the antibody. A CDR characterizing an antibody and/or altering (such as augmenting)The method of binding affinity of polypeptides such as antibodies is referred to as "library scanning mutagenesis". In general, library scanning mutagenesis was performed as follows. One or more amino acid positions in the CDRs are substituted with two or more (such as 3,4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids using methods established in the art. This results in a small pool of clones (in some embodiments, one clone for each amino acid position analyzed), each clone having a complexity of two or more members (if each position is substituted with two or more amino acids). In general, the libraries also include clones comprising natural (unsubstituted) amino acids. A small number of clones from each pool, for example about 20 to 80 clones (depending on the complexity of the pool) are screened for binding affinity to the target polypeptide (or other binding target) and candidate clones with increased, identical, decreased or no binding are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore surface plasmon resonance analysis, which detects about 2-fold or greater differences in binding affinity. Biacore is particularly useful when the starting antibody has bound with a relatively high affinity, e.g., a K of about 10nM or lessD. Examples are described herein using Biacore surface plasmon resonance screening.
Binding affinity can be determined using a Kinexa biosensor, proximity scintillation assay, ELISA, ORIGEN Immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinity may also be screened using an appropriate bioassay.
In some embodiments, each amino acid position in a CDR is substituted with all 20 natural amino acids using mutation-inducing methods known in the art (some methods are described herein). This results in a small pool of clones (in some embodiments, one clone for each amino acid position analyzed), each clone having a complexity of 20 members (if each position is substituted with all 20 amino acids).
In some embodiments, the library to be screened comprises substitutions at two or more positions, which substitutions may be in the same CDR or in two or more CDRs. Thus, the library may comprise substitutions at two or more positions in one CDR. The library may comprise substitutions at two or more positions in two or more CDRs. The library may comprise substitutions at 3,4, 5 or more positions found in two, three, four, five or six CDRs. Such substitutions may be made using low-repeat codons. See, e.g., Table 2 of Balitetal, 1993, Gene137(1): 109-18.
The CDRs may be CDRH3 and/or CDRL 3. The CDR may be one or more CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH 3. The CDRs may be kappa CDRs, cauchy CDRs or extended CDRs.
Candidates with enhanced binding may be sequenced, thereby identifying CDR substitution mutations (also referred to as "enhanced" substitutions) that result in enhanced affinity. Bound candidates can also be sequenced, thereby identifying CDR substitutions that remain bound.
Multiple screens can be performed. For example, candidates with enhanced binding (each containing an amino acid substitution at one or more positions of one or more CDRs) are also useful for designing a second library containing at least the original and substituted amino acids at each enhanced CDR position (i.e., a substitution mutation at an amino acid position of the CDR shows enhanced binding). Preparation, screening and selection of the libraries are discussed further below.
Library scanning mutagenesis also provides a means to characterize the CDRs, with enhanced binding, identical binding, reduced frequency of bound or unbound clones also providing information about the importance of each amino acid position in the stability of the antibody-antigen complex. For example, if the position of a CDR is changed to all 20 amino acids and binding is maintained, that position is considered likely not to be the position required for antigen binding. Conversely, a position of a CDR is considered to be functionally important if it remains bound only for a low percentage of substitutions. Thus, the library scanning mutagenesis approach yields information about positions in the CDRs that can be changed to many different amino acids (including all 20 amino acids) and positions in the CDRs that cannot be changed or can be changed to only a few amino acids.
Candidates with enhanced affinity may be combined in a second library comprising the enhanced amino acid, the original amino acid, and possibly additional substitutions at that position, depending on the complexity of the library desired or allowed using the desired screening or selection method. In addition, adjacent amino acid positions can be randomized to at least two or more amino acids, if desired. Randomization of adjacent amino acids may allow additional conformational plasticity of the mutated CDRs, which may in turn allow or facilitate the introduction of a larger number of enhancing mutations. The library may also comprise substitutions at positions that do not show enhanced affinity at the first screening.
The second library is screened or selected for library members with enhanced and/or altered binding affinity using any method known in the art, including screening using Biacore surface plasmon resonance analysis and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display.
The invention also encompasses fusion proteins comprising one or more fragments or regions from an antibody or polypeptide of the invention. In one embodiment, a fusion polypeptide is provided comprising at least 10 contiguous amino acids of the light chain variable region shown as seq id No. 53, 16, 17, 18 or 19 and/or at least 10 contiguous amino acids of the heavy chain variable region shown as seq id No. 54, 20, 21, 22 or 23. In other embodiments, fusion polypeptides comprising at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the light chain variable region and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the heavy chain variable region are provided. In another embodiment, the fusion polypeptide comprises a light chain variable region and/or a heavy chain variable region as set forth in any pair of sequences selected from SEQ ID NOs 53 and 54, 16 and 20, 17 and 21, 18 and 22, and 19 and 23. In other embodiments, the fusion protein comprises one or more CDRs. In other embodiments, the fusion polypeptide comprises CDRH3(VHCDR3) and/or CDRL3(VLCDR 3). For the purposes of the present invention, a fusion protein comprises one or more amino acid sequences to which an antibody and another native molecule are not linked, such as a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, a "tag" such as a FLAG tag or a 6His tag. Labels are well known in the art.
Fusion polypeptides may be produced by methods known in the art, such as synthesis or recombination. Typically, fusion proteins of the invention are prepared by expressing polynucleotides encoding them using recombinant methods described herein, although they may also be prepared by other methods known in the art, including, for example, chemical synthesis.
The invention also provides compositions comprising an antibody or polypeptide conjugated (e.g., linked) to an agent to facilitate coupling to a solid support, such as biotin or avidin. For simplicity, antibodies will generally be referred to and it is understood that these methods are applicable to any of the PCSK9 binding and/or antagonist embodiments described herein. Conjugation generally refers to linking these components as described herein. Attachment (attachment generally provides for securing the components in adjacent association at least when applied) can be achieved in any number of ways. For example, when the reagent and the antibody each have a substituent capable of reacting with each other, a direct reaction between the two is possible. For example, a nucleophilic group such as an amine or thiol group on one component may be capable of reacting with a carbonyl-containing group such as an anhydride or acyl halide group on another component, or with an alkyl group containing a good leaving group (e.g., a halide group).
The antibody or polypeptide of the invention may be linked to a labeling agent such as a fluorescent molecule, a radioactive molecule or any other label known in the art. The labels are known in the art and typically provide signals (either directly or indirectly).
The invention also provides compositions (including pharmaceutical compositions) and kits comprising any or all of the antibodies and/or polypeptides described herein, as the disclosure clearly appears.
The invention also provides isolated polynucleotides encoding the antibodies and peptides of the invention, and vectors and host cells comprising the polynucleotides.
Accordingly, the present invention provides a polynucleotide (or composition, including pharmaceutical compositions) comprising a polynucleotide encoding any of: antibodies 4a5, 5a10, 6F6, 7D4, L1L3, or any fragment or portion thereof having the ability to antagonize PCSK 9.
In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides having impaired effector function. Polynucleotides can be prepared and expressed using methods known in the art.
In another aspect, the invention provides a composition (such as a pharmaceutical composition) comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding an antibody described herein. In other embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In other embodiments, the composition comprises a polynucleotide shown by either or both of SEQ ID NO. 25 and SEQ ID NO. 26. Expression vectors and administration of polynucleotide compositions are further described herein.
In another aspect, the invention provides a method of making any of the polynucleotides described herein.
Polynucleotides complementary to any of these sequences are also encompassed by the invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond to DNA in a one-to-one manner, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the invention, and the polynucleotides may, but need not, be linked to other molecules and/or support materials.
The polynucleotide may comprise the native sequence (i.e., the endogenous sequence encoding the antibody or a portion thereof) or may comprise a variant of that sequence. Polynucleotide variants comprise one or more substitutions, additions, deletions and/or insertions such that the immunological activity of the encoded polypeptide is not diminished relative to the native immunologically active molecule. The variant is preferably tested for its effect on the immunological activity of the encoded polypeptide, as described herein, to exhibit at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity to the polynucleotide sequence encoding the native antibody or a portion thereof.
Two polynucleotide or polypeptide sequences are said to be "identical" if they are aligned for optimal correspondence and the nucleotide or amino acid sequences are identical. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, typically from 30 to about 75, or from 40 to about 50, where two sequences can be compared after optimal alignment with a reference sequence having the same number of contiguous positions.
Optimal alignment of sequences for comparison can be performed using preset parameters using the Megalign program in the Lasergene bioinformatics package (DNASTAR, Madison, Wis.). This procedure embodies several permutations described in the following references: dayhoff, M.O.,1978, Amodel of organic chemistry and transforming technologies, InDayhoff, M.O. (ed.) atlas of protein sequence and structure (National biomedicalresearch Foundation, Washington DC), Vol.5, Suppl.3, pp.345-358, HeinJ.,1990, Unifield Biochemical analysis and Phytogene p.626-Methods Enzylogvol.183, (Acemic Press, SanDiego, CA), Higgins, D.G.and P.M.,1989, CABS 83, 151, P.153, Sandyo, Numbe.78, Sangyo, S.103, Sandyno, N.103, Morgan. J.S.19811, Morgan. S.19811, Morgan. J.S.103, Morpho.103, Morgan. J.S.103, Morgan. 19811, Morpho.S.103, Morpho.S.S.103, Morgan. J.S.S.S.103, Sandyno. K.S.103, Sandyno.S.S.S.S.S.S.103, M.S.S.S.S.S.S.S.S.S.103, M.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.No. No. 33, S. 11, M.S.S.S.S. 23, S.S.S.S.S.S.S.S.S.S.S.S.
Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise 20 percent or less, typically 5 to 15 percent, or 10 to 12 percent additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) when the two sequences are optimally aligned. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the result by 100 to yield the percentage of sequence identity.
Variants may also be substantially homologous to the native gene or to portions or complements thereof, either simultaneously or selectively. These polynucleotide variants hybridize under moderately stringent conditions to naturally occurring DNA sequences (or complements) encoding the natural antibodies.
Suitable "moderately stringent conditions" include pre-washing in a solution of 5-fold SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); 5-fold SSC overnight hybridization at 50 ℃ to 65 ℃; followed by two washes in 2-, 0.5-and 0.2-fold SSC each containing 0.1% SDS at 65 ℃ for 20 minutes.
As used herein, "highly stringent conditions" or "high stringency conditions" are as follows: (1) low ionic strength and high temperature for cleaning, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate at 50 ℃; (2) during the hybridization at 42 ℃ with a denaturing agent, such as formamide, for example 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH6.5 containing 750mM sodium chloride, 75mM sodium citrate; or (3) a high stringency wash with 0.1 SSC containing EDTA at 55 ℃ with 50% formamide, 5 times SSC (0.75 moles NaCl, 0.075 moles sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 times danardt's solution, sonicated salmon sperm DNA (50 μ g/ml), 0.1% SDS and 10% dextran sulfate, and washing with 0.2 times SSC (sodium chloride/sodium citrate) at 42 ℃ and 50% formamide at 55 ℃. One skilled in the art will know how to adjust the temperature, ionic strength, etc. necessary to accommodate factors such as probe length and the like.
One of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that differ due to differences in codon usage are specifically contemplated by the present invention. In addition, allele lines of genes comprising the polynucleotide sequences provided herein are within the scope of the invention. A duality gene is an endogenous gene that is altered due to one or more mutations, such as deletions, additions, and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. The allele can be identified using standard techniques such as hybridization, amplification and/or database sequence comparison.
The polynucleotides of the invention may be obtained by chemical synthesis, recombinant methods or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One skilled in the art can use the sequences provided herein and a commercially available DNA synthesizer to generate the desired DNA sequence.
In preparing polynucleotides using recombinant methods as discussed further herein, a polynucleotide comprising the desired sequence may be inserted into an appropriate vector, which in turn is introduced into an appropriate host cell for replication and amplification. The polynucleotide may be inserted into the host cell by any method known in the art. Cells are transformed by direct uptake, phagocytosis, transfection, F mating (F-mating), or electroporation to introduce exogenous polynucleotides. When introduced, the exogenous polynucleotide may be maintained in the cell non-integrated into the vector (such as a plastid) or integrated into the genome of the host cell. The amplified polynucleotide may be isolated from the host cell using methods well known in the art. See, e.g., sambrookoketal, 1989, supra.
Alternatively, PCR allows for the replication of DNA sequences. PCR techniques are well known in the art and are described in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR, the polymeraseChemaine reaction, Mullseal, 1994, eds. (BirkauswerPress, Boston, MA).
RNA can be obtained by isolating DNA for use in an appropriate vector and inserting it into an appropriate host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, Sambrookettal, 1989, supra.
Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. Although the selected cloning vector may vary depending on the intended host cell, suitable cloning vectors will generally have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and/or may carry a marker gene that can be used to select for clones containing the vector. Suitable examples include plastids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBSSK +) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT 28. These and many other cloning vectors are available from commercial vendors such as Bayer corporation (BioRad), Strategene, and Invitrogen.
Expression vectors are typically replicable polynucleotide constructs comprising a polynucleotide of the invention. This suggests that the expression vector must be able to replicate in the host cell either as episomes (episomes) or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plastids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), cosmids, and expression vectors disclosed in PCT publication No. WO 87/04462. Carrier components generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; appropriate transcriptional control components (such as promoters, enhancers, and terminators). One or more translational control elements, such as ribosome binding sites, translation initiation sites, and stop codons, are also typically required for expression (i.e., translation).
The vector containing the polynucleotide of interest may be introduced into the host cell by any suitable method, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, ballistic bombardment, lipofection (lipofection), and infection (e.g., the vector is a pathogenic agent such as vaccinia virus). The choice of vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.
The invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell that overexpresses heterologous DNA can be used to isolate the gene encoding the antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, NSO, and CHO cells. See also PCT publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes such as E.coli or Bacillus subtilis, and yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe (S.pombe), or Kluyveromyces lactis. Preferably, the host cell expresses cDNA in an amount that is about 5-fold, more preferably 10-fold, even more preferably 20-fold greater than the corresponding endogenous antibody or protein of interest (if present in the host cell). Screening for host cell lines that specifically bind to PCSK9 or PCSK9 domain is performed by immunoassay or FACS. Cells over-expressing the antibody or protein of interest can be identified.
C. Composition comprising a metal oxide and a metal oxide
The compositions used in the methods of the invention comprise an effective amount of a PCSK9 antagonist antibody, a PCSK9 antagonist antibody-derived polypeptide, or other PCSK9 antagonist discussed herein. These compositions and examples of how they are formulated are also described in earlier sections and below. In one embodiment, the composition further comprises a PCSK9 antagonist. In another embodiment, the composition comprises one or more PCSK9 antagonist antibodies. In other embodiments, the PCSK9 antagonist antibody recognizes human PCSK 9. In other embodiments, the PCSK9 antagonist antibody is a humanized antibody. In other embodiments, the PCSK9 antagonist antibody comprises a constant region that does not elicit an unwanted or undesired immune response (such as antibody-mediated lysis or ADCC). In other embodiments, the PCSK9 antagonist antibody comprises one or more CDRs (such as one, two, three, four, five, or in some embodiments all six CDRs) of an antibody. In some embodiments, the PCSK9 antagonist antibody is a human antibody.
It is to be understood that the composition may comprise more than one PCSK9 antagonist antibody (e.g., a mixture of PCSK9 antagonist antibodies that recognize different epitopes of PCSK 9). Other exemplary compositions comprise more than one PCSK9 antagonist antibody recognizing the same epitope, or different classes of PCSK9 antagonist antibodies that bind to different epitopes of PCSK 9.
The compositions used in the present invention may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer (Remington: the science and practice of pharmacy 20) in the form of a lyophilized preparation or an aqueous solutionthEd, 2000, lippincott williamsa and wilkins, ed.k.e.hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol alcohols, butanol or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues)) A polypeptide; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine (glutamine), aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and/or non-ionic surfactants such as TWEENTM、PLURONICSTMOr polyethylene glycol (PEG). Pharmaceutically acceptable excipients are described further herein.
In one embodiment, the antibody is administered in a formulation of a sterile aqueous solution having a pH of between about 5.0 and about 6.5 and comprising from about 1mg/ml to about 200mg/ml of the antibody, from about 1mM to about 100mM histidine buffer, from about 0.01mg/ml to about 10mg/ml polysorbate 80, from about 100mM to about 400mM trehalose, and from about 0.01mM to about 1.0mM disodium EDTA dihydrate.
The PCSK9 antagonist antibodies and compositions thereof can also be used with other agents to augment and/or supplement the effectiveness of these agents.
D. Reagent kit
The invention also provides kits for use in the rapid methods. The kits of the invention comprise one or more containers containing a PCSK9 antagonist antibody (such as a humanized antibody) or a peptide described herein and instructions for use according to any of the methods of the invention described herein. In general, these instructions comprise instructions for administering a PCSK9 antagonist antibody, peptide, or aptamer for use in the therapeutic treatment described above.
In some embodiments, the anti-system is a humanized antibody. In some embodiments, the antibody is a human antibody. In other embodiments, the antibody is a monoclonal antibody. Instructions for use of PCSK9 antagonist antibodies typically include information on the dosage, administration regimen, and route of administration for the intended treatment. The container may be a unit dose, a bulk package (e.g. a multi-dose package) or a sub-unit dose. The instructions provided by the kits of the present invention are typically written instructions on labels or packaging coupons (inserts), such as paper included in the kit, but machine-readable instructions, such as instructions carried by magnetic or optical storage disks, are also acceptable.
The kit of the invention is suitably packaged. Suitable packaging includes, but is not limited to, glass vials (teal), bottles (bottle), jars (jar), plastic packaging (e.g., sealed mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a particular device, such as an inhaler, a nasal administration device (e.g., a nebulizer), or an infusion device such as a mini-pump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a PCSK9 antagonist antibody. The container (e.g., a pre-filled syringe or an auto-injector) may further comprise a second pharmaceutically active agent.
The kit may optionally provide additional components such as buffers and instructional information. Normally, the kit comprises a container and a label or package copy on or associated with the container.
Mutations and modifications
The PCSK9 antibody to be used in the practice of the invention encodes VHAnd VLThe DNA fragment of the region may be obtained by any of the methods described above. Various modifications, such as mutations, deletions and/or additions, may also be introduced into the DNA sequence using standard methods known to those skilled in the art. For example, mutagenesis can be performed using standard methods, such as PCR-mediated mutagenesis, in which the mutated nucleotides are incorporated into PCR primers such that the PCR product comprises the desired mutation or site-directed mutagenesis.
One substitution that may be made is, for example, altering one or more cysteines in the antibody, which may be chemically reactive to another residue such as, but not limited to, alanine or serine. For example, substitution of an unregulated cysteine may be performed. The substitution can occur in the CDRs or framework regions of the variable domain or in the constant domain of the antibody. In some embodiments, the cysteine is canonical (canonical).
Variable domains of antibodies, e.g., heavy and/or light chains, may also be modified, e.g., to alter the binding characteristics of the antibodies. For example, mutations can be made in one or more CDR regions to increase or decrease the K of the antibody to PCSK9DIncreasing or decreasing KoffOr altering the binding specificity of the antibody. Techniques for site-directed mutagenesis are well known in the art. See, e.g., sambrooketal, and ausubeletal, supra.
Modifications or mutations may also be made in the framework regions or constant domains to increase the half-life of the PCSK9 antibody. See, e.g., PCT publication No. WO 00/09560. Mutations in the framework or constant domains can also be made to alter the immunogenicity of the antibody, provide a site for covalent or non-covalent binding to another molecule, or alter such properties as complement fixation, FcR binding, and antibody-dependent cell-mediated cytotoxicity. According to the invention, a single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant domain.
In a process called "germ cell" at VHAnd VLSpecific amino acids in the sequence may be mutated to fit those in germ cell VHAnd VLNaturally found in the sequence. In particular, at VHAnd VLThe amino acid sequence of the framework regions in the sequence may be mutated to conform to germ cell sequences to reduce the risk of immunogenicity when the antibody is administered. Human VHAnd VLThe germ cell DNA sequence of a gene is known in the art (see, e.g., Vbase human germ cell sequence database; see also Kabat, E.A., et al (1991) sequence of protein of immunological interest, FifthEdion, U.S. Departmentof health and HumanServces, NIHPubl.No.91-3242, Tomlinsonet al, 1992, J.Mol.biol.227: 776-.
Another amino acid substitution that may be made is the removal of potential proteolytic sites in the antibody. These sites may occur in the CDRs or framework regions or in the constant domains of the variable domains of the antibodies. Substitution of cysteine residues and removal of proteolytic sites may reduce the risk of heterogeneity and thus increase the isotypes of the antibody product. Another type of amino acid substitution clears the asparagine-glycine pair by altering one or both residues to form a potential deamidation site. In another example, the C-terminal lysine of the heavy chain of the PCSK9 antibody of the invention can be cleaved. In various embodiments of the invention, the heavy and light chains of the PCSK9 antibody optionally include signal sequences.
When V of the present invention is obtained as a codeHAnd VLAfter the DNA fragments of the segments, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, into a Fab fragment gene, or into a scFv gene. In these operations, VLOr VHThe encoding DNA segment is operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that two DNA segments are joined such that the amino acid sequences encoded by the two DNA segments are in frame order.
Code VHIsolated DNA of a region may be obtained by operably linking the VHThe encoding DNA and another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3) were converted into the full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat, e.a., et al, 1991, sequence of proteins of immunologica interest, FifthEdion, U.S. department of health and HumanServices, NIHPubl. No.91-3242) and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is IgG1 or IgG2 constant region. The IgG constant region sequence can be any of a variety of alleles or allotypes known to occur in different individuals, such as Gm (1), Gm (2), Gm (3), and Gm (17). These isoforms represent amino acid substitutions that occur naturally in the constant region of IgG 1. In the case of the heavy chain gene of the Fab fragment, this VHThe encoding DNA may be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region. The CH1 heavy chain constant region may be derived from any heavy chain gene.
Code VLIsolated DNA of a region may be obtained by operably linking the VLThe encoding DNA is combined with another DNA molecule encoding a light chain constant region (CL) to be converted into the full-length light chain gene (and the Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (see, e.g., Kabat, e.a., et al, 1991, sequence of proteins of immunologica interest, FifthEdion, U.S. department of health and HumanServices, NIHPubl. No.91-3242) and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region may be any of a variety of allele known to occur in different individuals, such as Inv (1), Inv (2), and Inv (3). The lambda constant region can be derived from any three lambda genes.
To generate the scFv gene, the VHAnd VLThe coding DNA segment is operably linked to a coding flexible linker, e.g., a coding amino acid sequence (Gly)4-Ser)3Is linked so that said VHAnd VLThe sequence may be represented as having V's connected by flexible linkersLAnd VHContinuous single-stranded proteins of domains (see, e.g., Birdetal, 1988, Science242: 423-; Hustonet, 1988, Proc. Natl. Acad. Sci. USA85: 5879-; McCaffertyetal, 1990, Nature348: 552-) -554). The single chain antibody may be monovalent (if only a single V is used)HAnd VL) Double (if two V's are used)HAnd VL) Or multivalent (if more than two V are used)HAnd VL). Bivalent or multivalent antibodies may be generated to specifically bind PCSK9 to another molecule.
In another embodiment, the fusion antibody or immunoadhesin may be formulatedComprising all or part of a PCSK9 antibody of the invention linked to another polypeptide. In another embodiment, only the variable domain of the PCSK9 antibody is linked to the polypeptide. In another embodiment, V of the PCSK9 antibodyH(ii) Domain is linked to a first polypeptide, and V of a PCSK9 antibodyLThe domain is linked to a second polypeptide which is linked to the first polypeptide such that the VHAnd VLThe domains are associated in such a way that they interact with each other to form an antigen binding site. In another preferred embodiment, said VHDomains with VLThe domains are separated by a linker to allow the VHAnd VLThe domains may interact with each other. The V isHlinker-VLThe antibody is then linked to the polypeptide of interest. In addition, fusion antibodies can be generated in which two (or more) single chain antibodies are linked to each other. This is useful if it is desired to produce bi-or multivalent antibodies on a single polypeptide chain or if it is desired to produce bispecific antibodies.
In other embodiments, other modified antibodies can be made using PCSK9 antibodies that encode nucleic acid molecules. For example, "kappa antibodies" (Illetal.,1997, ProteinEng.10:949-57), "miniantibodies" (Martinetal.,1994, EMBO J.13:5303-9), "diabodies" (Holligeretal, 1993, Proc. Natl.Acad.Sci.USA90: 6444-.
Bispecific antibodies or antigen-binding fragments can be produced by a variety of methods, including fusion of hybridomas or linking Fab' fragments. See, e.g., Songsivilai & Lachmann,1990, Clin.exp.Immunol.79: 315-. In addition, bispecific antibodies can be formed as "dimeric antibodies" or "hausman antibodies". In some embodiments, the bispecific antibody binds to two different epitopes of PCSK 9. In some embodiments, the modified antibodies described above are prepared using one or more variable domains or CDR regions from human PCSK9 antibodies provided herein.
Generation of antigen-specific antibodies
The ability of more than 500 polyclonal and monoclonal antibodies raised against recombinant full-length human PCSK9, recombinant full-length murine PCSK9, and various synthetic peptides to down-regulate total LDLR protein in Huh7 human liver cell culture was evaluated. One group of these antibodies is raised against and reacts with a group of 12 to 20 amino acid residue polypeptides that are expected to cover most of the surface of the protein, according to the structure of PCSK 9. In the highest concentration, the optimal antibody exhibits only about 60% blocking activity.
Therefore, alternative and previously undiscovered methods were employed, namely the generation of monoclonal antibodies by immunization of PCSK9 nude mice with recombinant full-length PCSK9 protein. Antagonist antibodies generated by this antibody generation showed complete blockade of PCSK9 binding to LDLR, complete blockade of PCSK 9-mediated reduction of LDLR levels in Huh7 cells, and reduction of LDLc in vivo including mice to levels equivalent to PCSK 9-/-mice, as shown in example 7.
Representative antibodies (hybridomas) of the present invention were deposited at the American Type Culture Collection (ATCC) on day 28, 2.2008 and assigned the accession numbers of table 3. Hybridomas producing antibodies 4a5, 5a10, 6F6, and 7D4 were deposited.
TABLE 3