CN101325968B - Anti-CTLA-4 antibody compositions - Google Patents

Anti-CTLA-4 antibody compositions Download PDF

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CN101325968B
CN101325968B CN200680015670.4A CN200680015670A CN101325968B CN 101325968 B CN101325968 B CN 101325968B CN 200680015670 A CN200680015670 A CN 200680015670A CN 101325968 B CN101325968 B CN 101325968B
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antibody
ctla
compositions
amino acid
seq
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CN101325968A (en
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J·D·阿贝特
T·K·达斯
C·M·艾略特
K·W·穆图拉尼亚
S·尼玛
S·K·辛格
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Pfizer Products Inc
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Pfizer Products Inc
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Abstract

The present invention provides for compositions of anti-M-CSF antibodies comprising a chelating agent. Also provided are methods of treating diseases and disorders (including various neoplasia disorders) by using the compositions..

Description

Anti-CTLA-4 antibody compositions
To the cross reference of Patents and patent application
The application requires the U.S. Provisional Patent Application series number 60/659 of submitting on March 8th, 2005,766, the U.S. Provisional Patent Application series number 60/728 of submitting on October 19th, 2005,165, the U.S. Provisional Patent Application series number 60/752 of December in 2005 submission on the 20th, the U.S. Provisional Patent Application series number 60/762 that on January 26th, 712 and 2006 submits to, 456 rights and interests, it is all incorporated by reference in this text and examines with it at this.
Background of invention
Cytotoxic lymphocyte antigen-4 (" CTLA-4 ") are the members of immunoglobulin (" the Ig ") superfamily of protein.CTLA-4 plays and lowers t cell activation and keep immune homeostatic effect.Show, in animal model, block CTLA-4 (for example, by use CTLA-4 antibody) and can improve the effect of immunotherapy for cancer.
In document, reported in conjunction with CTLA-4 and suppressed its active antibody.For example, belong to Pfizer, Inc. and Abgenix, the United States Patent (USP) 6,682,736 of Inc. has been reported the human monoclonal antibodies of several anti-CTLA-4, comprises that having antibody 11.2.1 (is now called ticilimumab tM) heavy chain and the CTLA-4 antibody of light-chain amino acid sequence.The hybridoma that produces antibody 11.2.1 ties up to preservation under ATCC accession number PTA-5169.The United States Patent (USP) 5,977,318 that belongs to Bristol-Myers Squibb Company has been reported another kind of monoclonal antibody, its identification and the extracellular domain in conjunction with CTLA-4, thereby the combination of prevention CTLA-4 and B7 antigen.Belong to Medarex, the U.S. of Inc. openly applies for that 20050201994 have reported several human sequence's antibody of anti-CTLA-4, comprises and is now called ipilimumab tMa kind of antibody.
A kind of possible mode of using this type of CTLA-4 antibody is to use by parenteral route.For example, U.S. Patent application 6,682,736 have reported the iv formulation of anti-CTLA-4 antibody, and said preparation is the sterile liquid solution (pH5.5) that comprises anti-CTLA-4 antibody, 20mM sodium acetate, 0.2mg/ml polyoxyethylene sorbitan monoleate and 140mM sodium chloride.
The same with other protein formulations, for CTLA-4 antibody preparation, also there is identical consideration, the antibody in compositions is along with chemistry and the mechanical degradation of time.Usually, CTLA-4 antibody preparation should show acceptable chemistry and physical stability within the scope of the storage of expection and service condition, and CTLA-4 antibody preparation should have enough storage lives and still keep having biologic activity.Under given production CTL-4 antibody necessary time of product and resource situation, the preparation that reduces product loss is desirable.Therefore, the present patent application discloses to show and has compared the chemistry of disclosed CTLA-4 antibody preparation raising and/or the new CT LA-4 antibody preparation of physical stability in former document.
Summary of the invention
In one aspect, the invention provides the composition of liquid medicine that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise and the light-chain amino acid sequence at least 95% homogeneity aminoacid sequence shown in SEQ ID NO:4, wherein said antibodies people CTLA-4.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody is IgG2 antibody.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody is people's antibody.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises and utilizes people V hthe V of 3-33 germline gene haminoacid sequence.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody has and comprises the people FR1, the FR2 that by reading frame and CDR1, CDR2 and CDR3 sequence, are effectively connected and the V of FR3 sequence haminoacid sequence, described people FR1, FR2 and FR3 sequence are utilized people V h3-33 gene family.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody is the antibody separating.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody is recombinant antibodies.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody specificity ground is in conjunction with the comformational epitope on people CTLA-4 polypeptide.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises with SEQ ID NO:2 and has the heavy chain amino acid sequence of at least 95% sequence homogeneity and have the light-chain amino acid sequence of at least 95% sequence homogeneity with SEQID NO:4.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises with SEQ ID NO:2 and has the heavy chain amino acid sequence of at least 99% sequence homogeneity and have the light-chain amino acid sequence of at least 99% sequence homogeneity with SEQID NO:4.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises heavy chain amino acid sequence and light-chain amino acid sequence, the variable region that described heavy chain amino acid sequence contains SEQID NO:2, the variable region of containing SEQ ID NO:4 with described light-chain amino acid sequence.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises weight chain variable region amino acid sequence and light chain variable region amino acid sequence, described weight chain variable region amino acid sequence comprises SEQ ID NO:5, comprise SEQID NO:6 with described light chain variable region amino acid sequence.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises heavy chain amino acid sequence and light-chain amino acid sequence, described heavy chain amino acid sequence comprises SEQID NO:2, and described light-chain amino acid sequence comprises SEQ ID NO:4.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and the C-end lysine of the heavy chain of wherein said antibody does not exist.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises having the heavy chain of antibody 11.2.1 and the anti-CTLA-4 antibody of the monoclonal IgG2 of light-chain amino acid sequence.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody has heavy chain and the light-chain amino acid sequence identical with antibody by hybridoma cell line 11.2.1.4 (preservation under the ATCC accession number PTA-5169) generation.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody is ticilimumab.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said chelating agen is selected from glycine that amino polybasic carboxylic acid, hydroxyl amino carboxylic acid, edta salt and derivant, N-replace, deferoxamine derivant and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said chelating agen is selected from ethylenediaminetetraacetic acid, DTPA, nitrilotriacetic acid(NTA), N-2-acetylaminohydroxyphenylarsonic acid 2-iminodiacetic acid, two (aminoethyl) glycol ether, N, N, N ', N '-tetraacethyl, trans-diamino-cyclohexane tetraacethyl (DCTA), glutamic acid, aspartic acid, N hydroxyethyliminodiacetic acid, N, N-bis--ethoxy glycine, N-(trihydroxy methyl methyl) 10 glycine, glycylglycine, 2-(2-amino-2-oxoethyl) aminoethane sulphonic acid, deferoxamine, deferoxamine mesylate, EDTAP dipotassium ethylene diamine tetraacetate, disodium edetate, CaEDTA, edetate sodium, edetate trisodium, edetic acid potassium, citric acid, sodium citrate, anhydrous citric acid, citrate trisodium dihydrate, nicotiamide, NaTDC and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said chelating agen is EDTA.
The present invention also provides the compositions that comprises at least one antibody and chelating agen and further comprise buffer agent, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4.
The present invention also provides the compositions that comprises at least one antibody and chelating agen and further comprise buffer agent, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said buffer agent is selected from acetate, succinate, gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, Ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazoles, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate, imidazoles, tris, phosphate and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen and further comprise buffer agent, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said buffer agent comprises histidine.
The present invention also provides the compositions that comprises at least one antibody and chelating agen and further comprise histidine, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said histidine comprises L-Histidine or D-His.
The present invention also provides the compositions that comprises at least one antibody and chelating agen and further comprise histidine, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said histidine comprises L-Histidine.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of about 0.1 to about 200mg/ml.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody that concentration is about 20mg/ml.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from the excipient of tonicity agent, surfactant and buffer agent.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least two kinds of excipient that are selected from tonicity agent, surfactant and buffer agent.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises tonicity agent, surfactant and buffer agent.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises tonicity agent, antioxidant, surfactant and buffer agent.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from the excipient of tonicity agent, surfactant and buffer agent, and wherein said tonicity agent comprises saccharide.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from tonicity agent, the excipient of surfactant and buffer agent, wherein said tonicity agent comprises that at least one is selected from fructose, glucose, mannose, sorbose, xylose, lactose, maltose, sucrose, glucosan, pullulan, dextrin, cyclodextrin, soluble starch, hetastarch, the excipient of water-soluble glucan and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from the excipient of tonicity agent, surfactant and buffer agent, and wherein said tonicity agent comprises polyhydric alcohol.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from tonicity agent, the excipient of surfactant and buffer agent, wherein said polyhydric alcohol is selected from mannitol, trehalose, Sorbitol, erythritol, isomalt, lactose, maltose alcohol, xylitol, glycerol, lactitol, propylene glycol, Polyethylene Glycol, inositol and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from the excipient of tonicity agent, surfactant and buffer agent, and wherein said tonicity agent comprises nonreducing sugar.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from tonicity agent, the excipient of surfactant and buffer agent, wherein said tonicity agent comprises nonreducing sugar, wherein said nonreducing sugar comprises that at least one is selected from sucrose, the excipient of trehalose and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from the excipient of tonicity agent, surfactant and buffer agent, wherein said tonicity agent comprises nonreducing sugar, and wherein said nonreducing sugar is trehalose.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from tonicity agent, the excipient of surfactant and buffer agent, wherein said surfactant is selected from Polysorbate, poloxamer, tritons, sodium lauryl sulphate, sodium lauryl sulfate, OG sodium (sodium octyl glycoside), lauryl-sulfobetaines, myristyl-sulfobetaines, sub-oil base (linoleyl)-sulfobetaines, stearyl-sulfobetaines, lauryl-sarcosine, myristyl-sarcosine, sub-oil base-sarcosine, stearyl-sarcosine, sub-oil base-betanin, myristyl-betanin, cetyl-betanin, dodecanamide propyl-betanin, cocamidopropyl propyl amide-betanin, sub-oleamide propyl group-betanin, myristamide propyl group-betanin, palmidopropyl-betanin, isostearoyl amine propyl group-betanin, myristamide propyl group-dimethylamine, palmidopropyl-dimethylamine, isostearoyl amine propyl group-dimethylamine, sodium methylcocoyltaurate, methyl oil base taurine disodium, chlorination dihydroxypropyl peg 5 linoleammonium, Polyethylene Glycol, polypropylene glycol and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from tonicity agent, the excipient of surfactant and buffer agent, wherein said surfactant is selected from polysorbate 20, Polysorbate 21, polysorbate 40, polysorbate 60, Polysorbate 61, polysorbate 65, polyoxyethylene sorbitan monoleate, sorbimacrogol oleate 100, polysorbate 85 and composition thereof.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from the excipient of tonicity agent, surfactant and buffer agent, and wherein said surfactant is polyoxyethylene sorbitan monoleate.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises at least one and is selected from the excipient of tonicity agent, surfactant and buffer agent, and wherein said buffer agent comprises histidine.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises polyoxyethylene sorbitan monoleate.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions further comprises trehalose.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein said compositions has about 5.0 to about 6.5 pH.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of histidine is that about 1mM is to about 50mM.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of histidine is about 20mM.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of polyoxyethylene sorbitan monoleate is that about 0.01mg/ml is to about 10mg/ml.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of polyoxyethylene sorbitan monoleate is about 0.2mg/ml.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of EDTA is that about 0.001mg/ml is to about 10mg/ml.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of EDTA is about 0.1mg/ml.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of trehalose is that about 10mg/ml is to about 100mg/ml.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein the concentration of trehalose is about 84mg/ml.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein said compositions comprises the antibody of about 0.1mg/ml to about 100mg/ml; Approximately 0.001mg/ml is to the EDTA of about 1.0mg/ml; Approximately 1mM is to the histidine of about 50mM; Approximately 0.01mg/ml is to the polyoxyethylene sorbitan monoleate of about 10mg/ml; With the trehalose of about 10mg/ml to about 100mg/ml.
The present invention also provides the compositions that comprises at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions also comprises histidine, trehalose, polyoxyethylene sorbitan monoleate and EDTA, and wherein said compositions comprises: the approximately antibody of 20mg/ml; The approximately EDTA of 0.1mg/ml; The approximately histidine of 20mM; The approximately polyoxyethylene sorbitan monoleate of 0.2mg/ml; Trehalose with about 84mg/ml.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the heavy chain amino acid sequence that contains SEQ ID NO:2 and the light-chain amino acid sequence that contains SEQ ID NO:4, wherein said antibodies people CTLA-4, and described antibody was stable at the temperature of about 5 ℃ at least about 26 weeks.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the heavy chain amino acid sequence that contains SEQ ID NO:2 and the light-chain amino acid sequence that contains SEQ ID NO:4, wherein said antibodies people CTLA-4, and described antibody was stable at the temperature of about 25 ℃ at least about 26 weeks.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the heavy chain amino acid sequence that contains SEQ ID NO:2 and the light-chain amino acid sequence that contains SEQ ID NO:4, wherein said antibodies people CTLA-4, and described antibody was stable at the temperature of about 40 ℃ at least about 26 weeks.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody at least one of described compositions, freezing and cycle period of thawing is stable.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody at least six of described compositions, freezing and cycle period of thawing is stable.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area (aggragate chromatogram peak area) of described compositions and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of described compositions and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen, wherein said chromatographic isolation comprises SE-HPLC.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of described compositions and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen, wherein with ultraviolet detection, measure the amount of the antibody of having assembled.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of described compositions and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen, wherein in 214 nanometers, carry out described ultraviolet detection.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of described compositions and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen, wherein described compositions is being stored at the temperature of about 40 ℃ after about 24 weeks, it is limpid and colourless that described compositions still keeps substantially.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of described compositions and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen, and wherein as with after lysyl endopeptidase digestion promoting then by reversed-phase HPLC separate measure, with compared with not containing the antibody in the compositions of chelating agen, when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, total oxidation percentage ratio of the methionine residues at amino acid position 432 places reduces the amount that is equal to or greater than 2.2%.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of described compositions and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen, and wherein as with after lysyl endopeptidase digestion promoting then by reversed-phase HPLC separate measure, with compared with not containing the antibody in the compositions of chelating agen, when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, total oxidation percentage ratio of the methionine residues at amino acid position 256 places reduces the amount that is equal to or greater than 4.2%.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody is substantially by forming with the same aminoacid sequence of the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2 and further substantially by forming with the same aminoacid sequence of the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4.
The present invention also provides the compositions that comprises at least one antibody and chelating agen, described antibody is by forming with the same aminoacid sequence of the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2 and further by forming with the same aminoacid sequence of the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4.
The present invention also provides the method for the preparation of stable composition of liquid medicine, it comprises monoclonal anti CTLA-4 antibody is mixed with the pharmaceutically acceptable chelating agen that reduces the instable amount of described antibody, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of the described stable composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and chelating agen and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen.
The present invention also provides the method for the monoclonal anti CTLA-4 antibody of stable liquid pharmaceutical composition, it comprises the fluid composition that formation comprises described antibody and pharmaceutically acceptable chelating agen, wherein when described compositions is stored to about 24 weeks at the temperature of about 40 ℃, the aggregation chromatogram peak area of the described stable composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and chelating agen and at the temperature of about 40 ℃, store about 24 weeks not containing being reduced at least about 2% between the aggregation chromatogram peak area of the same combination of chelating agen.
The present invention also provides the method for the neoplasia condition of illness being used for the treatment of in experimenter, it comprises that described compositions comprises to described experimenter's applicating liquid pharmaceutical composition: monoclonal anti CTLA-4 antibody ticilimumab and the pharmaceutically acceptable chelating agen for the treatment of effective dose.
The present invention also provides the method for the neoplasia condition of illness being used for the treatment of in experimenter, it comprises to described experimenter's applicating liquid pharmaceutical composition, described compositions comprises: monoclonal anti CTLA-4 antibody ticilimumab and the pharmaceutically acceptable chelating agen for the treatment of effective dose, wherein through intravenous route, give described experimenter's applying said compositions.
The present invention also provides the method for the neoplasia condition of illness being used for the treatment of in experimenter, it comprises to described experimenter's applicating liquid pharmaceutical composition, described compositions comprises: monoclonal anti CTLA-4 antibody ticilimumab and the pharmaceutically acceptable chelating agen for the treatment of effective dose, wherein said experimenter need to treat neoplasia condition of illness.
The present invention also provides the method for the neoplasia condition of illness being used for the treatment of in experimenter, it comprises to described experimenter's applicating liquid pharmaceutical composition, described compositions comprises: monoclonal anti CTLA-4 antibody ticilimumab and the pharmaceutically acceptable chelating agen for the treatment of effective dose, wherein said neoplasia condition of illness is the cancer that is selected from the brain cancer, squamous cell cancer, bladder cancer, gastric cancer, cancer of pancreas, breast carcinoma, a cancer, neck cancer, esophageal carcinoma, carcinoma of prostate, colorectal carcinoma, pulmonary carcinoma, renal carcinoma, renal cancer, ovarian cancer, gynecological cancer and thyroid carcinoma.
The present invention also provides the test kit for the preparation of the fluid composition of the antibody through stable, and it comprises: the first container and the second container that pharmaceutically acceptable chelating agen is housed that monoclonal anti CTLA-4 antibody ticilimumab solution is housed.
The present invention also provides goods, and it comprises the container that the anti-CTLA-4 antibody of at least one heavy chain with ticilimumab and light-chain amino acid sequence and the mixture of chelating agen are housed.
The present invention also provides the composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen about 0.00001 to about 450 scope.
The present invention also provides the composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen is about 0.00001 to about 450 scope, and wherein said antibody comprises having the heavy chain of antibody ticilimumab and the monoclonal anti CTLA-4 antibody of light-chain amino acid sequence.
The present invention also provides the composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen about 0.0001 to about 100 scope.
The present invention also provides the composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen about 0.001 to about 10 scope.
The present invention also provides the composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen about 0.1 to about 1 scope.
The present invention also provides the composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen is about 0.00001 to about 450 scope, and wherein antibody is about 0.5 to the mol ratio of chelating agen.
The present invention also provides the composition of liquid medicine that comprises at least one human monoclonal anti-CTLA-4 antibodies and chelating agen, wherein said antibodies people CTLA-4.
The present invention also provides the pharmaceutical composition that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody that concentration is at least about 10mg/ml.
The present invention also provides the pharmaceutical composition that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 25mg/ml of about 10mg/ml.
The present invention also provides the pharmaceutical composition that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 200mg/ml of about 10mg/ml.
The present invention also provides the pharmaceutical composition that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody that concentration is about 20mg/ml.
The present invention also provides the compositions that comprises at least one chelating agen and at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2 and the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4.
The present invention also provides the compositions that comprises at least one chelating agen and at least one antibody, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 90% shown in SEQ ID NO:2 and the aminoacid sequence same with the light-chain amino acid sequence at least 90% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, and wherein said antibody comprises having the heavy chain of ticilimumab and the anti-CTLA-4 antibody of the monoclonal IgG2 of light-chain amino acid sequence.
The present invention also provides the method for the preparation of composition of liquid medicine, and it comprises at least one being had to the heavy chain of ticilimumab and the anti-CTLA-4 antibody of light-chain amino acid sequence mixes in solution with at least one chelating agen.
The present invention also provides the method for the neoplasia condition of illness being used for the treatment of in experimenter, it comprises that described compositions comprises to described experimenter's applicating liquid pharmaceutical composition: at least one for the treatment of effective dose has the heavy chain of ticilimumab and the anti-CTLA-4 antibody of light-chain amino acid sequence; With pharmaceutically acceptable chelating agen.
The present invention also provides the test kit for the preparation of the fluid composition of the antibody through stable, it comprises: the first container of the anti-CTLA-4 antibody-solutions of at least one heavy chain with ticilimumab and light-chain amino acid sequence is housed, and the second container of pharmaceutically acceptable chelating agen is housed.
The present invention also provides the composition of liquid medicine that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody that concentration is at least about 10mg/ml.
Accompanying drawing summary
Fig. 1 is bar diagram, its shown at 40 ℃ store reach within 7 weeks, by size exclusion chromatography (SEC), record afterwards at the various gathering percentage ratio being subject in test preparation;
Fig. 2 is bar diagram, its shown at 40 ℃ store reach after 7 weeks, by reduced form SDSPAGE (rSDSPAGE), record at various total (hydrolysis) impurity formation percentage ratio being subject in test preparation;
Fig. 3 is line chart, its shown when under the condition of accelerating at 40 ℃, store by SEC, record while reaching 24 weeks at the various gathering percentage ratio being subject in test preparation;
Fig. 4 is line chart, its shown when under the condition of accelerating at 40 ℃, store by rSDSPAGE, record while reaching 24 weeks at various total (hydrolysis) impurity formation percentage ratio being subject in test preparation;
Fig. 5 is line chart, its shown when under the condition of accelerating at 40 ℃, store by SEC, record while reaching 24 weeks at the various gathering percentage ratio being subject in test preparation;
Fig. 6 is line chart, its shown when under the condition of accelerating at 40 ℃, store by rSDSPAGE, record while reaching 24 weeks at various total (hydrolysis) impurity formation percentage ratio being subject in test preparation;
Fig. 7 is bar diagram, and it has shown when storing while reaching 24 weeks under the condition of accelerating at 40 ℃, as the function of EDTA level by SEC, record at the various gathering percentage ratio being subject in test preparation;
Fig. 8 is bar diagram, and it has shown when storing while reaching 24 weeks under the condition of accelerating at 40 ℃, as the function of EDTA level by rSDSPAGE, record at various total (hydrolysis) impurity formation percentage ratio being subject in test preparation;
Fig. 9 is line chart, its shown when under the condition of accelerating at 40 ℃, store by SEC, record while reaching 13 weeks at the various gathering percentage ratio being subject in test preparation;
Figure 10 is line chart, its shown when under the condition of accelerating at 40 ℃, store by rSDSPAGE, record while reaching 13 weeks at various total (hydrolysis) impurity formation percentage ratio being subject in test preparation; With
Figure 11, comprises Figure 11 A-11D, and it has shown nucleotide and the aminoacid sequence of anti-CTLA 4 antibody 11.2.1 (being now called ticilimumab).Figure 11 A has shown the full length nucleotide sequence (SEQ ID NO:1) of 11.2.1 heavy chain.Figure 11 B has shown the full length amino acid sequence (SEQ ID NO:2) of 11.2.1 heavy chain, and is shown in the aminoacid sequence (SEQ ID NO:5) of the 11.2.1 variable region of heavy chain between bracket " [] ".The aminoacid sequence of each 11.2.1 heavy chain CDR indicates with underscore.Described CDR sequence is as follows: CDR1:GFTFSSYGMH (SEQ ID NO:7); CDR2:VIWYDGSNKYYADSV (SEQ ID NO:8); And CDR3:DPRGATLYYYYYGMDV (SEQ ID NO:9).Figure 11 C has shown the nucleotide sequence (SEQ ID NO:3) of 11.2.1 light chain.Figure 11 D has shown the aminoacid sequence (SEQ ID NO:4) of total length 11.2.1 light chain, and is shown in the variable region of light chain (SEQ ID NO:6) between bracket " [] ".The aminoacid sequence of each CDR is as follows: CDR1:RASQSINSYLD (SEQ ID NO:10); CDR2:AASSLQS (SEQ ID NO:11); And CDR3:QQYYSTPFT (SEQ ID NO:12).
Detailed Description Of The Invention
Except as otherwise noted, conventionally according to the conventional method described in the various common and more professional list of references of knowing in this area and quote and discuss, carry out method of the present invention and technology in whole description.Referring to, for example, the people such as Sambrook, Molecular Cloning:A Laboratory Manual, the 2nd edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989); With the people such as Ausubel, CurrentProtocols in Molecular Biology, Greene Publishing Associates (1992); With Har low and Lane, Antibodies:ALaboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).That as this area, conventionally implement or as described herein, according to manufacturers instruction, carry out enzymatic reaction and purification technique.Analytical chemistry, synthetic organic chemistry and medical science and the pharmaceutical chemical term and laboratory procedure and technology of relating to described herein is in this area, to know and conventional.By the technology of standard, for chemosynthesis, chemical analysis, the preparation of medicine, prepares and sends, and experimenter's treatment.
Definition:
In order to help the following detailed description of reader understanding, provide following definition:
As used herein, terms " formulation " or " compositions ", when it relates to anti-CTLA-4 antibody, be the described antibody that hope is described and the pharmaceutically acceptable excipient including chelating agen is combined.For example, preparation of the present invention has storage life and/or the stability of raising compared with art-recognized preparation.
As used herein, term " antibody " refers to complete antibody or carries out the antigen-binding portion thereof of specific binding with complete antibody competition.Generally referring to, fundamental immunology, Ch.7 (Paul, W., ed., the 2nd edition .Rayen Press, N.Y. (1989)).Can produce antigen-binding portion thereof by recombinant DNA technology or by enzymatic or the chemical cleavage of complete antibody.In some embodiments, antigen-binding portion thereof comprises Fab, Fab ', F (ab ') 2, Fd, Fv, dAb and complementarity-determining region (CDR) fragment, single-chain antibody (scFv), chimeric antibody, double antibody and polypeptide, described polypeptide comprises to be enough to specific antigen in conjunction with at least part of antibody of giving this polypeptide.From N-end to C-end, ripe light chain and the equal inclusion region FR1 of weight chain variable domain, CDR1, FR2, CDR2, FR3, CDR3 and FR4.According to Kabat, Sequences of Proteins of Immunological Interest (Natiomal Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J.Mol.Biol.196:901-917 (1987), or the people such as Chothia, the definition of Nature 342:878-883 (1989), distributes to each domain by aminoacid.
As used herein, term " polypeptide " comprises the polypeptide analog of natural or artificial protein, protein fragments and protein sequence.Polypeptide can be monomer or polymer.
As used herein, Fd fragment refers to by V hand C hthe antibody fragment of 1 domain composition; Fv fragment is by the V of the single armed of antibody land V hdomain composition; DAb fragment (people such as Ward, Nature 341:544-546 (1989)) is by V hdomain composition.
Term " or its antigen-binding portion thereof ", when using together with term " antibody ", refers to and has amino terminal and/or carboxyl-terminal deletion but the identical polypeptide in relevant position in the sequence of remaining aminoacid sequence and natural generation wherein.In some embodiments, fragment length is at least 5,6,8 or 10 aminoacid.In other embodiments, the length of described fragment is at least 14, at least 20, at least 50 or at least 70,80,90,100,150 or 200 aminoacid.
As used herein, term " monoclonal antibody " refers to that available from the antibody of homogenizing be substantially the antibody of the colony of monospecific antibody, and described colony comprises the sudden change of the possible natural generation except existing on a small quantity or lacks identical colony C-end lysine.Monoclonal antibody is high degree of specificity, and it is for single antigen site.In addition, contrary from conventional (polyclone) antibody preparation (it comprises the different antibodies for different determinants (epi-position) conventionally), each monoclonal antibody is for the single determinant on antigen.Modifier " monoclonal " has indicated available from the feature of the antibody of the antibody colony of homogenizing substantially, but it is not interpreted as producing this antibody by any specific method.For example, monoclonal antibody used according to the invention can, by the earliest by Kohler, wait people, prepared by the described hybridoma method of Nature 256:495 (1975), or can pass through recombinant DNA method (for example, referring to, United States Patent (USP) 4,816,567) prepare.For example also can use at Clackson, Deng people, Nature 352:624-628 (1991) and Marks, wait people, and the technology of describing in J.Mol.Biol.222:581-597 (1991) separates " monoclonal antibody " from phage antibody library.
As used herein, term " antibody of separation " or " antibody of purification " refer to the antibody according to its origin or origin with 1 to 4 following aspect: (1) not with the natural combination component combination of accompanying with it under its native state, (2) not containing other albumen from same species, (3) by the cellular expression from different plant species, or (4) are not natural generations.Therefore, chemosynthesis or in the cell system different from the cell of its natural origin synthetic antibody combination component natural with it be separated and purification.Also can use protein purification technology well known in the art, by separation and purification, make antibody not basically contain natural combination component.Separate/example of the antibody of purification comprises and uses CTLA-4 through the anti-CTLA-4 antibody of affinity purification, in vitro by hybridoma or the synthetic anti-CTLA-4 antibody of other cell line with derive from the anti-CTLA-4 antibody of people of transgenic mice.
Separate/example of the antibody of purification comprises and uses CTLA-4 through the anti-CTLA-4 antibody of affinity purification, in vitro by hybridoma or the synthetic anti-CTLA-4 antibody of other cell line with derive from the anti-CTLA-4 antibody of people of transgenic mice.Therefore, in preferred embodiments, the purity that described anti-CTLA-4 antibody has is at least about 95% (weight of other components of the weight of the anti-CTLA-4 antibody of w/w-/except pharmaceutically acceptable excipient), and in further embodiment, described anti-CTLA-4 antibody has the purity of about 95%w/w to about 99.5%w/w.
When at least about sample display of 60 to 75% goes out the antibody of single kind, antibody is " substantially pure ", " homogenizing substantially " or " purification substantially ".Described antibody can be monomer or polymer.Substantially pure antibody can comprise about 50%, 60%, 70%, 80% or the antibody sample of 90%w/w conventionally, more generally about 95%, and preferably exceedes 99% purity.Can indicate by the method for knowing in many this areas purity or the homogeneity of antibody, for example, by the polyacrylamide gel electrophoresis of antibody sample, the dyestuff of then knowing in this area is to manifesting single polypeptide band after gel-colored.For some object, can be by obtaining higher resolution with the additive method for purification of knowing in HPLC or this area.
As used herein, term " people's antibody " wishes to comprise having and derive from the variable region of people's germline immunoglobulin sequences and the antibody of constant region.People's antibody of the present invention can comprise in for example CDR (particularly CDR3) can't help the amino acid residue (for example, by external random or direct mutagenesis or the sudden change that in by body, somatic mutation is introduced) of people's germline immunoglobulin sequences coding.But term " people's antibody ", as used herein, does not wish to comprise such antibody, wherein the CDR sequence of the germline that derives from for example mice of another kind of mammalian species is grafted on people's frame sequence.
As used herein, term " recombinant human antibody " hope comprises by recombination method to be prepared, express, everyone antibody that produces or separate, for example use the expressed antibody of recombinant expression carrier that is transfected into host cell, the antibody separating from the combination people antibody library of restructuring, from for human immunoglobulin gene, be for example, genetically modified animal (mice) separate antibody (referring to, for example, Taylor, L.D., Deng people (1992) Nucl.Acids Res.20:6287-6295), or by involving any other method preparation that the montage of human immunoglobulin gene's sequence is become to other DNA sequence, express, the antibody that produces or separate.Such recombinant human antibody has the variable region and the constant region that derive from people's germline immunoglobulin sequences.But, in certain embodiments, make such recombinant human antibody experience in vitro mutagenesis (or, when use is genetically modified animal for people Ig sequence, carry out body endosome cell mutation), thereby the aminoacid sequence in the VHHe VL district of recombinant antibodies is such sequence, although this sequence derives from people's germline VH and VL sequence associated, may not to be present in natively in people's antibody germline bank in vivo.
As used herein, the term " polynucleotide " being herein used interchangeably or " nucleic acid " refer to that length is the polymer form of the nucleotide of at least 10 bases, and described nucleotide can be the modified form of ribonucleotide or deoxyribonucleotide or any nucleotide.This term comprises strand and double chain form.Unless otherwise noted, " polynucleotide " or " nucleic acid " sequence comprises its complementary series.Therefore,, when mentioning the nucleic acid with particular sequence, should be understood to comprise its complementary strand with its complementary series.
As used herein, term " polynucleotide of separation " refers to polynucleotide or its some combinations in genome, cDNA or synthetic source, according to its origin or origin, the polynucleotide that separate have 1 to 3 following aspect: (1) does not combine with all or part of of polynucleotide existing together with described " polynucleotide of separation " natively, (2) be effectively connected to not and its natural polynucleotide that are connected, or (3) are not as natural generation of part of larger sequence.
As used herein, term " nucleotide of natural generation " comprises deoxyribonucleotide and ribonucleotide.Term " nucleotide of modification ", as used herein, comprises the nucleotide with glycosyl group etc. modification or that replace.Term " oligonucleotide key " comprises the oligonucleotide key such as thiophosphate, phosphorodithioate, seleno phosphate ester (phosphoroselenoate), two seleno phosphate esters (phosphorodiselenoate), phosphoroanilothioate, phoshoraniladate, phosphoramidate (phosphoroamidate) etc. herein.Referring to for example, the people such as LaPlanche, Nucl.Acids Res.14:9081 (1986); The people such as Stec, J.Am.Chem.Soc.106:6077 (1984); The people such as Stein, Nucl.Acids Res.16:3209 (1988); The people such as Zon, Anti-Cancer Drug Design6:539 (1991); The people such as Zon, Oligonucleotides and Analogues:APractical Approach, pp.87-108 (F.Eckstein, Ed., 0xfordUniversity Press, Oxford England (1991)); United States Patent (USP) 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), their disclosure is incorporated herein by reference.If need, oligonucleotide can comprise for detection of labelling.
" effectively connect " sequence comprise the expression control sequenc adjacent with genes of interest and with trans or in telekinesy to control the expression control sequenc of genes of interest.Term " expression control sequenc ", as used herein, refers to and realizes the expression of connected coded sequence and process necessary polynucleotide sequence.Expression control sequenc comprises suitable transcription initiation, termination, promoter and enhancer sequence; The effectively for example montage of RNA processing signal and polyadenylation signal; Stablize the sequence of kytoplasm mRNA; Strengthen the sequence (that is, Kozak consensus sequence) of translation efficiency; The sequence of Enhancin stability; With when wanting, increase the sequence of protein excretion.The character of this type of sequence depends on host living beings and difference; In prokaryote, this type of control sequence generally includes promoter, ribosome binding site and transcription terminator; In eukaryote, this type of control sequence generally includes promoter and transcription terminator.Term " control sequence " wish comprise (bottom line) its existence for express and processing be essential all component, the existence that also can comprise it is favourable additional assemblies, for example, targeting sequencing and fusion partners sequence.
As used herein, term " carrier " refers to the nucleic acid molecules that can transport coupled another kind of nucleic acid.In some embodiments, carrier is plasmid, extra DNA section can be connected into circular double stranded DNA ring wherein.In some embodiments, carrier is viral vector, wherein extra DNA section can be connected in viral genome.In some embodiments, carrier can import self-replicating (for example, having bacteria carrier and the additive type mammal carrier of antibacterial replication origin) in their host cell therein.In other embodiments, importing after host cell, carrier (for example, non-add type mammal carrier) can be integrated in the genome of described host cell, thereby described carrier can copy with host genome.In addition, some carrier can instruct and the expression of its gene being effectively connected.Such carrier is called " recombinant expression carrier " (or referred to as " expression vector ") herein.
As used herein, term " recombinant host cell " (or abbreviation " host cell ") refers to recombinant expression carrier is imported to cell wherein.Should the key to exercises, " recombinant host cell " and " host cell " not only refers to specific experimenter's cell, but also refers to the offspring of this type of cell.Because due to sudden change or environmental effect, some modification can occur in the successive generation, therefore such offspring in fact may be not identical with parental cell, but still within being included in the scope of term used herein " host cell ".
As used herein, term " combination specifically " refers to when antibody is with≤1 μ M, preferably≤1nM, and the dissociation constant of most preferably≤10pM is when antigen is combined.
As used herein, term " optionally hybridization " refer to can detect and combination specifically.Polynucleotide of the present invention, oligonucleotide and fragment thereof are optionally hybridized with nucleic acid chains under hybridization and wash conditions, and described condition makes the detectable amount of the detected combination to non-specific nucleic acid be reduced to minimum." high stringency " or " highly tight " condition can be used for obtaining selective cross condition known in the art and discussed herein.An example of " high stringency " or " highly tight " condition be under the hybridization temperature of 42 ℃ in the hybridization buffer of the salmon sperm DNA of the fragmentation of 6X SSPE or SSC, 50% Methanamide, 5X Denhardt ' s reagent, 0.5%SDS, 100 μ g/ml degeneration by polynucleotide incubation 12 to 16 hours (wherein polynucleotide can be fixed to the surface of solids for example film on) together with another kind of polynucleotide, then use 1X SSC, the lavation buffer solution of 0.5%SDS washs 2 times at 55 ℃.Also can be referring to people such as Sambrook, the same, pp.9.50-9.55.
Term " sequence homogeneity percentage ratio " refers to when just maximum corresponding when first continuous sequence and second continuous sequence are compared and compared in nucleotide sequence situation, the percentage ratio of corresponding residue.The length of sequence homogeneity comparison can be at least about 9 nucleotide, conventionally at least about 18 nucleotide, more commonly at least about 24 nucleotide, typically at least about 28 nucleotide, more typically at least about 32 nucleotide, and preferably at least about 36,48 or whole one section of sequence of more nucleotide.There are many algorithms of different known in the art that can be used for measuring nucleotide sequence homology.For example, can use FASTA, Gap or BESTFIT (it is Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, the program in Wisconsin) to carry out many nucleotide sequences.Comprise that the FASTA of for example program FASTA2 and FASTA3 provides comparison and sequence homogeneity percentage ratio (Pearson, the Methods Enzymol.183:63-98 (1990) of the best overlapping region between search sequence and search sequence; Pearson, Methods Mol.Biol.132:185-219 (2000); Pearson, Methods Enzymol.266:227-258 (1996); Pearson, J.Mol.Biol.276:71-84 (1998)).Except as otherwise noted, use the default parameter of specific program or algorithm.For example, (word length is as 6 take its default parameter can to use FASTA, with the NOPAM factor for rating matrix), or use Gap with its default parameter (as provided in GCG Version 6.1), measure the sequence homogeneity percentage ratio between nucleotide sequence.
Unless otherwise expressly stated, otherwise when mentioning " polynucleotide " or " nucleic acid " sequence, comprise its complementary series.Therefore,, when mentioning the nucleic acid with particular sequence, should be understood to comprise its complementary strand with its complementary series.
Term " similarity substantially " or " sequence similarity substantially ", when relating to nucleic acid or its fragment, refer to when using suitable nucleotide insertion or disappearance and another nucleic acid (or its complementary strand) to carry out the best comparison, at least about 85%, preferably at least about 90%, more preferably at least about nucleotide base of 95%, 96%, 97%, 98% or 99%, have nucleotide sequence homology, this is measured by any sequence homogeneity algorithm example FASTA described above, BLAST or Gap knowing.
When being applied to polypeptide, term " homogeneity substantially ", " homogeneity percentage ratio " or " % is same " refer to, two peptide sequences, when the best is compared, for example, by program GAP or BESTFIT and while using default breach weight (providing with program) to compare, total at least 70%, 75% or 80% sequence homogeneity, preferably at least 90% or 95% sequence homogeneity, and at least 97%, 98% or 99% sequence homogeneity more preferably.In certain embodiments, the difference of not same residue position is conservative amino acid displacement." conservative amino acid displacement " to be one of them amino acid residue had by another the have similar chemical property alternative displacement of amino acid residue of side chain R group of (for example, electric charge or hydrophobicity).Usually, conservative amino acid displacement does not change the functional characteristic of protein substantially.Two or more aminoacid sequences difference is each other in the situation of preservative replacement therein, can adjust upward sequence homogeneity percentage ratio to proofread and correct for the conservative character of displacement.For the method for carrying out this adjustment, know to those skilled in the art.Referring to, for example, Pearson, Methods Mol.Biol.243:307-31 (1994).The example with the aminoacid group of the side chain that has similar chemical property comprises 1) aliphatic lateral chain: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chain: serine and threonine; 3) side chain that comprises amide: agedoite and glutamine; 4) aromatic series side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acid side-chain: aspartic acid and glutamic acid; With 7) sulfur-containing side chain: cysteine and methionine.Conservative amino acid set of permutations is: Val-Leu-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and agedoite-glutamine.
Conventionally with sequence analysis software, measure the sequence homogeneity of polypeptide.Protein analysis software application distribute to various displacements, disappearance and other modify (comprising conservative amino acid displacement) similarity measure matching sequence.For example, GCG comprises the program such as " Gap " and " BESTFIT ", can with default parameter (as described in the specified default parameter of program) with as described in program determine that the polypeptide being closely related is for example from the sequence homology between the homeopeptide of different living species or between wild-type protein and its mutant or sequence homogeneity.Referring to for example GCG Version 6.1.Also can and adopt parameter default or that recommend to carry out many peptide sequences with FASTA, referring to GCG Version 6.1.(University of Wisconsin WI) FASTA (for example, FASTA2 and FASTA3) provides comparison and the sequence homogeneity percentage ratio of the best overlapping region between search sequence and search sequence.(Pearson，MethodsEnzymol.183：63-98(1990)；Pearson，Methods Mol.Biol.132：185-219(2000))。When sequence of the present invention is compared from the data base who comprises a large amount of sequences from different biologies, another kind of preferred algorithm is computer program BLAST, especially blastp or tblastn, it uses default parameter, as with as described in the default parameter that provides of program.Referring to, for example, the people such as Altschul, J.Mol.Biol.215:403-410 (1990); The people such as Altschul, Nucleic Acids Res.25:3389-402 (1997).The length of the peptide sequence comparing with regard to homology is generally at least about 16 amino acid residues, at least about 20 residues conventionally, and more generally at least about 24 residues, typically at least about 28 residues, and preferably exceed about 35 residues.When search comprises the data base from many different biological sequences, preferably comparing amino acid sequence.
The therapeutic outcome (it comprises treatment or the prevention of neoplasia condition of illness) of wanting for acquisition when " treatment effective dose " refers according to dosage administration and pass through the essential time period, effectively measure.Be noted that dose value can change with the severity of the condition of illness that will be alleviated.It is also understood that; for any specific experimenter; should and use compositions or the people's that supervision group compound is used professional judgement according to individual need; along with the time, adjust specifically according to dosage dosage regimen; and be appreciated that dosage range shown here is only exemplary and do not wish to limit scope or the enforcement of compositions required for protection.Similarly, the treatment effective dose of antibody or antibody moiety can be according to factor for example morbid state, age, sex and the weight of individuality, antibody or antibody moiety cause the ability of the reaction of wanting in individuality, and the route of administration of the described antibody preparation of wanting and changing.Treatment effective dose or such amount, any toxicity of wherein said antibody or antibody moiety or ill-effect are treated useful effect and exceed.
As used herein, for therapeutic purposes, term " experimenter " comprises any experimenter, preferably needs to treat the experimenter of neoplasia condition of illness.For prevention object, described experimenter is any experimenter, preferably in the risk in developing neoplasia condition of illness or be easy to develop the experimenter of neoplasia condition of illness.Organism alive, for example prokaryote and eukaryote wished to comprise in term " experimenter ".Experimenter's example comprises mammal, for example people, Canis familiaris L., cattle, horse, pig, sheep, goat, cat, mice, rabbit, rat and transgenic nonhuman animal.In particular of the present invention, described experimenter is people.
As used herein, the term " neoplasia " being herein used interchangeably and " neoplasia condition of illness " refer to because the new Growth of Cells causing is lost in the response for normal growth control, for example, refer to " superfluous natural disposition " Growth of Cells.Herein, neoplasia also can be exchanged and use with term " cancer ", and for object of the present invention; Cancer is neoplastic a kind of hypotype.As used herein, term " neoplasia condition of illness " also comprises other cellular abnormalities, for example hyperplasia, metaplasia (metaplasia) and abnormal development.Term neoplasia, metaplasia, abnormal development and hyperplasia are used interchangeably herein, and typically refer to the cell of experience abnormal cell growth.
As used herein, term " treatment " refers to therapeutic treatment and preventative or preventive measures, and wherein object is prevention or slows down (minimizing) by the pathological condition of targeting or condition of illness.Need the experimenter for the treatment of to comprise the experimenter who there is the experimenter of described condition of illness and tend to there is the experimenter of described condition of illness or will prevent therein described condition of illness.
When introducing element of the present invention or its preferred embodiment, article " a ", " an ", " the " and " described " wish to refer to exist one or more described elements.Term " comprises ", " comprising ", " containing ", " including " and " having " be to wish to refer to be included, and represent also to exist other elements except listed element.
Anti-CTLA-4 antibody:
According to the present invention, found that the stability of some monoclonal anti CTLA-4 antibody described herein can be by mixing described anti-CTLA-4 antibody to be improved with for example ethylenediaminetetraacetic acid of pharmaceutically acceptable chelating agen (" EDTA ") mutually.
Although be not wishing to be bound by theory, the existence that it is believed that chelating agen in compositions of the present invention contributes to improve the stability of described antibody polypeptides by the generation of one or more situations below reducing: gathering, fragmentation, oxidation, freeze/thaw unstability, variable color and/or the desamidation of anti-CTLA-4 antibody.The present invention includes and there is the chemistry that Comparatively speaking improves with previously disclosed antibody compositions and/or the anti-CTLA-4 antibody preparation of physical stability.
Therefore, in some aspects, the invention provides the compositions that comprises for example EDTA of pharmaceutically acceptable chelating agen and monoclonal anti CTLA-4 antibody or its antigen-binding portion thereof.In other respects, the anti-CTLA-4 antibody compositions of the aforementioned liquid that comprises chelating agen can comprise other pharmaceutically acceptable excipient, includes but not limited to that one or more are selected from the excipient of buffer agent, antioxidant, tonicity agent, surfactant and composition thereof.
The invention provides the new formulation of anti-CTLA-4 antibody.As used herein, phrase " anti-CTLA-4 antibody " refers to can be in conjunction with any antibody or its any part of any part of cytotoxic T lymphocyte associated protein 4 (" CTLA-4 ") polypeptide, and described CTLA-4 polypeptide can be present in any animal or therefrom separation.In certain embodiments, described CTLA-4 polypeptide is people CTLA-4 polypeptide.
For suitable anti-CTLA-4 antibody of the present invention, can be selected from polyclonal antibody or monoclonal antibody.In some aspects, monoclonal anti CTLA-4 antibody can be rodent antibody, chimeric antibody, humanized antibody or people's antibody.In other embodiments, monoclonal anti CTLA-4 antibody is the anti-CTLA-4 antibody of human monoclonal.
In certain embodiments, be suitable for anti-CTLA-4 antibody of the present invention and be included in those anti-CTLA-4 antibody of describing in the U.S. Patent number that belongs to the people such as Hanson 6,682,736 that December in 1999 submits on the 23rd and the method for the preparation of them.In other embodiments, be suitable for anti-CTLA-4 antibody of the present invention and comprise having at U.S. Patent number 6,682, the heavy chain of antibody and those anti-CTLA-4 monoclonal antibodies of light-chain amino acid sequence of called after 11.2.1 in 736.In other embodiments, be suitable for those anti-CTLA-4 monoclonal antibodies that anti-CTLA-4 antibody of the present invention comprises heavy chain and the light-chain amino acid sequence with antibody ticilimumab and ipilimumab.In other embodiments, be suitable for those anti-CTLA-4 monoclonal antibodies that anti-CTLA-4 antibody of the present invention comprises heavy chain and the light-chain amino acid sequence with antibody ticilimumab.
As used herein, by the antibody of coded representation, there is identical heavy chain and the light-chain amino acid sequence of monoclonal antibody obtaining with the hybridoma from there being same code.For example, monoclonal antibody 11.2.1 has heavy chain and the light-chain amino acid sequence identical with the monoclonal antibody obtaining from hybridoma 11.2.1.Therefore,, when mentioning antibody 11.2.1, comprise ticilimumab tM, it has the weight chain variable domain shown in the heavy chain shown in SEQ ID NO.2 and 4 and light-chain amino acid sequence and SEQ ID NO.5 and the light chain variable domain shown in SEQ ID NO.6.It is also included in the antibody that lacks end lysine on heavy chain, because this lysine is lost conventionally in preparation process in a certain proportion of antibody.
The difference of the aminoacid sequence in addition, can the CH based on them is selected this type of anti-CTLA-4 antibody.For example, anti-CTLA-4 antibody can be selected from the IgG classification with " γ " type heavy chain.Can determine by any method known in the art class and the subclass of anti-CTLA-4 antibody.Usually, can use antibody special for the antibody of certain kinds or subclass to determine class and the subclass of antibody.This antibody-like be purchased obtainable.Can determine class and subclass by ELISA or Western blotting and other technologies.Alternatively, can determine class and subclass by following manner: the heavy chain of antagonist and/or all or part of of the constant domain of light chain are checked order, the known amino acid sequence of the immunoglobulin of its aminoacid sequence and various types of and subclass is compared, thus class and the subclass of definite described antibody.
Anti-CTLA-4 antibody can be IgG, IgM, IgE, IgA or IgD molecule.In other embodiments, described anti-CTLA-4 antibody is IgG, and is IgG1, IgG2, IgG3 or IgG4 subclass.But, as what will appreciate that, conventionally do not wish to kill the cell of expressing CTLA-4.On the contrary, thus people just hope that will suppress CTLA-4 and its ligand binding slows down T cell and lower conventionally.A main mechanism of antibody killing cell is by complement combination and participates in CDC.The constant region of antibody is at the ability important role of antibodies complement and participation CDC.Therefore, people select the isotype of antibody the ability of complement combination to be provided or this ability is not provided conventionally.In situation of the present invention, usually, as mentioned above, using the antibody of cell killing is not preferred conventionally.There are many isotypes that can carry out the antibody of complement combination and CDC, include but not limited to following: muroid IgM, muroid IgG2a, muroid IgG2b, muroid IgG3, people IgM, human IgG1 and human IgG 3.The isotype that on the contrary, preferably can not carry out complement combination and CDC includes, but not limited to human IgG2 and human IgG 4.Mutually unusual except sequence of heavy chain, the difference of IgG antibody in its subclass is the number of disulfide bond and the length of hinge region.For example, IgG2 subclass has the several and distinct difference of other subclass.Known IgG2 and IgG4 subclass have 4 disulfide bond in its hinge region, and IgG1 has 2 disulfide bond, and IgG3 has 11 disulfide bond.Other differences of IgG2 antibody comprise the ability through Placenta Hominis of its minimizing, and IgG2 antibody can not bind lymphocytes Fc receptor.Therefore, in certain embodiments, anti-CTLA-4 antibody is subclass IgG2 or IgG4.In another preferred embodiment, anti-CTLA-4 antibody is subclass IgG2.
The difference of heavy chain amino acid sequence that in other embodiments, can be based on them is selected suitable anti-CTLA-4 antibody.For example, anti-CTLA-4 antibody of the present invention can have the following people V of use hthe people γ type heavy chain of arbitrary gene: V in germline gene h1, V h2, V h3, V h4 or V h5.In certain embodiments, anti-CTLA-4 antibody end user V h3 germline genes.In further embodiment, anti-CTLA-4 antibody end user V h3 germline genes and people DP-50 or DP-46 variable region of heavy chain, and in other embodiments, described anti-CTLA-4 antibody end user DP-50 variable region of heavy chain.DP-50 gene is also referred to as V h3-33 family gene.DP-46 gene is also referred to as V h3-30.3 family gene.In embodiment further, described anti-CTLA-4 antibody is used and is selected from D hthe people D of gene D1-26, DIR4 and DIR3 hgene, and in other embodiments, described anti-CTLA-4 antibody is used D1-26 people D hgene.In embodiment further, described anti-CTLA-4 antibody is used and is selected from J h4 and J h6 people J hgene, and in other embodiments, described anti-CTLA-4 antibody is used J h6 people J hgene.
In further embodiment, the difference of light-chain amino acid sequence that can be based on them is selected anti-CTLA-4 antibody.For example, suitable anti-CTLA-4 antibody can have lambda light chain or κ light chain.But in certain embodiments, anti-CTLA-4 antibody of the present invention has κ light chain.In some embodiments that described anti-CTLA-4 antibody comprises κ light chain therein, the polynucleotide of the variable domains of coding light chain comprise people V κl5, O12, L2, B3, L15 or A27 gene and people J κ 1, J κ 2, J κ 3, J κ 4 or J κ 5 genes.In some embodiments that described antibody comprises κ light chain therein, light chain variable domain (V l) partly by people V κo12 or V κa27 gene and people J κ3 or J κ4 gene codes.In particular of the present invention, light chain variable domain is by people V κo12/J κ 3 gene codes.
In addition, described antibody can comprise such heavy chain amino acid sequence, and described heavy chain amino acid sequence comprises and derives from people V hpeople's cdr amino acid sequence of 3-30 or 3-33 gene or preservative replacement wherein or somatic mutation.Be appreciated that described V hthe FR1 to FR3 of the variable region of heavy chain of 3-33 gene code antibody molecule.Therefore, the present invention includes such antibody, the sequence of the FR1 to FR3 of described antibody and antibody ticilimumab shares at least 85%, and more preferably at least 90%, more preferably at least 91%, more preferably at least 94%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and 100% homogeneity most preferably.
Described antibody also can further comprise and derive from A27 or O12 gene CDR district in its light chain, or it can comprise antibody ticilimumab CDR district.
In other embodiments of the present invention, described antibody suppression CTLA4 and B7-1, with B7-2 or with combination between the two.Preferably, described antibody can suppress the combination with B7-1, IC 50for about 100nM or lower, more preferably, approximately 10nM or lower, for example about 5nM or lower, more preferably, approximately 2nM or lower, or more preferably, approximately 1nM or lower.Similarly, described antibody can suppress the combination with B7-2, IC 50for about 100nM or lower, more preferably, 10nM or lower, for example, more preferably, approximately 5nM or lower, more preferably, approximately 2nM or lower, or more preferably, 1nM or lower.
In addition, in another embodiment, anti-CTLA 4 antibody is about 10 for the binding affinity of CTLA4 -8or larger affinity, more preferably, about 10 -9or larger affinity, more preferably, about 10 -10or larger affinity, and more preferably, about 10 -11or larger affinity.
Described anti-CTLA 4 antibody comprises and the antibody that has the heavy chain of antibody ticilimumab and the antibody competition of light-chain amino acid sequence and be combined.In addition, described anti-CTLA 4 antibody can be competed combination with antibody ipilimumab.
In another embodiment, described antibody preferably with there is the heavy chain of antibody ticilimumab and the antibody cross competition of sequence of light chain, variable heavy chain and variable sequence of light chain and/or heavy chain and light chain CDR sequence.For example, described antibody can be in conjunction with the epi-position with the heavy chain of antibody ticilimumab and the antibody institute combination of light-chain amino acid sequence, variable sequence and/or CDR sequence.In another embodiment, described antibody with there is the heavy chain of MDX-D010 and the antibody cross competition of sequence of light chain or antigen binding sequence.
In another embodiment, the heavy chain that use comprises antibody ticilimumab (comprises CDR-1, the aminoacid sequence of CDR-2 and CDR-3) and light chain (comprise CDR-1, the aminoacid sequence of CDR-2 and CDR-3) or comprise and there is the anti-CTLA-4 antibody of sequence that CDR sequence changes and implement the present invention, described CDR sequence changes to be selected to guard and changes, wherein said conservative change is selected from non-polar residue and is substituted by other non-polar residues, polarity charged residue is substituted by other polarity neutral residues, polarity charged residue is substituted by other polarity charged residues, displacement with residue like structure, non-conservation displacement, wherein said non-conservation displacement is selected from that polarity charged residue is replaced into polarity neutral residue and non-polar residue is replaced into polar residues, adds and disappearance.
In further embodiment of the present invention, described antibody comprises 10,7,5 or 3 amino acid changes that are less than of germline sequence in framework region HeCDR district.In another embodiment, described antibody comprises to be less than in 5 amino acid change He CDR districts and comprises and be less than 10 changes in framework region.In a preferred embodiment, described antibody comprises to be less than in 3 amino acid change He CDR districts and comprises and be less than 7 changes in framework region.In preferred embodiments, the change in framework region is guarded, and the change in He CDR district is somatic mutation.
More preferably, described antibody on heavy chain and light chain, or respectively with heavy chain or the light chain of antibody ticilimumab, share 100% sequence homogeneity or sequence similarity.
In another embodiment, described antibody is on heavy chain and light chain full length sequence, or respectively on heavy chain or light chain, with germline V κa27, germline V κ(it is V for O12 and germline DP50 hthe allele of 3-33 locus) sequence share at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, more preferably at least 99% sequence homogeneity or sequence similarity.More preferably, the sequence of heavy chain of described antibody and germline DP50 and/or share 100% sequence homogeneity or sequence similarity with the sequence of light chain of germline A27 or germline O12.
In one embodiment, described antibody is on heavy chain and light chain variable region sequence or respectively on heavy chain or light chain variable region sequence, with antibody 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, ticilimumab, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, the sequence of ipilimumab shares at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, more preferably at least 99% sequence (for example, aminoacid, nucleic acid or both) homogeneity or sequence similarity.More preferably, described antibody is shared 100% sequence homogeneity or sequence similarity with the heavy chain or the sequence of light chain that are selected from following antibody on heavy chain and light chain variable region sequence or respectively: 3.1.1,4.1.1,4.8.1,4.10.2,4.13.1,4.14.3,6.1.1, ticilimumab, 11.6.1,11.7.1,12.3.1.1,12.9.1.1, ipilimumab.
In another embodiment, on weight chain variabl area sequence, (it is V to described antibody with heavy chain germline DP50 hthe allele of 3-33 locus) weight chain variabl area sequence or with germline V κa27 or germline V κit is few 80% that the light chain variable region sequence of O12 is shared to, and more preferably at least 85%, more preferably at least 90%, more preferably at least 94%, more preferably at least 95%, more preferably at least 99% sequence homogeneity or sequence similarity.More preferably, the sequence of described heavy chain of antibody region sequence and germline DP50 or have 100% sequence homogeneity or sequence similarity with the sequence of light chain of germline A27 or germline O12.
In one embodiment of the invention, described antibody shares at least 80% with FR1 to the FR4 region sequence of antibody ticilimumab aspect the heavy chain from FR1 to FR4, light chain or both sequences, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence homogeneity or sequence similarity.More preferably, described antibody is shared 100% sequence homogeneity or sequence similarity in the heavy chain from FR1 to FR4, light chain or both sequences with antibody ticilimumab.
In another embodiment of the invention, described antibody shares at least 80% with FR1 to the FR3 region sequence of germline DP50 aspect the sequence of heavy chain from FR1 to FR3, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably about 100% sequence homogeneity or sequence similarity.
In another embodiment of the invention, described antibody aspect the sequence of light chain from FR1 to FR4 with germline V κa27 or germline V κfR1 to the FR4 region sequence of O12 shares at least 80%, and more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably about 100% sequence homogeneity or sequence similarity.
In one embodiment of the invention, the heavy chain of described antibody and antibody ticilimumab, light chain or both, CDR-1, CDR-2 and CDR-3 sequence share at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence homogeneity or sequence similarity.More preferably, described antibody is shared 100% sequence homogeneity or sequence similarity with antibody ticilimumab in heavy chain, light chain or both, CDR-1, CDR-2 and CDR-3 sequence.
In another embodiment of the invention, described antibody shares at least 80% with CDR-1 and the CDR-2 sequence of germline DP50 aspect heavy chain CDR-1 and CDR-2 sequence, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably about 100% sequence homogeneity or sequence similarity.
In another embodiment of the invention, described antibody aspect light chain CDR-1, CDR-2 and CDR-3 sequence with germline V κa27 or germline V κcDR-1, the CDR-2 of O12 and CDR-3 sequence share at least 80%, more preferably at least 85%, and more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably about 100% sequence homogeneity or sequence similarity.
In one embodiment, described anti-CTLA-4 antibody is the antibody that is called ticilimumab.
Heavy chain and light chain people germline gene that table 1 has been listed anti-CTLA-4 monoclonal antibody 11.2.1 (that is, ticilimumab) derive.
Table 1:
Figure 2006800156704A00800011
By biasing toward, utilize DP-50 variable region of heavy chain to produce some anti-CTLA-4 antibody of the present invention.DP-50 gene is also referred to as V h3-33 family gene.At XenoMouse tMin mice, exist and exceed 30 different functional weight chain variable genes for generation of antibody.Therefore, with the combination of antigen and the combinatorial property of functional activity aspect, skewed popularity shown antibody-AI preferably in conjunction with primitive.
In some embodiments, antibody is single-chain antibody (scFv), wherein V land V hdomain matches formation monovalent molecule by synthetic connector, and described synthetic connector can produce with the form of single protein chain them.The people such as Bird, Science 242:423-426 (1988); With the people such as Huston, Proc.Natl.Acad.Sci.USA85:5879-5883 (1988).In some embodiments, antibody is double antibody, that is, be bivalent antibody, wherein V hand V ldomain is expressed on single polypeptide chain, can not allow to carry out two pairings between domain on same chain, thereby force the complementary structure territory of described domain and another chain to match and produce the connector of two antigen binding sites but use is too short.Referring to for example, the people such as Holliger P., Proc.Natl.Acad Sci.USA 90:6444-6448 (1993), and the people such as Poljak R.J., Structure 2:1121-1123 (1994).In some embodiments, can will from one or more CDR of antibody of the present invention, covalently or non-covalently be integrated in molecule to become the immunoadhesin in conjunction with CTLA-4 specifically.In this type of embodiment, described CDR can be integrated into the part of larger polypeptide chain, it covalently can be connected to another polypeptide chain, or can noncovalently integrate it.
In another embodiment, anti-CTLA-4 antibody has the selectivity (or specificity) for CTLA-4, at least large 100 times of its selectivity for any other polypeptide of described selectivity ratios.In some embodiments, anti-CTLA-4 antibody is not shown any detectable specific binding to any other protein except CTLA-4.According to the instruction of this description, can measure the selectivity of anti-CTLA-4 antibody for CTLA-4 by the method for knowing in this area.For example, can measure selectivity with Western blotting, FACS, ELISA or RIA.Therefore, in some embodiments, monoclonal anti CTLA-4 antibody can be specifically in conjunction with CTLA-4.
In some embodiments, the C-end lysine of the heavy chain of anti-CTLA-4 antibody of the present invention does not exist.In some embodiments, the C-end lysine of the heavy chain of anti-CTLA-4 antibody of the present invention does not exist.Of the present invention, aspect some, anti-CTLA-4 antibody does not comprise signal polypeptide conventionally, because signal polypeptide is removed in post translational modification process.In various embodiments of the present invention, in the heavy chain of anti-CTLA-4 antibody and light chain one or both comprise signal sequence (or part of signal sequence).In other embodiments of the present invention, the heavy chain of anti-CTLA-4 antibody and light chain do not comprise signal sequence.
Table 2 has been listed the sequence flag symbol (SEQ IDNO) of nucleic acid of the variable region of encoding heavy chain and light chain and the aminoacid sequence of the anti-CTLA-4 monoclonal antibody 11.2.1 of corresponding prediction.
Table 2:
Figure 2006800156704A00800021
In some embodiments, the V that described nucleic acid molecules comprises coding monoclonal antibody 11.2.1 lthe nucleotide sequence of aminoacid sequence (SEQ ID NO:4) or its part.In some embodiments, described part comprises at least CDR2 district.In some embodiments, the aminoacid sequence of the light chain CDR of antibody described in described nucleic acid coding.In some embodiments, described part is the continuous part that comprises CDR1-CDR3.In some aspects, light chain CDR1 aminoacid sequence is indicated by SEQ ID NO:10, and light chain CDR2 aminoacid sequence is indicated by SEQ ID NO:11, and light chain CDR3 aminoacid sequence is indicated by SEQ ID NO:12.
In other embodiments, the V of described nucleic acid molecule encoding and SEQ ID NO:4 laminoacid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% same V laminoacid sequence.In other embodiments, the light-chain amino acid sequence that described nucleic acid molecules comprises coding SEQ IDNO:4 or the nucleotide sequence of its part.Nucleic acid molecules of the present invention is included in the nucleic acid of the nucleic acid array hybridizing of the lower light-chain amino acid sequence with the SEQ ID NO:4 that encodes of highly tight condition (for example above-described those).
In further embodiment, described nucleic acid molecules comprises nucleotide sequence, the V of described nucleotide sequence coded 11.2.1 haminoacid sequence (SEQ ID NO:2) or there is conservative amino acid sudden change and/or altogether 3 or non-conservation amino acid replacement still less described sequence at least partly.In various embodiments, the one or more CDR of described sequential coding district, preferably CDR3 district, all 3 GeCDR districts, the continuous part that comprises CDR1-CDR3, or complete V hdistrict.In some aspects, heavy chain CDR1 aminoacid sequence is indicated by SEQ ID NO:7, and heavy chain CDR2 aminoacid sequence is indicated by SEQ ID NO:8, and heavy chain CDR3 aminoacid sequence is indicated by SEQ ID NO:9.
In some embodiments, the V of described nucleic acid molecule encoding and SEQ ID NO:2 haminoacid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% same V haminoacid sequence.In embodiment further, the heavy chain amino acid sequence that described nucleic acid molecules comprises coding SEQ ID NO:2 or the nucleotide sequence of its part.Nucleic acid molecules of the present invention is included in the nucleic acid of the nucleotide sequence hybridization of the lower heavy chain amino acid sequence with the SEQ IDNO:2 that encodes of highly tight condition (for example above-described those).
In some aspects, the invention provides the composition of liquid medicine that comprises at least one people's antibody in conjunction with the separation of CTLA-4 and pharmaceutically acceptable excipient (comprising chelating agen), wherein said antibody comprises employing people V hthe V of 3-33 germline gene haminoacid sequence.
In other respects, the invention provides and comprise at least one composition of liquid medicine in conjunction with people's antibody of the separation of CTLA-4, wherein said antibody comprises with SEQ ID NO:2 and has the heavy chain amino acid sequence of at least 90% sequence homogeneity and have the light-chain amino acid sequence of at least 90% sequence homogeneity with SEQ ID NO:4.
In other respects, the invention provides and comprise at least one composition of liquid medicine in conjunction with people's antibody of the separation of CTLA-4, wherein said antibody comprises with SEQ ID NO:2 and has the heavy chain amino acid sequence of at least 95% sequence homogeneity and have the light-chain amino acid sequence of at least 95% sequence homogeneity with SEQ ID NO:4.
In other respects, the invention provides and comprise at least one composition of liquid medicine in conjunction with people's antibody of the separation of CTLA-4, wherein said antibody comprises with SEQ ID NO:2 and has the heavy chain amino acid sequence of at least 99% sequence homogeneity and have the light-chain amino acid sequence of at least 99% sequence homogeneity with SEQ ID NO:4.
In other respects, described antibody comprises heavy chain amino acid sequence and light-chain amino acid sequence, the variable region that described heavy chain amino acid sequence comprises SEQ ID NO:2, and the described light-chain amino acid sequence variable region that comprises SEQ ID NO:4.Aspect further, described antibody comprises the heavy chain amino acid sequence that contains SEQ ID NO:5 and the light-chain amino acid sequence that contains SEQ ID NO:6.Aspect further, described antibody comprises the heavy chain amino acid sequence that contains SEQ ID NO:2 and the light-chain amino acid sequence that contains SEQ ID NO:4.In other respects, described antibody comprises and contains the people FR1, the FR2 that by reading frame and CDR1, CDR2 and CDR3 sequence, are effectively connected and the V of FR3 sequence haminoacid sequence, described people FR1, FR2 and FR3 sequence are utilized people V h3-33 gene family.
In one embodiment, described anti-CTLA-4 antibody is ticilimumab (also referred to as CP-675,206), and it has heavy chain and the light-chain amino acid sequence of antibody ticilimumab.
In one embodiment of the invention, described anti-CTLA-4 antibody specificity ground is in conjunction with the comformational epitope on people CTLA-4.In other embodiments, described anti-CTLA-4 antibody suppresses people's tumor growth being administered to after experimenter.
The preparation of monoclonal anti CTLA-4 antibody preparation:
Conventionally anti-CTLA-4 antibody is formulated as for the pharmaceutical composition to experimenter's parenteral administration.In one embodiment, described pharmaceutical composition is fluid composition.In another embodiment, described pharmaceutical composition is fluid composition.
Compositions of the present invention comprises one or more anti-CTLA-4 monoclonal antibody of the present invention and pharmaceutically acceptable excipient, and described excipient comprises histidine and/or chelating agen.Liquid preparation of the present invention comprises one or more anti-CTLA-4 monoclonal antibody of the present invention and pharmaceutically acceptable excipient, and described excipient comprises histidine and/or chelating agen.
The preparation that term " pharmaceutical composition " refers to for example to allow the form of the biologic activity performance effect of active component to exist." pharmaceutically acceptable excipient " (vehicle, additive) be moderately (that is, safely) to experimenter, use those excipient of the effective dose of the active component that provides used.Term " excipient " or " carrier " are used in reference to inert substance herein, and it is typically used as diluent, vehicle, antiseptic, binding agent or the stabilizing agent of medicine.As used herein, term " diluent " refers to pharmaceutically acceptable (being safe and avirulent for using to people) solvent, and can be used for preparation liquid preparation herein.Exemplary diluent includes, but not limited to sterilized water and water for injection,bacteriostatic (BWFI).
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody and pharmaceutically acceptable chelating agen.In another embodiment, the present invention relates to the composition of liquid medicine that comprises anti-CTLA-4 antibody and EDTA.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody and DTPA.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen and pharmaceutically acceptable buffer agent.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen and histidine.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, EDTA and histidine.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, DTPA and histidine.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen and pharmaceutically acceptable tonicity agent.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen and trehalose.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, EDTA and trehalose.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, DTPA and trehalose.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen and pharmaceutically acceptable surfactant.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, EDTA and pharmaceutically acceptable surfactant.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, DTPA and pharmaceutically acceptable surfactant.In another embodiment, the present invention relates to comprise anti-CTLA-4 antibody, be selected from the pharmaceutically acceptable chelating agen of EDTA and DTPA and the compositions of polyoxyethylene sorbitan monoleate.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable buffer agent and pharmaceutically acceptable surfactant.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, histidine and pharmaceutically acceptable surfactant.In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, histidine and polyoxyethylene sorbitan monoleate.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen, pharmaceutically acceptable buffer agent and pharmaceutically acceptable surfactant.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen, pharmaceutically acceptable buffer agent and pharmaceutically acceptable tonicity agent.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody, pharmaceutically acceptable chelating agen, pharmaceutically acceptable buffer agent, pharmaceutically acceptable surfactant and pharmaceutically acceptable tonicity agent.
In another embodiment, the present invention relates to the compositions that comprises anti-CTLA-4 antibody and histidine.
The anti-CTLA-4 antibody being present in compositions can be previously described antibody in the application.In one embodiment, described compositions comprises anti-CTLA-4 antibody, and described antibody comprises and the V shown in SEQ ID NO:4 lthe V that aminoacid sequence 90%, 95% or 99% is same laminoacid sequence, also comprises and the V shown in SEQ ID NO:2 hthe V that aminoacid sequence 90%, 95% or 99% is same haminoacid sequence.In another embodiment, described compositions is included as the anti-CTLA-4 antibody of monoclonal anti CTLA-4 antibody 11.2.1.
The anti-CTLA-4 antibody being present in composition of liquid medicine can be previously described antibody in the application's book.In one embodiment, described composition of liquid medicine comprises anti-CTLA-4 antibody, and described antibody comprises and the V shown in SEQ ID NO:4 lthe V that aminoacid sequence 90%, 95% or 99% is same laminoacid sequence, also comprises and the V shown in SEQ ID NO:2 hthe V that aminoacid sequence 90%, 95% or 99% is same haminoacid sequence.In another embodiment, described composition of liquid medicine is included as the anti-CTLA-4 antibody of monoclonal anti CTLA-4 antibody 11.2.1.
Normally at least about 0.1 mg/ml of the concentration of anti-CTLA-4 antibody (mg/ml) or higher, at least about 1.0mg/ml or higher, at least about 10mg/ml or higher, at least about 50mg/ml or higher, at least about 100mg/ml or higher or at least about 200mg/ml or higher in composition of liquid medicine of the present invention.In certain embodiments, the concentration of anti-CTLA-4 antibody conventionally at about 0.1mg/ml to about 200mg/ml, approximately 0.5mg/ml is to about 100mg/ml, approximately 1mg/ml is to about 70mg/ml, approximately 2.0mg/ml is to about 65mg/ml, approximately 5.0mg/ml is to about 50mg/ml, approximately 10mg/ml is to about 35mg/ml, approximately 15mg/ml, to the scope of about 25mg/ml, or is about 20mg/ml.In one embodiment, in composition of liquid medicine the concentration of anti-CTLA-4 antibody at about 50mg/ml to the scope of about 100mg/ml.In some embodiments, when wanting can to use higher antibody concentration when compositions is carried out to subcutaneous delivery.
As used herein, term " chelating agen " typically refers to the excipient that can form with metal ion at least one key (for example, the key of covalent bond, ionic bond or other types).Chelating agen is multidentate ligand normally, and it can be used as and the stabilizing agent that may promote that instable substance classes is mutually compound in selected fluid composition.Conventionally, the compound that can be used as chelating agen has the functional group of being rich in electronics.The suitable functional group of being rich in electronics comprises hydroxy-acid group, hydroxyl and amino.The arrangement of these groups in amino polybasic carboxylic acid, hydroxyl polycarboxylic acid, hydroxyl amino carboxylic acid etc. causes forming the part having in conjunction with the ability of metal.
But the present invention wishes to be not limited to the main chelating agen working by the ability of chelating agen and metal ion formation key.Therefore, the present invention wishes not to be subject to any specific mechanism (by described mechanism, chelating agen works in preparation of the present invention) restriction, and the excipient that is called chelating agen herein can be realized its characteristic by forming with described chelating agen and metal ion the mechanism that the ability of key has nothing to do completely.
Being suitable for chelating agen of the present invention comprises, but be not limited to glycine, 2-(2-amino-2-oxoethyl) aminoethane sulphonic acid (BES), deferoxamine (DEF), citric acid, nicotiamide and dexycholate that amino polybasic carboxylic acid, hydroxyl amino carboxylic acid, N-replace.The example of suitable amino polybasic carboxylic acid comprises ethylenediaminetetraacetic acid (EDTA), DTPA (DTPA), nitrilotriacetic acid(NTA) (NTA), N-2-acetylaminohydroxyphenylarsonic acid 2-iminodiacetic acid (ADA), two (aminoethyl) glycol ether, N, N, N ', N '-tetraacethyl (EGTA), trans-diamino-cyclohexane tetraacethyl (DCTA), glutamic acid and aspartic acid.The example of suitable hydroxyl amino carboxylic acid comprises N hydroxyethyliminodiacetic acid (HIMDA), N, N-bis--ethoxy glycine (bicine) and N-(trihydroxy methyl methyl) 10 glycine (tricine).The example of the glycine that suitable N-replaces is glycylglycine.The example of suitable dexycholate is NaTDC.The present invention also comprises the mixture of two or more chelating agen.
For chelating agen of the present invention, (for example can be used as the free acid of this compound or free alkali form, " EDTA " being herein used interchangeably or " edetic acid ") or as corresponding salt form (for example, corresponding acid-addition salts or base addition salts, for example disodium edetate) exist.Suitable acid-addition salts for example comprises alkali metal salt (for example, sodium or potassium salt), alkali salt (for example, calcium salt), and can prepare salt with other weak binding metal ions.As known in the art, the character of salt and depend on the carboxyl number of existence and residing pH when stability chelating agen is provided wait the electric charge number being neutralized.As also known in this area, chelating agen has variable and bond strength particular target ion.As further illustrating, the suitable salt of EDTA comprises EDTAP dipotassium ethylene diamine tetraacetate, disodium edetate, CaEDTA, edetate sodium, edetate trisodium and edetic acid potassium; And the salt of suitable deferoxamine (DEF) is deferoxamine mesylate (DFM).
For chelating agen of the present invention, can exist with the form anhydrous, solvation or hydration of this compound or corresponding salt.When chelating agen exists with form solvation or hydration, its can solvation or the various states of hydration (comprise, for example, the form of anhydrous, hydration, two hydrations and three hydrations) exist.As further illustrating, the suitable hydrates of EDTA is EDETATE SODIUM dihydrate; And the suitable form of citric acid comprises anhydrous citric acid, citric acid monohydrate compound and citrate trisodium dihydrate.
For the suitable chelating agen of antibody compositions of the present invention, also comprise, for example, bind metal ion in solution so that its can not with obtainable O 2thereby reaction minimize or prevent hydroxyl radical free radical produce those chelating agen, thereby described hydroxyl radical free radical be dissociate with described antibody response the described antibody of degrading.Chelating agen can reduce the formation of reduced form oxygen kind in compositions of the present invention, reduces the formation of sour kind (for example, desamidation), reduces antibody aggregation, and/or reduces antibody fragment.This type of chelating agen can reduce or prevent the degraded of the antibody of preparing in the situation that protecting without chelating agen.
When mentioning the concentration of chelating agen, wish that described concentration represents the molar concentration of free acid or the free alkali form of described chelating agen.For example, the concentration of the chelating agen in some composition of liquid medicine is conventionally in the scope of the extremely about 5.0mM of extremely about 10.0mM, about 15 μ M of about 0.01 μ M extremely about 50mM, about 1 μ M, the extremely about 1.0mM of about 0.01mM or the extremely about 0.5mM of about 0.03mM.In certain embodiments, in composition of liquid medicine, the concentration of chelating agen can be about 0.01mM, 0.02mM, 0.027mM, 0.03mM, about 0.04mM, about 0.05mM, about 0.06mM, about 0.07mM, about 0.10mM, about 0.20mM, about 0.26mM, about 0.27mM, about 0.30mM, about 0.31mM, about 0.34mM, about 0.40mM, about 0.50mM, about 1.0mM.In certain embodiments, the concentration of chelating agen is about 0.027mM, about 0.05mM, approximately 0.13mM or about 0.27mM.In one embodiment, the concentration of chelating agen is about 0.05mM.In another embodiment, the concentration of chelating agen is about 0.13mM.
Unless otherwise noted, listed concentration is the concentration under environmental condition (that is, 25 ℃ and atmospheric pressure) herein.Scope between above-mentioned chelating agen concentration is also wished as a part of the present invention.For example, wish to comprise that the combination of using above-mentioned any value is as the scope of the value of the upper limit and/or lower limit.
In one embodiment, described chelating agen is selected from EDTA, DTPA, DFM and composition thereof.In another embodiment, described chelating agen is DFM.In another embodiment, described chelating agen is EDTA.In another embodiment, described chelating agen is DTPA.In another embodiment, described composition of liquid medicine comprises EDTA, and it measures general in the scope of the extremely about 10.0mM of extremely about 20.0mM, about 15 μ M of about 0.01 μ M extremely about 50mM, about 1 μ M, the extremely about 5.0mM of about 0.01mM or the extremely about 1mM of about 0.03mM.In certain embodiments, the EDTA concentration in composition of liquid medicine can be about 0.01mM, 0.02mM, 0.027mM, 0.03mM, about 0.04mM, about 0.05mM, about 0.06mM, about 0.07mM, about 0.10mM, about 0.20mM, about 0.26mM, about 0.27mM, about 0.30mM, about 0.31mM, about 0.34mM, about 0.40mM, about 0.50mM or about 1.0mM.In certain embodiments, the concentration of EDTA is about 0.027mM, about 0.05mM, approximately 0.13mM or about 0.27mM.In one embodiment, the concentration of EDTA is about 0.05mM.In another embodiment, the concentration of EDTA is about 0.13mM.In another embodiment, composition of liquid medicine comprises EDTA with the amount of about 0.27mM.
As noted above, except chelating agen, compositions of the present invention optionally also can comprise pharmaceutically acceptable buffer agent.As used herein, term " buffer agent " refers to and allows liquid antibody formulation can resist the compositions adding of the variation of pH.In certain embodiments, the buffer agent adding allows liquid antibody formulation can resist the variation of pH by the effect of its Acid-Base conjugation component.
For example, can prepare the preparation through buffering by the L-Histidine-HCl (L-Histidine-hydrogen chloride) and the L-Histidine that add amount suitable for reaching required pH.But in other embodiments, the buffer agent adding allows liquid antibody formulation can resist the variation of pH by the effect of its Acid-Base conjugation component.As the second example, can prepare the preparation through buffering by for example hydrochloric acid of acid and the L-Histidine that add amount suitable for reaching required pH.
The example of suitable buffer agent comprises, but be not limited to, acetate (for example sodium acetate), succinate (for example sodium succinate), gluconate, citrate and other organic acid buffer agents, such as aminoacid (for example include but not limited to, histidine), acetic acid, phosphoric acid and phosphate, Ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazoles, carbonic acid and bicarbonate, succinic acid, sodium benzoate and benzoate, gluconate, edetate (EDTA), acetate, malate, imidazoles, tris, the buffer agent of phosphate and composition thereof.In one embodiment, described buffer agent is acetate.
In another embodiment, described buffer agent is histidine.Histidine parent material for the preparation of compositions of the present invention can exist with different forms.For example, described histidine can be histidine enantiomer (for example, L-or D-enantiomer) or the salt form of the free acid of racemic form, histidine or free alkali form, histidine is (for example, mono-hydrochloric salts, dihydrochloride, hydrobromate, sulfate or acetate), the solvation form of histidine, the hydrated form (for example, monohydrate) of histidine or the anhydrous form of histidine.For the preparation of the histidine alkali of described compositions and/or the purity of salt, can be generally at least about 98%, at least about 99% or at least about 99.5%.As used herein, the in the situation that of histidine, term " purity " refers to the chemical purity of histidine, this is to understand in this area, for example The MerckIndex, the 13rd edition, described in the people ed. such as O ' Neil (Merck & Co., 2001).
When mentioning the concentration of buffer agent, wish that described concentration represents the molar concentration of free acid or the free alkali form of described buffer agent.For example, when being present in some composition of liquid medicine, the concentration of buffer agent can be at about 0.1mM to the scope of about 100mM.In one embodiment, the concentration of buffer agent is that about 1mM is to about 50mM.In another embodiment, the concentration of buffer agent is that about 5mM is to about 30mM.In multiple embodiments, the concentration of buffer agent is about 1mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM or about 100mM.In one embodiment, in pharmaceutical composition, the concentration of histidine is about 10mM.In another embodiment, the L-Histidine (existing with alkali form) that pharmaceutical composition comprises about 10mM.In another embodiment, in pharmaceutical composition, the concentration of histidine is about 20mM.In another embodiment, the L-Histidine (existing with base form) that pharmaceutical composition comprises about 20mM.Scope between above-mentioned chelating agen concentration is also wished for part of the present invention.For example, wish to comprise that the combination of using above-mentioned any value is as the scope of the value of the upper limit and/or lower limit.
Usually, use buffer agent to keep acceptable pH level (described pH level can affect the stability of antibody) in composition of liquid medicine.Composition of liquid medicine is conventionally buffered to and makes pH remain on about 4 to about 8, about 4.5 to about 7, about 5.0 to 6.5 or about 5.3 to about 6.3 scope.Scope between above-mentioned pH is also wished as a part of the present invention.For example, wish to comprise that the combination of using above-mentioned any value is as the scope of the value of the upper limit and/or lower limit.In one embodiment, composition of liquid medicine is buffered to and makes pH remain on about 5.5.In another embodiment, composition of liquid medicine is buffered to and makes pH remain on about 6.0.
As noted above, except chelating agen, compositions of the present invention also can comprise pharmaceutically acceptable tonicity agent alternatively.As used herein, term " tonicity agent " refers to the excipient of the osmotic pressure of scalable liquid antibody formulation.In certain embodiments, tonicity agent can be adjusted to the osmotic pressure of liquid antibody formulation etc. and to ooze state, and described like this antibody preparation and experimenter's systemic cell is at physical compatibility.In other embodiments, " tonicity agent " can contribute to the raising of the stability of any anti-CTLA-4 antibody described herein." waiting and ooze " preparation is the preparation with the osmotic pressure substantially the same with human blood.Deng the osmotic pressure that oozes preparation and conventionally have about 250 to 350 mOsm.The osmotic pressure that the has preparation lower than the osmotic pressure of human blood described in term " hypotonic ".Correspondingly, term " height oozes " is for describing the preparation of had osmotic pressure higher than the osmotic pressure of human blood.Can use for example vapour pressure or freezing type permeability manometer (ice-freezing type osmometer) to measure isotonicity.
Tonicity agent for the preparation of compositions of the present invention can exist with multi-form.When mentioning tonicity agent, wish that this title of tonicity agent comprises the form that all these are different.For example, tonicity agent can exist with following form: enantiomeric form (for example, L-or D-enantiomer) or racemic form; For example α of isomer or β, comprise α, α or β, β, or α, β or β, α; Free acid or free alkali form; Hydrate forms (for example, monohydrate); Or anhydrous form.
In one embodiment, tonicity agent is saccharide.As used herein, term " saccharide " refers to the molecule into polyhydroxy-alcohol.Saccharide is commonly referred to carbohydrate, and can comprise not commensurability sugar (saccharide) unit, for example, and monosaccharide, disaccharide and polysaccharide.The saccharide that is suitable for use in the present invention as tonicity agent comprises, but be not limited to, be selected from the sugar of fructose, glucose, mannose, sorbose, xylose, lactose, maltose, sucrose, glucosan, pullulan, dextrin, cyclodextrin, soluble starch, hetastarch, water-soluble glucan and its mixture.
In another embodiment, tonicity agent is polyhydric alcohol.As used herein, term " polyol " refers to the excipient with multiple hydroxyls, and comprises sugar (reproducibility and nonreducing sugar), sugar alcohol and saccharic acid.In one embodiment, described polyhydric alcohol has for example, molecular weight lower than about 600kD (, in about scope of 120 to about 400kD)." reducing sugar " be comprise reducible metal ion or with albumen in lysine or the sugar of the hemiacetal group of other aminoacid covalent reaction, and " nonreducing sugar " is the sugar without these character of reducing sugar.The polyhydric alcohol that is suitable for use in the present invention as tonicity agent comprises, but be not limited to, be selected from the polyhydric alcohol of mannitol, trehalose, Sorbitol, erythritol, isomalt, lactose, maltose alcohol, xylitol, glycerol, lactitol, propylene glycol, Polyethylene Glycol, inositol and composition thereof.In one embodiment, tonicity agent is the nonreducing sugar that is selected from trehalose, sucrose and composition thereof.
In one embodiment, tonicity agent is mannitol.In another embodiment, tonicity agent is PEARLITOL 25C.In another embodiment, tonicity agent is trehalose.In another embodiment, tonicity agent is α α-trehalose dihydrate compound.In another embodiment, tonicity agent is sucrose.
In one embodiment, in composition of liquid medicine the concentration of tonicity agent about 1mM to about 600mM, approximately 1mM to approximately 400mM, 1mM to approximately 300mM or 200mM to the scope of about 275mM.In another embodiment, tonicity agent is mannitol, and is present in composition of liquid medicine with the concentration of about 247mM.In another embodiment, tonicity agent is trehalose, and is present in composition of liquid medicine with the concentration of about 222mM.In another embodiment, tonicity agent is trehalose, and is present in composition of liquid medicine with the concentration of about 238mM.In another embodiment, tonicity agent is sucrose, and is present in composition of liquid medicine with the concentration of about 263mM.
In one embodiment, in composition of liquid medicine the concentration of tonicity agent about 1mg/ml to about 300mg/ml, approximately 1mg/ml to about 200mg/ml or approximately 50mg/ml to the scope of about 150mg/ml.In another embodiment, tonicity agent is mannitol, and is present in composition of liquid medicine with the concentration of about 45mg/ml mM.In another embodiment, tonicity agent is trehalose, and is present in composition of liquid medicine with the concentration of about 84mg/ml.In another embodiment, tonicity agent is trehalose, and is present in composition of liquid medicine with the concentration of about 90mg/ml.In another embodiment, tonicity agent is sucrose, and is present in composition of liquid medicine with the concentration of about 90mg/ml.
In one embodiment, tonicity agent is salt, for example sodium chloride.In one embodiment, when tonicity agent is salt, in composition of liquid medicine, the concentration of salt is in the scope of the extremely about 20mg/ml of about 1mg/ml.In another embodiment, tonicity agent is sodium chloride, and in composition of liquid medicine, the concentration of sodium chloride is about 8.18mg/ml.
Scope between above-mentioned tonicity agent concentration is also wished as a part of the present invention.For example, wish to comprise that the combination of using above-mentioned any value is as the scope of the value of the upper limit and/or lower limit.
Scope between above-mentioned tonicity agent concentration is also wished as a part of the present invention.For example, wish to comprise that the combination of using above-mentioned any value is as the scope of the value of the upper limit and/or lower limit.
As noted above, except chelating agen, compositions of the present invention also can comprise pharmaceutically acceptable surfactant alternatively.As used herein, term " surfactant " refers to the capillary excipient that can change liquid antibody formulation.In certain embodiments, described surfactant reduces the surface tension of liquid antibody formulation.In other embodiments, " surfactant " can contribute to the raising of the stability of any anti-CTLA-4 antibody described herein.For example, surfactant can reduce preparation antibody gathering and/or minimize the granule in preparation formation and/or reduce absorption.Surfactant also can improve the stability of antibody during freeze/thaw and afterwards.
Suitable surfactant comprises Polysorbate surfactant, poloxamer (for example poloxamer 18 and 407), for example Triton X-100 of triton surfactant
Figure 2006800156704_0
, for example Tween 20 of Polysorbate surfactant with Tween 80
Figure 2006800156704_2
, sodium lauryl sulphate, sodium lauryl sulfate, OG sodium (sodium octyl glycoside), lauryl-sulfobetaines, myristyl-sulfobetaines, sub-oil base (linoleyl)-sulfobetaines, stearyl-sulfobetaines, lauryl-sarcosine, myristyl-sarcosine, sub-oil base-sarcosine, stearyl-sarcosine, sub-oil base-betanin, myristyl-betanin, cetyl-betanin, dodecanamide propyl-betanin, cocamidopropyl propyl amide-betanin, sub-oleamide propyl group-betanin, myristamide propyl group-betanin, palmidopropyl-betanin, isostearoyl amine propyl group-betanin, myristamide propyl group-dimethylamine, palmidopropy1-dimethylamine, isostearoyl amine propyl group-dimethylamine, sodium methylcocoyltaurate, methyl oil base taurine disodium, chlorination dihydroxypropyl peg 5 linoleammonium, Polyethylene Glycol, polypropylene glycol and its mixture.
In one embodiment, surfactant is Polysorbate surfactant, and described Polysorbate surfactant comprises that at least one is selected from the excipient of polysorbate 20, Polysorbate 21, polysorbate 40, polysorbate 60, Polysorbate 61, polysorbate 65, polyoxyethylene sorbitan monoleate, sorbimacrogol oleate 100, polysorbate 85 and its mixture.In another embodiment, described composition of liquid medicine comprises polyoxyethylene sorbitan monoleate.
When being present in compositions, the concentration of surfactant is conventionally in the scope of the extremely about 10mg/ml of about 0.01mg/ml, the extremely about 5.0mg/ml of about 0.05mg/ml, the extremely about 1.0mg/ml of about 0.1mg/ml or the extremely about 0.7mg/ml of about 0.2mg/ml.In another embodiment, surfactant exists with the amount of about 0.2mg/ml.In another embodiment, surfactant exists with the amount of about 0.5mg/ml.In one embodiment, the polyoxyethylene sorbitan monoleate that composition of liquid medicine comprises about 0.2mg/ml.In another embodiment, the polyoxyethylene sorbitan monoleate that composition of liquid medicine comprises about 0.4mg/ml.In another embodiment, the polyoxyethylene sorbitan monoleate that composition of liquid medicine comprises about 0.5mg/ml.
Scope between above-mentioned surfactant concentration is also wished as a part of the present invention.For example, wish to comprise that the combination of using above-mentioned any value is as the scope of the value of the upper limit and/or lower limit.
Except chelating agen, compositions of the present invention also can comprise pharmaceutically acceptable antioxidant alternatively.Suitable antioxidant includes, but not limited to methionine, sodium thiosulfate, catalase and platinum.For example, composition of liquid medicine can comprise the methionine of concentration in the scope of the extremely about 100mM of 1mM, and the concentration of methionine is about 27mM especially.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and chelating agen, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4.
In one embodiment, the present invention includes the compositions that comprises at least one human monoclonal anti-CTLA-4 antibodies and chelating agen, wherein said antibodies people CTLA-4.
In one embodiment, the present invention includes the composition of liquid medicine that comprises at least one human monoclonal anti-CTLA-4 antibodies and chelating agen, wherein said antibodies people CTLA-4.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody that concentration is at least about 10mg/ml, at least approximately 15mg/ml, at least about 20mg/ml or at least about 25mg/ml.
In one embodiment, the present invention includes the composition of liquid medicine that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 200mg/ml of about 10mg/ml.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 200mg/ml of about 15mg/ml.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 200mg/ml of about 20mg/ml.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 200mg/ml of about 50mg/ml.
In one embodiment, the present invention includes the composition of liquid medicine that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 200mg/ml of about 100mg/ml.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody of concentration within the scope of the extremely about 25mg/ml of about 10mg/ml.
In one embodiment, the present invention includes the compositions that comprises at least one antibody and pharmaceutically acceptable excipient, described antibody comprises the aminoacid sequence same with the heavy chain amino acid sequence at least 95% shown in SEQ ID NO:2, and also comprise the aminoacid sequence same with the light-chain amino acid sequence at least 95% shown in SEQ ID NO:4, wherein said antibodies people CTLA-4, wherein said compositions comprises the antibody that concentration is about 20mg/ml.
In one embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.01mg/ml to about 200mg/ml, and about 0.3 μ M is to the chelating agen of about 50mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and about 3 μ M are to the chelating agen of about 5.0mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and the chelating agen of about 0.27mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and about 0.3 μ M is to the EDTA of about 50mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and about 3 μ M are to the EDTA of about 10.0mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and about 0.1mM is to the EDTA of about 1.0mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and the EDTA of about 0.27mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and about 3 μ M are to the DTPA of about 5.0mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 100mg/ml, and about 3 μ M are to the deferoxamine of about 5.0mM.
In one embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.01mg/ml to about 200mg/ml, and about 1mM is to the histidine of about 100mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, and about 3 μ M are to the chelating agen of about 5.0mM, and about 1mM is to the histidine of about 100mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, and about 3 μ M are to the chelating agen of about 5.0mM, and about 10mM is to the trehalose of about 400mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, about 3 μ M are to the chelating agen of about 5.0mM, the extremely approximately histidine of 100mM of the approximately extremely approximately trehalose of 400mM of 10mM, and about 1mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, about 3 μ M are to the chelating agen of about 5.0mM, approximately 10mM is to the trehalose of about 400mM, the extremely approximately polyoxyethylene sorbitan monoleate of 10mM of the approximately extremely approximately histidine of 100mM of 1mM, and about 0.005mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, about 3 μ M are to the EDTA of about 5.0mM, approximately 10mM is to the tonicity agent of about 400mM, the extremely approximately surfactant of 10mM of the approximately extremely approximately buffer agent of 100mM of 1mM, and about 0.005mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, about 3 μ M are to the EDTA of about 5.0mM, approximately 10mM is to the tonicity agent of about 400mM, the extremely approximately surfactant of 10mM of the approximately extremely approximately histidine of 100mM of 1mM, and about 0.005mM.
In another embodiment, described composition of liquid medicine comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, about 3 μ M are to the EDTA of about 5.0mM, approximately 10mM is to the trehalose of about 400mM, the extremely approximately surfactant of 10mM of the approximately extremely approximately histidine of 100mM of 1mM, and about 0.005mM.
Of the present invention aspect some, the anti-CTLA-4 antibody compositions of described liquid comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 0.1mg/ml to about 200mg/ml, approximately 1mM is to the histidine of about 100mM, approximately 0.005mM is to the polyoxyethylene sorbitan monoleate of about 10mM, about 3 μ M are to the EDTA of about 5.0mM, and about 10mM is to the trehalose of about 400mM.
In other aspects of the present invention, the anti-CTLA-4 antibody compositions of described liquid comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 1.0mg/ml to about 100mg/ml, approximately 10mM is to the histidine of about 50mM, approximately 0.01mM is to the polyoxyethylene sorbitan monoleate of about 1.0mM, about 3 μ M are to the EDTA of about 5.0mM, and about 100mM is to the trehalose of about 300mM.
In other aspects of the present invention, the anti-CTLA-4 antibody compositions of described liquid comprises the monoclonal anti CTLA-4 antibody ticilimumab of about 10mg/ml to about 50mg/ml, approximately 10mM is to the histidine of about 30mM, approximately 0.05mM is to the polyoxyethylene sorbitan monoleate of about 0.5mM, the extremely approximately trehalose of 250mM of the approximately extremely approximately EDTA of 1mM of 0.1mM, and about 200mM.
In other aspects of the present invention, the monoclonal anti CTLA-4 antibody ticilimumab that the anti-CTLA-4 antibody compositions of described liquid comprises about 20mg/ml, the approximately histidine of 20mM, the approximately polyoxyethylene sorbitan monoleate of 0.15mM, the approximately EDTA of 0.27mM, and the trehalose of about 222mM.
In another embodiment, the present invention relates to the stable composition of liquid medicine that comprises anti-CTLA-4 antibody and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of described chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen is about 0.00001 to about 450, about 0.0001 to about 100, about 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 within about 1 scope, or be about 0.5.
In another embodiment, the present invention relates to the stable composition of liquid medicine that comprises ticilimumab and pharmaceutically acceptable chelating agen, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, with the molar concentration of described chelating agen at about 0.003mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen is about 0.00001 to about 450, about 0.0001 to about 100, about 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 within about 1 scope, or be about 0.5.
In another embodiment, the present invention relates to comprise ticilimumab, the stable composition of liquid medicine of pharmaceutically acceptable chelating agen and histidine, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, the molar concentration of described chelating agen is in the scope of the extremely about 50mM of about 0.003mM, with the molar concentration of histidine at about 1mM to the scope of about 100mM, and wherein the mol ratio of antibody and chelating agen is about 0.00001 to about 450, about 0.0001 to about 100, about 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 within about 1 scope, or be about 0.5.
In another embodiment, the present invention relates to comprise ticilimumab, the stable composition of liquid medicine of pharmaceutically acceptable chelating agen and histidine, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, the molar concentration of described chelating agen is in the scope of the extremely about 50mM of about 0.003mM, with the molar concentration of histidine at about 10mM to the scope of about 50mM, and wherein the mol ratio of antibody and chelating agen is about 0.0001 to about 100, about 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 within about 1 scope, or be about 0.5.
In another embodiment, the present invention relates to comprise ticilimumab, the stable composition of liquid medicine of pharmaceutically acceptable chelating agen and histidine, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, the molar concentration of described chelating agen is in the scope of the extremely about 50mM of about 0.003mM, with the molar concentration of histidine at about 10mM to the scope of about 30mM, and wherein the mol ratio of antibody and chelating agen is about 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 within about 1 scope, or be about 0.5.
In another embodiment, the present invention relates to comprise ticilimumab, the stable composition of liquid medicine of pharmaceutically acceptable chelating agen and histidine, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, the molar concentration of described chelating agen is in the scope of the extremely about 50mM of about 0.003mM, with the molar concentration of histidine at about 10mM to the scope of about 30mM, and wherein the mol ratio of antibody and chelating agen is about 0.001 to about 10, about 0.01 to about 5, about 0.1 within about 1 scope, or be about 0.5.
In another embodiment, the present invention relates to the stable composition of liquid medicine that comprises ticilimumab, pharmaceutically acceptable chelating agen and histidine, the molar concentration of wherein said antibody is in the scope of the extremely about 1.35mM of about 0.0006mM, the molar concentration of described chelating agen is in the scope of the extremely about 50mM of about 0.003mM, with the molar concentration of histidine be about 20mM, and wherein the mol ratio of antibody and chelating agen, about 0.001 to about 10, about 0.01 to about 5, about 0.1 within about 1 scope, or is about 0.5.
Produce method and the antibody-producting cell system of anti-CTLA-4 antibody:
Can be by prepare antibody of the present invention with transgenic mice, described transgenic mice has the genomic essential part of the generation people antibody of insertion, but described mice is caught producing aspect endogenous rodent antibody, is being defect.Therefore, such mice can produce human normal immunoglobulin's molecule and antibody, and is being defect aspect generation muroid immunoglobulin molecules and antibody.
Technology for obtaining such mice is described below.
May produce transgenic animal (for example, mice), it can produce the complete bank (repertoire) of people's antibody after immunity inoculation in the situation that not producing endogenous immunoglobulin.But, especially, the embodiment that transgenic produces mice and derives from its antibody is disclosed in the U.S. Patent number 6,682,736 that belongs to the people such as Hanson.By using this technology, can prepare in conjunction with the antibody of MAdCAM and the hybridoma of this antibody-like of generation.
People's antibody has been avoided some potential problems relevant to the antibody with muroid or rat variable region and/or constant region.The existence of the protein in this type of muroid or rat source can cause the quick removing of antibody maybe can cause the experimenter who uses who has accepted this antibody-like to produce the immunne response for described antibody.
Heavy chain of antibody bonding pad (J in chimeric and germ line mutation type mice for example, has been described h) inhibition completely that causes endogenous antibody to produce of the homozygous deletion of gene.People's germline immunoglobulin gene array (array) is transferred in such germ line mutation type mice and will causes for example, after antigen (, CTLA-4) is attacked generation people antibody.Referring to, for example, the people such as Jakobovits, Proc.Natl.Acad.Sci.USA, 90:2551 (1993); The people such as Jakobovits, Nature, 362:255-258 (1993); The people such as Bruggermann, Year in Immuno., 7:33 (1993); With the people such as Duchosal, Nature 355:258 (1992).People's antibody also can derive from phage display library (people such as Hoogenboom, J.Mol.Biol., 227:381 (1991); The people such as Marks, J.MoL Biol., 222:581-597 (1991); The people such as Vaughan, Nature Biotech 14:309 (1996)).
In some embodiments, can pass through such as XENOMOUSE of immunity inoculation non-human transgenic animal tMmice produces the anti-CTLA-4 antibody of people, and the genome of described mice comprises human immunoglobulin gene, and described like this recombined small-mouse can produce people's antibody.XENOMOUSE tMmice is through engineered mouse species, the large fragment that it comprises human immunoglobulin heavy chain and light chain gene seat, and be defect aspect mouse antibodies generation.XENOMOUSE tMmice produces one-tenth proper manners (adult-like) the people bank of complete people's antibody, and produces antigenic specificity people antibody.In some embodiments, described XENOMOUSE tMmice comprises people's antibody V gene bank of about 80% by germline configuration (germlineconfiguration) yeast artificial chromosome (YAC) fragment of the megabase size of importing people's heavy chain gene seat and κ light chain gene seat.In other embodiments, XENOMOUSE tMmice also comprises approximately all lambda light chain seats.Referring to, for example, the people such as Green, Nature Genetics 7:13-21 (1994) and United States Patent (USP) 5,916,771,5,939,598,5,985,615,5,998,209,6,075,181,6,091,001,6,114,598,6,130,364,6,162,963 and 6,150,584.Also can be referring to WO 91/10741, WO 94/02602, WO 96/34096, WO96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00/09560 and WO 00/037504.
In some embodiments, the non-human animal who comprises human immunoglobulin gene is the animal with human normal immunoglobulin " minigene seat (minilocus) ".In minigene seat method, by comprising from the independent gene of Ig locus, simulate external source Ig locus.Therefore, by one or more V hgene, one or more D hgene, one or more J hgene, μ constant domain and the second constant domain (preferably, γ constant domain) are formed into the construct for inserting animal.Except other, at United States Patent (USP) 5,545,807,5,545,806,5,569,825,5,625,126,5,633,425,5,661,016,5,770,429,5,789,650,5,814,318,5,591,669,5,612,205,5,721,367, in 5,789,215 and 5,643,763, the method has been described.
Therefore, in some embodiments, can be by producing people's antibody with CTLA-4 antigen immune inoculation non-human animal, described non-human animal comprises some or all of human immunoglobulin heavy chains and light chain gene seat in its genome.
In some embodiments, described CTLA-4 antigen is CTLA-4 that separate and/or purification.In preferred embodiments, described CTLA-4 antigen is people CTLA-4.In some embodiments, the fragment that described CTLA-4 antigen is CTLA-4.In some embodiments, at least one epi-position that described CTLA-4 fragment comprises CTLA-4.In other embodiments, described CTLA-4 antigen is to express in its surface or cross the cell of expressing CTLA-4 or its immunogenic fragments.In other embodiments, described CTLA-4 antigen is CTLA-4 fusion rotein.Can use known technology from natural origin, to be purified into CTLA-4.
In preferred embodiments, described non-human animal is XENOMOUSE tManimal (AbgenixInc., Fremont, CA).Spendable another kind of non-human animal is the transgenic mice of being produced by Medarex (Medarex, Inc., Princeton, NJ).
Can carry out immunity inoculation animal by any method known in the art.Referring to, for example, Harlow and Lane, Antibodies:A Laboratory Manual, New York:ColdSpring Harbor Press, 1990.For for example method of mice, rat, sheep, goat, pig, cattle and horse of immunity inoculation non-human animal, in this area, know.Referring to, for example, Harlow and Lane (the same), and United States Patent (USP) 5,994,619.In preferred embodiments, CTLA-4 antigen is used with immune stimulatory and replied together with adjuvant.Exemplary adjuvant comprises completely or incomplete Freund's adjuvant, RIBI (muramyldipeptide) or ISCOM (immunostimulating complex).This type of adjuvant can be by polypeptide is isolated in local deposits thing and protects it to avoid rapid dispersion, or they can comprise the material of stimulation of host excreted factor, and the described factor is the chemoattractant of macrophage and immune other components.Preferably, if using polypeptide, immunization scheme can comprise twice or the using of polypeptide described in more times within the period of several weeks so.
With after CTLA-4 antigen immune inoculation animal, can obtain antibody and/or antibody produced cell from described animal.In some embodiments, by blood-letting or put to death animal and obtain the serum that comprises anti-CTLA-4 antibody from animal.Because it is available from described animal, thus this serum can be used, can be from this serum adaptive immune globulin fraction, or can be purified into anti-CTLA-4 antibody from this serum.
In some embodiments, from separate hang oneself immunity inoculation animal cell Dispersal risk produce property immortalized cell line.After immunity inoculation, put to death animal, then make lymph node and/or spleen B cellular immortalization.The method of cellular immortalization is comprised, but be not limited to, use Oncogene Transfection cell, use carcinogenic viral infection cell, cultured cell under the condition of selecting immortalized cells, make cell accept carcinogenic or mutagenic compounds, cell and for example skeleton oncocyte of immortalized cells are merged, and make tumor suppressor gene inactivation.Referring to, for example, Harlow and Lane (the same).In preferred embodiments, through the animal of immunity inoculation, be to express human immunoglobulin gene's non-human animal, and by spleen B cell fusion to the myeloma cell line from the species identical with this non-human animal.In a more preferred embodiment, the described animal through immunity inoculation is XENOMOUSE tManimal, and described myeloma cell line is nonsecreting type mouse myeloma.In the embodiment being more preferably, described myeloma cell line P3-X63-AG8-653.If adopted with myeloma cell, merge, so described myeloma cell does not preferably secrete immunoglobulin polypeptides (nonsecreting type cell line).Screen immortalized cells with CTLA-4, its part or the cell of expressing CTLA-4.In preferred embodiment, use enzymoimmunoassay (ELISA) or radioimmunoassay to carry out Preliminary screening.The example of ELISA screening is provided in WO 00/37504.
Select, clone such as hybridoma of anti-CTLA-4 antibody produced cell, and the feature of just wanting (comprises surging growth, high antibody production and the antibody feature of wanting, further screens as discussed further below).Can be in syngeneic animal, lack immune animal for example in nude mice in body amplified hybridization tumor, or in cell culture in amplification in vitro hybridoma.The method of selection, clone and amplified hybridization tumor is known to those skilled in the art.
As will appreciate that, can be in the cell line except hybridoma cell line recombinant expressed antibody of the present invention.The coding cDNA of specific antibodies or the nucleotide sequence of genomic clone can be used for transforming suitable mammal or nonmammalian host cell.
The present invention also comprises the nucleic acid molecules of the anti-CTLA-4 antibody of encoding.In some embodiments, heavy chain and the light chain of the anti-CTLA-4 immunoglobulin of different nucleic acid molecule encodings.In other embodiments, heavy chain and the light chain of the anti-CTLA-4 immunoglobulin of same nucleic acid molecule encoding.In one embodiment, described nucleic acid coding anti-CTLA-4 antibody of the present invention.
Can separate the coding heavy chain of this antibody or the nucleic acid molecules of complete light chain or its part from any source that produces anti-CTLA-4 antibody.In various embodiment, from separate the B cell of animal of personal anti-CTLA-4 immunity inoculation or from derive from the immortalized cells of this type of B cell of expressing anti-CTLA-4 antibody, isolate described nucleic acid molecules.The method that separates the mRNA of encoding antibody is known in the art.Referring to, for example, Sambrook, waits people, Molecular Cloning, the 3rd edition, the 3rd volume (1989).Described mRNA can be used for being created in the cDNA using in the polymerase chain reaction (PCR) of antibody gene or cDNA clone.In preferred embodiments, separate described nucleic acid molecules from hybridoma, described hybridoma has from non-human transgenic animal's human normal immunoglobulin and produces cell as one of its fusion partners.In the embodiment being more preferably, described human normal immunoglobulin produces cell separation from XENOMOUSE tManimal.In another embodiment, described human normal immunoglobulin produces cell from above-mentioned inhuman, non-mice transgenic animal.In another embodiment, described separate nucleic acid is from inhuman, non-transgenic animal.The described nucleic acid molecules separating from non-human animal can be used for to for example humanized antibody.
In some embodiments, the encode nucleic acid of heavy chain of anti-CTLA-4 antibody of the present invention can comprise coding V of the present invention hthe nucleotide sequence of domain, this nucleotide sequence is connected to the nucleotide sequence of coding from the heavy chain constant domain in any source by reading frame.Similarly, the encode nucleic acid molecules of light chain of anti-CTLA-4 antibody of the present invention can comprise coding V of the present invention lthe nucleotide sequence of domain, this nucleotide sequence is connected to the nucleotide sequence of coding from the light chain constant domain in any source by reading frame.
Of the present invention further aspect, by encoding heavy chain variable domains (V h) and light chain variable domain (V l) nucleic acid molecules be " transformed " into full length antibody gene.In one embodiment, by following manner by coding V hor V lthe nucleic acid molecules of domain is transformed into full length antibody gene: V will encode hor V lthe nucleic acid molecules of domain inserts encoding heavy chain constant domain (C respectively h) or light chain constant domain (C l) expression vector in, thereby V hsection is effectively connected to the C in carrier hsection, and V lsection is effectively connected to the C in carrier lsection.In another embodiment, by following manner by coding V hand/or V lthe nucleic acid molecules of domain is transformed into full length antibody gene: use the Protocols in Molecular Biology of the standard V that will encode hand/or V lthe nucleic acid molecules connection of domain is for example linked to coding C hand/or C lthe nucleic acid molecules of domain.The nucleotide sequence of people's heavy chain and light chain immunoglobulin constant domains gene is known in this area.Referring to, for example, the people such as Kabat, Sequences of Proteins ofImmunological Interest, the 5th edition, NIH Publ.No.91-3242,1991.Then, can be from having imported the nucleic acid of total length heavy chain and/or light chain of encoding described in the cells of nucleic acid molecules of coding total length heavy chain and/or light chain, and separate anti-CTLA-4 antibody.
The present invention also provides the carrier that comprises code book and invent the heavy chain of anti-CTLA-4 antibody or the nucleic acid molecules of its antigen-binding portion thereof.The present invention also provides the carrier that comprises the encode light chain of this antibody-like or the nucleic acid molecules of its antigen-binding portion thereof.The present invention further provides the carrier of the nucleic acid molecules that comprises encoding fusion protein, modified antibody, antibody fragment and its probe.
In some embodiments, by following manner, express anti-CTLA-4 antibody of the present invention or antigen-binding portion thereof: the coded portion as above obtaining or the DNA of full-length light chains and heavy chain are inserted in expression vector so that described gene is effectively connected to essential expression control sequenc, for example, transcribe and translate control sequence.Expression vector comprises plasmid, retrovirus, and adenovirus, adeno associated virus (AAV), plant virus is cauliflower mosaic virus such as, tobacco mosaic virus (TMV), cosmid, YAC, the episome in EBV source etc.Described antibody gene is connected into carrier, so that transcribe and translate that control sequence performance wants that it regulates the function of transcribing and translating of described antibody gene in carrier.Select expression vector and expression control sequenc with compatible with the expression host cell being used.Light chain of antibody gene and heavy chain of antibody gene can be inserted in carrier separately.In preferred embodiments, two genes are all inserted in same expression vector.Method by standard (for example, connect the complementary restriction site on antibody gene fragment and carrier, if or there is no restriction site, carry out flush end connection) antibody gene is inserted in expression vector.
Carrier is to be coded in people C complete in function easily hor C lthe carrier of immunoglobulin sequences, it has the suitable restriction site through transformation, thereby makes any V hor V lsequence can easily be inserted and express, as described above.In examples of such carriers, conventionally between the donor splicing site in the J region of inserting and the montage acceptor site before people's C-structure territory, montage occurs, and montage also can be positioned at people C hmontage location in outer demonstration occurs.Polyadenylation and tanscription termination occur at the place, natural dyeing body position in downstream, described coding region.The described recombinant expression carrier signal peptide that also codified contributes to antibody chain to secrete from host cell.Antibody chain gene clone can be entered in carrier, so that signal peptide is connected to the amino terminal of immunoglobulin by reading frame.Described signal peptide can be immunoglobulin signal peptide or allos the signal peptide signal peptide of NIg (that is, from).
Except described antibody chain gene, recombinant expression carrier of the present invention is carried at the regulating and controlling sequence of controlling described antibody chain gene expression in host cell.The design (comprising the selection of regulating and controlling sequence) that one of skill in the art will appreciate that expression vector can be dependent on such as following factors: the selection of host cell to be transformed, the protein expression level of wanting etc.The regulating and controlling sequence of preferably expressing for mammalian host cell comprises, instruct the viral element of protein high level expression in mammalian cell, for example derive from retrovirus (for example retrovirus LTR), cytomegalovirus (CMV) (for example CMV promoter/enhancer), simian virus 40 (SV40) (for example SV40 promoter/enhancer), adenovirus (for example, adenovirus major late promoter (AdMLP)) promoter and/or enhancer, polyoma and strong for example native immunoglobulin of mammalian promoter and the promoter of actin.About further describing of viral controlling element and its sequence, referring to for example, United States Patent (USP) 5,168,062, United States Patent (USP) 4,510,245 and United States Patent (USP) 4,968,615.For expressing the method for antibody plant, comprise the description of promoter and carrier and Plant Transformation, in this area, be known.Referring to, for example, United States Patent (USP) 6,517,529 is quoted as a reference herein.Bacterial cell or fungal cell for example in yeast cells the method for express polypeptide in this area, also know.
Except described antibody chain gene and regulating and controlling sequence, the extra sequence of recombinant expression carrier portability of the present invention, for example, regulate and control the sequence copying (for example, origin of replication) and the selectable marker gene of carrier in host cell.Described selectable marker gene contributes to select the host cell that wherein imported described carrier (referring to for example, United States Patent (USP) 4,399,216,4,634,665 and 5,179,017).For example, conventionally, selectable marker gene provides for example resistance of G418, hygromycin or methotrexate of medicine for wherein having imported the host cell of described carrier.Preferred selectable marker gene comprises dihydrofolate reductase (DHFR) gene (for using select/amplification of the DHFR-host cell of methotrexate), neomycin resistance gene (selecting for G418) and glutamine synthetase gene.
Can transform suitable mammal, plant, antibacterial or yeast host cell with the nucleic acid molecules of the anti-CTLA-4 antibody of coding and the carrier that comprises these nucleic acid molecules.Can adopt transgenic method to produce antibody of the present invention by following manner: producing for object heavy chain immunoglobulin and sequence of light chain is genetically modified mammal or plant, and therefrom with callable form, produces antibody.
Can be by (comprising for any known method that polynucleotide is imported to host cell, for example polynucleotide are packaged into virus (or being packaged into viral vector), and with virus (or carrier) transduction host cell) or by transfection method known in the art (as United States Patent (USP) 4,399,216,4,912,040,4,740,461 and 4, the method exemplifying in 959,455) transform.The method for transformation using depends on host to be transformed.For the method that heterologous polynucleotide is imported to mammalian cell, in this area, know, and comprise, but be not limited to, the transfection of the transfection of glucosan mediation, calcium phosphate precipitation method, polybrene mediation, protoplast fusion, electroporation, particle bombardment, by polynucleotide encapsulation in liposome, peptide stop compound, dendritic and directly DNA microinjection is entered in nucleus.
Can obtain as the mammal cell line of the host for expressing and know in this area, it comprises many immortalized cell lines that can obtain from American type culture collection (ATCC), (for example include but not limited to Chinese hamster ovary (CHO) cell, NSO cell, HeLa cell, baby hamster kidney (BHK) cell, monkey-kidney cells (COS), human liver cell cancerous cell, Hep G2), and many other cell lines.Nonmammalian cell includes but not limited to antibacterial, yeast, insecticide and plant, and they also can be used for expressing recombinant antibody.In order to prevent the change due to immunogenicity, pharmacokinetics and/or effector functions that inhuman glycosylation causes, it may be preferred to remove deglycosylation that the CH2 domain of antagonist carries out site-directed mutation.By determining which kind of system produces high expression level and produces the antibody with composing type CTLA-4 binding characteristic, select expression.
In addition, can strengthen and from production cell line, express antibody of the present invention (or derive from its other parts) by many known technology.For example, glutamine synthetase and DHFR gene expression system are the common methods for strengthening under certain conditions expression.For example limit dilution cloning of useful routine techniques and microdrop technique are identified high expressed sexual cell clone.Complete or glutamine synthetase system has partly been discussed in European patent 0 216 846,0 256 055 and 0 323 997 and european patent application 89303964.4.
About the transgenic in mammal, produce, can also in goat, cattle or other mammals, produce antibody, and reclaim antibody from their milk.Referring to, for example, United States Patent (USP) 5,827,690,5,756,687,5,750,172 and 5,741,957.
Can be from relevant cell material purification and/or be separated in the anti-CTLA-4 antibody of above-mentioned cells.Antibody can be present in complete cell, in cell lysate, or with partial purification or substantially pure form exist.In order to remove nucleic acid or the protein of other cellular components or for example other cells of other pollutant, by standard technique, carry out purification, described standard technique comprises the additive method of knowing in alkali/SDS processing, column chromatography and this area.Referring to Ausubel, F., waits people, ed.Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York (1987).
In the present invention, by different cell line or the of the present invention anti-CTLA-4 antibody of expressing, may will there is each other different glycosylation patterns in transgenic animal.But the coded anti-CTLA-4 antibody of all nucleic acid providing from here and aminoacid is considered to a part of the present invention, regardless of its glycosylation pattern or its modification or disappearance.Therefore, for the purposes of the present invention, described anti-CLTA-4 antibody can be glycosylated or nonglycosylated.When described anti-CLTA-4 antibody is while being glycosylated, they can have any possible glycosylation pattern.In addition, every heavy chain in an antibody can have identical glycosylation pattern, or two heavy chains can have different glycosylation patterns.In order to prevent the change due to immunogenicity, pharmacokinetics and/or effector function that inhuman glycosylation causes, the present invention also comprises that the site-directed mutation of antibody CH2 domain is to eliminate glycosylation.
As used herein, term " glycosylation " refers to the pattern of covalent attachment to the sugar unit of antibody.When saying that anti-CTLA-4 antibody herein has specific glycosylation pattern, this is to represent that most of mentioned anti-CTLA-4 antibody has this specific glycosylation pattern.In other respects, when saying that anti-CTLA-4 antibody herein has specific glycosylation pattern, this is to represent to be more than or equal to 50%, 75%, 90%, 95%, 99% or 100% mentioned anti-CTLA-4 antibody to have this specific glycosylation pattern.
Anti-CTLA-4 antibody of the present invention also comprises its glycosylation variants (for example, by disappearance, insert or replace suitable amino acid residue insert glycosylation site or delete that any glycosylation site produces).
Normally N-connection or O-connection of the glycosylation of polypeptide.Normally N-connection of the glycosylation of antibody polypeptides, and form two days linear structures.N-connection refers to the side chain that sugar moieties is attached to asparagine residue.Tripeptide sequence agedoite-X-serine and agedoite-X-threonine, wherein X is any aminoacid except proline, is the recognition sequence for sugar moieties enzymatic being attached to agedoite side chain.Therefore, in antibody, the existence of these tripeptide sequences has produced potential glycosylation site.
Three kinds of different structures of two days line style polysaccharide are called " G0 ", " G1 " and " G2 ", and it has 0,1 or 2 terminal galactose residues at the non reducing end of polysaccharide respectively.Referring to people such as Jefferis, Biochem.J., 268,529-537 (1990).In some cases, glycan structures also can have the fucosyl residues that is connected to N-acetyl-glucosamine, and described N-acetyl-glucosamine is covalently bond to the amino acid asparagine (for example, site 297) of finding in antibody.When fucose (F) exists, depend on the number of terminal galactose residues, within two days, line style polysaccharide nomenclature is changed into " G0F ", " G1F " or " G2F ".Referring to, Teillaud, ExpertOpin.Biol.Ther., 5 (Suppl.1): S15-S27 (2005).In addition, when antibody comprises two heavy chains, for each the repetition polysaccharide nomenclature in two heavy chains." G0F, G0F " sugared shape is that wherein two heavy chains are all attached with G0 polysaccharide and each G0 polysaccharide and all have the kind of fucose (F) residue that is connected to N-acetyl-glucosamine." G0F, G1F " sugared shape is that wherein heavy chain is attached with G0 polysaccharide and another heavy chain is attached with the kind of G1 polysaccharide, and wherein each G0 polysaccharide and G1 polysaccharide have fucose (F) residue that is connected to N-acetyl-glucosamine.
In certain embodiments, described anti-CTLA-4 antibody has the glycosylation pattern that is selected from " G0F, G0F ", " G0F, G1F ", " G1F, G1F ", " G1F, G2F " and its mixture.In other embodiments, for the antibody producing that exceedes 50%, anti-CTLA-4 antibody has the glycosylation pattern for " G0F, G1F ".In other embodiments, for the antibody producing that is less than 50%, anti-CTLA-4 antibody has the glycosylation pattern for " G0F, G0F ".For example, in one embodiment, anti-CTLA-4 antibody 11.2.1 described herein has the glycosylation pattern of " G0F, G0F " or " G0F, G1F ".In some embodiments, produce the anti-CTLA-4 antibody (11.2.1) of the admixture with different glycosylation pattern.For example, in the sample of antibody (11.2.1), can there is the mixture of antibody (11.2.1), some in described antibody have the glycosylation pattern and other glycosylation patterns with " G0F, G0F " of " G0F; G1F ", and its ratio is 3: 2.
Route of administration and according to dosage administration:
Compositions of the present invention can for example, exist with liquid solution form (, injectable and solution that can infusion).Preferred form depends on desirable method of application and therapeutic application.Common preferred compositions exists with solution form injectable or can infusion, for example compositions similar to the compositions of the passive immunization for people.Preferred method of application is for example, through parenteral (, in intravenous, subcutaneous, intraperitoneal, intramuscular and breastbone), to use or use by infusion techniques with the form of aseptic parenteral solution or oil (olagenous) suspension.As what it will be recognized by those skilled in the art, the approach of using and/or mode will depend on the result of wanting and change.In preferred embodiments, by intravenous infusion or injection administration of antibodies.In a further preferred embodiment, by intramuscular or subcutaneous injection, use described antibody.
Therapeutic composition is normally aseptic with stable under the condition of producing and store.
Compositions can be formulated as to solution, microemulsion, dispersion or liposome.Can, by anti-CTLA-4 antibody is mixed in suitable diluent with (if needs) together with a kind of or its combination in the composition exemplifying with the amount of needs, then carry out sterilizing (for example, filtration sterilization) above, prepare Injectable solution.Usually, by reactive compound is mixed in aseptic vehicle and prepares dispersion, described vehicle comprises basic dispersion medium and needed those other compositions from exemplifying above.Can prepare this type of suspension according to those suitable dispersants of known utilization, wetting agent and suspending agent or other acceptable reagent.Sterile injectable preparation can be also for example, sterile injectable solution or suspension in the acceptable diluent of nontoxic parenteral or solvent (1,3 butylene glycol).Wherein, spendable acceptable vehicle and solvent are water, Ringer's mixture and isotonic sodium chlorrde solution.In addition, aseptic fixed oil is routinely as solvent or suspension media.For this object, can use the fixed oil of any gentleness, comprise synthetic monoglyceride or diglyceride.In addition, n-3 polyunsaturated fatty acid can be used for preparing injectable liquid.
In the case of the sterilized powder for the preparation of sterile injectable solution, preferred preparation method is vacuum drying and lyophilization, and described drying means produces active component and add the powder of any extra composition of wanting from the solution of its previous aseptic filtration.Can be by for example using such as lecithin of coating, by keeping required granular size the dispersion in the situation that, and by using surfactant, keep the suitable mobility of liquid.
Can be by comprise for example Monostearate of reagent and the gelatin that postpone absorption in compositions, or by described compositions is mixed with, extend for example depot formulation of absorpting form (depots), liposome, polymer microballoon, polymer gel and implant, the prolongation that produces Injectable composition absorbs.
For using the additive method of antibody described herein, comprise the skin patch that directly drug release is entered to experimenter's skin.This type of patch can be included in alternatively in the liquid solution of buffering, dissolve and/or be dispersed in binding agent or be dispersed in the antibody of the present invention in polymer.
The additive method of using antibody described herein comprises the eye drop for eyes.
Can use described antibody once, but more preferably use repeatedly.For example, can from once a day to every 6 months or longer time applied once described in antibody.Can use for 1 time, every 2 days 1 time, every 3 days 1 time, 1 time weekly, every 2 weeks 1 time, every 1 month 1 time, every 2 months 1 time, every 3 months 1 time and every 6 months 1 time for example every day 3 times, every day 2 times, every day by scheme.
Also can be by antibody described in micropump continuous administration.Antibody can be applied in to the site of the body part of tumor or inflammation, use in the body part of tumor or inflammation or be applied in the site away from the body part site of tumor or inflammation.Can use described antibody 1 time, at least 2 times, or use at least a period of time until condition of illness is treated, alleviates or cures.Conventionally as long as existing, tumor just can use described antibody, condition is that described antibody can make tumor or cancer stop growing or reduce weight or volume, or use described antibody until the body part of inflammation heals, the part using described antibody as above-described pharmaceutical composition is used conventionally.
Compositions of the present invention can comprise antibody of the present invention or the antigen-binding portion thereof for the treatment of effective dose or prevention effective dose.In the described preparation of preparation, can be by dose volume and the method for application for example considering to want, the character of condition of illness to be treated and severity, and experimenter's age and stature size, determine the treatment effective dose that is present in the anti-CTLA-4 antibody in preparation.
Exemplary, the indefiniteness dosage range of the pharmaceutical composition of the present invention of using to experimenter are the extremely about 200mg/kg of about 0.01mg/kg (milligram (mg) of the anti-CTLA-4 antibody of being used with every kilogram of (kg) experimenter body weight counts expression), the extremely about 100mg/kg of about 0.1mg/kg, the extremely about 50mg/kg of about 1.0mg/kg, the extremely about 20mg/kg of about 5.0mg/kg or about 15mg/kg.For the purposes of the present invention, average man experimenter body weight is about 70kg.
Also wish the scope between any dosage between described herein, for example, approximately 0.01mg/kg-199mg/kg, as a part of the present invention.For example, wish to comprise that the combination of using described any value is as the scope of the value of the upper limit and/or lower limit.
Also can adjust dosage regimen according to dosage thereby best (for example replying of wanting is provided by use several divided doses to experimenter within a period of time, therapeutic or preventative replying), or can reduce pari passu or increase dosage according to the urgency for the treatment of situation.Unified in order conveniently to use with dosage, with dosage unit form preparation parenteral composition, be especially favourable.
Dosage unit form used herein refers to the unit physically separating of the single dose that is suitable as mammalian subject to be treated; Each unit comprises as calculated and produces the reactive compound of scheduled volume of the therapeutic effect of wanting and required pharmaceutical carrier.The standard of dosage unit form of the present invention is by following factors regulation and directly depend on following factors: (a) specific characteristic of anti-CTLA-4 antibody or part and particular treatment or the preventive effect that will obtain, and (b) intrinsic restriction in the field of the such antibody that is used for the treatment of individual sensitivity of preparation.
Liquid preparation of the present invention can be formulated as to unit dosage forms.For example, every bottle of unit dose can comprise the anti-CTLA-4 antibody of the variable concentrations of 1 to 1000 milliliter (ml).In other embodiments, every bottle of unit dose can comprise the anti-CTLA-4 antibody of the variable concentrations of about 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml, 15ml, 20ml, 30ml, 40ml, 50ml or 100ml.If needed, can be by adding sterile diluent these preparations to be adjusted to the concentration of wanting in each bottle.Also liquid preparation of the present invention can be prepared as to the unit dosage forms in sterile bag or container, described sterile bag or container are applicable to being connected to intravenous and use line or conduit.
Stability assessment:
The present invention includes the stable composition of liquid medicine that comprises anti-CTLA-4 antibody described herein and pharmaceutically acceptable chelating agen.Expect stable compositions keep outward appearance and the integrity of for example product or resist they variation (comprise may cause biologic activity reduce physics or chemical degradation).Various analytical technologies and indicator for measuring protein stability have been reported in the literature, at Peptide andProtein Drug Delivery, 247-301, VincentLee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, summarized many these technology and indicator in A.Adv.Drug Delivery Rev.10:29-90 (1993).Usually, composition of liquid medicine of the present invention, when experiencing low storage temperature and/or show the stability of raising during one or more freeze/thaw when experience within a period of time.
In one embodiment, described compositions, when storing at least about 12 months at the temperature at about 2 ℃ to about 8 ℃, preferably at least about 18 months, more preferably at least about 24 months time, more stable than the same combination that lacks chelating agen of storing under the same conditions same time.
In another embodiment, described compositions, when storing at least about 3 months at the temperature at about 25 ℃ to about 30 ℃, preferably at least 6 months, more preferably at least about 12 months time, more stable than the same combination that lacks chelating agen of storing under the same conditions same time.
In another embodiment, described compositions, when storing at least about 1 month at the temperature at about 40 ℃, preferably at least about 2 months, more preferably at least about 3 months time, more stable than the same combination that lacks chelating agen of storing under the same conditions same time.
As used herein, term " freeze/thaw " refers to the technology of using liquid antibody sample after refrigerated storage, wherein the temperature of sample is reduced to 0 ℃ or lower temperature with freezing this fluid sample, then allow sample experience make it recover the temperature of liquid condition, and carry out time enough to allow to use sample, then returned to refrigerated storage, preferably at 0 ℃ or lower temperature, stored.As used herein, term " refrigerated storage " refers at 0 ℃ or lower temperature, preferably at-20 ℃ or lower temperature, freezing and keep previously liquid antibody sample.
In one embodiment, described compositions, when carrying out at least 1 freeze/thaw, preferably at least 2 freeze/thaw, more preferably at least 3 freeze/thaw, more preferably at least 4 freeze/thaw, more preferably at least 5 freeze/thaw, more preferably during at least 6 freeze/thaw, the same combination that lacks chelating agen of the freeze/thaw condition more identical than experience is more stable.
In another embodiment, described compositions meets two or more in following condition:
(a) described compositions, when storing at least about 12 months at the temperature at about 2 ℃ to about 8 ℃, preferably at least about 18 months, and more preferably at least about 24 months time, more stable than the same combination that lacks chelating agen of storing under the same conditions same time;
(b) described compositions, when storing at least about 3 months at the temperature at about 25 ℃ to about 30 ℃, preferably at least 6 months, and more preferably at least about 12 months time, more stable than the same combination that lacks chelating agen of storing under the same conditions same time;
(c) described compositions, when storing at least about 1 month at the temperature at about 40 ℃, preferably at least about 2 months, and more preferably at least about 3 months time, more stable than the same combination that lacks chelating agen of storing under the same conditions same time; Or
(d) described compositions, when carrying out at least 1 freeze/thaw, preferably at least 2 freeze/thaw, more preferably at least 3 freeze/thaw, more preferably at least 4 freeze/thaw, more preferably at least 5 freeze/thaw, and more preferably during at least 6 freeze/thaw, the same combination that lacks chelating agen of the freeze/thaw condition more identical than experience is more stable.
In another embodiment, described compositions meets three or more in the condition of just having discussed above.
For the purposes of the present invention, antibody aggregation, antibody fragment and/or compositions variable color for example can be used as the stability indicator of compositions.Usually, composition of liquid medicine of the present invention, when one or more above-mentioned storages of experience or freeze/thaw condition, for the same combination that lacks chelating agen of experience the same terms, show at least one in lower level antibody aggregation, antibody fragment and compositions variable color.
Can measure the protein aggregation in fluid composition by the whole bag of tricks known in the art.These methods comprise the gel permeation chromatography that carrys out isolated protein based on its molecular weight." gel " is the mixture of water and for example agarose of polymer or polyacrylamide.The present invention also comprises use gel filtration HPLC (high performance liquid chromatography).The method that other generally acknowledged measurements are assembled comprises cation-exchange chromatography, and the method is to use the general liquid chromatography (LC) technology of the ion-exchange chromatography of anion column.The cation of exchange is from protein molecule in the present invention.Because multivalent protein aggregation can have the net charge of multiple single chain antigen binding proteins, thereby aggregation can obtain stronger reservation, and can be separated with single chain molecule.Preferred cationite is poly-aspartate post.Therefore, can easily distinguish monomeric protein and aggregation.But, those skilled in the art will recognize that, aggregation assay of the present invention is not limited to any specific chromatographic column type, as long as it can separate the protein molecule of two kinds of forms.
Can measure the protein fragmentation in composition of liquid medicine by the whole bag of tricks known in the art.These methods comprise, for example, and size exclusion chromatography, UV detection (for example,, in 214 nanometers), SDS-PAGE and/or substance assistant laser desorpted ionized/time-of-flight mass spectrometry (TOFMS) (MALDI/TOF MS).Can assess the protein fragmentation that causes electric charge to change (for example,, because desamidation occurs) by for example ion-exchange chromatography or isoelectrofocusing (IEF).
Usually, can measure by the visual observations of compositions itself variable color of compositions.Usually, this composition of liquid medicine that comprises chelating agen, for not comprising the identical compositions of described chelating agen, reduce the variable color (for example, pink or yellow) of compositions and/or kept the clarity (for example turbidity, turbidity and/or granule form) of compositions.For the purposes of the present invention, term " variable color " refer to the change (for example,, from limpid and colourless to pink or yellow) of color and the change of clarity (for example from limpid and colourless to muddy, muddiness and/or there is granule).Usually, can use other technologies for example by carrying out UV detection in 214 nanometers and/or by the standard colour code of compositions and do not have with described chelating agen estimate to comparison, the variable color of measuring compositions.Referring to PhEur 5.0,2005Monograph 2.2.2.
In one embodiment, after experiencing at least one in following condition, described compositions measures antibody aggregation:
(a) described compositions is stored at least about 12 months at the temperature of about 2 ℃ to about 8 ℃, preferably at least about 18 months, and more preferably at least about 24 months;
(b) described compositions is stored at least about 3 months at the temperature of about 25 ℃ to about 30 ℃, preferably at least 6 months, and more preferably at least about 12 months;
(c) described compositions is stored at least about 1 month at the temperature of about 40 ℃, preferably at least about 2 months, and more preferably at least about 3 months; Or
(d) make at least 1 freeze/thaw of described compositions experience, preferably at least 2 freeze/thaw, more preferably at least 3 freeze/thaw, more preferably at least 4 freeze/thaw, more preferably at least 5 freeze/thaw, and at least 6 freeze/thaw more preferably.Then for example, by chromatography (using HPLC) by antibody aggregation body and monomer separation, and according to the chromatogram of gained determine assemble degree.That the aggregation peak area on chromatogram that stable composition of liquid medicine of the present invention has is usually less than is about 6%, lower than about 5%, lower than about 4%, lower than about 3%, lower than about 2% or lower than about 1.5% the total peak area on described chromatogram.At one of this technology for measuring gathering concrete example, described compositions is stored 24 weeks at 40 ℃, then use SE-HPLC to carry out chromatography and carry out UV detection in 214 nanometers.This technology in embodiment 11 for measuring antibody aggregation, in described embodiment, for example, No. 37 preparation (comprising chelating agen) shows about 1.1% aggregation peak area on chromatogram, and preparation 26 (lacking chelating agen) shows about 6.4% aggregation peak area on chromatogram.
Usually, the difference between the aggregation chromatogram peak area of the same combination that lacks described chelating agen of the aggregation chromatogram peak area of stable composition of liquid medicine of the present invention and experience the same terms is at least about 2%, at least about 3%, at least about 4% or at least about 4.5%.For example in embodiment 11, the difference between preparation 37 (about 1.1% aggregation peak area on chromatogram) and the preparation 26 (about 6.4% aggregation peak area on chromatogram) of test is about 5.3% as mentioned above.
In another embodiment, after experiencing at least one in following condition, described compositions measures antibody fragment:
(a) described compositions is stored at least about 12 months at the temperature of about 2 ℃ to about 8 ℃, preferably at least about 18 months, and more preferably at least about 24 months;
(b) described compositions is stored at least about 3 months at the temperature of about 25 ℃ to about 30 ℃, preferably at least 6 months, and more preferably at least about 12 months;
(c) described compositions is stored at least about 1 month at the temperature of about 40 ℃, preferably at least about 2 months, and more preferably at least about 3 months; Or
(d) make at least 1 freeze/thaw of described compositions experience, preferably at least 2 freeze/thaw, more preferably at least 3 freeze/thaw, more preferably at least 4 freeze/thaw, more preferably at least 5 freeze/thaw, and at least 6 freeze/thaw more preferably.Then pass through chromatography (for example, using gel filtration) antibody fragment is separation with described compositions, and according to the chromatogram of gained, determine the degree of fragmentation.The fragment band volume on chromatogram that stable composition of liquid medicine of the present invention has be generally lower than about 9%, lower than about 8%, lower than about 7%, lower than about 6%, lower than about 5% or lower than about 4.5% the total band volume on described chromatogram.At an instantiation of this technology for measuring fragmentation, described compositions is stored 24 weeks at 40 ℃, then use reduced form SDS-PAGE (rSDS-PAGE) to carry out chromatography, wherein by scanning to measure band volume with Molecular Dynamics Personal Densitometer PDQC-90 or Bio-Rad GS800 Imaging Densitometer.This technology in embodiment 11 for measuring antibody fragment, in described embodiment, for example, No. 37 preparation (comprising chelating agen) shows about 4.5% fragment band volume and preparation 26 (lacking chelating agen) shows about 10.1% fragment band volume on chromatogram on chromatogram.
Usually, the difference between the fragment band volume of the same combination that lacks described chelating agen of the fragment band volume of stable composition of liquid medicine of the present invention and experience the same terms is at least about 2%, at least about 3%, at least about 4% or at least about 5%.For example, in embodiment 11, the difference between preparation 37 (about 4.5% fragment band volume on chromatogram) and the preparation 26 (about 10.1% fragment band volume on chromatogram) of test is about 5.6% as mentioned above.
Therapeutic Method:
Can in treatment, use the antibody of any type described herein.In preferred embodiments, described anti-CTLA-4 antibody is people's antibody.In a further preferred embodiment, described CTLA-4 is people, and described experimenter is people experimenter.In a further preferred embodiment, described anti-CTLA-4 antibody is human IgG2's antibody.Alternatively, described experimenter can be the mammal of the CTLA-4 albumen of expression and described anti-CTLA-4 antibody cross reaction.Described antibody can be administered to the non-human mammal (that is, primate) of expressing with the CTLA-4 of described antibody cross reaction, for veterinary's object or as human disease's animal model.This type of animal model can be used for assessing the therapeutic efficiency of antibody of the present invention.
The invention provides the method for the neoplasia condition of illness being used for the treatment of in experimenter, it comprises to described experimenter uses the composition of liquid medicine that comprises anti-CTLA-4 antibody and chelating agen (independent or combined with other excipient that are selected from buffer agent, tonicity agent or surfactant and composition thereof).In other embodiments, above-mentioned experimenter is the experimenter who needs prevention or treatment neoplasia condition of illness.
In another embodiment, the invention provides the method for the neoplasia condition of illness being used for the treatment of in experimenter, it comprises to experimenter uses the composition of liquid medicine that comprises anti-CTLA-4 antibody and pharmaceutically acceptable excipient, and described pharmaceutically acceptable excipient comprises chelating agen (independent or combined with other excipient that are selected from buffer agent, tonicity agent or surfactant and composition thereof).
Term " neoplasia " and " neoplasia condition of illness " all refer to it can is optimum, premalignant, transfer or pernicious " vegetation " or tumor.The present invention also comprises optimum, premalignant, that shift or pernicious neoplasia.The present invention also comprises optimum, premalignant, that shift or pernicious tumor.Therefore, all optimum, premalignant, shift or pernicious neoplasia or tumor be included in the present invention, and be called neoplasia interchangeably, vegetation or neoplasia related pathologies.Tumor is commonly called neoplasia piece (mass ofneoplasia) or or " superfluous natural disposition " cell in this area.However, be appreciated that for the purposes of the present invention, even if a neoplastic cell is also considered to vegetation or alternatively neoplasia.
Can any tissue or organ be can relate to by the neoplasia condition of illness of anti-CTLA-4 Antybody therapy of the present invention, and osteocarcinoma, the brain cancer, pulmonary carcinoma, squamous cell cancer, bladder cancer, gastric cancer, cancer of pancreas, breast carcinoma, a cancer, neck cancer, hepatocarcinoma, renal carcinoma, ovarian cancer, carcinoma of prostate, colorectal carcinoma, the esophageal carcinoma, gynecological cancer (for example cervical cancer and ovarian cancer), nasopharyngeal carcinoma or thyroid carcinoma included but not limited to.Especially, anti-CTLA-4 antibody preparation of the present invention is used for the treatment of breast carcinoma, carcinoma of prostate, colon cancer and pulmonary carcinoma.
In other embodiments, method and composition of the present invention comprises prevention and treatment neoplasia condition of illness, and described neoplasia condition of illness is selected from: acra mottle sample melanoma, actinic keratosis, adenocarcinoma, cystadenocarcinoma, adenoma, familial adenomatous polyposis, familial polyposis, polyp of colon, polyp, sarcoadenoma, adenosquamous carcinoma, adrenocortical carcinoma, AIDS dependency lymphoma, anus cancer, astrocytoma, bartholin gland carcinoma, basal cell carcinoma, cancer of biliary duct, bladder cancer, brain stem glioma, the cerebral tumor, breast carcinoma, bronchial gland carcinoma, capillary tube cancer, carcinoid, cancer, fallopian tube carcinoma, carcinoma of endometrium, carcinosarcoma, spongy lymphoma, central nervous system lymphoma, large cerebral astrocytoma, cancer of biliary duct, chondrosarcoma, papilloma of choroid plexus/cancer, clear cell carcinoma, skin carcinoma, the brain cancer, colon cancer, colorectal carcinoma, T-cell lymphoma,cutaneous, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependyma cancer, Epithelial cancer, the esophageal carcinoma, Ewing sarcoma, Extaagonactal perm celi tumors, fibre board stratotype cancer, Focal nodular hyperplasia, carcinoma of gallbladder, gastrinoma, germinoma, gestational trophoblastic neoplasms, glioblastoma, glioma, glucagonoma of pancreas, hemangioblastoma, hemangioendothelioma, hemangioma, adenoma of liver, adenoma of liver disease, hepatocarcinoma, hodgkin's lymphoma, hypopharyngeal cancer, hypothalamus and visual pathway glioma, insulinoma, intraepithelial neoplasia forms, upper Intradermal squamous cytoma forms, ophthalmic melanoma, wellability squamous cell carcinoma, large cell carcinoma, islet-cell carcinoma, Kaposi sarcoma, renal carcinoma, laryngeal carcinoma, leiomyosarcoma, pernicious lentigo type melanoma, leukemia related pathologies, lip and oral cancer, hepatocarcinoma, pulmonary carcinoma, lymphoma, malignant mesothe, malignant thymoma, medulloblastoma, medulloepithelioma, melanoma, meninges cancer, Merkel cell cancer, carcinoma mesothelial, metastatic carcinoma, mucoepidermoid carcinoma, multiple myeloma/plasma cell vegetation, mycosis fungoides, myelodysplastic syndrome, bone marrow proliferative condition of illness, nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, neuroepithelium adenocarcinoma, nodositas malignant melanoma, central nervous system's vegetation (for example, primary central nervous system lymphoma, spinal column axis tumor (spinal axis tumor), brain stem glioma or pituitary adenoma), non Hodgkin lymphoma, oat-cell carcinoma, oligodendroglia cancer, oral cancer, oropharynx cancer, osteosarcoma, pancreatic polypeptide, ovarian cancer, ovarian germ cell tumor, cancer of pancreas, papillary serous adenocarcinoma, pinealocyte, pituitary tumor, plasmocytoma, false sarcoma, pulmonary blastoma, parathyroid carcinoma, carcinoma of penis, pheochromocytoma, primitive neuroectodermal tumor on pinus and the canopy of the heavens, pituitary tumor, plasma cell vegetation, pleura pulmonary blastoma, carcinoma of prostate, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma (serous carcinoma), small cell carcinoma, carcinoma of small intestine, soft tissue cancer, Somatostatin Secretion type tumor (somatostatin-secreting tumor), scale cancer, squamous cell carcinoma, between subcutaneous, superficial spreading melanoma, primitive neuroectodermal tumor on the canopy of the heavens, thyroid carcinoma, undifferentiated carcinoma, carcinoma of urethra, uterus carcinoma, uvea melanoma, verrucous carcinoma, cancer of vagina, vasoactive intestinal polypeptide tumor, carcinoma vulvae, macroglobulinemia Waldenstron, high score voltinism cancer and wilms' tumor.
In a more preferred embodiment, use described anti-CTLA-4 antibody to the experimenter who suffers from breast carcinoma, carcinoma of prostate, pulmonary carcinoma or colon cancer.In the embodiment being more preferably, described method makes cancer stop abnormality proliferation, or does not gain in weight or volume or minimizing weight or volume.
Goods:
In another embodiment of the invention, the goods that comprise container are provided, described container is mounted with liquid pharmaceutical formulation and its operation instructions is optionally provided, described liquid pharmaceutical formulation comprise independent or with the preparation of the combined chelating agen of other pharmaceutically acceptable excipient in comprise at least one monoclonal anti CTLA-4 antibody of the present invention.Suitable container comprises, for example, and bottle, phial, sack and syringe.Can manufacture described container from for example glass of multiple material or plastics.Exemplary container is the glass vial that the single of 3-20cc is used.Alternatively, for multi-dose formulation, described container can be the glass vial of 3-100cc.Described container loads described preparation, and can indicate operation instruction on described container or with the label of its combination.Goods also can comprise the other materials of wanting from business and user perspective, comprise other buffers, diluent, filter, syringe needle, syringe, and have the package insert of operation instruction, contraindication and/or potential side effect list.
The present invention also provides the test kit for the preparation of the fluid composition of the antibody through stable, described test kit comprises the first container that monoclonal anti CTLA-4 antibody 11.2.1 solution is housed, and second container of the chelating agen that being enough in solution stablize the amount of described antibody (independent or combined with other excipient) is housed.
The following example has been described embodiment of the present invention.According to explanation of the present invention or practice disclosed herein, other embodiments within the scope of claims are obvious to those skilled in the art herein.Wish that description and embodiment are just considered to exemplary, scope and spirit of the present invention are indicated by embodiment following claim book.In described embodiment, unless otherwise noted, all percentage ratio all provides based on weight.Those skilled in the art will recognize that, use the molecular weight of art-recognized described composition to convert the weight described in embodiment and/or weight-volume ratio to molal quantity and/or molarity.The weight that herein exemplifies (for example, gram) is for example, for described volume (, the volume of buffer, antibody preparation etc.).Those skilled in the art will recognize that, when wanting different volumes of formulation, can adjust in proportion weight.
embodiment 1
The present embodiment has shown the generation of the lymphoma cell line that is created in the anti-CTLA-4 antibody described in the United States Patent (USP) 6,682,736 that belongs to the people such as Hanson.
Be prepared as follows, select and measure antibody of the present invention:
Antigen preparation: prepare three kinds of different immunogens for immunity inoculation XenoMouse tMmice: (i) CTLA-4-IgG fusion rotein, (ii) CTLA-4 peptide, and (iii) 300.19 muroid lymphoma cells of CTLA-4 mutant (Y201V) transfection expressed on cell surface with composing type ground.
CTLA-4-IgG1 fusion rotein:
The structure of expression vector
Use is according to primer pcr amplification from people's thymus cDNA library (Clontech) of sequence (Eur.J Immunol 18:1901-1905 (the 1988)) design of announcing to encode cDNA in mature cell external structure territory of CTLA-4.By described fragment directed sub-clone between people's oncostatin M signal peptide and human IgG γ 1 (IgG1) CH1/CH2/CH3 domain enter pSR5 (sindbis virus's expression plasmid) (InVitrogen) in.Described fusion rotein does not comprise hinge region but in the extracellular domain of CTLA-4, comprises cysteine 120 to form covalent dimer.The carrier of gained is called CTLA-4-IgG1/pSR5.Two chains to the complete CTLA-4-IgG1 cDNA in carrier all carry out sequence confirmation.Shown the aminoacid sequence of CTLA4-Ig albumen below.From human lymphocyte library (Clontech), pcr amplification goes out the mature cell external structure territory of CD44, then its sub-clone is entered in pSinRep5, thereby produces the reference protein with identical IgG1 tail.
OM-CTLA4-IgG1 fusion rotein:
MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVR
VTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYIC
KVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDLEGAPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPTPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK
Underlined=signal peptide.
From human lymphocyte library (Clontech), pcr amplification goes out the cDNA in the mature cell external structure territory of CD28, and its sub-clone is entered in pCDM8 (J.Immunol.151:5261-71 (1993)), thereby produce the human IgG1's fusion rotein that comprises thrombin cutting area and hinge region.The Degenerate PCR of use standard is cloned the CTLA4 of Adeps seu carnis Rhiopithecus roxellanae monkey, machin and macaque from the mRNA of the PBMC that separates personal PHA and stimulate.Sequencing shows that the aminoacid sequence of macaque and machin is identical, and itself and ripe human CTLA 4 extracellular domain have 3 differences (S13N, I17T and L105M).Adeps seu carnis Rhiopithecus roxellanae monkey shows with ripe human CTLA 4 extracellular domain to have 10 aminoacid differences (V21A, V33I, A41T, A51G, 541, S71F, Q75K, T88M, L105M and G106S).Use site-directed mutation to make all different aminoacid in Adeps seu carnis Rhiopithecus roxellanae monkey CTLA4 produce a single point sudden change with the important aminoacid mapping of the interaction to for described antibody and human CTLA 4-IgG.By the site-directed mutation of matchmaker (Promega), produce for the people of epitope mapping and the sudden change of Adeps seu carnis Rhiopithecus roxellanae monkey CTLA-IgG.Transient transfection by Cos7 cell produces IgG fusion rotein, and uses the A albumen technology of standard to carry out purification.By immunoblotting with BIAcore, analyze the CTLA4-IgG albumen of just assessing sudden change with the combination of antibody.
Expression/the purification of recombiant protein
As described in InVitrogen, by using, through the auxiliary mRNA of the CTLA-4-IgG1/pSR5 of SP6 in vitro transcription mRNA and DH-26S, baby hamster kidney cell is carried out to electroporation (Gibco) and produce restructuring sindbis virus.After 48 hours, gather in the crops recombinant virus, and just titration is carried out in the expression of the optimum protein matter in Chinese hamster ovary cell (CHO-K1).By CHO-K1 cell suspension cultures in DMEM/F12 (Gibco), hyclone (Gibco), non essential amino acid (Gibco), 4mM glutamine (Gibco), penicillin/streptomycin (Gibco), 10mM Hepes pH7.5 (Gibco) that described DMEM/F12 comprises 10% heat inactivation.For producing CTLA-4-IgG, with 1 × 10 7individual cell/ml is resuspended in CHO-K1 cell in DMEM/F12, and by room temperature incubation 1 hour of itself and sindbis virus.Then cell is diluted to 1 × 10 in DMEM/F12 6/ ml, described DMEM/F12 comprises 1% hyclone (by using A Protein S epharose (Pharmacia) to remove cattle IgG), non essential amino acid (Gibco), 4mM glutamine, 12.5mM Hepes pH7.5 and penicillin/streptomycin (Gibco).Infect latter 48 hours sedimentation cells, results conditioned culture media, and supplement with adequate proteins enzyme inhibitor tablet, by pH regulator to 7.5, then by 0.2 μ (Nalgene) filtration.By use 5ml A albumen HiTrap post (Pharmacia) with the flow velocity of 10ml/ minute by FPLC (Pharmacia) for affinity purification fusion rotein.With the PBS washing pillar of 30 times of bed volumes, and with 0.1M glycine/HCl pH2.8 within 1ml/ minute, to carry out eluting.Use immediately Tris pH9 that fraction (1ml) is neutralized to pH7.5.By SDS-PAGE, identify the fraction that comprises CTLA-4-IgG1, then use centriplus 50 (Amicon) to concentrate, then use PBS as solvent within 1ml/ minute, to be applied to sepharose 200 posts (Pharmacia).Collecting the fraction that comprises CTLA-4-IgG1, then by 0.2 μ. (Millipore) filtration sterilization, by its decile, and is chilled at-80 ℃.Use identical method to express and purification CD44-IgG1.From the conditioned medium of the Cos7 cell from transient transfection, be purified into CD28-IgG.
Characterize CTLA-4-IgG1
Using, the SDS-PAGE of colloid coomassie dyeing (Novex) is upper, and the CTLA-4-IgG1 of purification is as single band migration.Under irreducibility condition, CTLA-4-IgG1 is dimer (100kDa), and when processing with 50mM DTT, this dimer is reduced into single aggressiveness of 50kDa.In solution, the determined amino acid sequence of the CTLA-4-IgG1 of purification has been confirmed the N-terminal (MHVAQPAVVLAS) of CTLA-4, and confirms that Oncostatin .-M signal peptide is cut away from ripe fusion rotein.CTLA-4-IgG1 is bonded to fixing B7.1-IgG in the mode of concentration dependent, and this combination is blocked by the anti-human anti-CTLA-4 antibody of hamster (BNI3:PharMingen).Aseptic CTLA-4-IgG does not contain endotoxin, and can use 1.4 as extinction coefficient, by OD280, to be undertaken quantitatively.The productive rate of the CTLA-4-IgG of purification rises in the scope of CHO-K1 cell 0.5 to 3mg/.
CTLA-4 polypeptide:
The following CTLA-4 peptide of preparation as described below:
NH 2：MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQ VNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPC-CONH 2。
Abbreviation/material:
NMP, N-Methyl pyrrolidone; TFE, 2,2,2-trifluoroethanol; DCM, dichloromethane; FMOC, fluorenylmethyloxycarbonyl.Except following exception, all reagent provides by Perkin Elmer: TFE, Aldrich Chemical, FMOC-PAL-PEG resin, PerseptiveBiosystems.Fmoc-Arg (PMC)-OH; FMOC-Asn (Trt)-OH, FMOC-Asp (tBu)-OH, FMOC-Cys (Trt)-OH, FMOC-Glu (tBu)-OH, FMOC-Gln (Trt)-OH, FMOC-His (Boc)-OH, FMOC-Lys (BOC)-OH, FMOC-Ser (tBu)-OH, FMOC-Thr (tBu)-OH and FMOC-Tyr (tBu)-OH are for needing those aminoacid of side chain protected group.
Synthesizing of peptide
On Perkin-Elmer 431A (it adopts the feedback of being undertaken by the ultraviolet absorptivity at 301nm place to monitor to improve (Perkin-Elmer 759A type detector)), carry out peptide synthetic.Service condition double couple crosslinking circulation (conditional double coupling cycle) secretory piece sequence on FMOC-PAL- PEG resin.In circulation 10,11,18,19,20 and 28 to 33, force double couple crosslinking (Forced double couplings).When having circulated, each acidylate with the 50% mixture washing resin of DCM and TFE, then in NMP, use acetic anhydride to add cap to unreacted amino.From reactor, remove resin after 49 completing circulation, then continued remaining reaction.On 415mg resin through 6 hours, use reagent K that peptide is cut down to people International Journalof Protein and Peptide Research 36:255-266 (1990) such as () King from described resin, thereby the rough CTLA-4 peptide of 186mg is provided.
The sign of peptide
The 25mg aliquot of rough CTLA-4 peptide is dissolved in to 5ml 6M guanidine hydrochloride/100mMK 2pO 3(pH6.4), in, then on Pharmacia Hi Load Superdex 75 16/60 posts, (16mm × 600mm, 120ml bed volume) uses 2M guanidine hydrochloride/100mM K 2pO 3(pH6.4) with the flow velocity eluting of 2ml/ minute 180 minutes, collect 5ml fraction.Fraction is analyzed in the following way: 1.7 μ l fraction are loaded on NuPAGE Laemeli gel, use the MES buffer that loses shape to lose shape, and use Daichii silver to dye scheme to manifest.Those fraction of showing 12KDa molecular weight (judging with reference to molecular weight standard) are pooled together and stored at 4 ℃.By UV and gel electrophoresis, analyze the fraction of merging.By 100 microlitre absorption of sample (are absorbed on pvdf membrane) and wash to remove buffer agent salt and measure aminoacid sequence in ProSorb cartridge case.On Applied Biosystems 420 sequenators, check order.Observe the N-end sequence (MHVAQPAVVLA) of expection.Immunoblotting confirmation, described peptide is identified by the anti-human CTLA-4 antibody of BNI3 (PharMingen).For desalination, the aliquot that comprises 648 μ g materials is placed in to 3500 Da MWCO bag filters, and at the situation subinverse 0.1%TFA/H20 stirring, dialyses 9 days at 4 ℃.All the elements thing in bag filter is lyophilized into powder.
With " 300.19 " cell of CTLA-4 (Y201V) peptide antigen transfection:
From people's thymus cDNA library (Stratagene), pcr amplification goes out total length CTLA-4cDNA, and its sub-clone is entered in pIRESneo (Clontech).Use MatchMaker mutation system (Promega) to introduce and cause the CTLA-4 of composing type cell surface expression to suddenly change.The sudden change (Y201 becomes valine) of tyrosine has suppressed the combination (Chuang waits people J.Immunol.159:144-151 (1997)) of the adaptin AP50 of the quick internalization of responsible CTLA-4.The 300.19 muroid lymphoma cells without mycoplasma are cultivated in the RPMI-1640 that comprises 10% hyclone, non essential amino acid, penicillin/streptomycin, 2mM glutamine, 12.5mM Hepes pH7.5 and 25 μ M beta-mercaptoethanols.Use 200V/1180uF (GibcoCellPorator), in 1ml cell, use 20ug CTLA-4-Y201V/pIRESneo to carry out electroporation (3 × 10 to cell 6/ 0.4ml serum-free RPMI).By standing cell 10 minutes, be then placed in the pre-warm complete RPMI culture medium of 8ml.In the time of 48 hours, cell is diluted to 0.5 × 10 in the complete RPMI culture medium that comprises 1mg/ml G418 (Gibco) 6/ ml.Resisting cell is increased, and shows that by the BNI3 antibody that use is conjugated with phycoerythrin (PharMingen) described cell expresses CTLA-4 on cell surface.By aseptic sorting, separate the cell of high level expression.
The generation of immunity inoculation and hybridoma:
(i) at tail base portion with 1 × 10 7individually be suspended in 300.19 cells of as mentioned above expressing CTLA-4 through transfection in the phosphate buffer (PBS) with complete Freund's adjuvant through subcutaneous route immunity inoculation XenoMouse tMmice (8 to 10 week age), or (ii) tail base portion with use (a) 10 μ g CTLA-4 fusion rotein of complete Freund's adjuvant emulsifying or (b) 10 μ g CTLA-4 peptides through mice described in subcutaneous route immunity inoculation.In each situation, in incomplete Freund's adjuvant, repeat described dose 3 or 4 times.In fusion first 4 days, described mice was accepted the last injection of described immunogen or the cell in PBS.The spleen of the mice of the immunity inoculation of hanging oneself in the future and/or lymph node lymphocyte and [muroid nonsecreting type myeloma P3 cell line] merge and experience HAT and select, (Galfre as discussed previously, G. and Milstein, C., " Preparation ofmonoclonal antibodies:strategies and procedures. " MethodsEnzymol.73:3-46 (1981)).Reclaim and all secrete CTLA-4 specificity human IgG 2large quantities of hybridomas of K antibody.
The hybridoma of the anti-CTLA-4 antibody of following generation of following name is deposited in American type culture collection on April 29th, 2003,10801 University Blvd.Manassas, and Va.20110-2209:
Figure 2006800156704A00800031
embodiment 2
This embodiment has shown the generation of the recombinant mammalian cells system that produces anti-CTLA-4 antibody.
The heavy chain of clones coding monoclonal antibody 11.2.1 and the DNA of light chain from each hybridoma cell line 11.2.1, then used method known to those skilled in the art to determine described DNA sequence.According to the aminoacid sequence of the nucleotide sequence of antibody 11.2.1 and prediction, determine the concordance of using for the gene of each antibody chain.
Then 11.2.1 DNA sequence Insert Fragment sub-clone is entered in expression vector.Described expression vector is transfected in mouse myeloma (NSO) host cell subsequently, thereby forms the various elementary transfectant cell line that produces anti-CTLA-4 antibodies.Based on growth and productivity analysis, select guide's cell line (lead cell line).Then described in sub-clone, guide produces cloned cell line.
By carry out cell culture in the bioreactor that comprises cell culture medium by described cell line, produce anti-CTLA 4 antibody.At production period to culture media supplemented nutrient.Reaching after results standard, by independent filtration or by filter to gather in the crops bioreactor after centrifugal.Then by three chromatographic step that comprise A albumen affinity column and two ion exchange column, carry out the supernatant of purification through clarification.In this process, also carry out low pH deactivation and virus filtration to remove any potential virus.Concentrated described product, then diafiltration in preparation buffer to produce drug substance.
embodiment 3
Carry out the research of the impact of the buffer agent antagonists gathering different for assessment of 4 kinds and fragmentation.
Particularly, preparation comprises anti-CTLA 4 antibody 11.2.1 4 kinds of liquid preparations with acetate, succinate, histidine and edta buffer.Then described preparation is stored at 40 ℃, and carried out the measurement of antibody aggregation and fragmentation in the time of the 0th, 2,5 and 7 weeks.
The preparation of buffer solution:
Described in table 3, prepare 4 kinds of buffer solution.By first a certain amount of buffer agent kind (listing in table 3) being dissolved in, in water, prepare every kind of solution (about 80% target).Then by adding the acid of specifying in the table 3 of q.s or aqueous slkali by the pH regulator to 5.5 of each buffer solution.Regulating after pH, add the water of additional quantity so that the whole buffer concentration of 20mM to be provided.Select the buffer concentration of 20mM to guarantee the suitable pH stability at selected pH5.5 place.Then buffer solution is filtered to by germ tight filter (hole size of 0.22 micron) in the container of sterilizing to use after treating.
Table 3: buffer solution
By water, suitably dilute (1ml to 100ml) glacial acetic acid (99.9%) and prepare 1%v/v glacial acetic acid solution.By 40g solid sodium hydroxide being dissolved in to the sodium hydroxide solution of preparing 1 molarity (M) in 1L water.By water, suitably dilute the hydrochloric acid solution that concentrated hydrochloric acid (37.8%) is prepared 5 molarities (M).
The preparation of antibody preparation:
Evaluated antibody preparation is listed in table 4 below.For preparing each preparation, in the buffer solution shown in first a certain amount of tonicity agent (representing with mg/ml in table 4) being added to, then agitating solution is until tonicity agent dissolving.The antibody bulk solution of the purification process of describing in obtaining from embodiment 2 with 13.2mg/mL in 20mM sodium acetate buffer pH5.5+140mM sodium chloride.In the formulation soln that uses Amicon Ultra 15 MWCO10K (UFC901024) centrifugal concentrating device that this bulk solution is carried out to buffer-exchanged on the Beckman Coulter Allegra21R centrifuge moving at 5 ℃ with 6500 RPM and to identify above entering.Carry out the exchange of about 8 times of volumes, and antibody-solutions is concentrated into 27 to 30mg/ml.Prepare about 3 to 4ml preparation 1-18.At 280nm place, use 1.43 (mg/ml) -1cm -1extinction coefficient by ultravioletvisible spectroscopy (UV-Vis), determine the concentration of antibody.
By preparing 20mg/ml polyoxyethylene sorbitan monoleate (PS80) solution with suitable preparation buffer dilution and the dissolving polyoxyethylene sorbitan monoleate of as above preparation.Then with the concentrate of 20mg/ml, the buffer agent of polyoxyethylene sorbitan monoleate and appropriate amount, antibody, tonicity agent and water are added in antibody soothing agent solution, thus the whole solution of the anti-CTLA-4 monoclonal antibody of the 20mg/ml of acquisition in the preparation corresponding to forming shown in table 4 below.
For the preparation No.2 in table 4, as the concentrate of 200mg/ml, add PEG3350 at that point.
Then described preparation is filtered and is filtered in bottle by the degerming level filter of 0.2 μ.In the 1 type vial of 2ml, use 0.5 to 1ml packing volume.Described bottle Flurotec through Daikyo 777-1
Figure 2006800156704_3
coated stopper is airtight, presses pleat (crimp) sealing, and is placed in stability test case, upright storage 2,5 and 7 weeks at 40 ℃.The horizontal high voltage sterilizing of going forward side by side of washing bottle, also washs and autoclaving for 13mm Daikyo 777-1 serum plug (serumstopper).With regard to the level of gathering and fragmentation, analyze bipartite bottle immediately.
Table 4: tested antibody preparation
Figure 2006800156704A00800051
Figure 2006800156704A00800061
Analysis of agglomeration:
The antibody preparation of table 4 is stored at the temperature of 40 ℃.At the 0th, 2,5 and 7 weeks, use size exclusion chromatography (SEC) just to assemble and analyze every kind of preparation.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the flow velocity of 1ml/ minute and the UV at 214nm place to detect to carry out size exclusion chromatography.Fig. 1 has shown the percentage ratio of the high molecular weight species (that is, the aggregation of anti-CTLA-4 monoclonal antibody 11.2.1) of the eluting recording in correlation time for every kind of preparation.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to Fig. 1) of total peak area.As seen in Fig. 1, through the preparation of edta buffer, demonstrate the gathering of floor level, by this order, be the preparation through histidine, acetate and succinate buffering subsequently.
Fragmentation assay:
As noted above, at the temperature of 40 ℃, store the antibody preparation of table 4.At the 0th, 2,5 and 7 weeks, also with rSDS-PAGE, with regard to fragmentation, analyze every kind of preparation.Use NuPAGE4-12%bis-Tris gel and colloid indigo plant (coomassie) dyestuff to carry out rSDS-PAGE analysis.For reduced form gel (rSDS-PAGE), pass through NuPAGE
Figure 2006800156704_4
reducing agent obtains reduction.By scan to estimate total hydrolysis impurity (that is, the fragment of anti-CTLA-4 monoclonal antibody 11.2.1) with Molecular Dynamics Personal Densitometer PDQC-90 or Bio-Rad GS800 Imaging Densitometer.Fig. 2 has shown the fragmentation percentage ratio recording in correlation time for every kind of preparation.The percentage ratio (referring to Fig. 2) that described fragmentation level calculation is total band volume.As what can see from Fig. 2, through the preparation of edta buffer, show minimum fragmentation level, by this order, be the preparation through histidine, acetate and succinate buffering subsequently.
Table 5 below (a) (0 week), table 5 (b) (2 weeks), table 5 (c) (5 weeks) and table 5 (d) (7 weeks) have been reported gathering and the fragmentation data of graphic extension in Fig. 1 and 2.
Table 5 (a): the gathering on 0 time point and fragmentation result
Table 5 (b): the gathering on the 2nd time-of-week point and fragmentation result
Figure 2006800156704A00800072
Table 5 (c): the gathering on the 5th time-of-week point and fragmentation result
Figure 2006800156704A00800082
Table 5 (d): the gathering on the 7th time-of-week point and fragmentation result
Figure 2006800156704A00800083
Figure 2006800156704A00800091
embodiment 4
Carry out the research for assessment of the ability of the multiple freezing and thaw cycles of the different liquids preparation tolerance that comprises monoclonal anti CTLA-4 antibody 11.2.1.
Conventionally the ability that assessment liquid preparation tolerates multiple freeze/thaw determining that described preparation whether can refrigerated storage (with, if wanted, transport), then thaw for future use.
Evaluated preparation is listed in table 6 below.Identical with the method for describing in embodiment 3 for the preparation of the method for preparation.Every kind of solution of 2.5mL is placed in to 5-mL 1 type vial, stopper beyond the Great Wall, then sealing.Be accredited as the 1st to 4,7 to 8,11 to 12 identical with the preparation in embodiment 3 with same numbers marker character with the preparation of 15 to No. 16 below.
Table 6: tested antibody preparation
Figure 2006800156704A00800092
Figure 2006800156704A00800101
By every kind of preparation through 6 continuous freeze/thaw of course.3 circulations before carrying out in speed controlled refrigerator.Rear 3 circulations are slower circulations of using many water filling bottles corresponding to high heat load (high thermal load) that are placed in refrigerator or refrigerator to carry out.For circulation 1,2 and 3, the bottle that preparation is housed is placed in to speed controlled refrigerator (PlanerKryo 560-16) and through the following circulation of course: with the cooling preparation of the speed of 0.2 ℃/min until reach the temperature of-70 ℃, remain at-70 ℃ 1.5 to 3 hours, then with the speed of 0.3 ℃/min, thaw preparation until reach the temperature of 5 ℃.For circulation 4,5 and 6, by bottle and other water filling bottles (for sample bottle of every kind of preparation; 17 preparation bottles and about 30 water filling bottles altogether) together be placed in box.Then first this box is placed in to refrigerator at the temperature that remains on-70 ℃ freezing about 17 hours, is then placed in refrigerator at the temperature that remains on 2-8 ℃ about 50 hours.The average cooldown rate that the recording heat sensor that is placed in box records refrigerating process is 0.09 ℃/min, and the average rate of heat addition of course of defrosting is 0.03 ℃/min.
At all after dates of each freeze/thaw, with regard to granule formation, change color and turbidity, change and carry out every kind of preparation of visually rank.When preparation still keeps ice-cold at every turn after thawing, under black and white background, in transparent box, carry out this visual observations of every kind of preparation.Table 7 (below) has been reported result.
Table 7: the visually rank of the freeze/thaw stability of anti-CTLA-4 antibody 11.2.1
Figure 2006800156704A00800111
After freeze/thaw, the preparation that only comprises sodium chloride (that is, chloride ion) shows the increase larger than the preparation that comprises trehalose, sucrose or Sorbitol in sol particle level.But, with respect to the corresponding preparations that does not comprise PEG, PEG is added in the preparation that comprises sodium chloride, seem to have reduced the sol particle level recording after freeze/thaw.
Analysis of agglomeration:
In addition,, after 6 continuous freeze/thaw, for every kind of preparation, use size exclusion chromatography to measure the increase percentage ratio of sol particle.
After the 6th freeze/thaw, just assemble and analyze every kind of preparation with size exclusion chromatography.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the flow velocity of 1ml/ minute and the UV at 214nm place to detect to carry out size exclusion chromatography.Table 7 has shown the percentage ratio of the high molecular weight species (that is, the aggregation of anti-CTLA-4 monoclonal antibody 11.2.1) of the eluting recording in correlation time for every kind of preparation.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to table 7) of total peak area.As seen in table 7, through the preparation of edta buffer, demonstrate the gathering of floor level, by this order, be the preparation through histidine, acetate and succinate buffering subsequently.
embodiment 5
Carry out the research for the impact of the variable color in the liquid preparation that comprises monoclonal anti CTLA-4 antibody 11.2.1 and gathering for assessment of EDTA, methionine and oxygen free condition.From the Huo Liangge aspect, integrity aspect of aesthetic aspect, product, the variable color in these liquid preparations and gathering are normally undesired.
Table 8 has below been listed evaluated preparation processing.Identical with the method for describing in embodiment 3 for the preparation of the conventional method of preparation.For the present embodiment, preparation comprises monoclonal anti CTLA-4 antibody 11.2.1 (5mg/ml), sodium acetate buffer (20mM), sodium chloride (8.2mg/ml) and polysorbate 80 (0.2mg/ml) and has the initial preparation of pH5.5, and is added to several comprising in the 10-mL vial that seals top to allow to carry out sterile sampling.
According to table 8 below, initial preparation is carried out to various processing.As pointed in table 8, methionine is added in some bottles.The EDTA of two kinds of variable concentrations is added in other bottles.Nitrogen is added to the headroom that EDTA or methionine bottle are housed through selecting.In addition before, in nitrogen is injected into its headroom, get rid of the air in some remaining untreated bottles.In addition, leave some remaining bottles and do not accept to process to be used as experiment contrast.
Two bottles from every kind of processing in table 8 are stored 0,2,4,6,8,10,14,16 and 18 week at 40 ℃.On each time point, one in the bottle of two storages is assessed for visual color, and another bottle is carried out to sterile sampling to measure the level of storing rear 11.2.1 antibody aggregation.Table 9 and 10 has been reported result.
Table 8: the processing of tested antibody preparation
Preparation visual analysis:
After 0 (initially), 2,4,6,8,10,14,16 and 18 weeks, with regard to granule formation, change color and turbidity, change every kind of preparation of visually rank.In table 9, reported visual observations result.
Table 9: the preparation visually rank after treatment in table 8
Figure 2006800156704A00800122
Figure 2006800156704A00800131
Result in table 9 shows, stores that in bottle, to form pink after at least 4 weeks painted without the preparation of EDTA and/or methionine at 40 ℃.But do not wish to be bound by any specific theory, and it is believed that in one embodiment of the invention, this color change possibility (at least in part) is because oxidizing process causes.But in other embodiments, described color change may be because many other processes that have nothing to do with oxidation cause.
Nitrogen is added in the headroom of bottle, compared with adding methionine and/or EDTA, seem to there is less impact aspect minimizing variable color.
Analysis of agglomeration:
At the temperature of 40 ℃, store the antibody preparation of processing according to table 8.At the 0th, 2,6,8,10,14,16 and 18 weeks, just assemble and analyze every kind of preparation with size exclusion chromatography.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the flow velocity of 1ml/ minute and the UV at 214nm place to detect to carry out size exclusion chromatography.Table 10 has shown the high molecular weight species (that is, the aggregation of anti-CTLA-4 monoclonal antibody 11.2.1) of the eluting recording in correlation time of percentage ratio process to(for) every kind of preparation.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to table 10) of total peak area.
Table 10: about the gathering percentage ratio of the preparation processing in table 8
Figure 2006800156704A00800141
Result in table 9 shows, stores that to start in bottle, to form pink after at least 4 weeks painted without the preparation of EDTA and/or methionine at 40 ℃.As seen in table 10, through the preparation of EDTA and/or methionine processing, show minimum gathering level, be then through nitrogen treatment and untreated control formulation.
embodiment 6
Study to be evaluated at as liquid preparation and store rear methionine and the EDTA impact for the Oxidation of some methionine residues in anti-CTLA-4 antibody 11.2.1.
Methionine Oxidation Analysis:
At 40 ℃, store after 8 weeks, by lysine-C graphing method, measure the oxidation level of the methionine residues at amino acid sites 256 and 432 places in anti-CTLA-4 antibody 11.2.1.
On the 8th time-of-week point to preparation No.26,29 and 33 vial (table 8) being housed and carrying out sterile sampling according to their the processing gains of embodiment 5.Then, under standard conditions in tris buffer (pH8.0) with sample described in Lyc-C enzymic digestion, and analyze by reversed phase high-performance liquid chromatography.Use Grace Vydac Protein C4 analytical column, the 0.1%TFA being used in water carries out separate with the 0.085%TFA gradient eluent in acetonitrile.
Table 11: after the processing in table 8, the oxidation percentage ratio of the methionine in anti-Anti-CTLA-4 antibody 11.2.1
Figure 2006800156704A00800151
Result in table 11 shows, in 11.2.1 antibody preparation, adds methionine or EDTA, compared with preparation without EDTA or methionine, has reduced the oxidation percentage ratio at the methionine residues place of described two appointments.
embodiment 7
Carry out the research for assessment of the oxidation of some tryptophan in anti-CTLA-4 antibody 11.2.1 and tyrosine residue.
The anti-CTLA-4 antibody 11.2.1 preparation that pink variable color occurs is in time found in and carries out at 500nm place, having distinctive absorption maximum after ultraviolet/visible light spectrometry (UV-Vis).
Identical with the method for describing in embodiment 3 for the preparation of the method for preparation.For the present embodiment, the preparation of the monoclonal anti CTLA-4 antibody 11.2.1 solution in 20mM sodium acetate buffer, 8.2mg/ml sodium chloride and 0.2mg/ml polysorbate 80 (pH5.5) that comprises 5mg/ml is stored 4 weeks in two vials at 40 ℃, in the described time, there is pink variable color in described preparation.
Then the solution experience molecular weight (cut-off) in one of little vial formulation of variable color is filtered, this allows formulation excipients by defecator, but antibody is stayed.It is limpid and colourless filtering eluent (for example, water and excipient), but the fraction (for example, antibody 11.2.1) of collecting keeps pink.Therefore, filtration experiment shows, described pink variable color is relevant to antibody 11.2.1 itself, rather than produces from the excipient of preparation.
Next, under standard conditions, with trypsinization, there is second bottle of pink variable color, and by analyzing with the reversed-phase high-performance liquid chromatography (LC-MS) of mass spectrum coupling.Use Grace Vydac Protein C4 analytical column, the 0.1%TFA being used in water carries out separate with the 0.085%TFA gradient eluent in acetonitrile.Under 500nm, monitoring is through the UV-Vis absorbance of the peptide of digestion, then based on the corresponding peptide of its molecular weight identification.
The tryptic digest peptide section relevant to 500nm absworption peak has aminoacid sequence: GLEWVAVIWYDGSNK.Then, under standard conditions, further use Asp-N protease digestion peptide sequence GLEWVAVIWYDGSNK, the peptide that 500nm absorption (UV-Vis) peak falls digestion in company with Asp-N protease moves together, and the peptide of described digestion has aminoacid sequence: GLEWVAVIWY.
Therefore, do not wish by any specific theoretical constraint, it is believed that any or two or tyrosine residue (Y) among two trp residues (W) in the described peptide through protease digestion (GLEWVAVIWY) are possible oxidation site, described oxidation can cause the pink variable color of the antibody 11.2.1 preparation in the present embodiment.In specific embodiment, it is believed that any or two among two trp residues (W) in the described peptide through protease digestion (GLEWVAVIWY) are possible oxidation site, described oxidation can cause pink variable color.
However, also possible that, other mechanism except oxidation can cause for example, in the specific variable color (pink and yellow) of seeing in the various preparations of assessment herein any one or more.
embodiment 8
Carry out the research for the impact of anti-CTLA-4 antibody 11.2.1 variable color, gathering and fragmentation for assessment of EDTA and DTPA.
Particularly, three kinds of liquid preparations that comprise antibody 11.2.1 that have and do not there is EDTA and DTPA of preparation.Described preparation is stored at 40 ℃, at the 0th, 2,4,6,8 and 10 weeks, carried out variable color, gathering and the fragmentation assessment of antibody.
For the present embodiment, as described in example 3 above, the 20mg/ml anti-CTLA-4 antibody-solutions of preparation in the 20mM sodium acetate buffer (pH5.5) with 8.2mg/ml sodium chloride and 0.2mg/ml polyoxyethylene sorbitan monoleate is also distributed in several vials, then by adding EDTA or DTPA to process.With solid form, in preparation bottle, add EDTA and DTPA.With regard to the level of variable color, gathering and fragmentation, several bottles are analyzed immediately, also by several other bipartite bottles upright storage 2,4,6,8 and 10 weeks at 40 ℃.
Then treated and untreated bottle are carried out to sterile sampling to measure antibody 11.2.1 in preparation assemble on the the 2nd, 4,6,8 and 10 time-of-week points and the level of fragmentation, and observe variable color.Table 12 and 13 has been reported result.
Preparation visual analysis:
After 0 (initially), 2,4,6,8 and 10 weeks, with regard to granule formation, change color and turbidity, change every kind of preparation of visually rank.In table 12, reported visualization result.
The visually rank of table 12:EDTA and the processing of DTPA preparation
Figure 2006800156704A00800161
Result in table 12 shows, at 40 ℃, stores after at least 6 weeks, without the preparation of EDTA or DTPA, pink variable color occurs in bottle.
Analysis of agglomeration:
At the temperature of 40 ℃, store the antibody preparation of processing according to table 12.At the 0th, 2,6,8 and 10 weeks, just assemble and analyze every kind of preparation with size exclusion chromatography.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the flow velocity of 1ml/ minute and the UV at 214nm place to detect to carry out size exclusion chromatography.
Table 13 has shown the high molecular weight species (that is, the aggregation of anti-CTLA-4 monoclonal antibody 11.2.1) of the eluting recording in correlation time of percentage ratio process to(for) every kind of preparation.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to table 13) of total peak area.
Table 13: about the gathering percentage ratio of EDTA and the processing of DTPA preparation
Figure 2006800156704A00800171
As seen in table 7, the preparation that comprises EDTA and DTPA all demonstrates the gathering level lower than the preparation without EDTA or DTPA.
embodiment 9
Carry out the research for the impact of anti-CTLA-4 antibody 11.2.1 stability for assessment of EDTA and nitrogen.
Particularly, in the preparation (it comprises trehalose and polyoxyethylene sorbitan monoleate) of histidine buffering, aspect the formation of variable color, gathering, oxidation, fragmentation and electrically charged kind, analyzing the impact for antibody 11.2.1 stability of EDTA and nitrogen.
Evaluated preparation is listed in table 13 below.Identical with the method for describing in embodiment 10 below for the preparation of the method for preparation.At 40 ℃, store described preparation, and carried out stability assessment at the 0th, 4,8,12 and 24 weeks.
For the present embodiment, prepare as in Example 10 the anti-CTLA-4 antibody-solutions of 20mg/ml in the 20mM histidine buffering liquid (pH5.5) with 84mg/ml trehalose and 0.2mg/ml polysorbate 80.By the liquid storage with trehalose and polyoxyethylene sorbitan monoleate, the concentrated liquid storage of antibody is diluted to a part of preparing preparation containing the final composition of the anti-CTLA-4 antibody of 20mg/mL.Except adding 10mg/mL Na 2eDTA.2H 2o concentrate, to obtain outside the additional step of final concentration of 0.1mg/mL, is prepared the Part II of preparation similarly.Then with the amount of every 2ml vial 1ml, distribute preparation.Then half bottle of every kind of preparation is placed in freeze dryer, after evacuation, headroom changed into nitrogen.With nitrogen, filling after bottle, the measured value of its oxygen level is reported as about oxygen of 1.5% to 1.6%, and headroom has the bottle of air and is reported as about oxygen of 19.7% to 20%.
With regard to the level of the formation of variable color, gathering, fragmentation, oxidation and electrically charged kind, analyze several bottles immediately, also by several other bipartite bottles upright storage 2,4,8,12 and 24 weeks at 40 ℃.On each time point, for every kind of condition, process for every kind and take out the level of two storage bottles with the formation of antibody 11.2.1 gathering in measurement preparation, fragmentation, oxidation and electrically charged kind, and observe variable color.Table 14 has been reported result to 18.
Table 13: tested antibody preparation
Figure 2006800156704A00800181
Preparation visual analysis:
After 0 (initially), 4,8,12 and 24 weeks, with regard to granule formation, change color and turbidity, change every kind of preparation of visually rank.In table 14, reported visual observations result.
Table 14: the preparation visually rank after treatment in table 13
Figure 2006800156704A00800182
Figure 2006800156704A00800191
Result in table 14 shows, without the preparation of EDTA or nitrogen, stores the pink variable color of rear generation in 4 weeks at 40 ℃.Table 14 also shows, the preparation that substitutes the air in bottle headroom with nitrogen makes pink variable color be delayed to generation in the 12nd week.Two kinds of preparations that comprise EDTA do not have visible variable color within least 24 weeks.
Analysis of agglomeration:
At the temperature of 40 ℃, store the antibody preparation of processing according to table 13.At the 0th, 4,8,12 and 24 weeks, just assemble and analyze every kind of preparation with size exclusion chromatography.At each time point, preparation bottle is carried out to sterile sampling.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the UV at the flow velocity of 1ml/ minute and 214nm place to detect described sample is carried out to size exclusion chromatography.Table 15 has shown the high molecular weight species (that is, the aggregation of anti-CTLA-4 monoclonal antibody 11.2.1) of the eluting recording in correlation time of percentage ratio process to(for) every kind of preparation.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to table 15) of total peak area.
Table 15: the gathering percentage ratio of the preparation in table 13
Figure 2006800156704A00800192
In at table 15, see, and compare with the preparation in headroom with air without EDTA, preparation, nitrogen preparation and the EDTA+ nitrogen that comprises EDTA is proposed agent and is demonstrated lower gathering level in time.
Fragmentation assay:
At the temperature of 40 ℃, store the antibody preparation of preparing according to table 13.At the 0th, 4,8,12 and 24 weeks, use the just total hydrolysis impurity (that is, fragmentation) of reduced form SDS-PAGE (rSDS-PAGE) to analyze every kind of preparation.At each time point, preparation bottle is carried out to sterile sampling, be then loaded on NuPAGE 4-12%bis-Tris gel, use colloid indigo plant (coomassie) dyestuff.By using NuPAGE
Figure 2006800156704_5
reducing agent obtains the reduction of gel.By impurity (that is, the fragmentation) percentage ratio of each sample strip in reduced form gel is estimated in MolecularDynamics Personal Densitometer PDQC-90 or the enterprising line scanning of Bio-Rad GS800 ImagingDensitometer.Fragmentation level calculation is the percentage ratio (referring to Figure 16) of total band volume.
Table 16: total (impurity) fragmentation percentage ratio of the preparation in table 13
Figure 2006800156704A00800201
In at table 16, see, and compare with the preparation in headroom with air without EDTA, preparation, nitrogen preparation and the EDTA+ nitrogen that comprises EDTA is proposed agent and is demonstrated lower fragmentation level in time.
The preparation of bronsted lowry acids and bases bronsted lowry kind:
At the temperature of 40 ℃, store the antibody preparation of preparing according to table 13.At the 0th, 4,12 and 24 weeks, use imaging capillary electrophoresis (Imaging Capillary Electrophoresis, iCE) to analyze every kind of preparation with regard to the formation of bronsted lowry acids and bases bronsted lowry kind.Use ConvergentBiosciences iCE 280analyser carries out imaging capillary electrophoresis with assessment electric charge inhomogeneity.Described Convergent iCE 280to allow user the sample separating comprising to be carried out to imaging capillary isoelectric focusing (IEF) device of image taking in capillary tube.
On each time point, preparation bottle is carried out to sterile sampling.Then electrophoresis ampholyte, prepare sample in the mixture of methylcellulose, collimating marks thing (calibration marker) and water.By Sample introduction iCE 280in, and apply high potential/voltage.Make the pH3-10.5 polyacrylamide gel of preparing by hand and use Coomassie blue dyestuff to carry out IEF mensuration.Relative isoelectric point, IP (pI) and their position based on them separate sample protein matter component.By imaging CCD photographing unit, observe the relative quantity of every kind of component of separating.Then with conventional chromatogram figure integration software, carry out process data, and described data are expressed as to the loss (that is, the formation of bronsted lowry acids and bases bronsted lowry kind) (referring to table 17) of main peak.
Table 17: the main peak loss of the preparation in table 13
Figure 2006800156704A00800211
As seen in table 17, and to compare with the preparation in headroom with air without EDTA, preparation, nitrogen preparation and the EDTA+ nitrogen that comprises EDTA is proposed agent and is demonstrated higher complete main peak level in time.Therefore, not containing EDTA and/or do not have in headroom in the preparation of nitrogen, along with time bronsted lowry acids and bases bronsted lowry kind forms in the past amount was larger.
Amino-acid oxidase is analyzed:
At 40 ℃, store after 12 weeks, by lysine-C graphing method, measure the oxidation level of the methionine residues at amino acid sites 256 and 432 places in anti-CTLA-4 antibody 11.2.1.
On the 12nd time-of-week point, the bottle of the preparation that table 13 is housed is carried out to sterile sampling.Then, under standard conditions, in tris buffer (pH8.0), with lysyl endopeptidase (Lys-C), digest described sample, and analyze by reversed phase high-performance liquid chromatography.Use GraceVydac Protein C4 analytical column, the 0.1%TFA being used in water carries out separate with the 0.085%TFA gradient eluent in acetonitrile.
Table 18: the percentage ratio oxidation of the methionine in the anti-CTLA-4 antibody in the preparation of table 13
Figure 2006800156704A00800221
Result in table 18 shows, to 11.2.1 antibody preparation, adds EDTA and/or adds nitrogen to the headroom of bottle, and compares with the preparation in headroom with air without EDTA, has reduced the oxidation percentage ratio at the methionine residues place of two appointments.
embodiment 10
Study to compare the impact for the stability of lower series preparation: the anti-CTLA-4 antibody 11.2.1 preparation that comprises sodium acetate buffer and sodium chloride (that is, chloride ion) is to comprising the preparation of histidine buffer and trehalose.
Particularly, aspect variable color, gathering and fragmentation, analyze the impact for antibody 11.2.1 stability.
Evaluated preparation is listed in table 19 below.Identical with the method for describing in embodiment 3 for the preparation of the method for described preparation.
By following manner, carry out the preparation in preparation table 19: be taken at the antibody 11.2.1 liquid storage of the 11.9mg/ml in 20mM sodium acetate buffer (pH5.5), 140mM sodium chloride, then it had to Pellicon XL PBTK 30K 50cm 2in the Millipore Lab Scale TFF system of film, experience ultrafiltration/diafiltration (UF/DF) step.Next, in 35 to 40mg/ml scope in 20mM sodium acetate or 20mM histidine buffering liquid the concentrated solution of Dispersal risk 11.2.1.
With 3 times of target final concentrations, in sodium acetate or histidine buffering liquid, prepare the concentrate of tonicity agent.In every kind of buffer, with 20mg/ml, prepare the concentrated solution of polyoxyethylene sorbitan monoleate, and prepare Na with 10mg/ml 2eDTA.2H 2the concentrated solution of O.By suitably diluting concentrated solution, prepare single preparation.Then preparation is filtered by 0.2 μ degerming level filter, and inject several bipartite bottles.In 2-ml 1 type vial, use the packing volume of 1ml.Described bottle Flurotec through Daikyo 777-1
Figure 2006800156704_6
coated stopper is airtight, presses pleat (crimp) sealing, and it is uprightly stored at 25 ℃ and 40 ℃ in stability test case.Also another group bottle is placed 4 weeks at-20 ℃, and another is organized to experience 4x freeze/thaw (water filling bottle box) as described in Example 4.All preparations have 5.5 pH and the anti-CTLA-4 antibody 11.2.1 concentration of 20mg/ml.
With regard to the level of variable color, gathering and fragmentation, analyze several bottles immediately, also by several other bipartite bottles upright storage 4,8,12,18,24 and 36 weeks at 25 ℃ and 40 ℃.On each time point, for every kind of condition, every kind of preparation takes out two and stores bottle to measure the level of antibody 11.2.1 gathering, fragmentation, and observes variable color.Table 20 to 24 and Fig. 3 and 4 reported result.
Table 19: tested antibody preparation
Figure 2006800156704A00800231
Preparation visual analysis:
1) after initial mixing preparation, 2) frozen preparation after 4 weeks at-20 ℃, with 3) every kind of preparation of visually rank after 4 freeze/thaw (as described in Example 4, in box together with water filling bottle, in-70 ℃ to 5 ℃).After the 0th (initially), 8,12 and 24 weeks, with regard to granule formation, change color and turbidity, change every kind of preparation of visually rank.With regard to granule formation, change color and turbidity, change preparation is assessed, and be reported in table 20 (freeze/thaw), table 21 (storing at 25 ℃) and table 22 (storing at 40 ℃).
Table 20: the visually rank of the preparation after freeze/thaw in his-and-hers watches 19
Figure 2006800156704A00800232
Figure 2006800156704A00800241
Table 21: the visually rank of the preparation after storing at 25 ℃ in his-and-hers watches 19
Figure 2006800156704A00800242
Table 22: the visually rank of the preparation of his-and-hers watches 19 after storing at 40 ℃
Figure 2006800156704A00800243
Figure 2006800156704A00800251
Table 20 to the result in table 22 shows, the antibody 11.2.1 preparation that comprises EDTA has variable color, the turbidity of minimizing and the granule of minimizing compared without those preparation minimizings of EDTA and forms.Generally, the preparation that comprises sodium chloride has compares the variable color, turbidity and the granule formation that contain EDTA but do not contain the preparation increase of sodium chloride.
Analysis of agglomeration:
At the temperature of 25 ℃ and 40 ℃, store the antibody preparation of preparing according to table 19.The 0th (initially), 4,8,12,24 and 36 weeks, just assemble and analyze 25 ℃ of preparations with size exclusion chromatography.At the 4th, 8,12 and 24 weeks, just assemble and analyze 40 ℃ of preparations with size exclusion chromatography.At each time point, preparation bottle is carried out to sterile sampling.Just assemble and analyze every kind of preparation with size exclusion chromatography.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the UV at the flow velocity of 1ml/ minute and 214nm place to detect described sample is carried out to size exclusion chromatography.Table 23 (a) and 23 (b) have shown that for every kind of preparation, processing the antibody 11.2.1 recording in correlation time assembles percentage ratio.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to table 23 (a) and 23 (b)) of total peak area.
Table 23 (a): after storing at 25 ℃ in table 19 the percentage ratio of agent assemble
Figure 2006800156704A00800252
Figure 2006800156704A00800261
Table 23 below (b) reported aggregate data, and it carries out graphic extension in Fig. 3.
Table 23 (b): the gathering percentage ratio of the preparation after storing at 40 ℃ in table 19
Figure 2006800156704A00800262
As seen in table 23 (a), 23 (b) and Fig. 3, after storing at 25 ℃ and 40 ℃, and lack EDTA but there is acetate buffer and the preparation of sodium chloride is compared, the preparation that comprises EDTA demonstrates the gathering level of minimizing.In addition, and lack EDTA but comprise acetate buffer and the preparation of sodium chloride is compared, the preparation that comprises histidine buffer (without EDTA) has the aggregate amount of minimizing.
Fragmentation assay:
At the temperature of 25 ℃ and 40 ℃, store the antibody preparation of preparing according to table 19.The 0th (initially), 4,8,12,18 and 36 weeks, use the just total hydrolysis impurity (that is, fragmentation) of reduced form SDS-PAGE (rSDS-PAGE) to analyze every kind of preparation.At each time point, preparation bottle is carried out to sterile sampling, be then loaded on NuPAGE 4-12%bis-Tris gel, use colloid indigo plant (coomassie) dyestuff.By using NuPAGE
Figure 2006800156704_7
reducing agent obtains the reduction of gel.By impurity (that is, the fragmentation) percentage ratio of each sample strip in reduced form gel is estimated in Molecular Dynamics Personal Densitometer PDQC-90 or the enterprising line scanning of Bio-Rad GS800 Imaging Densitometer.Fragmentation level calculation is the percentage ratio (referring to table 24 (a) and 24 (b)) of total band volume.
Table 24 (a): the fragmentation percentage ratio of the preparation after storing at 25 ℃ in table 19
Figure 2006800156704A00800271
Table 24 below (b) reported fragmentation data, and it carries out graphic extension in Fig. 4.
Table 24 (b): the percentage ratio fragmentation of the preparation after storing at 40 ℃ in table 19
Figure 2006800156704A00800272
As seen in table 24 (a), 24 (b) and Fig. 4, after storing at 25 ℃ and 40 ℃, and lack EDTA but there is acetate buffer and the preparation of sodium chloride is compared, the preparation that comprises EDTA demonstrates the fragmentation level of minimizing.
embodiment 11
Carry out the research for the impact of anti-CTLA-4 antibody 11.2.1 preparation stability for the EDTA of variable concentrations relatively.By replacing part trehalose with mannitol, also tested another selection to histidine buffer-trehalose preparation.
Particularly, aspect variable color, gathering, fragmentation and oxidation, analyze the impact for antibody 11.2.1 stability.
Evaluated preparation is listed in table 25 below.Identical with the method for describing in embodiment 10 for the preparation of the method for described preparation.
By following manner, carry out the preparation in preparation table 25: be taken at the antibody 11.2.1 liquid storage of the 11.9mg/ml in 20mM sodium acetate buffer (pH5.5), 140mM sodium chloride, then it had to Pellicon XL PBTK 30K 50cm 2in the Millipore Lab Scale TFF system of film, experience ultrafiltration/diafiltration (UF/DF) step.Next, in 35 to 40mg/ml scope in 20mM sodium acetate or 20mM histidine buffering liquid the concentrated solution of Dispersal risk 11.2.1.
With 3 times of target final concentrations, in sodium acetate or histidine buffering liquid, prepare the concentrate of tonicity agent.In every kind of buffer, with 20mg/ml, prepare the concentrated solution of polyoxyethylene sorbitan monoleate, and prepare Na with 10mg/ml 2eDTA.2H 2the concentrated solution of O.By suitably diluting concentrated solution, prepare single preparation.On inspection, the concentration of EDTA is (as Na 2eDTA.2H 2o) in the scope of 0-0.1mg/ml.Then preparation is filtered by 0.2 μ degerming level filter, and inject several bipartite bottles.In 2-ml 1 type vial, use the packing volume of 1ml.
Described bottle Flurotec through Daikyo 777-1
Figure 2006800156704_8
coated stopper is airtight, presses pleat (crimp) sealing, and it is uprightly stored at 25 ℃ and 40 ℃ in stability test case.By another group bottle experience 4x freeze/thaw as described in Example 10.All preparations have 5.5 pH and the anti-CTLA-4 antibody 11.2.1 concentration of 20mg/ml.
With regard to the level of variable color, gathering, fragmentation and oxidation, analyze several bottles immediately, also by several other bipartite bottles upright storage 4,8,13,18 and 24 weeks at 25 ℃ and 40 ℃.On each time point, for every kind of condition, every kind of preparation takes out two and stores bottle to measure the level of antibody 11.2.1 gathering, fragmentation, and observes variable color.Table 26 to 31 and Fig. 5 and 10 reported result.
Table 25: tested antibody preparation
Preparation visual analysis:
1) after initial mixing preparation, 2) after 4 freeze/thaw, and 3) at 25 ℃ and 40 ℃, store every kind of preparation of visually rank after 4,8,13,18 and 24 weeks.With regard to granule formation, change color and turbidity, change the described preparation of assessment, and be reported in table 26 in 28.
Table 26: the visually rank of the preparation of table 25 after freeze/thaw
Figure 2006800156704A00800292
Figure 2006800156704A00800301
Table 27: the visually rank of the preparation of table 25 after storing at 25 ℃
Figure 2006800156704A00800302
* Y6 and Y4 are the colour code symbols in the yellow scale of EP.Y6 is more shallow than the yellow of Y4.(reference: PhEur5.0,2005 Monograph 2.2.2).
Table 28: the visually rank of the preparation of table 25 after storing at 40 ℃
* Y6 and Y4 are the colour code symbols in the yellow scale of EP.Y6 is more shallow than the yellow of Y4.(reference: PhEur5.0,2005 Monograph 2.2.2).
Table 26 to the result in 28 shows, the antibody 11.2.1 preparation that comprises all tested EDTA concentration is compared the variable color, the turbidity of minimizing and the granule of minimizing that without those preparations of EDTA, have a minimizing and formed.
In a word, and have EDTA but do not have compared with the preparation of sodium chloride, the preparation that comprises sodium chloride has the freeze/thaw protective effect that is formed proved minimizing by the variable color increasing, turbidity and granule.
Analysis of agglomeration:
At the temperature of 25 ℃ and 40 ℃, store the antibody preparation of preparing according to table 25.The 0th (initially), 4,8,13,18 and 24 weeks, just assemble and analyze 25 ℃ and 40 ℃ of preparations with size exclusion chromatography.At each time point, preparation bottle is carried out to sterile sampling.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the UV at the flow velocity of 1ml/ minute and 214nm place to detect described sample is carried out to size exclusion chromatography.Table 29 (a) has shown that the antibody 11.2.1 recording in correlation time for every kind of preparation after storage at 25 ℃ assembles percentage ratio.Table 29 (b) has shown that the antibody 11.2.1 recording after storage at 40 ℃ assembles percentage ratio.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to table 29 (a) and 29 (b)) of total peak area.
Table 29 (a): the gathering percentage ratio of the preparation after storing at 25 ℃ in table 25
Figure 2006800156704A00800321
Table 29 below (b) reported aggregate data, and it is graphic extension in Fig. 5.
Table 29 (b): the gathering percentage ratio of the preparation after storing at 40 ℃ in table 25
Figure 2006800156704A00800331
As seen in table 29 (a), 29 (b) and Fig. 5, after storing at 25 ℃ and 40 ℃, with lack EDTA but there is acetate buffer and the preparation of sodium chloride is compared, the preparation that comprises EDTA demonstrates the gathering level of minimizing in all tested EDTA concentration.The minimizing as the gathering percentage ratio of the preparation in the table 25 of the function of EDTA concentration is summarized in Fig. 7 diagram.
Fragmentation assay:
At the temperature of 25 ℃ and 40 ℃, store the antibody preparation of preparing according to table 25.The 0th (initially), 4,8,13,18 and 24 weeks, use the just total hydrolysis impurity (that is, fragmentation) of reduced form SDS-PAGE (rSDS-PAGE) to analyze 25 ℃ and 40 ℃ of preparations.At each time point, preparation bottle is carried out to sterile sampling, be then loaded on NuPAGE 4-12%bis-Tris gel, use colloid indigo plant (coomassie) dyestuff.By using NuPAGE
Figure 2006800156704_9
reducing agent obtains the reduction of gel.By impurity (that is, the fragmentation) percentage ratio of each sample strip in reduced form gel is estimated in Molecular Dynamics Personal Densitometer PDQC-90 or the enterprising line scanning of Bio-Rad GS800 Imaging Densitometer.Fragmentation level calculation is the percentage ratio (referring to Figure 30 (a) and 30 (b)) of total band volume.
Table 30 (a): the fragmentation percentage ratio of the preparation after storing at 25 ℃ in table 25
Figure 2006800156704A00800341
Table 30 below (b) reported fragmentation data, and it is graphic extension in Fig. 6.
Table 30 (b): the fragmentation percentage ratio of the preparation after storing at 40 ℃ in table 25
Figure 2006800156704A00800342
As seen in table 30 (a), 30 (b) and Fig. 6, after storing at 25 ℃ and 40 ℃, and lack EDTA but there is acetate buffer and the preparation of sodium chloride is compared, the preparation that comprises EDTA demonstrates the fragmentation level of minimizing.In addition, comprise histidine with trehalose but without the preparation of EDTA, demonstrate to compare and comprise sodium chloride but without the fragmentation of the preparation minimizing of EDTA.
The minimizing as the fragmentation percentage ratio of the preparation in the table 25 of the function of EDTA concentration has been summarized in Fig. 8 diagram.
Amino-acid oxidase is analyzed:
By lysine-C graphing method, measure the oxidation level of some methionine residues at amino acid sites 256 and 432 places in anti-CTLA-4 antibody.After storing, on the 12nd week and the 24th time-of-week point, the bottle of the preparation that table 25 is housed is carried out to sterile sampling at 40 ℃.Then, under standard conditions, in tris buffer (pH8.0), with lysyl endopeptidase (Lys-C), digest described sample, and analyze by reversed phase high-performance liquid chromatography.Use GraceVydac Protein C4 analytical column, the 0.1%TFA being used in water carries out separate with the 0.085%TFA gradient eluent in acetonitrile.Table 31 has been reported result.
Table 31: the oxidation percentage ratio of the methionine of anti-CTLA-4 antibody 11.2.1 in the preparation of table 25
Figure 2006800156704A00800351
Figure 2006800156704A00800361
As seen in table 31, the EDTA percentage ratio in antibody 11.2.1 preparation has reduced in time and the level of the methionine oxidation that occurs.
embodiment 12
Carry out for comparing the research for the impact of anti-CTLA-4 antibody 11.2.1 preparation stability of mannitol and Sorbitol.In the present embodiment, by the mannitol with variable concentrations and/or Sorbitol, replace part trehalose (table 32), detected another selection to histidine-trehalose preparation.On inspection, the concentration of EDTA is (as Na 2eDTA.2H 2o) in the scope of 0-0.1mg/ml.
Particularly, aspect variable color, gathering, fragmentation and oxidation, analyze the impact of antagonist 11.2.1 stability.
Evaluated preparation is listed in table 32 below.Identical with the method for describing in embodiment 10 for the preparation of the method for described preparation.
By following manner, carry out the preparation in preparation table 32: be taken at the antibody 11.2.1 liquid storage of the 11.9mg/ml in 20mM sodium acetate buffer (pH5.5), 140mM sodium chloride, then it had to Pellicon XL PBTK 30K 50cm 2in the Millipore Lab Scale TFF system of film, experience ultrafiltration/diafiltration (UF/DF) step.Next, in 35 to 40mg/ml scope in 20mM sodium acetate or 20mM histidine buffering liquid the concentrated solution of Dispersal risk 11.2.1.
With 3 times of target final concentrations, in sodium acetate or histidine buffering liquid, prepare the concentrate of tonicity agent.In every kind of buffer, with 20mg/ml, prepare the concentrated solution of polyoxyethylene sorbitan monoleate, and prepare Na with 10mg/ml 2eDTA.2H 2the concentrated solution of O.By suitably diluting concentrated solution, prepare single preparation.Then preparation is filtered by 0.2 μ degerming level filter, and inject several bipartite bottles.In 2-ml 1 type vial, use the packing volume of 1ml.
Described bottle Flurotec through Daikyo 777-1
Figure 2006800156704_10
coated stopper is airtight, presses pleat (crimp) sealing, and it is uprightly stored at 25 ℃ and 40 ℃ in stability test case.By another group bottle experience 4x freeze/thaw as described in Example 10.All preparations have 5.5 pH and the anti-CTLA-4 antibody 11.2.1 concentration of 20mg/ml.
With regard to the level of variable color, gathering, fragmentation and oxidation, analyze several bottles immediately, also by several other bipartite bottles upright storage 4,8,13,18 and 24 weeks at 25 ℃ and 40 ℃.On each time point, for every kind of condition, every kind of preparation takes out two and stores bottle to measure the level of antibody 11.2.1 gathering, fragmentation, and observes variable color.Table 33 to 37 and Figure 10 and 11 reported result.
Table 32: tested antibody preparation
Figure 2006800156704A00800371
Preparation visual analysis:
1) after initial mixing preparation, 2) 4 freeze/thaw (according to embodiment 4, in box together with water filling bottle, in-70 ℃ to 5 ℃) after, and 3) at 25 ℃ and 40 ℃, store every kind of preparation of visually rank after 8,13 and 24 weeks.With regard to granule formation, change color and turbidity, change the described preparation of assessment, and be reported in table 33 in 35.
Table 33: the visually rank of the preparation of table 32 after freeze/thaw
Figure 2006800156704A00800372
Figure 2006800156704A00800381
Table 34: the visually rank of the preparation of table 32 after storing at 25 ℃
Figure 2006800156704A00800382
Figure 2006800156704A00800391
Table 35: the visually rank of the preparation of table 25 after storing at 40 ℃
Figure 2006800156704A00800392
Table 33 to the result in 35 shows, and has EDTA but compared with the preparation of non-sodium chloride, comprises sodium chloride but have without the antibody 11.2.1 preparation of EDTA the freeze/thaw protective effect that is formed proved minimizing by the variable color increasing, turbidity and granule.Described result also shows, compared with preparation without EDTA, variable color, the turbidity of minimizing and the granule of minimizing that the antibody 11.2.1 preparation that comprises all tested EDTA concentration has minimizing form.
Analysis of agglomeration:
At the temperature of 25 ℃ and 40 ℃, store the antibody preparation of preparing according to table 32.The 0th (initially), 4,8,13,18 and 24 weeks, just assemble and analyze 25 ℃ and 40 ℃ of preparations with size exclusion chromatography.At each time point, preparation bottle is carried out to sterile sampling.Use tsk gel G3000SWXL-G2000SWXL post, mobile phase 0.2M sodium phosphate buffer (pH7.0), adopts the UV at the flow velocity of 1ml/ minute and 214nm place to detect described sample is carried out to size exclusion chromatography.Table 36 (a) has shown that the antibody 11.2.1 recording in correlation time for every kind of preparation after storage at 25 ℃ assembles percentage ratio.Table 36 (b) has shown that the antibody 11.2.1 recording after storage at 40 ℃ assembles percentage ratio.So calculate gathering level, the area being about under the chromatogram peak of every kind of preparation carries out integration, and the area through integration under the peak of high molecular weight species is reported as to the percentage ratio (referring to table 36 (a) and 36 (b)) of total peak area.
Table 36 (a): the gathering percentage ratio of the preparation after storing at 25 ℃ in table 32
Figure 2006800156704A00800401
Table 36 below (b) reported aggregate data, and it is in Fig. 9 graphic extension.
Table 36 (b): the gathering percentage ratio of the preparation after storing at 40 ℃ in table 32
Figure 2006800156704A00800402
As seen in table 36 (a), 36 (b) and Fig. 9, after storing at 25 ℃ and 40 ℃, compare with the preparation of sodium chloride (being chloride ion) with lacking EDTA but have acetate buffer, the preparation that comprises EDTA demonstrates the gathering level of minimizing in all tested EDTA concentration.The minimizing of the gathering percentage ratio of preparation in table 32 is summarized in Fig. 9 diagram.
Fragmentation assay:
At the temperature of 25 ℃ and 40 ℃, store the antibody preparation of preparing according to table 32.The 0th (initially), 4,8,13,18 and 24 weeks, use the just total hydrolysis impurity (that is, fragmentation) of reduced form SDS-PAGE (rSDS-PAGE) to analyze 25 ℃ and 40 ℃ of preparations.At each time point, preparation bottle is carried out to sterile sampling, be then loaded on NuPAGE 4-12%bis-Tris gel, use colloid indigo plant (coomassie) dyestuff.By using NuPAGE
Figure 2006800156704_11
reducing agent obtains the reduction of gel.By impurity (that is, the fragmentation) percentage ratio of each sample strip in reduced form gel is estimated in Molecular Dynamics Personal Densitometer PDQC-90 or the enterprising line scanning of Bio-Rad GS800 Imaging Densitometer.Fragmentation level calculation is the percentage ratio (referring to table 37 (a) and 37 (b)) of total band volume.
Table 37 (a): the fragmentation percentage ratio of the preparation after storing at 25 ℃ in table 32
Figure 2006800156704A00800412
Figure 2006800156704A00800421
Table 37 below (b) reported fragmentation data, and it is graphic extension in Figure 10.
Table 37 (b): the fragmentation percentage ratio of the preparation after storing at 40 ℃ in table 32
Figure 2006800156704A00800422
As seen in table 37 (a), 37 (b) and Figure 10, after storing at 25 ℃ and 40 ℃, with lack EDTA but there is acetate buffer and the preparation of sodium chloride (that is, chloride ion) is compared, the preparation that comprises EDTA demonstrates the fragmentation level of minimizing.
embodiment 13
The present embodiment illustrates the production of the composition of liquid medicine that comprises anti-CTLA-4 antibody ticilimumab, L-Histidine mono-hydrochloric salts monohydrate, edta disodium dihydrate, α α-trehalose dihydrate compound and polyoxyethylene sorbitan monoleate.
By obtaining following component, form composition of liquid medicine of the present invention: anti-CTLA-4 antibody ticilimumab (can obtain from the hybridoma cell line 11.2.1.4 of preservation under ATCC accession number PTA-5169 according to embodiment 1, or prepare from mammal cell line restructuring according to embodiment 2), L-Histidine mono-hydrochloric salts monohydrate (can be from Ajinomoto, Raleigh, NC obtains), L-Histidine (can be from Ajinomoto, Raleigh, NC obtains), edta disodium dihydrate (can be from Merck KgaA, Darmstadt, Germany obtains as Titriplex III), α α-trehalose dihydrate compound (can be from FerroPfanstiehl, Waukegan IL with production number T-104-1-MC obtain) and polyoxyethylene sorbitan monoleate (can be from Croda Inc., Mill Hall PA obtains as Crillet 4 HP).
By several liquid storages of first preparing anti-CTLA-4 antibody ticilimumab, L-Histidine mono-hydrochloric salts monohydrate, edta disodium dihydrate, α α-trehalose dihydrate compound and polyoxyethylene sorbitan monoleate, prepare described composition of liquid medicine.By 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine are dissolved in to the histidine buffering liquid pH5.5 for preparing 20mM in water.By being dissolved in, 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine, 84mg/mL (222mM) α α-trehalose dihydrate compound, 0.2mg/mL polyoxyethylene sorbitan monoleate and 0.1mg/mL (0.268mM) edta disodium dihydrate in water, prepare 1 × preparation buffer.By being dissolved in, 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine, 168mg/mL (444mM) α α-trehalose dihydrate compound, 0.4mg/mL polyoxyethylene sorbitan monoleate and 0.2mg/mL (0.536mM) edta disodium dihydrate in water, prepare 2 × preparation buffer.According to embodiment 2, prepare the liquid storage of anti-CTLA-4 antibody ticilimumab, then adopt and use the ultra-filtration process of 50kD type film (Biomax PES) it to be concentrated in histidine buffering liquid to 42 to 55mg/ml (target 45mg/mL).
For pharmaceutical compositions, to the concentrated liquid storage and the 2 × preparation buffer that are suitable for adding in the container of abundant mixing material compositions isopyknic anti-CTLA-4 antibody ticilimumab.After mixing, take out the solution of a small amount of volume, and use 1.43 (mg/mL) -1cm -1extinction coefficient by ultravioletvisible spectroscopy (UV-Vis), determine the concentration (expection in 21 to 27.5mg/mL scope, target 22.5mg/mL) of antibody.Finally, add the 1 × preparation buffer through the volume of suitable calculating, and mix so that antibody reaches the target level of 20mg/mL (scope is between 18-22mg/mL).
Then described pharmaceutical composition filtered by 0.2 μ degerming level filter and inject bottle.In 20 milliliter of 1 type vial, use the nominal packing volume of 20 milliliters.Described bottle Flurotec through Daikyo 777-1 coated stopper is airtight, and presses pleat sealing.By vial sterilizing, for 20mm Daikyo 777-1 serum plug, also carry out sterilizing.
Each single bottle unit comprises the poly-Sorbitol 80 of the anti-CTLA-4 antibody of about 400mg ticilimumab, 65.4mg L-Histidine mono-hydrochloric salts monohydrate, 13.6mg L-Histidine, 2mg edta disodium dihydrate, 1680mg α α-trehalose dihydrate compound and 4mg.
embodiment 14
The present embodiment for example understands the promising production of the composition of liquid medicine that comprises anti-CTLA-4 antibody ticilimumab, L-Histidine mono-hydrochloric salts monohydrate, calcium disodium chelate, α α-trehalose dihydrate compound and polyoxyethylene sorbitan monoleate.
Can form composition of liquid medicine of the present invention by obtaining following component: anti-CTLA-4 antibody ticilimumab (can obtain from the hybridoma cell line 11.2.1.4 of preservation under ATCC accession number PTA-5169 according to embodiment 1, or prepare from mammal cell line restructuring according to embodiment 2), L-Histidine mono-hydrochloric salts monohydrate (can be from Ajinomoto, Raleigh, NC obtains), L-Histidine (can be from Ajinomoto, Raleigh, NC obtains), calcium disodium chelate (can be from Sigma-Aldrich, St.Louis, MO obtains), α α-trehalose dihydrate compound (can be from Ferro Pfanstiehl, Waukegan IL with production number T-104-1-MC obtain) and polyoxyethylene sorbitan monoleate (can be from Croda Inc., Mill Hall PA obtains as Crillet 4 HP).
Can prepare described composition of liquid medicine by several liquid storages of first preparing anti-CTLA-4 antibody ticilimumab, L-Histidine mono-hydrochloric salts monohydrate, edta disodium dihydrate, α α-trehalose dihydrate compound and polyoxyethylene sorbitan monoleate.Can be by 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine be dissolved in to the histidine buffering liquid pH5.5 for preparing 20mM in water.Can in water, prepare 1 × preparation buffer by 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4 mM) L-Histidine, 84mg/mL (222mM) α α-trehalose dihydrate compound, 0.2mg/mL polyoxyethylene sorbitan monoleate and 0.1003mg/mL (0.268mM) calcium disodium chelate are dissolved in.Can in water, prepare 2 × preparation buffer by 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine, 168mg/mL (444mM) α α-trehalose dihydrate compound, 0.4mg/mL polyoxyethylene sorbitan monoleate and 0.2006mg/mL (0.536mM) calcium disodium chelate are dissolved in.Can prepare according to embodiment 2 liquid storage of anti-CTLA-4 antibody ticilimumab, then adopt and use the ultra-filtration process of 50kD type film (Biomax PES) it to be concentrated in histidine buffering liquid to 42 to 55mg/ml (target 45mg/mL).
For pharmaceutical compositions, can be to the concentrated liquid storage and the 2 × preparation buffer that are suitable for adding in the container of abundant mixing material compositions isopyknic anti-CTLA-4 antibody ticilimumab.After mixing, can take out the solution of a small amount of volume, and use 1.43 (mg/mL) -1cm -1extinction coefficient by ultravioletvisible spectroscopy (UV-Vis), determine the concentration (expection in 21 to 27.5mg/mL scope, target 22.5mg/mL) of antibody.Finally, can add the 1 × preparation buffer through the volume of suitable calculating, and mix so that antibody reaches the target level of 20mg/mL (scope is between 18-22mg/mL).
Then described pharmaceutical composition can be filtered and injects bottle by 0.2 μ degerming level filter.In 20 milliliter of 1 type vial, can use the nominal packing volume of 20 milliliters.Described bottle can be used the Flurotec through Daikyo 777-1 coated stopper is airtight, and presses pleat sealing.Can, by vial sterilizing, for 20mm Daikyo 777-1 serum plug, equally also carry out sterilizing.
Each single bottle unit will comprise the poly-Sorbitol 80 of the anti-CTLA-4 antibody of about 400mg ticilimumab, 65.4mg L-Histidine mono-hydrochloric salts monohydrate, 13.6mg L-Histidine, 2.006mg calcium disodium chelate, 1680mg α α-trehalose dihydrate compound and 4mg.
embodiment 15
The present embodiment for example understands the promising production of the composition of liquid medicine that comprises anti-CTLA-4 antibody ticilimumab, L-Histidine mono-hydrochloric salts monohydrate, sodium versenate, α α-trehalose dihydrate compound and polyoxyethylene sorbitan monoleate.
By obtaining following component, form composition of liquid medicine of the present invention: anti-CTLA-4 antibody ticilimumab (can obtain from the hybridoma cell line 11.2.1.4 of preservation under ATCC accession number PTA-5169 according to embodiment 1, or prepare from mammal cell line restructuring according to embodiment 2), L-Histidine mono-hydrochloric salts monohydrate (can be from Ajinomoto, Raleigh, NC obtains), L-Histidine (can be from Ajinomoto, Raleigh, NC obtains), sodium versenate (can be from Sigma-Aldrich, St.Louis, MO obtains), α α-trehalose dihydrate compound (can be from Ferro Pfanstiehl, Waukegan IL with production number T-104-1-MC obtain) and polyoxyethylene sorbitan monoleate (can be from Croda Inc., Mill Hall PA obtains as Crillet 4 HP).
By several liquid storages of first preparing anti-CTLA-4 antibody ticilimumab, L-Histidine mono-hydrochloric salts monohydrate, sodium versenate, α α-trehalose dihydrate compound and polyoxyethylene sorbitan monoleate, prepare described composition of liquid medicine.By 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine are dissolved in to the histidine buffering liquid pH5.5 for preparing 20mM in water.By being dissolved in, 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine, 84mg/mL (222mM) α α-trehalose dihydrate compound, 0.2mg/mL polyoxyethylene sorbitan monoleate and 0.096mg/mL (0.268mM) sodium versenate in water, prepare 1 × preparation buffer.By being dissolved in, 3.27mg/mL (15.6mM) L-Histidine HCl monohydrate and 0.68mg/mL (4.4mM) L-Histidine, 168mg/mL (444mM) α α-trehalose dihydrate compound, 0.4mg/mL polyoxyethylene sorbitan monoleate and 0.192mg/mL (0.536mM) sodium versenate in water, prepare 2 × preparation buffer.According to embodiment 2, prepare the liquid storage of anti-CTLA-4 antibody ticilimumab, then adopt and use the ultra-filtration process of 50kD type film (Biomax PES) it to be concentrated in histidine buffering liquid to 42 to 55mg/ml (target 45mg/mL).
For pharmaceutical compositions, to the concentrated liquid storage and the 2 × preparation buffer that are suitable for adding in the container of abundant mixing material compositions isopyknic anti-CTLA-4 antibody ticilimumab.After mixing, take out the solution of a small amount of volume, and use 1.43 (mg/mL) -1cm -1extinction coefficient by ultravioletvisible spectroscopy (UV-Vis), determine the concentration (expection in 21 to 27.5mg/mL scope, target 22.5mg/mL) of antibody.Finally, add the 1 × preparation buffer through the volume of suitable calculating, and mix so that antibody reaches the target level of 20mg/mL (scope is between 18-22mg/mL).
Then described pharmaceutical composition filtered by 0.2 μ degerming level filter and inject bottle.In 20 milliliter of 1 type vial, use the nominal packing volume of 20 milliliters.Described bottle Flurotec through Da ikyo 777-1
Figure 2006800156704_14
coated stopper is airtight, and presses pleat sealing.By vial sterilizing, for 20mm Daikyo 777-1 serum plug, also carry out sterilizing.
Each single bottle unit comprises the poly-Sorbitol 80 of the anti-CTLA-4 antibody of about 400mg ticilimumab, 65.4mg L-Histidine mono-hydrochloric salts monohydrate, 13.6mg L-Histidine, 1.92mg sodium versenate, 1680mg α α-trehalose dihydrate compound and 4mg.
Sequence
Ticilimumab (11.2.1) heavy chain DNA (SEQ ID NO:1)
atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60
gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120
tgtgcagcgt ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180
ggcaaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca 240
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300
caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag agatccgagg 360
ggagctaccc tttactacta ctactacggt atggacgtct ggggccaagg gaccacggtc 420
accgtctcct cagcctccac caagggccca tcggtcttcc ccctggcgcc ctgctccagg 480
agcacctccg agagcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 540
gtgacggtgt cgtggaactc aggcgctctg accagcggcg tgcacacctt cccagctgtc 600
ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcaacttc 660
ggcacccaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 720
acagttgagc gcaaatgttg tgtcgagtgc ccaccgtgcc cagcaccacc tgtggcagga 780
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840
gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcca gttcaactgg 900
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cacgggagga gcagttcaac 960
agcacgttcc gtgtggtcag cgtcctcacc gttgtgcacc aggactggct gaacggcaag 1020
gagtacaagt gcaaggtctc caacaaaggc ctcccagccc ccatcgagaa aaccatctcc 1080
aaaaccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1140
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc 1200
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac acctcccatg 1260
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380
cagaagagcc tctccctgtc tccgggtaaa tga 1413
Ticilimumab (11.2.1) heavy chain protein matter (SEQ ID NO:2)
[QVQLVESGGG VVQPGRSLRL SCAAS GFTFS SYGMHWVRQA PGKGLEWVA V IWYDGSNKYY 60
ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAR DP RGATLYYYYY GMDV WGQGTT 120
VTVSS]ASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA 180
VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA 240
GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF 300
NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTKGQPREP QVYTLPPSRE 360
EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR 420
WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K 451
Variable region (SEQ ID NO:5) is shown in [between bracket], and CDRs uses underscoreindicate.CDR1 is indicated by SEQ ID NO:7, and CDR2 is indicated by SEQ ID NO:8, and CDR3 is indicated by SEQ ID NO:9.
Ticilimumab (11.2.1) light chain DNA (SEQ ID NO:3)
atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60
agatgtgacatccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120
gtcaccatca cttgccgggc aagtcagagc attaacagct atttagattg gtatcagcag 180
aaaccaggga aagcccctaa actcctgatc tatgctgcat ccagtttgca aagtggggtc 240
ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 300
caacctgaag attttgcaac ttactactgt caacagtatt acagtactcc attcactttc 360
ggccctggga ccaaagtgga aatcaaacga actgtggctg caccatctgt cttcatcttc 420
ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480
ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600
ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660
cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta gtga 714
Ticilimumab (11.2.1) light chain protein matter (SEQ ID NO:4)
[DIQMTQSPSS LSASVGDRVT ITC RASQSIN SYLDWYQQKP GKAPKLLIY A ASSLQSGVPS 60
RFSGSGSGTD FTLTISSLQP EDFATYYC QQ YYSTPFTFGP GTKVEIK]RTV AAPSVFIFPP 120
SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180
LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214
Variable region (SEQ ID NO:6) is shown in [between bracket], and CDRs uses underscoreindicate.CDR1 is indicated by SEQ ID NO:10, and CDR2 is indicated by SEQ ID NO:11, and CDR3 is indicated by SEQ ID NO:12.
Sequence table
<110>Pharmacia and Upjohn Company，LLC
Das，Tapan
Elliott，Carrie
Muthurania，Kevin
Abate，Justin
Nema，Sandeep
Singh，Satish
The anti-CTLA-4 antibody compositions of <120>
<130>PC 33042
<150>60/659,766
<151>2005-03-08
<150>60/728,165
<151>2005-10-19
<150>60/752,712
<151>2005-12-20
<150>60/762,456
<151>2005-01-26
<160>12
<170>PatentIn version 3.3
<210>1
<211>1413
<212>DNA
<213> homo sapiens
<400>1
atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60
gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120
tgtgcagcgt ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180
ggcaaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca 240
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300
caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag agatccgagg 360
ggagctaccc tttactacta ctactacggt atggacgtct ggggccaagg gaccacggtc 420
accgtctcct cagcctccac caagggccca tcggtcttcc ccctggcgcc ctgctccagg 480
agcacctccg agagcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 540
gtgacggtgt cgtggaactc aggcgctctg accagcggcg tgcacacctt cccagctgtc 600
ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcaacttc 660
ggcacccaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 720
acagttgagc gcaaatgttg tgtcgagtgc ccaccgtgcc cagcaccacc tgtggcagga 780
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840
gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcca gttcaactgg 900
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cacgggagga gcagttcaac 960
agcacgttcc gtgtggtcag cgtcctcacc gttgtgcacc aggactggct gaacggcaag 1020
gagtacaagt gcaaggtctc caacaaaggc ctcccagccc ccatcgagaa aaccatctcc 1080
aaaaccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1140
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc 1200
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac acctcccatg 1260
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380
cagaagagcc tctccctgtc tccgggtaaa tga 1413
<210>2
<211>451
<212>PRT
<213> homo sapiens
<400>2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ash Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
130 135 140
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys
195 200 205
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu
210 215 220
Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
290 295 300
Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210>3
<211>714
<212>DNA
<213> homo sapiens
<400>3
atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60
agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120
gtcaccatca cttgccgggc aagtcagagc attaacagct atttagattg gtatcagcag 180
aaaccaggga aagcccctaa actcctgatc tatgctgcat ccagtttgca aagtggggtc 240
ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 300
caacctgaag attttgcaac ttactactgt caacagtatt acagtactcc attcactttc 360
ggccctggga ccaaagtgga aatcaaacga actgtggctg caccatctgt cttcatcttc 420
ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480
ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600
ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660
cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta gtga 714
<210>4
<211>214
<212>PRT
<213> homo sapiens
<400>4
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr
20 25 30
Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210>5
<211>125
<212>PRT
<213> homo sapiens
<400>5
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210>6
<211>107
<212>PRT
<213> homo sapiens
<400>6
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr
20 25 30
Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys
100 105
<210>7
<211>10
<212>PRT
<213> homo sapiens
<400>7
Gly Phe Thr Phe Ser Ser Tyr Gly Met His
1 5 10
<210>8
<211>15
<212>PRT
<213> homo sapiens
<400>8
Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
1 5 10 15
<210>9
<211>16
<212>PRT
<213> homo sapiens
<400>9
Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val
1 5 10 15
<210>10
<211>11
<212>PRT
<213> homo sapiens
<400>10
Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asp
1 5 10
<210>11
<211>7
<212>PRT
<213> homo sapiens
<400>11
Ala Ala Ser Ser Leu Gln Ser
1 5
<210>12
<211>9
<212>PRT
<213> homo sapiens
<400>12
Gln Gln Tyr Tyr Ser Thr Pro Phe Thr
1 5

Claims (11)

1. fluid composition, it comprises:
EDTA, its concentration is 0.01mM to 5.0mM;
Histidine, its concentration is 1mM to 100mM;
Polyoxyethylene sorbitan monoleate, its concentration is 0.01mg/ml to 10mg/ml;
Trehalose, its concentration is 100mM to 300mM; With
At least one human IgG antibody, its concentration is 1mg/ml to 200mg/ml, wherein said antibodies people CTLA-4 and comprising:
I) heavy chain, its varistructure domain amino acid sequence is the weight chain variable domain aminoacid sequence shown in SEQ ID NO:2; With
Ii) light chain, its varistructure domain amino acid sequence is the light chain variable domain aminoacid sequence shown in SEQ ID NO:4;
Wherein, compared with lacking the same combination of EDTA, described fluid composition has the characteristic that is selected from least one following improvement:
A) antibody aggregation reducing;
B) antibody fragment reducing;
C) the antibody freeze/thaw unstability reducing;
D) the compositions variable color reducing; With
The desamidation of the antibody e) reducing.
2. the compositions of claim 1, wherein said antibody is IgG2 antibody.
3. the compositions of claim 1, wherein said compositions comprises:
Antibody, its concentration is 1mg/ml to 100mg/ml;
EDTA, its concentration is 0.01mM to 1.0mM;
Histidine, its concentration is 1mM to 100mM;
Polyoxyethylene sorbitan monoleate, its concentration is 0.01mg/ml to 10mg/ml; With
Trehalose, its concentration is 100mM to 300mM.
4. the compositions of claim 1, wherein said compositions comprises:
Antibody, its concentration is 0.1mg/ml to 100mg/ml;
EDTA, its concentration is 0.001mg/ml to 1.0mg/ml;
Histidine, its concentration is 1mM to 50mM;
Polyoxyethylene sorbitan monoleate, its concentration is 0.01mg/ml to 5mg/ml; With
Trehalose, its concentration is 10mg/ml to 200mg/ml.
5. the compositions of claim 1, wherein said compositions comprises:
20mg/ml antibody;
0.27mM EDTA；
20mM histidine;
0.2mg/ml polyoxyethylene sorbitan monoleate; With
222mM trehalose.
6. the compositions of claim 1, wherein said compositions comprises:
20mg/ml antibody;
0.1mg/ml EDETATE SODIUM dihydrate;
20mM histidine;
0.2mg/ml polyoxyethylene sorbitan monoleate; With
84mg/ml trehalose.
7. the compositions of claim 1, the molar concentration of wherein said antibody is in the scope of 0.0006mM to 1.35mM, with the molar concentration of described EDTA in the scope of 0.003mM to 50mM, and wherein the mol ratio of antibody and EDTA in the scope of 0.00001:1 to 450:1.
8. the compositions of claim 1, wherein described compositions is being stored after 24 weeks at the temperature of 40 ℃, the aggregation chromatogram peak area of the stable composition of liquid medicine that comprises monoclonal anti CTLA-4 antibody and EDTA and at the temperature of 40 ℃, store 24 weeks not containing being reduced at least 2% between the aggregation chromatogram peak area of the same combination of EDTA.
9. fluid composition, it comprises:
Antibody, concentration is 20mg/ml, the heavy chain amino acid sequence of wherein said antibody is that the light-chain amino acid sequence of SEQ ID NO:2 and described antibody is SEQ ID NO:4;
0.27mM EDTA；
20mM histidine;
0.2mg/ml polyoxyethylene sorbitan monoleate; With
222mM trehalose.
10. fluid composition, it comprises:
Antibody, concentration is 20mg/ml, the heavy chain amino acid sequence of described antibody is that the light-chain amino acid sequence of SEQ ID NO:2 and described antibody is SEQ ID NO:4;
0.1mg/ml EDETATE SODIUM dihydrate;
20mM histidine;
0.2mg/ml polyoxyethylene sorbitan monoleate; With
84mg/ml trehalose.
11. for the preparation of according to the method for the fluid composition of any one in claim 1-10.
CN200680015670.4A 2005-03-08 2006-03-02 Anti-CTLA-4 antibody compositions Active CN101325968B (en)

Applications Claiming Priority (9)

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US65976605P 2005-03-08 2005-03-08
US60/659,766 2005-03-08
US72816505P 2005-10-19 2005-10-19
US60/728,165 2005-10-19
US75271205P 2005-12-20 2005-12-20
US60/752,712 2005-12-20
US76245606P 2006-01-26 2006-01-26
US60/762,456 2006-01-26
PCT/US2006/007555 WO2006096491A2 (en) 2005-03-08 2006-03-02 Anti-ctla-4 antibody compositions

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EP3053572A1 (en) * 2015-02-06 2016-08-10 Ares Trading S.A. Liquid pharmaceutical composition
PT3283107T (en) * 2015-04-17 2020-08-31 Bristol Myers Squibb Co Compositions comprising a combination of an anti-pd-1 antibody and another antibody
CN106620691B (en) * 2015-11-04 2020-08-21 信达生物制药(苏州)有限公司 Recombinant fully human anti-CTLA-4 monoclonal antibody preparation and application thereof
CN106913869B (en) * 2017-03-17 2020-07-28 信达生物制药(苏州)有限公司 anti-CT L A-4 monoclonal antibody preparation and application thereof
KR20200028006A (en) * 2017-07-14 2020-03-13 화이자 인크. Antibodies to MAdCAM
TW202110885A (en) * 2019-05-20 2021-03-16 美商潘迪恩治療公司 Madcam targeted immunotolerance
WO2021129775A1 (en) * 2019-12-25 2021-07-01 百奥泰生物制药股份有限公司 Anti-ctla-4 monoclonal antibody, preparation method therefor, and application thereof
CN111410688A (en) * 2020-03-13 2020-07-14 苏州智享众创孵化管理有限公司 Separation and purification method for improving purity of bispecific antibody
JP2024500308A (en) * 2020-12-03 2024-01-09 江▲蘇▼恒瑞医▲薬▼股▲フン▼有限公司 Anti-TSLP antibody pharmaceutical composition and its use
CN112569183B (en) * 2020-12-11 2022-12-09 上海赛金生物医药有限公司 Preparation of anti-CTLA-4 antibody and fusion protein
CN115282281B (en) * 2022-09-28 2022-12-13 智泽童康(广州)生物科技有限公司 Application of iron death inhibitor in preparation of medicine for preventing and treating biliary tract diseases

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