Variants of tissue inhibitor of metalloproteinase type three (timp-3), compositions and methods

Abstract

There are disclosed TIMP-3 muteins, variants and derivatives, nucleic acids encoding them, and methods of making and using them.

What is claimed is: 
     
         1 . An isolated TIMP-3 mutein having a mature region that is at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2, having at least one mutation, the mutation being selected from the group consisting of K45E; K45N; K45S; V47T; K49N; K49E; K49S; K50N; L51T; L51N; V52T; K53T; P56N; F57N; G58T; T63E; T63N; K65T; K65N; M67T; K68S; T74E; K75N; P77T; H78D; H78E; H78N; Q80E; Q80T; K94N; E96T; E96N; V97N; N98T; K99T; D110N; K112T; Q126N; K133S; R138T; R138N; H140T; T158N; K160T; T166N; M168T; G173T; H181N; A183T; R186N; R186Q, R186E, K188T; K188Q, K188E, P201N; K203T; I205F, I205Y, A208G, A208V, and A208Y. 
     
     
         2 . An isolated TIMP-3 mutein having a mature region that is at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2, having, having the mutation F57N and at least one further mutation, wherein the mutation is substitution at one or more of K residues of TIMP-3. 
     
     
         3 . The TIMP-3 mutein of  claim 2  wherein the further mutation introduces an N-linked glycosylation site into the amino acid sequence. 
     
     
         4 . An isolated TIMP-3 mutein having a mature region that is at least 90% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2, wherein the mutein has at least one mutation that introduces at least one N-linked glycosylation site into the amino acid sequence. 
     
     
         5 . The TIMP-3 mutein of  claim 4 , wherein two, three, four, five, or six N-linked glycosylation sites are introduced. 
     
     
         6 . The TIMP-3 mutein of  claim 3 , wherein the N-linked glycosylation site is introduced at a region of the TIMP-3 amino acid sequence selected from the group consisting of: the region comprising amino acids 48-54; the region comprising amino acids 93-100; the region comprising amino acids 121-125; the region comprising amino acids 143-152; amino acids 156-164; the region comprising amino acids 183-191; and combinations thereof. 
     
     
         7 . The TIMP-3 mutein of  claim 6 , wherein two, three, four, or five or six (or more) N-linked glycosylation sites are introduced. 
     
     
         8 . An isolated TIMP-3 mutein having a mature region that is at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2, wherein the mutein has at least one mutation selected from the group consisting of:
 (a) one or more mutations in a TIMP-3 surface exposed positively charged patch that results in changes in the characteristics of the TIMP-3 charged patch that mimic a TIMP-1, TIMP-2 or TIMP-4 charged surface;   (b) one or more mutations that reduce susceptibility to proteolytic cleavage;   (c) one or more mutations that result in decreased interaction of the TIMP-3 mutein with the scavenger receptor LRP-1;   (d) one or more mutations that result in decreased interaction of the TIMP-3 mutein heparin or extracellular matrix components;   (e) addition of one or more cysteinyl residues to the native TIMP-3 sequence;   (f) improved pharmacokinetic and/o pharmacodynamic properties;   (g) one or more mutations that introduce at least one N-linked glycosylation site; and   (h) combinations of the mutations set forth in (a)-(g).   
     
     
         9 . The TIMP-3 mutein of  claim 8 , which is selected from the group consisting of:
 i. K45E, K49S; (SEQ ID NO: 5)   ii. K45E, K49E; (SEQ ID NO: 6)   iii. K45E, T63E; (SEQ ID NO: 7)   iv. K45E, Q80E; (SEQ ID NO: 8)   v. K45E, T63E, H78E; (SEQ ID NO: 10)   vi. T63E, H78E, Q80E; (SEQ ID NO: 11)   vii. K45E, T63E, H78E, Q80E; (SEQ ID NO: 12)   viii. T63E, T74E, H78E; (SEQ ID NO: 13)   ix. T63E, T74E, H78D; (SEQ ID NO: 14)   x. L51T, T74E, H78D; (SEQ ID NO: 53)   xi. T74E, H78E, Q80E; (SEQ ID NO: 16)   xii. T74E, H78D, Q80E; (SEQ ID NO: 17)   xiii. K45N, V47T; (SEQ ID NO: 26)   xiv. K65N, M67T; (SEQ ID NO: 37)   xv. K45N, V47T, T63E, T74E, H78E; (SEQ ID NO: 18)   xvi. K49N, L51T, T63E, T74E, H78E; (SEQ ID NO: 19)   xvii. K45E, K49N, L51T, T63E; (SEQ ID NO: 20)   xviii. K49N, L51T, T74E, H78E; (SEQ ID NO: 21)   xix. K49N, L51T; (SEQ ID NO: 27)   xx. K50N, V52T; (SEQ ID NO: 30)   xxi. L51N, K53T; (SEQ ID NO: 54)   xxii. F57N; (SEQ ID NO: 33)   xxiii. P56N, G58T; (SEQ ID NO: 31)   xxiv. T63N, K65T; (SEQ ID NO: 36)   xxv. P56N, G58T, T63N, K65T; (SEQ ID NO: 32)   xxvi. K75N, P77T; (SEQ ID NO: 38)   xxvii. H78N, Q80T; (SEQ ID NO: 39)   xxviii. K94N, E96T; (SEQ ID NO: 40)   xxix. E96N, N98T; (SEQ ID NO: 41)   xxx. V97N, K99T; (SEQ ID NO: 42)   xxxi. D110N, K112T; (SEQ ID NO: 43)   xxxii. Q126N; (SEQ ID NO: 44)   xxxiii. R138N, H140T; (SEQ ID NO: 46)   xxxiv. R138T; (SEQ ID NO: 45)   xxxv. T158N, K160T; (SEQ ID NO: 47)   xxxvi. T166N, M168T; (SEQ ID NO: 48)   xxxvii. G173T; (SEQ ID NO: 49)   xxxviii. H181N, A183T; (SEQ ID NO: 50)   xxxix. R186N, K188T; (SEQ ID NO: 51)   xl. P201N, K203T; (SEQ ID NO: 52)   xli. A208Y; (SEQ ID NO: 55)   xlii. A208V; (SEQ ID NO: 56)   xliii. K45S, F57N; (SEQ ID NO: 23)   xliv. K49S, F57N; (SEQ ID NO: 28)   xlv. K68S, F57N; (SEQ ID NO: 34)   xlvi. K133S, F57N; (SEQ ID NO: 35)   xlvii. K45S, K133S, F57N; (SEQ ID NO: 24)   xlviii. K49S, K68S, F57N (SEQ ID NO: 29).   xlix. K45S, F57N, I205F, A208G (SEQ ID NO:57)   l. K45S, F57N, A208G (SEQ ID NO:58)   li. K45S, F57N, I205Y (SEQ ID NO:59)   lii. K45S, F57N, I205Y, A208G (SEQ ID NO:60)   liii. K45N, V47T, F57N, K75N, P77T, K94N, E96T, R138T, G173T (SEQ ID NO:61)   liv. K45N, V47T, F57N, K94N, E96T, R138T, G173T (SEQ ID N062):   lv. K45N, V47T, K50N, V52T, F57N, V97N, K99T (SEQ ID NO:63)   lvi. K45S, K50N, V52T, F57N, V97N, K99T, R186N, K188T (SEQ ID NO:64)   lvii. K45S, F57N, K94N, E96T, D110N, K112T, R138T, G173T (SEQ ID NO:65)   lviii. K45S, F57N, T63N, K65T, K94N, E96T, G173T (SEQ ID NO:66)   lix. K45N, V47T, K50N, V52T, F57N, V97N, K99T, R138T, R186N, K188T (SEQ ID NO:67)   lx. K45S, F57N, T63N, K65T, K94N, E96T, Q126N, R138T (SEQ ID NO:68)   lxi. K45N, V47T, K50N, V52T, F57N, V97N, K99T, R186N, K188T (SEQ ID NO:69)   lxii. K45N, V47T, K50N, V52T, V97N, K99T, R138T, R186N, K188T (SEQ ID NO:70)   lxiii. K45S, F57N, H78N, Q80T, K94N, E96T, R138T, G173T (SEQ ID NO:71)   lxiv. K45S, F57N, K75N, P77T, K94N, E96T, R138T, G173T (SEQ ID NO:72)   lxv. K45N, V47T, K50N, V52T, V97N, K99T, G173T, R186N, K188T (SEQ ID NO:73)   lxvi. K45E, F57N, Q126N, R138T, G173T (SEQ ID NO:74)   lxvii. K45S, F57N, T63N, K65T, K94N, E96T, R138T, G173T (SEQ ID NO:75)   lxviii. K45S, K50N, V52T, F57N, V97N, K99T, R138T, R186N, K188T (SEQ ID NO:76)   lxix. K45S, K50N, V52T, F57N, V97N, K99T, G173T, R186N, K188T (SEQ ID NO:77)   lxx. K45N, V47T, F57N, K94N, E96T, G173T, R186N, K188T (SEQ ID NO:78)   lxxi. K45N, V47T, F57N, K94N, E96T, D110N, K112T, R186N, K188T (SEQ ID NO:79)   lxxii. K45N, V47T, F57N, V97N, K99T, R138T, G173T (SEQ ID NO:80)   lxxiii. K45N, V47T, F57N, K99E G173T, R186N, K188T (SEQ ID NO:81)   lxxiv. K45E, K49E, F57N, K94N, E96T, D110N, K112T, G173T, R186N, K188T (SEQ ID NO:82)   lxxv. K50N, V52T, K94N, E96T, R138T, G173T (SEQ ID NO:83)   lxxvi. K45E, K50N, V52T, K94N, E96T, D110N, K112T, R138T, G173T (SEQ ID NO:84)   lxxvii. K50N, V52T, K94N, E96T, R138T, G173T, R186N, K188T (SEQ ID NO:85)   lxxviii. K45E, F57N, T63N, K65T, K94N, E96T, G173T, R186N, K188T (SEQ ID NO:86)   lxxix. K45N, V47T, F57N, K94N, E96T, D110N, K112T, G173T, R186Q, K188Q (SEQ ID NO:87)   lxxx. K45S F57N K94N, E96T R138T G173T (SEQ ID NO:88)   lxxxi. K45E F57N K94N, E96T R138T G173T (SEQ ID NO:89)   lxxxii. K45E F57N K94N, E96T D110N, K112T R138T G173T (SEQ ID NO:90)   lxxxiii. K45E F57N K94N, E96T R138T G173T R186Q, K188Q (SEQ ID NO:91)   lxxxiv. K45E F57N K94N, E96T R138T G173T R186E (SEQ ID NO:92)   lxxxv. K45E F57N K94N, E96T R138T G173T K188E (SEQ ID NO:93)   lxxxvi. K45E F57N K94N, E96T R138T G173T R186N, K188T (SEQ ID NO:94)   lxxxvii. K45E K50N, V52T K94N, E96T D110N, K112T R138T G173T (SEQ ID NO:95)   lxxxviii. K45E K50N, V52T K94N, E96T R138T G173T K188E (SEQ ID NO:96)   lxxxix. K50N, V52T F57N K94N, E96T R138T G173T (SEQ ID NO:97)   xc. K50N, V52T F57N K94N, E96T D110N, K112T R138T (SEQ ID NO:98) and   xci. K45E F57N K94N, E96T D110N, K112T R138T (SEQ ID NO:99).   
     
     
         10 . The TIMP-3 mutein of  claim 8 , which is selected from the group consisting of: K49E, K50E, K53E, K99E, R186Q, K188Q; K49S, K50N/V52T, K53E, V97N/K99T, R186N/K188T; K50N/V52T, V97N/K99T, R186N/K188T; K49E, K53E, K188Q; K50N/V52T, R186N/K188T; K50N/V52T, F57N, R186N/K188T; K45S, K50N/V52T, F57N, R186N/K188T; K50N/V52T, F57N, T63N/K65T, R186N/K188T; K45S, K50N/V52T, F57N R186N/K188T; K45S, K49S, K50N/V52T, F57N R186N/K188T; K49S, K50N/V52T, F57N, V97N/K99T, R186N/K188T; K45S, K50N/V52T, F57N, V97N/K99T, R186N/K188T; K45S, F57N, D110N, K112T; K45S, F57N, H78N, 080T, D110N, K112T; K45S, F57N, H78N, Q80T, D110N, K112T, Q126N; K45S, F57N, H78N, Q80T, K94N, E96T Q126N; K45S, F57N, H78N, Q80T, Q126N, G173T; K45S, F57N, T63N, K65T; K45S, F57N, T63N, K65T, K94N, E96T; K45S, F57N, T63N, K65T, R138T, G173T; K45N, V47T, F57N, T63N, K65T, R138T, G173T; K45S, F57N, T63N, K65T, K94N, E96T, R138T; K45N, V47T, F57N, T63N, K65T, K94N, E96T, R138T; K45S, F57N, Q126N, R138T, G173T; P56N, G58T, T63N, K65T, K94N, E96T, Q126N, G173T; P56N, G58T, T63N, K65T, D110N, K112T, Q126N, G173T; K49S, K50N, V52T, K53E, V97N, K99T, R186N, K188T; K50N, V52T, V97N, K99T, R186N, K188T; K49E, K53E, K188Q; K50N, V52T, R186N, K188T; K50N, V52T, F57N, R186N, K188T; K45S, K50N, V52T, F57N, R186N, K188T; K50N, V52T, F57N, T63N, K65T, R186N, K188T; K45S, K50N, V52T, F57N R186N, K188T; K45S, K49S, K50N, V52T, F57N R186N, K188T; K49S, K50N, V52T, F57N, V97N, K99T, R186N, K188T; K45S, K50N, V52T, F57N, V97N, K99T, R186N, K188T; K45E, K50N, V52T, D110N, K112T, R138T, G173T, K188E; K45E, F57N, D110N, K112T, R138T, G173T, K188E; K45E, K50N, V52T, K94N, E96T, D110N, K112T, G173T, K188E; K45E, F57N, K94N, E96T, D110N, K112T, G173T, K188E; K45E, K50N, V52T, D110N, K112T, R138T, G173T, R186N, K188T; K45E, F57N, D110N, K112T, R138T, G173T, R186N, K188T; K45E, K50N, V52T, K94N, E96T, D110N, K112T, G173T, R186N, K188T; K45E, F57N, K94N, E96T, D110N, K112T, G173T, R186N, K188T; K45E, K50N, V52T, D110N, K112T, R138T, G173T, R186Q, K188Q; K45E, F57N, D110N, K112T, R138T, G173T, R186Q, K188Q; K45E, K50N, V52T, K94N, E96T, D110N, K112T, G173T, R186Q, K188Q; K45E, F57N, K94N, E96T, D110N, K112T, G173T, R186Q, K188Q; K45E, K50N, V52T, D110N, K112T, R138T, K188E; K45E, F57N, D110N, K112T, R138T, K188E; K45E, K50N, V52T, K94N, E96T, D110N, K112T, K188E; K45E, F57N, K94N, E96T, D110N, K112T, K188E; K45E, K50N, V52T, D110N, K112T, R138T, R186N, K188T; K45E, F57N, D110N, K112T, R138T, R186N, K188T; K45E, K50N, V52T, K94N, E96T, D110N, K112T, R186N, K188T; K45E, F57N, K94N, E96T, D110N, K112T, R186N, K188T; K45E, K50N, V52T, D110N, K112T, R138T, R186Q, K188Q; K45E, F57N, D110N, K112T, R138T, R186Q, K188Q; K45E, K50N, V52T, K94N, E96T, D110N, K112T, R186Q, K188Q; K45E, F57N, K94N, E96T, D110N, K112T, R186Q, K188Q; K50N, V52T, D110N, K112T, R138T, G173T, K188E; K45S, F57N, D110N, K112T, R138T, G173T, K188E; K50N, V52T, K94N, E96T, D110N, K112T, G173T, K188E; K45S, F57N, K94N, E96T, D110N, K112T, G173T, K188E; K50N, V52T, D110N, K112T, R138T, G173T, R186N, K188T; K45S, F57N, D110N, K112T, R138T, G173T, R186N, K188T; K50N, V52T, K94N, E96T, D110N, K112T, G173T, R186N, K188T; K45S, F57N, K94N, E96T, D110N, K112T, G173T, R186N, K188T; K50N, V52T, D110N, K112T, R138T, G173T, R186Q, K188Q; K45S, F57N, D110N, K112T, R138T, G173T, R186Q, K188Q; K50N, V52T, K94N, E96T, D110N, K112T, G173T, R186Q, K188Q; K45S, F57N, K94N, E96T, D110N, K112T, G173T, R186Q, K188Q; K50N, V52T, D110N, K112T, R138T, K188E; K45S, F57N, D110N, K112T, R138T, K188E; K50N, V52T, K94N, E96T, D110N, K112T, K188E; K45S, F57N, K94N, E96T, D110N, K112T, K188E; K50N, V52T, D110N, K112T, R138T, R186N, K188T; K45S, F57N, D110N, K112T, R138T, R186N, K188T; K50N, V52T, K94N, E96T, D110N, K112T, R186N, K188T; K45S, F57N, K94N, E96T, D110N, K112T, R186N, K188T; K50N, V52T, D110N, K112T, R138T, R186Q, K188Q; K45S, F57N, D110N, K112T, R138T, R186Q, K188Q; K50N, V52T, K94N, E96T, D110N, K112T, R186Q, K188Q; K45S, F57N, K94N, E96T, D110N, K112T, R186Q, K188Q; K50N, V52T, K94N, E96T, D110N, K112T, R138T, G173T; K50N, V52T, K94N, E96T, R138T, G173T; K45E, F57N, K94N, E96T, D110N, K112T, R138T, G173T; K45E, F57N, K94N, E96T, R138T, G173T; K45S, F57N, K94N, E96T, D110N, K112T, R138T, G173T; K45S, F57N, K94N, E96T, R138T, G173T; K45N, V47T, H78N, Q80T, Q126N, R186Q, K188Q; K45N, V47T, F57N, H78N, Q80T, Q126N, R186Q, K188Q; K45N, V47T, F57N, H78N, Q80T, K94N, E96T, Q126N, K45N, V47T, F57N, H78N, Q80T, Q126N, R138T; K45N, V47T, F57N, H78N, Q80T, R138T, R186Q, K188Q; K45N, V47T, F57N, H78N, Q80T, K94N, E96T, D110N, K112T, R186Q, K188Q; K50N, V52T, K94N, E96T, H78N, Q80T, R138T; K50N, V52T, K94N, E96T, H78N, Q80T, R138T, R186Q, K188Q; K45E, F57N, Q126N, R138T, R186Q, K188Q; K45N, V47T, F57N, Q126N, R138T, R186Q, K188Q; K45N, V47T, F57N, H78N, Q80T, R186Q, K188Q; K45S, F57N, H78N, Q80T, Q126N, R138T, R186Q, K188Q; K45S, F57N, H78N, Q80T, K94N, E96T, R138T, R186Q, K188Q; K50N, V52T, K94N, E96T, H78N, Q80T, R138T, K45N, V47T, F57N, K94N, E96T, D110N, K112T, R186Q, K188Q; and K45S, F57N, H78N, Q80T, K94N, E96T, R138T; K45N, V47T, F57N, K94N, E96T 
     
     
         11 . An isolated nucleic acid that encodes a TIMP-3 mutein according to any one of  claims 1 - 10 . 
     
     
         12 . An expression vector comprising the isolated nucleic acid of  claim 11 . 
     
     
         13 . An isolated host cell transformed or transfected with the expression vector of  claim 12 . 
     
     
         14 . A method of producing a recombinant TIMP-3 mutein comprising culturing the transformed or transfected host cell of  claim 13  under conditions promoting expression of the TIMP-3 mutein, and recovering the TIMP-3 mutein. 
     
     
         15 . A composition comprising the TIMP-3 mutein of any one of  claims 1 - 10  and a physiologically acceptable diluent, excipient or carrier. 
     
     
         16 . A method of treating a condition in which matrix metalloproteases (MMPs) and/or other proteinases that are inhibited or inhibitable by TIMP-3 play a causative or exacerbating role, comprising administering to an individual afflicted with such a condition, an amount of composition according to  claim 15  sufficient to treat the condition. 
     
     
         17 . The method of  claim 16  wherein the condition is selected from the group consisting of inflammatory conditions, osteoarthritis, myocardial ischemia, reperfusion injury, and progression to congestive heart failure. 
     
     
         18 . The method of  claim 16 , wherein the condition is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (for example, ulcerative colitis, Crohn's disease, and celiac disease), psoriasis, myocarditis including viral myocarditis, inflammation related to atherosclerosis, and arthritic conditions including rheumatoid arthritis and psoriatic arthritis. 
     
     
         19 . The method of  claim 16 , wherein the condition is selected from the group consisting of dystrophic epidermolysis bullosa, osteoarthritis, pseudogout, rheumatoid arthritis including juvenile rheumatoid arthritis, ankylosing spondylitis, periodontal disease, ulceration including corneal, epidermal, or gastric ulceration, wound healing after surgery, restenosis, emphysema, Paget's disease of bone, osteoporosis, scleroderma, pressure atrophy of bone or tissues as in bedsores, cholesteatoma, abnormal wound healing, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myolitis, polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Celiac disease (nontropical Sprue), enteropathy associated with seronegative arthropathies, microscopic or collagenous colitis, eosinophilic gastroenteritis, or pouchitis resulting after proctocolectomy and ileoanal anastomosis, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, multiple sclerosis (MS), asthma (including extrinsic and intrinsic asthma as well as related chronic inflammatory conditions, or hyperresponsiveness, of the airways), chronic obstructive pulmonary disease (COPD. i.e., chronic bronchitis, emphysema), Acute Respiratory Disorder Syndrome (ARDS), respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, acute lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonia, eosinophilic pneumonia, bronchitis, allergic bronchitis bronchiectasis, tuberculosis, hypersensitivity pneumonitis, occupational asthma, asthma-like disorders, sarcoid, reactive airway disease (or dysfunction) syndrome, byssinosis, interstitial lung disease, hyper-eosinophilic syndrome, rhinitis, sinusitis, and parasitic lung disease, airway hyperresponsiveness associated with viral-induced conditions (for example, respiratory syncytial virus (RSV), parainfluenza virus (PIV), rhinovirus (RV) and adenovirus), Guillain-Barre disease, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft versus host disease (GVHD), cerebral ischemia, traumatic brain injury, multiple sclerosis, neuropathy, myopathy, spinal cord injury, and amyotrophic lateral sclerosis (ALS).