Compositions and methods for treating stomatitis diarrhea

Abstract

The present invention provides compositions and methods for treating stomatitis diarrhea.

Description

[0001] The present application is a divisional application of application number 201480050953.7, application date August 13, 2014, and invention name “Compositions and methods for treating sprue”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 61 / 865,787, filed on August 14, 2013, the entire contents of which are incorporated herein by reference.

[0004] Federal Funding Statement

[0005] This invention was made with Government support under Grant HR0011-08-1-0085 awarded by the Defense Advanced Research Projects Agency, which has certain rights in this invention. Background Art

[0006] Celiac sprue is an extremely common disease caused by an immune response in the small intestine of genetically susceptible individuals to dietary proteins called "gluten" found in wheat, barley, and rye products. The resulting inflammation causes degradation of the villi in the small intestine, impeding nutrient absorption. Symptoms can appear in early childhood or later in life and range in severity from diarrhea, fatigue, and weight loss to bloating, anemia, and neurological symptoms. There is currently no effective treatment for this lifelong disease other than complete elimination of gluten from the diet. Although a large proportion of sprue cases remain undetected, the prevalence of the disease in the United States and Europe is estimated to be 0.5-1.0% of the population. Finding suitable naturally occurring enzymes for oral therapy of sprue is difficult due to the stringent physical and chemical requirements for specific and efficient degradation of gluten-derived peptides in the harsh and highly acidic environment of the human digestive tract. Summary of the invention

[0007] In one aspect, the present invention provides a method for treating sprue, comprising administering to a patient suffering from sprue an effective amount of one or more polypeptides for treating sprue, wherein the one or more polypeptides comprise or consist of an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOS: 75, 74, 76-89, 95, 97-98, 102-111, or a processed version thereof. In one embodiment, the one or more polypeptides include one or more polypeptides comprising or consisting of an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 75, 74, 77, 78, 82, 88, 98, 105, 111 or a processed version thereof. In a further embodiment, the one or more polypeptides include a polypeptide comprising the amino acid sequence of SEQ ID NO: 89.

[0008] In another aspect, the present invention provides an isolated polypeptide selected from the group consisting of the following polypeptides or processed versions thereof:

[0009] (a) SEQ ID NO:95, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 116 is V or D; (ii) AA residue 255 is S, K, or G; (iii) AA residue 284 is D; (iv) AA residue 285 is T; (v) AA residue 286 is A, T, or N; (vi) AA residue 312 is S; (vii) AA residue 347 is N; (viii) AA residue 350 is T or A; (ix) AA residue 351 is N or G; (x) AA residue 354 is D; and (xi) AA residue 361 is S or H;

[0010] (b) SEQ ID NO:75, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is D; (ii) AA residue 246 is S, K, or G; (iii) AA residue 275 is D; (iv) AA residue 276 is S; (v) AA residue 277 is A, T, or N; (vi) AA residue 303 is S; (vii) AA residue 338 is S; (viii) AA residue 341 is T or A; (ix) AA residue 342 is N or G; (x) AA residue 345 is Q or D; and (xi) AA residue 352 is S or H;

[0011] (c) SEQ ID NO:76, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 120 is D; (ii) AA residue 259 is S, K, or G; (iii) AA residue 288 is D; (iv) AA residue 289 is T; (v) AA residue 290 is A, T, or N; (vi) AA residue 316 is S; (vii) AA residue 351 is S or N; (viii) AA residue 354 is A; (ix) AA residue 355 is N or G; (x) AA residue 358 is D; and (xi) AA residue 365 is S or H;

[0012] (d) SEQ ID NO: 78;

[0013] (e) SEQ ID NO:79, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 107 is V or D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is T; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is S or N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H;

[0014] (f) SEQ ID NO:80, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 76 is V or D; (ii) AA residue 206 is S, K, or G; (iii) AA residue 235 is D; (iv) AA residue 236 is S; (v) AA residue 237 is A, T, or N; (vi) AA residue 262 is S; (vii) AA residue 297 is S or N; (viii) AA residue 300 is T or A; (ix) AA residue 301 is N or G; (x) AA residue 302 is Q or D; and (xi) AA residue 309 is S or H;

[0015] (g) SEQ ID NO:81, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 105 is D; (ii) AA residue 244 is S or K; (iii) AA residue 272 is D; (iv) AA residue 273 is S; (v) AA residue 274 is A, T, or N; (vi) AA residue 299 is S; (vii) AA residue 334 is N; (viii) AA residue 337 is T or A; (ix) AA residue 338 is N or G; (x) AA residue 341 is Q or D; and (xi) AA residue 348 is S or H;

[0016] (h) SEQ ID NO:82, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is V or D; (ii) AA residue 244 is S, K, or G; (iii) AA residue 273 is D; (iv) AA residue 274 is T; (v) AA residue 275 is A, T, or N; (vi) AA residue 301 is S; (vii) AA residue 336 is N; (viii) AA residue 339 is T or A; (ix) AA residue 340 is N or G; (x) AA residue 343 is D; and (viii) AA residue 350 is S or H;

[0017] (i) SEQ ID NO:83, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 107 is V or D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is T; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H;

[0018] (j) SEQ ID NO:84, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 104 is V or D; (ii) AA residue 241 is S, K, or G; (iii) AA residue 270 is D; (iv) AA residue 271 is S; (v) AA residue 272 is D, A, T, or N; (vi) AA residue 398 is S; (vii) AA residue 33 is S; (viii) AA residue 336 is A; (ix) AA residue 337 is N or G; (x) AA residue 340 is D; and (xi) AA residue 347 is S or H;

[0019] (k) SEQ ID NO:85, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 104 is D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is S; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is S or N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H;

[0020] (l) SEQ ID NO:86, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 118 is V or D; (ii) AA residue 250 is K or G; (iii) AA residue 279 is D; (iv) AA residue 280 is S; (v) AA residue 281 is A, T, or N; (vi) AA residue 307 is S; (vii) AA residue 342 is S or N; (viii) AA residue 345 is A; (ix) AA residue 346 is N or G; (x) AA residue 349 is Q or D; and (xi) AA residue 356 is S or H;

[0021] (m) SEQ ID NO:87, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 121 is V or D; (ii) AA residue 253 is S, K, or G; (iii) AA residue 282 is D; (iv) AA residue 283 is S; (v) AA residue 284 is A, T, or N; (vi) AA residue 310 is S; (vii) AA residue 345 is S; (viii) AA residue 348 is T or A; (ix) AA residue 349 is N or G; (x) AA residue 352 is Q or D; and (xi) AA residue 357 is S or H;

[0022] (n) SEQ ID NO:88, wherein one, two, three, four, five, six, seven, eight, nine, or all ten of the following are true: (i) AA residue 111 is S, K, or G; (ii) AA residue 139 is D; (iii) AA residue 140 is T or S; (iv) AA residue 141 is D, A, T, or N; (v) AA residue 164 is S; (vi) AA residue 199 is S or N; (vii) AA residue 202 is T or A; (viii) AA residue 203 is N or G; (ix) AA residue 204 is Q or D; and (x) AA residue 211 is S or H; and

[0023] (o) SEQ ID NO:89.

[0024] In other aspects, the present invention provides a nucleic acid encoding a polypeptide of the present invention, a nucleic acid expression vector comprising the isolated nucleic acid of the present invention, and a recombinant host cell comprising the nucleic acid expression vector of the present invention.

[0025] In yet another aspect, the present invention provides a composition comprising:

[0026] (a) one or more polypeptides comprising an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 74-89, 95, 97-99 and 102-111 or processed versions thereof; and

[0027] (b) one or more other polypeptides comprising an amino acid sequence selected from the group consisting of:

[0028] (A) an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID No: 35, wherein:

[0029] (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and

[0030] (ii) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp;

[0031] (B) an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID No: 1, wherein:

[0032] (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and

[0033] (ii) Residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp.

[0034] In yet another aspect, the present invention provides a pharmaceutical composition comprising the isolated polypeptide, nucleic acid, expression vector, host cell or composition of the present invention and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a graph showing the activity of various polypeptides in cleaving fluorescent analogs of gliadin to which a fluorescent group and a quenching group are attached. X-axis: number of specific homologues. KWT=Kumamolisin-As. Y-axis: arbitrary enzyme units.

[0036] Figure 2 It is a graph showing the activity of polypeptides in cleaving fluorescent analogs of gliadin at different pH levels. SC PEP: prolyl endopeptidase from Sphingomonas capsulata. WT: Kumamolisin-As. Max: KumaMax TM The blue bars represent optimal protease activity at the indicated pH levels.

[0037] Figure 3is a graph showing the activity of different polypeptides in cleaving two peptides as gluten degradation products. The X-axis is the time in minutes and the Y-axis is the residual peptide fragments. Max = KumaMax TM ; K3 = Kumamolisin-As active site mutant; H4M = homolog 4 (SEQ ID NO: 75) KumaMax TM - Mutant; 33 = 33mer peptide (SEQ ID NO: 72); 26 = 26mer peptide (SEQ ID NO: 73).

[0038] Figure 4 is a graph showing the activities of various other polypeptides in cleaving the 33mer peptide degradation product of gluten (SEQ ID NO: 72). Detailed description

[0040] The entire contents of all cited references are incorporated herein by reference. In this application, unless otherwise indicated, the techniques used can be found in any known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (MP Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2 ndEd. (RI Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0041] Unless otherwise expressly stated in the application, the singular forms "a", "an" and "the" used in the application include plural references. "And" used in the application can also be used interchangeably with "or" unless otherwise expressly stated.

[0042] The amino acid residues used in this application are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0043] All embodiments of any aspect of the invention can be used in combination, unless the application explicitly states otherwise.

[0044] In a first aspect, the present invention provides a method for treating sprue, comprising administering to a patient suffering from sprue an effective amount of one or more polypeptides for treating sprue, wherein the one or more polypeptides comprise or consist of an amino acid sequence of a polypeptide selected from the following groups or a processed version thereof.

[0045] Homolog 1

[0046] MAPSDVEIVDPVAPEERITVTVLLRRRSSIPDQLIEGPDTLSRAELADRHGADPADVEAVRVAMSGAGLTVVGTDLPSRRVTVAGTAEALMRTFGAELQIVRDASGFQHRARSGELRIPAALDGIVIAVLGLDNRPQAEARFRASQPEAARSFRPDALGRVYRFPANTDGTGQTIAIVELGGGFRQSELDTYFGGLGIPAPQVLAVGVDGGQNLPSGDAGSADGEVLLDIEVAGALAPGARQVVYFAPNTDRGFVDAVTTAVHADPTPAAVSISWGAPEDKWTAQARRAFDAALADAAALGVTVTAAAGDRGSADGEGGGGLHTDFPASSPHLLACGGTKLAVADGGTVASETVWNGGERGGATGGGVSVAFGLPAYQRNAGVDKRRKTGKPGRGVPDVAAVADPATGYEVLVDGEQLVFGGTSAVAPLWAALVARLTQALGRPLGLLNTALYDGAQPGRTQPGFRDVTEGDNDISGKHGPYPARAGWDACTGLGVPDGEALLAALRKPGKE(SEQ ID NO:74);

[0047] Homolog 2

[0048] MQRGTKEGLNMARHLQADREPRIVPESKCLGQCDPAERIHVTIMLRRQEEGQLDALVHQLATGDARAKPVSRDAFAQRFSANPDDIRKTEDFAHRHQLTVDRVDPVESVVVLSGT(I / V / D)AQFEAAFSVKLERFEHRSIGQYRGRSGPIVLPDDIGDAVTAVLGLDSRPQARPHFRFRPPFKPARGAAAVTFTPIQLASLYDFPAGDGAGQCIAIIELGGGYRAADIQQYFRGLGITTPPKLVDVNVGTGRNAPTGEP(N / S / K / G)GPDGEVALDIEIAGAIAPAAKIAVYFAP(N / D)(S / T)(D / A / T / N)AGFIQAVNAAVTDKTNQPSVISISW(G / S)GPEAIWQAQSAQAFNRVLQAAAAQGITVCAASGD(S / N)GS(G / T / A)(D / N / G)GL(Q / D)DGADHV(D / S / H)FPASSPYVLGCGGTQLDALPGQGIRSEVTWNDEASGGGAGGGGVSALFDLPAWQQGLKVARADGTTTPLAKRGVPDVAGDASPQTGYEVSVAGTPAVMGGTSAVAPLWAALIARINAANGASAGWINPVLYKHPGALRDITKGSNGTYAAASGWDACTGLGSPNGAQLATILARKPSS(SEQ ID NO:95);

[0049] Homolog 4

[0050] MANHPLNGSERECLKDAQPIGKADPNERLEVTMLVRRRSHDAFEKHISALAAQGASAKHIDHDEFTKHFGADSADLAAVHAFAQKHGLSVVESHEARRAVVLSGT(V / D)AQFDAAFGVSLQQYEHDGGTYRGRTGPIHLPDELNGVVDAVMGLDNRPQARPSFRTRAQGNVRWTARAAGASTFTPVQLASLYDFPQGDGQNQCIGIIELGGGYRPADLKTYFASLNMKAPSVTAVSVDHGRNHPTGDP(N / S / K / G)GPDGEVMLDIEVAGAVAPGAKIVVYFAP(N / D)(T / S)(D / A / T / N)AGFIDAIGTAIHDTKNKPSVISISW(G / S)GPESAWTQQAMNAFDQAFQSAAALGVTICAASGD(N / S)GS(G / T / A)(D / N / G)GV(G / Q / D)DGADHV(D / S / H)FPASSPYALGCGGTSLQASGNGIASETVWNDGANGGATGGGVSSFFALPAWQEGLRVTRAGGAHSPLAMRGVPDVAGNADPVTGYEVRVDGHDMVIGGTSAVAPLWAGLIARINAIKGAPVGYINPHLYKDPLALVDITKGNNDDFHATAGWDACTGLGRPDGKKVKDAVS(SEQ ID NO:75);

[0051] Homolog 5

[0052] MNHDHSPTGGELSNWVRVPGSERAAVQGSRKVGPADPNEQMSVTVVVRRPAADTAVTSMIEKVGAQPLSERRHLTREEFASTHGANPADLSKVEKFAHEHNLQVKEVNAAAGTMVLSGT(V / D)TSFSKAFGVELSTYEHPDFTYRGRIGHVHIPDYLADTIQSVLGLDNRPQASPRFRVLKEEGGVTTAHAGRTSYTPLEVAALYNFPSIHCKDQCIGILELGGGYRPADLQTYFNGLGIPQPNITDVSVGGAANRPTGDP(N / S / K / G)GPDGEVVLDIEVAAAVTPGAKIAVYFAD(N / D)(S / T)(D / A / T / N)DGFLNAITTAIHDTRNKPSVISISW(G / S)KAEIGWTPQAINAMNQAFRDAAALGVTICCASGD(D / S / N)GS(T / A)(D / / N / G)RV(Q / D)DGRYHV(D / S / H)FPASSPYVLACGGTRLESSGSTITQEVVWNEGALGGGATGGGVSDVFDRPNWQANANVPTSANPERRIGRGVPDWAGNADPATGYQILVDGTRAVIGGTSAVAPLFAGLIAIINQKLGHSVGFINPILYNLSAQHNVFHDITSGNNDMSGQNGPYEAQPGWDACTGLGSPDGTKLMNAISEAHRLVSVG(SEQ ID NO:76);

[0053] Homolog 6

[0054] MAPEERRTLPGSAMPRPAGAQVLGQIPDDERVEVTVVLQPRAPLPEPGPTPMSRAELADLRSPPEGALEAIARYVAGQGLEVIAADAPRRRIVLAGSAARIAALFGISFVRLQLEGRRYRTYEGEISLPAELAPLVVAVLGLDTRPFARSHRRPAVAPNAPTTAPTVARAYDFPTAYDGRGTTIGFIELGGGFQESDLVRYCEGLGLSTPQVSVVGVDGARNAPTGDPNGPDAEVMLDLEVATGVANGADLVLYMAANTDAAFYSAIATALRDATHAPVAISISWGAPEESYPATTIAAFESVLEEAVHVGVTVLVAAGDQGSTDGVDDGRAHVDYPAASPYVLACGGTRLDLDGTTIVAETVWNDLPNGGATGGGISALFPVPSWQAGIAMPPSANPGAGPGRGVPDVAGNADPDTGYRIVVDGVATVVGGTSAVAPLWAGLVARCHQAGARGGFWNPLLYAARGSSAFHEITVGSNGAYDAGPIWNACCGLGSPNGTAILQTLRA(SEQ ID NO:77);

[0055] Homolog 6 mutant strain

[0056] MAPEERRTLPGSAMPRPAGAQVLGQIPDDERVEVTVVLQPRAPLPEPGPTPMSRAELADLRSPPEGALEAIARYVAGQGLEVIAADAPRRRIVLAGSAARIAALFGISFVRLQLEGRRYRTYEGEISLPAELAPLVVAVLGLDTRPFARSHRRPAVAPNAPTTAPTVARAYDFPTAYDGRGTTIGFIELGGGFQESDLVRYCEGLGLSTPQVSVVGVDGARNAPTGDPNGPDAEVMLDLEVATGVANGADLVLYMAANTDAAFYSAIATALRDATHAPVAISISWSAPEESYPATTIAAFESVLEEAVHVGVTVLVAAGDQGSTGGVDDGRAHVHYPAASPYVLACGGTRLDLDGTTIVAETVWNDLPNGGATGGGISALFPVPSWQAGIAMPPSANPGAGPGRGVPDVAGNADPDTGYRIVVDGVATVVGGTSAVAPLWAGLVARCHQAGARGGFWNPLLYAARGSSAFHEITVGSNGAYDAGPIWNACCGLGSPNGTAILQTLRA(SEQ ID NO:78);

[0057] Homolog 9

[0058] MTKQPVSGSSDKIHPDDAKCIGDCDPSEQIEVIVMLRRKDEAGFRQMMSRIDAGEAPGQAVSREEFDRRFTASDEDIDKVKAFAKQYGLSVERAETETRSVVLKGT(I / V / D)EQFQKAFDVKLERFQHHNIGEYRGRTGPVNVPDEMHDAVTAVLGLDSKPQARPHFRFRPPFKPLRGAAPASFSPVDLAKLYDFPDGDGAGQCIAIIELGGGYRDSDLSAYFSKLGVKAPTVVPVGVDGGKNAPTGNP(N / S / K / G)GPDGEVTLDIEIAGAIAPGARIAVYFAP(N / D)(S / T)(D / A / T / N)AGFVDAVNRALHDAANKPSVISISW(G / S)GPESNWSPQSMSAFNDVLQSAAALGVTVCAASGD(G / S / N)GS(A / T)(D / N / G)GV(G / Q / D)DGADHV(D / S / H)FPASSPYVLGCGGTSLAASGAGIAKEVVWNDGDQGGAGGGGVSGTFALPVWQKGLSVTRNGKHIALAKRGVPDVAGDASPQTGYEVLIDGEDTVVGGTSAVAPLWAALIARINAIDASPAGFVNPKLYKAKTAFRDITEGNNGSFSAAAGWDACTGMGSPDGGKIAAALKPAKPSQSAGQQ(SEQ ID NO:79);

[0059] Homolog 10

[0060] MGRLQGSYRPSLGTPVGPVPDDQPIDVTVVLRPTAADDFRADPDDVAAVRAFAGRAGLDVAEVDEPARTVRLRGP(A / V / D)AAARTAFDTPLALYDSGGRAIRGREGDLGLPDELDDRVVAVLGLDERPAARPRFQPAASARQGLTALQVARAYDFPAATGEGQTIAIIELGGGFGQADLDTYFGGLDLPTPAVSAVGVQGAANVPGGDP( / S / K / G)DGADGEVLLDIEVAGAVAPGAAQVVYFAP(N / D)(T / S)(D / A / T / N)AGFLAAINAAAAATPRPAAISISWG(G / S)PESSWTAQAMRAYDQAFAAARAAGITVLAAAGD(A / S / N)GA(D / T / A)(D / S / N / G)(A / Q / D)TDRLVA(D / S / H)FPAGSPNVIACGGTKLTLDAAGARASEVVWNEAADSATGGGYSATFTRPAWQPAAVGRYRGLPDISGNADPQTGYRVVVDGQPTVVGGTSAVAPLLAGLVARLAQLTGAPVADLAAVAYANPAAFTDITAGDNQGYPARSGWDPASGLGSPVGTKLLTAVGGPTPPPTTPPPTTPPPTTPPPTIPPPTTPPTQTVDAADRALWSAVATWAGGTHTGANARAAKAVRAWAQAKSLA(SEQ ID NO:80);

[0061] Homolog 12

[0062] MTQPRYTPLPGSEREAPLLAARSNATAARASRAQTASATVVLRRRSELPEALVLDQQFISSDELAARYGADPVDIEKVRSVLERFKVSVVEVDAASRRVKVEGA(V / D)ADIERAFNIALHSASGTDPHSGRGFEYRYRTGVLSVPAELGGIVTAVLGLDNRRQAETRLRVVPAAALGSSYTPVQLGEIYNFPQDATGAGQRIAIIELGGGYTPAGLRRYFASLGVVPPKVAAVSVDGAQNAPGPDP(G / S / K / G)ADGEVQLDVEVAGALAPGAHVLVYFAP(N / D)(T / S)(D / A / T / N)QGFLDAVSQAAHATPPPTAISISW(G / S)ASEDSWTASARDALNQALRDAAALGVTVTAAAGD(S / N)GS(S / T / A)(D / N / G)GV(P / Q / D)DRRAHV(D / S / H)FPASSPYVLATGGTSLRADPATGVVQSETVWNDSQGSTGGGVSDVFPRPAWQAHVDVPHAGRGVPDVSAVADPATGYQVLVDNQPAVIGGTSAVAPLWAALVARLAESLGRPLGLLQPLVYPRTPGSTAYPGFRDITIGNNGAYKAGKGWDAATGLGVPDGTELLAHLRGLNGSE(SEQ ID NO:81);

[0063] Homolog 13

[0064] MARHLHAGSEPKVITESKCIGACDPAERIHVTVMLRREGEQALDALVDKLASGDPAAKPVSREDFAKRFGARADDIQHTEAFAKRHQLTVERVDPVQSVVELAGT(I / V / D)AQFENAFGVKLEKYEHHAIGSFRARTGAIALPDELHDAVTAVLGLDTRPQAHPHFRFRPPFQPARSGAGTSYTPLQLASIYNFPEGDGAGQCIALVELGGGYRAADIRQYFEQLGVKPPKLVDVSVNGGRNAPTDDP(N / S / K / G)GPDGEVALDIEVAGAIAPGATIAVYFAG(N / D)(S / T)(D / A / T / N)AGFIQSVNQAIHDSTNRPSVVSISW(G / S)GPEASWTQQSITAFNNVLKTAASLGVTVCAASGD(S / / N)GS(S / T / A)(D / N / G)GL(Q / D)DGSNHV(D / S / H)FPASSPYVLACGGTTLDAQAGQGIRREVVWNDEAASGGAGGGGVSAVFPAPSYQKGLSAKATGGGSTPLSQRGVPDVAGDASPTTGYIISIAGTTAVLGGTSAVAPLWAALIARINANGKSPVGWANPKLYAQPGAFHDITQGNNGAFAASEGWDACTGLGSPDGAKVAAALQGASGGSQQGRATGA(SEQ ID NO:82);

[0065] Homolog 14

[0066] MTKHPLPGSERVLAPGSKVVAQCDPSETIEVVVVLRRKNEQQFAQMMKTIEAGAAGARPLTREELEQRFGALPEDIAKLKAFAAQHGLSVVREDASARTVVLSGR(I / V / D)EQFQQAFDVQLQHYEHQSMGRFRGRTGAISVPDELHGVVTAVLGLDDRPQARPHFRIRPPFQPARAQSASSFTPLQLASLYRFPQGDGSGQCIGIVELGGGYRTADLDSYFSSLGVGSPKVVAVGVDQSGNQPTGDP(N / S / K / G)GPDGEVTLDIEIAGALAPAATIAVYFTT(N / D)(S / T)(D / A / T / N)AGFIDAVSQAVHDRTNQPSVISISW(G / S)APESMWTAQSMKALNDVLQSAAAIGVTVCAASGD(S / N)GS(S / T / A)(D / N / G)GV(G / Q / D)DGRDHV(D / S / H)FPASSPYVLACGGTSLQGSGRTVAHEVVWNDGSNGGATGGGVSGAFPVPAWQEGLSTSAAQGGQRALTGRGVPDVAGDASPLTGYDVIVDGNNTVIGGTSAVAPLWAALIARINGAKGAPVGFVNPKLYKASACNDITQGNNGSYAATTGWDACTGLGSPDGVKVAAAL(SEQ ID NO:83);

[0067] Homolog 15

[0068] MSPIASRRSALPLSERPAPENARALAAVEPDRTMTVSVLVRRKKPLVLADLEGKKLTHREFERRYGASEKDFATIAKFAAGHGLAVDHHASSLARRTVVLRGT(A / V / D)RQMQQAFGVTLHDYEDSETQQRYHSFTGAITVPAAHARIIESVLGLDARPIAKPHFRVRKRSAAATGAVSFNPPQVASLYSFPTGVDGSGETIGILELGGGYETSDIQQYFSGLGIQPPTVVAVSVDGAVNAPGNP(N / S / K / G)GADGEVALDIQVAGSIAPGAKLAVYFAP(N / D)(T / S)(E / D / A / T / N)QGFVDAITTAVHDTANKPSVLSISW(G / S)GPESSWPQAAAQSLNNACESAAALGVTITVASGD(N / S)GS(T / A)(D / N / G)GV(Q / D)DGQNHV(D / S / H)FPASSPYVLACGGTYLAAVNNGVPQESVWDDLASGGGATGGGVSALFPLPAWQTGANVPGGSMRGVPDVAGDASPESGYNVLVDGQPQVVGGTSAVAPLWAALIALVNQQKGEAAGFVNAALYQNPSAFHDITQGSNGAYAAAPGWDPCTGLGSPMGTAIAKILA(SEQ ID NO:84);

[0069] Homolog 16

[0070] MSAFDQLVPLPGSEKTVPDAAPSQTLDPNEVLTVTIRIRRKRTLASLVSTTAPVTEVVSRSEYASRFGADPAIVKQVEAFASAYDLSLVEQSLARRSVLLRGT(V / D)AQMEQAFGVSLANYQLADGTVFRGRTGVVNVPSELVEHIEGVFGLDNRPQARAHFQVYKPEKGTKVAPRAGGISYTPPQLARLYNFPTGVTGKGQCIAIIELGGGFRTADIKTYFGGLGLKPPTVVAVSVDGGHNAPSTA(D / S / K / G)SADGEVMLDIDVAGGVAPGAKIVVYFAP(N / D)(T / S)(D / A / T / N)QGFLDAITTAMHDTKNKPSVISISW(G / S)AAESNWTPQALTSFNQAFQAAAALGITVCAAAGD(T / S / N)GS(D / T / A)(D / N / G)SV(G / Q / D)DGKAHV(D / S / H)FPASSPFVLACGGTKLTATDNVIASEVVWHESKTSATGGGVSDVFDLPDYQQKSHVPPSVNDKTRIGRGVPDVAAVADPVTGYAVRVDGSNLVFGGTSAVAPLMAGLIALINQQRGKAVGFIHPLIYANPSAFRDITQGNNTTTTGNKGYAATTGWDACTGLGVADGKKLASVLTATPVA(SEQ ID NO:85);

[0071] Homolog 17

[0072] MAATPRFASQPRVTLPGSQKHPLTTDTEVPPPAPVKAAATKLSATPFTVTVIVKRKNPLNLKQVLKPAGRLTHAAFAKAHGPSPDGVKLVKAFAKEFGLTVAPAPGQGRRALYLTGT(A / V / D)AAMQTAFGVTFATKIMEGTKYRVREGDICLPKELIGHVDAVLGLDNRPQAKPHFRHHKPAATSVSYTPVQVGQLYGFPSGAKATGQTIGLIELGGGFRAADITAYFKTLGQTAPKVTAVLVDKAKNTPTTS(S / K / G)SADGEVMLDIEVAAAVAPGANIAVYFAP(N / D)(T / S)(D / A / T / N)QGFIDAISQAVHDTVNKPSVISISW(G / S)GPESTWTAQSLAALDAACQSAAALGITITVAAGD(D / S / N)GS(T / A)(D / N / G)GV(K / Q / D)GTVNHV(D / S / H)FPASSPHVLGCGGTKLLGSGTTITSEVVWNELTANEGATGGGVSNVFPLPTWQAKSNVPKPTVAAGGRGVPDVSGNADPSTGYTVRVDGSTFPIGGTSAVAPLWAGLIALCNAQNKTTAGFINPALYAAAAAKSFRDITSGNNGGFKAGPGWDACTGLGSPIGTAIAKTLAPATKSTSKTAVKNAPEIRFRPHKKAPTKTAAKTPALRRLK(SEQ ID NO:86);

[0073] Homolog 19

[0074] MPTSSRFASQSRVPLPGSERKPFVPAGAPKAAKTPKVSTAVKTVPATGRIRVSLIVPPKQPLDTKRLGKLDARLSRAQFAARHGADPASVRLVKAFAKEFGLTVEPITQPGRCTVQLSGT(C / V / D)AAMRKAFAISLVEHTTEQGKFRLREGEISLPAELEGHVLAVLGLDNRPQAKPHFRIAKPRATNVSYTPVQVAQMYGFPAGATATGQTIGIIELGGGYRAADLTAYFKTLGLPAPTVTAVPIDGGKNTPGNA(N / S / K / G)GADGEVMLDIEVCAAVAQGAKIAVYFTT(N / D)(T / S)(D / A / T / N)QGFIDAITTAVHDSTNKPSVISISW(G / / S)GPESSWTEQSMTALDAACQAAAAVGVTITVAAGD(N / S)GS(S / T / S)(D / N / G)GA(S / Q / D)GDNV(D / S / H)FPASSPHVLACGGTKLVGSGSTITSEVVWDETSNDEGATGGGVSTVFALPTWQKNANVPSPTTSAGGRGVPDVSGDADPSTGYTIRVDSETTVIGGTSAVAPLWAGLIALANAQNKVAAGFVNPALYAAGAKKAFRDITQGNNGSFSAGPGWDACTGLGSPVGNLVIQAVAPKSTTTKKAKKGKTK(SEQ ID NO:87); and

[0075] Homolog 26

[0076] MHSYLKQQSHMQSYLEQENHMRSYLEMRKKPYFDDLANIRPGGLTPAQVCQAYQFAKVQPVRPVKLGIVSLAGQYLSSDMSKAFTGYGLPTPVVSTAGSQVLGDLWSNVE( N / S / K / G)MMDIEIAGAAWAYATGTAATLLMQFEP(N / D)(N / T / S)(E / D / A / T / N)TGIPNAINALVAAGCEVISISW(G / S)APANLQTMEAITARKEACKQAAVQNV HVFAASGD(E / S / N)SL(N / T / A)(D / N / G)(G / Q / D)TNSRTP(D / S / H)DPCCDPNVWGVGGTRLVLQADGSIAQESAWGDGNAADKGGGGGFDSREPLPDYQVGVVHS EHRGSPDSSANADPGTGYAIVANGQWLIGGGTSASAPLTAGYVAAILSTLPGPISQSVLQRKLYTAHKTAFRDILLGSNGAPARPGWEEATGLGSINGPGLAAALQS(SEQ IDNO:88).

[0077] The polypeptides disclosed in the present application are Kumamolisin homolog polypeptides and modified versions thereof in gluten rich in hydroxyproline (P) and glutamine (Q) components known as "gliadins", which are identified as having similar, improved or complementary activities to Kumamolisin-As, which are believed to be the culprit for most immune responses in most patients with sprue. Many other Kumamolisin homologs tested by the inventors have little or no such gliadin hydrolysis activity. Therefore, the polypeptides disclosed in the present application can be used to treat sprue. The amino acid sequences disclosed in the present application are for preprocessed versions of the polypeptides, which can hydrolyze their substrates in a processed form. Therefore, the present application also covers the use of processed versions of the polypeptides. Those skilled in the art will understand that the specific processing of the polypeptides is different between one cell type or a set of conditions and another cell type or another set of conditions. In one embodiment, the processed form of the homologue lacks the residues shown in Table 1 below, which is a control of the residues of Kummamolisin and each homologue disclosed in the present application in a pre-processed form rather than a processed form.

[0078] Table 1:

[0079]

[0080] In one embodiment, the one or more polypeptides are selected from the group consisting of the following homologs or processed versions thereof.

[0081] Homolog 1 (NCBI YP_005536585)

[0082] MAPSDVEIVDPVAPEERITVTVLLRRRSSIPDQLIEGPDTLSRAELADRHGADPADVEAVRVAMSGAGLTVVGTDLPSRRVTVAGTAEALMRTFGAELQIVRDASGFQHRARSGELRIPAALDGIVIAV LGLDNRPQAEARFRASQPEAARSFRPDALGRVYRFPANTDGTGQTIAIVELGGGFRQSELDTYFGGLGIPAPQVLAVGVDGGQNLPSGDAGSADGEVLLDIEVAGALAPGARQVVYFAPNTDRGFVDAV TTAVHADPTPAAVSISWGAPEDKWTAQARRAFDAALADAAALGVTVTAAAGDRGSADGEGGGGLHTDFPASSPHLLACGTKLAVADGGTVASETVWNGGERGGATGGGVSVAFGLPAYQRNAGVDKRR KTGKPGRGVPDVAAVADPATGYEVLVDGEQLVFGGTSAVAPLWAALVARLTQALGRPLGLLNTALYDGAQPGRTQPGFRDVTEGDNDISGKHGPYPARAGWDACTGLGVPDGEALLAALRKPGKE(SEQ ID NO:74);

[0083] Homolog 2 (NCBI ZP_04943175)

[0084] MQRGTKEGLNMARHLQADREPRIVPESKCLGQCDPAERIHVTIMLRRQEEGQLDALVHQLATGDARAKPVSRDAFAQRFSANPDDIRKTEDFAHRHQLTVDRVDPVESVVVLSGTIAQFEAAFSVKLERFEHRSIGQYRGRSGPIVLPDDIGDAVTAVLGLDSRPQARPHFRFRPPFKPARGAAAVTFTPIQLASLYDFPAGDGAGQCIAIIELGGGYRAADIQQYFRGLGITTPPKLVDVNVGTGRNAPTGEPNGPDGEVALDIEIAGAIAPAAKIAVYFAPNSDAGFIQAVNAAVTDKTNQPSVISISWGGPEAIWQAQSAQAFNRVLQAAAAQGITVCAASGDSGSGDGLQDGADHVDFPASSPYVLGCGGTQLDALPGQGIRSEVTWNDEASGGGAGGGGVSALFDLPAWQQGLKVARADGTTTPLAKRGVPDVAGDASPQTGYEVSVAGTPAVMGGTSAVAPLWAALIARINAANGASAGWINPVLYKHPGALRDITKGSNGTYAAASGWDACTGLGSPNGAQLATILARKPSS(SEQ ID NO:97);

[0085] Homolog 4 (NCBI ZP_10028298)

[0086] MANHPLNGSERECLKDAQPIGKADPNERLEVTMLVRRRSHDAFEKHISALAAQGASAKHIDHDEFTKHFGADSADLAAVHAFAQKHGLSVVESHEARRAVVLSGTVAQFDAAFGVSLQQYEHDGGTYRGRTGPIHLPDELNGVVDAVMGLDNRPQARPSFRTRAQGNVRWTARAAGASTFTPVQLASLYDFPQGDGQNQCIGIIELGGGYRPADLKTYFASLNMKAPSVTAVSVDHGRNHPTGDPNGPDGEVMLDIEVAGAVAPGAKIVVYFAPNTDAGFIDAIGTAIHDTKNKPSVISISWGGPESAWTQQAMNAFDQAFQSAAALGVTICAASGDNGSGDGVGDGADHVDFPASSPYALGCGGTSLQASGNGIASETVWNDGANGGATGGGVSSFFALPAWQEGLRVTRAGGAHSPLAMRGVPDVAGNADPVTGYEVRVDGHDMVIGGTSAVAPLWAGLIARINAIKGAPVGYINPHLYKDPLALVDITKGNNDDFHATAGWDACTGLGRPDGKKVKDAVS(SEQ ID NO:98);

[0087] Homolog 5 (NCBI ZP_09078202)

[0088] AGRTSYTPLEVAALYNFPSIHCKDQCIGILELGGGYRPADLQTYFNGLGIPQPNITDVSVGGAANRPTGDPNGPDGEVVLDIEVAAAVTPGAKIAVYFADNSDDGFLNAITTAIHDTRNKPSVISISWGKAEIGWTPQAINAMNQAFRDAAALGVTICCASGDDGSTDRVQDGRYHVDFPASSPYVLACGGTRLESSGSTITQEVVWNEGALGGGATGGGVSDVFDRPNWQANANVPTSANPERRIGRGVPDWAGNADPATGYQILVDGTRAVIGGTSAVAPLFAGLIAIINQKLGHSVGFINPILYNLSAQHNVFHDITSGNNDMSGQNGPYEAQPGWDACTGLGSPDGTKLMNAISEAHRLVSVG(SEQID NO:99);

[0089] Homolog 6 (NCBI YP_003109679)

[0090] MAPEERRTLPGSAMPRPAGAQVLGQIPDDERVEVTVVLQPRAPLPEPGPTPMSRAELADLRSPPEGALEAIARYVAGQGLEVIAADAPRRRIVLAGSAARIAALFGISFVRLQLEGRRYRTYEGEISLPAELAPLVVAVLGLDTRPFARSHRRPAVAPNAPTTAPTVARAYDFPTAYDGRGTTIGFIELGGGFQESDLVRYCEGLGLSTPQVSVVGVDGARNAPTGDPNGPDAEVMLDLEVATGVANGADLVLYMAANTDAAFYSAIATALRDATHAPVAISISWGAPEESYPATTIAAFESVLEEAVHVGVTVLVAAGDQGSTDGVDDGRAHVDYPAASPYVLACGGTRLDLDGTTIVAETVWNDLPNGGATGGGISALFPVPSWQAGIAMPPSANPGAGPGRGVPDVAGNADPDTGYRIVVDGVATVVGGTSAVAPLWAGLVARCHQAGARGGFWNPLLYAARGSSAFHEITVGSNGAYDAGPIWNACCGLGSPNGTAILQTLRA (SEQ ID NO:77);

[0091] Homolog #6 mutant strain:

[0092] MAPEERRTLPGSAMPRPAGAQVLGQIPDDERVEVTVVLQPRAPLPEPGPTPMSRAELADLRSPPEGALEAIARYVAGQGLEVIAADAPRRRIVLAGSAARIAALFGISFVRLQLEGRRYRTYEGEISLPAELAPLVVAVLGLDTRPFARSHRRPAVAPNAPTTAPTVARAYDFPTAYDGRGTTIGFIELGGGFQESDLVRYCEGLGLSTPQVSVVGVDGARNAPTGDPNGPDAEVMLDLEVATGVANGADLVLYMAANTDAAFYSAIATALRDATHAPVAISISWSAPEESYPATTIAAFESVLEEAVHVGVTVLVAAGDQGSTGGVDDGRAHVHYPAASPYVLACGGTRLDLDGTTIVAETVWNDLPNGGATGGGISALFPVPSWQAGIAMPPSANPGAGPGRGVPDVAGNADPDTGYRIVVDGVATVVGGTSAVAPLWAGLVARCHQAGARGGFWNPLLYAARGSSAFHEITVGSNGAYDAGPIWNACCGLGSPNGTAILQTLRA(SEQ ID NO:78);

[0093] Homolog 9 (NCBI YP_005042475)

[0094] MTKQPVSGSSDKIHPDDAKCIGDCDPSEQIEVIVMLRRKDEAGFRQMMSRIDAGEAPGQAVSREEFDRRFTASDEDIDKVKAFAKQYGLSVERAETETRSVVLKGTIEQFQKAFDVKLERFQHHNIGEYRGRTGPVNVPDEMHDAVTAVLGLDSKPQARPHFRFRPPFKPLRGAAPASFSPVDLAKLYDFPDGDGAGQCIAIIELGGGYRDSDLSAYFSKLGVKAPTVVPVGVDGGKNAPTGNPNGPDGEVTLDIEIAGAIAPGARIAVYFAPNSDAGFVDAVNRALHDAANKPSVISISWGGPESNWSPQSMSAFNDVLQSAAALGVTVCAASGDGGSADGVGDGADHVDFPASSPYVLGCGGTSLAASGAGIAKEVVWNDGDQGGAGGGGVSGTFALPVWQKGLSVTRNGKHIALAKRGVPDVAGDASPQTGYEVLIDGEDTVVGGTSAVAPLWAALIARINAIDASPAGFVNPKLYKAKTAFRDITEGNNGSFSAAAGWDACTGMGSPDGGKIAAALKPAKPSQSAGQQ(SEQ ID NO:102);

[0095] Homolog 10 (NCBI YP_711059)

[0096] MGRLQGSYRPSLGTPVGPVPDDQPIDVTVVLRPTAADDFRADPDDVAAVRAFAGRAGLDVAEVDEPARTVRLRGPAAAARTAFDTPLALYDSGGRAIRGREGDLGLPDELDDRVVAVLGLDERPAARPRFQPAASARQGLTALQVARAYDFPAATGEGQTIAIIELGGGFGQADLDTYFGGLDLPTPAVSAVGVQGAANVPGGDPDGADGEVLLDIEVAGAVAPGAAQVVYFAPNTDAGFLAAINAAAAATPRPAAISISWGGPESSWTAQAMRAYDQAFAAARAAGITVLAAAGDAGADDATDRLVADFPAGSPNVIACGGTKLTLDAAGARASEVVWNEAADSATGGGYSATFTRPAWQPAAVGRYRGLPDISGNADPQTGYRVVVDGQPTVVGGTSAVAPLLAGLVARLAQLTGAPVADLAAVAYANPAAFTDITAGDNQGYPARSGWDPASGLGSPVGTKLLTAVGGPTPPPTTPPPTTPPPTTPPPTIPPPTTPPTQTVDAADRALWSAVATWAGGTHTGANARAAKAVRAWAQAKSLA(SEQ ID NO:103);

[0097] Homolog 12 (NCBI YP_003658449)

[0098] MTQPRYTPLPGSEREAPLLAARSNATAARASRAQTASATVVLRRRSELPEALVLDQQFISSDELAARYGADPVDIEKVRSVLERFKVSVVEVDAASRRVKVEGAVADIERAFNIALHSASGTDPHSGRGFEYRYRTGVLSVPAELGGIVTAVLGLDNRRQAETRLRVVPAAALGSSYTPVQLGEIYNFPQDATGAGQRIAIIELGGGYTPAGLRRYFASLGVVPPKVAAVSVDGAQNAPGPDPGADGEVQLDVEVAGALAPGAHVLVYFAPNTDQGFLDAVSQAAHATPPPTAISISWGASEDSWTASARDALNQALRDAAALGVTVTAAAGDSGSSDGVPDRRAHVDFPASSPYVLATGGTSLRADPATGVVQSETVWNDSQGSTGGGVSDVFPRPAWQAHVDVPHAGRGVPDVSAVADPATGYQVLVDNQPAVIGGTSAVAPLWAALVARLAESLGRPLGLLQPLVYPRTPGSTAYPGFRDITIGNNGAYKAGKGWDAATGLGVPDGTELLAHLRGLNGSE(SEQ ID NO:104);

[0099] Homolog 13 (NCBI YP_004348568)

[0100] MARHLHAGSEPKVITESKCIGACDPAERIHVTVMLRREGEQALDALVDKLASGDPAAKPVSREDFAKRFGARADDIQHTEAFAKRHQLTVERVDPVQSVVELAGTIAQFENAFGVKLEKYEHHAIGSFRARTGAIALPDELHDAVTAVLGLDTRPQAHPHFRFRPPFQPARSGAGTSYTPLQLASIYNFPEGDGAGQCIALVELGGGYRAADIRQYFEQLGVKPPKLVDVSVNGGRNAPTDDPNGPDGEVALDIEVAGAIAPGATIAVYFAGNSDAGFIQSVNQAIHDSTNRPSVVSISWGGPEASWTQQSITAFNNVLKTAASLGVTVCAASGDSGSSDGLQDGSNHVDFPASSPYVLACGGTTLDAQAGQGIRREVVWNDEAASGGAGGGGVSAVFPAPSYQKGLSAKATGGGSTPLSQRGVPDVAGDASPTTGYIISIAGTTAVLGGTSAVAPLWAALIARINANGKSPVGWANPKLYAQPGAFHDITQGNNGAFAASEGWDACTGLGSPDGAKVAAALQGASGGSQQGRATGA(SEQ ID NO:105);

[0101] Homolog 14 (NCBI YP_001861188)

[0102] HMTKHPLPGSERVLAPGSKVVAQCDPSETIEVVVVLRRKNEQQFAQMMKTIEAGAAGARPLTREELEQRFGALPEDIAKLKAFAAQHGLSVVREDASARTVVLSGRIEQFQQAFDVQLQHYEHQSMGRFRGRTGAISVPDELHGVVTAVLGLDDRPQARPHFRIRPPFQPARAQSASSFTPLQLASLYRFPQGDGSGQCIGIVELGGGYRTADLDSYFSSLGVGSPKVVAVGVDQSGNQPTGDPNGPDGEVTLDIEIAGALAPAATIAVYFTTNSDAGFIDAVSQAVHDRTNQPSVISISWGAPESMWTAQSMKALNDVLQSAAAIGVTVCAASGDSGSSDGVGDGRDHVDFPASSPYVLACGGTSLQGSGRTVAHEVVWNDGSNGGATGGGVSGAFPVPAWQEGLSTSAAQGGQRALTGRGVPDVAGDASPLTGYDVIVDGNNTVIGGTSAVAPLWAALIARINGAKGAPVGFVNPKLYKASACNDITQGNNGSYAATTGWDACTGLGSPDGVKVAAAL(SEQ ID NO:106);

[0103] Homolog 15 (NCBI YP_002754884)

[0104] MSPIASRRSALPLSERPAPENARALAAVEPDRTMTVSVLVRRKKPLVLADLEGKKLTHREFERRYGASEKDFATIAKFAAGHGLAVDHHASSLARRTVVLRGTARQMQQAFGVTLHDYEDSETQQRYHSFTGAITVPAAHARIIESVLGLDARPIAKPHFRVRKRSAAATGAVSFNPPQVASLYSFPTGVDGSGETIGILELGGGYETSDIQQYFSGLGIQPPTVVAVSVDGAVNAPGNPNGADGEVALDIQVAGSIAPGAKLAVYFAPNTEQGFVDAITTAVHDTANKPSVLSISWGGPESSWPQAAAQSLNNACESAAALGVTITVASGDNGSTDGVQDGQNHVDFPASSPYVLACGGTYLAAVNNGVPQESVWDDLASGGGATGGGVSALFPLPAWQTGANVPGGSMRGVPDVAGDASPESGYNVLVDGQPQVVGGTSAVAPLWAALIALVNQQKGEAAGFVNAALYQNPSAFHDITQGSNGAYAAAPGWDPCTGLGSPMGTAIAKILA(SEQ ID NO:107);

[0105] Homolog 16 (NCBI YP_003387700)

[0106] MSAFDQLVPLPGSEKTVPDAAPSQTLDPNEVLTVTIRIRRKRTLASLVSTTAPVTEVVSRSEYASRFGADPAIVKQVEAFASAYDLSLVEQSLARRSVLLRGTVAQMEQAFGVSLANYQLADGTVFRGRTGVVNVPSELVEHIEGVFGLDNRPQARAHFQVYKPEKGTKVAPRAGGISYTPPQLARLYNFPTGVTGKGQCIAIIELGGGFRTADIKTYFGGLGLKPPTVVAVSVDGGHNAPSTADSADGEVMLDIDVAGGVAPGAKIVVYFAPNTDQGFLDAITTAMHDTKNKPSVISISWGAAESNWTPQALTSFNQAFQAAAALGITVCAAAGDTGSDDSVGDGKAHVDFPASSPFVLACGGTKLTATDNVIASEVVWHESKTSATGGGVSDVFDLPDYQQKSHVPPSVNDKTRIGRGVPDVAAVADPVTGYAVRVDGSNLVFGGTSAVAPLMAGLIALINQQRGKAVGFIHPLIYANPSAFRDITQGNNTTTTGNKGYAATTGWDACTGLGVADGKKLASVLTATPVA(SEQ ID NO:108);

[0107] Homolog 17 (NCBI YP_004216463)

[0108] MAATPRFASQPRVTLPGSQKHPLTTDTEVPPPAPVKAAATKLSATPFTVTVIVKRKNPLNLKQVLKPAGRLTHAAFAKAHGPSPDGVKLVKAFAKEFGLTVAPAPGQGRRALYLTGTAAAMQTAFGVTFATKIMEGTKYRVREGDICLPKELIGHVDAVLGLDNRPQAKPHFRHHKPAATSVSYTPVQVGQLYGFPSGAKATGQTIGLIELGGGFRAADITAYFKTLGQTAPKVTAVLVDKAKNTPTTSSSADGEVMLDIEVAAAVAPGANIAVYFAPNTDQGFIDAISQAVHDTVNKPSVISISWGGPESTWTAQSLAALDAACQSAAALGITITVAAGDDGSTDGVKGTVNHVDFPASSPHVLGCGGTKLLGSGTTITSEVVWNELTANEGATGGGVSNVFPLPTWQAKSNVPKPTVAAGGRGVPDVSGNADPSTGYTVRVDGSTFPIGGTSAVAPLWAGLIALCNAQNKTTAGFINPALYAAAAAKSFRDITSGNNGGFKAGPGWDACTGLGSPIGTAIAKTLAPATKSTSKTAVKNAPEIRFRPHKKAPTKTAAKTPALRRLK(SEQ ID NO:109);

[0109] Homolog 19 (NCBI YP_005056054)

[0110] MPTSSRFASQSRVPLPGSERKPFVPAGAPKAAKTPKVSTAVKTVPATGRIRVSLIVPPKQPLDTKRLGKLDARLSRAQFAARHGADPASVRLVKAFAKEFGLTVEPITQPGRCTVQLSGTCAAMRKAFAISLVEHTTEQGKFRLREGEISLPAELEGHVLAVLGLDNRPQAKPHFRIAKPRATNVSYTPVQVAQMYGFPAGATATGQTIGIIELGGGYRAADLTAYFKTLGLPAPTVTAVPIDGGKNTPGNANGADGEVMLDIEVCAAVAQGAKIAVYFTTNTDQGFIDAITTAVHDSTNKPSVISISWGGPESSWTEQSMTALDAACQAAAAVGVTITVAAGDNGSSDGASGDNVDFPASSPHVLACGGTKLVGSGSTITSEVVWDETSNDEGATGGGVSTVFALPTWQKNANVPSPTTSAGGRGVPDVSGDADPSTGYTIRVDSETTVIGGTSAVAPLWAGLIALANAQNKVAAGFVNPALYAAGAKKAFRDITQGNNGSFSAGPGWDACTGLGSPVGNLVIQAVAPKSTTTKKAKKGKTK(SEQ ID NO:110); and

[0111] Homolog 26 (NCBI YP_004030750)

[0112] MHSYLKQQSHMQSYLEQENHMRSYLEMRKKPYFDDLANIRPGGLTPAQVCQAYQFAKVQPVRPVKLGIVSLAGQYLSSDMSKAFTGYGLPTPVVSTAGSQVLGDLWSNVENMMDIEIAGAAWAYATGTAATLLMQFEPNNETGIPNALVAAGCEVISISWGAPANLQTMEAITARKEACKQAAVQNVH VFAASGDESLNDGTNSRTPDDPCCDPNVWGVGGTRLVLQADGSIAQESAWGDGNAADKGGGGGFDSREPLPDYQVGVVHSEHRGSPDSSANADPGTGYAIVANGQWLIGGGTSASAPLTAGYVAAILSTLPGPISQSVLQRKLYTAHKTAFRDILLGSNGAPARPGWEEATGLGSINGPGLAAALQS(SEQ ID NO:111).

[0113] In one embodiment, the one or more polypeptides include one or more polypeptides comprising or consisting of an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 74, 75, 77, 78, 82, 88, 98, 105, 111 or a processed version thereof. In a further embodiment, the one or more polypeptides include a polypeptide comprising or consisting of the following amino acid sequence:

[0114] Homolog 4 mutant

[0115] (SEQ ID NO:89), or a processed version of that sequence.

[0116] The method comprises administering the one or more polypeptides and any other suitable active agent to treat sprue. In various non-limiting embodiments, the method further comprises administering to the subject an amount of one or more other polypeptides, the polypeptide comprising an amino acid sequence selected from the group consisting of the following amino acid sequences:

[0117] (A) an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID No: 35, wherein:

[0118] (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and

[0119] (ii) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp;

[0120] (B) an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID No: 1, wherein:

[0121] (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and

[0122] (ii) Residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp.

[0123] The one or more other polypeptides have been disclosed for treating sprue (see WO2013 / 023151). The other polypeptides are either processed versions of Kumamolisin-As (SEQ ID No: 67), or processed versions of Kumamolisin-As (SEQ ID No: 33), or modified versions thereof, which are known to be members of the sedolisin family of serine carboxypeptidases, utilizing the key processed form of the catalytic triad Ser 278 -Glu 78 -Asp 82 to hydrolyze its substrate (pre-processed form of Ser 467 -Glu 267 -Asp 271 ). Its optimal activity is at pH ~4.0. Although the natural substrate of Kumamolisin-As is still unknown, it has been previously shown to degrade collagen under acidic conditions. In addition, the enzyme has been shown to be thermostable, with an ideal temperature of 60°C, but still showing high activity at 37°C.

[0124] The other polypeptides may include one or more amino acid changes at one or more residues of SEQ ID NO: 67 (wild-type processed Kumamolisin-As), the residues selected from the group consisting of residues 73, 102, 103, 104, 130, 165, 168, 169, 172 and 179 (numbering based on the wild-type processed Kumamolisin-As amino acid sequence). In a non-limiting embodiment, the one or more changes associated with the wild-type processed Kumamolisin-As amino acid sequence (SEQ ID No: 67) may be selected from the group comprising the following changes:

[0125]

[0126] In various non-limiting embodiments, the one or more changes associated with the wild-type processed Kumamolisin-As amino acid sequence may include at least N102D. In another non-limiting embodiment, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may include at least N102D and D169N or D169G. In another non-limiting embodiment, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may include at least N102D, D169G and D179H. In another non-limiting embodiment, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may include at least S73K, D104T, N102D, G130S, D169G and D179H.

[0127] The other polypeptides may include one or more amino acid changes at one or more residues of SEQ ID NO: 33 (wild-type pre-processed Kumamolisin-As), the residues selected from the group consisting of residues 119, 262, 291, 292, 293, 319, 354, 357, 358, 361 and 368 (numbering based on the wild-type pre-processed Kumamolisin-As amino acid sequence). In a non-limiting embodiment, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may be selected from the group consisting of:

[0128]

[0129]

[0130] In various non-limiting embodiments, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may include at least N291D. In another non-limiting embodiment, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may include at least N291D and 358N or 358G. In another non-limiting embodiment, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may include at least N291D, 358G and 368H. In another non-limiting embodiment, the one or more changes associated with the wild-type Kumamolisin-As amino acid sequence may include at least V119D, S262K, D293T, N291D, G319S, D358G and D368H.

[0131] As used in this application, "at least 75% identical" means that the polypeptide has 25% or less differences (including any amino acid substitution, deletion, addition or insertion) from the polypeptide defined by SEQ ID No: 1 or 35 over its entire amino acid sequence length.

[0132] In various further embodiments, the one or more other polypeptides comprise or consist of an amino acid sequence that is at least 75% identical to any one of SEQ ID NOS: 2-33 or 36-67, or as an alternative 2-32 or 36-66. Each specific polypeptide example of the various polypeptides represented by SEQ ID NOS has increased protease activity against oligopeptide PQPQLP (SEQ ID NO: 34) (representing a substrate of gliadin) at pH 4 compared to wild-type Kumamolisin-As. In preferred embodiments, the one or more additional polypeptides comprise an amino acid sequence that is at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence corresponding to any one of SEQ ID NOS:36-66, or consist of at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence corresponding to any one of SEQ ID NOS:36-66. In various further embodiments, the one or more other polypeptides comprise or consist of an amino acid sequence that is identical to an amino acid sequence that corresponds to any one of SEQ ID NOS: 2-33 or 36-67, or as an alternative to any one of 2-32 or 36-66. In various further embodiments, the one or more other polypeptides comprise or consist of an amino acid sequence that corresponds to any one of SEQ ID NOS: 2-33 or 36-67, or as an alternative to any one of 2-32 or 36-66.

[0133] In one embodiment, the one or more other polypeptides include an amino acid sequence as shown below (KumaMax TM ) or a polypeptide comprising the amino acid sequence shown below (KumaMax TM) is composed of a polypeptide consisting of: (SEQ ID NO: 90), or a processed version of the sequence.

[0134] In one embodiment, the method comprises administering homolog 4 or full-length mutant homolog 4 (SEQ ID NOs: 75, 89 and / or 98) or a processed version thereof, and one or more other polypeptides disclosed herein, including but not limited to KumaMa TM x (SEQ ID NO: 90) or a processed version thereof. As described in the examples shown below, homolog 4 (SEQ ID NO: 98) has enhanced anti-γ-gliadin peptide (amino acid sequence IQPQQPAQL (SEQ ID NO: 92)) activity compared to Kumamolisin polypeptides. Therefore, administration of homolog 4 (SEQ ID NO: 98) or a processed version thereof and one or more Kumamolisin polypeptides (such as KumaMax TM(SEQ ID NO:90) or a processed version thereof) can provide improved treatment for gluten digestion. In further embodiments, the Homolog 4 polypeptide comprises or consists of the full-length Homolog 4 mutant (SEQ ID NO:89) or a processed version thereof, wherein the following examples show that it can significantly improve the activity against gastric gluten degradation products, which have been specifically linked to sprue, the gluten degradation products being: the 33mer peptide (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:72)) and the 26mer peptide (FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO:73)).

[0135] In another embodiment, the method comprises administering homolog 1 (SEQ ID NO: 74), homolog 6 (SEQ ID NO: 77), and / or homolog 6 mutant (SEQ ID NO: 78), or processed versions thereof, and one or more other polypeptides disclosed herein, including but not limited to KumaMax TM (SEQ ID NO:90) or a processed version thereof. In the following demonstrated example, homolog 1 (SEQ ID NO:74) was optimally active when pH reached 5, and homolog 6 mutant was demonstrated to have optimal activity at a lower pH level than other homologs and other Kumamolisin-related polypeptides. Therefore, homolog 1 (SEQ ID NO:74), homolog 6 (SEQ ID NO:77), and / or homolog 6 mutant (SEQ ID NO:78), or a processed version thereof can be used alone in a suitable pH environment, or in combination with one or more other polypeptides to expand the pH range of the one or more other polypeptides, for example, to more accurately simulate the pH of the stomach.

[0136] In further embodiments, the method comprises administering homolog 26 (SEQ ID NO: 88 or 111) or a processed version thereof, and one or more other polypeptides disclosed herein, including but not limited to KumaMax TM (SEQ ID NO: 90) or a processed version thereof. As described in the examples shown below, homolog 26 (SEQ ID NO: 111) has a very strong activity in cleaving the 33mer gliadin peptide, and thus it can be used alone or in combination with one or more other polypeptides to treat sprue, including but not limited to KumaMax TM (SEQ ID NO:90).

[0137] Sprue (also known as celiac disease or gluten intolerance) is an extremely common disease in which dietary proteins called "gluten" found in wheat, barley, and rye products cause an immune response in the small intestine of genetically susceptible individuals. The resulting inflammation causes the degeneration of the villi in the small intestine, which hinders the absorption of nutrients. Its symptoms can appear in early childhood or later in life and range in severity from diarrhea, fatigue, weight loss, abdominal pain, bloating, excess gas, indigestion, constipation, bloating, nausea / vomiting, anemia, easy bruising, depression, anxiety, slow growth in children, hair loss, dermatitis, irregular menstruation, mouth ulcers, joint pain, nosebleeds, seizures, tingling or numbness in the hands or feet, delayed puberty, tooth enamel defects to neurological symptoms such as ataxia or paresthesia. There is currently no effective treatment for this lifelong disease, which can only be eliminated from the diet. Although a large proportion of celiac disease remains undetected, its prevalence in the United States and Europe is estimated to be 0.5-1.0% of the population.

[0138] As used herein, "treating sprue" means achieving one or more of the following: (a) reducing the severity of sprue; (b) limiting or preventing the development of typical symptoms of sprue; (c) preventing the worsening of typical symptoms of sprue; (d) limiting or preventing the recurrence of sprue in patients who have previously suffered from the disease; (e) limiting or preventing the recurrence of symptoms in patients who previously had symptoms of sprue; and (f) limiting the development of sprue in subjects who are at risk of developing sprue or who have not yet developed clinical manifestations of sprue.

[0139] The subject to be treated according to the method of the present invention can be any subject suffering from celiac sprue, including human subjects. The subject can be suffering from some symptoms or have no symptoms.

[0140] In one embodiment, the subject may be of HLA-DQ2 serotype; in another embodiment, the subject may be of HLA-DQA serotype. Polypeptides with enhanced resistance to γ-gliadin (homologs 1, 4, 5 and 9 (SEQ ID NOs: 74, 75, 76, 79, 89, 98, 99 and 102) are particularly effective in treating patients with HLA-DQ8 serotype. Polypeptides with enhanced resistance to gluten degradation products α2-gliadin and α9-gliadin and / or 33-mer, 26-mer (homolog 4 mutant (SEQ ID NO: 89) and homologs 13 and 26 (SEQ ID NOs: 82, 88, 105 and 111)) described herein are particularly effective in treating patients with HLA-DQ2 serotype.

[0141] As used herein, "effective amount" refers to the amount of a polypeptide that is effective in treating sprue. The polypeptide is typically formulated into a pharmaceutical composition, such as those disclosed above, and can be administered by a suitable route, including oral, parenteral, by aerosol inhalation or topical application in a unit dosage form containing conventional pharmaceutically acceptable carriers, adjuvants and excipients. In a preferred embodiment, the pharmaceutical composition and formulation are administered orally, such as in the form of tablets, pills, lozenges, elixirs, suspensions, emulsions, solutions or syrups.

[0142] The dosage regimen can be adjusted to provide the best desired effect (e.g., therapeutic effect or preventive effect). Suitable dosage ranges can be, for example, 0.1 ug / kg-100 mg / kg body weight; alternatively, 0.5 ug / kg-50 mg / kg; 1 ug / kg-25 mg / kg, or 5 ug / kg-10 mg / kg body weight. The polypeptide can be delivered by rapid perfusion, or can be administered multiple times (e.g., 2, 3, 4, 5 or more times), which is determined by the attending physician.

[0143] In another aspect, the present invention provides an isolated polypeptide selected from the group consisting of the following polypeptides or processed versions thereof: Homolog 2

[0144] MQRGTKEGLNMARHLQADREPRIVPESKCLGQCDPAERIHVTIMLRRQEEGQLDALVHQLATGDARAKPVSRDAFAQRFSANPDDIRKTEDFAHRHQLTVDRVDPVESVVVLSGT(I / V / D)AQFEAAFSVKLERFEHRSIGQYRGRSGP IVLPDDIGDAVTAVLGLDSRPQARPHFRFRPPFKPARGAAAVTFTPIQLASLYDFPAGDGAGQCIAIIELGGGYRAADIQQYFRGLGITTPPKLVDVNVGTGRNAPTGEP(N / S / K / G)GPDGEVALDIEIAGAIAPAAKIAVYFAP(N / D )(S / T)(D / A / T / N)AGFIQAVNAAVTDKTNQPSVISISW(G / S)GPEAIWQAQSAQAFNRVLQAAAAQGITVCAASGD(S / N)GS(G / T / A)(D / N / G)GL(Q / D)DGADHV(D / S / H)FPASSPYVLGCGGTQLDALPGQGIRSEVTWN DEASGGGAGGGGVSALFDLPAWQQGLKVARADGTTTPLAKRGVPDVAGDASPQTGYEVSVAGTPAVMGGTSAVAPLWAALIARINAANGASAGWINPVLYKHPGALRDITKGSNGTYAAASGWDACTGLGSPNGAQLATILARKPSS(SEQ ID NO:95),

[0145] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 116 is V or D; (ii) AA residue 255 is S, K, or G; (iii) AA residue 284 is D; (iv) AA residue 285 is T; (v) AA residue 286 is A, T, or N; (vi) AA residue 312 is S; (vii) AA residue 347 is N; (viii) AA residue 350 is T or A; (ix) AA residue 351 is N or G; (x) AA residue 354 is D; and (xi) AA residue 361 is S or H;

[0146] Homolog 4

[0147] MANHPLNGSERECLKDAQPIGKADPNERLEVTMLVRRRSHDAFEKHISALAAQGASAKHIDHDEFTKHFGADSADLAAVHAFAQKHGLSVVESHEARRAVVLSGT(V / D)AQFDAAFGVSLQQYEHDGGTYRGRTGPIHLPDELNG VVDAVMGLDNRPQARPSFRTRAQGNVRWTARAAGASTFTPVQLASLYDFPQGDGQNQCIGIIELGGYRPADLKTYFASLNMKAPSVTAVSVDHGRNHPTGDP(N / S / K / G)GPDGEVMLDIEVAGAVAPGAKIVVYFAP(N / D)(T / S)(D / A / T / N)AGFIDAIGTAIHDTKNKPSVISISW(G / S)GPESAWTQQAMNAFDQAFQSAAALGVTICAASGD(N / S)GS(G / T / A)(D / N / G)GV(G / Q / D)DGADHV(D / S / H)FPASSPYALGCGGTSLQASGNGIASETV WNDGANGGATGGGVSSFFALPAWQEGLRVTRAGGAHSPLAMRGVPDVAGNADPVTGYEVRVDGHDMVIGGTSAVAPLWAGLIARINAIKGAPVGYINPHLYKDPLALVDITKGNNDDFHATAGWDACTGLGRPDGKKVKDAVS(SEQ ID NO:75);

[0148] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is D; (ii) AA residue 246 is S, K, or G; (iii) AA residue 275 is D; (iv) AA residue 276 is S; (v) AA residue 277 is A, T, or N; (vi) AA residue 303 is S; (vii) AA residue 338 is S; (viii) AA residue 341 is T or A; (ix) AA residue 342 is N or G; (x) AA residue 345 is Q or D; and (xi) AA residue 352 is S or H;

[0149] Homolog 5

[0150] MNHDHSPTGGELSNWVRVPGSERAAVQGSRKVGPADPNEQMSVTVVVRRPAADTAVTSMIEKVGAQPLSERRHLTREEFASTHGANPADLSKVEKFAHEHNLQVKEVNAAAGTMVLSGT(V / D)TSFSKAFGVELSTYEHPDFTYRGRIGHVHI PDYLADTIQSVLGLDNRPQASPRFRVLKEEGGVTTAHAGRTSYTPLEVAALYNFPSIHCKDQCIGILELGGGYRPADLQTYFNGLGIPQPNITDVSVGGAANRPTGDP(N / S / K / G)GPDGEVVLDIEVAAAVTPGAKIAVYFAD(N / D)(S / T )(D / A / T / N)DGFLNAITTAIHDTRNKPSVISISW(G / S)KAEIGWTPQAINAMNQAFRDAAALGVTICCASGD(D / S / N)GS(T / A)(D / / N / G)RV(Q / D)DGRYHV(D / S / H)FPASSPYVLACGGTRLESSGSTITQEVVWNEGALGGGA TGGGVSDVFDRPNWQANANVPTSANPERRIGRGVPDWAGNADPATGYQILVDGTRAVIGGTSAVAPLFAGLIAIINQKLGHSVGFINPILYNLSAQHNVFHDITSGNNDMSGQNGPYEAQPGWDACTGLGSPDGTKLMNAISEAHRLVSVG(SEQ ID NO:76);

[0151] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 120 is D; (ii) AA residue 259 is S, K, or G; (iii) AA residue 288 is D; (iv) AA residue 289 is T; (v) AA residue 290 is A, T, or N; (vi) AA residue 316 is S; (vii) AA residue 351 is S or N; (viii) AA residue 354 is A; (ix) AA residue 355 is N or G; (x) AA residue 358 is D; and (xi) AA residue 365 is S or H;

[0152] Homolog #6 mutant

[0153] MAPEERRTLPGSAMPRPAGAQVLGQIPDDERVEVTVVLQPRAPLPEPGPTPMSRAELADLRSPPEGALEAIARYVAGQGLEVIAADAPRRRIVLAGSAARIAALFGISFVRLQLEGRRYRTYEGEISLPAELAPLVVAVLGLDTRPFARSHRRPAVAPNAPTTAPTVARAYDFPTAYDGRGTTIGFIELGGGFQESDLVRYCEGLGLSTPQVSVVGVDGARNAPTGDPNGPDAEVMLDLEVATGVANGADLVLYMAANTDAAFYSAIATALRDATHAPVAISISWSAPEESYPATTIAAFESVLEEAVHVGVTVLVAAGDQGSTGGVDDGRAHVHYPAASPYVLACGGTRLDLDGTTIVAETVWNDLPNGGATGGGISALFPVPSWQAGIAMPPSANPGAGPGRGVPDVAGNADPDTGYRIVVDGVATVVGGTSAVAPLWAGLVARCHQAGARGGFWNPLLYAARGSSAFHEITVGSNGAYDAGPIWNACCGLGSPNGTAILQTLRA(SEQ ID NO:78);

[0154] Homolog 9

[0155] MTKQPVSGSSDKIHPDDAKCIGDCDPSEQIEVIVMLRRKDEAGFRQMMSRIDAGEAPGQAVSREEFDRRFTASDEDIDKVKAFAKQYGLSVERAETETRSVVLKGT(I / V / D)EQFQKAFDVKLERFQHHNIGEYRGRTGPVNVPDEMH DAVTAVLGLDSKPQARPHFRFRPPFKPLRGAAPASFSPVDLAKLYDFPDGDGAGQCIAIIELGGGYRDSDLSAYFSKLGVKAPTVVPVGVDGGKNAPTGNP(N / S / K / G)GPDGEVTLDIEIAGAIAPGARIAVYFAP(N / D)(S / T)(D / A / T / N)AGFVDAVNRALHDAANKPSVISISW(G / S)GPESNWSPQSMSAFNDVLQSAAALGVTVCAASGD(G / S / N)GS(A / T)(D / N / G)GV(G / Q / D)DGADHV(D / S / H)FPASSPYVLGCGGTSLAASGAGIAKEVVWNDGDQG GAGGGGVSGTFALPVWQKGLSVTRNGKHIALAKRGVPDVAGDASPQTGYEVLIDGEDTVVGGTSAVAPLWAALIARINAIDASPAGFVNPKLYKAKTAFRDITEGNNGSFSAAAGWDACTGMGSPDGGKIAAALKPAKPSQSAGQQ(SEQ IDNO:79),

[0156] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 107 is V or D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is T; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is S or N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H;

[0157] Homolog 10

[0158] MGRLQGSYRPSLGTPVGPVPDDQPIDVTVVLRPTAADDFRADPDDVAAVRAFAGRAGLDVAEVDEPARTVRLRGP(A / V / D)AAARTAFDTPLALYDSGGRAIRGREGDLGLPDELDDRVVAVLGLDERPAARPRFQPAASARQGLTALQVAR AYDFPAATGEGQTIAIIELGGGFGQADLDTYFGGLDLPTPAVSAVGVQGAANVPGGDP( / S / K / G)DGADGEVLLDIEVAGAVAPGAAQVVYFAP(N / D)(T / S)(D / A / T / N)AGFLAAINAAAAATPRPAAISISWG(G / S)PESSWTAQAM RAYDQAFAAARAAGITVLAAAGD(A / S / N)GA(D / T / A)(D / S / N / G)(A / Q / D)TDRLVA(D / S / H)FPAGSPNVIACGGTKLTLDAAGARASEVVWNEAADSATGGGYSATFTRPAWQPAAVGRYRGLPDISGNADPQTGYRVVVDGQPTV VGGTSAVAPLLAGLVARLAQLTGAPVADLAAVAYANPAAFTDITAGDNQGYPARSGWDPASGLGSPVGTKLLTAVGGPTPPPTTPPPTTPPPTTPPPTIPPPTTPPTQTVDAADRALWSAVATWAGGTHTGANARAAKAVRAWAQAKSLA(SEQ ID NO:80),

[0159] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 76 is V or D; (ii) AA residue 206 is S, K, or G; (iii) AA residue 235 is D; (iv) AA residue 236 is S; (v) AA residue 237 is A, T, or N; (vi) AA residue 262 is S; (vii) AA residue 297 is S or N; (viii) AA residue 300 is T or A; (ix) AA residue 301 is N or G; (x) AA residue 302 is Q or D; and (xi) AA residue 309 is S or H;

[0160] Homolog 12

[0161] MTQPRYTPLPGSEREAPLLAARSNATAARASRAQTASATVVLRRRSELPEALVLDQQFISSDELAARYGADPVDIEKVRSVLERFKVSVVEVDAASRRVKVEGA(V / D)ADIERAFNIALHSASGTDPHSGRGFEYRYRTGVLSVP AELGGIVTAVLGLDNRRQAETRLRVVPAAALGSSYTPVQLGEIYNFPQDATGAGQRIAIIELGGGYTPAGLRRYFASLGVVPPKVAAVSVDGAQNAPGPDP(G / S / K / G)ADGEVQLDVEVAGALAPGAHVLVYFAP(N / D)(T / S) (D / A / T / N)QGFLDAVSQAAHATPPPTAISISW(G / S)ASEDSWTASARDALNQALRDAAALGVTVTAAAGD(S / N)GS(S / T / A)(D / N / G)GV(P / Q / D)DRRAHV(D / S / H)FPASSPYVLATGGTSLRADPATGVVQSETVWN DSQGSTGGGVSDVFPRPAWQAHVDVPHAGRGVPDVSAVADPATGYQVLVDNQPAVIGGTSAVAPLWAALVARLAESLGRPLGLLQPLVYPRTPGSTAYPGFRDITIGNNGAYKAGKGWDAATGLGVPDGTELLAHLRGLNGSE(SEQ ID NO:81),

[0162] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 105 is D; (ii) AA residue 244 is S or K; (iii) AA residue 272 is D; (iv) AA residue 273 is S; (v) AA residue 274 is A, T, or N; (vi) AA residue 299 is S; (vii) AA residue 334 is N; (viii) AA residue 337 is T or A; (ix) AA residue 338 is N or G; (x) AA residue 341 is Q or D; and (xi) AA residue 348 is S or H;

[0163] Homolog 13

[0164] MARHLHAGSEPKVITESKCIGACDPAERIHVTVMLRREGEQALDALVDKLASGDPAAKPVSREDFAKRFGARADDIQHTEAFAKRHQLTVERVDPVQSVVELAGT(I / V / D)AQFENAFGVKLEKYEHHAIGSFRARTGAIALPDELHDAV TAVLGLDTRPQAHPHFRFRPPFQPARSGAGTSYTPLQLASIYNFPEGDGAGQCIALVELGGGYRAADIRQYFEQLGVKPPKLVDVSVNGGRNAPTDDP(N / S / K / G)GPDGEVALDIEVAGAIAPGATIAVYFAG(N / D)(S / T)(D / A / T / N)AGFIQSVNQAIHDSTNRPSVVSISW(G / S)GPEASWTQQSITAFNNVLKTAASLGVTVCAASGD(S / / N)GS(S / T / A)(D / N / G)GL(Q / D)DGSNHV(D / S / H)FPASSPYVLACGGTTLDAQAGQGIRREVVWNDEAASGGAGGG GVSAVFPAPSYQKGLSAKATGGGSTPLSQRGVPDVAGDASPTTGYIISIAGTTAVLGGTSAVAPLWAALIARINANGKSPVGWANPKLYAQPGAFHDITQGNNGAFAASEGWDACTGLGSPDGAKVAAALQGASGGSQQGRATGA(SEQID NO:82);

[0165] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is V or D; (ii) AA residue 244 is S, K, or G; (iii) AA residue 273 is D; (iv) AA residue 274 is T; (v) AA residue 275 is A, T, or N; (vi) AA residue 301 is S; (vii) AA residue 336 is N; (viii) AA residue 339 is T or A; (ix) AA residue 340 is N or G; (x) AA residue 343 is D; and (viii) AA residue 350 is S or H;

[0166] Homolog 14

[0167] MTKHPLPGSERVLAPGSKVVAQCDPSETIEVVVVLRRKNEQQFAQMMKTIEAGAAGARPLTREELEQRFGALPEDIAKLKAFAAQHGLSVVREDASARTVVLSGR(I / V / D)EQFQQAFDVQLQHYEHQSMGRFRGRTGAISVPDE LHGVVTAVLGLDDRPQARPHFRIRPPFQPARAQSASSFTPLQLASLYRFPQGDGSGQCIGIVELGGGYRTADLDSYFSSLGVGSPKVVAVGVDQSGNQPTGDP(N / S / K / G)GPDGEVTLDIEIAGALAPAATIAVYFTT(N / D)( S / T)(D / A / T / N)AGFIDAVSQAVHDRTNQPSVISISW(G / S)APESMWTAQSMKALNDVLQSAAAIGVTVCAASGD(S / N)GS(S / T / A)(D / N / G)GV(G / Q / D)DGRDHV(D / S / H)FPASSPYVLACGGTSLQGSGRTVAHE VVWNDGSNGGATGGGVSGAFPVPAWQEGLSTSAAQGGQRALTGRGVPDVAGDASPLTGYDVIVDGNNTVIGGTSAVAPLWAALIARINGAKGAPVGFVNPKLYKASACNDITQGNNGSYAATTGWDACTGLGSPDGVKVAAAL(SEQ ID NO:83),

[0168] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 107 is V or D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is T; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H;

[0169] Homolog 15

[0170] MSPIASRRSALPLSERPAPENARALAAVEPDRTTMTVSVLVRRKKPLVLADLEGKKLTHREFERRYGASEKDFATIAKFAAGHGLAVDHHASSLARRTVVLRGT(A / V / D)RQMQQAFGVTLHDYEDSETQQRYHSFTGAITVPA AHARIIESVLGLDARPIAKPHFRVRKRSAAATGAVSFNPPQVASLYSFPTGVDGSGETIGILELGGGYETSDIQQYFSGLGIQPPTVVAVSVDGAVNAPGNP(N / S / K / G)GADGEVALDIQVAGSIAPGAKLAVYFAP(N / D) (T / S)(E / D / A / T / N)QGFVDAITTAVHDTANKPSVLSISW(G / S)GPESSWPQAAAQSLNNACESAAALGVTITVASGD(N / S)GS(T / A)(D / N / G)GV(Q / D)DGQNHV(D / S / H)FPASSPYVLACGGTYLAAVNNGVPQ ESVWDDLASGGGATGGGVSALFPLPAWQTGANVPGGSMRGVPDVAGDASPESGYNVLVDGQPQVVGGTSAVAPLWAALIALVNQQKGEAAGFVNAALYQNPSAFHDITQGSNGAYAAAPGWDPCTGLGSPMGTAIAKILA(SEQ ID NO:84),

[0171] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 104 is V or D; (ii) AA residue 241 is S, K, or G; (iii) AA residue 270 is D; (iv) AA residue 271 is S; (v) AA residue 272 is D, A, T, or N; (vi) AA residue 398 is S; (vii) AA residue 33 is S; (viii) AA residue 336 is A; (ix) AA residue 337 is N or G; (x) AA residue 340 is D; and (xi) AA residue 347 is S or H;

[0172] Homolog 16

[0173] MSAFDQLVPLPGSEKTVPDAAPSQTLDPNEVLTVTIRIRRKRTLASLVSTTAPVTEVVSRSEYASRFGADPAIVKQVEAFASAYDLSLVEQSLARRSVLLRGT(V / D)AQMEQAFGVSLANYQLADGTVFRGRTGVVNVPSELVEHIEG VFGLDNRPQARAHFQVYKPEKGTKVAPRAGGISYTPPQLARLYNFPTGVTGKGQCIAIIELGGGFRTADIKTYFGGLGLKPPTVVAVSVDGGHNAPSTA(D / S / K / G)SADGEVMLDIDVAGGVAPGAKIVVYFAP(N / D)(T / S)(D / A / T / N)QGFLDAITTAMHDTKNKPSVISISW(G / S)AAESNWTPQALTSFNQAFQAAAALGITVCAAAGD(T / S / N)GS(D / T / A)(D / N / G)SV(G / Q / D)DGKAHV(D / S / H)FPASSPFVLACGGTKLTATDNVIASEVVWHESKT SATGGGVSDVFDLPDYQQKSHVPPSVNDKTRIGRGVPDVAAVADPVTGYAVRVDGSNLVFGGTSAVAPLMAGLIALINQQRGKAVGFIHPLIYANPSAFRDITQGNNTTTTGNKGYAATTGWDACTGLGVADGKKLASVLTATPVA(SEQ IDNO:85),

[0174] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 104 is D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is S; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is S or N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H;

[0175] Homolog 17

[0176] MAATPRFASQPRVTLPGSQKHPLTTDTEVPPPPAPVKAAATKLSATPFTVTVIVKRKNPLNLKQVLKPAGRLTHAAFAKAHGPSPDGVKLVKAFAKEFGLTVAPAPGQGRRALYLTGT(A / V / D)AAMQTAFGVTFATKIMEGTKYRVREGDICLPKE LIGVDAVLGLDNRPQAKPHFRHHKPAATSVSYTPVQVGQLYGFPSGAKATGQTIGLIELGGGFRAADITAYFKTLGQTAPKVTAVLVDKAKNTPTTS(S / K / G)SADGEVMLDIEVAAAVAPGANIAVYFAP(N / D)(T / S)(D / A / T / N)QGFIDA ISQAVHDTVNKPSVISISW(G / S)GPESTWTAQSLAALDAACQSAAALGITITVAAGD(D / S / N)GS(T / A)(D / N / G)GV(K / Q / D)GTVNHV(D / S / H)FPASSPHVLGCGGTKLLGSGTTITSEVVWNELTANEGATGGGVSNVFPLPTWQAKSN VPKPTVAAGGRGVPDVSGNADPSTGYTVRVDGSTFPIGGTSAVAPLWAGLIALCNAQNKTTAGFINPALYAAAAAKSFRDITSGNNGGFKAGPGWDACTGLGSPIGTAIAKTLAPATKSTSKTAVKNAPEIRFRPHKKAPTKTAAKTPALRRLK(SEQ ID NO:86),

[0177] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 118 is V or D; (ii) AA residue 250 is K or G; (iii) AA residue 279 is D; (iv) AA residue 280 is S; (v) AA residue 281 is A, T, or N; (vi) AA residue 307 is S; (vii) AA residue 342 is S or N; (viii) AA residue 345 is A; (ix) AA residue 346 is N or G; (x) AA residue 349 is Q or D; and (xi) AA residue 356 is S or H;

[0178] Homolog 19

[0179] MPTSSRFASQSRVPLPGSERKPFVPAGAPKAAKTPKVSTAVKTVPATGRIRVSLIVPPKQPLDTKRLGKLDARLSRAQFAARHGADPASVRLVKAFAKEFGLTVEPITQPGRCTVQLSGT(C / V / D)AAMRKAFAISLVEHTTEQGKFRLRE GEISLPAELEGHVLAVLGLDNRPQAKPHFRIAKPRATNVSYTPVQVAQMYGFPAGATATGQTIGIIELGGGYRAADLTAYFKTLGLPAPTVTAVPIDGGKNTPGNA(N / S / K / G)GADGEVMLDIEVCAAVAQGAKIAVYFTT(N / D)(T / S) (D / A / T / N)QGFIDAITTAVHDSTNKPSVISISW(G / / S)GPESSWTEQSMTALDAACQAAAAVGVTITVAAGD(N / S)GS(S / T / S)(D / N / G)GA(S / Q / D)GDNV(D / S / H)FPASSPHVLACGGTKLVGSGSTITSEVVWDETSNDEG ATGGGVSTVFALPTWQKNANVPSPTTSAGGRGVPDVSGDADPSTGYTIRVDSETTVIGGTSAVAPLWAGLIALANAQNKVAAGFVNPALYAAGAKKAFRDITQGNNGSFSAGPGWDACTGLGSPVGLVIQAVAPKSTTTKKAKKGKTK(SEQ ID NO:87),

[0180] wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 121 is V or D; (ii) AA residue 253 is S, K, or G; (iii) AA residue 282 is D; (iv) AA residue 283 is S; (v) AA residue 284 is A, T, or N; (vi) AA residue 310 is S; (vii) AA residue 345 is S; (viii) AA residue 348 is T or A; (ix) AA residue 349 is N or G; (x) AA residue 352 is Q or D; and (xi) AA residue 357 is S or H; and

[0181] Homolog 26

[0182] MHSYLKQQSHMQSYLEQENHMRSYLEMRKKPYFDDLANIRPGGLTPAQVCQAYQFAKVQPVRPVKLGIVSLAGQYLSSDMSKAFTGYGLPTPVVSTAGSQVLGDLWSNVE( N / S / K / G)MMDIEIAGAAWAYATGTAATLLMQFEP(N / D)(N / T / S)(E / D / A / T / N)TGIPNAINALVAAGCEVISISW(G / S)APANLQTMEAITARKEACKQAAVQNV HVFAASGD(E / S / N)SL(N / T / A)(D / N / G)(G / Q / D)TNSRTP(D / S / H)DPCCDPNVWGVGGTRLVLQADGSIAQESAWGDGNAADKGGGGGFDSREPLPDYQVGVVHS EHRGSPDSSANADPGTGYAIVANGQWLIGGGTSASAPLTAGYVAAILSTLPGPISQSVLQRKLYTAHKTAFRDILLGSNGAPARPGWEEATGLGSINGPGLAAALQS(SEQ IDNO:88),

[0183] wherein one, two, three, four, five, six, seven, eight, nine, or all ten of the following are true: (i) AA residue 111 is S, K, or G; (ii) AA residue 139 is D; (iii) AA residue 140 is T or S; (iv) AA residue 141 is D, A, T, or N; (v) AA residue 164 is S; (vi) AA residue 199 is S or N; (vii) AA residue 202 is T or A; (viii) AA residue 203 is N or G; (ix) AA residue 204 is Q or D; and (x) AA residue 211 is S or H.

[0184] The polypeptide may be a processed version of each listed polypeptide; the polypeptide claimed in the present application includes any such processed version of each listed polypeptide. The processed version of the polypeptide is as defined above.

[0185] In one embodiment, the isolated polypeptide comprises an amino acid sequence selected from homolog 4, homolog 6 mutant, homolog 13 and 26 or a processed version thereof. In another embodiment, the isolated polypeptide comprises an amino acid sequence selected from the full-length sequence of homolog 4 mutant:

[0186] (SEQ ID NO:89), or a processed version of that sequence.

[0187] The polypeptides disclosed in the present application have been identified as having similar, improved or complementary activities to polypeptides related to Kumamolisin in gluten, a hydroxyproline (P) and glutamine (Q) rich component known as "gliadin", which is believed to be the culprit for most of the immune response in most patients with sprue. Many other Kumamolisin homologs tested by the inventors have little or no such gliadin hydrolytic activity. Therefore, the polypeptides can be used to treat sprue. The polypeptides of this aspect of the invention are capable of degrading gliadin at various pH values. This degradation occurs under the conditions disclosed in the following examples.

[0188] As used throughout this application, the term "polypeptide" is understood in its broadest sense to refer to a sequence of subunit amino acids, whether naturally occurring or synthetically derived. The polypeptides of the present invention include L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D-amino acids and L-amino acids. The polypeptides described herein can be chemically synthesized or recombinantly expressed. The polypeptides can be linked to other compounds to promote their extended half-life in vivo, such as by pegylation, HESylation, PASylation or glycosylation. Such linkage can be covalent or non-covalent as understood by those skilled in the art. The polypeptides can be linked to any other suitable linker, including but not limited to any linker that can be used for purification or detection (such as FLAG or His tag).

[0189] In yet another aspect, the present invention provides a composition comprising:

[0190] (a) one or more polypeptides comprising an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 74-78, 80-88, 95, 97-99 and 102-111 or processed versions thereof; and

[0191] (b) one or more other polypeptides comprising an amino acid sequence selected from the group consisting of:

[0192] (A) an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID No: 35, wherein:

[0193] (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and

[0194] (ii) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp;

[0195] (B) an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID No: 1, wherein:

[0196] (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and

[0197] (ii) Residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp.

[0198] The one or more other polypeptides may be any of the polypeptides described above. For example, the one or more other polypeptides may include an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to any one of SEQ ID NOS: 2-33 or 36-67, or as an alternative to any one of 2-32 or 36-66, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to any one of SEQ ID NOS: 2-33 or 36-67, or as an alternative to any one of 2-32 or 36-66. In another embodiment, the one or more other polypeptides include KumaMax TM (SEQ ID NO: 90) or a processed version of the sequence, or by KumaMax TM (SEQ ID NO:90) or a processed version of that sequence.

[0199] In one embodiment, the composition includes homolog 4 (SEQ ID NO: 75 or 98) or full-length mutant homolog 4 (SEQ ID NO: 89) or a processed version thereof, and one or more other polypeptides disclosed in the present application, including but not limited to KumaMax TM (SEQ ID NO: 90) or a processed version thereof. In another embodiment, the composition comprises SEQ ID NO: 74, SEQ ID NO: 77 and / or SEQ ID NO: 78 (homolog 1, homolog 6 and / or homolog 6 mutant) or a processed version thereof, and one or more other polypeptides disclosed in the present application, including but not limited to KumaMax TM (SEQ ID NO: 90) or a processed version thereof. In a further embodiment, the method comprises administering SEQ ID NO: 88 and / or 111 (homolog 26) or a processed version thereof, and one or more other polypeptides disclosed herein, including but not limited to KumaMax TM (SEQ ID NO:90) or a processed version thereof.

[0200] On the other hand, the present invention provides isolated nucleic acids encoding the polypeptides of any aspect or embodiment of the present invention. The isolated nucleic acid sequence may include RNA or DNA. "Isolated nucleic acids" as used in this application refer to those that have been removed from their normal surrounding nucleic acid sequences in a genomic or cDNA sequence. Such isolated nucleic acid sequences may include other sequences for promoting expression and / or purification of encoded proteins, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings of this application, nucleic acid sequences that can be used to encode the polypeptides of the present invention are obvious to those skilled in the art.

[0201] In a further aspect, the present invention provides nucleic acid expression vectors, including the isolated nucleic acid of any embodiment of the present invention operatively linked to a suitable control sequence. "Recombinant expression vectors" include vectors that operatively link a nucleic acid coding region or gene to any control sequence that can affect the expression of a gene product. "Control sequences" operatively linked to the nucleic acid sequence of the present invention are nucleic acid sequences that can affect the expression of nucleic acid molecules. The control sequences do not need to be linked to the nucleic acid sequence, as long as they can guide the expression of the nucleic acid sequence. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence is still considered to be "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including, but not limited to, plasmid vectors and viral expression vectors. The control sequences used to drive the nucleic acid sequences in the disclosed mammalian systems can be constitutive sequences (driven by any of a variety of promoters (including but not limited to CMV, SV40, RSV, actin, EF)) or inducible sequences (driven by any of several inducible promoters (including but not limited to tetracycline-sensitive, ecdysone-sensitive, hormone-sensitive)). The construction of expression vectors for transfecting prokaryotic cells is also well known in the art and can be achieved using standard techniques. (See, for example, Sambrook, Fritsch and Maniatis: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ), and the Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector must be able to replicate in the host organism either as an episome or by integrating the gene into the host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, the invention is intended to include other expression vectors that serve equivalent functions (eg, viral vectors).

[0202] In another aspect, the present invention provides a recombinant host cell comprising a nucleic acid expression vector of the present invention. The host cell may be prokaryotic or eukaryotic. The cell may be capable of transient or stable transfection or transduction. Any technique known in the art may be used to achieve such transfection and transduction of the expression vector into prokaryotic and eukaryotic cells, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation or liposome-mediated transfection, DEAE dextran-mediated transfection, polycation-mediated transfection or virus-mediated transfection. (See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2 nd Ed. (RI Freshney. 1987. Liss, Inc. New York, NY). A method for producing a polypeptide of the invention is another aspect of the invention. The method comprises the steps of: (a) culturing a host according to this aspect of the invention under conditions conducive to expression of the polypeptide; and (b) optionally, recovering the expressed polypeptide. The expressed polypeptide may be recovered from a cell-free extract, a cell pellet, or from a culture medium. Methods for purifying recombinantly expressed polypeptides are also well known to those skilled in the art.

[0203] In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide, a nucleic acid, a nucleic acid expression vector, a recombinant host cell, or a composition in any aspect or embodiment of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be used, for example, in the method described below of the present invention. In addition to the polypeptide, nucleic acid, etc. of the present invention, the pharmaceutical composition may include (a) a lyoprotectant; (b) a surfactant; (c) a filler; (d) a tension regulator; (e) a stabilizer; (f) a preservative and / or (g) a buffer.

[0204] In certain embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a freeze-drying protective agent, such as sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative, such as benzalkonium chloride, phenylethylammonium, chlorhexidine, phenol, metacresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid and their various mixtures. In other embodiments, the pharmaceutical composition includes a filler, such as glycine. In other embodiments, the pharmaceutical composition includes a surfactant, for example, polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or a combination thereof. The pharmaceutical composition may also include a tension modifier, for example, a compound that makes the formulation substantially isotonic or isotonic with human blood. Exemplary tension modifiers include sucrose, sorbitol, glycine, methionine, mannitol, glucose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, for example, which, when combined with the target protein, can substantially prevent or reduce the chemical and / or physical instability of the target protein in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0205] The polypeptide, nucleic acid, etc. of the present invention may be the only active agent in the pharmaceutical composition, or the composition may further include one or more other active agents suitable for the intended use.

[0206] The pharmaceutical composition described in the present application mainly includes a combination of compounds described in the present application and a pharmaceutically acceptable carrier, diluent or excipient. This pharmaceutical composition is substantially free of pharmaceutically unacceptable components, that is, the pharmaceutically unacceptable components contained are lower than the values ​​allowed by the U.S. regulations during the period of submission of the application. In some embodiments in this regard, if the compound can be dissolved in water or suspended in water, the composition optionally also includes other pharmaceutically acceptable carriers, diluents or excipients. In other embodiments, the pharmaceutical composition described in the present application is a solid pharmaceutical composition (e.g., tablets, capsules, etc.).

[0207] These compositions can be prepared by methods known in the pharmaceutical field and can be administered by any suitable route. In a preferred embodiment, the pharmaceutical composition and formulation are designed for oral administration. Conventional pharmaceutical carriers, aqueous bases, powder bases or oily bases, thickeners and similar substances are necessary or suitable.

[0208] The pharmaceutical composition can be in any suitable form, including, but not limited to, tablets, pills, powders, lozenges, capsules, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or in liquid media), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions and sterile packaged powders.

[0209] Examples

[0210] Sprue is an autoimmune disorder that affects approximately 1% of the population (1, 2). The disease is characterized by an inflammatory response to gluten, the major protein in wheat flour, and related proteins in barley and rye (2). Gluten consists of a heterogeneous mixture of the glycoproteins α-gliadins and glutenins (3). During digestion, part of the α-gliadins are degraded by pepsin and intestinal proteases into oligopeptides that are resistant to further proteolysis due to their abnormally high proline and glutamine content. The immunogenic oligopeptides generated by incomplete proteolysis are enriched in the PQ motif (4, 5), which causes inflammation and damage in the intestines of people with sprue. Currently, the only treatment for this disease is to completely eliminate gluten from the diet, which is difficult to achieve due to the ubiquitous presence of this protein in modern food products.

[0211] Oral enzyme therapy (OET), which uses oral proteases to hydrolyze immunogenic peptides before they can cause inflammation, is a treatment approach currently under development for the treatment of gluten intolerance. Several different proteases have been considered for use for this purpose, as they are either specific for cleaving proline residues or glutamine residues. However, these enzymes have often been shown to have characteristics that hinder their use for gluten degradation in OET. Most of these peptides have optimal catalytic activity at neutral pH; however, the pH range in the human stomach is 2-4. Therefore, these enzymes are most active when they reach the pH-neutral small intestine, but this is too late to effectively prevent celiac disease, as this is where gluten-derived pathologies develop. In addition, several of these enzymes have been shown to be unstable at this lower pH in the human stomach, are easily hydrolyzed by digestive proteases, or require extensive renaturation processes during their purification, all of which hinder their use in the clinic.

[0212] An ideal protease for treating gluten intolerance using OET should have the following properties: optimal activity at lower pH, easy purification, stable under conditions in the human stomach, and high specificity for amino acid motifs found in gluten-derived immunogenic oligopeptides. We previously identified a protease with high activity under acidic conditions, Kumamolisin-As (KumaWT) from the acidophilic bacterium Alicyclobacillus sendaiensis, and used computer modeling tools to alter its structure toward the desired oligopeptide specificity. An exemplary computer-designed enzyme (named KumaMax) showed a high activity under acidic conditions compared to wild-type KumaWT. TM ) has a more than 100-fold increase in proteolytic activity and an 800-fold increase in substrate specificity for the targeted PQ motif. TM It was shown to be resistant to common pepsin and has a very high yield in E. coli without the need for renaturation. The above designed protein was evaluated for its catalytic activity against PQLP (SEQ ID No: 68) peptide; the exemplary results are listed in Table 2.

[0213] Table 2:

[0214]

[0215]

[0216] Table 2. Fold changes in hydrolysis activity of the PQ motif of all purified, sequenced mutants compared to wild-type Kumamolysin-As. These are the fold changes of all purified, sequenced and tested mutants compared to wild-type Kumamolysin in a pure protein assay. The assay was performed at pH 4, the final enzyme concentration was 0.0125 mg / mL, and the substrate concentration was 5 μM.

[0217] In this study, the inventors tested a large number of Kumamolisin homologues obtained from various organisms to determine their activity in degrading gliadin. They all have the catalytic triad present in Kumamolisin-As (Ser in the unprocessed form of Kumamolisin-As 467 -Glu 267 -Asp 271 To assess the ability of these homologues to respond to the target gluten, the purified homologues were incubated with the protein with purified peptides representing immunogenic regions distributed across gliadin (the part of gluten that is problematic for celiac disease patients).

[0218] The ability of each homologue to cleave a fluorescent analog of gliadin, a hexapeptide (QPQLPY (SEQ ID NO: 91)) that combines a fluorescent group and a quenching group, was evaluated under conditions that simulate gastric acid in an experiment. The degradation rate can be calculated by measuring the fluorescence signal over time. This activity is compared to the activity of Kumamolisin (expressed as KWT below). Kumamolisin has some activity in cleaving these gliadin substrates. Exemplary results are shown in Figure 1 As can be seen, under these conditions, only a portion of the tested homologues (1, 2, 4, 8 and 9) had comparable or better activity than Kumamolisin.

[0219] In further studies, the pH level was varied and the activity of each homologue was tested at different pH levels. Figure 2 Most of the tested homologues were expressed in Kumamolisin (hereinafter referred to as "WT") and KumaMax TM (denoted by "Max") were shown to be active in the pH range of both. Interestingly, two of the homologues have an extended pH range compared to Kumamolisin. Homologue 1 was optimally active at pH 5, and homologue 6 was shown to be optimally active at a pH level lower than the other homologues and the value of Kumamolisin / KumaMax, which explains why no activity was observed for this homologue in the fluorescence experiments performed at pH = 4. This suggests that homologues 1 and 6 can be used, for example, to expand the pH range of Kumamolisin-related polypeptides in order to more accurately simulate the pH conditions of the stomach.

[0220] We further tested the ability of these homologues to cleave different non-fluorescently labeled peptides associated with celiac disease. The results are listed in Table 3, where + and - represent a visual indication of the ability of the homologues to cleave the indicated peptides. -<+ / -<++ / -<+<++<++++<++++. The numbers represent the % of peptide degraded by the homologues after 80 minutes of incubation (thus the numbers are The smaller , the more effective the homologue); (undetected) means that the peptide was below the detection limit after 80 minutes.

[0221] It can be seen that Kumamolisin and KumaMax TM The activity against γ-gliadin peptide was similarly low, while homologue 4 showed increased activity against α-gliadin peptide, suggesting that homologue 4 alone or in combination with kumamolisin-related peptides could be effective in treating gluten digestion problems.

[0222] Table 3:

[0223]

[0224] Since the sequence identity percentage of these Kumamolisin homologs is low, we made the same mutations in each homolog as we did with Kumamolisin to make KumaMax. We tested the activity of these homologs against two peptides that are products of gluten degradation in the stomach and have a clear association with sprue: a 33mer peptide ((LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:72)) and a 26mer peptide (FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO:73)). In particular, the 33mer peptide was strongly associated with sprue. In addition, the KumaMax of each homolog TM The mutation inspired us to make these 3 active site mutations individually on the Kumamolisin background (note, KumaMax TM The mutant strain containing only these active site mutations is referred to as the K3 mutant strain below. The data are listed in Tables 4 and Figure 3 We found that among the several homologues that were shown to have activity against these two very important peptides, it is interesting that KumaMax TM (SEQ ID NO:90) mutation had enhanced activity against both peptides; in fact, the homolog 4 mutant (SEQ ID NO:89) was the best enzyme overall tested in the assay shown below, including KumaMax TM (SEQ ID NO: 90). This also shows that the K3 mutant can also bind to KumaMax TM (SEQ ID NO: 90) combination, resulting in a stronger therapeutic effect. (NA = no activity).

[0225] Table 4

[0226] Homologs 33mer 26mer KumaMax 0.1％ 26％ K3 mutant 26％ 5％ KumaWT 18％ NA Homolog #1 46％ NA Homolog #1 Max NA NA Homolog #2 31％ NA Homolog #2 Max 29％ NA Homolog #4 38％ NA Homolog #4 Max 10％ 19％ Homolog #5 NA NA Homolog #5 Max NA NA Homolog #9 NA 52％ Homolog #9 Max NA 60％ Homolog #10 NA NA Homolog #10 Max NA NA

[0227] Further studies were performed using other homologues of Kumamolisin. Some of these homologues (homologues 13 and 26) were shown to have high activity against 33mer peptides; see Figure 4 In particular, the wild-type homologue 26 has similarities to KumaMax in the cut-off 33mer peptide. TM Quite super active.

[0228] We then observed the digestion of the 33mer by HPLC after 60 minutes of incubation; degradation of the 33mer at more than one position indicated a clear therapeutic advantage. Wild-type Kumamolysin only degraded the 33mer at one position (WT-like), while KumaMax TM 33mer (Max-like) was degraded at multiple positions. The data are shown in Table 5.

[0229] Table 5

[0230]

[0231]

[0232] The overall statistical result of the data is that several homologs (including homolog 4 (SEQ ID NO: 75) and homolog 4 mutant (SEQ ID NO: 89), 13 and 26) can be used as therapeutic agents to treat sprue. Homolog 26 (SEQ ID NO: 88) is almost identical to KumaMax TM (SEQ ID NO: 90) was equally effective and this was without any design, homolog 4 was shown to be effective in KumaMax with a constructed TM In addition, the pH profiles of these homologues (particularly homologue 6 (including homologue 6 mutants) and homologue 1) indicate that these homologues (used alone or in combination) can expand the pH range of therapeutic efficacy in the human stomach.

[0233] method

[0234] Protein expression and purification

[0235] The gene encoding each target protein contained in the pET29b plasmid was transformed into Escherichia coli BL21 (DE3) cells. A single colony was picked and inoculated into Terrific Broth containing 50 μg / μL kanamycin (TB+Kan). TMIncubate overnight at 37°C. 500uL of overnight culture is added to 500mL autoinduction medium (5g tryptic peptone, 2.5g yeast extract, 465mL ddH2O), shaken vigorously at 37°C for 4 hours, and then autoinduction components (500uL MgSO4, 500uL 1000x trace metals, 25mL 20x NPS, 10mL 20x 5052, 500uL 50mg / mL Kan) are added. Then the culture is shaken at 18°C ​​for 30 hours before centrifugation. The cell mass is resuspended in 10mL 1x PBS, then 5mL lysis buffer (50mM HEPES, 500mM NaCl, 1mM bME, 2mg / mL lysozyme, 0.2mg / mL DNase, ddH2O) is ultrasonically lysed and centrifuged. The protein is then purified through a 1mL TALON cobalt affinity column. KumaMax, KumaWT and SC Pep were washed three times with 20 mL of wash buffer (10 mM imidazole, 50 mM HEPES, 500 mM NaCl, 1 mM bME, ddH2O) and then diluted in 15 mL of dilution buffer (200 mM imidazole, 50 mM HEPES, 500 mM NaCl, 1 mM bME). EP-B2 needs to be refolded on the column, so after lysis, the cell pellet is resuspended in 10 mL of EP-B2 buffer, where the difference between EP-B2 buffer and wash buffer is only that it is diluted in guanidine hydrochloride instead of ddH2O to achieve denaturation of EP-B2 inclusion bodies. The resuspension was precipitated, and the supernatant (containing denatured EP-B2) was loaded onto the column and filtered using a 0.8 μm filter. EP-B2 was washed once with 20 mL of EP-B2 buffer and then washed twice with 20 mL of wash buffer to refold the protein on the column. The protein was diluted with 15 ml of dilution buffer. All proteins were concentrated from 15 mL to ~500 uL and then dialyzed once in 1 L of dialysis buffer (20% glycerol, 50 mM HEPES, 500 mM NaCl, 1 mM bME). The protein concentration was calculated spectrophotometrically, and the extinction coefficient for KumaWT and all KumaWT variants was 53,985 M -1 cm -1 For SC Pep, the extinction coefficient is 152,290M -1 cm -1 For EP-B2, the extinction coefficient is 58,245M -1 cm -1 .

[0236] Purified enzyme analysis

[0237] The Kumamolisin-As variants that showed the best activity on the FQ substrate in the activity screening were arranged in order and purified on a small scale. 500uL of TB+Kan overnight culture was added to 50mL TB+Kan and grown at 37°C until an optical density of 0.5-0.8 was reached. IPTG was added to 0.5mM and the culture was expressed at 22°C for 16-24 hours. The cells were centrifuged, resuspended in 500uL of rinsing buffer (1x PBS, 5mM imidazole, ddH2O), transferred to a 2mL centrifuge tube, and lysed in 1mL lysis buffer (1x PBS, 5mM imidazole, 2x Bug Buster TM , 2mg / mL lysozyme, 0.2mg / mL DNase, ddH2O). After centrifugation, transfer the supernatant to a fresh-keeping tube. Place a column with 200uL TALON cobalt resin in a centrifuge tube, pour the supernatant on the column and shake for 20 minutes, then centrifuge and discard the flow-through. Wash the protein three times with 500uL rinse buffer, and discard the flow-through between washes. Dilute the enzyme with 200uL dilution buffer (1x PBS, 200mM imidazole, dd H2O) and calculate the concentration by spectrophotometry. The extinction coefficient is 53,985M -1 cm -1 .

[0238] In this assay, Kumamolisin-As mutants were incubated for 15 minutes in 100 mM sodium acetate buffer at pH 4. The enzyme was added to 5 μM substrate so that the final protein concentration was 0.0125 mg / mL. Fluorescence intensity was measured every 30 seconds for 1 hour.

[0239] Kinetic characterization

[0240] The enzyme variability in gluten degradation was measured by hydrolyzing the fluorescent quenched α-gliadin hexapeptide analog QXL520-PQPQLP-K(5-FAM)-NH2(FQ) (SEQ ID NO:69) as substrate. Each enzyme was incubated in 100 mM sodium acetate buffer, pH 4, at room temperature for 15 minutes. After 15 minutes, 50 uL of fluorescent substrate was added, with a final concentration distribution of 100, 50, 25, 12.5, 6.25 and 0 μM peptide, maintaining 0.05 μM KumaMax for all changes in substrate concentration. TM , 0.5 μM KumaWT, 0.5 μM SC Pep and 0.5 μM EP-B2. The plate was immediately placed on a spectrophotometer and read for 1 hour using an excitation wavelength of 455 nm and reading at an emission wavelength of 485 nm.

[0241] The specificity of each enzyme for different dipeptide motifs was also tested using various chromogenic substrates that release p-nitroaniline (pNA) upon hydrolysis: [Suc-APQ-pNA], [Suc-AQP-pNA], [Suc-APE-pNA], and [Suc-APR-pNA]. Again, each enzyme was incubated in 100 mM sodium acetate buffer, pH 4, at room temperature for 15 minutes. After 15 minutes, 20 uL of substrate was added to the enzyme incubation to give a final concentration distribution of 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 0 μM, with a final concentration of 0.5 μM for all tested enzymes. The plate was immediately placed on a spectrophotometer and read for 1 hour, simulating the absorbance of the reaction at 385 nm.

[0242] The standard curve for the fluorescent peptide was prepared by mixing different concentrations of substrate and product in a buffer at pH 4. The substrate concentrations were 100, 50, 25, 12.5, 6.25 and 0 μM, and the product concentrations were 20, 5, 1.25, 0.3125, 0.078125, 0 μM.

[0243] The standard curve for the absorbent peptide used product concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625 and 0 μM diluted in pH 4 buffer.

[0244] Protease stability

[0245] Enzyme stability was determined in the presence of digestive proteases, pepsin, and trypsin. KumaWT, KumaMax, and TM , SC Pep and EP-B2. Each enzyme was pre-incubated with 100 mM sodium acetate at pH 3.5 for pepsin digestion analysis and a dialysis buffer at pH 7.5 for trypsin (see "Protein Expression and Purification") digestion analysis. Each enzyme for each experiment was incubated in each buffer at 37°C for 15 minutes at a concentration of 0.2 mg / mL.

[0246] After pre-incubation in the appropriate buffer, 0.1 mg / mL digestion protease was added. The reaction was repeated three times and incubated at 37°C for 30 minutes. SDS was added and boiled for 5 minutes to ensure inactivation of the digestion protease. Quantification of enzyme degradation was achieved using ImageJ and SDS-PAGE gel.

[0247] The autohydrolysis rate of the protein was determined at pH 4 and 7.5 in the absence of pepsin or trypsin. Each enzyme (at a concentration of 0.2 mg / mL) was incubated in 100 mM sodium acetate buffer at pH 4 and dialysis buffer at pH 7.5. Samples were taken at 20 minutes, 40 minutes, and 60 minutes. SDS was added and the samples were boiled for 5 minutes to ensure enzyme denaturation and inhibit further autohydrolysis. Similarly, SDS-PAGE gels and ImageJ were used in combination for quantification of enzyme autohydrolysis.

[0248] LCMS Gliadin Degradation Analysis

[0249] The enzymatic activity of full-length α9-gliadin was determined by high performance liquid chromatography-mass spectrometry. For each enzyme, 7 μL of 1M sodium acetate buffer at pH 4 was added to 28 μL of 5 μM enzyme, and the separate test tubes were incubated side by side at 37°C for 15 minutes. Then, 27 μL of each enzyme mixture was added to each gliadin test tube with 27 μL of dialysis buffer as a control. Incubate again at 37°C, and take out 5 μL samples at 10 minutes, 20 minutes, 30 minutes, 40 minutes and 50 minutes. The samples at each time point were quenched in 95 μL of 80% acetonitrile and 1% formic acid and approximately 33 μM leupeptin. Each sample was analyzed on HPLC to compare the degradation of gliadin by different proteases over time.

[0250] References:

[0251] 1. Armstrong, MJ, Hegade, VS & Robins, G. Advances in coeliacdisease. Curr Opin Gastroenterol 28, 104-12 (2012).

[0252] 2. Sollid, LM Coeliac disease: dissecting a complex inflammatory disorder. Nat Rev Immunol 2, 647-55 (2002).

[0253] 3. Wieser, H. Chemistry of gluten proteins. Food Microbiol 24, 115-9 (2007).

[0254] 4. Shan, L. et al. Structural basis for gluten intolerance in celiacsprue. Science 297, 2275-9 (2002).

[0255] 5.Shan,L.Identification and analysis of multivalent proteolyticallyresistant peptides from gluten:implicatoins for celiac sprue.Journal ofProteome Research (2005).

[0256] 6.Chand,N.&Mibas,A.A.Celiac disease:current concepts in diagnosis andtreatment.J Clin Gastroenterol 40,3-14(2006).

[0257] 7.Shan,L.,Marti,T.,Sollid,L.M.,Gray,G.M.&Khosla,C.Comparativebiochemical analysis of three bacterial prolyl endopeptidases:implicationsfor coeliae sprue.Biochem J 383,311-8(2004).

[0258] 8.Siegel,M.et al.Rational design of combination enzyme therapy forceliac sprue.Chem Biol 13,649-58(2006).

[0259] 9.Stepniak,D.et al.Highly efficient gluten degradation with a newlyidentified prolyl endoprotease:implications for celiac disease.Am J PhystolGastrointest Liver Physiol 291,G621-9(2006).

[0260] 10.Ehren,J.et al.A food-gradeenzyme preparation with modest glutendetoxification properties.PLoS One 4,e6313(2009).

[0261] 11.Bethune,M.T.,Strop,P.,Tang,Y.,Sollid,L.M.&Khosla,C.Heterologousexpression,purification,refolding,and structural-functional characterizationof EP-B2,a self-activating barley cysteine endoprotease.Chem Biol 13,637-47(2006).

[0262] 12.Okubo,A.et al.Processing,catalytic activity and crystal structuresof kumamolisin-As with an engineered active site.FEBS J 273,2563-76(2006).

[0263] 13.Gardner,J.D.,Ciociola,A.A.&Robinson,M.Measurement of meal-stimulated gastric acid secretion by in vivo gastric autotitration.J ApplPhysiol 92,427-34(2002).

[0264] 14.Wlodawer,A.et al.Crystallographic and biochemical investigationsof kumamolisin-As,a serine-carboxyl peptidase with collagenase activity.JBiol Chem279,21500-10(2004).

[0265] 15.Ehren,J.,Govindarajan,S.,Moron,B.,Minshull,J.&Khosla,C.Proteinengineering of improved prolyl endopeptidases for celiac spruetherapy.Protein Eng Des Sel 21,699-707(2008).

[0266] 16.Bethune,M.T.&Khosla,C.Oral enzyme therapy for celiac sprue.MethodsEnzymol 502,241-71(2012).

[0267] 17.Gass,J.,Vora,H.,Bethune,M.T.,Gray,G.M.&Khosla,C.Effect of barleyendoprotease EP-B2 on gluten digestion in the intact rat.J Pharmacol Exp Ther318,1178-86(2006).

[0268] 18.Vora,H.,Mclntire,J.,Kumar,P.,Deshpande,M.&Khosla,C.A scaleablemanufacturing process for pro-EP-B2,a cysteine protease from barley indicatedfor celiac sprue.Biotechnol Bioeng 98,177-85(2007).

Claims

1. A method for treating sprue, comprising administering to a patient suffering from sprue an effective amount of one or more polypeptides for treating sprue, wherein the one or more polypeptides comprise or consist of an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOS: 75, 74, 76-89, 95, 97-98, 102-111 or a processed version thereof.

2. The method of claim 1 , wherein the one or more polypeptides comprise one or more polypeptides comprising an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 75, 74, 77, 78, 82, 88, 98, 105, 111 or a processed version thereof, or one or more polypeptides consisting of an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 75, 74, 77, 78, 82, 88, 98, 105, 111 or a processed version thereof.

3. The method of claim 1 or 2, wherein the one or more polypeptides include a polypeptide comprising the amino acid sequence of SEQ ID NO:

89.

4. The method according to any one of claims 1 to 3, further comprising administering to the subject an amount of one or more other polypeptides, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of the following amino acid sequences: (A) an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID No: 35, wherein: (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and (ii) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp; (B) an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID No: 1, wherein: (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and (ii) Residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp.

5. The method of claim 4, wherein the one or more other polypeptides comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID No: 1 or SEQ ID NO:

35.

6. The method of claim 5, wherein the one or more other polypeptides comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID No: 1 or SEQ ID NO:

35.

7. The method according to claim 6, wherein the one or more other polypeptides comprise the amino acid sequence of SEQ ID No: 1 and / or SEQ ID NO:

35.

8. The method according to any one of claims 4-7, wherein the one or more other polypeptides comprise the amino acid sequence of any one of SEQ ID No: 2-33 or 36-67.

9. The method according to any one of claims 4 to 8, wherein the one or more additional polypeptides comprise the amino acid sequence of SEQ ID NO:

90.

10. The method according to any one of claims 4 to 9, wherein the one or more polypeptides comprise SEQ ID NO: 75, SEQ ID NO: 89 and / or SEQ ID No: 98, or processed versions thereof.

11. The method of any one of claims 4-10, wherein the one or more polypeptides comprise SEQ ID NO: 74, 77 and / or 78, or a processed version thereof.

12. The method of any one of claims 4-10, wherein the one or more polypeptides comprise SEQ ID NO: 88 and / or SEQ ID NO: 111, or a processed version thereof.

13. The method of any one of claims 1-12, wherein the polypeptide and other polypeptides (when present) are administered orally.

14. An isolated polypeptide selected from the group consisting of polypeptides comprising an amino acid sequence selected from the following groups or processed versions thereof: (a) SEQ ID NO:95, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 116 is V or D; (ii) AA residue 255 is S, K, or G; (iii) AA residue 284 is D; (iv) AA residue 285 is T; (v) AA residue 286 is A, T, or N; (vi) AA residue 312 is S; (vii) AA residue 347 is N; (viii) AA residue 350 is T or A; (ix) AA residue 351 is N or G; (x) AA residue 354 is D; and (xi) AA residue 361 is S or H; (b) SEQ ID NO:75, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is D; (ii) AA residue 246 is S, K, or G; (iii) AA residue 275 is D; (iv) AA residue 276 is S; (v) AA residue 277 is A, T, or N; (vi) AA residue 303 is S; (vii) AA residue 338 is S; (viii) AA residue 341 is T or A; (ix) AA residue 342 is N or G; (x) AA residue 345 is Q or D; and (xi) AA residue 352 is S or H; (c) SEQ ID NO:76, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 120 is D; (ii) AA residue 259 is S, K, or G; (iii) AA residue 288 is D; (iv) AA residue 289 is T; (v) AA residue 290 is A, T, or N; (vi) AA residue 316 is S; (vii) AA residue 351 is S or N; (viii) AA residue 354 is A; (ix) AA residue 355 is N or G; (x) AA residue 358 is D; and (xi) AA residue 365 is S or H; (d) SEQ ID NO: 78; (e) SEQ ID NO:79, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 107 is V or D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is T; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is S or N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H; (f) SEQ ID NO:80, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 76 is V or D; (ii) AA residue 206 is S, K, or G; (iii) AA residue 235 is D; (iv) AA residue 236 is S; (v) AA residue 237 is A, T, or N; (vi) AA residue 262 is S; (vii) AA residue 297 is S or N; (viii) AA residue 300 is T or A; (ix) AA residue 301 is N or G; (x) AA residue 302 is Q or D; and (xi) AA residue 309 is S or H; (g) SEQ ID NO:81, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 105 is D; (ii) AA residue 244 is S or K; (iii) AA residue 272 is D; (iv) AA residue 273 is S; (v) AA residue 274 is A, T, or N; (vi) AA residue 299 is S; (vii) AA residue 334 is N; (viii) AA residue 337 is T or A; (ix) AA residue 338 is N or G; (x) AA residue 341 is Q or D; and (xi) AA residue 348 is S or H; (h) SEQ ID NO:82, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is V or D; (ii) AA residue 244 is S, K, or G; (iii) AA residue 273 is D; (iv) AA residue 274 is T; (v) AA residue 275 is A, T, or N; (vi) AA residue 301 is S; (vii) AA residue 336 is N; (viii) AA residue 339 is T or A; (ix) AA residue 340 is N or G; (x) AA residue 343 is D; and (viii) AA residue 350 is S or H; (i) SEQ ID NO:83, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 107 is V or D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is T; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H; (j) SEQ ID NO:84, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 104 is V or D; (ii) AA residue 241 is S, K, or G; (iii) AA residue 270 is D; (iv) AA residue 271 is S; (v) AA residue 272 is D, A, T, or N; (vi) AA residue 398 is S; (vii) AA residue 33 is S; (viii) AA residue 336 is A; (ix) AA residue 337 is N or G; (x) AA residue 340 is D; and (xi) AA residue 347 is S or H; (k) SEQ ID NO:85, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 104 is D; (ii) AA residue 245 is S, K, or G; (iii) AA residue 274 is D; (iv) AA residue 275 is S; (v) AA residue 276 is A, T, or N; (vi) AA residue 302 is S; (vii) AA residue 337 is S or N; (viii) AA residue 340 is T or A; (ix) AA residue 341 is N or G; (x) AA residue 344 is Q or D; and (xi) AA residue 351 is S or H; (l) SEQ ID NO:86, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 118 is V or D; (ii) AA residue 250 is K or G; (iii) AA residue 279 is D; (iv) AA residue 280 is S; (v) AA residue 281 is A, T, or N; (vi) AA residue 307 is S; (vii) AA residue 342 is S or N; (viii) AA residue 345 is A; (ix) AA residue 346 is N or G; (x) AA residue 349 is Q or D; and (xi) AA residue 356 is S or H; (m) SEQ ID NO:87, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 121 is V or D; (ii) AA residue 253 is S, K, or G; (iii) AA residue 282 is D; (iv) AA residue 283 is S; (v) AA residue 284 is A, T, or N; (vi) AA residue 310 is S; (vii) AA residue 345 is S; (viii) AA residue 348 is T or A; (ix) AA residue 349 is N or G; (x) AA residue 352 is Q or D; and (xi) AA residue 357 is S or H; (n) SEQ ID NO:88, wherein one, two, three, four, five, six, seven, eight, nine, or all ten of the following are true: (i) AA residue 111 is S, K, or G; (ii) AA residue 139 is D; (iii) AA residue 140 is T or S; (iv) AA residue 141 is D, A, T, or N; (v) AA residue 164 is S; (vi) AA residue 199 is S or N; (vii) AA residue 202 is T or A; (viii) AA residue 203 is N or G; (ix) AA residue 204 is Q or D; and (x) AA residue 211 is S or H; and (o) SEQ ID NO:

89.

15. The isolated polypeptide according to claim 14, selected from the group consisting of: (i) SEQ ID NO:75, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is D; (ii) AA residue 246 is S, K, or G; (iii) AA residue 275 is D; (iv) AA residue 276 is S; (v) AA residue 277 is A, T, or N; (vi) AA residue 303 is S; (vii) AA residue 338 is S; (viii) AA residue 341 is T or A; (ix) AA residue 342 is N or G; (x) AA residue 345 is Q or D; and (xi) AA residue 352 is S or H; (ii) SEQ ID NO:78; (iii) SEQ ID NO:82, wherein one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following are true: (i) AA residue 106 is V or D; (ii) AA residue 244 is S, K, or G; (iii) AA residue 273 is D; (iv) AA residue 274 is T; (v) AA residue 275 is A, T, or N; (vi) AA residue 301 is S; (vii) AA residue 336 is N; (viii) AA residue 339 is T or A; (ix) AA residue 340 is N or G; (x) AA residue 343 is D; and (viii) AA residue 350 is S or H; (iv) SEQ ID NO:88, wherein one, two, three, four, five, six, seven, eight, nine, or all ten of the following are true: (i) AA residue 111 is S, K, or G; (ii) AA residue 139 is D; (iii) AA residue 140 is T or S; (iv) AA residue 141 is D, A, T, or N; (v) AA residue 164 is S; (vi) AA residue 199 is S or N; (vii) AA residue 202 is T or A; (viii) AA residue 203 is N or G; (ix) AA residue 204 is Q or D; and (x) AA residue 211 is S or H; and (v) SEQ ID NO:

89.

16. The isolated polypeptide according to claim 14 or 15, comprising the amino acid sequence of SEQ ID NO: 89 or a processed version thereof.

17. The isolated polypeptide of claim 14, comprising the amino acid sequence of SEQ ID NO: 78 or a processed version thereof.

18. A nucleic acid encoding the polypeptide of any one of claims 14-17.

19. A nucleic acid expression vector comprising the isolated nucleic acid of claim 18.

20. A recombinant host cell comprising the nucleic acid expression vector of claim 19.

21. A composition comprising: (a) one or more polypeptides comprising an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 74-89, 95, 97-99 and 102-111 or processed versions thereof; and (b) one or more other polypeptides comprising an amino acid sequence selected from the group consisting of: (A) an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID No: 35, wherein: (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and (ii) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp; (B) an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID No: 1, wherein: (i) the polypeptide degrades PQPQLP (SEQ ID NO: 34) at pH 4; and (ii) Residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp.

22. The composition of claim 21, wherein the one or more polypeptides comprise SEQ ID NO:75, SEQ ID NO:89 and / or SEQ ID NO:98, or processed versions thereof.

23. The composition of claim 21, wherein the one or more polypeptides comprise SEQ ID NO: 74, 77 and / or 78, or a processed version thereof.

24. The composition of claim 21, wherein the one or more polypeptides comprise SEQ ID NO: 88 and / or SEQ ID NO: 111, or processed versions thereof.

25. The composition of any one of claims 21-24, wherein the one or more other polypeptides comprise the amino acid sequence of SEQ ID No: 1 and / or SEQ ID NO:

35.

26. The composition of any one of claims 21-25, wherein the one or more other polypeptides comprise the amino acid sequence of any one of SEQ ID Nos: 2-33 or 36-67.

27. The composition of any one of claims 21-26, wherein the one or more other polypeptides comprise the amino acid sequence of SEQ ID No: 90 or a processed version thereof.

28. A pharmaceutical composition comprising the isolated polypeptide of any one of claims 14 to 17 or the composition of any one of claims 21 to 27 and a pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, wherein the composition is formulated for oral administration.

Images