IL4/IL13 receptor molecule for veterinary use
12428466 ยท 2025-09-30
Assignee
Inventors
- Hangjun Zhan (Foster City, CA, US)
- Lam Nguyen (Union City, CA, US)
- Fawn Qian (Burlingame, CA, US)
- Shyr Jiann Li (Millbrae, CA, US)
Cpc classification
A61K45/06
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
C07K2319/32
CHEMISTRY; METALLURGY
International classification
C07K14/715
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
Provided are various embodiments relating to IL13R/IL4R heterodimeric proteins derived from companion animal species and that bind to IL13 and/or IL4. Such heterodimeric proteins can be used in methods to treat IL13 and/or IL4-induced conditions in companion animals, such as canines, felines, and equines.
Claims
1. A heterodimeric protein comprising: a) a first contiguous polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide, wherein the amino acid sequence of the at least one IL13R ECD is SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, or SEQ ID NO: 34, SEQ ID NO: 36, and b) a second contiguous polypeptide comprising at least one IL4R ECD and a second Fc polypeptide, wherein the amino acid sequence of the at least one IL4R ECD is SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, wherein the IL13R ECD and/or the IL4R ECD are derived from a companion animal species.
2. The heterodimeric protein of claim 1, wherein the first contiguous polypeptide and/or the second contiguous polypeptide comprises one, two, three, or four IL4R ECDs and/or one, two, three, or four IL13R ECDs.
3. The heterodimeric protein of claim 1, wherein the first contiguous polypeptide and/or the second contiguous polypeptide further comprises at least one binding partner other than IL4R ECD or IL13R ECD, and wherein the at least one binding partner comprises IL5, IL6, IL17, IL22, IL31, LFA-1, TNF-, TSLP, and/or IgE.
4. The heterodimeric protein of claim 1, wherein the heterodimeric protein binds to IL13 and/or IL4 with a dissociation constant (Kd) of less than 510.sup.6 M, less than 110.sup.6 M, less than 510.sup.7 M, less than 110.sup.7 M, less than 510.sup.8 M, less than 110.sup.8 M, less than 510.sup.9 M, less than 110.sup.9 M, less than 510.sup.10 M, less than 110.sup.10 M, less than 510.sup.11 M, less than 110.sup.11 M, less than 510.sup.12 M, or less than 110.sup.12 M, as measured by biolayer interferometry; and/or wherein the heterodimeric protein reduces IL13 and/or IL4 signaling in a companion animal species.
5. The heterodimeric protein of claim 1, wherein the companion animal species is canine, feline, or equine.
6. The heterodimeric protein of claim 1, wherein the first Fc polypeptide and/or the second Fc polypeptide is: a) a canine IgG-A, IgG-B, IgG-C, or IgG-D Fc polypeptide; b) a feline IgG1a, IgG1b, or IgG2 Fc polypeptide; and/or c) an equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7 Fc polypeptide.
7. The heterodimeric protein of claim 1, wherein the first Fc polypeptide or the second Fc polypeptide comprises a knob mutation; and/or wherein the first Fc polypeptide or the second Fc polypeptide comprises a hole mutation.
8. The heterodimeric protein of claim 1, wherein the first Fc polypeptide or the second Fc polypeptide comprises: a) SEQ ID NO: 38 with an amino acid substitution at position 138, SEQ ID NO: 39 with an amino acid substitution at position 137, SEQ ID NO: 40 with an amino acid substitution at position 137, or SEQ ID NO: 41 with an amino acid substitution at position 138; and/or b) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46, each with an amino acid substitution at position 154; and/or c) SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, each with an amino acid substitution at position 130.
9. The heterodimeric protein of claim 1, wherein the first Fc polypeptide or the second Fc polypeptide comprises: a) SEQ ID NO: 38 with a tryptophan at position 138, SEQ ID NO: 39 with a tryptophan at position 137, SEQ ID NO: 40 with a tryptophan at position 137, or SEQ ID NO: 41 with a tryptophan at position 138; and/or b) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46, each with a tryptophan at position 154; and/or c) SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, each with a tryptophan at position 130.
10. The heterodimeric protein of claim 1, wherein the first Fc polypeptide or the second Fc polypeptide comprises: a) SEQ ID NO: 38 or SEQ ID NO: 41, each with an amino acid substitution at position 138 and/or position 140 and/or position 181; ID NO: 39 or SEQ ID NO: 40, each with an amino acid substitution at position 137 and/or position 139 and/or position 180; and/or b) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46, each with an amino acid substitution at position 154 and/or position 156 and/or position 197; and/or c) SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, each with an amino acid substitution at position 130 and/or position 132 and/or position 173.
11. The heterodimeric protein of claim 1, wherein the first Fc polypeptide or the second Fc polypeptide comprises: a) SEQ ID NO: 38 with a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181; SEQ ID NO: 39 with a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180; SEQ ID NO: 40 with a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180; or SEQ ID NO: 41 with a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181; and/or b) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46, each with a serine at position 154 and/or an alanine at position 156 and/or a threonine at position 197; and/or c) SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, each with a serine at position 130 and/or an alanine at position 132 and/or a threonine at position 173.
12. The heterodimeric protein of claim 1, wherein the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101.
13. The heterodimeric protein of claim 1, wherein the first contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113; and/or wherein the second contiguous polypeptide comprises the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
14. A pharmaceutical composition comprising the heterodimeric protein of claim 1 and a pharmaceutically acceptable carrier.
15. A heterodimeric protein comprising: a) a first polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide, and b) a second polypeptide comprising at least one IL4R ECD and a second Fc polypeptide; wherein the IL13R ECD and/or the IL4R ECD are derived from a canine; wherein the IL13R ECD sequence is SEQ ID NO: 22; wherein the IL4R ECD sequence is SEQ ID NO: 23; wherein the first polypeptide is SEQ ID NO: 103; wherein the second polypeptide is SEQ ID NO: 102; wherein the first Fc polypeptide is SEQ ID NO 55; and wherein the second Fc polypeptide is SEQ ID NO 59.
16. The heterodimeric protein of claim 15, wherein the first polypeptide and/or the second polypeptide further comprises at least one binding partner other than IL4R ECD or IL13R ECD, and wherein the at least one binding partner comprises IL31.
17. The heterodimeric protein of claim 15, wherein the heterodimeric protein binds to IL13 and/or IL4 with a dissociation constant (Kd) of less than 510.sup.6 M as measured by biolayer interferometry.
18. The heterodimeric protein of claim 15, wherein the heterodimeric protein reduces IL13 and/or IL4 signaling in a companion animal species.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
DESCRIPTION OF CERTAIN SEQUENCES
(4) Table 1 provides a listing of certain sequences referenced herein.
(5) TABLE-US-00001 TABLE1 DescriptionofCertainSequences SEQID NO: SEQUENCE DESCRIPTION 1 MGLTSQLIPTLVCLLALTSTFVHGHNFNITIKEII Canislupus KMLNILTARNDSCMELTVDVFTAPKNTSDKEIFCR interleukin-4precursor AATVLRQIYTHNCSNRYLRGLYRNLSSMANKTCSM NEIKKSTLKDFLERLKVIMQKKYYRH 2 MDLTSQLIPALVCLLAFTSTFVHGQNFNNTLKEII Feliscatus KILNILTARNDSCMELTMDVLAAPKNTSDKEIFCR interleukin-4precursor ATTVLRQIYTHHNCSTKFLKGLDRNLSSMANRTCS VNEVKKCTLKDFLERLKAIMQKKYSKH 3 MGLTYQLIPALVCLLACTSNFIQGCKYDITLQEII Equuscaballusinterleukin-4 KTLNNLTDGKGKNSCMELTVADAFAGPKNTDGKEI precursor CRAAKVLQQLYKRHDRSLIKECLSGLDRNLKGMAN GTCCTVNEAKKSTLKDFLERLKTIMKEKYSKC 4 MALWLTVVIALTCLGGLASPSPVTPSPTLKELIEE Canislupus LVNITQNQASLCNGSMVWSVNLTAGMYCAALESLI interleukin-13precursor NVSDCSAIQRTQRMLKALCSQKPAAGQISSERSRD TKIEVIQLVKNLLTYVRGVYRHGNFR 5 MWFLDSTRQSGDQGGRRHTWPIKATARGQGHKPLS Feliscatus LGQPTCPLLAPPVLALGSMALWLTVVIALTCLGGL interleukin-13precursor ASPGPHSRRELKELIEELVNITQNQVSLCNGSMVW SVNLTTGMYCAALESLINVSDCTAIQRTQRMLKAL CTQKPSAGQTASERSRDTKIEVIQLVKNLLNHLRR NFRHGNFK 6 MALWLTAVIALACLGGLASPAPLPSSMALKELIKE Equuscaballusinterleukin- LVNITQNQAPLCNGSMVWSVNLTADTYCRALESLS 13precursor NVSTCSAIQNTRKMLTKLCPHQLSAGQVSSERARD TKIEVIVLVKDLLKNLRKIFHGGKHVDA 7 MGRLCSGLTFPVSCLVLVWVASSGSVKVLHEPSCF Canislupus SDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSE interleukin-4receptor NHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWA subunitalpha GQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWL LMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVY NVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNS TWSDWSPSTTWLNYYEPWEQHLPLGVSISCLVILA ICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQD SQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEH GLGREEESPKTAKNGPLQGPGKPAWCPVEVSKTIL WPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCP SLEGSGGSFQEGREGIVARLTESLFLDLLGGENGG FCPQGLEESCLPPPSGSVGAQMPWAQFPRAGPRAA PEGPEQPRRPESALQASPTQSAGSSAFPEPPPVVT DNPAYRSFGSFLGQSSDPGDGDSDPELADRPGEAD PGIPSAPQPPEPPAALQPEPESWEQILRQSVLQHR AAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGP SGEAGYKAFCSLLPGGATCPGTSGGEAGSGEGGYK PFQSLIPGCPGAPTPVPVPLFTFGLDTEPPGSPQD SLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDP LRDDLASSIVYSALTCHLCGHLKQWHDQEERGKAH IVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLE ASLSPASLVPSGVSKEGKSSPFSQPASSSAQSSSQ TPKKLAVLSTEPTCMSAS 8 MGRLCSGLTFPVSCLILMWAAGSGSVKVLRAPTCF Feliscatus SDYFSTSVCQWNMDAPTNCSAELRLSYQLNFMGSE interleukin-4receptor NRTCVPENGEGAACACSMLMDDFVEADVYQLHLWA subunitalpha GTQLLWSGSFKPSSHVKPRAPGNLTVHPNVSHTWL LRWSNPYPPENHLHAELTYMVNISSEDDPTDVSVC ASGFLCHLLGLRRVETGAPGARLPPWLCAPRPRRV PGSQCAVISCCRWVLIALTSRGGRWRLTPGLRSQT RYVSVAEGLFGATPRVLCPGTQAGLASAAREQMSP DPSAFHSIDYEPWEQHLPLGVSISCLVILAVCLSC YLSVIKIKKEWWDQIPNPAHSHLVAIVIQDPQVSL WGKRSRGQEPAKCPHWKTCLRKLLPCLLEHGMERK EDPSKIARNGPSQCSGKSAWCPVEVSKTILWPESI SVVRCVELLEAPVESEEEEEEEEDKGSFCPSPVNL EDSFQEGREGIAARLTESLFMDLLGVEKGGFGPQG SLESWFPPPSGSAGAQMPWAEFPGPGPQEASPQGK EQPFDPRSDPLATLPQSPASPTFPETPPVVTDNPA YRSFGTFQGRSSGPGECDSGPELAGRLGEADPGIP AAPQPSEPPSALQPEAETWEQILRQRVLQHRGAPA PAPGSGYREFVCAVRQGSTQDSGVGDFGPSEEAGY KAFSSLLTSGAVCPESGGEAGSGDGGYKPFQSLTP GCPGAPAPVPVPLFTFGLDAEPPHCPQDSPLPGSS PEPAGKAQDSHKTPPAPEQAADPLRDDLASGIVYS ALTCHLCGHLKQCHGQEEGGEAHPVASPCCGCCCG DRSSPLVSPLRAPDPLPGGVPLEASLSPASPAPLA VSEEGPPSLCFQPALSHAHSSSQTPKKVAMLSPEP TCTMAS 9 MGCLCPGLTLPVSCLILVWAAGSGSVKVLHLTACF Equuscaballus SDYISASTCEWKMDRPTNCSAQLRLSYQLNDEFSD interleukin-4receptor NLTCIPENREDEVCVCRMLMDNIVSEDVYELDLWA subunitalpha GNQLLWNSSFKPSRHVKPRAPQNLTVHAISHTWLL TWSNPYPLKNHLWSELTYLVNISKEDDPTDFKIYN VTYMDPTLRVTASTLKSRATYSARVKARAQNYNST WSEWSPSTTWHNYYEQPLEQRLPLGVSISCVVILA ICLSCYFSIIKIKKEWWDQIPNPAHSPLVAIVLQD SQVSLWGKQSRGQEPAKCPRWKTCLTKLLPCLLEH GLQKEEDSSKTVRNGPFQSPGKSAWHTVEVNHTIL RPEIISVVPCVELCEAQVESEEEEVEEDRGSFCPS PESSGSGFQEGREGVAARLTESLFLGLLGAENGAL GESCLLPPLGSAHMPWARISSAGPQEAASQGEEQP LNPESNPLATLTQSPGSLAFTEAPAVVADNPAYRS FSNSLSQPRGPGELDSDPQLAEHLGQVDPSIPSAP QPSEPPTALQPEPETWEQMLRQSVLQQGAAPAPAS APTGGYREFAQAVKQGGGAAGSGPSGEAGYKAFSS LLAGSAVCPGQSGVEASSGEGGYRPYESPDPGAPA PVPVPLFTFGLDVEPPHSPQNSLLPGGSPELPGPE PTVKGEDPRKPLLSAQQATDSLRDDLGSGIVYSAL TCHLCGHLKQCHGQEEHGEAHTVASPCCGCCCGDR SSPPVSPVRALDPPPGGVPLEAGLSLASLGSLGLS EERKPSLFFQPAPGNAQSSSQTPLTVAMLSTGPTC TSAS 10 MERPARLCGLWALLLCAAGGRGGGVAAPTETQPPV Canislupus TNLSVSVENLCTVIWTWDPPEGASPNCTLRYFSHF interleukin-13receptor DNKQDKKIAPETHRSKEVPLNERICLQVGSQCSTN subunitalpha-1 ESDNPSILVEKCTPPPEGDPESAVTELQCVWHNLS YMKCTWLPGRNTSPDTNYTLYYWHSSLGKILQCED IYREGQHIGCSFALTNLKDSSFEQHSVQIVVKDNA GKIRPSFNIVPLTSHVKPDPPHIKRLFFQNGNLYV QWKNPQNFYSRCLSYQVEVNNSQTETNDIFYVEEA KCQNSEFEGNLEGTICFMVPGVLPDTLNTVRIRVR TNKLCYEDDKLWSNWSQAMSIGENTDPTFYITMLL ATPVIVAGAIIVLLLYLKRLKIIIFPPIPDPGKIF KEMFGDQNDDTLHWRKYDIYEKQTKEETDSVVLIE NLKKASQ 11 MMTKCSSDRNVFKRKWFLFPASQYTFRPIHQARPC Feliscatus EVPAVHLEPSPPWEVGLGLLNLESEFRKLGLRGRR interleukin-13receptor LAAAPPDSRAEAASQTQPPVTNLSVSVENLCTVIW subunitalpha-1 TWDPPEGASPNCTLRYFSHFDNKQDKKIAPETHRS KEVPLNERICLQVGSQCSTNESDNPSILVEKCTPP PEGDPESAVTELQCVWHNLSYMKCTWLPGRNTSPD TNYTLYYWHSSLGKILQCENIYREGQHIGCSFALT NLKDSSFEQHSVQIVVKDNAGKIRPSFNIVPLTSH VKPDPPHIKRLFFQNGNLYVQWKNPQNFYSRCLSY QVEVNNSQTETHDIFYVEEAKCQNSEFEGNLEGTI CFMVPGILPDTLNTVRIRVRTNKLCYEDDRLWSNW SQAMSIGENTDPTFYITMLLATPVIVAGAIIVLLL YLKRLKIIIFPPIPDPGKIFKEMFGDQNDDSLHWK KYDIYEKQTKEETDSVVLIENASQ 12 MYFLCLIWTESQPPVTNLSVSVENLCTVIWTWNPP Equuscaballus EGVSPNCSLWYFSHFGNKQDKKIAPETHRSKEVPL interleukin-13receptor NERICLQVGSQCSTNESDNPSILVEKCISPPEGDP subunitalpha-1 ESAVTELQCVWHNLSYMKCTWLPGKNASPDTNYTL YYWHSSLGKILQCEDIYREGQHIGCSFALTEVKDS IFEQHSVQIMVKDNAGKIRPFFNIVPLTSHVKPDP PHIKKLFFQNGDLYVQWKNPQNFYSRCLSYQVEVN NSQTETRDIFSVEEAKCQNPEFEGDLEGTICFMVP GVLPDTVNTVRIRVKTNKLCYEDDKLWSNWSQAMS IGKKADPTFYIAMLLIIPVIVAGAIIVLLLYLKRL KIIMFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIY EKQTKEETDSVVLIENLKRASQ 13 TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT ExemplaryCanislupus LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV IL13RECD-IL4RECD- GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ IgGAFc CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG (withoutsignalsequence) KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT GGGSGSGSVKVLHEPSCFSDYISTSVCQWKMDHPT NCSAELRLSYQLDFMGSENHTCVPENREDSVCVCS MPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVK PRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSEL TYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASILK SGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEP KRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPK DILLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQ MQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQ FTCKVNNKALPSPIERTISKARGQAHQPSVYVLPP SREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQE PESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGD TFICAVMHEALHNHYTQESLSHSPGK 14 SGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAE Exemplarycanine LRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDD IL4RECD-IL13RECD- AVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPG IgGAFc(withoutsignal NLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVN sequence) VSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASY SARVRAWAQTYNSTWSDWSPSTTWLNYYEPGGGSG TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT FNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILR ITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTA KTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCR VNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKE LSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPER KHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFT CAVMHETLQNHYTDLSLSHSPGK 15 SGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAE Exemplarycanine LRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDD IL4RECD-IL13RECD- AVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRITG IgGBFc NLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVN (withoutsignalsequence) VSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASY SARVRAWAQTYNSTWSDWSPSTTWLNYYEPGGGSG TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT PKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKP KDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGK QMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGK QFTCKVNNKALPSPIERTISKARGQAHQPSVYVLP PSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQ EPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRG DTFICAVMHEALHNHYTQESLSHSPGK 16 SGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAE Exemplarycanine LRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDD IL4RECD-IL13RECD- AVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPG IgGC(withoutsignal NLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVN sequence) VSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASY SARVRAWAQTYNSTWSDWSPSTTWLNYYEPGGGSG TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT AKECECKCNCNNCPCPGCGLLGGPSVFIFPPKPKD ILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQV QTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQF KCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPS RDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDT FICAVMHEALHNHYTQISLSHSPGK 17 SGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAE Exemplarycanine LRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDD IL4RECD-IL13RECD- AVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPG IgGDFc(withoutsignal NLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVN sequence) VSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASY SARVRAWAQTYNSTWSDWSPSTTWLNYYEPGGGSG TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT PKESTCKCISPCPVPESLGGPSVFIFPPKPKDILR ITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTA KTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCR VNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKE LSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPES KYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFT CAVMHEALQNHYTDLSLSHSPGK 18 SGSVKVLRAPTCFSDYFSTSVCQWNMDAPTNCSAE ExemplaryFeline LRLSYQLNFMGSENRTCVPENGEGAACACSMLMDD IL4RECD-IL13RECD FVEADVYQLHLWAGTQLLWSGSFKPSSHVKPRAPG (withoutsignalsequence) NLTVHPNVSHTWLLRWSNPYPPENHLHAELTYMVN ISSEDDPTDVSVCASGFLCHLLGLRRVETGAPGAR LPPWLCAPRPRRVPGSQCAVISCCRWVLIALTSRG GRWRLTPGLRSQTRYVSVAEGLFGATPRVLCPGTQ AGLASAAREQMSPDPSAFHSIDYEPGGGSGSQTQP PVTNLSVSVENLCTVIWTWDPPEGASPNCTLRYFS HFDNKQDKKIAPETHRSKEVPLNERICLQVGSQCS TNESDNPSILVEKCTPPPEGDPESAVTELQCVWHN LSYMKCTWLPGRNTSPDTNYTLYYWHSSLGKILQC ENIYREGQHIGCSFALTNLKDSSFEQHSVQIVVKD NAGKIRPSFNIVPLTSHVKPDPPHIKRLFFQNGNL YVQWKNPQNFYSRCLSYQVEVNNSQTETHDIFYVE EAKCQNSEFEGNLEGTICFMVPGILPDTLNTVRIR VRTNKLCYEDDRLWSNWSQAMSIGENTDPT 19 SGSVKVLHLTACFSDYISASTCEWKMDRPINCSAQ Exemplaryequine LRLSYQLNDEFSDNLTCIPENREDEVCVCRMLMDN IL4RECD-IL13RECD IVSEDVYELDLWAGNQLLWNSSFKPSRHVKPRAPQ (withoutsignalsequence) NLTVHAISHTWLLTWSNPYPLKNHLWSELTYLVNI SKEDDPTDFKIYNVTYMDPTLRVTASTLKSRATYS ARVKARAQNYNSTWSEWSPSTTWHNYYEQPGGGSG TESQPPVTNLSVSVENLCTVIWTWNPPEGVSPNCS LWYFSHFGNKQDKKIAPETHRSKEVPLNERICLQV GSQCSTNESDNPSILVEKCISPPEGDPESAVTELQ CVWHNLSYMKCTWLPGKNASPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTEVKDSIFEQHSVQ IMVKDNAGKIRPFFNIVPLTSHVKPDPPHIKKLFF QNGDLYVQWKNPQNFYSRCLSYQVEVNNSQTETRD IFSVEEAKCQNPEFEGDLEGTICFMVPGVLPDTVN TVRIRVKTNKLCYEDDKLWSNWSQAMSIGKKADPT 20 MAVLGLLFCLVTFPSCVLSTETQPPVTNLSVSVEN Exemplarycanine LCTVIWTWDPPEGASPNCTLRYFSHFDNKQDKKIA IL13RECD-IL4RECD- PETHRSKEVPLNERICLQVGSQCSTNESDNPSILV IgGBFc EKCTPPPEGDPESAVTELQCVWHNLSYMKCTWLPG (withsignalsequence) RNTSPDTNYTLYYWHSSLGKILQCEDIYREGQHIG CSFALTNLKDSSFEQHSVQIVVKDNAGKIRPSFNI VPLTSHVKPDPPHIKRLFFQNGNLYVQWKNPQNFY SRCLSYQVEVNNSQTETNDIFYVEEAKCQNSEFEG NLEGTICFMVPGVLPDTLNTVRIRVRTNKLCYEDD KLWSNWSQAMSIGENTDPTGGGSGSGSVKVLHEPS CFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMG SENHTCVPENREDSVCVCSMPIDDAVEADVYQLDL WAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHT WLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFK VYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTY NSTWSDWSPSTTWLNYYEPKRENGRVPRPPDCPKC PAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER TISKARGQAHQPSVYVLPPSREELSKNTVSLTCLI KDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDG SYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPGK 21 MAVLGLLFCLVTFPSCVLSSGSVKVLHEPSCFSDY Exemplarycanine ISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHT IL4RECD-IL13RECD- CVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQ IgGBFc LLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMW (withsignalsequence) TNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVT YMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWS DWSPSTTWLNYYEPGGGSGTETQPPVTNLSVSVEN LCTVIWTWDPPEGASPNCTLRYFSHFDNKQDKKIA PETHRSKEVPLNERICLQVGSQCSTNESDNPSILV EKCTPPPEGDPESAVTELQCVWHNLSYMKCTWLPG RNTSPDTNYTLYYWHSSLGKILQCEDIYREGQHIG CSFALTNLKDSSFEQHSVQIVVKDNAGKIRPSFNI VPLTSHVKPDPPHIKRLFFQNGNLYVQWKNPQNFY SRCLSYQVEVNNSQTETNDIFYVEEAKCQNSEFEG NLEGTICFMVPGVLPDTLNTVRIRVRTNKLCYEDD KLWSNWSQAMSIGENTDPTPKRENGRVPRPPDCPK CPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVV VDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNG TYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIE RTISKARGQAHQPSVYVLPPSREELSKNTVSLTCL IKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDED GSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHY TQESLSHSPGK 22 TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT ExemplarycanineIL13R LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV extracellulardomain GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ (withoutsignalsequence) CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT 23 SGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAE ExemplarycanineIL4R LRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDD extracellulardomain(ECD; AVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPG withoutsignalsequence) NLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVN VSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASY SARVRAWAQTYNSTWSDWSPSTTWLNYYEP 24 SQTQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT ExemplaryfelineIL13R LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV extracellulardomain(ECD; GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ withoutsignalsequence) CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCENIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETHD IFYVEEAKCQNSEFEGNLEGTICFMVPGILPDTLN TVRIRVRTNKLCYEDDRLWSNWSQAMSIGENTDPT 25 SGSVKVLRAPTCFSDYFSTSVCQWNMDAPTNCSAE ExemplaryfelineIL4R LRLSYQLNFMGSENRTCVPENGEGAACACSMLMDD extracellulardomain(ECD; FVEADVYQLHLWAGTQLLWSGSFKPSSHVKPRAPG withoutsignalsequence) NLTVHPNVSHTWLLRWSNPYPPENHLHAELTYMVN ISSEDDPTDVSVCASGFLCHLLGLRRVETGAPGAR LPPWLCAPRPRRVPGSQCAVISCCRWVLIALTSRG GRWRLTPGLRSQTRYVSVAEGLFGATPRVLCPGTQ AGLASAAREQMSPDPSAFHSIDYEP 26 TESQPPVTNLSVSVENLCTVIWTWNPPEGVSPNCS ExemplaryequineIL13R LWYFSHFGNKQDKKIAPETHRSKEVPLNERICLQV extracellulardomain(ECD; GSQCSTNESDNPSILVEKCISPPEGDPESAVTELQ withoutsignalsequence) CVWHNLSYMKCTWLPGKNASPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTEVKDSIFEQHSVQ IMVKDNAGKIRPFFNIVPLTSHVKPDPPHIKKLFF QNGDLYVQWKNPQNFYSRCLSYQVEVNNSQTETRD IFSVEEAKCQNPEFEGDLEGTICFMVPGVLPDTVN TVRIRVKTNKLCYEDDKLWSNWSQAMSIGKKADPT 27 SGSVKVLHLTACFSDYISASTCEWKMDRPTNCSAQ ExemplaryequineIL4R LRLSYQLNDEFSDNLICIPENREDEVCVCRMLMDN extracellulardomain(ECD; IVSEDVYELDLWAGNQLLWNSSFKPSRHVKPRAPQ withoutsignalsequence) NLTVHAISHTWLLTWSNPYPLKNHLWSELTYLVNI SKEDDPTDFKIYNVTYMDPTLRVTASTLKSRATYS ARVKARAQNYNSTWSEWSPSTTWHNYYEQP 28 SQTQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT Exemplaryfeline LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV IL13RECD-IL4RECD-IgG2 GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ Fc(withoutsignalsequence) CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCENIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETHD IFYVEEAKCQNSEFEGNLEGTICFMVPGILPDTLN TVRIRVRTNKLCYEDDRLWSNWSQAMSIGENTDPT GGGSGSSGSVKVLRAPTCFSDYFSTSVCQWNMDAP TNCSAELRLSYQLNFMGSENRTCVPENGEGAACAC SMLMDDFVEADVYQLHLWAGTQLLWSGSFKPSSHV KPRAPGNLTVHPNVSHTWLLRWSNPYPPENHLHAE LTYMVNISSEDDPTDVSVCASGFLCHLLGLRRVET GAPGARLPPWLCAPRPRRVPGSQCAVISCCRWVLI ALTSRGGRWRLTPGLRSQTRYVSVAEGLFGATPRV LCPGTQAGLASAAREQMSPDPSAFHSIDYEPSPKT ASTIESKTGECPKCPVPEIPGAPSVFIFPPKPKDT LSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMH TAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFK CKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQ EELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPE NNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTY TCSVSHEALHSHHTQKSLTQSPGK 29 SGSVKVLRAPTCFSDYFSTSVCQWNMDAPTNCSAE Exemplaryfeline LRLSYQLNFMGSENRTCVPENGEGAACACSMLMDD IL4RECD-IL13RECD-IgG2 FVEADVYQLHLWAGTQLLWSGSFKPSSHVKPRAPG (withoutsignalsequence) NLTVHPNVSHTWLLRWSNPYPPENHLHAELTYMVN ISSEDDPTDVSVCASGFLCHLLGLRRVETGAPGAR LPPWLCAPRPRRVPGSQCAVISCCRWVLIALTSRG GRWRLTPGLRSQTRYVSVAEGLFGATPRVLCPGTQ AGLASAAREQMSPDPSAFHSIDYEPGGGSGSSQTQ PPVTNLSVSVENLCTVIWTWDPPEGASPNCTLRYF SHFDNKQDKKIAPETHRSKEVPLNERICLQVGSQC STNESDNPSILVEKCTPPPEGDPESAVTELQCVWH NLSYMKCTWLPGRNTSPDTNYTLYYWHSSLGKILQ CENIYREGQHIGCSFALTNLKDSSFEQHSVQIVVK DNAGKIRPSFNIVPLTSHVKPDPPHIKRLFFQNGN LYVQWKNPQNFYSRCLSYQVEVNNSQTETHDIFYV EEAKCQNSEFEGNLEGTICFMVPGILPDTLNTVRI RVRTNKLCYEDDRLWSNWSQAMSIGENTDPTSPKT ASTIESKTGECPKCPVPEIPGAPSVFIFPPKPKDT LSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMH TAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFK CKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQ EELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPE NNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTY TCSVSHEALHSHHTQKSLTQSPGK 30 TESQPPVTNLSVSVENLCTVIWTWNPPEGVSPNCS Exemplaryequine LWYFSHFGNKQDKKIAPETHRSKEVPLNERICLQV IL13RECD-IL4RECD-IgG2 GSQCSTNESDNPSILVEKCISPPEGDPESAVTELQ Fc(withoutsignalsequence) CVWHNLSYMKCTWLPGKNASPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTEVKDSIFEQHSVQ IMVKDNAGKIRPFFNIVPLTSHVKPDPPHIKKLFF QNGDLYVQWKNPQNFYSRCLSYQVEVNNSQTETRD IFSVEEAKCQNPEFEGDLEGTICFMVPGVLPDTVN TVRIRVKTNKLCYEDDKLWSNWSQAMSIGKKADPT GGGSGSSGSVKVLHLTACFSDYISASTCEWKMDRP TNCSAQLRLSYQLNDEFSDNLTCIPENREDEVCVC RMLMDNIVSEDVYELDLWAGNQLLWNSSFKPSRHV KPRAPQNLTVHAISHTWLLTWSNPYPLKNHLWSEL TYLVNISKEDDPTDFKIYNVTYMDPTLRVTASILK SRATYSARVKARAQNYNSTWSEWSPSTTWHNYYEQ PDMSKCPKCPAPELLGGPSVFIFPPNPKDTLMISR TPVVTCVVVNLSDQYPDVQFSWYVDNTEVHSAITK QREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTN VGVPQPISRAISRGKGPSRVPQVYVLPPHPDELAK SKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYST TPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVM HEALHNHYTKTDISESLGK 31 SGSVKVLHLTACFSDYISASTCEWKMDRPTNCSAQ Exemplaryequine LRLSYQLNDEFSDNLICIPENREDEVCVCRMLMDN IL4RECD-IL13RECD-IgG2 IVSEDVYELDLWAGNQLLWNSSFKPSRHVKPRAPQ Fc(withoutsignalsequence) NLTVHAISHTWLLTWSNPYPLKNHLWSELTYLVNI SKEDDPTDFKIYNVTYMDPTLRVTASTLKSRATYS ARVKARAQNYNSTWSEWSPSTTWHNYYEQPGGGSG STESQPPVTNLSVSVENLCTVIWTWNPPEGVSPNC SLWYFSHFGNKQDKKIAPETHRSKEVPLNERICLQ VGSQCSTNESDNPSILVEKCISPPEGDPESAVTEL QCVWHNLSYMKCTWLPGKNASPDTNYTLYYWHSSL GKILQCEDIYREGQHIGCSFALTEVKDSIFEQHSV QIMVKDNAGKIRPFFNIVPLTSHVKPDPPHIKKLF FQNGDLYVQWKNPQNFYSRCLSYQVEVNNSQTETR DIFSVEEAKCQNPEFEGDLEGTICFMVPGVLPDTV NTVRIRVKTNKLCYEDDKLWSNWSQAMSIGKKADP TDMSKCPKCPAPELLGGPSVFIFPPNPKDTLMISR TPVVTCVVVNLSDQYPDVQFSWYVDNTEVHSAITK QREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTN VGVPQPISRAISRGKGPSRVPQVYVLPPHPDELAK SKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYST TPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVM HEALHNHYTKTDISESLGK 32 QPPVTNLSVSVENLCTVIWTWDPPEGASPNCTLRY Exemplarycaninemini- FSHFDNKQDKKIAPETHRSKEVPLNERICLQVGSQ IL13RECD CSTNESDNPSILVEKCTPPPEGDPESAVTELQCVW HNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLGKIL QCEDIYREGQHIGCSFALTNLKDSSFEQHSVQIVV KDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFFQNG NLYVQWKNPQNFYSRCLSYQVEVNNSQTETNDIFY VEEAKCQNSEFEGNLEGTICFMVPGVLPDTLNTVR IRVRTNKLCYEDDKLWSNWSQAMSI 33 KVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLS Exemplarycaninemini- YQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEA IL4RECD DVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTV HPNISHTWLLMWTNPYPTENHLHSELTYMVNVSND NDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARV RAWAQTYNS 34 QPPVTNLSVSVENLCTVIWTWDPPEGASPNCTLRY Exemplaryfelinemini- FSHFDNKQDKKIAPETHRSKEVPLNERICLQVGSQ IL13RECD CSTNESDNPSILVEKCTPPPEGDPESAVTELQCVW HNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLGKIL QCENIYREGQHIGCSFALTNLKDSSFEQHSVQIVV KDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFFQNG NLYVQWKNPQNFYSRCLSYQVEVNNSQTETHDIFY VEEAKCQNSEFEGNLEGTICFMVPGILPDTLNTVR IRVRTNKLCYEDDRLWSNWSQAMSI 35 KVLRAPTCFSDYFSTSVCQWNMDAPTNCSAELRLS Exemplaryfelinemini-IL4R YQLNFMGSENRTCVPENGEGAACACSMLMDDFVEA ECD DVYQLHLWAGTQLLWSGSFKPSSHVKPRAPGNLTV HPNVSHTWLLRWSNPYPPENHLHAELTYMVNISSE DDPTDVSVCASGFLCHLLGLRRVETGAPGARLPPW LCAPRPRRVPGSQCAVISCCRWVLIALTSRGGRWR LTPGLRSQTRYVSVAEGLFGATPRVLCPGTQAGLA SAAREQMSPDPSAFHSIDYEP 36 QPPVINLSVSVENLCTVIWTWNPPEGVSPNCSLWY Exemplaryequinemini- FSHFGNKQDKKIAPETHRSKEVPLNERICLQVGSQ IL13RECD CSTNESDNPSILVEKCISPPEGDPESAVTELQCVW HNLSYMKCTWLPGKNASPDTNYTLYYWHSSLGKIL QCEDIYREGQHIGCSFALTEVKDSIFEQHSVQIMV KDNAGKIRPFFNIVPLTSHVKPDPPHIKKLFFQNG DLYVQWKNPQNFYSRCLSYQVEVNNSQTETRDIFS VEEAKCQNPEFEGDLEGTICFMVPGVLPDTVNTVR IRVKTNKLCYEDDKLWSNWSQAMSI 37 KVLHLTACFSDYISASTCEWKMDRPTNCSAQLRLS Exemplaryequinemini- YQLNDEFSDNLTCIPENREDEVCVCRMLMDNIVSE IL4RECD DVYELDLWAGNQLLWNSSFKPSRHVKPRAPQNLTV HAISHTWLLTWSNPYPLKNHLWSELTYLVNISKED DPTDFKIYNVTYMDPTLRVTASTLKSRATYSARVK ARAQNYNSTWSEWSPSTTWHNYYEQP 38 PVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVL Exemplarywild-typecanine DLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGT IgG-AFc YRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIER TISKARGRAHKPSVYVLPPSPKELSSSDTVSITCL IKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDED GSYFLYSKLSVDKSRWQQGDPFICAVMHETLQNHY TDLSLSHSPGK 39 PAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV Exemplarywild-typecanine DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGT IgG-BFc YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER TISKARGQAHQPSVYVLPPSREELSKNTVSLTCLI KDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDG SYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPGK 40 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVV Exemplarywild-typecanine DLDPENPEVQISWFVDSKQVQTANTQPREEQSNGT IgG-CFc YRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEE IISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLV KDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDG SYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QISLSHSPGK 41 PVPESLGGPSVFIFPPKPKDILRITRTPEITCVVL Exemplarywild-typecanine DLGREDPEVQISWFVDGKEVHTAKTQPREQQFNST IgG-DFc YRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIER TISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCL IKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDED GSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHY TDLSLSHSPGK 42 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplarywild-typefeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP PAQEELSENKVSVTCLIKSFHPPDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 43 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplarywild-typefeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP PAQEELSENKVSVTCLIKSFHPPDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 44 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplarywild-typefeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP PAQEELSENKVSVTCLIEGFYPSDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 45 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplarywild-typefeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP PAQEELSENKVSVTCLIEGFYPSDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 46 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKP Exemplarywild-typefeline KDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNT IgG2Fc EMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGK EFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLP PTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQP EPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 47 GPSVFIFPPNPKDTLMITRTPEVTCVVVDVSQENP Exemplarywild-typeequine DVKFNWYMDGVEVRTATTRPKEEQFNSTYRVVSVL IgG1Fc RIQHQDWLSGKEFKCKVNNQALPQPIERTITKTKG RSQEPQVYVLAPHPDELSKSKVSVTCLVKDFYPPE INIEWQSNGQPELETKYSTTQAQQDSDGSYFLYSK LSVDRNRWQQGTTFTCGVMHEALHNHYTQKNVSKN PGK 48 GPSVFIFPPNPKDALMISRTPVVTCVVVNLSDQYP Exemplarywild-typeequine DVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVL IgG2Fc PIQHQDWLSGKEFKCSVTNVGVPQPISRAISRGKG PSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPD ISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSK LSLETSRWQQVESFTCAVMHEALHNHFTKTDISES LGK 49 GPSVFIFPPKPKDVLMITRMPEVTCLVVDVSHDSS Exemplarywild-typeequine DVLFTWYVDGTEVKTAKTMPNEEQNNSTYRVVSVL IgG3Fc RIQHQDWLNGKKFKCKVNNQALPAPVERTISKATG QTRVPQVYVLAPHPDELSKNKVSVTCLVKDFYPPD ITVEWQSNEHPEPEGKYRTTEAQKDSDGSYFLYSK LTVEKDRWQQGTTFTCVVMHEALHNHVMQKNISKN PGK 50 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplarywild-typeequine DVQFNWYVDGVETHTATTEPKQEQFNSTYRVVSVL IgG4Fc PIQHKDWLSGKEFKCKVNNKALPAPVERTISAPTG QPREPQVYVLAPHRDELSKNKVSVTCLVKDFYPPD IDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSK LIVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKS PGK 51 GPSVFIFPPKPKDVLMISRKPEVTCVVVDLGHDDP Exemplarywild-typeequine DVQFTWFVDGVETHTATTEPKEEQFNSTYRVVSVL IgG5Fc PIQHQDWLSGKEFKCSVTSKALPAPVERTISKAKG QLRVPQVYVLAPHPDELAKNTVSVTCLVKDFYPPE IDVEWQSNEHPEPEGKYSTTPAQLNSDGSYFLYSK LSVETSRWKQGESFTCGVMHEAVENHYTQKNVSHS PGK 52 GRPSVFIFPPNPKDTLMISRTPEVTCVVVDVSQE Exemplarywild-typeequine NPDVKFNWYVDGVEAHTATTKAKEKQDNSTYRVV IgG6Fc SVLPIQHQDWRRGKEFKCKVNNRALPAPVERTIT KAKGELQDPKVYILAPHREEVTKNTVSVTCLVKD FYPPDINVEWQSNEEPEPEVKYSTTPAQLDGDGS YFLYSKLTVETDRWEQGESFTCVVMHEAIRHTYR QKSITNFPGK 53 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplarywild-typeequine DVQFNWYVDGVETHTATTEPKQEQNNSTYRVVSIL IgG7Fc AIQHKDWLSGKEFKCKVNNQALPAPVQKTISKPTG QPREPQVYVLAPHPDELSKNKVSVTCLVKDFYPPD IDIEWKSNGQPEPETKYSTTPAQLDGDGSYFLYSK LIVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKS PGK 54 PVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVL Exemplaryvariantcanine DLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGT IgG-AFc YRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIER Heterodimerknob TISKARGRAHKPSVYVLPPSPKELSSSDTVSIWCL T(138)W IKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDED GSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHY TDLSLSHSPGK 55 PAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV Exemplaryvariantcanine DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGT IgG-BFc YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER Heterodimerknob TISKARGQAHQPSVYVLPPSREELSKNTVSLWCLI T(137)W KDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDG SYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPGK 56 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVV Exemplaryvariantcanine DLDPENPEVQISWFVDSKQVQTANTQPREEQSNGT IgG-CFc YRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEE Heterodimerknob IISKIPGQAHQPNVYVLPPSRDEMSKNTVTLWCLV T(137)W KDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDG SYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QISLSHSPGK 57 PVPESLGGPSVFIFPPKPKDILRITRTPEITCVVL Exemplaryvariantcanine DLGREDPEVQISWFVDGKEVHTAKTQPREQQFNST IgG-DFc YRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIER Heterodimerknob TISKARGQAHQPSVYVLPPSPKELSSSDTVTLWCL T(138)W IKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDED GSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHY TDLSLSHSPGK 58 PVPEPLGGPSVLIFPPKPKDILRITRTPEVPCVVL Exemplaryvariantcanine DLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGT IgG-AFc YRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIER Heterodimerhole TISKARGRAHKPSVYVLPPSPKELSSSDTVSISCA T(138)S IKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDED L(140)A GSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHY TDLSLSHSPGK 59 PAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV Exemplaryvariantcanine DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGT IgG-BFc YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER Heterodimerhole TISKARGQAHQPSVYVLPPSREELSKNIVSLSCAI T(137)S KDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDG L(139)A SYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QESLSHSPGK 60 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVV Exemplaryvariantcanine DLDPENPEVQISWFVDSKQVQTANTQPREEQSNGT IgG-CFc YRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEE Heterodimerhole IISKTPGQAHQPNVYVLPPSRDEMSKNTVTLSCAV T(137)S KDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDG L(139)A SYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYT QISLSHSPGK 61 PVPESLGGPSVFIFPPKPKDILRITRTPEITCVVL Exemplaryvariantcanine DLGREDPEVQISWFVDGKEVHTAKTQPREQQFNST IgG-DFc YRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIER Heterodimerhole TISKARGQAHQPSVYVLPPSPKELSSSDTVTLSCA T(138)S IKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDED L(140)A GSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHY TDLSLSHSPGK 62 PVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVL Exemplaryvariantcanine DLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGT IgG-AFc YRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIER Heterodimerhole TISKARGRAHKPSVYVLPPSPKELSSSDTVSISCA T(138)S IKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDED L(140)A GSYFLTSKLSVDKSRWQQGDPFTCAVMHETLQNHY Y(181)T TDLSLSHSPGK 63 PAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVV Exemplaryvariantcanine DLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGT IgG-BFc YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIER Heterodimerhole TISKARGQAHQPSVYVLPPSREELSKNTVSLSCAI T(137)S KDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDG L(139)A SYFLTSKLSVDKSRWQRGDTFTCAVMHEALHNHYT Y(180)T QESLSHSPGK 64 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVV Exemplaryvariantcanine DLDPENPEVQISWFVDSKQVQTANTQPREEQSNGT IgG-CFc YRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEE Heterodimerhole IISKIPGQAHQPNVYVLPPSRDEMSKNTVTLSCAV T(137)S KDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDG L(139)A SYFLTSKLSVDKSRWQRGDTFICAVMHEALHNHYT Y(180)T QISLSHSPGK 65 PVPESLGGPSVFIFPPKPKDILRITRTPEITCVVL Exemplaryvariantcanine DLGREDPEVQISWFVDGKEVHTAKTQPREQQFNST IgG-DFc YRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIER Heterodimerhole TISKARGQAHQPSVYVLPPSPKELSSSDTVTLSCA T(138)S IKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDED L(140)A GSYFLTSKLSVDKSRWQQGDTFTCAVMHEALQNHY Y(181)T TDLSLSHSPGK 66 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerknob EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP T(154)W PAQEELSENKVSVWCLIKSFHPPDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 67 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerknob EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP T(154)W PAQEELSENKVSVWCLIKSFHPPDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 68 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerknob EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP T(154)W PAQEELSENKVSVWCLIEGFYPSDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 69 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerknob EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP T(154)W PAQEELSENKVSVWCLIEGFYPSDIAVEWEITGQP EPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 70 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNT IgG2Fc EMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerknob EFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLP T(154)W PTQEELSENKVSVWCLIKGFHPPDIAVEWEITGQP EPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 71 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIKSFHPPDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 72 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIKSFHPPDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 73 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIEGFYPSDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 74 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIEGFYPSDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 75 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNT IgG2Fc EMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLP T(154)S PTQEELSENKVSVSCAIKGFHPPDIAVEWEITGQP L(156)A EPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRG NTYTCSVSHEALHSHHTQKSLTQSPGK 76 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIKSFHPPDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFVTSKLSVDRSHWQRG Y(197)T NTYTCSVSHEALHSHHTQKSLTQSPGK 77 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1aFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIKSFHPPDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFVTSKLSVDRSHWQRG Y(197)T NTYTCSVSHEALHSHHTQKSLTQSPGK 78 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIEGFYPSDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFLTSRLSVDRSRWQRG Y(197)T NTYTCSVSHEALHSHHTQKSLTQSPGK 79 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNT IgG1bFc QVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLP T(154)S PAQEELSENKVSVSCAIEGFYPSDIAVEWEITGQP L(156)A EPENNYRTTPPQLDSDGTYFLTSRLSVDRSRWQRG Y(197)T NTYTCSVSHEALHSHHTQKSLTQSPGK 80 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKP Exemplaryvariantfeline KDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNT IgG2Fc EMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGK Heterodimerhole EFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLP T(154)S PTQEELSENKVSVSCAIKGFHPPDIAVEWEITGQP L(156)A EPENNYQTTPPQLDSDGTYFLTSRLSVDRSHWQRG Y(197)T NTYTCSVSHEALHSHHTQKSLTQSPGK 81 GPSVFIFPPNPKDTLMITRTPEVTCVVVDVSQENP Exemplaryvariantequine DVKFNWYMDGVEVRTATTRPKEEQFNSTYRVVSVL IgG1Fc RIQHQDWLSGKEFKCKVNNQALPQPIERTITKTKG Heterodimerknob RSQEPQVYVLAPHPDELSKSKVSVWCLVKDFYPPE T(130)W INIEWQSNGQPELETKYSTTQAQQDSDGSYFLYSK LSVDRNRWQQGTTFTCGVMHEALHNHYTQKNVSKN PGK 82 GPSVFIFPPNPKDALMISRTPVVTCVVVNLSDQYP Exemplaryvariantequine DVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVL IgG2Fc PIQHQDWLSGKEFKCSVINVGVPQPISRAISRGKG Heterodimerknob PSRVPQVYVLPPHPDELAKSKVSVWCLVKDFYPPD T(130)W ISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSK LSLETSRWQQVESFTCAVMHEALHNHFIKTDISES LGK 83 GPSVFIFPPKPKDVLMITRMPEVTCLVVDVSHDSS Exemplaryvariantequine DVLFTWYVDGTEVKTAKTMPNEEQNNSTYRVVSVL IgG3Fc RIQHQDWLNGKKFKCKVNNQALPAPVERTISKATG Heterodimerknob QTRVPQVYVLAPHPDELSKNKVSVWCLVKDFYPPD T(130)W ITVEWQSNEHPEPEGKYRTTEAQKDSDGSYFLYSK LTVEKDRWQQGTTFTCVVMHEALHNHVMQKNISKN PGK 84 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplaryvariantequine DVQFNWYVDGVETHTATTEPKQEQFNSTYRVVSVL IgG4Fc PIQHKDWLSGKEFKCKVNNKALPAPVERTISAPTG Heterodimerknob QPREPQVYVLAPHRDELSKNKVSVWCLVKDFYPPD T(130)W IDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSK LTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKS PGK 85 GPSVFIFPPKPKDVLMISRKPEVTCVVVDLGHDDP Exemplaryvariantequine DVQFTWFVDGVETHTATTEPKEEQFNSTYRVVSVL IgG5Fc PIQHQDWLSGKEFKCSVTSKALPAPVERTISKAKG Heterodimerknob QLRVPQVYVLAPHPDELAKNTVSVWCLVKDFYPPE T(130)W IDVEWQSNEHPEPEGKYSTTPAQLNSDGSYFLYSK LSVETSRWKQGESFTCGVMHEAVENHYTQKNVSHS PGK 86 RPSVFIFPPNPKDTLMISRTPEVTCVVVDVSQENP Exemplaryvariantequine DVKFNWYVDGVEAHTATTKAKEKQDNSTYRVVSVL IgG6Fc PIQHQDWRRGKEFKCKVNNRALPAPVERTITKAKG Heterodimerknob ELQDPKVYILAPHREEVTKNTVSVWCLVKDFYPPD T(130)W INVEWQSNEEPEPEVKYSTTPAQLDGDGSYFLYSK LTVETDRWEQGESFTCVVMHEAIRHTYRQKSITNF PGK 87 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplaryvariantequine DVQFNWYVDGVETHTATTEPKQEQNNSTYRVVSIL IgG7Fc AIQHKDWLSGKEFKCKVNNQALPAPVQKTISKPTG Heterodimerknob QPREPQVYVLAPHPDELSKNKVSVWCLVKDFYPPD T(130)W IDIEWKSNGQPEPETKYSTTPAQLDGDGSYFLYSK LTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKS PGK 88 GPSVFIFPPNPKDTLMITRTPEVTCVVVDVSQENP Exemplaryvariantequine DVKFNWYMDGVEVRTATTRPKEEQFNSTYRVVSVL IgG1Fc RIQHQDWLSGKEFKCKVNNQALPQPIERTITKTKG Heterodimerhole RSQEPQVYVLAPHPDELSKSKVSVSCAVKDFYPPE T(130)S INIEWQSNGQPELETKYSTTQAQQDSDGSYFLYSK L(132)A LSVDRNRWQQGTTFTCGVMHEALHNHYTQKNVSKN PGK 89 GPSVFIFPPNPKDALMISRTPVVTCVVVNLSDQYP Exemplaryvariantequine DVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVL IgG2Fc PIQHQDWLSGKEFKCSVINVGVPQPISRAISRGKG Heterodimerhole PSRVPQVYVLPPHPDELAKSKVSVSCAVKDFYPPD T(130)S ISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSK L(132)A LSLETSRWQQVESFTCAVMHEALHNHFIKTDISES LGK 90 GPSVFIFPPKPKDVLMITRMPEVTCLVVDVSHDSS Exemplaryvariantequine DVLFTWYVDGTEVKTAKTMPNEEQNNSTYRVVSVL IgG3Fc RIQHQDWLNGKKFKCKVNNQALPAPVERTISKATG Heterodimerhole QTRVPQVYVLAPHPDELSKNKVSVSCAVKDFYPPD T(130)S ITVEWQSNEHPEPEGKYRTTEAQKDSDGSYFLYSK L(132)A LTVEKDRWQQGTTFTCVVMHEALHNHVMQKNISKN PGK 91 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplaryvariantequine DVQFNWYVDGVETHTATTEPKQEQFNSTYRVVSVL IgG4Fc PIQHKDWLSGKEFKCKVNNKALPAPVERTISAPTG Heterodimerhole QPREPQVYVLAPHRDELSKNKVSVSCAVKDFYPPD T(130)S IDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSK L(132)A LTVETNRWQQGTTFACAVMHEALHNHYTEKSVSKS PGK 92 GPSVFIFPPKPKDVLMISRKPEVTCVVVDLGHDDP Exemplaryvariantequine DVQFTWFVDGVETHTATTEPKEEQFNSTYRVVSVL IgG5Fc PIQHQDWLSGKEFKCSVTSKALPAPVERTISKAKG Heterodimerhole QLRVPQVYVLAPHPDELAKNTVSVSCAVKDFYPPE T(130)S IDVEWQSNEHPEPEGKYSTTPAQLNSDGSYFLYSK L(132)A LSVETSRWKQGESFTCGVMHEAVENHYTQKNVSHS PGK 93 RPSVFIFPPNPKDTLMISRTPEVTCVVVDVSQEN Exemplaryvariantequine PDVKFNWYVDGVEAHTATTKAKEKQDNSTYRVVS IgG6Fc VLPIQHQDWRRGKEFKCKVNNRALPAPVERTITK Heterodimerhole AKGELQDPKVYILAPHREEVTKNTVSVSCAVKDF T(130)S YPPDINVEWQSNEEPEPEVKYSTTPAQLDGDGSY L(132)A FLYSKLTVETDRWEQGESFTCVVMHEAIRHTYRQ KSITNFPGK 94 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplaryvariantequine DVQFNWYVDGVETHTATTEPKQEQNNSTYRVVSIL IgG7Fc AIQHKDWLSGKEFKCKVNNQALPAPVQKTISKPTG Heterodimerhole QPREPQVYVLAPHPDELSKNKVSVSCAVKDFYPPD T(130)S IDIEWKSNGQPEPETKYSTTPAQLDGDGSYFLYSK L(132)A LTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKS PGK 95 GPSVFIFPPNPKDTLMITRTPEVTCVVVDVSQENP Exemplaryvariantequine DVKFNWYMDGVEVRTATTRPKEEQFNSTYRVVSVL IgG1Fc RIQHQDWLSGKEFKCKVNNQALPQPIERTITKTKG Heterodimerhole RSQEPQVYVLAPHPDELSKSKVSVSCAVKDFYPPE T(130)S INIEWQSNGQPELETKYSTTQAQQDSDGSYFLTSK L(132)A LSVDRNRWQQGTTFTCGVMHEALHNHYTQKNVSKN Y(173)T PGK 96 GPSVFIFPPNPKDALMISRTPVVTCVVVNLSDQYP Exemplaryvariantequine DVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVL IgG2Fc PIQHQDWLSGKEFKCSVTNVGVPQPISRAISRGKG Heterodimerhole PSRVPQVYVLPPHPDELAKSKVSVSCAVKDFYPPD T(130)S ISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLTSK L(132)A LSLETSRWQQVESFTCAVMHEALHNHFIKTDISES Y(173)T LGK 97 GPSVFIFPPKPKDVLMITRMPEVTCLVVDVSHDSS Exemplaryvariantequine DVLFTWYVDGTEVKTAKTMPNEEQNNSTYRVVSVL IgG3Fc RIQHQDWLNGKKFKCKVNNQALPAPVERTISKATG Heterodimerhole QTRVPQVYVLAPHPDELSKNKVSVSCAVKDFYPPD T(130)S ITVEWQSNEHPEPEGKYRTTEAQKDSDGSYFLTSK L(132)A LTVEKDRWQQGTTFTCVVMHEALHNHVMQKNISKN Y(173)T PGK 98 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplaryvariantequine DVQFNWYVDGVETHTATTEPKQEQFNSTYRVVSVL IgG4Fc PIQHKDWLSGKEFKCKVNNKALPAPVERTISAPTG Heterodimerhole QPREPQVYVLAPHRDELSKNKVSVSCAVKDFYPPD T(130)S IDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLTSK L(132)A LTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKS Y(173)T PGK 99 GPSVFIFPPKPKDVLMISRKPEVTCVVVDLGHDDP Exemplaryvariantequine DVQFTWFVDGVETHTATTEPKEEQFNSTYRVVSVL IgG5Fc PIQHQDWLSGKEFKCSVTSKALPAPVERTISKAKG Heterodimerhole QLRVPQVYVLAPHPDELAKNTVSVSCAVKDFYPPE T(130)S IDVEWQSNEHPEPEGKYSTTPAQLNSDGSYFLTSK L(132)A LSVETSRWKQGESFTCGVMHEAVENHYTQKNVSHS Y(173)T PGK 100 RPSVFIFPPNPKDTLMISRTPEVTCVVVDVSQENP Exemplaryvariantequine DVKFNWYVDGVEAHTATTKAKEKQDNSTYRVVSVL IgG6Fc PIQHQDWRRGKEFKCKVNNRALPAPVERTITKAKG Heterodimerhole ELQDPKVYILAPHREEVTKNTVSVSCAVKDFYPPD T(130)S INVEWQSNEEPEPEVKYSTTPAQLDGDGSYFLTSK L(132)A LTVETDRWEQGESFTCVVMHEAIRHTYRQKSITNF Y(173)T PGK 101 GPSVFIFPPKPKDVLMISRTPTVTCVVVDVGHDFP Exemplaryvariantequine DVQFNWYVDGVETHTATTEPKQEQNNSTYRVVSIL IgG7Fc AIQHKDWLSGKEFKCKVNNQALPAPVQKTISKPTG Heterodimerhole QPREPQVYVLAPHPDELSKNKVSVSCAVKDFYPPD T(130)S IDIEWKSNGQPEPETKYSTTPAQLDGDGSYFLTSK L(132)A LTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKS Y(173)T PGK 102 SGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAE CanineIL4RECDcanine LRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDD IgG-BFcknob AVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPG NLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVN VSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASY SARVRAWAQTYNSTWSDWSPSITWLNYYEPGGGSG GGGSGGGGSGGGGSGGGGSGPAPEMLGGPSVFIFP PKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFV DGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWL KGKQFTCKVNNKALPSPIERTISKARGQAHQPSVY VLPPSREELSKNTVSLWCLIKDFFPPDIDVEWQSN GQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRW QRGDTFICAVMHEALHNHYTQESLSHSPGK 103 TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT CanineIL13RECDcanine LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV IgG-BFchole GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT GGGSGGGGSGGGGSGGGGSGGGGSGPAPEMLGGPS VFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQ ISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIG HQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLSCAIKDFFPPDIDV EWQSNGQQEPESKYRTTPPQLDEDGSYFLTSKLSV DKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK 104 SGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAE CanineIL4RECDcanine LRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDD IgG-BFchole AVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPG NLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVN VSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASY SARVRAWAQTYNSTWSDWSPSTTWLNYYEPGGGSG GGGSGGGGSGGGGSGGGGSGPAPEMLGGPSVFIFP PKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFV DGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWL KGKQFTCKVNNKALPSPIERTISKARGQAHQPSVY VLPPSREELSKNTVSLSCAIKDFFPPDIDVEWQSN GQQEPESKYRTTPPQLDEDGSYFLTSKLSVDKSRW QRGDTFICAVMHEALHNHYTQESLSHSPGK 105 TETQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT CanineIL13RECDcanine LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV IgG-BFcknob GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETND IFYVEEAKCQNSEFEGNLEGTICFMVPGVLPDTLN TVRIRVRTNKLCYEDDKLWSNWSQAMSIGENTDPT GGGSGGGGSGGGGSGGGGSGGGGSGPAPEMLGGPS VFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQ ISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIG HQDWLKGKQFTCKVNNKALPSPIERTISKARGQAH QPSVYVLPPSREELSKNTVSLWCLIKDFFPPDIDV EWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSV DKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK 106 SGSVKVLRAPTCFSDYFSTSVCQWNMDAPTNCSAE FelineIL4RECDfeline LRLSYQLNFMGSENRTCVPENGEGAACACSMLMDD IgG-2Fcknob FVEADVYQLHLWAGTQLLWSGSFKPSSHVKPRAPG NLTVHPNVSHTWLLRWSNPYPPENHLHAELTYMVN ISSEDDPTDVSVCASGFLCHLLGLRRVETGAPGAR LPPWLCAPRPRRVPGSQCAVISCCRWVLIALTSRG GRWRLTPGLRSQTRYVSVAEGLFGATPRVLCPGTQ AGLASAAREQMSPDPSAFHSIDYEPGGGSGGGGSG GGGSGGGGSGGGGSGPKTASTIESKTGEGPKCPVP EIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLG PDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRV VSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTIS KAKGQPHEPQVYVLPPTQEELSENKVSVWCLIKGF HPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYF LYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKS LTQSPGK 107 SQTQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT FelineIL13RECDfeline LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV IgG-2Fchole GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCENIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETHD IFYVEEAKCQNSEFEGNLEGTICFMVPGILPDTLN TVRIRVRTNKLCYEDDRLWSNWSQAMSIGENTDPT GGGSGGGGSGGGGSGGGGSGGGGSGPKTASTIESK TGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTP EVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPR EEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKS LPSAMERTISKAKGQPHEPQVYVLPPTQEELSENK VSVSCAIKGFHPPDIAVEWEITGQPEPENNYQTTP PQLDSDGTYFLTSRLSVDRSHWQRGNTYTCSVSHE ALHSHHTQKSLTQSPGK 108 SGSVKVLRAPTCFSDYFSTSVCQWNMDAPTNCSAE FelineIL4RECDfeline LRLSYQLNFMGSENRTCVPENGEGAACACSMLMDD IgG-2Fchole FVEADVYQLHLWAGTQLLWSGSFKPSSHVKPRAPG NLTVHPNVSHTWLLRWSNPYPPENHLHAELTYMVN ISSEDDPTDVSVCASGFLCHLLGLRRVETGAPGAR LPPWLCAPRPRRVPGSQCAVISCCRWVLIALTSRG GRWRLTPGLRSQTRYVSVAEGLFGATPRVLCPGTQ AGLASAAREQMSPDPSAFHSIDYEPGGGSGGGGSG GGGSGGGGSGGGGSGPKTASTIESKTGEGPKCPVP EIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLG PDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRV VSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTIS KAKGQPHEPQVYVLPPTQEELSENKVSVSCAIKGF HPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYF LTSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKS LTQSPGK 109 SQTQPPVTNLSVSVENLCTVIWTWDPPEGASPNCT FelineIL13RECDfeline LRYFSHFDNKQDKKIAPETHRSKEVPLNERICLQV IgG-2Fcknob GSQCSTNESDNPSILVEKCTPPPEGDPESAVTELQ CVWHNLSYMKCTWLPGRNTSPDTNYTLYYWHSSLG KILQCENIYREGQHIGCSFALTNLKDSSFEQHSVQ IVVKDNAGKIRPSFNIVPLTSHVKPDPPHIKRLFF QNGNLYVQWKNPQNFYSRCLSYQVEVNNSQTETHD IFYVEEAKCQNSEFEGNLEGTICFMVPGILPDTLN TVRIRVRTNKLCYEDDRLWSNWSQAMSIGENTDPT GGGSGGGGSGGGGSGGGGSGGGGSGPKTASTIESK TGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTP EVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPR EEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKS LPSAMERTISKAKGQPHEPQVYVLPPTQEELSENK VSVWCLIKGFHPPDIAVEWEITGQPEPENNYQTTP PQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHE ALHSHHTQKSLTQSPGK 110 SGSVKVLHLTACFSDYISASTCEWKMDRPTNCSAQ EquineIL4RECDequine LRLSYQLNDEFSDNLTCIPENREDEVCVCRMLMDN IgG-2Fcknob IVSEDVYELDLWAGNQLLWNSSFKPSRHVKPRAPQ NLTVHAISHTWLLTWSNPYPLKNHLWSELTYLVNI SKEDDPTDFKIYNVTYMDPTLRVTASTLKSRATYS ARVKARAQNYNSTWSEWSPSTTWHNYYEQPGGGSG GGGSGGGGSGGGGSGGGGSGGPSVFIFPPNPKDAL MISRTPVVTCVVVNLSDQYPDVQFSWYVDNTEVHS AITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKC SVTNVGVPQPISRAISRGKGPSRVPQVYVLPPHPD ELAKSKVSVWCLVKDFYPPDISVEWQSNRWPELEG KYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFT CAVMHEALHNHFTKTDISESLGK 111 TESQPPVTNLSVSVENLCTVIWTWNPPEGVSPNCS EquineIL13RECDequine LWYFSHFGNKQDKKIAPETHRSKEVPLNERICLQV IgG-2Fchole GSQCSTNESDNPSILVEKCISPPEGDPESAVTELQ CVWHNLSYMKCTWLPGKNASPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTEVKDSIFEQHSVQ IMVKDNAGKIRPFFNIVPLTSHVKPDPPHIKKLFF QNGDLYVQWKNPQNFYSRCLSYQVEVNNSQTETRD IFSVEEAKCQNPEFEGDLEGTICFMVPGVLPDTVN TVRIRVKTNKLCYEDDKLWSNWSQAMSIGKKADPT GGGSGGGGSGGGGSGGGGSGGGGSGGPSVFIFPPN PKDALMISRTPVVTCVVVNLSDQYPDVQFSWYVDN TEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSG KEFKCSVTNVGVPQPISRISRGKGPSRVPQVYVL PPHPDELAKSKVSVSCAVKDFYPPDISVEWQSNRW PELEGKYSTIPAQLDGDGSYFLTSKLSLETSRWQQ VESFTCAVMHEALHNHFTKTDISESLGK 112 SGSVKVLHLTACFSDYISASTCEWKMDRPINCSAQ EquineIL4RECDequine LRLSYQLNDEFSDNLTCIPENREDEVCVCRMLMDN IgG-2Fchole IVSEDVYELDLWAGNQLLWNSSFKPSRHVKPRAPQ NLTVHAISHTWLLTWSNPYPLKNHLWSELTYLVNI SKEDDPTDFKIYNVTYMDPTLRVTASTLKSRATYS ARVKARAQNYNSTWSEWSPSTTWHNYYEQPGGGSG GGGSGGGGSGGGGSGGGGSGGPSVFIFPPNPKDAL MISRTPVVTCVVVNLSDQYPDVQFSWYVDNTEVHS AITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKC SVTNVGVPQPISRAISRGKGPSRVPQVYVLPPHPD ELAKSKVSVSCAVKDFYPPDISVEWQSNRWPELEG KYSTTPAQLDGDGSYFLTSKLSLETSRWQQVESFT CAVMHEALHNHFTKTDISESLGK 113 TESQPPVTNLSVSVENLCTVIWTWNPPEGVSPNCS EquineIL13RECDequine LWYFSHFGNKQDKKIAPETHRSKEVPLNERICLQV IgG-2Fcknob GSQCSTNESDNPSILVEKCISPPEGDPESAVTELQ CVWHNLSYMKCTWLPGKNASPDTNYTLYYWHSSLG KILQCEDIYREGQHIGCSFALTEVKDSIFEQHSVQ IMVKDNAGKIRPFFNIVPLTSHVKPDPPHIKKLFF QNGDLYVQWKNPQNFYSRCLSYQVEVNNSQTETRD IFSVEEAKCQNPEFEGDLEGTICFMVPGVLPDTVN TVRIRVKTNKLCYEDDKLWSNWSQAMSIGKKADPT GGGSGGGGSGGGGSGGGGSGGGGSGGPSVFIFPPN PKDALMISRTPVVTCVVVNLSDQYPDVQFSWYVDN TEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSG KEFKCSVTNVGVPQPISRAISRGKGPSRVPQVYVL PPHPDELAKSKVSVWCLVKDFYPPDISVEWQSNRW PELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQ VESFTCAVMHEALHNHFTKTDISESLGK
DESCRIPTION OF THE EMBODIMENTS
(6) IL13R/IL4R heterodimeric proteins that bind canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4 are provided. In some embodiments, the IL13R/IL4R heterodimeric protein comprises a first contiguous polypeptide comprising an extracellular domain of an IL13R polypeptide and an Fc polypeptide and a second contiguous polypeptide comprising an extracellular domain of an IL4R polypeptide and an Fc polypeptide. Methods of producing or purifying IL13R/IL4R heterodimeric proteins and contiguous polypeptides are also provided. Methods of treatment using IL13R/IL4R heterodimeric proteins to bind IL13 and/or IL4 and inhibit IL13- and/or IL-4-mediated signaling are provided. Such methods include, but are not limited to, methods of treating IL13- and/or IL4-induced conditions in companion animal species. Methods of detecting IL13 and/or IL4 in a sample from a companion animal species are also provided.
(7) For the convenience of the reader, the following definitions of terms used herein are provided.
(8) As used herein, numerical terms such as Kd are calculated based upon scientific measurements and, thus, are subject to appropriate measurement error. In some instances, a numerical term may include numerical values that are rounded to the nearest significant figure.
(9) As used herein, a or an means at least one or one or more unless otherwise specified. As used herein, the term or means and/or unless specified otherwise. In the context of a multiple dependent claim, the use of or when referring back to other claims refers to those claims in the alternative only.
(10) Exemplary IL13R/IL4R Heterodimeric Proteins
(11) Novel IL13R/IL4R heterodimeric proteins are provided, for example, heterodimeric proteins that bind canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4.
(12) Amino acid sequence, means a sequence of amino acids residues in a peptide or protein. The terms polypeptide and protein are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present disclosure, a polypeptide refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
(13) The term contiguous polypeptide herein is used to mean an uninterrupted sequence of amino acids. A contiguous polypeptide is typically translated from a single continuous DNA sequence. It can be made by genetic engineering, for example, by removing the stop codon from the DNA sequence of the first protein, then appending the DNA sequence of the second protein in frame, so that the DNA sequence is expressed as a single protein, Typically, this is accomplished by cloning a cDNA into an expression vector in frame with an existing gene
(14) IL4R, as used herein, is a polypeptide comprising the entirety or a fragment of IL4 receptor subunit alpha that bind to IL-4.
(15) For example, IL4R refers to an IL4R polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. In some embodiments, IL4R is an extracellular domain fragment that binds IL4. In some such embodiments, the IL4R may be referred to as an IL4R extracellular domain (ECD). In some embodiments, IL4R comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
(16) IL13R, as used herein, is a polypeptide comprising the entirety or a portion of IL13 receptor subunit alpha-1 that binds to IL-13.
(17) For example, IL13R refers to an IL13R polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. In some embodiments, IL13R is an extracellular domain fragment that binds IL13. In some such embodiments, the IL13R may be referred to as an IL13R extracellular domain (ECD). In some embodiments, the IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36.
(18) The term companion animal species refers to an animal suitable to be a companion to humans. In some embodiments, a companion animal species is a small mammal, such as a canine, feline, dog, cat, horse, rabbit, ferret, guinea pig, rodent, etc. In some embodiments, a companion animal species is a farm animal, such as a horse, cow, pig, etc.
(19) An extracellular domain (ECD) is the portion of a polypeptide that extends beyond the transmembrane domain into the extracellular space. The term extracellular domain, as used herein, may comprise a complete extracellular domain or may comprise a truncated extracellular domain missing one or more amino acids, that binds to its ligand. The composition of the extracellular domain may depend on the algorithm used to determine which amino acids are in the membrane. Different algorithms may predict, and different systems may express, different extracellular domains for a given protein.
(20) An extracellular domain of an IL4R polypeptide may comprise a complete extracellular domain or a truncated extracellular domain of IL4R that binds IL4. As used herein, the terms extracellular domain of an IL4R polypeptide, IL4R ECD, and similar terms refer to an IL4R polypeptide that does not comprise a transmembrane domain or cytoplasmic domain, even if the term follows an open transitional word, such as comprising, comprises, and the like. In some embodiments, an extracellular domain of an IL4R polypeptide is an extracellular domain of an IL4R polypeptide derived from a companion species animal. For example, in some embodiments, an extracellular domain of an IL4R polypeptide is derived from canine IL4R, feline IL4R or equine IL4R. In some embodiments, an extracellular domain of an IL4R polypeptide comprises the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, or any fragment thereof. In some embodiments, an extracellular domain of an IL4R polypeptide comprises the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, or any fragment thereof.
(21) An extracellular domain of an IL13R polypeptide may comprise a complete extracellular domain or a truncated extracellular domain of IL13R that binds IL13. As used herein, the terms extracellular domain of an IL13R polypeptide, IL13R ECD, and similar terms refer to an IL13R polypeptide that does not comprise a transmembrane domain or cytoplasmic domain, even if the term follows an open transitional word, such as comprising, comprises, and the like. In some embodiments, an extracellular domain of an IL13R polypeptide is an extracellular domain of an IL13R polypeptide derived from a companion species animal. For example, in some embodiments, an extracellular domain of an IL13R polypeptide is derived from canine IL13R, feline IL13R or equine IL13R. In some embodiments, an extracellular domain of an IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26, or any fragment thereof. In some embodiments, an extracellular domain of an IL13R polypeptide comprises the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36, or any fragment thereof.
(22) The terms IL13R/IL4R heterodimeric protein and IL4R/IL13R heterodimeric protein are used interchangeably to refer to a heterodimeric protein comprising a first contiguous polypeptide comprising an IL13R polypeptide and a second contiguous polypeptide comprising an IL4R polypeptide.
(23) In some embodiments, the first contiguous polypeptide and/or second contiguous polypeptide comprises an Fc polypeptide.
(24) The IL13R/IL4R heterodimeric protein of the invention may comprise an extracellular domain of a IL13R polypeptide and/or an extracellular domain of a IL4R polypeptide, wherein the polypeptides are derived from a companion animal species. For example, a heterodimeric protein may comprise an extracellular domain of an IL4R polypeptide from a dog, cat, or horse and/or may comprise an extracellular domain of an IL13R polypeptide from a dog, cat, or horse.
(25) Wild-type refers to a non-mutated version of a polypeptide that occurs in nature, or a fragment thereof. A wild-type polypeptide may be produced recombinantly. A wildtype IL13R ECD or a wildtype IL4R ECD refers to a protein having an amino acid sequence that is identical to the same portion of an extracellular domain of an IL13R or IL4R that occurs in nature.
(26) A variant is a nucleic acid molecule or polypeptide that differs from a referent nucleic acid molecule or polypeptide by single or multiple amino acid substitutions, deletions, and/or additions and substantially retains at least one biological activity of the referent nucleic acid molecule or polypeptide.
(27) A biologically active entity, or an entity having biological activity, is an entity having any function related to or associated with a metabolic or physiological process, and/or having structural, regulatory, or biochemical functions of a naturally-occurring molecule. Biologically active polynucleotide fragments are those exhibiting similar activity, but not necessarily identical, to an activity of a polynucleotide of the present invention. A biologically active polypeptide or fragment thereof includes one that can participate in a biological reaction, including, but not limited to, a ligand-receptor interaction or antigen-antibody binding. The biological activity can include an improved desired activity, or a decreased undesirable activity. An entity may demonstrate biological activity when it participates in a molecular interaction with another molecule, such as hybridization, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, when it has diagnostic and/or prognostic value in determining the presence of a molecule, such as a biologically active fragment of a polynucleotide that may be detected as unique for the polynucleotide molecule, and when it can be used as a primer in a polymerase chain reaction (PCR).
(28) As used herein, percent (%) amino acid sequence identity and homology with respect to a nucleic acid molecule or polypeptide sequence are defined as the percentage of nucleotide or amino acid residues in a referent sequence that are identical with the nucleotide or amino acid residues in the specific nucleic acid molecule or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of sequences being compared.
(29) In some embodiments, a variant has at least about 50% sequence identity with the referent nucleic acid molecule or polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, deleted, at the N- or C-terminus of the polypeptide. In some embodiments, a variant has at least about 50% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 98% sequence identity with the sequence of the referent nucleic acid or polypeptide.
(30) In some embodiments, a contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide having at least 85%, at least 90%, at least 95%, at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36. In some embodiments, a contiguous polypeptide comprises an extracellular domain of an IL4R polypeptide having at least 85%, at least 90%, at least 95%, at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
(31) As used herein, position corresponding to position n, wherein n is any number, refers to an amino acid position of a subject polypeptide that aligns with position n of a reference polypeptide after aligning the amino acid sequences of the subject and reference polypeptides and introducing gaps. Alignment for purposes of whether a position of a subject polypeptide corresponds with position n of a reference polypeptide can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, CLUSTAL OMEGA, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any parameters needed to achieve maximal alignment over the full length of two sequences being compared. In some embodiments, the subject polypeptide and the reference polypeptide are of different lengths.
(32) In some embodiments, the contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine at a position corresponding to position 18 of SEQ ID NO: 22, at a position corresponding to position 18 of SEQ ID NO: 24, or at a position corresponding to position 18 of SEQ ID NO: 26. In some embodiments, the contiguous polypeptide comprises an extracellular domain of an IL13R polypeptide comprising a cysteine at position 18 of SEQ ID NO: 22, at position 18 of SEQ ID NO: 24, at position 18 of SEQ ID NO: 26, at position 15 of SEQ ID NO: 32, at position 15 of SEQ ID NO: 34, or at position 15 of SEQ ID NO: 36.
(33) A point mutation is a mutation that involves a single nucleotide or amino acid residue. The mutation may be the loss of a nucleotide or amino acid, substitution of one nucleotide or amino acid residue for another, or the insertion of an additional nucleotide or amino acid residue.
(34) An amino acid substitution may include but is not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 2. Amino acid substitutions may be introduced into a molecule of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC or enhanced pharmacokinetics.
(35) TABLE-US-00002 TABLE 2 Original Exemplary Residue Substitutions Ala (A) Val; Leu; Ile Arg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp; Lys; Arg Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu; Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) Thr Thr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V) Ile; Leu; Met; Phe; Ala; Norleucine
(36) Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
(37) Non-conservative substitutions will entail exchanging a member of one of these classes with another class.
(38) A fusion partner, as used herein, refers to an additional component of an IL13R/IL4R contiguous polypeptide, such as an additional polypeptide, such as albumin, an albumin binding fragment, or a fragment of an immunoglobulin molecule. A fusion partner may comprise an oligomerization domain such as an Fc domain of a heavy chain immunoglobulin.
(39) The term IgX Fc or IgX Fc polypeptide means the Fc region is derived from a particular antibody isotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where X denotes the antibody isotype. Thus, IgG or IgG Fc denotes the Fc region of a chain, IgA or IgA Fc denotes the Fc region of an chain, IgD or IgD Fc denotes the Fc region of a chain, IgE or IgE Fc denotes the Fc region of an chain, IgM or IgM Fc denotes the Fc region of a chain, etc.
(40) In some embodiments, the Fc polypeptide or the IgG Fc region comprises CH1, hinge, CH2, CH3, and CL1. In some embodiments, the IgG Fc polypeptide comprises the hinge, CH2, and CH3, but does not comprise CH1 or CL. In some embodiments, the IgG Fc polypeptide comprises CH2 and CH3, but does not comprise CH1, the hinge, or CL. In some embodiments, the IgG Fc polypeptide comprises CH1, hinge, CH2, and CH3, with or without CL1.
(41) IgXN Fc or IgXN Fc polypeptide denotes that the Fc region is derived from a particular subclass of antibody isotype (such as canine IgG subclass A, B, C, or D; feline IgG subclass 1, 2a, or 2b; or equine IgG subclass IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7, etc.), where N denotes the subclass.
(42) In some embodiments, IgX or IgXN regions are derived from a companion animal, such as a dog, a cat, or a horse. In some embodiments, IgG regions are isolated from canine heavy chains, such as IgGA, IgGB, IgGC, or IgGD. In some instances, IgG Fc regions are isolated from feline heavy chains, such as IgG1a, IgG1b, or IgG2. In other instances, IgG regions are isolated from equine heavy chains, such as IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, or IgG7. Polypeptides comprising an Fc region of IgGA, IgGB, IgGC, or IgGD may provide for higher expression levels in recombination production systems.
(43) In some embodiments, a contiguous polypeptide comprises a first variant IgG Fc polypeptide comprising a knob mutation and a second variant IgG Fc polypeptide comprising a hole mutation. Nonlimiting exemplary knob and hole mutations are described, for example, in Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat Biotechnol, 16(7):677-81 (1998).
(44) A knob mutation, as used herein, refers to an interfacing mutation of a molecule (e.g., an Fc polypeptide) that comprises a bulky amino acid.
(45) A hole mutation, as used herein, refers to an interfacing mutation of a molecule (e.g., an Fc polypeptide) that comprises one or more smaller amino acids.
(46) In some embodiments, a variant IgG Fc polypeptide comprises a knob mutation. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39, position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(47) In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(48) In some embodiments, a variant IgG Fc polypeptide comprises a tryptophan at a position corresponding to position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41, or position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(49) In some embodiments, a variant IgG Fc polypeptide comprises a tryptophan at position 138 of SEQ ID NO: 38; position 137 of SEQ ID NO: 39; position 137 of SEQ ID NO: 40; position 138 of SEQ ID NO: 41; position 154 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(50) In some embodiments, a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 54, 55, 56, 57, 66, 67, 68, 69, 70, 81, 82, 83, 84, 85, 86, or 87.
(51) In some embodiments, a variant IgG Fc polypeptide comprises a hole mutation. In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at a position corresponding to position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40; position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; and/or position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(52) In some embodiments, a variant IgG Fc polypeptide comprises an amino acid substitution at position 138 and/or position 140 and/or position 181 of SEQ ID NO: 38; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 39; position 137 and/or position 139 and/or position 180 of SEQ ID NO: 40; position 138 and/or position 140 and/or position 181 of SEQ ID NO: 41; position 154 and/or position 156 and/or position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or position 130 and/or position 132 and/or position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(53) In some embodiments, a variant IgG Fc polypeptide comprises a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position corresponding to position 181 of SEQ ID NO: 38; a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 39; a serine at a position corresponding to position 137 and/or an alanine at a position corresponding to position 139 and/or a threonine at a position corresponding to position 180 of SEQ ID NO: 40; a serine at a position corresponding to position 138 and/or an alanine at a position corresponding to position 140 and/or a threonine at a position corresponding to position 181 of SEQ ID NO: 41; a serine at a position corresponding to position 154 and/or an alanine at a position corresponding to position 156 and/or a threonine at a position corresponding to position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or a serine at a position corresponding to position 130 and/or an alanine at a position corresponding to position 132 and/or a threonine at a position corresponding to position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(54) In some embodiments, a variant IgG Fc polypeptide comprises a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 38; a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 39; a serine at position 137 and/or an alanine at position 139 and/or a threonine at position 180 of SEQ ID NO: 40; a serine at position 138 and/or an alanine at position 140 and/or a threonine at position 181 of SEQ ID NO: 41; a serine at position 154 and/or an alanine at position 156 and/or a threonine at position 197 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46; or a serine at position 130 and/or an alanine at position 132 and/or a threonine at position 173 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.
(55) In some embodiments, a variant IgG Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 101.
(56) A signal sequence refers to a sequence of amino acid residues or polynucleotides encoding such, which facilitates secretion of a polypeptide of interest and is typically cleaved upon export of the polypeptide to the outside of the cell surface membrane.
(57) A linker refers to one or more amino acid residues that connects a first polypeptide with a second polypeptide.
(58) In some embodiments, the linker is a glycine-rich and/or serine-rich, flexible, non-structural linker. In some embodiments, a linker comprises the amino acids G (Gly) and/or S (Ser). For example, a linker may comprise G or a repeat of G (e.g., GG, GGG, etc.); GS or a repeat of GS (e.g., GSGS (SEQ ID NO: 114), GSGSGS (SEQ ID NO: 115), etc.); GGS or a repeat of GGS (e.g., GGSGGS (SEQ ID NO: 116), GGSGGSGGS (SEQ ID NO: 117), etc.); GGGS (SEQ ID NO: 118) or a repeat of GGG-S(SEQ ID NO: 119) (e.g., GGGSGGGS (SEQ ID NO: 120), GGGSGGGSGGGS (SEQ ID NO: 121), etc.); GSS or a repeat of GSS (e.g., GSSGSS (SEQ ID NO: 122), GSSGSSGSS (SEQ ID NO: 123), etc.); or GGSS (SEQ ID NO: 124) or a repeat of GGSS (SEQ ID NO: 125) (e.g., GGSSGGSS (SEQ ID NO: 126) GGSSGGSSGGSS (SEQ ID NO: 127), etc.).
(59) In some embodiments, the contiguous polypeptide comprises at least one linker. In some embodiments, the contiguous polypeptide comprises an optional signal sequence, and at least one optional linker. In some embodiments, the contiguous polypeptide does not comprise a signal sequence, or a linker. In some embodiments, the contiguous polypeptide is translated with a signal sequence, but the signal sequence is cleaved from the contiguous polypeptide.
(60) In some embodiments, a heterodimeric protein comprises a) a first contiguous polypeptide comprising at least one IL13R extracellular domain (ECD) and a first Fc polypeptide and b) a second contiguous polypeptide comprising at least one IL4R ECD and a second Fc polypeptide, wherein the IL13R ECD and/or the IL4R ECD are derived from a companion animal species.
(61) In some embodiments, a first contiguous polypeptide or a second contiguous polypeptide has the formula:
IL13R(n)-L-Fc or
IL4R(n)-L-Fc, wherein IL13R(n) is at least one IL13R extracellular domain (ECD) polypeptide derived from a companion animal species, IL4R(n) is at least one IL4R ECD polypeptide derived from a companion animal species, (n) is one, two, three, four, or more ECD polypeptides, L is an optional linker, Fc is a variant Fc polypeptide, such as a variant Fc polypeptide comprising knob or a hole mutation.
(62) In addition, other binding partner(s) may be included in the contiguous polypeptide before, after, and/or between any one or more IL13R or IL4R ECD polypeptide(s). Other potential binding partners include: IL5, IL6, IL17, IL22, IL31, LFA-1, TNF-, TSLP, and/or IgE.
(63) In some embodiments, the heterodimeric protein comprises a first contiguous polypeptide comprising the amino acid sequence of SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113.
(64) In some embodiments, the heterodimeric protein comprises a second contiguous polypeptide comprising the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
(65) Exemplary Expression and Production
(66) Polynucleotide sequences that encode all or part (e.g., the extracellular domain) of a contiguous polypeptide with or without a signal sequence are provided. If a homologous signal sequence (i.e., a signal sequence of native IL-4R or IL13R) is not used in the construction of the nucleic acid molecule, then another signal sequence may be used, for example, any one of the signal sequences described in PCT/US06/02951.
(67) Typically, nucleotide sequence encoding the polypeptide of interest, such as a contiguous polypeptide, is inserted into an expression vector, suitable for expression in a selected host cell.
(68) A vector is a plasmid that can be used to transfer DNA sequences from one organism to another or to express a gene of interest. A vector typically includes an origin of replication and regulatory sequences which regulate the expression of the gene of interest, and may or may not carry a selective marker gene, such as an antibiotic resistance gene. A vector is suitable for the host cell in which it is to be expressed. A vector may be termed a recombinant vector when the gene of interest is present in the vector.
(69) A host cell refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Nonlimiting exemplary mammalian cells include, but are not limited to, NS0 cells, PER.C6 cells (Crucell), 293 cells, and CHO cells, and their derivatives, such as 293-6E, DG44, CHO-S, and CHO-K cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) encoding an amino acid sequence(s) provided herein.
(70) The term isolated as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as isolated when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be isolating the polypeptide. Similarly, a polynucleotide is referred to as isolated when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as isolated.
(71) In some embodiments, the heterodimeric protein or contiguous polypeptide is isolated using chromatography, such as size exclusion chromatography, ion exchange chromatography, protein A column chromatography, hydrophobic interaction chromatography, and CHT chromatography.
(72) The terms label and detectable label mean a moiety attached to a IL13R/IL4R contiguous polypeptide to render it detectable. In some embodiments, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (for example, .sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, or .sup.153Sm); chromogens, fluorescent labels (for example, FITC, rhodamine, lanthanide phosphors), enzymatic labels (for example, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (for example, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, for example, acridinium compounds, and moieties that produce fluorescence, for example, fluorescein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety.
(73) Exemplary IL13R/IL4R Heterodimeric Proteins as Decoy Receptor Traps
(74) The IL13R/IL4R heterodimeric proteins of the invention can function as decoy receptors for trapping IL13 and/or IL4 and inhibiting their interaction with IL13R and/or IL4R on cell surfaces. Decoy receptors, such as those of the invention, recognize their ligands with high affinity and specificity but are structurally incapable of signaling. They compete with wild-type receptors for ligand binding and participate in ligand/receptor interactions, thus modulating the activity of or the number of functioning receptors and/or the cellular activity downstream from the receptors. Decoy receptors can act as molecular traps for agonist ligands and thereby inhibit ligand-induced receptor activation.
(75) IL13 as used herein refers to any native IL13 that results from expression and processing of IL13 in a cell. The term includes IL13 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. The term also includes naturally occurring variants of IL13, e.g., splice variants or allelic variants.
(76) In some embodiments, a canine IL13 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, a feline IL13 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, an equine IL13 comprises the amino acid sequence of SEQ ID NO: 6.
(77) IL4 as used herein refers to any native IL4 that results from expression and processing of IL4 in a cell. The term includes IL4 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), and companion animals (e.g., dogs, cats, and equine), unless otherwise indicated. The term also includes naturally occurring variants of IL4, e.g., splice variants or allelic variants.
(78) In some embodiments, a canine IL4 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, a feline IL4 comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, an equine IL4 comprises the amino acid sequence of SEQ ID NO: 3.
(79) The invention provides IL13R/IL4R heterodimeric proteins as therapeutic agents. The IL13R/IL4R heterodimeric proteins of the invention bind to IL13 and/or IL4, described in more detail herein, which have been demonstrated to be associated with allergic diseases. In various embodiments, IL13R/IL4R heterodimeric proteins can bind IL13 and/or IL4 with very high affinity. In various embodiments, the IL13R/IL4R heterodimeric proteins can interfere with IL13 and/or IL4 signaling.
(80) The term affinity means the strength of the sum total of noncovalent interactions between a single binding site of a molecule (for example, a receptor) and its binding partner (for example, a ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K.sub.D). Affinity can be measured by common methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), or surface plasmon resonance devices.
(81) The terms K.sub.D, K.sub.d, Kd or Kd value as used interchangeably to refer to the equilibrium dissociation constant of a receptor fusion-ligand interaction. In some embodiments, the K.sub.d of the fusion molecule to its ligand is measured by using biolayer interferometry assays using a biosensor, such as an Octet System (Pall ForteBio LLC, Fremont, CA) according to the supplier's instructions. Briefly, biotinylated antigen is bound to the sensor tip and the association of fusion molecule is monitored for ninety seconds and the dissociation is monitored for 600 seconds. The buffer for dilutions and binding steps is 20 mM phosphate, 150 mM NaCl, pH 7.2. A buffer only blank curve is subtracted to correct for any drift. The data are fit to a 2:1 binding model using ForteBio data analysis software to determine association rate constant (k.sub.on), dissociation rate constant (k.sub.off), and the K.sub.d. The equilibrium dissociation constant (K.sub.d) is calculated as the ratio of k.sub.off/k.sub.on. The term k.sub.on refers to the rate constant for association of a molecule X to its partner Y and the term k.sub.off refers to the rate constant for dissociation of a molecule X or partner Y from the molecule X/partner Y complex.
(82) The term binds to a substance is a term that is well understood in the art, and methods to determine such binding are also well known in the art. A molecule is said to exhibit binding if it reacts, associates with, or has affinity for a particular cell or substance and the reaction, association, or affinity is detectable by one or more methods known in the art, such as, for example, immunoblot, ELISA KD, KinEx A, biolayer interferometry (BLI), surface plasmon resonance devices, or etc.
(83) Surface plasmon resonance denotes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26.
(84) Biolayer interferometry refers to an optical analytical technique that analyzes the interference pattern of light reflected from a layer of immobilized protein on a biosensor tip and an internal reference layer. Changes in the number of molecules bound to the biosensor tip cause shifts in the interference pattern that can be measured in real-time. A nonlimiting exemplary device for biolayer interferometry is an Octet system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal. Biochem. 377: 209-277.
(85) In some embodiments, an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, or equine IL13 and/or IL4 with a dissociation constant (Kd) of less than 510.sup.6 M, less than 110.sup.6 M, less than 510.sup.7M, less than 110.sup.7M, less than 510.sup.8 M, less than 110.sup.8 M, less than 510.sup.9 M, less than 110.sup.9 M, less than 510.sup.10 M, less than 110.sup.10 M, less than 510.sup.11 M, less than 110.sup.11 M, less than 510.sup.12 M, or less than 110.sup.12M, as measured by biolayer interferometry. In some embodiments, an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, or equine IL13 and/or IL4 with a Kd of between 510.sup.6 M and 110.sup.6 M, between 510.sup.6 M and 510.sup.7M, between 510.sup.6 M and 110.sup.7 M, between 510.sup.6 M and 510.sup.8 M, 510.sup.6 M and 110.sup.8 M, between 510.sup.6 M and 510.sup.9 M, between 510.sup.6 M and 110.sup.9 M, between 510.sup.6 M and 510.sup.10 M, between 510.sup.6 M and 110.sup.10 M, between 510.sup.6 M and 510.sup.11M, between 510.sup.6 M and 110.sup.11 M, between 510.sup.6 M and 510.sup.12M, between 510.sup.6 M and 110.sup.12M, between 110.sup.6 M and 510.sup.7 M, between 110.sup.6 M and 110.sup.7 M, between 110.sup.6 M and 510.sup.8M, 110.sup.6 M and 110.sup.8M, between 110.sup.6 M and 510.sup.9M, between 110.sup.6 M and 110.sup.9 M, between 110.sup.6 M and 510.sup.10 M, between 110.sup.6 M and 110.sup.10 M, between 110.sup.6 M and 510.sup.11M, between 110.sup.6M and 110.sup.11M, between 110.sup.6M and 510.sup.12 M, between 110.sup.6 M and 110.sup.12M, between 510.sup.7 M and 110.sup.7 M, between 510.sup.7M and 510.sup.8M, 510.sup.7M and 110.sup.8M, between 510.sup.7M and 510.sup.9M, between 510.sup.7M and 110.sup.9 M, between 510.sup.7M and 510.sup.10 M, between 510.sup.7 M and 110.sup.10 M, between 510.sup.7M and 510.sup.11 M, between 510.sup.7 M and 110.sup.11M, between 510.sup.7 M and 510.sup.12M, between 510.sup.7M and 110.sup.12M, between 110.sup.7M and 510.sup.8 M, 110.sup.7 M and 110.sup.8 M, between 110.sup.7 M and 510.sup.9 M, between 110.sup.7 M and 110.sup.9 M, between 110.sup.7 M and 510.sup.10 M, between 110.sup.7 M and 110.sup.10 M, between 110.sup.7 M and 510.sup.11 M, between 110.sup.7 M and 110.sup.11 M, between 110.sup.7 M and 510.sup.12 M, between 110.sup.7 M and 110.sup.12 M, between 510.sup.8M and 110.sup.8M, between 510.sup.8M and 510.sup.9M, between 510.sup.8M and 110.sup.9 M, between 510.sup.8M and 510.sup.10 M, between 510.sup.8M and 110.sup.10 M, between 510.sup.8M and 510.sup.11 M, between 510.sup.8 M and 110.sup.11 M, between 510.sup.8M and 510.sup.12M, between 510.sup.8M and 110.sup.12 M, 110.sup.8M and 510.sup.9M, between 110.sup.8M and 110.sup.9 M, between 110.sup.8M and 510.sup.10 M, between 110.sup.8 M and 110.sup.10 M, between 110.sup.8M and 510.sup.11 M, between 110.sup.8 M and 110.sup.11 M, between 110.sup.8 M and 510.sup.12M, between 110.sup.8M and 110.sup.12M, between 510.sup.9M and 110.sup.9M, between 510.sup.9 M and 510.sup.10 M, between 510.sup.9 M and 110.sup.10 M, between 510.sup.9 M and 510.sup.11M, between 510.sup.9 M and 110.sup.11 M, between 510.sup.9 M and 510.sup.12 M, between 510.sup.9 M and 110.sup.12 M, between 110.sup.9 M and 510.sup.10 M, between 110.sup.9 M and 110.sup.10 M, between 110.sup.9M and 510.sup.11 M, between 110.sup.9M and 110.sup.11 M, between 110.sup.9M and 510.sup.12 M, between 110.sup.9M and 110.sup.12 M, between 510.sup.10 M and 110.sup.10 M, between 510.sup.10 M and 510.sup.11 M, between, 110.sup.10 M and 510.sup.11 M, 110.sup.10 M and 110.sup.11 M, between 110.sup.10 M and 510.sup.12 M, between 110.sup.10 M and 110.sup.12 M, between 510.sup.11 M and 110.sup.12 M, between 510.sup.11 M and 510.sup.12 M, between 510.sup.11 M and 110.sup.12 M, between 110.sup.11 M and 510.sup.12 M, or between 110.sup.11 M and 110.sup.12 M, as measured by biolayer interferometry. In some embodiments, an IL13R/IL4R heterodimeric protein binds to canine IL13 and/or IL4, feline IL13 and/or IL4, and/or equine IL13 and/or IL4.
(86) To reduce or inhibit means to decrease, reduce, or arrest an activity, function, or amount as compared to a reference. In some embodiments, by reduce or inhibit is meant the ability to cause an overall decrease of 20% or greater. In some embodiments, by reduce or inhibit is meant the ability to cause an overall decrease of 50% or greater. In some embodiments, by reduce or inhibit is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time. A reference as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy or non-diseased sample. In some examples, a reference is obtained from a non-diseased or non-treated sample of a companion animal. In some examples, a reference is obtained from one or more healthy animals of a particular species, which are not the animal being tested or treated.
(87) The term substantially reduced, as used herein, denotes a sufficiently high degree of reduction between a numeric value and a reference numeric value such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values. In some embodiments, the substantially reduced numeric values is reduced by greater than about any one of 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.
(88) In some embodiments, an IL13R/IL4R heterodimeric protein may reduce IL13 and/or IL4 signaling in a companion animal species by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL13 and/or IL4 signaling in the absence of the fusion molecule. In some embodiments, signaling is measured by a reduction in IL4-dependent TF-1 cell proliferation. In some embodiments, the reduction in IL13 and/or IL4 signaling or the reduction in proliferation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, between 25% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, between 40% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.
(89) Exemplary Pharmaceutical Compositions
(90) The terms pharmaceutical formulation and pharmaceutical composition refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.
(91) A pharmaceutically acceptable carrier refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a pharmaceutical composition for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin, canine or other animal albumin; buffers such as phosphate, citrate, tromethamine or HEPES buffers; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, or magnesium trisilicate; polyvinyl pyrrolidone, cellulose-based substances; polyethylene glycol; sucrose; mannitol; or amino acids including, but not limited to, arginine.
(92) The pharmaceutical composition can be stored in lyophilized form. Thus, in some embodiments, the preparation process includes a lyophilization step. The lyophilized composition may then be reformulated, typically as an aqueous composition suitable for parenteral administration, prior to administration to the dog, cat, or horse. In other embodiments, particularly where the fusion molecule is highly stable to thermal and oxidative denaturation, the pharmaceutical composition can be stored as a liquid, i.e., as an aqueous composition, which may be administered directly, or with appropriate dilution, to the dog, cat, or horse. A lyophilized composition can be reconstituted with sterile Water for Injection (WFI). Bacteriostatic reagents, such benzyl alcohol, may be included. Thus, the invention provides pharmaceutical compositions in solid or liquid form.
(93) The pH of the pharmaceutical compositions may be in the range of from about pH 5 to about pH 8, when administered. The compositions of the invention are sterile if they are to be used for therapeutic purposes. Sterility can be achieved by any of several means known in the art, including by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Sterility may be maintained with or without anti-bacterial agents.
(94) Exemplary Uses of IL13R/IL4R Heterodimeric Proteins and Pharmaceutical Compositions
(95) The IL13R/IL4R heterodimeric proteins or pharmaceutical compositions comprising the IL13R/IL4R heterodimeric proteins of the invention may be useful for treating an IL13- and/or IL4-induced condition. As used herein, an IL13 or IL4-induced condition means a disease associated with, caused by, or characterized by, elevated levels or altered distribution of IL13 or IL4. Such IL13 and/or IL4-induced conditions include, but are not limited to, a pruritic or an allergic disease. In some embodiments, the IL13- and/or IL4-induced condition is atopic dermatitis, pruritus, asthma, psoriasis, scleroderma, or eczema. An IL13- or IL4-induced condition may be exhibited in a companion animal, including, but not limited to, canine, feline, or equine.
(96) As used herein, treatment is an approach for obtaining beneficial or desired clinical results. Treatment as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a companion animal. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). Also encompassed by treatment is a reduction of pathological consequence of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one-hundred percent removal of all aspects of the disorder.
(97) In some embodiments, an IL13R/IL4R heterodimeric proteins or pharmaceutical compositions comprising it can be utilized in accordance with the methods herein to treat IL13- or IL4-induced conditions. In some embodiments, an IL13R/IL4R heterodimeric proteins or pharmaceutical compositions is administered to a companion animal, such as a canine, a feline, or equine, to treat an IL13- and IL4-induced condition.
(98) A therapeutically effective amount of a substance/molecule, agonist or antagonist may vary according to factors such as the type of disease to be treated, the disease state, the severity and course of the disease, the type of therapeutic purpose, any previous therapy, the clinical history, the response to prior treatment, the discretion of the attending veterinarian, age, sex, and weight of the animal, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the animal. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
(99) In some embodiments, IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered parenterally, by subcutaneous administration, intravenous infusion, or intramuscular injection. In some embodiments, an IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered as a bolus injection or by continuous infusion over a period of time. In some embodiments, an IL13R/IL4R heterodimeric protein or pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein is administered by an intramuscular, an intraperitoneal, an intracerebrospinal, a subcutaneous, an intra-arterial, an intrasynovial, an intrathecal, or an inhalation route.
(100) An IL13R/IL4R heterodimeric protein described herein may be administered in an amount in the range of 0.1 mg/kg body weight to 100 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 0.1 mg/kg body weight to 50 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 1 mg/kg body weight to 10 mg/kg body weight per dose. In some embodiments, an IL13R/IL4R heterodimeric protein may be administered in an amount in the range of 0.5 mg/kg body weight to 100 mg/kg body, in the range of 1 mg/kg body weight to 100 mg/kg body weight, in the range of 5 mg/kg body weight to 100 mg/kg body weight, in the range of 10 mg/kg body weight to 100 mg/kg body weight, in the range of 20 mg/kg body weight to 100 mg/kg body weight, in the range of 50 mg/kg body weight to 100 mg/kg body weight, in the range of 1 mg/kg body weight to 10 mg/kg body weight, in the range of 5 mg/kg body weight to 10 mg/kg body weight, in the range of 0.5 mg/kg body weight to 10 mg/kg body weight, or in the range of 5 mg/kg body weight to 50 mg/kg body weight.
(101) An IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein can be administered to a companion animal at one time or over a series of treatments. For example, IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein may be administered at least once, more than once, at least twice, at least three times, at least four times, or at least five times.
(102) In some embodiments, the dose is administered once per week for at least two or three consecutive weeks, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more weeks of no treatment. In other embodiments, the therapeutically effective dose is administered once per day for two to five consecutive days, and in some embodiments, this cycle of treatment is repeated two or more times, optionally interspersed with one or more days or weeks of no treatment.
(103) Administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order. The term concurrently is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered with a time separation of no more than about a specified number of minutes. The term sequentially is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s), or wherein administration of one or more agent(s) begins before the administration of one or more other agent(s). For example, administration of the two or more therapeutic agents are administered with a time separation of more than about a specified number of minutes. As used herein, in conjunction with refers to administration of one treatment modality in addition to another treatment modality. As such, in conjunction with refers to administration of one treatment modality before, during or after administration of the other treatment modality to the animal.
(104) In some embodiments, the method comprises administering in combination with an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein, a Jak inhibitor, a PI3K inhibitor, an AKT inhibitor, or a MAPK inhibitor. In some embodiments, the method comprises administering in combination with an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R contiguous polypeptide, an anti-IL17 antibody, an anti-TNF antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD25 antibody, an anti-IL31 antibody, an anti-IL23 antibody, an anti-IgE antibody, an anti-CD11 antibody, anti-IL6R antibody, anti-4-Intergrin antibody, an anti-IL12 antibody, an anti-IL1 antibody, or an anti-BlyS antibody.
(105) Provided herein are methods of exposing to a cell an IL13R/IL4R heterodimeric protein or a pharmaceutical composition comprising an IL13R/IL4R heterodimeric protein under conditions permissive for binding to IL13 and/or IL4. In some embodiments, the cell is exposed to the IL13R/IL4R heterodimeric protein or pharmaceutical composition ex vivo. In some embodiments, the cell is exposed to the IL13R/IL4R heterodimeric protein or pharmaceutical composition in vivo. In some embodiments, a cell is exposed to the IL13R/IL4R heterodimeric protein. In some embodiments, a cell is exposed to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition under conditions permissive for binding of the heterodimeric protein to extracellular IL13 and/or IL4. In some embodiments, a cell may be exposed in vivo to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition by any one or more of the administration methods described herein, including but not limited to, intraperitoneal, intramuscular, intravenous injection into the subject. In some embodiments, a cell may be exposed ex vivo to the IL13R/IL4R heterodimeric protein or the pharmaceutical composition by exposing the cell to a culture medium comprising the heterodimeric protein or the pharmaceutical composition. In some embodiments, the permeability of the cell membrane may be affected using any number of methods understood by those of skill in the art (such as electroporating the cells or exposing the cells to a solution containing calcium chloride) before exposing the cell to a culture medium comprising the fusion molecule or the pharmaceutical composition.
(106) In some embodiments, the exposure results in a reduction of IL13 and/or IL4 signaling function by the cell. In some embodiments, an IL13R/IL4R heterodimeric protein may reduce IL13 and/or IL4 signaling in a cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to IL13 and/or IL4 signaling function in the absence of the IL13R/IL4R heterodimeric protein. In some embodiments, the reduction in IL13 and/or IL4 signaling and/or the reduction in TF-1 proliferation is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 10% and 60%, between 10% and 70%, between 10% and 80%, between 10% and 90%, between 10% and 100%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 15% and 60%, between 15% and 70%, between 15% and 80%, between 15% and 90%, between 15% and 100%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 40%, between 20% and 45%, between 20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and 80%, between 20% and 90%, between 20% and 100%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 25% and 45%, between 25% and 50%, between 25% and 60%, between 25% and 70%, between 25% and 80%, between 25% and 90%, between 25% and 100%, between 30% and 35%, between 30% and 40%, between 30% and 45%, between 30% and 50%, between 30% and 60%, between 30% and 70%, between 30% and 80%, between 30% and 90%, between 30% and 100%, between 35% and 40%, between 35% and 45%, between 35% and 50%, between 35% and 60%, between 35% and 70%, between 35% and 80%, between 35% and 90%, between 35% and 100%, between 40% and 45%, between 40% and 50%, between 40% and 60%, between 40% and 70%, between 40% and 80%, between 40% and 90%, between 40% and 100%, between 45% and 50%, between 45% and 60%, between 45% and 70%, between 45% and 80%, between 45% and 90%, between 45% and 100%, between 50% and 60%, between 50% and 70%, between 50% and 80%, between 50% and 90%, between 50% and 100%, between 60% and 70%, between 60% and 80%, between 60% and 90%, between 60% and 100%, between 70% and 80%, between 70% and 90%, between 70% and 100%, between 80% and 90%, between 80% and 100%, or between 90% and 100%.
(107) Provided herein are methods of using the IL13R/IL4R heterodimeric protein, polypeptides and polynucleotides for detection, diagnosis and monitoring of an IL13- or IL4-induced condition. Provided herein are methods of determining whether a companion animal will respond to IL13R/IL4R heterodimeric protein therapy. In some embodiments, the method comprises detecting whether the animal has cells that express IL13 or IL4 using an IL13R/IL4R heterodimeric protein. In some embodiments, the method of detection comprises contacting the sample with an antibody, polypeptide, or polynucleotide and determining whether the level of binding differs from that of a reference or comparison sample (such as a control). In some embodiments, the method may be useful to determine whether the IL13R/IL4R heterodimeric protein described herein are an appropriate treatment for the subject animal.
(108) In some embodiments, the sample is a biological sample. The term biological sample means a quantity of a substance from a living thing or formerly living thing. In some embodiments, the biological sample is a cell or cell/tissue lysate. In some embodiments, the biological sample includes, but is not limited to, blood, (for example, whole blood), plasma, serum, urine, synovial fluid, and epithelial cells.
(109) In some embodiments, the cells or cell/tissue lysate are contacted with an IL13R/IL4R heterodimeric protein and the binding between the IL13R/IL4R heterodimeric protein and the cell is determined. When the test cells show binding activity as compared to a reference cell of the same tissue type, it may indicate that the subject would benefit from treatment with an IL13R/IL4R heterodimeric protein. In some embodiments, the test cells are from tissue of a companion animal.
(110) Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays which can be conducted include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). An indicator moiety, or label group, can be attached to the subject antibodies and is selected to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. Appropriate labels include, without limitation, radionuclides (for example .sup.125I, .sup.131I, .sup.35S, .sup.3H, or .sup.32P), enzymes (for example, alkaline phosphatase, horseradish peroxidase, luciferase, or p-galactosidase), fluorescent moieties or proteins (for example, fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (for example, Qdot nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). General techniques to be used in performing the various immunoassays noted above are known to those of ordinary skill in the art.
(111) For purposes of diagnosis, the IL13R/IL4R heterodimeric protein can be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to polypeptides are known in the art. In some embodiments, the IL13R/IL4R heterodimeric protein need not be labeled, and the presence thereof can be detected, for example, using an antibody that binds to the IL13R/IL4R heterodimeric protein. In some embodiments, the IL13R/IL4R heterodimeric protein can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). The anti-IL13 and IL4 antibodies and polypeptides can also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody or the polypeptide is labeled with a radionuclide (such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I, .sup.3H, or any other radionuclide label, including those outlined herein) so that the cells or tissue of interest can be localized using immunoscintiography. The IL13R/IL4R heterodimeric protein may also be used as staining reagent in pathology using techniques well known in the art.
(112) In some embodiments, a IL13R/IL4R heterodimeric protein is used for a diagnostic and a IL13R/IL4R heterodimeric protein is used as a therapeutic. In some embodiments, the first and second IL13R/IL4R heterodimeric proteins are different.
(113) The following examples illustrate particular aspects of the disclosure and are not intended in any way to limit the disclosure.
EXAMPLES
Example 1
(114) Expression and Purification of Canine IL4 and IL13
(115) A nucleotide sequence encoding canine IL13 protein (SEQ ID NO: 4) was synthesized with poly-His tag on the C-terminal end and cloned into a mammalian expression vector and transfected to 293 cells or CHOS. The same method was used to clone and express a nucleotide sequence encoding canine IL4 protein (SEQ ID NO: 1) with a poly-His tag on the C-terminal end.
(116) The supernatant containing canine IL13 protein was collected and filtered. Canine IL13 was affinity purified using Ni-NTA column (CaptivA Protein A Affinity Resin, Repligen). The same method was used to purify canine IL4.
Example 2
(117) Extracellular Domains of IL13R and IL4R
(118) Extracellular domains of canine, feline, and equine IL4R that are responsible for binding canine, feline and equine IL4 and/or IL13 were identified and boundaries were defined. Full-length extracellular domains of canine IL4R, feline IL4R, and equine IL4 were identified as SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27, respectively. Extracellular domain fragments of canine IL4R, feline IL4R, and equine IL4R postulated to retain biological activity were identified as SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 37, respectively.
(119) Extracellular domains of canine, feline, and equine IL13R that are responsible for binding canine, feline, and equine IL4 and/or IL13 were identified and boundaries were defined. Full length extracellular domains of canine IL13R, feline IL13R, and equine IL13R were identified as SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, respectively. Extracellular domain fragments of canine IL13R, feline IL13R, and equine IL13R postulated to retain biological activity were identified as SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, respectively.
(120) An unpaired cysteine (Cys) in canine IL13R (at position 18 of SEQ ID NO: 22), feline IL13R (at position 18 of SEQ ID NO: 24), and equine IL13R (at position 18 of SEQ ID NO: 26) was identified informatically and determined as embedded (unexposed) based on 3-D modeling. It is unlikely that the unpaired cysteine will form disulfide bonds and the likelihood of aggregation is low. Thus, site-directed mutagenesis of this Cys residue was not introduced.
Example 3
(121) Expression and Purification of Canine IL13R/IL4R Contiguous Polypeptides from CHO Cells
(122) Nucleotide sequences encoding canine IL13R ECD/IL4R ECD contiguous polypeptides linked to an IgGB Fc polypeptide were designed with a signal sequence. For contiguous polypeptide IL13RECD-IL4RECD-IgGB Fc (SEQ ID NO: 20), an extracellular domain of IL13R (SEQ ID NO: 22) precedes an extracellular domain of IL4R (SEQ ID NO: 23). For contiguous polypeptide IL4RECD-IL13RECD-IgGB Fc (SEQ ID NO: 21), an extracellular domain of IL4R precedes an extracellular domain of IL13R.
(123) The nucleotide sequences were synthesized chemically and inserted into an expression vector suitable for transfection into a CHO host cell. After transfection into CHO cells, the fusion proteins were secreted from the cell. For example, fusion protein was purified by single step Protein A column chromatography.
(124) Each of IL13RECD-IL4RECD-IgGB Fc and IL4RECD-IL13RECD-IgGB Fc may be expressed and purified in a single step with a protein A column or other chromatographic methods, such as ion exchange column chromatography, hydrophobic interaction column chromatography, mixed mode column chromatography such as CHT, or multimodal mode column chromatography such as CaptoMMC. Low pH or other viral inactivation and viral removal steps can be applied. The purified protein may be admixed with excipients, and sterilized by filtration to prepare a pharmaceutical composition of the invention. The pharmaceutical composition may be administered to a dog with an atopic dermatitis or asthma in an amount sufficient to bind and/or inhibit either IL13 and/or IL4.
(125) The vectors were then used to perform pilot-scale transfection in CHO-S cells using the FreestyleMax transfection reagent (Life Technologies). The supernatant was harvested by clarifying the conditioned media. Protein was purified with a single pass Protein A chromatography step and used for further investigation.
Example 4
(126) Demonstration of IL13 and IL4 Binding Activity
(127) This example demonstrates that both IL13RECD-IL4RECD-IgGB Fc (SEQ ID NO:20) and IL4RECD-IL13RECD-IgGB Fc (SEQ ID NO:21) bind canine IL4 and IL13 with kinetics requisite for therapeutic activity.
(128) The binding analysis was performed using a biosensor Octet as follows. Briefly, canine IL4 (produced using 293 cells) was biotinylated. The free unreacted biotin was removed from biotinylated IL4 by extensive dialysis. Biotinylated canine IL4 was captured on streptavidin sensor tips. The IL4 association with various concentrations (12, 16, and 44 nM) of IL13RECD-IL4RECD-IgGB Fc (SEQ ID NO:20) was monitored for ninety seconds. Dissociation was monitored for 600 seconds. A buffer only blank curve was subtracted to correct for any drift. The data were fit to a 1:1 binding model using ForteBio data analysis software to determine the k.sub.on, k.sub.off, and the Kd. The buffer for dilutions and all binding steps was: 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd for IL13RECD-IL4RECD-IgGB Fc and ligand IL4 was 810.sup.11.
(129) The canine IL4 association with various concentrations (40.7, and 140 nM) of IL4RECD-IL13RECD-IgGB Fc (SEQ ID NO:21) was monitored for ninety seconds. Dissociation was monitored for 600 seconds. A buffer only blank curve was subtracted to correct for any drift. The data were fit to a 1:1 binding model using ForteBio data analysis software to determine the k.sub.on, k.sub.off, and the Kd. The buffer for dilutions and all binding steps was: 20 mM phosphate, 150 mM NaCl, pH 7.2. The Kd for IL4RECD-IL13RECD-IgGB Fc and ligand IL4 was 1.110.sup.11.
(130) Canine IL4 and canine IL13 with C-terminal polyHis tag was expressed and purified from 293 cells. EZ-Link NHS-LC-biotin was obtained from Thermo Scientific (Cat. #21336), and Streptavidin biosensors was obtained from ForteBio (Cat. #18-509).
(131) IL4 and IL13 sequential binding experiments with IL13R-IL4R-IgGB (SEQ ID NO:20) were performed. Biotinylated canine IL13R-IL4R-IgGB was captured on streptavidin sensor tips. Canine IL13R-IL4R-IgGB was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4 using concentrations of 30 g/mL of IL4 and IL13 in PBS (
(132) IL4 and IL13 sequential binding experiments with IL4R-IL13R-IgGB (SEQ ID NO:21) were performed. Biotinylated canine IL4R-IL13R-IgGB was captured on streptavidin sensor tips. Canine IL4R-IL13R-IgGB was exposed to either (1) canine IL4 followed by IL13 or (2) canine IL13 followed by IL4 using concentrations of 30 ug/mL of IL4 and IL13 in PBS (
(133) The tight binding of IL13RECD-IL4RECD-IgGB Fc and IL4RECD-IL13RECD-IgGB Fc to IL4 or IL13 is thought to be due to simultaneous binding contributions made by both IL4RECD and IL13RECD.
Example 5
(134) Cellular Functional Activity of Canine IL4RECD-IL13RECD-Fc (SINK)
(135) TF1 cells (ATCC cat #CRL-2003), a human Erythroleukemia cell line which expresses endogenous interleukin 4 receptors on cell surface, was used in a proliferation assay. Cells grown in RPMI1640 (Gibco, Cat #11875) supplemented with 10% Fetal Bovine Serum, heat inactivated (Sigma, Cat #2868) and 2 nM/ml Human GM-CSF (R&D System, Cat #215-GM-010) at exponential growth phase were used for the assay. Cells were washed with PBS twice and resuspended in above medium without GM-CSF. 20,000 cells per well were plated in a 96-well plate (Corning, Cat #3610). Canine IL4RECD-IL13RECD-IgGB Fc (SINK) was added at a series of dilutions followed by addition of canine IL4 (Sino Biological Inc, Cat #70021-DNAE-5) at 50 ng/ml. The cells were incubated in 37 C., 5% CO2 for 48 hours in a total volume of 100 l. At the end of the incubation, the cells were cooled in room temperature and assayed for proliferation/variability by measuring cellular ATP content using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat #G7570).
(136) In this assay, 100 l premixed reagent A and B were added to each well. After shaking on an orbital shaker for 2 mins, the cells were lysed. Mono-oxygenation of luciferin was catalyzed by luciferase in the presence of Mg2+ and ATP that presented in cells, resulting in the generation of a luminescent signal proportional to the amount of ATP in the cells. The amount of ATP is directly proportional to the number of cells present in culture. The plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal and luminescence was detected using a Synergy HT microplate reader (Biotek, Winooski, VT).
(137) The data were analyzed using 4 parameter logistic fit and IC50 is 2.0 nM. See
Example 6
(138) Canine, Feline, and Equine IgG Fc Polypeptides for IL31R and IL4R Heterodimeric Proteins
(139) Pairs of variant canine IgG Fc polypeptides, variant feline IgG Fc polypeptides, and variant equine IgG Fc polypeptides were designed such that a knob-in-hole heterodimerization approach may be used to prepare heterodimeric proteins comprising at least one IL31R ECD and at least one IL4R ECD. First, pairing of two Fc polypeptides was designed by introducing CH3 interfacing mutations so that a first Fc polypeptide comprises a bulky amino acid (knob) and a second Fc polypeptide comprises smaller amino acids in the same general location (hole).
(140) An amino acid substitution of threonine to tryptophan at a position corresponding to position 138 of canine IgG-A (SEQ ID NO: 38), at a position corresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 39), at a position corresponding to position 137 of canine IgG-C Fc (SEQ ID NO: 40), or at a position corresponding to position 138 of canine IgG-D Fc (SEQ ID NO: 41) (T138W or T137W) can be introduced as a knob. Examples of amino acid sequences of a first variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptide comprising a knob mutation are SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively.
(141) An amino acid substitution of threonine to serine at a position corresponding to position 138 and/or of leucine to alanine at a position corresponding to position 140 and/or of tyrosine to threonine at a position corresponding to position 180 of canine IgG-A (SEQ ID NO: 38) or of IgG-D (SEQ ID NO: 41) (T138S, L140A, and/or Y180T); or of threonine to serine at a position corresponding to position 137 and/or of leucine to alanine at a position corresponding to position 139 and/or of tyrosine to threonine at a position corresponding to position 179 of canine IgG-B Fc (SEQ ID NO: 39) or of IgG-C(SEQ ID NO: 40) (T137S, L139A, and/or Y179T) can be introduced as a hole. Examples of amino acid sequences of a second variant canine IgG-A, IgG-B, IgG-C, and IgG-D Fc polypeptides comprising a hole mutation are SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65.
(142) An amino acid substitution of threonine to tryptophan at a position corresponding to position 154 of feline IgG1a Fc (SEQ ID NO: 42 or SEQ ID NO: 43), feline IgG1b Fc (SEQ ID NO: 44 or SEQ ID NO: 45), or of feline IgG2 (SEQ ID NO: 46) (T154W) can be introduced as a knob. Examples of amino acid sequences of a first variant feline IgG1a, and IgG1b, and IgG2 Fc polypeptide comprising a knob mutation are SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.
(143) An amino acid substitution of threonine to serine at a position corresponding to position 154 and/or of leucine to alanine at a position corresponding to position 156 and/or of tyrosine to threonine at a position corresponding to position 197 of feline IgG1a (SEQ ID NO: 42 or SEQ ID NO: 43), feline IgG-b Fc (SEQ ID NO: 44 or SEQ ID NO: 45), or feline IgG2 Fc (SEQ ID NO: 46) (T154S, L156A, and/or Y(197)T) can be introduced as a hole. Examples of amino acid sequences of a second variant feline IgG1a, IgG1b, IgG2 Fc polypeptide comprising a hole mutation are SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80.
(144) An amino acid substitution of threonine to tryptophan at a position corresponding to position 130 of equine IgG1 Fc (SEQ ID NO: 47), of equine IgG2 Fc (SEQ ID NO: 48), of equine IgG3 Fc (SEQ ID NO: 49), of equine IgG4 Fc (SEQ ID NO: 50), of equine IgG5 Fc (SEQ ID NO: 51), of equine IgG6 Fc (SEQ ID NO: 52), or of equine IgG7 Fc (SEQ ID NO: 53) (T130W) can be introduced as a knob. Examples of amino acid sequences of a first variant equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides comprising a knob mutation are SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively.
(145) An amino acid substitution of threonine to serine at a position corresponding to position 130 and/or of leucine to alanine at a position corresponding to position 132 and/or of tyrosine to threonine at a position corresponding to position 173 of equine IgG1 Fc (SEQ ID NO: 47), of equine IgG2 Fc (SEQ ID NO: 48), of equine IgG3 Fc (SEQ ID NO: 49), of equine IgG4 Fc (SEQ ID NO: 50), of equine IgG5 Fc (SEQ ID NO: 51), of equine IgG6 Fc (SEQ ID NO: 52), or of equine IgG7 Fc (SEQ ID NO: 53) (T130W, L(132)A, and/or Y(173)T) can be introduced as a hole. Examples of amino acid sequences of a second variant equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, and IgG7 Fc polypeptides comprising a hole mutation are SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101.
Example 7
(146) IL13R/IL4R ECD Heterodimeric Proteins
(147) In addition to contiguous IL13R/IL4R ECD polypeptide formats, heterodimeric protein pairs may have the following formats: Heterodimeric protein A: Polypeptide 1: IL13R(n)-L-Fc1 and Polypeptide 2: IL4R(n)-L-Fc2; or Heterodimeric protein B: Polypeptide 1: IL4R(n)-L-Fc1 and Polypeptide 2: IL13R(n)-L-Fc2, wherein IL13R(n) is at least one IL13R extracellular domain (ECD) polypeptide derived from a companion animal species, IL4R(n) is at least one IL4R ECD polypeptide derived from a companion animal species, (n) is one, two, three, four, or more ECD polypeptides, L is an optional linker, Fc1 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising knob mutation, Fc2 is a variant Fc polypeptide, such as a variant Fc polypeptide comprising a hole mutation. An optional linker could also be used between multiple ECD polypeptides. In addition, other binding partner(s) may be included before, after, and/or between any one or more ECD polypeptide(s). Other potential binding partners include: IL5, IL6, IL17, IL22, IL31, LFA-1, TNF-, TSLP, and/or IgE.
(148) Examples of pairs of contiguous polypeptide 1 and 2 that may form a heterodimeric protein include SEQ ID NOs 102 and 103, SEQ ID NOs: 104 and 105, SEQ ID NOs: 106 and 107, SEQ ID NOs: 108 and 109, SEQ ID NOs: 110 and 111, and SEQ ID NOs: 112 and 113. A host cell may be cotransfected with vectors expressing these contiguous polypeptide pairs to produce the heterodimeric proteins described.