NZ739130B2

NZ739130B2 - Multivalent and multispecific gitr-binding fusion proteins

Info

Publication number: NZ739130B2
Application number: NZ739130A
Authority: NZ
Inventors: Quinn Deveraux; Brendan P Eckelman; Abrahim Hussain; Kyle S Jones; Amir S Razai; John C Timmer; Katelyn M Willis
Original assignee: Inhibrx Inc
Filing date: 2016-07-22
Publication date: 2023-11-28

Abstract

This disclosure generally provides molecules that specifically engage glucocorticoid-induced TNFR-related protein (GITR), a member of the TNF receptor superfamily (TNFRSF). More specifically, the disclosure relates to multivalent and/or multispecific molecules that bind at least GITR.

Description

Mlli'lfl‘lVALENT ANB hr’lllljl'lSl’EClFlC Gl'T'R—BlNlHNG El?Sl?N PRG'E‘ElNS RELATEEE Al’l’LlCATlONS {Glltlll This application claims the benefit of US, Provisional Application No. streets; filed July 23, , the ts of Whi eh are incorporated herein by reference in their entirety.

GRATION OF SEQUENCE LESTENG {than} The contents ott‘ne text file named "lNlllOZZthllWQ_ST25 tat," which was created on July 22"! 20l t3 and is 209 KB in size; are hereby incorporated by reference in their entirety.

EIELB OF THE lNVENTlON {6993} This sure generally provides molecules that specifically engage glueoeorticold—induced TNFR—related protein (GITRL a member of the TNE receptor superfamily {TNT'RSF}. More ically the disclosure relates to rnultiv'alerit and/or innltispeeitic molecules that bind at least Gl'l‘R BACKGROUNE 0E THE lNVENTlON While} The tumor necrosis factor receptor superrarnily consists of several structurally related cell sur?tee ors Activation by multimerie ligands is a common feature of many ofthese receptors. Many members of 'tlie TNFRSF have therapeutic utility in numerous pathologies? it‘aetivated properly. lmportantly, to properly agoriize this reeeptor family often requires higher order clustering, and conventional nt antibodies are not ideal for this. ore, there exists a therapeutic need for more potent agonist molecules of the TNFESE Stilt/EVIAR‘Y OE THE ENVENTIQN liltltlSl The disclosure provides niultiyalent "l‘NF receptor snperfamily (TNFRSF) binding ?lSlOl’l polypeptides that bind at least Ol’tlCilld—lllélllced TNFR—related protein [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW (GETR, also known as tumor necrosis factor receptor an’iily member l8 (TNFRSFl8) and/or activationwindncihle TNFR family receptor tAlTR». These molecules that bind at least Gills, are referred to herein as l‘trtmgeting molecules" or l'l‘lii~targeti ng fusions or "GlTR—targeting proteins or "‘{i‘lTRstargeting fusion polypeptides" or "GTTR- targeting fusion proteins," in some embodiments, the argeting molecule is a inultivalent molecule, for example, a multivalent GER—targeting fusion protein, in some embodiments, the Gl'l'R—targeting molecule is a specific molecule, for example, a multispecil'ic GER-targeting iusion protein. in $01116 embodiments, the GER—targeting molecule is a multivalent and multispecilic le, for example, a multiyalent and inultispecitie GlT‘thargeting fusion protein As used herein, the term "fusion protein" or "fusion polypeptide" or "GTTR—targeting fusion protein" or "GlTR~targeting fusion ptide," unless otherwise speci?cally denoted, refers to any fusion protein embodiment of the disclosure, ing, but not limited to, multivalent ?rsion proteins, ntultispecitic fusion proteins, or inultiyalent and specifie ?ision proteins.

E?lllltél These GlTR~‘targeting molecules include at least one domain that binds GlTR referred to herein as a "GlTRminding " (GlTlhBD). These GlTRwBBs include a polypeptide sequence that speci?cally binds to Gl'l‘R, in some ments, the GlTR—BD includes a polypeptide sequence that is or is derived from an antibody or antibody fragment including, for example, scFv, Fahs, single domain antibodies tsdAh), VNAR, or V’Hlls. ln some embodiments, the GlTR~BD includes a human or humanized sdAh. {6997} The argeting molecules of the disclosure overcome problems and limitations from convention antibodies that target s of the TNF receptor superfamily ("l‘NFRSFL including Gl'l'R, Conventional antibodies targeting members oftlie 'l‘NFRSF have been shown to require an exogenous crosslinking to achieve sufficient agonist activity; as evidenced by the necessity for Fc—garnma Receptor (FcyRs) for the activity antibodies to Blot, 3R5, Silk and 0X40 awa e: a! ZOOl al Nat. Med. '7, i 0, Li et a," 2008 Drug Dev Res. 69, 69‘32; Pukac et al 2005 Br. 3. Cancer 92, l/Jldll—lll-ilvl; Yanda eta] 2008 Ann, Gncol, l9, liloil—l 067; Yang er a! 2007 Cancer Lett, 25 l : £46457; Bulliard at all 2013: llEi‘s/‘l Zliltg): l635; Bulliard et a! 20% li’nmunol and Cell Biol 92: 475—480). in addition to crosslinlting via Fey-Rs other exogenous agents including addition of the oligomeric ligand 01‘ antibody g entities (e5.4 (1‘ , protein A and secondary antibodies) have he demonstrated to enhance anti—TNFRSF antibody clustering and ream signaling For example, the [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW addition of the DRS ligand TRAE L enhanced the apoptosis ng ability of an anti-DRE antibody (Graves 617.527 2i714 Cancer Cell76: 9) these findings suggest the need tor clustering of "l‘NFRSFs beyond a dimer. {(79578} The present disclosure provides isolated polypeptides that speci?cally hind GlTR, ln some embodiments, the isolated polypeptide is d from antibodies or antibody ii'agnients including scli‘v, Fabs, single domain antibodies (sdAh), \I’NAR, or Vlllis. in some embodiments, the isolated polypeptide is human or humanized sdAh. The sdAh fragments can be derived from Vl-ll-l, VNAR, engineered VH or Vii domains. "Vi-ills can be generated from canielid heavy chain only antibodies. VNARs can be generated from cartilaginous fish heavy chain only antibodies Various s have been implemented to generate monomeric sdAhs from tionally heterodimeric VH and VK domains, including interface engineering and ion ofspecil'lc germlme lainilies. in other embodiments, the ed ptides are derived from non-antibody scaffold proteins for example but not limited to designed anl lin/lipocalins, centyiins and fynoniers.

E6999} in some embodiments, the isolated polypeptide includes an amino acid sequence selected from the group consisting of SEQ ll) NS: l969i7 in some embodiments, the ed polypeptide es an amino acid sequencecS‘clected from the group consisting of SEQ lD Ni): 42-62. in some embodiments, the isolated polypeptide includes an amino acid sequence selected from the group consisting of SEQ EB NU: 63—80, {(7919} in some embodiments, the isolated polypeptide includes an amino acid sequenceethatis at least 509 609,6 6594,7t796, 759,6 8094533 596,9. ‘56 9l94, 996, 93%, 9494;, 95% 9696, 9 ,or 9994:- identical to an amino acid sequence selected from the group consisting of SEQ ll} NO: l9~8t7 in some embodiments, the isolated polypeptide includes an amino acid sequence that is at least 50%, 699-6, 6596, 7096 7594,,79796,859/6, 90%, 9196, 299,6 93‘56, 9494, 959,6 9656, 97946,9996,or 9976 identical to an amino acid sequence selected from the group consisting of SEQ ll) NO: 42—62.. in some embodiments, the isolated polypeptide includes an amino acid sequence that is at least 5996, 6096, 65%, 709i), 759A7 8057/o \590,0. ll/6, 9i0/6, 9296,57 .3 /0, 943/6.I959'Q} 9570/0, 97/69930//o, CFO.946 identical to an amino acid sequence selected from the group consisting of SEQ lD NO: 63—80.

Etl?lll in some ments, the isolated polypeptide comprises a complementarity ining region 7 (CDRl) comprising an amino acid sequence selected from the group consisting lasso in no: ms, 109, ii2, ll’7, izn, i25, isi, ms, 143, 148, and 749; 6 [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW coinplen’rentarity determining region 2 {(313122) con’iprisii’ig an amino acid sequence se1ected from the group consisting ofSEQ 11) NO: 107, 110, 113, 115, 118, 121, 123, 128, 130, 132, 1341, 136., 137, 139, 141, 144, and 14?, and a compiementarity determining region 3 (C1518) comprising an amino acid sequence seiected from the group consisting of SEQ 11) NO: 108, 111,114,116,l19,122,124,126,127,129, 33,135,141},142,145,146, and 150. {111112} The present disc1osure a1so provides multiva1ent 111th} binding fusion ns, which comprise two or more "i‘NFRSF binding domains ("E‘B?st Where at1east one TED binds GER, referred to herein as a (i’iTR—binding domain (631111—813). in some embodiments, the fusion proteins of the present osure have y in treating neop1asnis 1n some embodiments, the fusion proteins ofthe present disclosure bind TNFRSF member expressed on a tumor ceii, for example, at 1east GlTR {111113} in some embodiments, BDS of the present disciosure are derived from antibodies or antibody fragments ineiuding scf'v, f‘abs, sing1e domain antibodies (sdAb), V1mg or VHHs, in some embodiments, the G11‘R~Bf,ls are human or humanized sdAb, The sdAb fragments can be derived from VHH, VNAR, engineered VH or Vb: domains, VHHs can be generated from carnehd heavy chain only antibodies. VNARs can be generated from carti1aginous fish heavy chain only antibodies Various s have been impieinented to generate monomeric sdAbs front conventionaily heterodinieric VH and VK domains, including interface engineering and selection ofspecii'ic germline tarni1ies, in other embodiments, the Gi'l'R—BDS are derived from nonwantihody scatfo1d proteins for examine but not limited to designed ankyrin repeat proteins (darpins’), ayimers, antieaiin/lipocalins, centyrins and iynoiners.

Emu} Genera11y, the niu1tivalent fusion proteins of the present disc1osure ine1ude at least two or more GiTRwBi‘k operably linked Via a linker po1ypeptide, The utilization of sdAb fragments as the ic GiTR~BD sequences within the multiyaient fusion ns of the present disclosure has the benefit of avoiding the heavy chain : 1ight chain mis— pairing problem common to many hi/niu1tispeei?c antibody ches. in on, the niuitivaient fusion proteins of the present disc1osure avoid the use of 1ong linkers necessitated by many bispecific dies. 191115} in some embodiments, the niu1tivalent fusion protein contains two or more different Gi'i‘ibBDs, in some ments, the niu1tivalent fusion n contains three or more different {iiTRsBDs in some embodiments, the mul ti vai ent fusion n ns four or more different (i’ETR—B 131st in some embodiments, the mu1tiva1ent fusion protein [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW contains five or more ent GiTR-BDs, in some en’ihodimei’its, the m uitivaient fusion protein contains six or more different STIR—EDS, {(3916} in some embodiments, the aient fusion protein contains muitipie copies of a (EITR—BI} For example, in some embodiments, the niuitivaient fusion protein contains at least two copies of a GiTR—BD in some diinei’its, the m uitivaient fusion protein contains at ieast three copies ofa Gi'i‘RwBB. in some embodiments, the niuitivaient fusion protein contains at ieast four copies or" a GT5i3R~BD in some embodiments, the niuitivaient fusion protein contains at ieast five copies oi’a GiTR-BD, in some embodiments, the niuitivaient titsion protein contains at least six copies ofa GiTRwBD. in some embodiments, the muitivaient fusion protein contains six or more copies of a GETR— {0917} In some ments, the multivaient tusion protein contains at ieast one Gi'i'R—BD that comprises an amino acid seq eiected from thegroup consisting oi SEQ 13.3 NS: i9—8i’). in some embodiments, the muttivaient fusion protein contains two or more copies ot‘a GTTR—BD that ses an amino acid sequence seiected from the group consisting of SEQ TD NO: 1980. in some embodiments, the muitivaient fusion protein contains three or more copies or" a GT'i‘RBD that comprises an amino acid sequence seiected from the group consisting of SEQ it) Ni}: £980 in some ments, the inuitivaient fusion protein contains four or more copies oi’a GiTR-BD that comprises an amino acid sequence seiected from the group consisting ofSEQ it) NO: 1980. in some embodiments, the muitivaient fusion protein contains five or more copies of a —BD that comprises an amino acid sequence seiected from the group consisting of SEQ ED NO: 19—80. in some embodiments, the vaient fusion protein contains six or more copies of a GiTRrBD that comprises an amino acid sequence selected from the group consisting of SEQ Ti") NO: 19—530, {0918} In some embodiments, the multivaient fusion protein contains at ieast one GHR—8D that comprises an amino acid ce thatis at ieast 50%,60"/Ei, 65°43, 70%, 8,90%' 75%, 809%} 85 5,91% 2%, 93%, 94"A}, 95%, 9 ,%, S97‘32}, 9830, or 99‘"/0 identical to an amino acid sequence‘tsciected from the group consisting of SEQ ID NO: 3.9980, In some embodiments, the niuitivaient fusion protein contains two or more copies of a GiTR—BD that comprises an amino acid sequence that is at ieast 50%, 60%, 65 .4; 70‘3", ’5‘3’5,80‘34; 8.5%, 90%,91‘3", o, 933,,94‘3", 95‘3//,o 96"/o, ) ‘36, 98345, or 99% identical to an amino acid sequence selected from the group consisting of SEQ TD Ni): 19—80. in some embodiments, [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW the tnuitivalent fusion n contains three or more copies oi’a GITR-BD that comprises an amino acid sequence that is at least 5035, . 65%, 70‘35, .5 .4;. 80%,85‘3'5. 90‘34;. 91‘35, 92‘3593‘35,9/-1‘35,95‘3/5.96%, 97‘35, 98‘3/5. or 99% identical to an amino acid sequence 5elected from the group consisting of SEQ ll') N01980 in some embodiments, the multivalent fusion protein contains four or more copies oi’a GITR-BD that comprises an amino acid sequence that is at least 50‘35, 60‘3'5,65‘3/, 4;. 70%, 75%, 8034;. 85%,90‘3'5,_91%. 92%,93‘35. 94%. 95 ‘35, 96%. 97%, 98%, or 99‘3/5 identical to an amino acid sequence selected from the group consisting of SEQ 1D NQ: 19—50. in some embodiments, the multivalent hision protein contains ?ve or more copies ofa G1'l3R~BD that comprises an amino acid sequence that is at least50%. 609/5. 65%,'0‘3x5. 75‘34,30‘35,85‘35. 90%, 9i‘35, ,93‘35,94‘35, 9.5%, 96‘35, 9795, 93%, or 99% identical to an amino acid sequence selected from thegroup ting of SEQE D N0: 1980 in some embodiments, the multivalent fusion protein contains six or more copies of a GlTRwBD that comprises an amino acid seqnenee that is at least 50‘3" ,60‘35,65‘3/5. 70" 5,575" 5,580‘3/5. , ', 90‘35, . 9234;, 93%5, 9475 95‘34; 96"",97':/’5 98‘344,; or 99% identical toan amino acid sequence selected from the group consisting of SEQ 1D NQ: 19—80.

E0019} in some embodiments, the multivaient fusion protein contains at least one GlT‘R—BD that ses an amino acid sequence 5 elected from the group consisting of SEQ 1D NO: 42—62. in some embodiments, the m ultivalent fusion protein contains two or more copies ol’a Gl'i‘R~l3D that comprises an amino acid sequence selected from the group consisting oi SEQ 11.) N0: . in some ments, the ninitivalent fusion protein contains three or more copies of a (ilTR—BD that comprises an amino acid sequence selected from the group consisting of SEQ 1D NQ: 42—62. in some embodiments, the multivalent fusion protein contains four or more copies of a. Gl'l‘R—BD that comprises an amino acid sequence selected from the group consisting of SEQ ll?) NO: 42-62. lu some embodiments, the multivaient tusion protein contains five or more copies of a GER—ED that comprises an amino acid sequence selected from the group consisting of SEQ 113 NS: 4-2562. in some embodiments, the inuitivalent fusion n contains six or more copies of a GITRBD that comprises an amino acid sequence seiected from the group consisting of SEQ 51) ND: 42452.

E09230} in some embodiments, the va1ent1’usion protein contains at least one GlTR—BI) that comprises an amino acid sequence thatis at least 50%, 60‘1/35, 65"‘,4; 70%, 75%, 80%, 45‘3‘5 90%, 91‘35, 92%, 93%, 94‘34 9‘355, 96‘35, 97‘35, 9" ,or 99':/5identica1 to on [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW amino acid sequence seiected from the group consisting of SEQ 11) NO: 42—62. in some embodiments, the niuitivaient hision protein contains two or more copies ofa G1TR~BD that comprises an amino acid sequence thatis at 1east 5(3‘3',613‘%1,65‘311, 713‘3 11,75%,80941, 85‘311,9(3‘341,91‘3’, 9257,11 93‘341,94‘3", 9553/41, 960/11, 3 %, 98‘34'1, or 99‘341identica1 to an amino acid sequence selected from the group consisting of SEQ 1D NQ: 42—62. in some embodiments, the vaient ?rsion protein contains three or more copies of a Gi'i‘R—BD that comprises an amino acid sequence thatis at ieast 1 613341 56%. 71’3‘311 75‘311 84,"41. 85%, 913%, 91‘3’1'1. 92%, 93%, 94‘41, 95%, 96%, 97‘41, 98%, or 99% identica1 to an amino acid sequence seiected front the group consisting of SEQ 113 NO: 42—62. in some embodiments, the i11111tiva1entfusioi1 protein contains four or more copies of a. Gi'i‘R—BD that comprises an amino acid sequence that is st 513"41, 60541, 65%, 713941.33. /11,81331—1, 85%,90‘3/41, 91311, 92‘341,93‘34I1,9‘341, 9-. %, 96%, 97%, 38941.,or 99‘341identica1 to an amino acid sequence seiected from the group consisting of SEQ 113 NO: 42.162, in some ments. the niuitiva1ei1t fusion protein contains ?ve oi~ more copies of a. (11113—1313 that comprises an amino acid sequence thatis at ieast 50'41, 613%, 65%,70‘34.,75‘341, 89%,85‘341, 91.3341, 91%. 92‘341. 93%9,4‘,341, 95% 96%,97‘41, 98% or 990'41 identicai to an amino acid sequence seiected from the group consisting of SEQ 11.3 NC: 42—62.. in some embodiments, the. inuitiva1ent fusion protein contains six or more copies of a GER-BI) that comprises an amino acid sequence that is at 1east 51.3%, 65‘34,1 713%, 75%, 813%,85%11, 9(3‘741, 91‘341, 92%,9‘31, 94%, 95%,96‘4., 97%, 98%o,r 99‘341ident1ca1 to an amino acid sequence ted from the. group consisting of SEQ 1113 NQ: 2. 11113211 in some embodiments, the muitivaient fusion protein contains at 1east one (311713.1313 that ses an amino acid sequencems{163cted tiom the group consisting o1 SEQ 113 NO: 635313. 111 some embodiments, the iniiitivaient titsion protein contains two or more copies of a GER—13113111211comprises an amino acid sequence‘1'.se1e1'.‘ted troin the group ting of SEQ 113 NO: 63—89. 111 some embodiments, the m nitivaient fusion n contains three or more copies ofa (3117311813 that comprises an amino acid sequence seieeted from the. group consisting of SEQ 113 NQ: 631813. 111 some ments, the 111111 ti va1 ei1t fusion protein contains four or more copies of a GER—813 that comprises an amino acid ce seiected from the group consisting of SEQ 11) NO: 63—81). in some embodiments, the niuitivaient iiision n contains ?ve or more copies ofa D thatccomprises an amino acid sequecen"se1ected from the group consisting of SEQ 113 N13: 63430. in some embodiments, the inuitit'tient 1‘usion protein contains six or more [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW copies oi’a GlTR-BD that comprises an amino acid sequence ed from the group consisting of SEQ ll) NO: 63670, E9922} ln some embodiments, the vaient fusion protein contains at least one GlTR—Bl) that comprises an amino acid sequence thatis at least 5096, 607’6, 65°,6, 7096, 7596, 8094i, 6 59,6 9096, 9194, 92%/0, 9396, 9494, 95°6 9696, 9794i, 9996, or 999/6 identical to an amino acid sequence selected from the group consisting ofSEQ ll) NO: 6380. in some embodiments, the alent fusion protein contains two or more. copies of a Gl’l‘R—BD that comprises an amino acid sequence that is at least 5096, 6096, 6596, 7096, 7596,8096, 8594i, 90° 6,9l94;, 92966, 9936, ,9496, 9596, 9696, 9796, 989/ or 99% identical to an amino acid sequence selected from the group consisting of SEQ ll) NS: 63930 in some ments, the mid ti valent fusion protein ns three or more copies of a Gilli—Bl} that comprises an amino acid sequence thatis at least 50%, 6096, 6594.,7{396, 7596, 8094i, 8596, 9096, 91%, 9296, 9396, 94966, 9596,9694. 9796, 9896, or 999.4; identical to an amino acid sequence selected from the group consisting ot" SEQ ll) NS: 63980 in some ments, the inultivalent fusion protein contains four or l’l’lOl'G copies of a GlTR—BD that ses an amino acid sequence that is at least 5 96, 609/ 6596,7094. 7596, 80°/6, 8594- 9096, 9196, 9296, 9.3’96,94E96, 9596, 96.96, 9796, 989/6, or 9996 identical to an amino acid sequence 5elected from the group consisting of SEQ ll) Ni): 6380 in some embodiments, the inultivalent fusion protein contains ?ve or more copies of a Gilli—ED that comprises an amino acid sequence that is at least 5096,6096 65 94; , 7096, 75x6, 8094;, S956,9096, 919.43, 9296,9396, 9496, 9596, 9696, 9796, 9896, or 9996 cal to an amino acid sequence ed from the group consisting of SEQ 1D NQ: 63—90. in some embodiments, the inultivalent fusion protein contains six or more co:5ies of a Gl'l‘R—BD that comprises an amino acid sequence that is at least 5096 6096, 5 ,’096, 759'6, St096, 596, 9096, 9l96, 9296,9396,9496, 9596, 9696, 9796, .9396, or 9996 identical to an amino acid sequence selected front the group consisting of SEQ ED ND: 6380. {0923} in some embodiments, the multivaient tusion protein contains at least one Gl’l‘R—BD that comprises a complementarity determining region l (CDRl) comprising an amino acid sequence*6selected from the group consisting of SEQ lD NO: 306, E09, 3 l2, il7, l20, 325, l3l, 338, l43, 148, and 349;:1aconipiementaiity deteiiniiiing region7 {(IDR2) comprising an amino acid sequence seleeted from the Group consisting oi SEQ 113 NO: 107, lit), "3, H5, H8, lZE, l23,l28, l3 334, £36, 337, l39, l4i, Mid, and l47, and a. complementarity detemiiningregion 3 (ifDR3) coinpiisinO an amino acid sequence selected [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW from the group ting ot‘SEQ 113 NO: 108, 111, 114, 116, 119, 122, 124, 126, 127, 129, 133, 135, 140, 142, 145, 146, and 150. in some embodiments, the inu1tii/aient fusion protein contains two or more copies of n GYER,~B[) that comprises a £12111 comprising an amino acid sequence seiected from the group consisting of SEQ 11‘) N0: 1116, 109, 112, 1 17, 120, 125, 131, 133, 143, 148, and 149; a CD112 comprising an amino acid sequence se1ected from the group consisting of SEQ 1113 N0:1117, 110, 113, 115, 118, 121, 123, 128, 130, 132, 134, 136, 13?, 139, 141, 144, and 147'; and n CDR3 comprising; an amino acid sequence seiected from the group consisting ot‘SEQ 113 NQ: 1118, 111, 114, 116, 119, 122, 124, 126, 127, 129, 133, 135, 141), 142, 145, 146, and 1511. in some embodiments, the mn1tivaieiit fusion protein ns three or more copies of a G1’1‘Rs131‘} that comprises 2i (3131111 comprising an amino acid sequence seiected from the group consisting of SEQ 11) N1}: 106, 109, 112, 117, 121), 125, 131, 138, 143, 148, and 149, a CDR2 comprising an amino acid sequence se1ecte<1 from the group consisting of SEQ 11) NQ: 11.17, 1111, 113, 115, 118, 121, 123, 128, 130, 132, 134, 136, 137, 139, 141, 144, and 147;,anr1 21C13R3 comprising an amino acid sequence seiected from the group consisting ot‘SEQ 11) NO: 103, 111, 114, 116, 119, 122, 124, 126, 12.7, 129, 133:, 135, 1411, 142, 145, 146, and 15(11n some embodiments, the mnitivaient fusion protein contains four or more copies of 3 (11111431) that comprises a CDR1 comprising an amino acid sequence seiected from the group consisting of SEQ 11) N1): 106, 1119, 112, 117, 1211, 125, 131, 138, 143, 148, and 149, at 2 con’iprisii’ig an amino acid sequence seiected front the group consisting of SEQ 11) NO: 1117, 111), 113, 115, 118, 121, 123,128,131), 132, 134, 136, 137, 139, 141, 144, and 147; and a. 61.1113, comprising an amino acid sequence seiected from the group consisting of SEQ 11) NO: 1118, 111, 114, 116, 119, 122, 124, 126, 127, 129, 133, 135, 140, 142, 145, 146, 1.1nsonie embodiments, the nient fusion protein contains five or more copies of a wBD that comprises a, CDR1 comprising an amino acid sequence seiected from the group consisting ot‘SEQ 113 NO: 1116, 109, 112, 117, 120., 125, 131, 138, 143, 148, and 149; u C3112 comprising an amino acid ce seiected from the group consisting of SEQ 11) N13: 107,111),113,115,118,121,123,128,131),132,134,136,137,132141, 144, and 147, and 3,131,116 comprising an amino acid sequence se1ected from the group ting of SEQ 11} N0: 108,111,114,116,119,122,124,126,127,129,133,135,141),142,145, 146, and 1511. in some embodiments, the a1eiit fusion protein contains sis or more copies of a (211111—131) that comprises s (112111 comprising an amino acid sequence se1ectet1 from the group consisting of SEQ 1121 NO: 1116, 109, 112, 117, 120, 125, 131, 138, 143, 148, [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW ed set by CLNDW and 149; 211319112 comprising an amino acid sequence selected from the group consisting of SEQ 1113 NO: 1117,1111,113,115,118,121,123,128,1311,1323,,134,136,137,139,141, 1441, and 147'; and n (3111113 comprising an amino acid sequence selected from the group consisting o’ESEQ 111190: 11111, 111, 114, 1113, 119, 122, 124, 126, 127, 129, 133, 135, 140, 142, 145, 146, and 1511. {11112-41 in some embodiments, the inultiva1ent tusion n contains at least one Gi'l'R—BD that comprises an amino acid sequence ed from the group con sisting of SEQ 1123 N13: 19-80 and at least one iniinunog1ohulin Fc region polypeptide comprising an amino acid sequence seiected from the group consisting ot‘SEQ 1D NOs: 1'6. in some ments, the muitivalent Fusion protein contains two or more copies of n (1111171211) thatccomprises an amino acid sequecen"L.se1ected from the group consisting ot" SEQ 11) N1): l9~80 and at 1east one inirn unogiohulin Fc region poiypeptide comprising an amino acid ce selected front the group consisting of SEQ 11> NQs: 1—6. in some embodiments, the muitiva1ent fusion protein contains three or more copies of a G1’1‘RwBl) that comprises an amino acid sequence selected from the group consisting of SEQ 1131 NQ: 19—811 and at 1east one irmnunogiohuiin Fc region poiypeptide comprising an amino acid sequence se1ected from the group consisting of SEQ 11) NOS: 1—6. 1n some embodiments, the muitivalent fusion protein contains four or more copies of a (31119 ~81) that comprises an amino acid sequence seiected from the group consisting of SEQ 11) NO: l9w80 and at 1east one imniunogiohulin Fc region poiypeptide comprising an amino acid sequence selected from the group consisting of SEQ 11) N'Qs: he, in some embodiments, the Hittitl‘v’ai?ll‘i fusion protein contains live or more copies ot‘a GlTR~BD that comprises an amino acid sequence selected from the group consisting of SEQ 11.) N0: 198—11 and at 1east one iminnnogiohuhn Fc region poiypeptide comprising: an amino acid sequence selected from the group consisting of SEQ 11‘) NQs: 1—6. in some embodiments, the inu1tivalent tusion protein ns six or more copies of a —813 that comprises an amino acid ce selected from the group consisting of SEQ 11) N8: 19—811 and at least one iinninnogiobulin Fc region polypeptide comprising an amino acid ce etl from the group consisting ot" SEQ 1D NQs: 143. 11111251 in some embodiments, the niu1tivalent fusion protein contains at least one 431.) that comprises an amino acid sequence that is at1east5110/éi 6110/, 115% 7110/} 75%, 8091,8515?) (1019191,, SH 9 5,9491),95%,9o%, 97‘31 9110/,0, or 9913/5 identical to an amino acid seeonnence selected from the group consisting of SEQ 1131 N13: 19~80 and at least [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW one noglohulin Fc region polypeptide cornprising an amino acid sequence selected from the group consisting of SEQ ll} NQs: l66. lh some embodiments, the inultivalent fusion protein contains two or more copies of a Gl’lRwl?l.) that comprises an amino acid 3 3 sequence that is at least 50/6 6036,.5596 70/6 , 30‘}6 85"/6 90%, 9196. 2‘36 93%, 94%, 5‘3496%, 9796, 98‘34t, or 99‘"6 identical to an amino acid ce ed from the group ting of SEQ ll:3 NQ: l9wSO and at least one intintinoglohnlin E'c region polypeptide contprising an amino acid sequence ed front the group consisting of SEQ ED NQs: l—6. in some embodiments, the ntultivalent fusion n contains three or more copies ofa Gilli-Bl) that comprises an amino acid sequence that is at least 59‘3/ , 60%. 6533/6, 7(3‘3'75/6,/t') 85336. 353/, 3:30 /i), gig/65.4 923/, 3335/64, 94‘36. 953/, 3:36"/'{)4, 97‘36. 983/, Ol‘ 93"4o identical to an antino acid sequence selected front the group consisting of SEQ ll) NQ: l9~ 80 and at least one intrnunoglobulin Fc region polypeptide comprising an amino acid sequence selected front the group consisting of SEQ ll) NQs: l—6. in some embodiments. the inultivalent titsion n contains four or more copies of a Gl'l‘R—BD that coinpii ses an amino acid sequence tltatis at least 50':/6, 60534;, 65‘3'6. 70‘346,475‘36 85‘3/6, 99‘36, 9i‘3'6. 92%. 93% 94‘?6, 95%. 96,697‘6, 98%. or 99",6 identical to an amino acid sequence selected front the group consisting; of SEQ ll.) NQ: l9—8t’) and at least one iniinunoglohul in P6 region polypeptide comprising an amino acid sequence selected front the group consisting of SEQ ll) Nils: l 6 in some embodiments, the inulti I'alent fusion protein contains five or more copies ofa Gl'l‘R—BD that comprises an amino acid sequence that is at least 59%, 60%, 65946.4 79% 75%, 8936.48tS5"6, 90‘3'/’64, l‘I/6. 92%, 935%, 94946.4 95%, 96%, 97‘3/6, 98%, or 99% identical to an amino acid sequence selected item the group consistingof SEQ ll.) NQ: l9~8tl and at least one iinninnoglobulin Fc region polypeptide comprising an amino acid sequence selected fl’Om the group consisting of SEQ ll) NOs: l—t’5. in some embodiments, the m ultivalent fusion protein contains six or more copies of a (EUR—Bl) that comprises an amino acid ce that is at least 5096, 6(13‘36,65",6, 7945075% 89%, , 90%, 9l"/6. 92%6, 93‘36,.. 94"/6.495%, 9 ‘36,97‘345. 935%, or99‘3//6 identical to an amino acid sequence selected front the0group consisting of SEQ ll.) NQ: l9—86‘ and at least one iinntunoglohulin Fc region polypeptide comprising an antino acid sequence selected from the group consisting of SEQ ll} NQs; l—6 E9926} in some ernhodintents, the ntultivalent tiision n contains at least one QlT‘R—BD that comprises an amino acid sequence ed front the group consisting of SEQ lD NO: 42—62 and at least one iinrnurtogloliulin Fc region polypeptide comprising an [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW amino acid ce selected from the group consisting of SEQ lD NQs: HS. in some embodiments, the alent liision protein contains two or more copies ol’a (HIRED that comprises an amino acid sequence selected from the group consisting of SEQ ll) Ni}: 42~62 and at least one immunoglohulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ lD NQs: L6. in some embodiments, the multivalent rusion protein contains three or more copies or‘a Gl'l‘RwBD that comprises an amino acid sequence selected from the group consisting; of SEQ ll.) N0: 42—62 and at least one iinn’iunoglobulin Ec region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll) NQs: l—o, in some ments, the multivalent fusion protein contains four or more copies of a ED that comprises an amino acid sequence selected from the group consisting of SEQ ll) N0: 42 ~62 and at least one inirn unoglohulin Fc region polypeptide comprising an amino acid sequence selected front the group consisting of SEQ lit NQs: E6. in some embodiments, the multivalent fusion protein contains five or more copies ot" a Gl'l‘R—ED that comprises an amino acid sequence selected from the group consisting of SEQ ED NO: 42—62 and at least one oglohulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll) NQs: L6. in some embodiments, the multivalent fusion n ns six or more copies of a Gills—Bl) that comprises an amino acid sequence selected from the group consisting of SEQ lD NQ: 42—62 and at least one oglobulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting ot" SEQ ll) NQs: l—o. 399927} in some embodiments, the niultivalent fusion protein ns at least one (HIRED that comprises an amino acid sequence selected from the group consisting of SEQ ll) N0142w62 and at least one oglohnlin Ec region polypeptide comprising the amino acid sequence of SEQ ll) NQ: l, in some embodiments, the niultivalent fusion protein contains two or more copies oi’a GlTR~BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 42432 and at least one imrnunoglobulin Ec region polypeptide comprising the arnino acid ce of SEQ ll) N01l. in some embodiments, the multivalent fusion protein contains three or more copies of a GETRED that ses an amino acid sequence selected from the group consisting of SEQ ED NO: 42—62 and at least one innnunoglohulin Fc region polypeptide comprising the amino acid sequence of SEQ ll) NO: l, in some embodiments, the multivalent fusion protein contains four or more copies ot‘a GlTR—BD that comprises an amino acid ce selected [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW from the group consisting of SEQ ll) NO: 42—62 and at least one iinniunoglohulin Fc region polypeptide comprising the amino acid ce oi‘SEQ 1D NQ: 1, hi some embodiments the niultivalent fusion protein contains five or more copies of a Gl'l'R—Bl.) that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 42-62 and at least one imniunogloliulin Fc region polypeptide comprising the amino acid sequence of SEQ lD NO: l. in some embodiments, the multiyalent fusion protein contains six or more copies of a Gl’l‘R—Bl.) that comprises an amino acid sequence selected from the group consisting of SEQ ll} N0: 42-62 and at least one inimunoglohulin Fc region polypeptide comprising the amino acid sequence of SEQ ll) NQ: l. {6&8} in some embodiments, the alent fusion protein contains at least one GlT‘R—BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 42—62 and at least one iinniunoglohulm Fc region polypeptide sing the amino acid sequence of SEQ ll.) NO: 2, in some embodiments, the multiyalent fusion protein contains two or more copies of a Stilt-Bl) that comprises an amino acid sequence selected from the group consisting of SEQ ll) NQ: 42—62 and at least one imniunoglohulin Fc region polypeptide comprising the amino acid sequence of SEQ ll} NO: 2. ln some embodiments, the inultivalent fusion protein contains three oi~ more copies ot" a Gl'l‘R—BD that ses an amino acid ce selected from the group consisting of SEQ ll) N0: 42-62 and at least one inn’nunoglohnlin Fc region polypeptide comprising the amino acid sequence ofSEQ ll:s NO: 2. in some ments, the rnultivalent fusion protein contains four or more copies of a Gl’l‘R—Bl.) that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 42—62 and at least one imniunoglohulin Fc region ptide comprising the amino acid sequence oi‘SEQ 1D NQ: 2, hi some embodiments the niultivalent fusion protein contains five or more copies of a Gl'l'R—Bl.) that comprises an amino acid ce selected from the group consisting of SEQ ll) NO: 42-62 and at least one imniunogloliulin Fc region polypeptide comprising the amino acid sequence of SEQ lD NO: 2. in some embodiments, the multiyalent fusion protein contains six or more copies of a Gilli-Bl.) that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 42-62 and at least one inimunoglohulin Ec region polypeptide comprising the amino acid sequence of SEQ ll) N0: 2. some; in some embodiments, the multivalent fusion protein contains at least one BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 42—62 and at least one iinniunoglohulm Fc region polypeptide comprising the [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW amino acid sequence of SEQ 1D NQ; 3. in some embodiments; the multi Ialent tusion protein contains two or more copies of a Gl'l'R—BD that comprises an amino acid sequence selected front the group consisting of SEQ ll.) NO: 42—62 and at least one inununoglohul in Po region polypeptide comprising the amino acid sequence of SEQ ll) ND: 3:. in some embodiments, the multivalent tusion protein contains three or more copies ot‘a GlTR-BD that ses an amino acid sequence ed from the group consisting of SEQ ll) NS: 4-262 and at least one imntunoglobulin Fc region polypeptide comprising the amino acid sequence oi‘SEQ ll) NO: 3. in some embodiments, the niultivalent fusion protein contains four or more copies oi’a SHRED that comprises an amino acid sequence selected from the group consisting of SEQ ll} NO: 426.2 and at least one imniunoglo‘ouhn Ec region polypeptide sing the amino acid sequence of SEQ ll) NQ: 3. in some embodiments, the n’iultivalei’it fusion protein contains live or more copies ofa GlTR-BD that ses an amino acid ce ed from the group consisting of SEQ ll) NO: 42432 and at least one imniunogio‘oulin Ec region polypeptide comprising the antino acid sequence of SEQ ll.) N0: 3. in some embodiments the rnultivalent fusion n contains six or more copies of a SHRED that comprises an amino acid sequence selected from the group consisting of SEQ ll) 62 and at least one ininiunoglohulin Fe region polypeptide comprising the. amino acid sequence of SEQ ll) NQ: 3. {@939} in some embodiments, the inultivalent fusion n contains at least one Gilli—ED that ses an amino acid sequence selected from the group consisting of SEQ ll} NO: 42—62 and at least one nnmunoglo‘ouhn Fc region polypeptide comprising the amino acid sequence of SEQ 1D NQ; 4. in some embodiments the inultivalent tusion protein contains two or more copies of a Gl'l'R—BD that comprises an amino acid sequence selected front the group consisting of SEQ ll.) NO: 42—62 and at least one inununoglohul in Po region polypeptide comprising the amino acid sequence of SEQ ll) NO: 4. in some embodiments, the multivalent tusion protein contains three or more copies ot‘a GlTR-BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NS: 4-262 and at least one imntunoglobulin Fc region polypeptide comprising the amino acid sequence of SEQ ll) NO: 4. in some embodiments; the nuiltivalent fusion protein coi’itains four or more copies of a D that ses an amino acid sequence selected from the group consisting of SEQ ll} N0: 42~62 and at least one iinniunogiobulin Fc region polypeptide comprising the amino acid ce of SEQ ll) NQ: 4. in some embodiments, the n’iultivalei’it fusion protein contains live or more copies ofa GlTR-BD that comprises an [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW amino acid sequence selected from the group consisting of SEQ lD NO: 42—62 and at least one intinunogiohulin Ec region polypeptide comprising the antino acid sequence of SEQ ll) N0: 4 in some embodiments the rnnitivalent fusion protein contains six or more copies of a GlTRED that comprises an amino acid sequence selected from the group consisting of SEQ lD NO: 42—62 and at least one iinntunoglohuhn Ec region polypeptide comprising the amino acid sequence of SEQ it) hit): 4, {WISE} in some embodiments the vaient fusion protein contains at least one GlTR—BD that contpri ses an amino acid sequence selected from the group consisting of SEQ ll) NO: 42432 and at least one intinunoglohuiin Ec region polypeptide comprising the antino acid sequence of SEQ It.) NO: 5‘ in some embodiments the valent fusion protein contains two or more copies of a GITRED that comprises an antino acid ce selected front the group consisting of SEQ ll) NQ: 49:62 and at least one intm unoglohulin Ec region polypeptide comprising the amino acid sequence of SEQ ll) NO: 5. in some embodiments the niultivaient fusion protein contains three or more copies of a Gl’l‘RwBD that comprises an antino acid sequence selected front the group consisting of SEQ ED NO: 42—62 and at least one iininunoglohulin Ec region polypeptide comprising the antino acid sequence of SEQ ll} NO: 5; in some embodiments, the innitivalent titsion protein ns four or more copies of a GlT‘R—BD that comprises an amino acid sequence selected front the group ting of SEQ ll} NO: 42—62 and at least one iniinunogiohuiin Ec region polypeptide comprising the antino acid sequence of SEQ ll.) NO: 5 in some embodiments, the innitivalent fusion protein contains five or more copies of a Gl'l‘RrBD that comprises an amino acid sequence selected from the group consisting of SEQ lD NO: 42—62 and at least one intinunogio‘oulin Ec region polypeptide comprising the antino acid sequence of SEQ ii) N0: 5 in some embodiments, the rnnltivalent fusion protein contains six or more copies of a GlTRED that comprises an amino acid sequence selected from the group consisting of SEQ lD NO: 42—62 and at least one noglohuhn Ec region polypeptide comprising the amino acid sequence of SEQ it) hit): 5, {WISE} in some embodiments the niultivaient fusion protein contains at least one GlTR—BD that comprises an amino acid seque ice selected from the group consisting of SEQ ll} N0: 42~62 and at least one intinunoglohuhn Fc region polypeptide comprising the amino acid ce of SEQ 1D NQ: 6. in sonte embodiments the inuitivaient fusion protein contains two or more copies of a GITRED that comprises an antino acid ce ed front the group consisting of SEQ ll) NQ: 49:62 and at least one intm unoglohulin ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW Fc region polypeptide comprising the amino acid sequence of SEQ lD NO: 6. in some embodiments, the inultivalent liision protein contains three or more copies of a Gl'l‘R—ED that comprises an amino acid sequence selected from the group consisting of SEQ ll) NQ: 42~62 and at least one immunoglobulin Fc region polypeptide sing the amino acid sequence of SEQ ll} Ni}: 6‘ in some enihodiinei’its, the valent fusion protein contains four or more copies ot‘a Gilli—Bl) that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 42—62 and at least one ininiunogiohulin Fc region polypeptide comprising the amino acid sequence ot‘SEQ ll) N0: 6. in some embodiments, the rnultivalent fusion protein contains five or more copies ofa Gilli—8D that comprises an amino acid sequence selected from the group consisting of SEQ ll.) NQ: 42—62 and at least one intniunoglohul in Fe region polypeptide comprising the amino acid ce of SEQ ll} N0: 6. in some embodiments, the valent fusion protein contains siX or more copies of a Gilli—Bl) that comprises an amino acid sequence selected from the group ting of SEQ ll} NO: 42—62 and at least one iinmunoglo‘oulin Fc region polypeptide comprising the amino acid sequence of SEQ ll) N0: 6.

Etlmdt in some ments, the niultivalent fusion protein contains at least one Gl'l‘RrBD that comprises an anti no acid sequence selected from the group consisting of SEQ ll) NO: 634st) and at least one immunoglohulin Po region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll) NQs; 1—6, in some embodiments, the multivaient rusion protein contains two or more copies ot‘a Gilli—8D that coinpri ses an amino acid sequence selected from the group consisting; of SEQ ll.) NQ: 63—30 and at least one iinn’iunoglohulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll) NQs: l—o, in some embodiments, the multivaient fusion protein contains three or more copies ot" a Gills—Bl) that comprises an amino acid sequence selected from the group consisting of SEQ ll) N0: 63~80 and at least one imi’n unogloliulin Fc region ptide comprising an amino acid sequence selected front the group ting of SEQ if) NQs: l—o. in some embodiments, the alent fusion protein contains four or more copies of a Gilli—Bl.) that comprises an amino acid sequence selected from the group consisting of SEQ ll) NQ: 63—30 and at least one iinn’iunoglohulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll) NQs: l—o, in some embodiments, the multivalent fusion protein ns ?ve or more copies of a GETRED that ses an amino acid sequence selected from the group consisting of SEQ ll) [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW N0: 63-80 and at least one imn’iunoglobulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll) NQs: he, in some embodiments, the multivalent fusion protein ns six or more copies of a Gills-Bl) that ses an amino acid sequence selected from the group consisting of SEQ lD NO: 63-830 and at least one imm unoglobulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll.) NQs: l-o.

{Gm-4} in some embodiments", the valent fusion protein contains at least one GlTR-BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 63-80 and at least one immunoglobuhn Ec region polypeptide comprising the amino acid sequence of SEQ ll.) 'NQ: l A in some embodiments the inultivalent fusion protein contains two or more copies of a GlTR-Bl) that ses an amino acid sequence selected from ll’l? group consisting of SEQ ll) NQ: 63-80 and at least one imrn unoglohulin Ec region polypeptide comprising the amino acid sequence of SEQ ll) NO: l. in some embodiments the valent fusion protein contains three or more copies of a Gl’f‘R-BD that comprises an amino acid sequence selected from El’l? group consisting of SEQ ED NO: 63—80 and at least one immunoglobulin Ec region ptide comprising the amino acid sequence of SEQ ll} NO: it in some embodiments, the inultivalent titsion protein contains four or more copies of a GlTR-BD that comprises an amino acid sequence selected from the group consisting of SEQ ll} NO: 63-80 and at least one imrnunogloliulin Fc region polypeptide comprising the amino acid sequence of SEQ ll.) NO: 1. in some embodiments, the multivalent fusion protein contains five or more copies of a Gl'l‘R-BD that comprises an amino acid sequence selected from the group ting of SEQ lD NO: 63-80 and at least one iminunoglo‘oulin Ec region polypeptide comprising the amino acid sequence of SEQ ll) N0: l in some embodiments the mtiltivalent fusion n contains six oi~ more copies of a GlTR-BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 63-80 and at least one irnmunoglobulin Ec region ptide comprising the amino acid sequence of SEQ ll.) NO: 1, {@335} in some embodiments", the inultivalent fusion protein contains at least one Gilli-Bl) that comprises an amino acid sequence selected from the group consisting of SEQ ll} N0: 63-80 and at least one immunogloliulin Fc region polypeptide comprising the amino acid sequence of SEQ if) NU: 2. in some embodiments the inultivalent fusion protein contains two or more copies of a GlTR-BD that ses an amino acid sequence selected from ll’l? group consisting of SEQ ll) NQ: 63-80 and at least one imrn unoglohulin [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW Fc region ptide comprising the amino acid sequence of SEQ lD NO: 2. in some embodiments, the multivalent hision protein contains three or more copies of a Gl'l‘R—ED that ses an amino acid sequence selected from the group consisting of SEQ ll) NQ: 63~80 and at least one imin unoglohulin Fe region polypeptide comprising the amino acid ce of SEQ ll} N43: 2‘ in some embodiments, the valent fusion protein ns four or more copies ol‘a Gilli—ED that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 63—S0 and at least one imniunoglohulin Ec region polypeptide comprising the amino acid sequence oi’SEQ ll) NQ: 2. in some embodiments, the valent fusion protein contains five or more copies oi‘a Gilli—8D that comprises an amino acid sequence selected from the group consisting; of SEQ ll.) NO: 63—89 and at least one immunogiohul in Fc region polypeptide sing the amino acid sequence of SEQ ll} N0: 2. in some embodiments, the multiyalent fusion protein ns si\ or more copies of a Gilli—ED that comprises an amino acid sequence ed from the group consisting of SEQ ll} NQ: 63—89 and at least one inimunoglohulin Fe region polypeptide comprising the amino acid sequence of SEQ ll) NQ: 2. sense} in some ments, the niultiyalent fusion protein contains at least one Gl'l‘RrED that comprises an anti no acid sequence selected from the group consisting; of SEQ ll) NO: 634st) and at least one immunoglohulm Po region polypeptide comprising the amino acid sequence of SEQ ll) NO: 3. ln some ments, the niultiyalent fusion protein contains two or more copies ofa Gilli-Bl) that comprises an amino acid sequence selected from the group consisting of SEQ ll) NQ: 63*Stl and at least one immunoglohulin Fc region polypeptide comprising the amino acid sequence of SEQ ll) NO: 3. in some embodiments, the multivalent hision protein contains three or more copies of a Gl'l‘R—ED that comprises an amino acid sequence selected from the group consisting of SEQ ll) NQ: 63~80 and at least one imin unoglohulin Fe region polypeptide comprising the amino acid sequence of SEQ ll} N43: 3‘ in some embodiments, the rnultivalent fusion protein contains four or more copies ol‘a Gilli—ED that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 63—S0 and at least one imniunoglohulin Ec region polypeptide comprising the amino acid sequence of SEQ ll) NO: '3. in some embodiments, the in ultiyalent fusion protein contains five or more copies of a GlTR—BD that comprises an amino acid sequence selected from the group consisting of SEQ 11) NO: {33—80 and at least one oglohul in Ec region polypeptide comprising the amino aeid sequence of SEQ ll} N0: 3. in some embodiments, the multiyalent fusion protein contains si\ or more copies of ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW a GlTR—BD that comprises an amino acid sequence selected from the group ting of SEQ ll:l NO: 63w80 and at least one inimunoglobulin EC region polypeptide comprising the amino acid sequence of SEQ ll.) N0: 3, {@937} in some embodiments, the inultivalent fusion n contains at least one GlTR—BD that comprises an amino acid sequence selected from the group consisting of SEQ ll} NO: (33—80 and at least one iinmunoglobulin Ec region polypeptide comprising the amino acid sequence of SEQ ED NO: ll. in some embodiments, the inultivalent fusion protein contains two or more copies of a GlTR—BD that ses an amino acid sequence ed from the group consisting of SEQ 1D NQ: 63—80 and at least one immunoglohulin Fc region polypeptide comprising the amino acid sequence of SEQ ll} NQ: [it in some embodiments, the valent fusion protein contains three or more copies of a GiTR-BD that comprises an amino acid sequence selected from the group consisting of SEQ 113 NO: 6360 and at least one iminunoglobnlin Ec region polypeptide comprising the amino acid sequence of SEQ ll) N014. in some embodiments, the niultivalent fusion protein contains four or more copies of a GlTR—BD that comprises an amino acid sequence selected from the group consisting of SEQ ll} N0: 63~80 and at least one nogio‘oulin Fc region polypeptide sing the amino acid sequence of SEQ ll.) N0: 4 in some embodiments, the niul ti val ent fusion protein contains five or more copies ofa (SHRED that comprises an amino acid sequence selected from the group consisting of SEQ ll) NQ: 63-80 and at least one imniunoglo‘oulin Ec region polypeptide comprising the amino acid sequence of SEQ 13 N8: 4-. in some embodiments, the multivalent fusion protein contains six or more copies of a D that comprises an amino acid sequence selected from the group consisting of SEQ ll:l NO: 63w80 and at least one inimunoglobulin EC region polypeptide comprising the amino acid sequence of SEQ ll.) N0: 4, {@938} in some embodiments, the inultivalent fusion protein contains at least one GlTR—BD that comprises an amino acid sequence ed from the group consisting of SEQ ll} NO: (33—80 and at least one iinmunoglobulin Ec region polypeptide comprising the amino acid sequence of SEQ it) NO: 5. in some embodiments, the niultivalent fusion protein contains two or more copies of a GER—Bl) that comprises an amino acid sequence selected from the group consisting of SEQ 11) N0: 63—80 and at least one iinn’iunoglobulin Ec region polypeptide comprising the amino acid ce of SEQ ll} NO: 5. in some embodiments, the niultivalent fusion protein contains three or more copies of a GiTR-BD that comprises an amino acid sequence selected from the group consisting of SEQ H) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW N0: 63—80. and at least one imniunoglohulin Fc region polypeptide comprising the amino acid ce of SEQ it} NO: 5. in some embodimentsv the inultivalent ?rsion protein contains four or more copies of a ED that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 63—80 and at least one ininiunoglohuiin Ee region polypeptide sing the amino acid sequence of SEQ ED N0: 5. in some ments, the inultivalent fusion protein contains five or more copies ofa GlTFcBD that comprises an amino acid sequence selected from the group consisting of SFQ ll) NO: <33«‘§0 and at least one iniinunoglohulin Fc region polypeptide comprising the amino acid sequence of SEQ ll) NO: 5, in some embodiments the inultivalent fusion protein contains six or more copies of a Gl'l‘RrBD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 634st) and at least one nogloliulin Fc region polypeptide comprising the amino acid sequence of SEQ ED N0: 5. guess} in some embodiments, the inultivaient fusion protein contains at least one Gl’l'R—Bl.) that comprises an amino acid sequence selected from the group consisting of SEQ ll} N0: 63-80 and at least one immunoglohuhn Fe region polypeptide comprising the amino acid sequence of SEQ if) NU: 6. in some embodiments the inuitivalent fusion protein contains two or more copies of a. Gi'l'R—Bl} that comprises an arnino acid sequence selected front the group consisting of SEQ ll) Ni}: 63~80 and at least one inirnunoglohulin Fc region polypeptide comprising the amino acid sequence of SEQ ll} Ni}: 6‘ in some embodiments, the multivaient fusion protein contains three or more copies oi‘a Gl'l‘RwBD that i ses an amino acid sequence ed from the group consisting; of SEQ ll.) N0: 63—80. and at least one imniunoglohulin Fc region ptide comprising the amino acid sequence of SEQ it} NO: s. in some embodimentsv the inultivalent ?rsion protein contains four or more copies of a Gl'l‘RED that ses an amino acid sequence selected from the group consisting of SEQ ll) NO: 63—80 and at least one iinniunoglohuiin Ee region polypeptide comprising the amino acid sequence of SEQ ED N0: 6. in some ments, the valent fusion protein contains five or more copies ofa GlTFcBD that comprises an amino acid sequence selected from the group consisting of SFQ ll) NO: <33«‘§0 and at least one immunoglobulin Fe region polypeptide comprising the antino acid sequence of SEQ ll} N0: 6. in some embodiments; the valent fusion protein contains six or more copies of a SHRED that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 634st) and at least one iinniunogloliulin Fc region polypeptide comprising the amino acid sequence of SEQ ED N0: 6.

[Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 399946} in some ernhodinientsa the rnultivaient fusion protein contains at least one (HIRED that ses a complementarity determining region 1 {CDRi} comprising an amino acid sequence seiected from the group consisting; of SEQ it.) NO: the, 10% i123, 1171 129,i255131,£38,143e148,and£49;aconn?enunnangrdacnnnnngieguni2((33R2} conmngngmimnmoamdsmuwm?sekcwd?onnhegnmpcmmmnngofSEQiDh?}101 3:i15,1181121,123,128,130,132:i34,1361137,139,141,144,and147;anda coinpienientarity determining region 3 (CDRP?) comprising an amino acid sequence seiected ?ont?nzgnnu3connsong 133v BS, 140,, 142, 145,, 146, and 150 and at least one iinniunogio‘ouhn Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ 11"} NOs: i—rS. in some embodiments, the inuitivaient fusion protein ns two or more umkw?a??hd?)$?cmmnm?aiDRlmmmnwganmmmnw?sawmmewkaml ?cnrdn:gnmuicon?s?ngtniSEX}iiih?):106vi09,112i117,1205125,131,138,i43,i48i and 149; a, CDRZ comprising an amino acid sequence selected front the group consisting of SEt?iith?}:107,110,113;1159118,121,123,128,ESQ,i32;134,1367137,139,141, 1441v and 147; and a CDRB comprising an amino acid sequence seiected front the group con?snng 142,i45j146,muiiSGandathxmtoneinununoghnnuutiregnuipohqmpndeconqni?ng an amino acid sequence seiected front the group consisting of SEQ ED NOS: 1—6. in some embodiments, the rnultivaient tusion protein ns three or more copies oi‘a Gi'l‘RwBD ?mtammnwsaCDRdcommnnganmnmoaadsmmmmen?mmd?om?mgnmp connsnngrafSEKEEE)h?):1067109,112,117,120,125,£31,1381143,148,and.149;a {TERA comprising an arnino acid sequence selected from the group consisting of SEQ 11} ho):i0?,llOi113,1i5,118,i21,123e£28,13Gi132,134513o,137,t39,t41,144sand 347; and a CD123 comprising an amino acid sequence selected from the group consisting of SEi}lI)t£Ct i0$,111;114,116,119,122,1243126,127,£29,13BZ135,140,142,145, 146, and 15:0 and at least one iniinunoglohuiin Ec region poiypeptide comprising an amino acid ce selected from the group consisting of SEQ It) NQs: i—o‘ in some embodiments? the multivaient fusion protein contains four or more copies of a (itTR—BD that comprises a CDRE sing an amino acid ce seiected from the group connsnngofSEdgiDlsO:1061109,112,117,120,125:i31,1381143,l485and149;a CDRE comprising an amino acid ce selected from the group consisting of SEQ 11) NB: 10'7jllt),113,1153113,121,123,128;13t),132,1343136,137,139,141;Miami [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 147, and 21 (31318.3 comprising an amino acid sequence selected from the group consisting of SEQ 1113 NO: 1118, 111, 114, 116, 119, 122, 124, 126, 127, 129, 133, 135,141}, 142, 145, 146, and 1511 and at teast one imniunogiobniin Fc region potypeptide comprising an amino acid sequence selected from the group consisting of SEQ 113} N011: L6. In some embodiments, the mu1tiv111ent tusion protein contains ?ve or more copies of a {31111—1313 that comprises :24, (71313.1 comprising an amino acid se1111131101511ected from the group consisting of SEQ 11.) N0: 1116, 109, 112, 117, 1211, 125, 131, 138, 143, 148, and 149,21 {31313.2 comprising an amino acid sequence se1ected from the group consisting of SEQ 113 N8: 107,110,113,115,118,121,123,128,130,132,134,136,137,139,141,144, and 147; and a CD133 sing an amino acid sequence seiected from the group consisting of SEQ 11) NO: 108,111,114,116,119,122,124,126,127,129,133,135,140,142,145, 146, and 1513 and at least one intmunogiohuhn Fc region poiypeptide comprising an antino acid sequence seiected 110111 the group consisting of SEQ 11> N135: 1—6. 111 some embodiments, the ntuitivaient fusion protein contains six or more copies of a. (11111—1313 that comprises 11513111 comprising an amino acid sequence seiected front the group consisting of SEQ 11) NO: 106, 109, 112, 117, 120, 125, 131, 138, 143, 148, a11d149,a {313112 comprising an antino acid sequence seiected front the group consisting of SEQ 11.) N0: 1117 11t’1,113,115,118,121,13,128, 130,132,1~4 136,137,139,141,144,aitd147,and a. {313113 comprising an aminoacidse uenee seiected front the group consisting of SEQ 1121 NQ: 108,111, 114, 116, 119, 122, :24, 126,127, 129, 133, 13:5, 140, 5, 146, and 1511 and at 1eust one iinntunogiohuhn E1: region poiypeptide comprising an antino acid sequence se1ected from the group consisting of SEQ 1D NOS: 1—6 E13841} in some ments, the vaient hision protein ses an amino acid sequence seiected from the group consistingotSEQ 11) NO: 811 (‘15, 111 some embodiments, the in 1111111411tit tusion protein comprises an amino acid c seiected from the group consisting of SEQ 1123 NO: 81—93, in some embodiments, the uient 11151011 protein comprises an amino acid 81(111111103’1ected from the group con5isting of SEQ 0: 94—1415, {1111421 in some embodiments, the muitivaient fusion n coinpn ses an amino acid sequence thatis ntieast511‘341,60‘31 65'3/41,711‘344;, 75% 8O:4, 85‘34.1,30‘34 91:/41,92‘34,193%, 94‘3/44, 95%,9604;, 97‘34, 98%o,r 99‘/4:- 1111311t1ca1to an amino acid sequence seiected front t111 group consisting of SEQ 11) NO: 81-1 ()5. in some embodiments, the ntuhivaient fusion n comprises an amino acid sequence that is at least 50%, 6094, 65%, 7/0940 754,811‘341, [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW S5343;;, 9(‘30,9370 92%, 933'094.7100, 95%,,6%, 9770 98%, or 99% identical to an amino acid sequence seiected from the group ting of SEQ iD ND: 81993, in some embodiments, the muitivaient fusion n ses an amino acid senuen re that is at least 59%, 60%, 65%, 7035,, 35%,80%,h35 ‘3/,9t3%,93‘3", 923/5, 93%, 949/5, 953/5, 96‘3", 979/"/,o 9836,or 99% cal to an amino acid sequence seiected from the group consisting of SEQ ED N{)2 94- {(33343} in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ 133 NO: 83. in some embodiments, the muitivaient fusion protein comprises the amino acid sequence ofSEQ if) NU: 82. in some embodiments, the mnitivaient fusion protein comprises the amino acid sequence of SEQ ii) NO: 83 in some embodiments, the aient fusion protein comprises the amino acid sequence of SEQ 3D ND: 84. in some embodiments, the muitivaient fusion n comprises the amino acid sequence of SEQ if) Ni): 85. in some ments, the muttivaient fusion n compii ses the amino acid sequence of SEQ ED NO: 36, in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ ED NO: S7, in some embodiments, the mnitivaient fusion protein comprises the amino acid sequence of SEQ 1D N0: 88 hi some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ 31‘} NO: S9: in some embodiments, the muitivaient fusion protein comprises the amino acid ce of SEQ 3D N{)2 90: in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ 113 NO: 91, in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ ED NO: 92. in some embodiments, the inuitivaient fusion protein ses the amino acid sequence of SEQ H) NO: 93:. in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ It.) ND: 94, in some embodiments, the mnitivaient fusion protein comprises the amino acid sequence of SEQ 1D NO: .95. in some embodiments, the innitivaient fusion protein ses the amino acid sequence of SEQ ED ND: 9o in some embodiments, the aient fusion protein comprises the amino acid sequence of SEQ it} NO: 97. in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ if} N0: 953, in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ 133 N43: 99. in some embodiments, the mnitivaient fusion n comprises the amino acid sequence of SEQ 1D ND: 300. in some embodiments, the muitivaient fusion protein comprises the amino acid sequence of SEQ ED NO: 103. in some embodiments, the muitivaient fusion protein . is!) D r,| [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW comprises the amino acid ce ol’SEQ 18 NS; 102, in some embodiments, the alent ?rsion protein comprises the amino acid sequence of SEQ ll) NO: lll3, in some embodiments, the inultivalent fusion protein comprises the amino acid sequence of SEQ l1.) N9: 104. ln some embodiments, the multivalent fusion protein comprises the amino acid sequence ofSEQ ll} NO: lGS. {?ttest} in some embodiments, the multivalent Gl'l‘lhtargeting tusion protein is tetravalent. As used herein, a tetravalent Gl'l‘R—targeting le refers to two copies of a, Gilli—targeting tusion n that includes two GlTR—BDs. For example, in some embodiments, a tetravalent targeting molecule of the disclosure includes two copies of a Girl‘ltrtargeting fusion protein having the following structure: (Gilli—81’.))sl?inkerw (GlTR~89)~Linl molecule of the disclosure includes two copies of a GlTR—bll’ldlllg fusion protein having the following structure: BD)—Linl§er~(G1'l‘R~Bl3l—Linlrenllinge~Fc, Where the 8D is an isolated polypeptide sequence that binds Gl’l‘R. in some embodiments, the tetravalent Gilli—targeting molecule ofthe disclosure includes two copies of a inding fusion protein having the following structure: tGl'l'R—BD)—Linl Whe re the Gl'l‘R—BD is an sdAh sequence that binds Gl’l‘R. In some embodiments, the tetrayalent GllR~targeting molecule of the disclosure includes two copies of a GlTR- binding fusion protein having the tollowing structure: (GlTR—ED)—Linker—(G1TR—BD)— Linkerwllingewl‘c, where the —Bl) is a humanized or hilly human sdAb ce that binds GITR. ln some ments, the Gl’l‘RsBl} comprises a complementarity determining region l {(IDRl) comprising an amino acid sequence selected from the group consisting of SEQ ll.) NO: 106, 109, ill, ll7, lZO, 125, l3l, 138, 143, 148, and 149; a complementarity determining region 2 (CDR2) comprising an amino acid sequence sele tte£14 from the group consisting of SEQ ll) NO: l07, llfl, 113, 1,15, H8, 121, l2'3, l28, 1'39, 132 l34, 136, 137, 139, ldl, 144, and 147, and a coinplen’ientarity determining region 3 {(313133) sing an amino acid sequence ed from the group consisting of SEQ ll) NO: 108,, ill, llél, 116, 1,19, 122-), 122-1, l26, l27, l29, 133, l35, 140, 142, tilt"), 1/416, and 151‘), in some embodiments, the tetravalent Gills—targeting molecule contains at least one Gilli—BI} that comprises an amino acid sequence selected from the group consisting of SEQ ll) N0: 19~ till. in some embodiments, the tetravalent Gl'i‘R—targeting molecule contains at least one Gills—Bl) that comprises an amino acid sequence that is at least 50%, 60"?45, 65%, 70%, 75%, 80%, 3596, 90%,91%, 9296, 9395, 94%, 9596, 96%, 97%, 98%, or 99% identical to an [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW amino acid sequence selected from the group consisting of SEQ ID NO: lSl—tltl In some ments, the tetravalent Gl'I'R—targeting molecule contains at least one Gl'l‘R—BD that comprises an amino acid sequence selected from the group consisting of SEQ ID NS: 4-2" 62. in some embodiments; the tetiavalent GER-targeting molecule contains at least one GITR—BD that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63—80. In some embodiments, the tetravalent Gl'l‘Rdargeting le comprises two copies of an amino acid sequence selected from the group consisting of SEQ ED NO: 8lmtl3. guess} in some embodiments, the multivalent Gi'I'R—targeting fusion protein is hexavalent. As used herein, a hexavaleut Gilli—targeting molecule refers to two copies of a argeting fusion protein that includes three GITREDs For example, in some embodiments, a hexavalent GlTR~targeting molecule of the disclosure includes two copies of a Gl'l'R—targeting fusion n having the following structure: (Gl'l‘Rd?M—Linher— (GI'l'R—BINslsinkei‘dGI’l‘R—Bl?sliinker—HingesEci In some embodiments, the hexavalent Gilli—targeting molecule ofthe disclosure includes two copies ot‘a GlTR~targeting fusion protein has the following structure: (Gl'I‘R—BD)linker—(Gl'l‘R—BD)—Liiil l.inl in some embodiments, the hexavalent GlTR —tar$ eting molecule of the disclosure includes.4 two copies of a Gill‘s—targeting fusion protein has the ing structure: (Gilli—BB)— Linkerw(GI'll{—BD)"Linkerm{GI'lR—BD)"Linker—Hinge"EC, Where the Gl'l‘RddD is an sdAb sequence that binds Gl'I‘R in some embodiments the liexavalent Gl'l‘R—targetiug molecule of the disclosure includes two copies of a rgeting fusion protein has the following ure: (Gl'l‘R~l3D)—Linlter—(Gl"l‘l{~l3D:t—lsinlter—(GllR?D)—Liiilter—lliiige—Fc, where the Gl'l‘RrBl‘) is a zed or fully human sdAb ce in some embodiments the tetravalent GlTR~targeting molecule contains at least one GITR~BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) Ni}: l9~80 ln some embodiments, the tetravalent Gl'l‘Rdargeting molecule contains at least one Gl'l‘RIBB that comprises an amino acid sequence that is at least 5096, (it’ll/ti, 65%, 700/5, 75%, 39%, 850/5, 90%, gill/6n! 92% 9394;; 94%" 95% 96%; 97%" 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ED N0: 19~80In some embodiments, the tetrai/alent Glllhtargeting molecule contains at least one hBl) that comprises an amino acid sequence selected from the group consisting of SEQ II) NO: 42-62. In some ments, the tetravalent SITE—targeting molecule contains at least one GITR-BD that . Lit D r,| [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW comprises an antino acid sequence selected front the group consisting of SEQ ll) N0: 63~ lit}. in some ments, the tetravalent Gl'l‘Rdar teting niolecule comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 944,65. {6946} The niultivalent fusion proteins ofthe present disclosure are capable of enhanced clustering of TNFRSF nienihers ared to non—cross-linked bivalent antibodies. The enhanced clustered of 'leRSF members mediated by the niultivalent fusion proteins ofthe t disclosure induce enhanced "l‘NFRSF—dependent signaling compared to non-cross—linlred bivalent antibodies. ln niost embodiments, the multi A'alent fusion protein will incorporate more than two Gl'l'R—BDs, for example, three, four, five, or six. in these embodiments, the interaction of the non—"i‘NFRSF antigen is capable of providing the additional inlcing function and TNFR‘SF activation is achieved with only one or two TBDs. {lilitl‘il in some embodiments, the multivalent fusion protein also es one or more —Bl?s and one or more onal binding doniaints) that hind to a target other than GlTR. Tn some embodiments, the niultivalent, niultispecilic fusion protein also includes one or more GlTR—BDs and one or more additional binding dontaint?s) directed toward non—TNFRSF oer antigen. in any ofthese embodiments, the nuiltivalent, inultispecific fusion protein can also include one or more onal g domaint’s) directed to a TNFRSF member, referred to herein as a TNFRSF-hinding domain (TED). in any of these embodiments the interaction of the nonw'leRSF antigen is capable of providing; the additional crosslinlring function and 'l‘NlL'RSF activation is achieved with only one or two (i’lTR—BDs or only one or two GlTR—BDs and TBDs.

Etl?élSl in some embodiments, the nioltivalent, niultispecii‘ic fusion protein also includes one or more additional g doniaints’) directed to a SF nieniher, referred to herein as a. TNFRSF—binding domain (TED). in these embodiments, the niultivalent, inultispecific fusion protein is binds at least two distinct antigens. In some embodiments, all ofthe "l‘BDs of the multivalent, multispecilic fusion protein recognize the same e on the given SF ntenther. For example, the innltivalent, inultispecific fusion proteins of present disclosure may incorporate 2 3, 4, 5, or 6 TBDs with identical specificity to a given TNFRSF member. in other embodiments, the m ultivalent, m ecitic fusion n incorporates TBDs that recognize distinct epitopes on the given TNTRSF member. For example, the niultivalent, niultispecitic fusion proteins of present disclosure may incorporate 2, 3, 4, 5, or 6 TBDs with distinct recognition specificities toward various [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW epitopes on Gil—K, CD40 or CD137. in these embodiments, the n’iultivalent, n’iul ti specific tusion proteins of the present disclosure with contain multiple TBDs that target distinct regions of the particular 1N FRSF member in some embodiments, the Es may recognize different es on the same TNFRST member or recognize epitopes on distinct TNFR SF members, Eor example, the present sure provides multivalent, n’iultispecil'ic fusion proteins incorporating 'l‘BDs that bind GlTR and 0X40. {0049} in other embodiments, the fusion proteins oft‘ne present disclosure is a multispecii'ic fusion protein that binds GlTR and a second TNFRSF member expressed on a non-tumor cell such as, by way ot‘nonwlimiting example, 0X40, C927, H‘VEM, C1340, lymphotoxin beta. receptor (LTBR), ectodysplasin AZ receptor (El) 2R), ectodysplasin A receptor (EDAR), Titr'eakR, BCMA, BAFFR, 3R3, one or (DB? in some embodiments, the n’iultispecil'ic fusion protein is also rnultivalent. in some embodiments, the multispecil‘ic hision protein is ific. in these embodiments, the multispecitic fusion proteins ofthe present disclosure modulate immune cells leading to enhanced tumor ction. in other embodiments, the multispecil‘ic fusion proteins of the present disclosure have utility in treating inflammatory conditions. in these embodiments, the multispecitic fusion proteins of the present disclosure modulate immune cells leading to dampening, or the inflammatory insult. For example, speci?cally agonizing TNFRZ can enhance Treg proliferation leading to in’irn une suppression. } in some embodiments, the multispecitic fusion protein contains at least one Gl’l'R—BD that comprises an amino acid ce selected from the group consisting, of SEQ ll} Ni}: l9~80 in some embodiments, the n’iultispecil'ic fusion protein contains at least one Gl'l'R—BD that comprises an airiino acid sequence selected from the group consisting of SEQ l1) NO: 4362, in some embodiments, the multispecii‘ic fusion protein ns at least one GITR~BD that comprises an amino acid sequence selected from the group consisting of SEQ TD NO: 63—00, {01151} in some ments, the multispecitic fusion n contains at least one —BD that comprises an amino acid sequence that is at least 50%, 60%, , I030, 75%, 30%, 851/0, 90%, 91%, 2‘16, 93%, 940/0951/159,630, 97‘3’0, 989/0, or 99% identical to an amino acid sequence selected from the group consisting of SEQ 11) NO: l9—ti0 in some embodiments, the multispeci?c fusion protein contains at least one GlTRwBD that comprises an amino acid ce that is at least 5(W0, 601/0 0,70/1'0, 75‘",0 8090, 8511/0. 90‘3’0’, 9l‘30, ‘39’, 930/0, 949/, 95%, 96%, 97‘34, 9" ,or 99‘:/’0 identical to an amino acid [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW sequence seiected. from the group consisting of SEQ 11) ND: 42432. in some embodiments, the niultispecitic fusion protein contains at least one (31111—131) that comprises an amino acid sequence that is at least 509/15, 611%, 65%, 709/15, 75%, 80%, 859/15, 911%, 910/1}, 929/15, 93%, 94%, 95941, 96%, 97%, 98941, or 9.9% identica1 to an amino acid sequence seiected from the group consisting 3 11} NO: 63—80, {131152} in some embodiments, the multispecitic fusion protein ns at least one Gi'l'R—BD that comprises a complementarity determining region 1 (CD111) comprising an amino acid sequence seiected from the group consisting of SEQ 11) NO: 106, 109, 112, 117, 120, 125, l31, 138, 143:, 148, and 149; a complementarity determining region 2 (CDR2) comprising an amino acid sequence seiected from the group consisting of SEQ 11.) N0: 111?, 119,113,115, l18,121,127,128,130,132,1341, 136, 137,139,141,144,and147,and a. complementarity ining region 3 13) comprising an amino acid sequence seiected from the group consisting 018131} 1113 N0: 1118, 111, l14, 116, 119, 122, 124, 126, 127, l29, 133 l3 J}1 140, 1412, 145, 1/16, and 150, 1911531 in some embodiments, the peci?c fusion protein contains at least one (111193131) that comprises an amino acid sequence selected from the group consisting of SEQ 11) NO: 1931) and at least one inimunogiohuliu 13c region polypeptide comprising an amino acid sequence seiected from the group consisting of SEQ 11) NOs: 1—6, in some embodiments, the peciiic fusion protein contains at 1east one GiTR—BD that comprises an amino acid sequence selected from the group consisting of SEQ 11) NO: 42—62 and at 1east one immunog10huiin Fe region poiypeptide comprising an amino acid sequence seiected from the group ting ot‘SEQ 1D NQs: 1—6. in some embodiments, the muitispecii‘ic fusion protein contains at least one wBB that comprises an amino acid sequence selected from the group consisting of SEQ 11) NO: 63~811 and at least one. iniinunog1ohulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 143. {131154} in some embodiments, the multispecitic fusion protein contains at least one (EUR—81.) that comprises an amino acid sequence that is at least 511%, 60%, 659/15, 711%, 75%, 311%, 5, 90%, 91%, 211/15, 93%, 94%, 5, 96%, 97%, 98%, or 99% identical to an amino acid ce seiected from the group consisting of SEQ 11) NO: 19—h0 and at 1east one immunoglo‘ouiin Ec region polypeptide sing an amino acid sequence selected from the group consisting of SEQ 11) NQs: 1—6, in some embodiments, the mu1tispeci?c fusion protein contains at least one GITR—BD that comprises an amino acid. sequence that is D r,r [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW at least 50%, .0943, 65%, 70%, 75%, 80%, 85%, 90%, 9 %, 92%, 93%, 9' %, 95%, 96%, 97%, 93%, or 999/1:- identical to an amino acid ce selected front the group consisting of SEQ ll} NO: 42—62 and at least one immunoglohulin Fc region polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll) NOs: 1—6. in some embodiments, the pecilic fusion protein contains at least one GlTR—BD that comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9l9’él, 92%, 93‘5", 94%, 95%, 96%, 979/5, 98%, or990/13 identical to an amino acid sequence selected front the group consisting of SEQ ED Ni): 63-89 and at least one iinmnnoglohulin Fc region ptide comprising an amino acid sequence selected from the group consisting of SEQ ll)l NOS: 1—6. {@955} In some embodiments, the mnltispecific fusion protein ns at least one GlTR—BD that comprises a complementarity deten’nii’iing region l (CDRl) comprising an amino acid ce ed from the group consisting of SEQ ll) NO: 106, 109, ll2, 117, lZG, lZS, 131, 138, 143, MS, and 149; a complementarity determining region .2 {(71,}ng comprising an amino acid sequence selected from the group consisting of SEQ ll) NO: 197, 110,113,115,118,l21,123,128,130,132,134,136,137,139,l41,144, and 147, and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ll) NO: lllS, lll, llil, llti, ll9, lZZ, l24, l26, 127, l29, 3.l1») 3, 135, 141}, 142, 145, 146, and 150 and at least one irnmunoglohulin Fc region polypeptide comprising an amino acid ce selected from the group consisting of SEQ ll.) 'N’Qs: l~o 39956} The multispecilic fusion proteins ofthe present disclosure are capable of enhanced clustering of'l‘NFRSF mcmhers compared to non~cross—linlred bivalent antibodies, The enhanced clustered of "l‘NFRSF members mediated by the multispeci?c fusion ns of the present disclosure induce enhanced TNPRSF—dependent signaling con’ipared to lion—crossdinked bivalent antibodies. in most embodiments, the multispeciiic litsion protein will incorporate more than 2 TBDs, for example, three, four, ?V‘Q, or six, in some embodiments, the specific titsion protein will incorporate TBDS and a binding domain directed toward noneTNFRSF mernber antigen. in these embodiments, the interaction of the non—TNFRSF antigen is capable of providing the additional crosslinking rhnction and 'l‘NFRSF activation is ed with only one or two 'l'BDs. in these embodiments, the rnnltispecilic fusion protein is inultispecitlc, binding two distinct antigens.

[Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 39957} in some ments, TBDs of the present disclosure are derived from antibodies or antibody nents including scFv, Fahs, single domain antibodies {sdAb}, VNAR, or Vlllls ln some embodiments, the ’l'BDs are human or humanized sdAb. The sdAb fragments can be derived from Vl’ll’l, VNAR, engineered Vl’l or VB: domains. Vl’ll’ls can be generated from carnelid heavy chain only antibodies. Vmgs can be gei’ierated from cartilaginous ?sh heavy chain only antibodies. Various methods have been implemented to generate nionomeric sdAbs from conventionally heterodimeric VH and VK domains, including interface engineering and ion of specific germline families, in other embodiments, the "l‘DBs are derived from non—antibody scaffold ns for example but not limited to designed ankyri n repeat proteins (darpins), ayimers, anticalin/lipocalins, centyrins and fynomers. {6958} Generally the ninltispeciiic fusion proteins of the present disclosure consist of at least two or more 'l'BDs operahly linked via a linker polypeptide. The utilization of sdAb fragments as the speci tic TED Within the multi specific fusion the present disclosure has the benefit of avoiding the heavy chain : light chain inis-pairing problem common to many hi/multispecific antibody approaches. in addition, the inultispecil‘ic iiision proteins of the t disclosure avoid the use or" long linkers necessitated by many bispecifie antibodies, {@859} in some embodiments, all ofthe TBDs ol’the rnultispecific fusion protein recognize the same epitope on the given 'l‘NFRSF member. For e, the inultispeciflc fusion proteins of present disclosure may incorporate 2. 3, 4, 5, or 6 "l‘Bl‘ls with cal icity to GITR. in other embodimen ts, the inultispecilic liision protein orates TBBS that ize distinct epitopes on the given TNFRSF member. for e, the specitic fusion proteins of present sure may incorporate 2, 3, ll, 5, or {a T895 with distinct ition specificities toward various epitopes on Gilli, (Ball) or CDlL‘i? in these embodiments, the inultispecilic lhsion proteins of the present disclosure with contain multiple TBBs that target distinct s of the particular TNFRSF member, in some embodiments, the 'l'BDs may recognize different epitopes on the same TNFRSF member or recognize epitopes on distinct TNFRSF members, For example, the present disclosure provides multispecil'ic fusion proteins incorporating TBDs that bind GETR and ()X40.

Etl?élll in some embodiments, the iiision protein ot‘the present disclosure, eg. inultiyalent and/or niiiltisiiecitic fusion ns, is composed of a, single polypeptide. in other embodimen ts, the fusion protein ol’ the present disclosure is composed ol’rnore than ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW one polypeptide. For e, a odin’rerizatioi’r domain is orated into the fusion protein such that the construct is an asymmetric fusion protein, For example, if an iinmunogiohui in Fc region is incorporated into the fusion protein, the CH3 domain can be used as homodimerization domain, or the CH3 dimer interface region can he mutated so as to enahie heterodimeiization. {(3961} in some embodiments, the fusion protein eontains the T8533 and/or Gi'i‘R~ BBS at opposite ends of the fusion protein. For esampie, in some embodiments, the 118133 and/oi GiTR—BDs are ioeated on both the antinostenninai (N —ternnnai) portion ot‘the t‘nsioi’r protein and the carhoxy—terniinai (Oterminai) portion of the fusion protein. in other embodiments, ah the "fBDs and/or Gi’i‘RsBi‘h reside on the same end of the fusion protein For e, TBDs and/or GiTR-BDS reside on either the amino or carbonyl terminal portions of the fusion protein. {(3962} in some embodiments, the fusion protein contains an iinmunogiobniin Fe region in some embodiments, the immunogiohuiin Fc region is an igG isotype seiected from the group consisting ot‘igGi isotype, EgGZ isotype, igGB e, and F464 suheiass.

E6963} in some embodiments, the immunogiobuiin Fc region or imniunoiogicaiiy active fragment thereof is an igG isotype. For examp e, the nogiohniin Fe region of the fusion protein is ofhuman igGi isotype, having an amino acid sequence: >GPS VFLFPPKPKD TLMISRTPEV Te\VVHVSH3 DPEVKFNWYV NAKT KPREEQYEBT YRVVSVLTVL HQLn?" \EPQVY TLPPSRDELT ANCV\1[C]J KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK R.EiQQ GEVFSCOVMI EALiJHYTQK SLSLSPGK (SEQ ID NO: 1) {(3964} in some embodiments, the iinniunogiobuiin Fc region or iinmunoiogicaiiy active fragment thereof ses a human igGi poiypeptide sequence that is at least 50%, 609''o, 654':- 70‘36, 75%, 80%, 85%, 9 0,9104, 920/o, 93%, 94%, 95%, 9 "'6 9’04:- 980/, or "6 identicai to the amino aeid sequence of SFQ if) NO: i {6965} in some embodiments, the human igGi Fc region is modified at amino acid Asn297 (Boxed, Kahat Numbering) to toto giy'eso5/ia'tion of the fusion protein. 6 0., Aan97Aia (N297A) or Asn297Asp 3). in some embodiments, the Fe region of the fusion protein is modified at amino acid Leu235 (Boxed, Rabat Numbering) to aiter Fe receptor interactions, 62g, 1{31:25Ciiu (1423',9F) or Feu2.55Aia (F235A), in some [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW embodiments, the Fe region oi‘the fusion n is modified at amino acid Leu234 (Boxed, Rabat Numbering) to alter Fe receptor interactions, eg Len234Aia (L234A). in some embodiments, the Fc region ofthe fusion protein is i ed at amino acid Leanzil (Boxed, Kabat Numbering) to alter Fe receptor ctions, gig, LenZESGln (1.23513). in some embodiments, the Fe region of the fusion protein is altered at both amino acids 234 and 235, eg; , iAia and LenZBSAla (L234AfLZ35A) or LeuZ34Vai and LenZSSAla (i4234lvV'1’iJZ35A) in some embodiments, the Fc region ofthe fusion n is aitered at Gly235 to reduce Fe or binding. For example, wherein Gly235 is deleted from the fusion protein. in some embodiments, the human lgGi Fe region is modi?ed at amino acid Gift/236 to enhan ".6 the interaction with 0332/4, eg Giy236Ala (G2363A). in some embodiments, the human lgGl Fe region is lacks 143/5447 (EU index of Kabm er a! l99l Seqzren 'es ofProreirzs ofimmzmological Interest). {seen} in some embodiments, the Fe region of the fusion protein is d at one or more or"the following positions to reduce Fe receptor binding: Len 234 (L234), Len235 (L235), Asp265 (1)265), Asp270 (D270), Ser293 (S293), AanQTI’ (N297), Asn325 (N325) 327 (A327), For example, Len 234Ala (LT/EMA), 5Aia tLZ35A), Asplo?Asn (D2635N), AsplmAsn (l HUN), Sier293Asn ($298N), Asn297Ala (N297A), Asn325Ghi (N325l3) orAlaBZ’FSer (A3278). in preferred embodiments, modi?cations within the Fe region reduce binding to Fe-r‘eeeptor—gammn receptors while have minimal impact on binding to the neonatal Fe receptor {FoRn}.

{MW} in some embodiments, the Fe region oft‘ne titsion protein is lacking; an amino acid at one or more ofthe following positions to reduce Fe receptor binding: (i'lu233 (E233), LenZSLl (L234), or LenZ35 (L235). in these embodiments, Fe on e three tintino acids reduces the complement protein Clo binding. "i‘hese modified Fc region polypeptides are referred to herein as "Fe deletion" polypeptides.

GL SVE'L FL ti'it’KLIl'Li‘l‘l __. 'Rl'l. E‘VTJV TITVEVS E DEE VKFNWYVDGV EVHNAKTKPR EE'3 F4: r-v m is N P:3‘4 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI Eh.; SKAKGQ VSLTCLVKGF YPSDIAViWE SNGQPENNYK DSDG SEE YSYLTV DKSRWQQGNV F?' HNHYTQKSLS LSPGK (SEQ ID NO: 2) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 39968} in some embodiments, the immunoglohulin Fe region or immni’ioiogieaily active ii'agnient feonipi‘ises a human lgGl polypeptide sequence that is at least 51‘3‘343, 6934:1159, 700/93 75‘3L:.8(‘9‘3 5/L'3 9i‘)‘,34:. 9l‘3’ 92%. 93 ‘3L1.94% 95‘}/'L'3. 913%. 97‘3’0, 98993. or 990/4: identical to the amine acid nee of SEQ l1) NO: 2 {9969} In seme ments. the immunoglobulin Fe region or immunologically active fragment of the fusion protein is of human 5.ng isntype, having an amino acid SGQHQHCQI ) PVAG '5 5V E ‘L F 'c L LML S R'i' 3317:33V3T CVVVDV‘S HE D PEVQ FN‘LIL71"JD (3V {W3 E] NAKTK 3P RE} EC; 5&3 31.3 E3 [xV33k/‘t33x7 113133937132 Q Did L L'K‘L’ S NK(_33:: PL"H.

PiiEECTISKTK. GQEJ‘REPQVYT LPE’SREEMTK 13-2393 LTC VK GFYPSD: SVES DGL'S E‘E3_[.. YSKL '3_[3'3ifi39KSRVVIQTEG NV E'SCS‘WIHE 3.5 ID NO: 3) {9979} In seme embodiments. the fusion or immunologically active fragment thereofeompi'ises a human igG2 polypeptide sequence that is at least 50%6094: 65‘3’ . 7.5 90. ’9.9.%.‘339%). 91941. 95‘3 £3.96"A). 97‘ ’0.98% , 80,996. L3 L3.590%. 93‘3’ or 99% identical in the amino acid sequence 01‘ SEQ ID NO: 3. 39973} in some embodiments. the human 5 £15132 Fe region is modi?ed at amino acid Asn297 (Boxed, to prevent to glycosylation oftne antibody/.9 g, As11297Ala (N297A) or Asn297Asp ) in some embodiments the human lgts‘Z Fe 1egion is lacks l3Ls447 (EU index of Rabat 91 L21! 1991 Semwrzces ofProreim (3’I?’lii’lbir7(3")0’e£h Iizzeiesr). {9972} in some embodiments the immunoglnhulin Fe 1"gion or logically active fraqment of the fusion piotein is of human qu3 isotype.having an amino acid sequence: »_PEL GC " VFLFEPKL BID TLI: SR£53373 i'CVVX/‘DVSE’IE LLE’LVQFKYI‘JYV DGVEVHNAKT KE’REEQYEST FRVR’S’VLTVL KCKVS'J ALP K (2133:" \El [:12VY TL PPS RE) .lil’3' "Xi QVSLTC L317 KG E353 E’SD 3! NYN'I‘TPPMLD ‘3 [V313 E 53K EJ313VEEI’SRWQQ (3N 3LETSC SVW] :3 53.3. S PGK (S ET) I D NO' 4: I" ) {9973} in some embodiments. the antibody or immunologically active fragment thereof comprises a, human igGE polypeptide sequence that is atleast 500’LL), 66996. 6534:, [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 70‘3/13, 75%, 803/11 85%, 9(‘30,93%, 923:;3, 93‘3", 942/0, 953133, 9‘3097.71", 98‘3:3 or 99‘3" identical to the amino acid ce of SEQ ED NO: 4. {(3974} in some ments, the human igG3 Fe 112gion is modified at amino acid A511297 (Boxed, Kahat Numhennu) to t to glycosylation of the a11tihodv,e 1‘1an 97Ala (39297151) or 33151129'Asp ) in some ments, the human lgG3 Fc region is modified at amino acid 435 to extend the half—lite, cg SHiS (R435H), in some embodiments, the human qu3 Fe 1egion is lacks {1135417 (F.U index 0tKab11t 121* a! 1993 Sequences QfProlems 1'1flmmzmelogicm’ Interest).

E6975} in some e111hodinients, the imniunogiohtilin Fc region or immunologically active ?‘agnicnt of the fusion protein is of human lng?l isotypc, having an amino acid '98 C113 (311C362 V KD LLMTQRTBEV DGVEVHNAKT KPREEQFTTST YRVVSVLTVL KCQD\LLHVY TLPPOQLFJT KGFYPSDTAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKU)W 5 JJWQL GKVFSCTVMI EALQINHYT'TK SLSLSLGI~ (SEQ TD NO: 5) {(3976} in some embodiments, the antibody or immunologically active fragment thereofcompi’ises a liu1’11a1’1 1,1584 polypeptide sequence that is at least 50%, 60%, 65%, 70%,75‘3/1'. 800/ 85%, 90%, 91%, 92%,93‘3/1';_94%, 95%,96‘3/1'. 97%, 98%,01' 999.421 identical to the amino acid sequence of SEQ l1) NO: 5. } in some embodiments, the immunoglohnlin Fe 1eegion or immunologically active fragment ot‘the fusion n is o1" human lgG4 isotype, having an amino acid SEQHCHCCI PAPELLGGPS VFLFPPKPKD TLMTSRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNPKT KPREEQFW?T YRVVSVLTVL HQDvLNCKTY KCKVFSNKGLP CSTBKTTSKA LGCVt?PCVY T PiWC'39 KNQVSLTCLV [JF’CSTLAV EWISNGQPEN NYKTTPPVLD Cf) DGS.FFLY.5R LTVDLVRWQ: GNVFSCSVME EALTNHYTQK GK (SET ID NO: 6) {9978} in some embodiments, the antibody or immunologically active fi'agmeht fcoinpi'ises aliui11a11igG4 polypeptide sequence that is at least 50%6,094. 65‘3" , [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 7313343, 75%,89’37'33 85%, 99%,93':/'33 9234, 93%, 94'3/33 9534, 96%,9'7‘3:/'33 98343. or 99‘//0 identical to the amino acid ce of SEQ LU NO: 6. 393379} 3n other embodiments, the human igG/l Fe region is modified at amino acid 235 to a3te3’ Fc receptor interactions, (3.533.363.32358313 (LZ35E). in some embodiments, the human li§G4 Fe iegion is ed at amino acid [8533297' (Kabat Nuinheiing) to prevent to glycosy‘iation of the antibody, eg 383332971838. {N29728:} oi' 2853329728sp 3)). 333 some embodiments the human 333,64 Fe iegion is lacks ly-/s44 (353.33; miles ofKK32 2532‘ 1 l993 bun/39mm (3,21rates/25 0151321323233233303526231223636333 3339893 in some embodiments, the human 3gG Fe region is modified to enhance FoRn binding. Examples of Fe mutations that enhance binding to FeRn are MetZS Z’l'yi‘, 83332543333", "33256423333 (M25281 S254T, T256E, tespeetiveiy) (Kabat numbering, Dali’Acqua er a! 20.06, J. 8203’ 2: Vol 283(33) 235 34235 24), =‘s‘let428Leu and $e3 (M428L, N43483:) (Zines/sky 32/? a] 2030 Minn/"e Bio/eels, Vol. 28(2) l5 .7459), 03 lVietZSlee, "3’l3t256Asp, Met428Leu (3842523, T2563), M42833, respectively), (EU index of Anna! ’ l99l 8e5,223emes off)isle/"2'33 of[WW/22220309332: Interest) 3339833 in some embodiments where the thsion protein ot‘the disclosure includes an 33o polypeptide, the Fe polypeptide is mutated or modified. in these embodiments, the. mutated or moilii‘ie33 Fe ptide includes the foilowing mutations: Met252373373 and Met428Leti {384252332 M428L} using the Rabat numbeiing . {9982} in some embodiments, the human lgG Fe region is modi?ed to alter antibody/dependent echuiar cytotoxieity' (ADCC) anal/or conipleinent—depen dent eytotoxieity {CDC}, eg. , the amino acid modi?cations tleseiihetl in Natsu3ne et 313., 23308 Cancer Res, 68(30): 3863—72; ltlusogie et al, 2003 3 immunoi, 366(4): 25736; Moore ct £33., 23330 mAbs 2(2): l83 389; 3332233 et al. 283333 PNAS, 3030 3):340333, Shields et 33, 20333 38C, 2763693,3: 6593—6604; ltagen et at, 2007 Cancer Res, 67(38): 8882—88933; Slavenhagen et al, 2008 Advan. Enzyme , 48: 352~l 64; Alegte et al, 3992 3 3333333333303, 148: 68, ed in Kanehe and Niwa, 23333 Biotliugs, 25( 33: 3'31.

Examples of mutations that enhance ADCC include modi?cation at 8e3239 and 2, for example 83:32 '393/8333 and 333:."5173Clu (82393), 333233). Examples of mutations that enhance CDC iiielude inodii‘ieations at Lys26, and (333333:3’L)) 333 some embodiments, the Fe region is inoclii‘iecl at one or both 03these positions toi 3;sanipie 83a r {3333.333Ma (K323352533 and 37333.4.) using the Kahat numbering system.

Us; {.33 [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 399983} in some embodiments, the human 5 gt} Fc region is modified to induce heterodimerization, For example, having an amino acid modi?cation within the CH3 domain at "i‘lirS as, which when replaced with a more buiky amino acid, ag Try (T3new; is able to preterentiaiiy pair with a second CH} domain having amino acid itiodii‘ieations to less bulky amino acids at positions Thr366, Let1368, and Tyr407, e27 Ala. and Val, , Ser, respectively (1366811368Af‘i’4t‘i7‘v’). Heterodimerization via (‘83 modi?cations can be thither stabiiized by the introduction of a disnifide bond, for exampie by changing Ser’SSAE to {33's (SEMI) and Y349 to Cys (3734913) on te CH3 domains (Reviewed in Carter, 200i i of immunological Methods, 248: 7——-l5). {($84} in some embodiments, the human igG Fc region is d to prevent dinteiization. in these embodiments, the fusion proteins ofthe present disclosure are monomeric. For example modi?cation at e Thr366 to a ci’iarged residue, eg. 'lhr366Lys, 'i‘hr366Arg, 'i‘hrSeoAsp, or TindoeGin (Think T366312, T3661), or T366E, respectively), prevents CH3~CH3 dimerization, 399985} in some embodiments, the Fc region ot‘the fusion protein is d atone or more ofthe foliovving positions to reduce Fe receptor binding: Len 234 , Len235 (LL35), AspQ-lo? ), Asp27€3 , Sei298 (S298), Asn297 (N297), Asn3 25 (N325) oi‘Aia327 (A327). For exampie, Leu 234Aia 6.1234153), i.i€’313235Aia. (11.235153), AspZoSAsn (D265N), (LXspZYOi/Xsn (BEEN), Ser298Asn (SZQSN), Asn297Ala th29'7A), AsnEZSGln (NSZSE) orAia327Ser (A3278). in preferred embodiments, modi?cations within the Fc region reduce binding to Foreceptongamma receptors While have minimai impact on g to the neonatal Fe or (FcRii).

Eti?Sfi} in some embodiments, the n protein contains a polypeptide derived from an imninnogiobuiin hinge region. The hinge region can be seieeted from any of the human lgG snhciasses. For example, the fusion protein may contain a, modi?ed igGi hinge having the sequence of E3PKSSDK"]?HTCP PC: (SEQ El) Ni}: 7), where in tlie CysZZO that forms a distiitide with the C—terminai cysteine of the light chain is mutated to serine, e.g, Cst-ZZOSer ). in other embodiments, the fusion protein contains a ted hinge having a sequence CDK"]TH"]TCPPC (SEQ it} NO: 8).

EGQS’B in some embodiments, the fusion protein has a modi?ed hinge from ing, which is riioditied to prevent or reduce strand exchange, 2* U Ser228Pro ($122812), having the sequence E::~3K‘iGPPCiPPCZ (SEQ ED NS: 9). in some embodiments, the. fusion protein [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW ains linker polypeptides. in other embodiments, the fusion protein contains linker and hinge ptides. {($88} ln some embodiments, the fusion proteins ot" the present disclosure lack or have reduced lhmose ed to the N—linked glycan~chain at N297, There are numerous ways to prevent tiicosylation, including but not limited to production in a Fill—3 delicient cell line, addition inhibitors to the mammalian cell culture media, for example ospeimine; and metabolic engineering oftlie production cell line, 39989} in some embodiments, the TED is engineered to eliminate recognition by preexisting antibodies found in humans. in some embodiments, single domain antibodies ofthe t sure are modified by mutation of position Len l l, for example Leul lGlu (Ll ll?) or lieivil llnys (Ll lK). in other embodiments, single domain dies of the present disclosure are modified by changes in carboxy—terminal region, for example the terminal sequence consists ofGQG’l’LV'l’Vlil—‘GG (SEQ ll) N0: l0) or GQG'l'LVl‘VEPGG (SEQ ll.) NO: ll) or modification thereof, ln some embodiments, the single domain antibodies oftlie present disclosure are andlllSCl by mutation of position l l and by changes in carbosy—terniinal region. {($96} ln some embodiments, the "l‘BDs and/or Glll‘lerlls of the fusion proteins of the t disclosure are operably linked via amino acid linkers, in some embodiments, these linkers are composed inately ot‘the amino acids Glycine and Serine, denoted as (ES—linkers herein. The GSwlinkers ot‘ the fusion proteins of the present disclosure can be ofvarions lengths, for example, 5, s, 7, 8, 9, l0, ll, l2, l3, li-l, l5, l6, l7, l8, l9, 20 amino acids in length, Eti?Qll in some embodiments, the GS~linker comprises an amino acid sequence selected from the group consisting oi GGSGGS, i.e., (GGS); (SEQ ll) NU: l2); GGSGGSGGS, i.e., (808k (SEQ ll) N0: l3); GGSGGSGGSGGS, ie (GGSli (SEQ ll) N0: l4); and GGSGGSGGSGGSGGS, ie., ((346835 (SEQ ll) NO: l5). } in some ments, the linker is a flexible linker comprising Glycine es, such as, by way of nonslimiting example, GG, GGG, GGGG (SEQ ll.) 'NQ: lo), GGGGG (SEQ ll) N0: l7), and GGGGGG (SEQ ll} N0: its), 3995.33} in some embodiments, the GlTR-targeting thsion protein includes a combination of a GSwlinker and a Glycine linker.

Us) \l [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW ed set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW Brief Bescrintiun of Figures {6994} Figure 1A is schematic representation of exemplary multivalent fusion proteins of the present disclosure.

{M95} s 2A 28, and .2C are a series of graphs demonstrating the binding or" Gilli—targeting fusion proteins to (EUR expressed on Cl’lO cells as assessed by flow cyton’ietry. The GlTR antibody, TEX-518, was used as a control for these studies Eti?in} Figures 3A, 3B,, and 3C are a series cfgraphs demonstrating the ability of Gl’l‘R,~targeting fusion proteins to block the interaction between Gl’l‘Rl.. and Gl’l‘R. Herein, a flow cytometry assay using GITR expressing Cl’lO cells and recombinant GlTRli was used to assess blocking capacity. The GlTR antibody, TEX-518, was used as a l for these studies. {6897} Figures 14A, 48, 4C, ill), and Ill?) are a series ofgraphs depicting the g oftlie OUR—targeting molecules oi‘the disclosure ed to as lithlovl . l, ev l .2, lizCO?vl3, lizCllot/ld, hamster thOthZ, Z.3, lizCl_l6v2,4, lizClléir/S, eKl, lizCUéSv?» .2, hzt‘nrwss, thOon?xil, hZCOoVKS, liZCOG-V?) .6, lizCUéSv?» ,7, lizCOet/Ll8, liZCOl3‘l/‘39, thGfivB. ll"), . l l and h2C06v3lZ for human GITR and cynomolgus GETR {"cync Gilli") expressed on the surface of CHO cells, as measured by flow cytonietry. {6898} Figures 5A., SB, SC, SD, and 5E are a series ofgraphs depicting the binding oftlie OUR—targeting molecules oi‘the disclosure ed to as lithldvil. l, liz€2ll=lv4. l 2. lithlil-vdl, lizCllllvilQZ, lizCleS, thOAlv l .2. l, llZCO4V5. l, hZCOLlVSQ, l‘iZCOil—VS3, lith‘erS .4, lizCllzl-VS i lizCliéle . 6., thOAlVS . 7', ltZCO-"l-VS 8 lith‘erS .9, lizCi’?l-VS l0, lizCO4vSl l, and thdeS. l2 for human GITR and cyuomolgus GlTR ("cyno Gilli") expressed on the surface of CHO cells, as measured by llow cytornetryi } Figure 6 is a schematic representation of teti‘avalent anti—Gl'l‘R molecules of the disclosure, which are constructed with EWO tandem copies of a single~doniaiii variable region (sdAh) fused to a human lgtfll Fe . Surrogate molecules are constructed with Fc don’iains derived from mouse lgGZa.

Ell?llllll Figure 7 is a graph depicting the binding an anti—Gilli mo ecule oftlie disclosure, referred to herein as tetravalent lizCOd—hlgtfil, to pri mary human T cells.

Tetravalent lithlo—lilgGl is constructed with two copies cf the Gilli-binding molecule of SEQ ll} NO: 93, which, in turn, is constructed with two tandem copies of a siiiglewdornain [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW variable region (sdAh) of SEQ TD N0: 59 fused to a human lgGi Fe domain of SEQ TD NU: i.

Ellilllll} Figures 8A and 88 are a series of graphs depicting the y of tetravaient GlTR—targeting molecules ofthe disclosure to activate NF—kB signaling in reporter cell lines expressing (EUR {@162} FiguresSJA, 98, and 9C are a series of graphs depicting that treatment with a tetravaient Gl'l‘ltrtargeting molecule of the disclosure signi?cantly reduced eras tumor growth irrespective of day of administration Etl?lllgtl Figure l0 is a series of graphs depicting the dosendependent suppression of CF26. tumor growth by a tetravalent Gi'l‘R—targeti ng molecule of the disclosure {@9164} Figure ll is a series of graphs depicting the dose~dependent suppression of MCBS tumor growth by a tetravalent (i’lTR—targeting molecule ot‘the disclosure. l?l Figures HA, lZB, and lZC are a series ofgraphs depicting the impact och function on inhibition of C'l‘26 tumor growth.

Ellllltltil Figures BA" BB, and l3C are a. series of graphs depicting that treatment with a tetravaient Gl'l‘Rdargeting molecule had uence resistance to re "challenge with CF26. tumors {@9167} Figures MA, MB? and 14th are a series of graphs ing that treatment with a tetravaient GER—targeting molecule of the disclosure signi?cantly reduced ng frequency and altered the ratio oi’l‘mg to 'l'cmm— celis within the tumor microenvironinent, {lilting} Figures iSA and i738 are a series of graphs depicting that treatment with a tetravaient GlTR—targeting le of the disclosure signi?cantly induced CD8 T cell activation and eration.

Betailed ?escription {691%} The disclosure es molecules that specifically engage glucocoiticoide induced TNTR—reiated protein {GITRL a member of the TNF receptor ‘arniiy (TNFRSF). More speci?cally this disclosure relates to ivaient molecules that bind at least GlTRi These vaient TNFR SF binding fusion proteins comprise two or more TNTRSF binding domains (TEES), where at least one TED binds GlTR, referred to herein as a Rwhinding domain" (GER—BB). {llllilitll Gi'l‘Fi is a member of the TNFRSF and is tutivcty expressed on CDZ5+,I’Foxop3+ regulatory T-ceils (Treg) in a tumor and upreguiated on other T—cell [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW populations following activation. it is hypothesised to have and dominant role in Treg— mediated immunological selfltolerance, Gills agonists dampen the suppressive activities of "l‘regs and in mouse models have been shown to enhance. effector T—cell l lhei"eftire a functional GlTR agonist has great potential tumor immunotherapy.

{Willi} in some embodiments, the fusion proteins ofthe present sure incorporate at least one Gilli-Bl). in some embodiments, the fusion protein is a inultivalent fusion protein, in some embodiments, the fusion protein is a multispeeifie fusion protein that binds GlTR and a second n, such as, for example, any other TNFRSF member. in some embodiments, the fusion n is a multispecific and multivalent fusion protein.

Elltll l2} in some embodiments, the Gl'l‘RrBD binds human and cynomolgus monkey Gilli, in some embodiments, the GER-Bl} blocks, inhibits or otherwise modulates the interaction of GlTR and its ligand Gilli—Ligand {GlTR~L). in other embodiments, the Gilli—8D does not block, inhibit or otherwise modulate the interaction of Gl'l'R and Gl'l‘lh L in some embodiments, the fusion protein of the present disclosure incorporates multiple. copies ofthe same GlTR—BD. ln some embodiments, the fusion protein of the present disclosure incorporates multiple Gl'l‘R—BDS that recognize the same epitope on Gl'l‘R, in some embodiments, the fusion protein of the present disclosure incorporates multiple Gl’l‘Rs BBS that recognize distinct epitopes on Gilli. in some embodiments, the fusion protein of the t disclosure incorporates multiple (llTR—BDs, wherein some GlTR~BDs block the Gl'l'R—Gl'l‘R—L interaction and other do not block the Gl"l‘R~Gl'l‘R—L interaction. in preferred embodiments, Gl’l'R—targeting fusion proteins of the present disclosure induce direct cell death of tumor cells.

Ellhlld} in some embodiments, the Gl'l‘lhtargeting molecule includes at least one copy of a single~domain antibody (sdAb) ce that specifically binds Gl'l‘R, in some embodiments, the GlTR—targeting molecules include two or mor >- copies of an sdAb that specifically binds GlTR, for example, three or more, four or more, live or more, or six or more copies of an sdAb that specifically binds Gl'l‘R, {llllillé} A single—domain dy (sdAb) is an antibody nt ting of a. single monomeric variable antibody domain that is able to bind selectively to a specific antigen, With a lar weight of only l2-l5 kDa, single~domain antibodies are much smaller than common dies 050460 him) which are composed oftwo heavy n chains and two light , and even smaller than Fab fragments (~59 klla, one light chain [it 0 [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW and half a heavy chain} and single—chain A'ariable fragments (~25 kDaj two variable domains one from a light and one from a heavy chain). llltll l5} Single domain antibodies are dies whose compleinentaiy determining; regions are part of a single domain polypeptide. Examples include, but are not d to? heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from tional 4~eliain antibodies, ered antibodies and single domain scaffolds other than those derived from antibodies Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and/or bovine. in some embodiments, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain dy devoid of light chains. For clarity reasons this variable domain derived from a heavy chain dy naturally devoid ot‘light chain is known herein as a Vl-ll-l to distinguish it from the conventional VH oi‘t‘our chain imniunoglobulins. Such a VHH le can be derived from antibodies raised in Camelidae species for example in caniel, llania, dromedaiy, alpaca and oi Other species besides Carnelidae may produce heavy chain antibodies naturally devoid of light chain; such Vlllls are Within the scope of the disclosure. {hill in} Gl’l‘R \I’llli (llania~deriyed) and humanized sequences are shown below; and the CDR sequences are shown in the sequences presented below in some embodiments, the GlTR—binding sdAb is fused to an lgG Fc region and in these einbodinientsthe fusion protein is bivalent having two Gl'l‘ltwbiiiding domains per le. in some embodiments, two Gl'l'R—binding sdAbs (2X) are fused to an lgG Fc region and in these embodiments the thsion protein is tetiavalent haying four GlTR-binding domains per molecule. in some embodiments, three Gilli—binding sdAbs (3X) are fused to an lgG Fc region and in these embodiments, the fusion n is hexavalent haying sis Gl'l‘Rhinding domains per molecule Exemolary GEEK-Binding sdAhs SbSVESllNCWVRIFPChokEl‘\[SSGSBAYADSJ5 KPZDTKVV\CDVATGWGRDISAXUc/GTFVTVSC .EQ ID NO: 106) Al l, [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW n—m 3 y." 54—1I331,SCQGL‘YRLACGCLRLS ‘I’IVI’LVRQAE‘IJAIRILVIA71: T674S114'\1YLE‘3 CV (133191" I S [\G.1I:23:1I—‘.\I‘ ' nwn— - 1. \I' 1.1. 'IE AI‘xI’Y.’ (I11913117 @111 NnE‘" JY 111GQC (SEQ ID 1‘10: 201 r\ r~1R1. \/ 14 ?‘’ /\‘\‘ 7—,. 3/ V KJiJ\I . . GS I1R113? I. A ‘GSIFSII SVK «RF 'I: SRDS.ALI‘ mvc.L V \.1 RMNS_.E IE‘SI1I1‘1111’3I E S EEQ II D NC) : TCGLSTK (SI C D E<.3 [x ‘I\71\11EZGE'\E1.‘17\1I)Y 171337111 (SEQ ID NO: RLSCAASG8VPS I: IPXI‘TGVIJY RLAP"GQQRIELVAVLNGT SSEK‘IAL.

KNIAVY IJQIM IDGIEJ K1? {AEJVO'E’E1%1‘1L’.IEYWGQG'IE' \I’SS\3 \7 GSVE‘SIDAI‘i (b ‘7IE1IE\1CIESSE"\.E{ (S AASGNI7 IIE E‘SII "11711019: E‘CIR‘{IQRE‘IVIAI"C IEII—‘GGP. I'IDSVKC4 ._3\ T‘u'rx‘ 7r » (An ‘ V{'A7r;..

'[\ I'CIEVK 1".4 E. ’IVAO"gnu. ‘ ~30 .x);\»/ 72 1': (8113’ . 1—L \1 < I_/ 3 Ci} 17.1 r1 \_/ [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW QVQTQESGGGGV’DTGSGRLGCT" SIDSMSWFRQAPGNExaLVAP TGGD'leAD SX'CER FI"I "PAPARN"VVLQKNSIR}_I"VYYCN1VV°T"WG‘~ IDYWC.§QGTQVTVS ALO. ID NO: 24) DDR1: SGSJE'S' (SEQ CARE: LITGGIII (53 ID NO" CCI3: NAVVSHGWCRNAEDXW SEQ *_.C3C3C%E1‘7C2, L1}11:1CIZVAEES\TCI’I["531"J:1T*\ GAP("1'759RELVAVID :TUV C)..""‘P‘NK%[ \<*J».

KPEDTAVYYC GWGRETIHIVGDGTQVTVS . ""'?tSnL.'T.

'\"’TGTTJ QVQGQAQGGGGQPGGSTRLSCAASGSVFSIDSMSK:RQAEGNE LLVALITGGHIITYGD JV (GRF1‘ 1:5: 1T"AW 'T‘"INAT' (SEQ ID NO" :6) czR1: ID NO: 120) :R2: LTTGGHTTT (SEQ IF NO. 123) CLRE: DAAVSTGWRNALTTM (337 ID NO: 124) QDGESLRLSTDASGRV‘"ILSVOYY"GG’"{ER yVALITGGRTTTYAst KGRFTTGR_TNAEATVHLRMTGLKPTDTAVYTCNAAVGTGVGRNLDY"GCG"QVTV SEQ 19 ND: 27) 2 SGSVFS‘ 7 (SEQ CLRZ: LITGGRTTT (GEQ ID N 1 GW 1.RNA 3': )1’W 1’ S [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW or 0‘7 u'av « E‘SIDSNS1 {GR }.1r CIYZT‘L’H' Y CIA ’ 'Intrxl r3, ‘1"\7 1‘11.ij~ r\ ' 1-1/1» I. \f'z'ws >»»z_ 3;? ID 1‘10: 2'?) C391}. 1 :3C353‘x’" "3 I "I ’ I II D 110: 12 O) CT'RZ: LITGGRTTT (SEQ ID NO: 121) {DRE 2 .1AAL Li‘I'GIUQIhJASAYW (S E152. ID NO: .'.2 6) ;\\7 IGG71.11123 "3‘381.1R1'JSC3IETASC353CI1E‘SINI—1IV1A' \1"’CK‘‘ ‘ 'IVA:I.1.'I‘GC‘.ASI"1\YAD SVKGRE‘T:IbRDSALI FT‘TT'.‘ ,1 \a _'.V\_.'LEM ISD (SEQ I ID NC): 29 DR1: SGSIZFSINHLI (SLQ .1) NO: 112) CD112: (SEC! .11) NO: 113) CLI'E: AEVNEC'WITALYI131711 (SEQ ID NO: 123’) 91-QT QESGGGIVQAGGSLR SC?" ERS AS LIVMGWYRQAPGNQH*ILVAA ’EIYAG SV 3GP III: I ‘31? DNAE"1‘1'I‘VY1'1’)I\C1‘1II.I\1~ 1""7v YCIACSEI1.1C{...)I :31.‘ G {YIICI’TGL '1,\«'I"‘\.753 3Q ID NO: 30) (31,111: SPLIIJ?VAW1J1 ( SEQ 11) NC): 14:81 TTSGGSPN (SEQ 1D NO: 1.28) CIDR3: :R1Z)DS." ' " (SEQ: ID 1‘10: 129) . («r-«7 0'~r~ ":"T)' . .MDK/tx1‘1.\)"\3 C) \I’ E101 D:3.I :31'113E1? QI" 13":1‘ 1f {PEDTA7W CNAAVSTC ,7RN?»YDDYWGQGTQ\TVS b'1 E252.

[Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ’* O T 1') J K) 1.1 L.\ , VFW" ‘15 '1’: SJ1N‘GDE VKGRF' 'ISGDSAKNTVY I ,QM: RL: {SEC} ID I\1CL : 3,) L D 4,, GT4R1: S"L:S I CLDRZ : V L3 r- (J O r' LCJ 'L:.' L) x).

CLR:FA YP'Lt’ ISSG 1‘7S7‘JE'RQGPGNEFJLLVAT. TGGI’"M LYAD EEV 2",G1L [IT IE‘ ?ANA191 TV 13 1'J\/ll\.\l:;.[\‘f r \.),L_L =_. 1.17""! YC31\1Pv’\‘7E3'[C‘1LICLKSA')YWGC‘CT\I11V"; (SEQ ID NO: 3:3) LC" 1‘ TL? or ‘L-:01 LL-LJ I L i1. : LL) Li'xJ L’ .C' x). (SEQ NC) 2 K/L. TTT ID NO : 134) (\IR 1. {\DL-x‘, \J'L- I. 3: 1x13.LA‘] LTGW \jl\x? Li S E10. ID NC): 13C.) ).Jk\\/r\k1‘1\)\‘ ’ orn‘ "\Q 1|THI 7) LL .1? CDG PGl‘M 1.<[]1LV,AI,. I", ."L:L\T"1.~r). :L 11..

CNAA,7JTCTI GRN?»LDDYWCLCLGT’WVTV LEW/71:N *?OT.Q I #1:: ' ITC[5G1.<.L"7"1'1.‘ (S EC) ATADDY Al‘s/MSW: RDAEGNER''LV’L’L .. TGGR‘T T'TYA: LEV {GK E"1." IS' TREE—ll}\nr " C‘N.AV‘L/S‘ " G KNADDY MGCDC: CQJQ,or? IDT: NO : CDR1.: [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW Pillar-$20 C." GEVQ 1, "0-.— r Q.(‘4 0T7 x). "I .‘C J K) JJ 1\ IJx)'\_ . I). \/ ITS "‘me[wok/I] SvI'CGRFT :_g. RDIVAKITLYLI’QI/I. ' ‘TT CL: .LUALJT:TA\7YY’GNA GEQGTLVT7I~CP I’ S EQ I I) N f. ,r\ H.a 30) CI4R1: ,—<’\\‘rbVFSI DAM ( ID NO: 106) CIDICZ : TTCGRTTY (SEQ : 136) C) C -. '1rx ;; : NA\JSTGWGRNADDYw (SEQ II n. v42Q.1 EVQT LES.‘5GszrIGGSTRLSCT’ S T \II‘T‘"ITRQA I4IL \1 \JLI‘LYYI.'\T\ '1YYAE S‘I’GPIT" 'CZSR mtITL'JQMSSURAEIIIIVYIFIIXV‘7 ST CIT/\I CE I‘N ADDYW rw;KjI‘GTIA!"'Wk P IJLQ("I"I TT‘ .\ IS Ao: 37) (If-LII]. : GGVFGIII:M (SEQ ID NO: I06) 1*. 1~. ’1 K/L._I_ ; : AITGGRTTY {$3 I= NO" \ 137) CZIZI 3 : IIAV‘V7STGWGRNA I) I) .‘I’w SEQ ID NO: Hllvé?l aw ' - n C4 r~r rw-r ,_‘ I \/ .J'CIQKFL II)L S. C:AA‘ ‘ . I7ST, :I _ RQA PC ._ .Kji. J L SVKGRFTT SRI‘IIAKNTLYLQI’IS ST-PGAEITT1»_\/YYONAVVGTCI: GRNADDYWGQGTLVTVKP (S E152. ID NO : 38 ) CLICI: SGSVESIL SM (S‘IQ u NO: 120) CIIQI2: ' ITCI5GI DR3 YW (SEQ ID NO 12 2) Hi 1 . l T 7QTLESGGGTVQPGGST RLSC1»_r7»‘ISGSVFSGUS/MSW: ROAEGKGL"L‘v’CL-_ TGGR'II YA; CV CGRE‘I‘C’. SI‘ ‘A'I.A'VY "Cf-NAV‘V’ GWC}k'\IABIA IAIGQGT I‘M/7'_I"‘I7'I’\’P (SEQ ID NO: 39) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW Bibi-$21 I C‘Gix I‘VE} 1', CI 'I‘GGR'1I C SVKGRFTLSRDRAKITLYLIQIV'ISSL QAELTA/LYY’"NAVVSTGIJCRI'xJAD:DYIJG0CwrxTLI/"T‘x,7ECP { SEQ I ID NC): 40) GDRI: SGSVRETIDAIVI (SEQ ID NO: 138‘ CIDRZ: (SEC). IID NC): L17) CL RS: ITAYVSTGIJGRNADDYW (SE IL IIC‘: 122) H11v421 . :1.

EVQT LESIn'GGEVCPGGST RLSCAAGCS»ECLDAMLSII‘JERQA GE"TT.«LVCI TGGI’ "IYY?‘nE SV C’CGR EC"I' C S RC 3 IIxAE L. '1"AVY S'I' CIVIC I‘N; I: IDY 1111w1 3I‘ LV'IVE P or y;\§1'r‘o- k)L:'KJ \’ .C' x). ..

CLIQ: LITGGRTTY (SC : NAVVS. GWGRNA' h2606v1.1 aw ' - r1 -~4r:(~¢:«E.‘v\/f\"‘r:r~¢ «n7 ‘.~r~ \/ .J'CIQKFK'1‘": g?kjkj LAC:midi:SV CEC‘S C1'CD2—I Q I'JYFQAC‘GRGI.311' TI .SJRI‘NAKNTLYLQI’IS ST RAE'CTZ»- S E152. IID NO: 4'2) (‘1 r .‘1 1' l—‘ U) CD 7/1 ,4 \1 13'] U) -+ 1- PH #5:: U) :93 C r-/44O l-—‘ UV (‘0 /x LG I 1"1 U r-/L;O 1-" }_\ .)\ haCO?vl.2 E JQILESGGGTVQPGGST RLSCIEISGSVFS; All-wIJYROIIEKGREL‘V’SI» LSGISSATYAE CV (G'KE‘1‘ I"JI‘ D'I n‘v’D.’"rALA‘DVS _'C41/\11<1DA:1GY EIIGQG’I' IN'IV (SEQ ID NO: 43) CERT . GSVES [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW thGév1.3 ‘IIRVCI ITS {FLVSALSGISSAKIAE SVKGRFTISRDNAKITVLQVSCFRMHLTAVYYCYADVSTCWRDn€GYTQCITLV"V :SEQ ID NO: 44) CDRI: GSVFSI DAM (SEQ ID NO: 106) CDRZ: LSGISSAT (SEQ ID NO: 139) CLRE: YALVSTquRFA1GYW (SEQ IL NO: 116) th66v1.4 SGTGSVVPGTSIRLSCAAJCS»FIDAMDIYRQA Gg?RELVSAL-SGISSA:YAE SV >DKKERIAVYY‘Y‘I°ICWCIDA.EYWGC.TI’ h2606v2.1 aw ' - r1 "(f‘r‘t »« .J'CIQKFKK:1I:I,3vI‘" Q'Huk:f\"‘f~r~( :wacIII—ma:0V I153]:C4 rw 7 0'~ n \/ , I'I‘fI?C223i‘C:1\(§I. ]]J\KA\‘ SVKGKFTISRI‘NAKNTLILQMSSIRAEFTAVYYCYADVSTGII SEW ID NO: x. 46) (3 r "J I—‘ U) 6‘) U1 ,4 ‘4 13'] U) a r- PH #5:: U) :93 C r-/44O l-—‘ UV (‘0 /x ’\O , I-I U r-/L;O I-‘ I—‘ .)\ th96v2.2 '7 7QTLESGGGTVQPGGST RLSCIEISGSVFSI )AM..,IIIVROAEKGREL‘V’A.

TI"F ‘InVV’"§DVSJGWRPAWQXWGQGJL"IX (SEQ ID NO: I7) CCRI: [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW thGév2.3 ’9' TQ‘I"‘""‘<\CEQ1 r 7‘ \/ 1'21 1 , 4:0(34530. H. "1‘1" SVKGRRTZSRDNRKIT REC—QGTLV' "V {SEQ ID NO: 48:) CDRl: SGSR7FSEDAM (SLR "D NO: 138) CDRZ: LSRISSAT (SEQ i0 NO: 133) CLEE: YALVSTwWGRRAHGYW (SEQ IL NO: 116) jPGG‘TRLSC"’JCb»F:DAML JYRQA VAVLSLLQSALYAE /NTL' ]'QMS {n>LRAEL. PAVYY"’Y‘[OTKWJu11H£EYVW%:Z..T'" .GWCRRV11 ".1 (SEQ ID 1...\ 1—3 (71 haCOévB aw ' - r1 l‘rlfttwi‘v\/f\"‘r:r~¢ » ~r~7 or" \/ .J'CIQKFK'1‘": \ngKFKIL mbw? new . x1‘1.\)'\. MV 1‘ 1 J1- WYFQ?RCRVR1LVW‘ SVKGRFTESRDNAKNTLYLQWSSTRAERTAVYYCYADVJTGT{ SEQ ID NO: 50) "’1: 1—3 x." 1-4 U r-/L;O 1-" }_\ .)\ th06v3.l R 7QTLESGGGRVQPGGST RLSCff?GSVFS; )AM..,wWVRDLxE-RQC‘RAL‘V’A 1 ,4 \4'03KFT1"? I )11n‘v’7t'" x"M-YZA‘DVS JGWCRPAWQEWGQGJL"IX Cf) 3Q ID NO: 51) CDRl: [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW thGév3.2 \/ ’9' Tom-I»l "< 1 1 , .LIC)(:I,I,IJI.)\CEQ;_ _ AI) T I IT XII—QCITLV' "V {SEQ II) NC): CI'Rl : th56v3.3 EVQI EVC’FG’TSI RLSCAASGSVFS:DAIVIE ELVAV'_L. SGI {‘3 SAKYAE SVGR III I SRDSAIINRCIYIIQIVIDGIJK]:I.IIAVY 1"YDIDVS'I' CIII‘IICIRIDA} GYWGI’,1 3I‘ .I,I\I"'IIII' . (SWC" ‘7‘\‘1I".1 (SEQ II:l I.__I H (71 h2606v3.4 ..v ‘1".‘r‘m?I‘U.J'CIQKFK'1‘": IIILS.CZ\ZI—ISG3V FE}IJ2— VIIYFC)?I3I~1\VI SVKG ._~\_FTI F'3RDNAKNTVYL’.L’IS S T RAE FT1»_VYYCYADVQT GI " SEQ. ID NO: 541) CLI’I: SGSVFSILPM (S‘IQ u NO: 158) It: H x." I"! U r-/L;O I-‘ I—‘ .)\ th06v3.5 F 7QILESGGGFVQPGGSI RLSCIJ‘ISGSIITFS; IAII-IIIIVRQLIEKQREL‘V’A 1 ,4 w. CG'KFII"R1387"; ' ‘ I )‘In‘II'Y" C31%DVS .'C41IIIk [‘AVIQE II‘IIGCDGI INT ‘7' Cf) IQ ID NO: 55:) CIIRI : [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW \/ ’9' TO"T*Q4:01:14 thug/Ah SVKGRFTESRASAPNLVLLQM. MQrTLV"V 'SEQ ID NO: 36) CVRl: TDAM (3:? "D NO: 138) th56v3.7 EVQTLESGGGEV’A?uSERLbCT’JCEVFSEDAME ~ELVAVESQISSA YAA SATG FTISRJA/NmVV]QESSURAERTAVYYFY‘[°1"WE[DA.EYWGC IULVWIR' (SEQ IF NO: :7 . (SEMCEETI) (SEQ ID }.__\ 1—3 (71 h2606v3.8 —?0hcaw(?g.J'Cugkjk:1 or~7 o.~r~v [‘10 >1"):— . Jx?kztx1‘1.\)'\3 k) WYFQ?7"RQR[LVA‘ ; LAE SAL GRFT : SRDA‘V _-‘(? Z w (A\4m ti:0H 1->« U) m A RAEFT1»_VYYCYADVJTGT " "’1: 1—3 x." F"! U r-/L;O F" }_\ .)\ th06v3.9 E 7QTLESGGGTVQPGGST RLSCZJAASGSVFS; IAlI-wWVROAEKQRELVA 1. v 1.. ‘1nVV’"§DVS JGWCRPAdQEWGQGJLVIX CCRl: ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW thGévS . 10 ’9' T 0-"; x 1 \/ ml? 1 , .L.L>(3,.,::E;Z—\. .1—h) AVKGRFT I SRASAI—‘N I V I LCQIVI. "GQLGTLV' V L’ SEQ ID NO: ’60) CDRI.x SGSVEVIDAM (3:? ID NO: 138‘ CIDRZZ I..SGI[ISSAI‘\’I (SEQ II) NC): I-"I’II CLRE : . ADV‘WG'WwRLEIHGIII (SEQ ID NW: 142:) th56v3.11 EVQLLESGGGEVCQ3C~uSSRLbCT’ SGSV’E‘SIDAI’ES DxV’ El 3R[I‘TI"PJYFAIPI‘I'I‘V’IVI C21ESE} \.EL 'lIAVYYCIYAMEST DAILC (SEQ ID NO: 61, . GIN C3RI) (SEQ ID F-‘ H (71 112606in . 12 - r1 -‘(f~r~( .JL:.buLL3L3E ‘v "C" "'3"V’ F‘SIICDE— . Jx?kztx1‘1.\)'\3 k) 1}'\TY}?C)2—I""I\CJ}<[]J\«Z\‘ -. {DYE— QL’IS S I RAEFTAVYYCYADVQIT GI " C) r" "J I_L U) 6‘) If.) ,4 ‘4 hr] U) -+ r- PH #1:: U) :93 C r-/44O l._‘. CA, CO "’1: H x." I"! U r-/L;O I'" }_\ .)\ th04vl F‘ 7QIALERUCYCDG,‘(IN I «.TT’QL Gem." INLD." {Cal—h IGMLJW : RQAEGKDLLWVSAI NETS-G SI/‘C I :3YA .C3}? E‘TIIE)‘ I<'.I)I\U.—\i LN’I'IJY LC; VY WCEQN RV'1GOG.

NO: 63) CIIZRI : [if ‘H‘l‘ f‘U.’ , J. V1.31 I. III N NGC [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW thG-?vl .2 '1 PS T11G: 1‘1m ‘EA Gbr’E" ': SRDNAKB TLYLQMS SLRAEDTAVYYCQNRVTKGQGTEVTV 1: S Q T) CT'Rl.x TT’"1 (SEQ .JD NO.. 143‘ GDRZ: 5%. N'ExTGGS‘J’TTS (SEQ :1) NC): 145—1) CLRE: QNRVTR (SEQ :D IQ: 146) ["1,QTL"1"C2C)3V1"?"GSLRLSJAA'WFTE‘ST"" WERQ" GET‘ITMT'JVSA NQGGSWTS A,.

SSVV""111f’ ""’I""VPVT"""’AGQG TV ' S 130 I 1) NO: 65) CL' (SEQ : 1431 CLI’C: I IQGTSWTS (SE Tn 110" 147) TD N0: 1451 h2604v9 LUSC’3GE WQPCG: 1.1118(22115‘113": ["‘1‘ 1" 1 WWW)C‘(3‘\GI.3151\7SP1: N IZGM 'I‘S‘YE—x SSF"1~1GRFTZ GRD1\11K§ T1 YLQMSST REEL.1AVYWCQNRVTRGQGTLVTV (SLQ .1) ‘ {1.2: 7" TNNGGSWTS th04v2 .2 E 7Q1LESGGGTVQPGGST RLSCZJXSGFTFST11G11VLJW.1 RQAEGKGL51’1‘VTSA-_1\11TGGS1‘C1 STA SS" .G}? E‘TTS‘ .12131'115—‘11 INTLY T1211115 .‘1’CQN RV'1GQG’' Ci:R1 : SGFT ‘n‘w f‘U.’ .

, J. V11": #1 \_/ n\J c ,._1 "1.. VJ 7. II N NGC ("‘2 r. fr) (J) J1v xi 43 50 5 1 1:3 Z0 / /U D ["1 IO 1 H 13 G" (J) ’4‘) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 1 c1 ITK J .L Elrwowz'.\)>1\ .S 1T’FT77.S RDNAK} TLYLQMS TAVYENTwl.'\r\\I\Ir\. ‘J'\IRVT-hL:‘JLJ.L.LJAr "Im’r v'1'" CFRT x .

, SGF' F‘TTI': '1A .3 .0. ) n ~«'J.J ?x .. a CL. R": : "F" VTQ41 wT-r —.— ?qr." .7: I" "0' Lb": ’CJLTH~-».'3":,—.GSLRLAAWSFTFST"" FFRQ" '2EDE SSVKIL:~I’ET [5: ’CQNF\? 1') r" fxr[\KZKKZ NO: 69) - r~<:- QLijkj,‘ L}I K«n7 7’\C .J'CIQKFK'1‘": O "r‘ .'73FT FaF T ITIC‘ V’ J x.‘- \/Ifx1‘1.\) W 17"}?0317P(:T\7DI.HIM7113 (x31\. 7\)\"K’Fx .L ,J ._ l"; SSF"IT'IGRFT :.o RDN11K}-TT‘T‘ .i.7‘1v7‘7‘171EYCQE\TR‘V7TRaJQGHAyTiVT,? {MTV .T CLTLJ SGFTF ' ,,, -n1I/\y;\ .. dik/Q th04v5.3 F 7QTLESGGGFVQPGG; C-FTFS’THuMDW : RQAEGKDL S S‘I’ G 1%? FT"To .7<'. D NAT 'n 1-1v J.J.JYD 1-1111‘-1J.J.L\'1LJ 1 . 'r‘L‘JL- V5 WxT A} RUKJL71I" «1 CERT : [if r\M f\ «M . .QJ CD‘J ". ’QNRVTR [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW th94v5.4 'T E‘S'I‘HG Z—\ 1Q IITS‘K} SS\xKC"FI"SRDNAKBTLYLCNLQSERPEITAR/Y‘CZ‘JRVIKCZGTEVI" (S'Q I) NC): 72) CT'RI: iSGE‘"EJTT'"I: (SEQ JD NO: 143) CIDRI: (S510 ID NC): 147) CLRE: QNLVTR (SEQ ID IQ: 146) thQ4v5.5 EVQT ESG’TGEVCP-.‘GSLRLSCAA"’"FIE‘ST'" IFL—RQ" mI'IVSA QSGGSNISYA 5153\7I’"I’}'1 IS RA I‘25‘1."V’I’W(""\IP"'I 3(3in 'SHIQ II) NO: 73) CIX (S BC) I 4: '3 ) CLICE: RIQS‘WC‘SW'IS (S mQ u NO: I47) I) IMO: 1116) h2604v5.6 EV ’IGE VQPCJG: :‘\CZZIA.C3":VII" T WIPOPGEIDI.II«:\7SP\I<‘S\:C1.C"I‘SYE—x SSICCRFIZVRDRAKE TI YLQI‘IQLIT RRELI-‘7»‘VYIYCQNRVIRCQCTLVI'V (SEQ -_L) NO: /4) CLICL: SGFTESTHGM (SmQ u NO: I43) : AIQSGSSWIS I) NO: 1.417) CERB: (SEQ I ) NO: I '10) th04v5.7 E 7QTLESGGGT‘WPGGST RLSCA IGMUW: RQAEGKDLLWVSAI ‘TSGGSWI SEA S SI’ C‘ 7? E‘"["Io m 1- .C‘:.' . J. J.J Y IQ;IV] VY INCEQN RV ’1'}?)2" .’\C: \/ L7: ‘IW‘I‘ r;\/C311 , J. : 1.413) . .Q SG 147) CLFE: ’QNRVTR (SE-Q ID NO: I (J) {Jr [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW thGév5.8 ''T E‘S THG: Wm "'K133‘{A ISRDNAKB TLYLQI -TLERAEDTAVY'V'CQLIRVT WCQGTEV’'1'" CT'RT.x SGE‘"T7JTT’"I: (SEQ JD NO.. 143) (J "r, \D a CLRE : QNL VTR (SEQ :13 1'0: 146) EVQT ESG’TGEVC'P'"GSLRLSCAA’."’"E‘TE‘"T"" WERQ" GEI‘ITMT'J‘JSA QSGGSNTSYA 5157\7I’7I’}'1 If: I" "'1’"V"\IP"'[I"7"’— .VI. I'V' 1&3ka NO : 7 7 :J (S 510 I. 4; '3‘: CLI’Q: RTQSTC'SEYTS (S‘VQ u NO: I47) 4: T) 1M0: 1.45) haCOév? . 1C2 EV :JIISCI’3GE V’QPCSG? {I}IJSCIZIACB":["I'F" T INTRO7""DI.IE’\:\7SP\I<‘S\:C1.C"I‘S‘YE—x SSTvaFT’- L1:'<: TT Y LQMC‘DT 7‘VYEYCQNRVTRGQC‘TLVTV (SEQ ._L) CLTCL: SGFTESTHGM (S ‘VQ u NO: I43) : ATQSGSSW«ITS D NO: I47) CFRB: (SEQ : ) NO: I is) thO4v5 . 11 E 7QTLESGGGTVQPGGST RL IGMUW: RQAEGKDLLWVSAH '"ISGGSWT STA SST’ TE}? E‘TT" RIDNN \NTLY 1; VY WCEQN RVT}.CéQCj NO: 79, CERT : [if ‘H‘l‘ f‘U.’ ..

, J. V1.31 r1 \_/ n\J c ,._A ,4); (A) f\ «M , .\: CD‘J ". ’QNRVTR (SEQ :D NO: 146) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW hZC94V5 . 12 IGEVQI "01.1:.7K)J:JL\]JE)7I\_I- 14": I. 137S'I'HGIV 'I~< ‘x I "\PGRTZ I'SRDNAE’L TLY LC;MSITRAEDTALVY'17CQ11RVTKGQGTLVTV 1: S Q T) <3 ".1 >0 H J L:7K) 1 13 Zo }_\ 1.11: (A) n1 .3 .0. ) n ~«'J.J 1x .1 I. (7) r" [3.7 C.1‘ IO '7/--1 r_ .1 \1-3 50 1 1 7‘3 C [:1 (j 7 /--1C.) 1.1. 11> 171 2:14 Hllv420 + F1: on palypeptides MRWVTFE. ‘LL FLFS. 'AII.I "I.7QLLESGGGEIVQPGCSLRLSGAASGSVF. S111 FRC‘APGR G IL‘V SAT'I'GGP''I']I"1.Y21III:3VRG R I"? ,1I S R" 7\11—1.,{N1_1I1‘1 :3 S 11 )II‘AVY1. YCLIAVVSTG.. 3.

RNA )DYII ’T\1\P3CSC'SEVQLLRSGGC\7':PGGSLRLSGAASGSVFSTLEXIST/‘1:R ‘ GG1‘177‘1'I1’Y‘E .7 V IRGI FT I ' .1 1IJI§1ARNIFILI I1’IL.QPV1ISII711 )’I‘I‘AV I1’Y CNAY V C'QGTLVT'VRIPGdGGDR‘T’I’ITCI’L CPAPGGL S11.17FLF 1:' 1. RI:RDTLML SRTPF- I H Q1311! 1I.I1.\1GRE'II1’ :‘1 KFIVWY 17DG‘17E H. [\1AI’JT‘ R1? R n P1?I:1~"‘V‘.'I'L P.S R1,IIL177/113?" L ’ I’GFY I7 S DTA. I 7:7? WESIIGQP LYSVLTVDKSLHQQGF" SCSVl‘ ETLHVHYTQCSL MRWVT FII .SLL FL FSS CAASSISVFS II FAMSW FROAP IF ‘ II ‘I'1’I S RIDNARN ’1' LE YISSLRAEDTAWW Y'GI\'AVV’I Y1" GQGTLI’T\1< SCSCGSEVQLLS QAPSII‘IG ELV AITC'GRTTYYAESVRG' FTCSRIDNA V'IVKITIGGSD'1. [I1CP I‘G "APGSIS‘1. 1.7 ' ‘ K13’I‘1311'IIIII S RT P 1 VL I7‘V’IDVSILLDPI:IVRFN1"Y\7LDGI17I'3'I17IINAFL RPRE FQYNSTY RVVSVLTVL IIQDWLNGREY KGKVS \R'AIL P A. PI 3.1?Q‘17Y’II‘LP P3111)'E'II1’1I' KNQ‘ZS’'C-IL. VRCI'I‘Y 1IJSDI1IA‘7IF.

WI I‘lIQQ'-1\1'V FSCSVMHEATIIIIII‘IYTCLIISL 2x H11v42<§ IgleFc: 11’1IK‘171‘17II' F III)S .‘L F S Sr7\‘1' S11"3V1,; IRLSI \ASGSV1"SII D ‘1 GLET I17SALTGGR'T'TYIYIAL'SVV1\GRFTI SRL1117‘AIKNTLYLQMSSLRAEDTAVYYCNAVVST AIWG .I1.\7'1."’1KPII%GIGCISE‘VC>1I1 1S SGGIIIVQI-‘GESL I LISC ASG S‘1 [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW CEAI-I‘GKGLEIMSAITGG .<‘I".I' Y YALE/SE"GR .‘TI I I’ISSLIKAIII .5 1_/ K {"1 x:% <3. /‘ S. GI"? C1 P NAI{DIYI T I? EVTCVVVDVSI E 1" R:VKEINI‘IY\7DGVEVHNAIII'IKER.4':YNSTYRVVSV1m 7LL KVSI AL fIA.f‘:1;{TIIf" I KGQ fixI'I‘I"Sm/11* (IICD V SI:I ’ICZ I..VKCw E‘Y TI’PVIDSI‘GL FE‘LY 'DE~‘1 3\II"Q-."‘GI\II9IITSC1SIv’IIVIHEALHNHYTQ D NC): 83)- W’IKI‘IVIII E‘LSIFEES—1.515"VCI-I.EISGGCIEV’IPCTIISII GI" LVL"I 'I'I'YYA. I JVAKIVTLYLQMSC II‘IG —1.IDIDY I‘1IC3_’ITIE.1\/ II"9IIKIEGG SCECI S III/QI I.-IEI SG r~lr~ lerpo II 1'" > IL‘SAC/K:{3'73ch :I,1I\:II«(az- 79JLYK/J..L1IC)'\Z"L)\/ .L K) QAPGKC 1ET17GAI GGRTTYYAESVKGRFILSRLNIYKTTLYIImLRIIIELPIWI\7YY{LII AVV S"? GISCZRNAI'D‘.\I II:VI ‘~’ "\VUCJL‘JLDI 1. I‘HTGI? PCDICI‘ ? 51L IIGGI-‘SIVIET‘I E‘I‘ I—‘K I.

'I' '8 EV I'CVI‘7I97IDI17SI .V?II\IIIYVI/\1I\7EI\7I‘1N‘III'IKIUR. .LQYINSTYRVVSI‘JL-IVLIIQDALI: KEI Y KL\ I’LDI\IK.I1I?.AI?II EIICIII I SKAKG; I? REE. KNICD'V'SI"'II DLVKGEIY‘ AVIWII ‘IN.x‘1:.LusI ~..I’I‘ fIfIVL IDS IDGS EII‘IIQ‘II KL I'.‘97 IDKS ?WCLQG} I'IIS’C'EAIII—IEA\LI—INHYIIIQ SPCK (SEQ ID NO: 84} 2x Hilvé?l + Fa daleticn palypeptide MKIINIV'I' EI I SLL III EI’SISAY IIIE/SIIGLIII9 '\J. GGQII{\r {\L-n7I/\ )f\r1‘< ' SGSV ESI SI'IEI‘IC A?I21KCI " LI\ GT-ELVSLITGGR'I’I'Y'YA.LSI7KG- 'ITICRJQAKKILYIQESI» IIII‘TIAYYI C'IIAVVSTGI‘IG RNALIIYIGCDG‘I'IV'.‘9IKI’GGSGCISEEVC D'L ‘I 3.I9 0f I;1\"-r1:13'§"r qmgwc1x. _ #1. r35)»: 1L; 9’ .L 1.) .L "I I ELEL .713LI TL1GRTTIYIYA: IDNIYKETTLYIII‘IISSLRAE L \Itl7‘ S 'T T‘vI \/ STF‘IYCRDI?DDVIV\I'IQ'IDTLI/TICKL’HLJFGLKTF.I-IPGPAE’G/GPSVEIIIIPKEKLTLMISRTPE ‘97'I‘CI7L"V‘97SK. E7 KIEII:III’\1Y VIDC.VIVI] NP.\KT KI RE‘I‘ICDII'IYIS'I YRII'VS‘ .1 1.1P vLT '. 'i' II")1ILNGKI‘I Y KC XIIITKNQVSL' CLVKIFY VT7: TEES: 'IIII’I: 1/IIDSI C"\‘\'\‘71- -.- ". I": IT'VIIDK’; C'NCDtICINV9 E":CSVE'EII‘AIHNHY'I"KSL ST-S GK {SE ID NO: 85) 2X Hllv481.l + Fe deletimn pclypeptide LIKWVIE‘ZHLLTLE‘SAYSEVQ 1LEE’4G' EVQPGCSIRLCLAASGSVF.)6 C1) C1111 FRAE’GK ILI'I‘GGE'I'.I'Y. .Y.I"I\1HISVKGI II’I‘C'SR‘I IiI?IAQCK‘IIIIII-Y 'IIE’ISSIEI—YEI DII'AVY. YCIIA\7\I’ G GOGTLVTYEPGF‘"CwGSEIV’IQLLESGGGEVQELGES'RLLSGI IAISCS7’ FSI DSI‘IS'I‘II'FE .'YYAEJSYKGI FTC. . .I.I.I.’1"I.1O:‘IS.- I'I‘AV.’Y CNA‘ IIV [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW gym r\r\r\rr1‘r \"rr ‘I I w- -- L; .L LCZrLC I RNAI IC:‘VCW Lj'L/L: .L J.J \/ . ('k (:LijJ'i‘I.H‘I.7 EICJP \7 IINAK'IK 3L.\ .4 ITKLQV - wrcTGQ"‘L \- rmx—ycn \/ 1.‘ .L\L_ ;_<_‘CC IV IC‘SCSVC.7. SCI] MI’LWV'CI‘ I1C' CIC C 'EJL)LC1CL‘:'L:! L‘J \ g‘[?k1\dts.[ [\iJk)‘-.'PL .010; {\r‘ \‘1\r - r \1- or 7\ n(:IEJV(JV? .1 0 .IL) CIC ‘DSI‘VS 7.C‘I‘LQA IC-‘GK GLEL?SC..I EGRITYE WAKNTIYLO33.3AEMTLV.2G \"V°"GNG RNADDYWG‘IGTLVTVKPGGS~I EVQL QPGCILRLSQAAC"SVFSTDSMSWER Q.zLPG TGG?TTYY‘F"VKGRPTILRDN GLLIGRNTLDTWLG THTGI I'C C CC.‘C\7C I! Vn"- DV E'CEYKGK 7.‘SNKALIA:-..LI SNCZQIC-‘E'IILIC SIiIC ’CI' LI’DI C, {WQQG‘C .C.HI.‘IH‘.I.’ C 2 (E C) IO: 87) 2x Hllvéml . l IgGich :‘IvIVTFC SLT "LIT.3L--YSE‘J L. LLVQPGCSLFL SCZL‘LSGSC‘V’CFSCCL SLJISI'IITI'.Q1»-PGK r: -r n- \,7’ L0..JLJ‘LI L..LJ .xJ.J CC’CI’C.1: CI 1.7 GG CT1. TI u1.." RAIAED‘III.p.‘\QG LLCCVTTT‘JIKE’GG EVQLLESGGC QAI‘GKGIIE' 7C C; ITGG I’HHSVCGI‘ ‘ ’ISSIC I RAED‘IIAC‘JY YCZNA7‘! ALIC‘C‘CT’V'LIJ-: .LEL CGI:SWLFchL’KJIL‘ILSR TL EV‘ 'GVV7DVS HET'LEVNENHVVEGVEC‘7HN7‘7C' ICER.L'.LQY1\ISTYR.VVS‘L7'--T 7L VSNK7LL CPAI‘C CCJC AVEWESNGQPENI'I‘f r(IC‘I'IIE‘PI’IIIDGSFFLY SKCLTVTALKS LII’VQQ CI V: SS SVT’IHEALHNHYTQ \::"‘3)CD13")C‘I \ u 17m - -TGGR'I‘TYI I .3‘ RDNAKNTLYLQIIS SLR" HIKFVKr~ r\ rm IC C7 K]?C~,~ NII'LrLo :I'LZL/"x ZKJL.) }.1 \CVCQ' IC. '3‘7 7L "(woxrn-Io» . .uku.LixLJL. V’V’ .L L) .L. .I QAPGKG IELVSLITGGRTTYYA EGR NAIIN:mL'J. LYLGMSSLRAE mmYY'LAV YYCNA SVFW ("I Tf"(j‘w?kjlRNA.CI)ID‘II "'rr"f\‘7‘! C 7‘1C: ‘15,; C1 VCI C‘LI’ "\VCL‘JCJJLJLCJA GI?I? C3 ICC" I I?L, L7 C CIE‘P IJNI/K ,. ICZRTPE [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW KFEVTM JDGVII7EZVE1N;'—E[""[\P ETEQT FEPQ\ ITK"JIPIIQ1 .. CL JCJ _ xii._1\ KN QV7 E. EITI‘ WESHGQP LTHT’LTVDKHPWQQG TV SCSV’1\'&EY‘LH1VHTTQ5 CSL_[ ESL-F5. S'E’ {SEQ 2x Hllv421 l + PC delet npolypeptide MKJVTFIISLLPLFCSATISE7Q .LESGGGEVCUP'JIGTRACTAAS’TSVFSIIFPQVISTTFRCI‘PGK\Jf; ‘ IEI'T’II SRDIII"EnKNT L L_ V15 S1I;RA1§;['I[,AV’T V CNIr‘V V’S’I aIPATKIEJKFKZ‘ED UVKL'1IJ r1 «who *1*~f\ LI" E1r~<£§f~~ ‘\7'r’\"3 :1‘3014 Iu’.<.1I:-UK. _ EIDUDVN?n-wn -c~1r~4n «?x 7 QAPHE’IG L TC'RTTTTADSVKG FTCSR:TNAKEV'TLTLC‘VISéLbA-DTAV’TTCIE‘TAVV’I CQC'E‘LV’T\7KE‘(33GGDK'I1IEE1CP PC I‘APGE"P3V7}? H1~P/EE41I3"1I‘1I;1'IE [IIE‘~["‘I1 P VHNAELTKPRE- TTRVVSVLTVTHQDWLNC‘KET 140 K"7S\= ~rn— P P,-: v ‘ PO VT;. .LiJ I) E) L 1‘1 Q‘] CT I I. V’KGFT PS T‘ QQ'.NV FSC’SV’IE 1EALIENITTQEECIL 2x H11v421 IgleFc MI K‘IN"7T F I E .§.. CA; at;1‘ 17 wow- E. II. RE; 8 VASGSV’FEI DIE—\M SW FRQAPGA ET T7SLIITGGK1'TTTA 3WCCCTTII‘E7I11VKPII1‘CHCCQ3 IG3GIIEVQ E-‘G3S L E L S CI QAPGKG ELTVCSLETEGGYRTTTCYLLIEFIT‘JCCECICRFT:_\.IR31.\TL"KL\IITILTL‘OLTSSLRT’AEDLZATTYYC:I1?T‘JCCTVT LIIE‘GUqRNJAD1.3TENCQC’IE‘LV'IE' I’I14E GGGGIE14'I1. HT 3P1 ‘ I) *I11I;[.C~CII":3V’ ‘ILE PEV’ITCW7VVD‘V’I‘SHEL PEV’IK NWTVDGVI’ K 1'E'I': RI; TTR\7VSV IL' KT T SEAKGQ PRE PQV'\41—4 H 1—1 1.51 h. ELTKATQV’LTCLVKGFTPSDT . PV1I;DS1I3 3:3 F13TIT-F5 KL '1. ’IEDKF'RWQQCKD 5:r‘ 5;.VM KST-SLSPGK (SEQ IID NO: 91.) For{\r‘ \‘1\r_ .JK)L1K:\:!EJ\ jGGSIEI. RE;S~’II,AC"ISGS‘I\7 ‘SIEI DAi‘vSW I'RQAPGK GRT"I1. T I (a C" ’L‘I 0‘ \/ D. I/IEI )[v RIB».

RNADDTWC{GTLV’TV’KPGCEGCDEVQLLESGGC;7C‘PGGSLRLSCAA‘RG. In- ‘ ..

,EH 'T‘ I KJKGPIL I TY‘EI‘F"" ,I‘T '18}\DNA\Ki .1I; T T FEM-F5 S1I;E’\A} STGWGRNADDTTNGCCTLVTVE’PGGGI‘DE{THTCPPCAPPEL1:: CCIS‘V’FLF' [‘I‘E‘I‘I1'FI‘7V‘IDV781 EILFEIIVSFI‘NT‘I'TV’]:GII‘JEV’IIIEVIAIK'IE‘KPKE} I;Q.‘1’1.‘1ST VI1I<’.VI‘7"-"I'I ’I'I‘I7L; " [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW K TY KC\ L\V’1-3.'\1KJA...1 1-72' IE1 E’CT 1' ‘ J. S KAKGCQ [C‘l101.781."'13 :LVKGE" AV7 } S 1‘1 P1131‘1'N‘. 3 1? P 'v\ S DGS 1311‘ S1?151QQ EQ- :D N\u’: 92) v'KJL‘JCJJJ'w 01 RLDCAA‘NCIS'Eb1-4 ‘ 51L "\71.'—\VL.G TbSAKYAA T :. QRDKAKNTVVLM.5STLRAEDTI‘VYYCYP. VSTC--1/\1-.RDAHGYIJGC‘CTL\T14-: C5SGC5S. 3VC LE3 SC5GC5E3VQ1? r\r;-\j1.ij SCIAJASGS'\ E S1.DA1.V.1"'51'\1YRQA. .1)’AV1 AK.113 .3:AH"r'x. IT 0 C11: ~x ‘AUVSTGW .7 3.31»x) :1.'<.A1E';1 TAV n??mT T’" C» _ J .L 1-..1‘1‘JY 13: .l.1« TVI" GGGCDICTHTCEPCPAPELL-GG '3'17FLEPPKPKDLLMTSRTPEV'"1177\7DVSHEDE’3V C3V1'1‘v’711‘1AKTK PI\ E" E C‘V\1°'1 Y 1\‘\7"v'S"v’L "11' 7111-1121) 1'\i1.‘1\1C31<131 ' LIL-QT: \ SRDELTK \lQVSLTCLVKGEC‘ILSD--AV."1"1L'SNGCQE"KNYETTP ESECS» ' "13"KSTJV1JC QPGCS LRTL SC.AASG SVFS T L.1»_1‘1S 1’1 FT’.Q./»-PG K ‘1AK1"_ B.:.1ADDYE‘1!GQGTLVT\TCKPGE' ESGGG‘FVQPGGSLRLS SAGSVFSTDAMSWFR QAPGKG ELVSATTGGRTTYYAESVFGA TTSE’DNAICNTLYLQMS SL1 ~J 21 C1 -L ,__ 1-1:! ,4‘4 J:<11:1"4 SEI‘GWC1.1\1.‘1A1.31)‘\’1,3’\1GQ 1G1.*1r.~(\1'-7;.)1;EVQJ.{\1 3T1: \IO, LSG 11.1115: C—. :1V 13 S T 1:)A MSE‘JFRCAPGKGLELVSAT ' r'<,'\*\mm L—LL‘ YYZ-L"7\.—11." 'VI’CGRE‘T LSRDK‘"-KI TLLL(QT\/IAHC1' ‘TAEDTAVTY C3331AVV S'T C517‘1L'5 [\NL \‘1.)! }V ‘ C5'T .1.."/"13"\}"K1‘GC5GC5DK1H\ SF.TPE‘V7TG\.7‘,7‘V7D‘V’S113' DEE‘TKE"JW"LGXCLTVEEIAETKPRLECYVST V ESVLTVLHQ IWL NG [CEIY KC: KVS ' ESNGQE’ENNYKTT PPVL VDKSRE‘CQQGLTV TQKSLSLSP’AK (SEQ TD NO: -24) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 3x H11V§20.1 + Fe deletion polypeptide 7T E7 IE I L E7S Sxix)- 7r\ 3‘1:I‘d :1>'~< IIJL/I —.LL; 1- r~ -« no." "(L/.7LID1CJIJL) V T :3, (‘33. -1 L‘L ’ICII IITGGR'LI'YYAI FTCSRDNAKNTLYLQIVIS S'LFAEDTAV Y 7\III—:I3‘IIY WCQII’II‘LV'IIKE" AGEIGCISFVIL IMIIII‘I'LI‘I rxl »« V'wz'mr?f" ifrT‘IQ‘f'T MC. >=~ IuL?J :1'\:II\’ILV'P‘:1C:1?I IIIIL\IIIILI.K1K:H3‘L11L2b751V L K).1.L)Z‘\;1II.II\.IJK)7‘7 L‘ EL QAPGKG [ELVCATTGGRTTYYAIESVE’RFTISR’DNAIIXTLYL' ’ISSLRAEDTAVYYCNAVV STC3WG}.NAD31IIWIGQ JVTVKE .IGGEIVI SLJRLLICAASGSE7SIIDA LISWERQKUGECLLV ATTGGRLLYYAE S\,IEGRFTK/QRDE\L1' LALIA‘I‘IVLC'II GWTRNL 7'3YI WGIII’I'I II..I\I"1.7‘I\/7KI‘GGGGDK] HTCEDPC P.I""GGPSJ.

S..E YKCIVSNKALE’AE’TEK‘ 'IISKAKIIJQEREPQVYTLE’PST’DELTKT'QV.

D II AX "SNGQEINNYK’I‘T PPV - ISISEIIJGS YSKEHITVIJKSI’I‘IQQ' NV EI‘SCSVE’IIIII.

TQKSLSLSPGK (SEQ TD NO: 95) 3x Hllv42$ IgGlch NV‘II" E‘ III JV .7811AIVIE3W EDGK {NT [:11' 7-3 ID > 'LxI'LIu lJ.J «0.1L EGR'I"II Y1 ILQI‘I m: L L\:1.Q o]:.\ WIN1‘?"V \I 31/7ka~r7r~ RLIADDYWGGTLVTVKE’GGI I ITVG IL: GGGEVQPGGLRLSV'ALIIS" VSA I'TCG ?’3I‘T Y YIDEII‘ SV II"I‘IEL)3E\I3II\IIFI\LI< ’IS SLR;III. 3 7 AIR7Y YCNAVV .?SEALLLSGJC7—)1— «.TTIQLUGGS.hp "HI—n:x.L.SL.I/»_r»‘ISG-S\.IEKSLL‘A(In- T -,~—w7 I IT —\ CIT/I3‘7’ISTGWGRITALTYW II..IV’III] S}? T E" :1\\III.II ("I7\/I’IVDI‘\I’S H ’ HNAK73I' KI . [:3 R EII \=ST Y RVVS VIE:EII YECEIVSNIL" T PAE’I LEDSFL‘ALTKNF‘VSLTLVKGF GSE'E‘LY SKLTVLKSRII‘IQQGNVFSLI‘SI\I'"=I‘IIHEAT H (,3I\IS. III. SLL)EGK III) NO: 9(3) 3x Hllv429.l IgleFc MKII‘IVT FII SLL FL FSSSA A ITG 3}? T'II'YY .(II r~«g r‘ {w aJK.I:.:LFKI‘D r~ a I. L? L3 EI-IV’I'ITATTGGRTTYYAIESVKG' FTCSR:3NAKK‘TLYLFWISSLRAEDTAVYYCNAVV rt‘yfkj ILILJV IV VLL LzLIW'NK‘ITI'} \‘7r [‘I‘?MO" r‘ - r1 I‘r:(T:II‘ Iv/Ir/ I F‘\L11L41 VREEF MACI IIx‘IK/ItL1‘1.\)I\JIL/. vI I. ...

IJ'LIILJLFK'1‘": ‘1? .I MTSII‘LFR’QA’L-GKGLE'LJCII‘ I GRTTY‘A:SI\KGI’E‘TLSRDNAI’NIT-YLQE’ISSIRAEDTAVYY TGWGENAD I WGOG]. LV’I[777\PGGGG .7H'I‘LII? ’r‘PI—LE’EuI-LGGE S‘\7IELE E7I~‘: [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW II..IVIII] SRT E'Il‘x’I'IC‘VV .I’IDII 7SII I7HNA"’I'K.IK: IIIIE. QY‘IS'II Y R‘I77IV7SVLIVL IQ 1 KC KVSNV. I. C: LVKG E‘ YPSDIAI7ETESIIGQPEKLIYKTTRL’IILDSFCJSFFLYSKLTVLI'KSRIIQQI74NI.7FS"TSVI‘IIHEAIH (SI’I’ TI) NO: I; 97) 3x Hllvé?i + Fa delettion ptide BIKI."WT F: SLL FL FS.SAY SEVQLLE SCQGGEVC‘PCQGSLRL SCLPLAS WSVFS II FSIVISII FRQAPGK Q L‘ 1-.K)1\J.J. V ’S ’.

J L RAE. I'II'AII’Y ’"r‘ «who 1erI:1C..?)CF'\:‘-.J IIHIIIv»: Stash. 7\777\777 :1‘:1C)II.J.L<.I.:~..UK . »N»- LLVSLTI«CJRTTYYAESVKGRFTIISRINAKIVTLYLF‘VISSLRADTAVYYC'NAVV '1"! -~4r~r~<’-< w 03,50 .LLV .[\l /.3r~.T.1I-\-rr 7,» -«ol~f~ ~41:1~(r GQJSJV .J.'CJI)CJK:I‘:1.IJ‘./ r\ ~r~r~ MS". : RQAFC‘KGLE‘LVSLIT'. ’7 7F'JRL‘NAKNTLVLQL’ISSF'I- '. ANGKKAI. IA}. SOC 73F I-‘C‘I ' ’C5C5 I9S7‘7E].

I.7L.I7S E:13LJ‘PFI7'I ’ NQ I7; ICL IDIIIAVI.".'II' ISISDICVD PEN N id? \I..'I‘VID. 7C3RIIQ<"C3I\ 7 FSC‘ SVMI1..A7 SLSPGK. m .,J V.) -I U 7 C) (V, 3x Hllv4Q1.1 + Fe deletimn polypeptide MKFIIIVI' F I IFMqnv. ..- IJIJnI- "13(3ka 1;.-ppm/3 vv .m-\4 '31 K7} L1»; I .SC5SV‘E SILISIVIS‘: IQAFGK G-I-IELVCLLTGGR'I’I'Y IA‘JSI7KGRTT""RJVAKKTLY70M 'IJFTIIIYV. CLIP I7 7 "IC5CDC5‘I'I V'II'I/7KPGCSSGGS III =\I.SCJ.’.AI.I I.- v r C).n or" .-x-\-.r-.-o- .VC'LT TC:GRTTYYAI "/71LTLYLCDMSSLRALF TAVYYCNAV\7 SICII’"I’NAL‘DYI'Ix7‘QCwTLIITVKV‘CJJSCCTSEVQI GEVCLCJSJJRLSCI" SGSVFSTDS TTY7 .'CISR DN}N'I‘LYLQIIS 3I.I\Z"\.IC =7. TAVY Y CI _7:‘I7‘.7I7ST'-:‘IIIGRN:I:LYTAILS-QCTLVTVKE-wugu )KI‘H’I‘CPFCEAFGC tSIVFL.. r’t’KEKL‘TLIIL SKT 3737‘!ICVVVI 7‘.7SHE ID "n"CKFNII IC5 III’EIPK"'IIKI:9.7<'. 3r \.-:" "7‘.1 (/2TY19V II’SII’L’I' 7‘.7II..HQ DI"IL KVS "CAL PAP T I K; '7SKAKCQF .EPQVYTLPPSR[77 I'II TKI\IQVSLYCLVKSFY S. 7C§CDI9IE.NN.V K’I7"II' (R I’D3.307C5I\'7‘7 E'S CD SVIVIH.IIAL [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 3:: H1 1121401 IgGlch 7 'T F I 7r\ V1110 r~ «1 no." 1% 1 L IFS S xix)- LIL; —.1L3 1- my."L17DbeJL) V I‘ :3. (1'.w -1 ' "‘LV L--TGGR'I"I'Y‘LA. FTILRJWAKIVTLYLQII S'LFA3DTAVYYSLIAVVSTI/‘IG \I1—1I3I3‘II‘1ICJC3'I’I‘I1\1'IKP 33L)GC3SI"‘1I"L 1 on.ICNJ N,[:517377\V'PI3C31. :..I1L)KL. ,4 Q. '12- 7‘ 13¢ wrpo .v- :1'1: 3.1. .Ll‘LM’x: E) V .L K) L QAFGKG [ELVSL ITGGRTTYYAE SVI .GRFT T S:HDNAILNTLYL' ’ISSLRAEDTAV''TCNAVV STCI3% GI?I.\IAD31['IWGQ .1VI‘1'KV7GC3.-L1SL :GGEV SI1LKI1’LISAASCISFS I DS {:2 ,r'IIO:p'U C2 '63L" t" (33L I IAFSVKGRFT:SRDNAKNTLYLMSSL ‘AEDTAVLY I3Y1A1'GI3’7' "I..\I"I."1/’K[‘IACIGGDKI H .‘CI—IPC [:3.AIL"II1.L1.

IN I 5: RT F IIEV’I‘ GVI73] I.3\7 E‘IIL FNW117VDG 7L 7II I\I1—17I\7I.I"’I~" 1317. IS'I VSI17IUTV7I I'vLNGKDYKCKV'IN(ALPAPTE TISKAI’GQPREFQVYILPFSRD3LTKNQVSLTCLV*CGF 035'ng ' I‘TPI‘17II1'L SDG S?II I}L 753 KLT I17EK3RINC3'1,3GI\IL17' S 3S. \1 III] E1291 NIIYTQL'LSLSL SP'L (SLQ -_L) NO: 100) 3C:C:SI.1RI1S -. ALASCJ'S‘V’ .‘SI DS'IE3W I'I‘LQAI-IGK .‘GGR’I"I 171' 7LC3IV L". I. I\.—1.Q (11.x xv"; TEW ~wr‘\1 mm RNADDYWCGTLVTVKI’GG- - TVG .Lx LLTSGGGFRQFGGLRLSSALLSS 1 I'. 'CIG .3T'I' Y 17A.E‘ S317 FT (3 S R L3N11K L.33 3 ‘ ALVYYCINIAQFIJ or!SILT;LIL»D\_'JC7'"\T «.17 ' S.?lm "‘T":x.LSL../»_r»‘LSG-S\IFSLDL3«II—\,~—xr7 ‘T Q KGIRFI." G S I‘. 7IST-SWGRIIALFYI’II GSGSDI/{TIITCFPC PAP73LI I..IVIIII SI? T I? 3‘1"." C"7171133175 II ’ HIIAKII' KI . [:3 FL I33 1:S 'I 1: R‘177‘I11'7SVLI.

YICFVSNK." 3T PAP: L QVYTLEDSFFSLTKNC‘VSLTLVKGF SKLTV"IKSRIILIQQGNVFSC7‘SIL/"LVIH3AT H (,3KSI1SLL)I C1.K III) NO: IOI) MKII'LIVT F: SLL FL FSS . PISC:«rchq r~ a 1C1».K3'\Z";D .. K? x) QAL‘HL‘LG ELVSLTTISGRTTYYAESVKGRFTLSR:IDIF‘KAIFTLYLF‘A’ISC:LRAEDTAVTYYCFIAIIVT VINCWQUL 1.1V IV VLL LzCJ- \erT \‘rr DH10'~ ,~ - r1 -~1r:(:1:«E VC/ L V ‘r11. ..1 ‘5 F‘\1.11 C.41 PIS.. .J.'L.11_‘.C7K'1‘": MAC .. . .Ix‘lkufLITAKJLIKI MSII‘LIFRQA’FGKGLEL1SLLTGIRTTVYLL. S\KGI’FTTSRDNAYNIT-"XL’I’ISSLRAEDTAVYY "'I‘ GIN G I4N A I3 III I . (3C3GI. IN’.L"7 \PGGGC: .‘CIP I-IC‘PLWGC: "VII [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ’3 RT . V'I C3\I’V‘TvTDVS HE. D E «1‘9 DHTETII\ KT KDD. .K/ \1 ST". RII’VS‘»I VLI1 Q1.WL .KE‘. "QV‘T'T‘IP .E _.|IJ TAVE.’V.J.S\1C:QDNNYKT "LTI.ITDKSE’WEOQGNVE‘S_/‘\J\TTI'TT:AL INHY 'TQKELCIISPHK (SEQ ID ETC): 3x Hllv421 l + PC delet eptide MKEAITDTTSLLELDCSAYSE7Q .LESGGGEXCUPSGLREVAAS’ASVFSZDAMSEYFRQAPGK ‘ ET'TCSRIDI'TE+11in1.' L L_ YES S L RAEJE'.EAVY YCNAV.I’S’.

P‘: (I Q. r~ Ir" a - Or‘o K3'\Z";D 31(3chRLSCJAJT{JubV» QAE’HI’TG ELVC.TLTCRTTYYAJSVKCA FTCSR'VNAKNTLYLF‘VISELLADTAVYYC'NAVV I’Nr‘lTl'i' \- rr[1\7V:3(:Ir-«Q :T‘r‘ -~41:1~(r -.»-. -~4rIr~¢~ T‘\/fI ~ .JE.CJC3CCz‘JEJ rwc'r.’ ~» J'Ifukz-QJIJE .E.II.‘.\J.th1-1.I)'I. C;\’ET\‘F‘? [\Q‘~r~ J'3,_/CJ 'LJ.J\. v . .EJi". '. 31E1C21 VLVSEEDPETVKDNE‘TV"DTVEVEIK‘AKTKPRELTYNE:YRK7SVTTVLI1QDWL ‘ \1 Q V’I ‘CL DTJA T’TE.I'EE E S. N C3 (,1: E‘ EN N KKEET'TTET’E’VTI. ‘SI (3 SEE" "‘KEU:‘JEVDI ’" 9 m1"OCEN 71E"SCSVEV1I1..ATIJA DH 3/, SLSPGK 337 ID E0: 3x Hllv421 IgleFc MKWV’1.' E‘ I E )1...qnv' ..- IJ-IJnI- "13(3ka 1;.- V '3J. \3 L1»;{vb-n70 )r‘r .-.-1. SCAT.ESVES1 S -.-TGGR'E’E'Y IA‘JSVKG FITSRDNAIQQTLYLQMSS BLEDTAVYV. CITAV I‘ T‘ECQG’I'L \ITTIIHVKPGGSGCZS 3‘ ES GCSGET.".I I. .

IAIJI-,.o.,«-..- 7-mo-:J‘v Us.) Q1»; C .VIJLTTGGRTTYYAEQ v .E’TZSRLT‘IAE’L:TTLYL’QMSSLRJAIJE NA‘V’T\7 SiIT’TT’NADDYWCQ"TEX/TVKTV‘CJJCCGSEVQT LESC’TGEVT~’_ ".JCJSJJRLIECI" SCSVESTDA MSW E EIQAE‘GKGLE 1. . KGR ET'I‘1TSRJN} KN'I‘LYIQEES SLRAEIE- '1TAVYY CNAVVST'-WGR1\IZDLY.I\TGQGTLVT‘JK'CGDKTIITCPPCE’AEELLGGPS 7ELFE’PKPKDT V. KIT."WY.DC‘JVE‘VHNAE’TRPREIEIQ‘AT. T LTT'RQVYTLPTWRDJLTKNQ.SLTCL‘VTE’GF .J.C\’J_JVT\I1"JIT)I\'\J'71:3? f\f\'r\ /.L -‘VLDSI)CSSE TIE..YSKL’I".’TD ~3RNQQGE§1VETS ESVMH EIALELH CN 11 Y? ND: 1 104) ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW 3x HllVILiZl . 1 19131ch IVIKI‘IVT F I E. . IL FEES" . "CI-I[:1’3C1'3C3‘"\7’21-7C‘CE11' GLL‘LVCL ._TGGR'I"I'TTA.L SVKG ETC {‘3RDNAKNTLTLQIIS ESL ‘\IHL'2T G’3‘LVI"97K]? 3C EEGCS II 723536 3CIW/"9f1 3:.3:.LLRI E3 '1..7I2"\.E3C EZVI‘SII27\""A7 E9 QAPGKGLELVCLITGGRTTTTAEST{GRFTSKDNAKNTLTLCHTSLRLADTAVTTCNAVV TGEAICQN1:)[2""IIGCC" .‘I'mVGG‘miqS'iVQIIII EGSIRLSCMSCI’SVPS I I)'3 12'ISWLTRQAP GLIEILVCLITGGRTTTTAE SI/KGRETCS \I‘NAKNTLTLQZ‘ISCLRAEDT‘VTT 7"I7"! 8'I CI‘IGRNAK '7‘97K [:‘GCGGDK’I‘HTCP PC- PAT: " P PKE‘KDT ,.‘Nn ICDT.tEAC'GI;>G'.‘L‘9"1. ".IGG :9 E19 ."c "L.\7131‘~1\7U1\/7:)\47S}II:3v II‘.E1\7K‘.:3].\3;lii‘x \Il‘GVI3"..]l\II"I’TI’} :1}.'3QT.3"1Y}<"U}II'\7I.IH’) II~KETKCKV£NIT‘LPHPTZKTTSKAGOPREPO‘,ITTLPPSRDELTKNQVSLTCLV'CGF SDI. L\\I‘."vIl}'Io[C—s;,2I‘[:1=.\i.‘:.\ITI'\I"ITPEVLIDS.2’3"" I" I. .7E3KL’I‘VI2 KE3 I‘WQ',2G ." S "WWII NHTTQKSLSLSPGI’I (SLQ -_L) NO: 105) {613117} En some embodiments the tiniest proteins tatgeting GIi.R ot"{ht t disclosure include two or more polypeptide sequences that are operahiy linked via. amino acid s. In some embodiments, these linkers are composed predominately of the amino acids Glycine and Seiine, d as GSniinhei's herein. The GSwiinkei‘s oi‘the fusion inottins oft‘ne wesent disclosnie eon he ofVaiious lengths, foi example":.. "7,3 9, l0, ii, 32, 13:, i4, l5, i6, l7, 18, I9, 20 amino acids in length. {@9118} in some embodiments, the (IS—tinker comprises an amino acid sequence ed from the group ting of GGSGGS, i.e., (GIGS); (SEQ 15.) NO: l2), GGSGGSGGS, ie., (668% (SEQ H) NO: l3); GGSGGSGGSGGS, i.e., ((308).; (SEQ 11) N0: l4); andGGSGGSGGSGGSGGS, i.e., ((3883):, (SEQ ED N0: 15).

EGMIF); in some embodiments, the imkei‘ is a, flexible linker comprising Glycine residues, such as. by way of non—inniting e, GG, GGG, GGGG (SEQ ED NO: l6), (IGGGG (SEQ ID NO: l7), and (IGGGGG (SEQ ID NO: '58}. {691299} in some embodiments, the GITR—hinding fusion protein incl odes a combination of a GS—iinhet and a Giyeine linker, {613123} in some ments, the muitivaient GHthargeting fusion protein is tetravaient In some embodiments, the tetiavatent GITR——tatgeting moieenle of the disclosure includes two copies of a Gi'I‘R—tziigeting fusion protein having the following structure: (GII‘RED')—Linl<;et—(Gi"i‘R~BI2)—Linket—Hinge—Ee. In some embodiments, the. teti‘zwaient CITR——ta.ttgeciting inoieeuie ot the diseiosnreincludes two copies of a GITRshinding fusion [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW protein having the thllowing structure: {CslTR—BD)linker—{CslTR—BD)-Linl where the Gl'l‘R—BD is an isolated polypeptide sequence that binds Gilli. in some embodiments, the teti‘avalent Gl'l'R—tnrgeting molecule ofthe disclosure includes two copies of a GlTR—hinding fusion protein having the following structure: (GlTR~Bl))—Linl B)linker-Plinge-Fc, where the GlTR—BD is an sdAb sequence that binds GITR. ln some embodiments, the tetravalent Gl'l'R—tnrgeting molecule of the disclosure includes two copies of a. Gl'l'R—hinding fusion protein having the following structure: (Gl"l‘l§>Bl‘))~ Linker—(GlTR—BD)—Linker—l—linge—Fc, where the GlTR—BD is a humanized or fully human sdAh sequence that binds Gl'l‘R. in some embodiments, the tetravalent hargeting molecule comprises a complementarity determining region l (CIERl') comprising an amino acid sequence ed from the group consisting of SEQ ll} N0: 106, lOQ, llZ, l l7, lZO, l25, l3 l, l33, l43, l48, and M9; :1 complementarity ining region 2 (CDRZ) comprising an amino acid sequence selected from the group consisting of SEQ ll) NO: l07i l£tl, ll}, £l,5, llS, l2l, lZS, l28, l3tl, l32 £34, l36, l37, l39, l4l, lzitd, and l4l7'; and a coinplementaritjt' determining region 3 (CURE) con’iprising an amino acid sequence selected from the group ting oi‘SEQ 1D NO: lOS, lll, ll4, lloi ll9, l22, l24, lZo, l27, l29, l3}, £35, ill-(l, HZ, lélvfi, lite}, and tilt) in some embodiments, the alent Gl"l‘R,~ targeting molecule contains at least one GlTR-Bl) that comprises an amino acid sequence selected from the group consisting of SEQ ED N0: l9~80. in some embodiments, the tetravnlent Gll‘lhtargeting molecule contains at least one GlTR~BD that comprises an amino acid sequence ed from the group consisting of SEQ ll) NO: 4362, in some embodiments, the tetrzurulent targeting molecule at least one GlTR-BD that comprises an amino acid sequence selected from the group consisting of SEQ ll) NO: 63' 80. in some embodiments, the tetravalent ?targeting molecule comprises an amino acid sequence selected from the group consisting of SEQ ll} N0: , {@9122} in some embodiments, the inultivztlent GlTR-turgeting fusion protein is hexavalent. in some embodiments, the hexavnlent Gll‘lhtargeting le of the disclosure includes two copies of a, Gl'l‘R—targetihg fusion protein hi» ving the following structure: (GER—EDNAnheintGlTR—BDHsinlcer4GllR~BDl"Linkeinl’linge—Fc, in some embodiments, the hesavalent GER-targeting le of the sure includes two copies ofa Gilli-targeting fusion protein having the following structure: (Gilli—8D} Linker-(GITR—BD)linker-(GlTR—BD)linker-Hinge~Fc, Where the GlTRsBl) is a humanized or an isolated polypeptide sequence that binds GETR. in some embodiments, the [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW hesavalent GER-targeting molecule of the disclosure includes two copies oi‘a GlTR— targeting fusion protein having the following structure: (GlTFcBDl—Linlrer—(Gl'l‘lt?llll— l4inker—(Gl'l‘RrBD)—l.inl Gills, in some embodiments, the liexavalent Gilli—targeting molecule of the disclosure includes two copies ol’a GlTRmtargeting tusion protein having the following structure: (Gilli—8D)"Linkerw(Gl'lR—BD}Linl is a humanized or fully human sdAb sequence that binds Gl'l'R. in some embodiments, the hesavalent GER-targeting niolecule comprises a complementarity determining region l (CDRl) comprising an amino acid sequence selected from the group consisting ofSEQ ll) N0: lilo, lili}, llZ, ll7, lift), l25, l3l, BS, MS, MS, and Mg; aconipleinentarity ining region 2 tCDRZ} comprising an amino acid sequence selected from the group consisting ot‘SEQ ll) NQ: ltll’, llO, ll3, llS, ll8, lZl, l23, tht, 80,132, l34, l36, l3’7, l39, l4l, ln’ld, and H7; and a ernentarig/ determining region 3 (CDRES) comprising an amino acid sequence ed from the group consisting ot" SFQ lllN01l03, ll l, ll/Jl, i, i :3, ll‘), l22, l24, lZo', l27, l29, l3’3, l3'3', MO, l42, MS, Md, and tilt). in some embodiments, the liexavalent Gl'l'R—targeting molecule contains at least one Gl'l'R—BD that comprises an amino acid sequence selected from the group consisting; of SEQ ll.) NO: ill—St). in some embodiments, the liexavalent {Ellis—targeting molecule contains at least one {llTRsBl} that comprises an amino acid ce selected from the group consisting of SEQ lD N0: 42— 62, in some embodiments, the liexavalent Gl'l‘Rdargeting molecule contains at least one Gl’l'R—Bl.) that comprises an amino acid sequence ed from the group ting of SEQ ll} Ni}: 63-80. in some embodiments, the nexavalent GlTR-targeting molecule comprises an amino acid sequence selected front the group consisting of SEQ ll) NO: 94- {@9123} The GlTR~targe ting proteins described herein are useful in a variety of therapeutic, diagnostic and prophylactic tions. For example, the GER-targeting proteins are useful in treating a Variety of diseases and disorders in a subj ect. in some embodiments the Gl'l'R—targeting proteins are useful in treating, alleviating a symptom or", rating and/or delaying the progression of a disease or disorder in a subject stiflering from or identi?ed as being at risk for an inilanunatory disease or disorder. in some embodiments, the Gll‘lhtargeting proteins are useful in treating, alleviating a symptom of, ameliorating and/or delaying the progression of a cancer or other stic ion In some ments, the cancer is bladder cancer, breast , e/cervical cancer, [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW o 'aiian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neclr cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, slrin cancer, neoplasm of the central nervous system, lymphoma, leukemia, inyeloma, sarcoma, and virus-related . in certain en’ibodiments, the cancer is a metastatic cancer, refractory cancer, or recurrent cancer in some embodiments, the Gll‘lbtargeting proteins are useful in reducing or depleting the number of "l‘ regulatory cells in a tumor of a t in need thereof. in some embodiments, the (i’lTR—targeting proteins are useful in stimulating an immune response in a subject. in some embodiments, the Gll‘lbtargeting proteins are useful in treating, alleviating a symptom of, ameliorating and/or delaying the progression of an autoimmune disease or er. ln some embodiments, the GlTRstargeting proteins are useful in treating, alleviating a symptom of, rating and/or delaying the ssion of viral, bacterial and parasitic infections. {@3124} 'l‘lierapeutic formulations of the disclosure, which include a Gl’l‘R—targeting molecule of the disclosure, are used to treat or alleviate a symptom associated with a disease or disorder associated with aberrant activity and/or sion ofGl'l‘R in a subiect, A therapeutic regimen is carried out by identifying a subj ect, r»: g, a human patient suffering from (or at risk of ping) a e or disorder associated with nt activity and/or expression of (EUR using standard methods, including any ofa variety of clinical and/or tory procedures, The term patient includes human and veterinary subj ects. The term subject includes humans and other mammals, EtltllZSl Ethcaciousness of treatment is ined in association with any known method for diagnosing or treating the ular disease or disorder associated with aberrant activity and/or sion of Gl'l'R, Alleviation ot" one or more symptoms of the disease or disorder associated with aberrant activity and/or sion of GlTR tes that the GER—targeting molecule confers a clinical benefit. {thlzol 'l‘lierapeutic uses of the Gills—targeting molecules oftlie disclosure can also include the administration of one or more additional agents. in some embodiments, the one or more additional agents is an lTR antibody or fusion protein, an anti —Pl)l antibody or fusion protein, a LAG-3 dy or fusion protein, a CTLA—zl antibody or tusioi’i protein, and/or a l’D~Ll antibody or fusion protein, {@127} The GlTR~targe ting molecules of the present invention may be stered alone or with other modes of treatment, They may be provided , substantially [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW cei’itei’npoi'aheous with, or after other medes cftreatment, for example, surgery, chemotherapy, radiation therapy, or the administration ofa biologic, such as another therapeutic polypeptitie/antibody, {@9128} In some embcdiments, the (EUR—targeting molecules of the present icn may he used in ation with a cl'ieh’iotherapeutic agent, Examples of chemotherapeutic agents include, but are not hunted to, aikyiating agents such as thiotepa and Cytoxan® cyclosphosphauude; alkyl sulfonates such as busul?au, iinprosulfau and piposultan; dines such as dcpa, cai'bcquune, nietui'edepa, and ui'edepa; ethylenimiues and niethyiaineiainines including tainine, triethyleueiuelamine, trietyiencphesphorauude, tiiethiyienethiophesphui‘amidc and trimethylolonielauuue; acctugcuius (especially bull ataein and acincne); a camptctheciu {including the synthetic analogue cah); hiyosiau'h; callystatii’i; (EC—1065 {including its adozeiesin, cai‘zeiesin and bizeiesiu synthetic analogues); ciyptophycins cularly ctyptophyciii i and eiyptophyciu 8); dolastatiu; duocaruiycin (inchidiug the synthetic analogues, K‘W—2l39 and CBLTM'S ), cleutherehiu; pauciatistauu; a sarcudictyiu; spengistatin, uitmgen rcls such as chloraiubucii, chioruaphazine, choiophospnainide, estraiuustine, ifosfainide, mechlotethaniine, mechlorethainine oxide hydrochloride, inelphalan, uevemhichiu, phenestariue, preduiuuistinc, ti‘cfosfamidc, uracil mustard; nitiesurcas such as cannustiue, chlorozotocih, toteinustiue, ioniusune, himustine, and rauihuiustine; antibiotics such as the enediyne antibiotics (e.g , cahcheamicin, especially calicheamicin gaunnail and calicheaniiciu omegaii (see, tag Agnew, {Chem [mt Ed Engi, 33: £83486 ( 994)); dyneniiein, including dyneniiein A; sphunates, such as hate, an espei'aniicin; as well as neocarzinostatin chromophore and related chroniopiotein enediyne antio‘oiotic chmmophores), aclacinomysins, aetinomyciu, authi'amyciu, azaserine, hieemycius, cactinomyciu, carahicin, mycin, carziuophilin, chromomyeinis, dactiuomycin, daunomhicin, detomhicin, 6—diazo~5 —oxu—L—norieueine, Adrianiyciu?k" donoruhiciu (including nioi’phoiino—doxombicin, cyanoniorpnolinodoxonibicin, 2—pyi‘i'olino— doxoiubiein and deoxydoxombiciu), epim‘oiciu, esoruhicin, idamhicin, niarcellouiycin, mitomyeius suc as mitomycin C, henolic acid, nogalantycin, olivontycins, nycin, po‘t?i‘omyeih, ciu, quelamycih, i'odotuhicin, unigrin, streptozochi, tubei'cidin, ubennnex, zinostatin, zorubicin; antiwinetabolites such as methotrexate and 5~ ?ueroutacil (S-PU); fehe acid analogues such as erin, methettexate, ptcrupteiin, trimetrexate; purine : such as tludarabine, 6—inei'eaptopui'ine, thianiipriue, [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW tl'iioguahine; pyrimidine anaiegs such as ancitahine, aaacitidine, 6-azauridine, fur, cytarahine, ditieoxyuricline, tiexi?uritiine, ahine, tloxundine; androgens such as caiusterone, tanolone pi'cpienate, epitiostauoi, mepitiostanei testolaetcne; anti— ailrenais such as aminoglutetliiniiile, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatoi’ie; aldophospharnide ide; aniinoievuiinic acid; eniiiiracii; anisacrine; bestrahucii; bisantrene; eclatraxate; det‘etainiiie; icine; diaziq uone; eifoinithine; elliptiniuin acetate; an epothilone; etoglucicl; gallium nitrate; hyth‘cxyurea; lentinan; inine; maytansinoids such as inay'iansii’ie and ansamitocins; initcguaaune; niitoxaiitrone; inopit‘ianinol; nitraeiiiie; peritestatin; iet; piranrhicin; losoxantrniie; yilinic acid; 2* ethylhych‘azide; procar‘oazine; PSKE?’ ccharide complex (Jill‘s-$1 Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogeimanium; tenuazcnic acid; triaziquoi’ie; 2,2,2"~‘trichloreti‘ietl’iylamine; trichetiiecenes (especially T—Z toxin, verracurin A roridin A and anguidine); uretlian; Vindesine; tiacarbazine; niustine; nutohronitol; mitoiactol; pipohmnian; gacytosine; oside ("Ara—C"); cyclephosphamide; tliictepa; taxoidsv eg. Taxoi® paclitaxel (Bristol- Myers Squibb tholcgy, Princeton, NJ ,3, Ahra‘xane® Crenrophcr—i’ree, albumin—engineered nanopartieie formulation of paeiitaxel (American Pharmaceutical ers, Schauhiberg, lllinois), and Taxotereigl doxetaxel (Rhonew Poulenc Rcren Antony, France); chioranlnicil; Gemzai?l genicitabine; 6—tiiieguanine; mereaptopurine; methotrexate; platinum anaicgs such as cispiatin, osaiipiatin and carbeplatin; vinhiastine; platinum; etoposide (VP—16); mide; mitoxahtrone; Vincristine; Naveihine® Vinoreibine; ncvantrone; tenipeside; xate; iycin; antinopterin; xelcda; ihandronate; innotecan (Caniptosar, CPT~E 1) (including the treatment regimen of iiinotecan with 5-H} and orii’igi; topoisenierase inhibitor RES 2000; difluoroinetihylornithine (DMFO); retincicis such as retinoic acid; capecitahine; eonihretastatin; leucm/ciin (EV); cxaliplatin, including the oxaiipiatin treatment regimen (FOLFOX); inhibitors of PRC—alpha; Raf, HmRas, EGFR (cg, erlutini‘o (latices/ail)» and VEGEA that reciuce cell proliteraticn and pliarniaceutically g cceptahle saltsv acids or derivatives of any cfthe above genres} Further ncnlimiting exemplars! herapeutic agents incl utie anti" hormonal agents that act to regulate er inhibit hormone action on cancers such as anti— estrogens and selective estrogen receptor modulators (SERh/ls), including, for e, tamoxifen (including Nolvadexa‘a tamoxifen), raloxifene, drolnxifenet 4—liydroxytanioxifen, trioxii’enea kecxifene, LYl l Mill, onapristune, and Fareston‘«59 ii'ene; arun’iatase [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW inhibitors that inhibit the engyn’re arornatase, which regulates estrogen tion in the l glands, such as, for example, 4(5)~iinidazoles, arninoglntethiinide, MegaseRD inegestrol icetate, Arornasin® exentestane, forinestanie, fadrozole, Rivisor?l vorozole, Peinarall] letrozole, and Arintidexll anastrozole; and anti —androgens such as tlutan'iide, nilutarnide, hicalutan’iide, leuprolide, and goserelin; as well as troxacitahine (a l,3— dioxolarie nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling, pathways implicated in abherant cell proliferation, such as, for example, PRC—alpha, nd l-l—Ras, ribozynies such as a VEGF expression llllllbl{01'{€_g., Angiozyine. . t . ® rihozyine) and a HERZ sion inhibitor; vaccines such as. _i . _ . i , .. _ _ _ . ‘ e, and Vaxid® gene therapy vaccines, for example, Allov'eetin® e, Leuvectin vaccine; Proleulcin® rill—2,; Lurtotecan® topoisornerase l inhihitor; Aharelix® rinRH; and ceutically acceptable salts, acids or tites of any of the above. {($136} in some embodiments, the Gl'l'R—targeting rnolecule ofthe present invention can he used together with an anti—angiogenesis agent, "the angiogenesis agent refers to a small molecular weight substance, a polynucleotide (including, eg an tory RNA (RNAi or siRNA», a polypeptide, an isolated protein, a inant protein, an antibody, or conjugates or fusion proteins thereof, that ts angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly. it should he understood that the anti—angiogenesis agent includes those agents that bind and block the angiogenic activity ofthe angiogenic factor or its receptor, For example, an anti—angiogenesis agent is an antibody or other nist to an angiogenic agent, 6.3 antibodies to VEGEA (rag, hevacizurnah {JIM/astinli’? or to the VEGF—A receptor (cg, KDR or or Flt-l receptor), anti~PDGFR inhibitors such as ldleevec® t:lrnatinib Mesylate}, small molecules that block \I’EGF or signaling (e,g irl‘K78'7/ZK2284, SU6668, SitteritElJ/SU l 1248 (snnitini‘o rnalate), AMG706, or those described in, cg, international patent application Wt} 2004/1 8304). Anti~angiogensis agents also include native angiogei’iesis inhibitors , cg, angiostatin, endostatin, etc, See, eg Klagshrun and re ( l99l) Anna. Reta Pi’sysz‘ol 53:2l7~39; Streit and Detinar (2003) rilnmgene 22:3 l 72—3 l 79 (eg, 'l‘ahle 3 listing anti— angiogenic therapy in malignant melanoma); Ferrara & Alital o (N99) Nature .Mer'rcirze StilZ}: 8594364, Tonini er of, (2003) Oncogene 216549—6556 (cg, Table 2 listing known antiwangiogenic factors}; and, Sate (2003) Int; J (Tim Oracol, 81200906 (cg Table l g anti—angiogenic agents used in clinical trials), [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW E?tll‘Sll in some embodiments, the (RTE-targeting molecule is used in con’ihination with other anti~tumor agents, such as antiwliER—Z antibodies, anti—CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist tag, a tyrosine l llERl/EGFR inhibitor (9.g, erlotinih (Tarceva.®), platelet derived growth factor inhibitors (cg, Gleevec® (lrnatinih te)), a CDX—Z inliihitor (9.33., celeccxih), interferons, "its BNlQ—mo‘i‘iS MPDL3ZSOA}, f " ., i, i" cytokines, antagonists (cg, neutralizing antibodies) that bind to one or more oftlie toliowing targets Ethf/Z, Eth3, Erhl‘lrl, l’DGFR—heta, BlyS, i, BCMA, l’ll—l or VEGF , l’Dlil, FDLZ, C'l‘LAd, receptoits), TRAlli/ApoE, and other hioactive and organic chemical agents, are {@9132} in some embodiments, the Gilli—targeting molecule is administered during and/or after treatment in ation with one or more additional agents. in some embodiments, the —targeting molecule and the additional agent are ated into a single therapeutic composition, and the GlTR-taigeting molecule and additional agent are administered siintiitaneousiy, Alternatively, the GER—targeting molecule and additional agent are separate from each other, eg, each is formulated into a separate therapeutic composition, and the targeting molecule and the additional agent are administered sim ultaneously, or the targeting molecule and the additional agent are administered at ent times during a treatment regimen. For example, the Gilli—targeting molecule is administered prior to the administration of the additional agent, the Gl’l‘thargeting le is administered subsequent to the stration of the additional agent, or the Gll‘lhtargeting molecule and the additional agent are administered in an alternating fashion. As described herein, the Gl’l‘thargeting molecule and additional agent are administered in single doses or in multiple doses, {@9133} in some embodiments, the Gilli—targeting molecule and the additional ’s) are administered aneously. For example, the Gl'llthai'geting molecule and the additional s) can be ated in a. single composition or administered as two or more separate compositions, in some embodiments, the rgeting molecule and the additional agentt's) are administered sequentially, or the (EUR—targeting molecule and the additional agent are administered at different times during a treatment regimen. {@9134} Methods for the screening of GER-targeting les that possess the desired speci?city include, but are not limited to, enzyme linked immunosorhent assay -j ’4‘) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW ation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW (ELlSA), enzymatic assays. flow cytometry, and other immunologically mediated techniques lcnovvn within the art.

Ellilldd} ’l‘he disclosure. further es nucleic acid sequences and particularly DNA sequences that encode the present fusion proteins. Preferably, the DNA sequence is carried by a vector suited for extr’acl’iromosomal ation such as a phage, virus, plasmid, phagemid, cosmid, YAC, or episornc. in particular, a DNA vector that encodes a desired fusion protein can he used to facilitate the methods of preparing the Gl’l‘thargeting molecules described herein and to obtain signi?cant quantities ot‘the fusion protein. The DNA sequence can be inserted into an appropriate expression vector, lo, a vector which ns the necessary ts for the transcription and translation ofthe inse ed protein« coding sequence. A variety of host—vector s may he utilized to express the protein— coding sequence. These include mammalian cell systems infected with virus leg, vacciriia virus, adenovirus, etc), insect cell s inlectcd with virus (cg, haculovirus); microorganisms such as yeast containing, yeast s, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Depending on the host—vector system utilized, any one ot‘a number ot‘suitablc transcription and translation elements may be used.

Wilma} ’l‘he disclosure. also provides methods of producing a Gl'l'R—targeting molecule by culturing a cell under conditions that lead to expression ofthe polypeptide, wherein the cell comprises an isolated nucleic acid molecule encoding a GER—targeting molecule bed herein, and/or vectors that include these isolated nucleic acid sequences.

The disclosure provides methods of producing a Gl'l‘llrtargeting molecule by culturing a cell under conditions that lead to expression ol’ the Gill‘s—targeting molecule, n the cell ses an isolated nucleic acid molecule encoding a Gll‘lhtargeting molecule described herein, and/or vectors that include these isolated nucleic acid sequences. {@9137} The fusion ns of the disclosure (also referred to herein as "active con’ipoundsl?, and derivatives, fragments, analogs and logs f, can be orated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the fusion protein and a pharmaceutical ly acceptable carrier, As use herein, the term "pharmaceutically acceptable carrier" is ed to include any and all solvents, dispersion media, coatings, antibacterial and antil‘ungal agents, isotonic and tion delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s ceutical Sciences, a standard reference text in the field, which is incorporated herein [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW by reference Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringerls solutions, dextrose solution, and 5% human serum albumin. l.iposome.s and non—aqueous vehicles such as fixed oils may also he used The use of such media and agents for pharmaceutically active substances is well lcnown in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also he incorporated into the compositions, SSl A ceutical composition of the disclosure is formulated to he compatible with its intended route of administration. Examples of routes of administration include. parenteral, sag, intravenous, intradermal, subcutaneous, intratumoral, oral (sag, inhalation), transdernial (Lei, topical), transniucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the ing components: a sterile diluent such as water for iniection, saline solution, lined oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as henzyl l or methyl ns, antioxidants such as ascorbic acid or sodium bisultite, chelating agents such as ethylenediaminetetraacetic acid (ED'I'A); buffers such as acetates, citrates or ates, and agents for the adjustrn ent of tonicity sue as sodium chloride or dextrose. The pll can be adjusted with acids or hases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can he ed in ampoules, disposable syringes or multiple dose vials made ol‘glass or plastic. {@3139} niaceutical compositions suitable for inie italile use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the estemporaneous preparation of sterile injectahle ons or dispersion. For enous stration, suitable carriers include physiological saline, bacteriostatic water, {Iremoplror Elam (BASE, l-larsippany, NJ.) or phosphate buffered saline (PBS), ln all cases, the composition must he sterile and should he tlui d to the extent that easy syringeability exists. it must be stable under the conditions ol’ manufacture and storage and must he ved against the contaminating action of microorganisms such as bacteria and tungi.

The carrier can he a solvent or dispersion medium containing, for e, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and le mixtures thereof The proper fluidity can be tained, for example, by the use ofa coating such as lecithin, by the maintenance of the required particle size in the case ot" sion and by the use. of surfactants Prevention of the action of microorganisms D I 5 [Annotation] CLNDW None set by CLNDW ation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thinierosal, and the like. ln many cases, it will be preferable to include ic agents, for example, sugars, polyalcohols such as manitol, sorhitol, sodium chloride in the composition. Prolonged absorption ofthe iniectable con’ipositions can he brought about by including in the con’iposition an agent which delays absorption, for example, aluminum monostearate and n.

{Gill-4h} Sterile inj ectable solutions can he prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a con’ibination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, sions are prepared by incorporating the active compound into a sterile vehicle that contains a basic sion medium and the i‘equi red other ingredients from those enumerated above. in the case of sterile powders for the preparation of sterile iniectable solutions, methods ol‘preparation are vacuum drying and freeze~drying that yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile~ ed on thereof. ll Oral compositions generally e an inert diluent or an edible carrier. "l‘hey can be enclosed in gelatin capsules or compressed into tablets For the purpose of oral therapeutic administration, the active compound can he incorporated with en ts and used in the l'bll’l’l oi‘tahlets, troches, or capsules. {Pral compositions can also he ed using a fluid carrier for use as a mouthwash wherein the compound in the fluid carrier is applied orally and svvished and expectorated or swallowed. l’harmaeeutically compatible binding agents, and/or adjuvant materials can be included as part of the con’iposition. The tablets, pills, capsules, troches and the lilre can n any of the following ingredients, or compounds of a similar nature: a binder such as mi crocrystalline. ose, gnni tragacanth or gelatin, an eseipient sue as starch or e, a disintegrating agent such as alginic acid, Primogel, or corn ; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccliarin; or a flavoring agent such as mint, methyl salicyl ate, or orange fl avoring {63142} For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, cg. , a gas such as carbon dioxide or a nebulizer. {@9143} Systemic administration can also he by transmucosal or ermal means.

For transmucosal or transdernial administration, penetrants appropriate to the barrier to be ..,,6 [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW permeated are used in the thirnulation. Such ants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid tives, smucosal administration can he accomplished through the use of nasal sprays or suppositories, For transderinal administration, the active nds are formulated into ointnients, salves, gels, or creams as generally known in the art {?lllds‘i} The nds can also he prepared in the form of suppositories (e.g with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

EllhldS} in one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release fonnulation, including implants and inicroencapsulate’ delivery systems.

Biodegradable, biocompatihle polymers can he used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collageni polyorthoesters, and polylactic acid, Methods for preparation of such hormulations will he apparent to those skilled in the art, The materials can also he obtained commercially from Alza Corporation and Nova ceuticals lnc. Liposomal suspensions can also he used as ceutically acceptable carriers. These can he prepared according to methods known to those d in the art, for example, as described in US. Patent No, 4,522,?" l , {@9146} it is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity ot‘dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the t to he treated, each unit containing a predetermined quantity ol’actiye compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier The specification for the dosage unit forms or" the sure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of nding such an active compound for the treatment ot‘indiyiduals.

{Gill-47} The pharmaceutical compositions can be included in a lcit, container, pack, or dispenser together with instructions for administration, These pharmaceutical compositions can be included in stic lrits with ctions for use.

Ell?ldél} l-l'harmaceutical compositions are administered in an amount etlective for treatment or prophylaxis ofthe specific indication, The therapeutically et‘ective amount is typically dependent on the weight of the subject being treated, his or her physical or health [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW condition, the extensiveness of the condition to be treated, or the age of the subject being treated in some embodiments, the phamiaceutical composition may be administered in an amount in the range of about fill ug/ g body weight to about 50 mg/kg; body weight per dose. ln some ments, the pharmaceutical composition may be administere' in an amount in the range of about 100 tag/kg body weight to about St} mgr/kg body weight per dose. in some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 ugflrg body weight to about 29 mg/lrg body weight per dose. ln some embodiments, the pharmaceutical osition may be administered in an amount in the range of about 0.5 rug/leg body weight to about 20 lug/leg body weight per close, {@9149} In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about it) mg to about LGOO mg per dose. ln some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. ln some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 300 mg per dose. in some embodiments, the pharmaceutical composition may be administered in an amount in the range ct" about 20 mg to about 200 mg per dose {@158} The pharmaceutical composition may be administered as needed to subjects. in some embodiments, an ettbctiye dose of the ceutical composition is stered to a subject one or more times. ln various embodiments an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, such as, for example, every two months, every three months, or eyery six . ln other embodiments, an et‘tectiye dose of the pharmaceutical composition is administered more than once a month, such as, for example, every two weeks, every week, twice per week, three times per week, daily, or multiple times per day. An ive dose of the pharmaceutical composition is administered to the subject at least once. in some ments, the et‘iectiye dose of the pharmaceutical composition may be stered multiple times, including for periods of at least a month, at least six months, or at least a year. In some embodiments, the pharmaceutical composition is administered to a subject as— needed to alleviate one or more symptoms oi‘a condition.

Etl?lSll Unless otherwise de?ned, scienti?c and cal terms used in connection with the present disclosure shall hate the meanings that are commonly understood by those of ordinary skill in the art. Further, unless ise required by context, singular terms [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW shall include pluralities and plural terms shall include the singular. Generally, nornenclatures utilized in connection with, and techniques of, cell and tissue culture, lar biology, and protein and oligo~ or polynucleotide chemistry and hybridization described herein are those well—known and commonly used in the art Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (egg electroporation, ction). Enzymatic reactions and ation techniques are performed according to cturer's specifications or as ly accornpli shed in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well linown in the art and as described in various general and more speci?c references that are cited and discussed throughout the present speci?cation. See 2. g. Sainbrook er til. Molecular g: A Laboratory Manual (2d ed, Cold Spring or Laboratory Press, Cold Spring l-lar'hor, NY, 0989)), The nonienclatures utilized in connection with, and the laboratory procedures and ques of, analytical chemistry, synthetic organic try, and medicinal and ceutical chemistry described herein are those nown and connnonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment ot" patients. The term t includes human and veterinary subjects. {@9152} As utilized in accordance with the present disclosure, the following terms, unless ise indicated, shall be understood to have the following meanings: {llllil53l As used , the terms "targeting fusion protein" and "antibody" can be synonyms. As used herein, the term "antibody" refers to immunoglohulin les and immunologically active portions ofirnnrunoglobulin (lg) inolecules, lie", molecules that contain an antigen binding site that specifically binds (imniunoreacts with) an antigen. By "specifically bind" or "immunoreacts with" "or directed against" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower allinity (lid > lt‘f?). Antibodies include, but are not limited to, polyclonal, onal, chinieric, dAb (domain antibody), single chain, Fab, Fabs and Ftab’); fragments, llv, scFys, an Fab expression library, and single domain antibody {sdAb} fragments, for example VHS-l, VNAR, engineered Vii or VK.

Ell?lfld} The basic antibody ural unit is linown to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 tht) and one "heavy" chain (about 50-70 hDa). The amino~terrninal [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW portion of each chain includes a variable region ot‘ahon‘t lOG to l ll} or more amino acids primarily responsible for antigen recognition, The y—terniinal portion of each chain defines a constant region primarily responsible for effector function in general, antibody molecules obtained front humans relate to any of the classes lgG, lgM, lgA, lgE and lgl), which differ from one another by the nature ol’ the heavy chain t in the le.

Certain classes have subclasses (also known as isotypes) as vvell, such as lqu, lgGg, and , Furthermore, in humans, the light chain may be a kappa chain or a lambda chain E?lllSSl The term "monoclonal antibody" (rnAli) or "n’ionoclonal an tihody composition", as used herein, refers to a population ofantihody molecules that contain only one molecular s of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product in particular, the complementarity determining regions tCDRs) of the monoclonal antibody are identical in all the molecules of the population. MAhs contain an antigen binding site capable ofiinninnoreacting with a particular epitope of the antigen characterized by a unique binding af?nity for it, E?lllStil The term "antigen—binding site" or "binding portion" refers to the part of the iinniunoglohulin molecule that participates in antigen binding. The antigen binding site is formed by antino acid residues of the N«terminal variable ("V") regions of the heavy ("H") and light ("14") chains, Three highly divergent stretches within the V regions of the heavy and. light chains, ed to as "hypervarialile regions," are interposed between more conserved tlanlting stretches ltnovvn as "ii‘anreworlt s," or ". Thus, the term "FR" refers to antino acid sequences which are lly found between, and adjacent to, hypervaiiahle regions in imniunoglohulins. in an antibody molecule, the three hypervariahle regions ot‘a light chain and the three variahle regions ot‘a heavy chain are disposed relative to each other in three—dimensional space to form an antigen~hinding sur?ace. The an tigen—hinding surface is complementary to the three—dimensional e of a hound antigen, and the three hypervariable regions of each ofthe heavy and light chains are referred to as "coniplernentarity~determining regions," or "CDRs." The assignrnent of amino acids to each domain is in accordance with the definitions of Kabat ces of Proteins ot" lmmunological st (National lnstitutes of Health, Bethesda, Md (l 987 and 199m, or Cliothia & Lesl: J, Mol, Biol. l—9l7 @987}, (Eliothia er al. Nature 342:878— 8833 0989).

[Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW E90915"? l The single domain an tihody (sdAh) fragments portions of the fusion proteins ofthe present disclosure are referred to hangeably herein as targeting polypeptides herein {@9158} As use herein? the term "epitope" includes any protein determinant capable of specific binding to/by an ii’nmunoglobulin or fragment thereof, or a T—cell receptor. The term "epitope" includes any protein deterniinant capable of specific binding to/by an ininiunoglobulin or "l‘wcell receptor. Epitopic determinants usually consist of chemically active e groupings ofinolecules such as antino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An dy is said to specifically bind an antigen when the dissociation constant is E l uh’l; cg :1 lOO nl‘vl, preferably S l0. nlvl and more preferably :1 l nlvl.

ElllllSEPl As used herein, the terms "immunological g" and "immunological g properties" and fic binding" refer to the non-covalent interactions of the type which occur bets ’eeri an irninunoglobulin molecule and an antigen for which the intinunoglobulin is specific. The strength, or af?nity of immunological binding interactions can be expressed in terms of the dissociation constant (Kg) of the interaction" wherein a smaller Kd represents a r affinity. immunological binding properties of selected polypeptides can be quanti?ed using methods well known in the art Qne such method s measuring the rates of antigen—bindins.4 site/an tigen complex formation and dissociatioi’r, n those rates depend on the concentrations of the complex partners, the affinity of the interaction and geometric parameters that equally influence the rate in both directions. s, both the "on rate constant" than) and the "off rate constant" (hog) can be determined by calculation ofthe trations and the actual rates of association and dissociation {See Nature 36l 186-87 {l 993)) The ratio of lean/hm, enables the cancellation of all ters not d to affinity, and is equal to the dissociation constant lid. (See, generally; Davies et al. ("390) Annual Rey Biocheni 59439—473). An antibody of the present disclosure is said to specifically bind to an antigen, when the bri urn binding constant (lid) is it tilt/l, preferably 5 100 rth more preferably 3 ll} nl‘vl, and most preferably 5; lilil pM to about 1 10M, as measured by assays such as radioligand binding assays, surface plasrnon resonance (SPR), tlow cytonretry binding assay, or similar assays known to those skilled in the art {@9168} Preferably; residue positions which are not identical differ by conservative amino acid substitutions.

W e...

[Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW Etlllltill Conseiyative amino acid substitutions refer to the intercl’iangeability of es having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphaticehydroxyl side chains is serine and ine, a group of amino acids haying arnide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group ol‘aniino acids having basic side chains is lysine, ne, and histirline; and a group of amino acids having sulfur— containing side chains is cysteine and methionine. Suitable conservative amino acids substitution groups are: valine—leucine~isoleucine, phenylalanine~tyrosine, ~arginine, e valine, glutainic— aspartic, and asparagine—glutaniine. {@9162} As discussed , minor variations in the amino acid sequences of antibodies or inimunoglohulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least Styli), 90%, 95%, and most preferably 99%, hi particular, conservative amino acid replacements are contemplated, Conservative replacements are those that tal Genetically encoded amino acids are generally divided into families: ( l) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginirie, histidine; (3:) hon-polar amino acids are e, valine, e, cine, proline, alanine, nine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutaniine, ne, serine, threonine, tyrosine. ’l‘he liydrophilic amino acids include arginine, asparagine, aspaitate, glutamine, ate, histidine, , serine, and threonine, The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and yaline Qtlier families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy ; (ii) asparagine and glutamine, which are the amide containing family, (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family, and (iv) phenylalanine, tiyptophan, and tyrosine, which are the aromatic family For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or yaline, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major el‘lect on the binding or properties ofthe resulting molecule, especially ifthe replacement does not involve an amino acid within a framework site, Whether an amino acid ge s in a onal peptide can readily be determined [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW by ng the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or imniunoglobulin molec ules can be readily prepared by those of ordinary skill in the art. Suitable — and carboxy~termini of fragments or analogs occur near boundaries of functional s. Stnictural and functional domains can be identified by comparison of the tide and/or amino acid sequence data to public or proprietary sequence databases. l’rel‘erably, computerized comparison methods are used to identify sequence motifs or ted protein conformation domains that occur in other proteins ol’lrnown ure and/or function. Methods to identify protein sequences that fold into a linown three~dirnensional structure are known. Bowie er al, Science 253: led (l 99hr "l‘hus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the disclosure. {($163} Preferred amino acid substitutions are those which: (l) reduce tibility to protcolysisr (2) reduce tibility to oxidation (3’) alter g y for fonning protein complexes, (4) alter binding ailinities, and (All conl'er or modify other pliysicochemical or functional properties of such analogs. Analogs can include various rnuteins of a sequence other than the naturally~occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions} may be made in the naturally— occurring sequence (preferably in the portion of the polypeptide outside the dornaint's) fonning intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics ofthe parent sequence (eg a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of a.rt«recogniaed polypeptide secondary and ry structures are bed in Proteins" Structures and Molecular Principles (Creighton, Edy W. ll.

Freeman and Company, New York @9843); lntroduc‘tion to Protein Structure (Ci n and l. loose eds", Garland Publishing, New Yorlc NY 09%)); and nton et al, Nature 354105 (rear).

Ellllltiéll The term eptide fragment" as used herein refers to a polypeptide that has an amino terminal and/or carboxy~teiininal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the lly—occurring sequence deduced; for example from a full length cDNA sequence Pragnien ts typically are at least 5, 6; 3 or l0 amino acids long? ably at least 14 amino acids long’ more preterably at least "3.x ’4‘) [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW amino acids long, usually at least 50 amino acids long? and. e A'en more preferably at least 70 amino acids long, The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial ty to a portion of a d amino acid sequence and which has c binding to GlTRy under suitable binding conditions. Typically, polypeptide s comprise a vative amino acid substitution tor addition or deletion) with respect to the naturally" occurring sequence.

Analogs typically are at least 29 amino acids long, preferably at least 50 amino acids long or longer, and. can often be as long as a ength naturally—occurring polypeptide.

Etl?lfiS} Feptide analogs are commonly used in the pharmaceutical industry as non" peptide drugs with properties analogous to those ofthe template peptide. These types of non—peptide compound are termed "peptide mimetics" or "peptidoniinietics’i Fauchere, ll Ady. Drug Res l5:29 H986), Veher and Freidinger TlNS p.392 (P985); and Eyans era]. l.

Merl. Chem. 30:1229 ( l987). Such compounds are often developed with the aid of coinputen zed molecular modeling l’eptide mimetics that are stiucturally similar to therapeutically usetul es may be used to produce an lent therapeutic or prophylactic effect. Generally omimetics are structurally similar to a paradigm polypeptide (:13, a polypeptide that has a. biochemical property or pliannacologieal activity); such as human an tibodyg but have one or more peptide es ally replaced by a linkage selected from the group consisting of: -_ Cl-lgNl-lu, ""(H‘igS‘; "Cl-lg,— CH2", "CHIClln—tcis and trans), "CDC/Hg", ClltillllCllz——, and ~CHgSOw, by methods well known in the art. Systematic substitution ot" one or more amino acids of a consensus sequence with a. D—amino acid of the sante type leg, D—lysine in place of ll—lysine} may be used to generate more stable peptides. ln addition; constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem, pl :337 (l 992)); for example, by adding internal ne residues capable ot‘ forming intrarnolecular disulllde s which e the peptide. {billed} The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds; a biological macrontolecule, and/or an extract made from biological materials.

Etl?lf?'} As used herein. the terms " or "labeled" refers to incorporation ofa able marker, eg, by incorporation of a radiolabeled antino acid or attachment to a polypeptide ofbiotinyl moieties that can be detected by marked avidin tag, streptayidii’i [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW containing a ?uorescent marker or ericymatic activity that can be ed by l or calor‘inietr'ic s). in n situations, the label or marlrer‘ can also he therapeutic.

Various methods of label mg polypeptides and glycoproteins are known in the art and may be used, Examples of labels for polypeptides include, but are not limited to, the following: r’adioisotopes or r‘adionticlides (8.3:, 3H, "C, 15N, 358, QOY, 9917c, 111in, 125 l, 131l), ?uorescent labels (cg. Fl'llC, r‘hodarnine, lanthanide phosphors), enzymatic labels (cg horseradish peroxidase, ll—galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e, g. , leucine zipper pair‘ sequences, binding sites for secondary dies, metal binding domains, epitope tags). ln some embodiments, labels are attached by spacer arms of s lengths to reduce potential steric hindrance The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or ition capable of inducing a desired therapeutic effect when properly administered to a patient. {?lling} As used herein, the terms "treat," treating," "treatment," and the like refer to reducing and/or ameliorating a disorder and/or symptoms associated tl’ierewith By "alleviate" and/or "alleviating" is meant decrease, suppress, ate, diminish, arrest, and/or stabilize the development or progression of a disease such as, for example, a cancer, it will he appreciated that, although not precluded, treating a, er or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. } in this disclosure, "comprises," "comprising," c"containing," "having," and the like can have the meaning ascribed to them in US Patent law and can mean "includes," "including," and the like; the terms "consisting essentially of" or "consists essentially" likewise have the meaning ascribed in US Fatent law and these terms are nded, allowing for the presence of more than that which is recited so long as basic or novel teristics of that which is recited are not d by the presence of more than that which is recited, but excludes prior art embodiments, {llllil’ftll By "effective amount" is meant the amount required to ameliorate the ms of a, disease relative to an ted patient, The effective amount of active cornponnd(sl used to practice the present disclosure for therapeutic treatment of a e varies depending upon the manner ofadrninistration, the age body weight, and general health of the subject tely, the attending physician or veterinarian will decide the "3.x {Jr [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW ation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount, {(3017 3,} EV "suhj eet" is meant a mammal, including, but not d to, a human or man mammal, such as a , equine, canine, rodent, ovine, primate, canielid, or feline. {09172} The term "administering," as used herein, reters to any mode ol‘transterring, ring, introducing, or transporting a therapeutic agent to a t in need of treatment with such an agent. Such modes include, but are not lirnited to, oral, l, intravenous, intraperitoneal, intramuscular, intradernial, intranasal, and subcutaneous administration. {60173} By "?‘agnient" is meant a portion of a polypeptide or nucleic acid molecule.

This portion contains, preferably, at least lO‘i/é, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 9 % ofthe entire length ol‘the nce nueleie acid molecule or polypeptide. A fragment may contain ll), 20, 3t), 40, 50, 60, 70, 80, 90, or l00, 200, 300, 400, 500, 600, 700, 800, 90C, or l000 nucleotides or amino acids 39991743 Ranges proxrr'ided herein are understood to he shorthand for all ol‘the values within the range, For e, a range of l to 50 is understood to include any number, combination ofnumhers, or sub—range from the group consisting of l, 2, 3, Al, 5, 6, 7, S, 9, l0, ll, l2, l3, l4, l5, l6, l7, l3, l9, 20, 2l, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3l, 32, 33, 34, 3'5, 36, ’37, 38, 39, 40., 4l, 42, 43, 44, 45, 46, 47, 48, 49, or '50.. {($175} Unless specifically stated or obvious from context, as used herein, the tenns "a," "an," and "the" are understood to he singular or plural. Unless speci?cally stated or obvious from context, as used herein, the term "or" is understood to he inclusive.

Etlhl’l'ti} Unless speci?cally stated or obvious front contest, as used herein, the term "about" is understood as Within a range of normal tolerance in the art, for example Within 2 rd deviations of the mean. About can he understood as within "3%, 9%, 3%, 7%, %, "or ,0/ new "0/ 3 /o, [/03 3 /o, A H), l‘fvli, 05%, Ol%, 0.03%), or 0.0l% ofthe stated value. Unless otherwise clear ii‘orn the context, all numerical values provided herein are modified by the term "about." {69177} The disclosure will he ?nther described in the following examples, which do not limit the scope of the disclosure described in the claims.

[Annotation] CLNDW None set by CLNDW [Annotation] CLNDW ionNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW EXAMPLES Exampie i. rgeting Moieeuies Eiiid GETR (69178} As shown in Figures 2A, 28, and 2C, varieiis GiTR—targeting fusien proteins ut‘the disclosure bind to (EUR expressed an CHO eeiis as assessed by ?ow eytumetry. The GiTR antihedy, TEX—5 18v was used as a control for these studies, {@3179} The binding af?nities of the GER—targeting ies referred to herein as 313(30de (SEQ 13D NQ: 42}, thOévi ,2 (SEQ ID NO: 4'3), thGévi .3 (SEQ ID NO: 44)? 11330on .4 (SEQ ED NO: 45}, .l (SEQ ID NO: 46), th?éx/ZQ (SEQ ED NE); 47)? thOovlS (SEQ ii) NQ: 48),, thOévZ.4 (SEQ 11) NO: 49), 112C06v3 (SEQ ii) N0: 50)v thGGVSi (SEQ RD NO: 5 i), th06V3i2 (SEQ ED N0: 52), h2CO<§v33 (SEQ H} N0: 53), h2CG6v34 (SEQ ID NO: 54)? lizCOévBS (SEQ ii) ND: 55); i1ZC06V'3.6 (SEQ if} NQ: 56}, in?rm}? (SEQ ID NO: 57), 11303668 (SEQ ED NO: 58)v thOfwB? (SEQ ED NQ: 59),, itsCtth?ii’) (SEQ H} NO: 6.0), thUt‘Sv3 ii (SEQ it) NO: (Si), (1203637312 (SEQ H.) Ni): 62), i1zCO4v4,i (SEQ ID NO: 6'3), thf?iv?iil (SEQ ID NO: 64), .2 (SEQ 1D NE); 65)? ligCMMQQ (SEQ 1D NE): 66)? ligCO?wS (SEQ ED NO: 67}, hzt304v12l (SEQ 1D NO: 68), iith_}4v5.i (SEQ ED N0: 69), thUAii/Sl (SEQ ii) N0: 70),, hZCO4V’53 (SEQ it.) NO: 7'} ), thO-tv?/t (SEQ ID NO: 72), thfiéivSiS (SEQ H} NS: 731 h2€94V5f§ (SEQ ii?) Ni): 74), hZCO4VS,7 (SEQ ID NO: 75), thMVS ‘8 (SEQ ID NO: 76), thMVSQ (SEQ ED Nt): 77); thOA’ivSlG (SEQ 11) NO: 78),}12CO4VSH (SEQ Ii} N0: 79), and iizCMvSiZ (SEQ ii) Ni): 80) for human and eyrieirioigus GETR expressed on the surfaee {if CHO eeiis were detemtirted by ?ow cytometry. Ere its are shown in Figures 4A~4E and SA—SE.

Exampie 2. Targeting Meieeuies Biock the interactien Between (SITE and GITR~L SENSE} As shewn in Figures 3A, 3B,, and 3C, various GER—targeting fusion proteins {if the disclosure were state to block the interaction between GE'I‘RL arid SEER. Brie?y, in these studies, a ?ow symmetry assay using GITR expressing CHO eeiis and recombinant GI'I'RL was used to implement to assess ng eupaeity, The GH‘R dy; TEX—518,, was used as a comic} for these studies.

[Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW Example 3. Binding Al‘l‘lnities of GlTl?Targeting Molecules for Human and Cynoniolgns Gl’l'R {?lllgll The binding af?nities oftlie Gl'l‘R—targeting molecule referred to herein as bivalent lizCO‘ovB?ahlgGl or 2x lizCOdt/Ll?dgGl Po (SEQ ll} NO: 93) for human and cynomolgus GETR extracellular domain human lgGl fusion protein {Gilli—Fe) were determined by surface plasmon resonance. Brie?y ylated human and eynomolgus Gl’l‘R—Fc were captured on the chip surface and then bivalent lieCGév39—ltlgGl was injec ie at ll") concentrations (0 nM — 600 nM) at 40nl/min for l20 seconds. iation was followed for 24h seeonds. ital, hell, and KDl are reported in the table below, r ‘\.r rr‘ l \ . t \ . \ ‘ t . t S : t \ \ . . ‘ ., _. \ § t l : l , , .. .a i a. i ; ‘ , l 'u ' ‘ t \ t .- ,. ‘ i \ t ‘ t t t : i l ‘l \ \ « \ l l t {\ :‘\ : l t M .*\‘ l t ‘ \\\\\\\\\\tccccccccccc\\\\\\\\\\\\\\\\\\\\\\\\ Example 4. g of GlTRKl’argeting Molecules for Primary Human T Cells {($182} ’l‘he ability ot" an anti~Gl"l‘R. le of the disclosure, referred to herein as tetravaleiit thOti—ldg?l to primary human T cells was evaluated herein Tetravalent lizCOe-hlgiill is constiucted with two copies of the GlTR—liinding molecule of SEQ ll) NO: 93: wliieli, in turn, is constmeted with two tandem copies ofa single—domain le region (sdAb) of SEQ ll) NO: 59 fused to a human lgGl Fe domain ot" SEQ ll) NS: l. {69183} Total PBMC or purified Treg isolated by seenee"activated cell sorting were prepared from healthy human donors The cells were ted in Vitro with anti—CD3 and anti~CD28 supplemented with recombinant human lllZl The cells were incubated with iriarying concentrations of tetravalent liszlti—hlgGl and a surface ypiiig antibody cocktail. Samples were then washed and stained with a llnoi'escently—laheled an ti —hlgG secondary antibody and then ed by flow cytonieti'y, Activated CD4 T cells were identi?ed by staining with CD3, (7134, and C925. Results ofthese studies are shown in Figure 7 for activated CD4 T cells from three donors (closed symbols, solid lines} and activated Treg from two donors (open symbols; dashed lines}.

Enamels". 5. GlTR~Targeting Molecules te NF—lill Signaling {Gilltlsl} 'l‘etravalent anti—Gl'l‘R—targeting les aetivated NF—RB signaling in reporter cell lines sing Gl'l‘R "the studies described herein used two tetravalent D i [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW Gilli—targeting molecules of the disclosure The first tetrayalent Gill‘s—targeting molecule es two copies ofthe Gl'l‘?hinding fusion protein referred to herein as 2);; thtlov39 lgGl—Fe (SEQ ll) NO: 93), which, in turn, includes two copies ofthe ltthlox-r3.9 Gl’l'liwl?l) (SEQ ll) NO: 59) and the lgGl Fe polypeptide of SEQ ll) NO: l. The second tetravalent GlTR—targeting molecule includes two copies of the GlTR—hinding fusion protein is referred to herein as 2x {:06 lgGl—Fc, which, in turnv includes two copies of the {:06 Gilli—8D (SEQ ll.) N0: 22’) and the lng l he polypeptide of SEQ ll.) N0: ll EtllllSSS llEK293 cell lines containing a NF—kB-driven secreted alkaline phosphatase (SEA?) reporter gene were stably transl‘ected with human Gl'l'R (Figure 8A) or cynoniolgus monkey Gl'l‘R (Figure 88) ’l‘he cell lines were incubated with titrating doses of tetrayalent Gilli antibodies oveinight at 37°C, SEAP reporter gene expression was quantiti ed by the hydrolysis of a substrate that is measured by optical density at o??nhl Example 6. Gl'l‘R—"l‘argeting Molecules in 'l'urnur Models SiZil As shown in Figures9A~9Q treatment with a GlTR—tarf eting molecule of the.4 disclosure significantly d orzs tumor growth irrespective of day of administration, BALE/c mice were ated subcutaneously with C'l‘Zti colorectal carcinoma cells and were administered tetravalent C(loshlgG l? which es two copies of the GlTR—hinding fusion protein referred to herein as 2x Clio—lgGl Fc which; in turn, includes two copies of the Gl'l‘R~l3l) of SEQ ll) N0: 22 and the human lgGl Fe polypeptide sequence ofSEQ ll) NS: l) or Human lgG l—l3c as a control on Day 7 (Figure 9A), Day 9 (Figure 98) or Day ll e 9C), at which points the mean tumor volumes were l25, 2309 or 3 l0 mni3( alent CGé—lg?l Fc ent resulted in significant reduction in tumor growth compared to Human Fe beginning 6~8 days after administration regardless of the day of treatment {,0 < 0.05, determined Via iled, ed t—test) {@9187} As shown in Figure 10, treatment with a GlTR~targeting molecule of the disclosure produced dose~tlependent suppression of Elf/36 tumor growth, BALE/c mice were inoculated subcutaneously with CF26 ctal carcinoma cells and were administered tetravalent CGti—nilgGan which includes two copies of the GlTR~hinding lusion n referred to herein as 2X COo—nilgGl 2;: Fe, which, in turn, includes two copies of the Gl'l'R—BD of SEQ 11) NO: 22 and a niurine lgGZa sequence or eci?c a as a control on Day 9 (approximate tumor volunte 266 131113), ’l'etrayalent C(lti~ mlgGZa treatment ed in signi?cant reduction in tumor volume compared to control [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW when administered at 2.5. 0.25, 0.08, or 0.025 mgfkg (p <1 0.05}. Tetravalent COES—mlgGZ-a dosed at 0,008 mg/kg did not signi?cantly suppress C'l‘lo tumor growth. Statistical signi?cance was determined via one—way ANGVA with multiple comparisons ofthe Tetrayaleut (Illo~rnlg(}2a groups to mlgGZa. {00188} As shown in Figures l lA—l l8? treatment with a GlTR-targeting molecule of the disclosure produced dosewdependent suppression oflVlCBX tumor growth. C57BL/6 mi ee were inoculated subcutaneously with MC38 colorectal carcinoma cells and were administered tetravalent C06—nilgG2a or ecific nilgGZa as a control on Day 7 {mean tumor volume ll0—l l5 3311113). Administration of tetravalent gG2a at doses of 0.08 or above resulted in significant tumor growth reduction compared to inlgGQ-la control ing on Day l4 (p < 0.05) (Figure l lA). Tetravaient COS—hilgGZa treatment at 0025 signi?cantly reduced tumor growth compared to mlgGZa control beginning on Day 18 (p <1 0.05). 'lletrayalent C06~nilgG2a dosed at 0,008 org/"leg did not significantly suppress MESS tumor growth. Statistical significance was determined Via one—way ANOVA with multiple comparisons of the C06 groups to lgG2a. dual tumor volumes on Day 20 after MCSS inoculation are shown in Figure llB. There is a similar reduction in tumor growth at this tiniepoint in the 2,5, 0,25, and 0.08 mg/kg ent groups. e 7. impact of Fe Function on inhibition of €126 Tumor Growth {00189} BALB/c mice were inoculated subcutaneously with eras colorectal carcinoma cells and were administered tetravalent (lOo—i’nlg?ila with either wild—type Fc or N297G mutation to hlocl< binding to Fc receptors (nilgG2a—silent) on Day 9 (mean tumor volume 260 min?) Non—speci?c mlgGZa, anti—Gilli. mA‘o l—mlgGZa, and anti—Gl'l‘lit control mAhl—mlgGZasilent were used as controls. As shown in Figure lEA, although tetrayalent C06 \ as most potent with wild—type Fca both wild—type and silent formats significantly reduced tumor growth compared to control (p < 0.05). mAhl only inhi‘oited C126 growth when administered in the wild—type Fe format Statistical signi?cance was determined via y ANOVA with multiple compari sons of the treatment groups to mlgGZa, individual tumor volumes on Day 22 alter CTZS inoculation are shown in Figure lZB. The ence in tumor growth between he wild~type and silent formats ayalent {306 is not significant, while format was signi?cant: for the ability of uiAhl to suppress tumor .

Kaplan—Meier is shows that treatment with tetrayalent C06 with wild-type Fe can significantly enhance the al ofCTRo—hcaring mice (Figure lEC). A single [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW ed set by CLNDW [Annotation] CLNDW None set by CLNDW [Annotation] CLNDW MigrationNone set by CLNDW [Annotation] CLNDW Unmarked set by CLNDW administration of tetra Ialent (Zile-inlgiiiZa on Day l0. extends median sunrival to 66 days, compared to 20 days for the migG2a control group, e 8. Treatment with GITR~Targeting Molecules Results in Resistance to Re" challenge {69199} Mice that had received tetravalent lgGQa—induced CT26 rejection were resistant to re—ehallenge. BALE/c mice that had ed CTZrS tumors upon treatment with tetravalent nlng/Za were re—inocuiated with CTZé, Renea, or EMTtS marine tumor cell lines, As shown in Figure 13A, mice that have previously rejected C'T'Zfi were completely ant to tumor growth upon subsequent re~moculation of this m odei importantly, naive, agen’natehed mice demonstrated CTZS tumor growth As shown in Figure BB, Renea tumors did not grow well in mice that had previously reieeted CTR}. indeed, two of four mice were completely resistant, and one mouse had marked reduction in Renea growth compared to naive, tched controls. Renca shares T cell epitopes with C7126, suggesting that T cell—mediated immunity is induced. As shown in Figure 13C, EM’TG tumors grow well in BALE/c mice whether they previously eliminated GTE-‘16 upon C06 treatment or were naive. EMTti does not share T cell epitopes with erze.

Examine 9. Effect of ent. with Gl'TRw'l‘argeting Molecules on T cells E69191} Treatment significantiy reduced ng frequency and altered the ratio to Tammy cells within the tumor mieroenvironment. BALE/e mice were inoculated subcutaneously with C’T'Zt’i coioreetai carcinoma cells and were. administered 25 trig/leg tetravalent C(36— mlgGZa with either wild—type Fe or N297G mutation to block binding to Fc receptors (migGZa—siient} on Day 9. Non-speci?c mlgGZa was used as a control. Peripheral blood and tumors were collected and analyzed by flow try 3 days atter treatment. As shown in Figure MA, treatment with alent Ctit’isniigGZa significantly reduced the frequency of circulating Tag, con vention a1 (TIM T cells {4Tcon}, and CD8 T cells (8T) (p < 0.05). No effect was observed with the mlgGZa—silent format. As shown in Figure MB, treatment with tetravalent Ctio—inlgGZa cantly reduced the frequency of intratumorai Treg and conventional (JIM T cells (p < (Midi), hut CD8 T ceiis were not changed. No effect was observed with the n’iigGZa-siiei’it format. As shown in Figure 14C, as a consequence of the potent reduction of Tm; by tetravaient Ct‘té—migGZa, the ratios ofei‘l’eetor T cells to "ll-?g were cantly increased in the tumor (p < (105). Statistical signi?cance was determined via iled, unpaired Nest.

Example10. Effect of GITR-Targeting Molecules on T Cell Activation and eration Treatment significantly d CD8 T cell activation and proliferation.

BALB/c mice were inoculated subcutaneously with CT26 colorectal carcinoma cells and were administered 2.5 mg/kg tetravalent C06-mIgG2a with either wild-type Fc or N297G mutation to block binding to Fc ors (mIgG2a-silent) on Day 9. Non-specific mIgG2a was used as a control. Peripheral blood was analyzed by flow cytometry 12 days after treatment. As shown in Figure 15A, treatment with tetravalent C06-mIgG2a significantly induced the frequency of circulating CD8 T cells (p < 0.005), but Treg and conventional CD4 T cells were not changed. This effect was not observed with the -silent format. As shown in Figure 15B, CD8 T cells also adopted an activated, proliferating ype (CD62L- Ki67+) following treatment with tetravalent C06-mIgG2a. Statistical significance was determined via two-tailed, unpaired t-test.

Definitions of the ic embodiments of the invention as claimed herein In a first aspect, the invention relates to an isolated polypeptide that binds a glucocorticoid-induced TNFR-related protein (GITR), comprising at least one inding domain (GITR-BD), sing a complementarity determining region 1 (CDR1); a complementarity determining region 2 (CDR2); and a complementarity determining region 3 (CDR3) comprising; the amino acid sequences set forth in SEQ ID NOS: 106, 107 and 108, respectively; the amino acid ces set forth in SEQ ID NOS: 109, 110 and 111, respectively; the amino acid sequences set forth in SEQ ID NOS: 112, 113 and 114, respectively; the amino acid sequences set forth in SEQ ID NOS: 106, 115 and 116, respectively; the amino acid sequences set forth in SEQ ID NOS: 117, 118 and 119, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 121 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 149, 123 and 150, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 123 and 124, respectively; the amino acid sequences set forth in SEQ ID NOS: 125, 121 and 124, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 121 and 126, respectively; the amino acid sequences set forth in SEQ ID NOS: 112, 113 and 127, tively; the amino acid sequences set forth in SEQ ID NOS: 148, 128 and 129, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 130 and 124, respectively; the amino acid sequences set forth in SEQ ID NOS: 131, 132 and 133, tively; the amino acid sequences set forth in SEQ ID NOS: 120, 134 and 135, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 121 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 106, 136 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 106, 137 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 137 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 137 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 139 and 116, respectively; the amino acid sequences set forth in SEQ ID NOS: 106, 139 and 116, respectively; the amino acid ces set forth in SEQ ID NOS: 138, 139 and 140, respectively; the amino acid sequences set forth in SEQ ID NOS: 109, 110 and 111, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 141 and 116, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 141 and 142, respectively; the amino acid sequences set forth in SEQ ID NOS: 143, 144 and 145, respectively; the amino acid sequences set forth in SEQ ID NOS: 143, 144 and 146, respectively; or the amino acid sequences set forth in SEQ ID NOS: 143, 147 and 146, respectively.

In a second aspect, the invention relates to an isolated nucleic acid encoding the polypeptide of the first aspect.

In a third aspect, the invention relates to an in vitro host cell comprising the c acid of the second aspect.

In fourth aspect, the ion relates to a pharmaceutical composition comprising the polypeptide of the first aspect.

In a fifth aspect, the invention relates to the use of an isolated polypeptide according to the first aspect for the manufacture of a medicament for treating cancer, optionally wherein the cancer is selected from the group ting of bladder cancer, breast cancer, uterine cancer, cervical , ovarian cancer, prostate cancer, ular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney , head and neck , lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous , lymphoma, leukemia, myeloma, a, and virus-related cancer, optionally, wherein the cancer is a metastatic cancer, refractory cancer, or recurrent cancer.In a sixth aspect, the invention s to the use according to the fifth aspect in combination with an anti-PD1 or anti-PDL1 antibody, optionally wherein the antibody is an anti-PD1 antibody and the anti-PD1 antibody is BMS-936558.

Claims

What is claimed is:

1. An isolated polypeptide that binds a glucocorticoid-induced TNFR-related protein (GITR), comprising at least one GITR-binding domain (GITR-BD), comprising a complementarity determining region 1 (CDR1); a complementarity determining region 2 (CDR2); and a mentarity determining region 3 (CDR3) comprising: the amino acid sequences set forth in SEQ ID NOS: 106, 107 and 108, respectively; the amino acid sequences set forth in SEQ ID NOS: 109, 110 and 111, tively; the amino acid sequences set forth in SEQ ID NOS: 112, 113 and 114, respectively; the amino acid sequences set forth in SEQ ID NOS: 106, 115 and 116, respectively; the amino acid sequences set forth in SEQ ID NOS: 117, 118 and 119, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 121 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 149, 123 and 150, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 123 and 124, respectively; the amino acid sequences set forth in SEQ ID NOS: 125, 121 and 124, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 121 and 126, respectively; the amino acid sequences set forth in SEQ ID NOS: 112, 113 and 127, respectively; the amino acid sequences set forth in SEQ ID NOS: 148, 128 and 129, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 130 and 124, tively; the amino acid sequences set forth in SEQ ID NOS: 131, 132 and 133, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 134 and 135, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 121 and 122, tively; the amino acid ces set forth in SEQ ID NOS: 106, 136 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 106, 137 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 120, 137 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 137 and 122, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 139 and 116, respectively; the amino acid sequences set forth in SEQ ID NOS: 106, 139 and 116, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 139 and 140, respectively; the amino acid sequences set forth in SEQ ID NOS: 109, 110 and 111, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 141 and 116, respectively; the amino acid sequences set forth in SEQ ID NOS: 138, 141 and 142, respectively; the amino acid sequences set forth in SEQ ID NOS: 143, 144 and 145, respectively; the amino acid ces set forth in SEQ ID NOS: 143, 144 and 146, respectively; or the amino acid sequences set forth in SEQ ID NOS: 143, 147 and 146, respectively.

2. The isolated polypeptide of claim 1, wherein the polypeptide comprises two or more GITR-binding domains (GITR-BDs) that specifically bind GITR.

3. The isolated polypeptide of claim 2, n said two or more GITR-binding domains are operably linked via a linker polypeptide.

4. The isolated polypeptide of claim 3, wherein the polypeptide comprises at least two or at least three copies of the same GITR-BD.

5. The isolated polypeptide of any one of claims 1-4, wherein the polypeptide is tetravalent for binding GITR, optionally wherein the polypeptide comprises two copies of a fusion protein that comprises the structure: (GITR-BD)-Linker-(GITR-BD)-Linker-Hinge-Fc.

6. The isolated polypeptide of any one of claims 1-4, wherein the polypeptide is lent for binding GITR, optionally n the polypeptide comprises two copies of a fusion protein that comprises the structure (GITR-BD)-Linker-(GITR-BD)-Linker-(GITR-BD)-Linker-Hinge-Fc.

7. The isolated polypeptide of any one of claims 1-6, wherein each GITR-BD is a sdAb and comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 138; a complementarity ining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 141; and a complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 116, optionally wherein each GITR-BD comprises the amino acid sequence ed from SEQ ID NOs: 50-59 and 61-62.

8. The isolated polypeptide of claim 7, wherein the ptide ses the amino acid sequence of SEQ ID NO: 93.

9. The isolated polypeptide of any one of claims 1-8, comprising one or more additional binding domain(s).

10. The isolated polypeptide of any one of claims 1-6 and 9, wherein the D comprises a single domain antibody (sdAb).

11. The isolated polypeptide of any one of claims 1-6 and 9, wherein each GITR-BD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-80, or a sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-80, wherein the GITR-BD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-62 and/or wherein the GITR-BD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-80.

12. The ed polypeptide of any one of claims 1-11, wherein the ed ptide comprises an immunoglobulin Fc region polypeptide.

13. The isolated polypeptide of any one of claims 1-12, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-105, or an amino acid sequence that is at least 95% identical to an amino acid ce selected from the group consisting of SEQ ID NOs: 81-105.

14. The isolated polypeptide of any one of claims 9-13, wherein the one or more additional binding domain binds to a target other than GITR, optionally wherein the one or more additional binding domains binds a Tumor Necrosis Factor receptor superfamily (TNFRSF), optionally wherein the TNFRSF member is selected from the group ting of OX40, CD27, herpesvirus entry mediator (HVEM), CD40, toxin beta receptor (LTBR), ectodysplasin A2 receptor (ED2R), ectodysplasin A receptor , TweakR, B cell maturation antigen (BCMA), B cell-activating factor receptor (BAFFR), death receptor 3 (DR3), death receptor 6 (DR6), and CD137.

15. The isolated polypeptide of any one of claims 9-13, wherein the one or more additional binding domains binds a non-TNFRSF-member.

16. The isolated ptide of any one of claims 9-15, wherein the one or more additional binding domain(s) comprises an antibody or n-binding fragment thereof, ally n the antibody or antigen-binding fragment thereof is a scFv, a Fab, a single domain antibody (sdAb), a VNAR, or a VHH.

17. An isolated nucleic acid encoding the polypeptide of any one of claims 1-16.

18. An in vitro host cell comprising the nucleic acid of claim 17.

19. A pharmaceutical composition comprising the polypeptide of any one of claims 1-16.

20. Use of an isolated ptide according to any of claims 1-16 for the manufacture of a medicament for treating cancer, ally n the cancer is selected from the group consisting of bladder cancer, breast , e cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, a, and virus-related cancer, optionally, wherein the cancer is a metastatic cancer, refractory cancer, or recurrent cancer.

21. The pharmaceutical composition according to claim 19 further sing an anti-PD1 or anti-PDL1 antibody, optionally n the antibody is an D1 antibody and the anti- PD1 antibody is BMS-936558.

22. Use according to claim 20 in combination with an anti-PD1 or anti-PDL1 antibody, optionally n the antibody is an anti-PD1 antibody and the anti-PD1 antibody is BMS-

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