WO2014165573A1 - Regulating transplant rejection of donor and embryonic stem cell-derived tissues and organs - Google Patents

Abstract

The invention provides methods and cells for improved transplantation of donor transplants to subjects in need thereof.

Description

REGULATING TRANSPLANT REJECTION OF DONOR AND EMBRYONIC STEM CELL-DER!VED TISSUES AND ORGANS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No: 61/807,632, filed Aprii 02, 2013, the disclosure of which is explicitly incorporated by reference herein in its entirety.

BACKGROUND

Stem cell therapy is a rapidly advancing field where huge strides have been made in the repair of critical organs. Unfortunately , as with any other graft, stem ceil transplants have a high probability of developing immune-mediated complications ultimately resulting in rejection. While strategies such as host immune suppression or use of host-derived ceils address the issue of rejection, they are not idea! due to compromised patient health or the prohibitive cost of individualized treatment.

identification of key triggers and development of novel, broadly applicable strategies to specifically control the immune response is imperative for wide scale

implementation of cell therapies. Aianyi (membrane) aminopepiidase (ANPEP) is a ce!i surface protease expressed by monocytes, macrophages, dendritic ceils, among many others. The inventors have recently identified ANPEP as a regulator of receptor-mediated antigen upiake and presentation in dendritic ceils, an inflammatory adhesion molecule, a regulator of innate immunity, and a regulator of endocytosis, and tested whether ANPEP was involved in immune responses elicited by stem cell transplantation.

SUMMARY OF THE INVENTION

fn a first aspect the invention provides a method of limiting development of transplant rejection, comprising administering to a subject in need thereof and/or treating a donor transplant with an effective amount of an inhibitor of aianyi {membrane) aminopeptidase (ANPEP) to treat or limit development of transplant rejection. The inhibitor can prevent the expression, activity and/or function of ANPEP; any suitable ANPEP inhibitor can be used, as deemed most appropriate for an intended use. In exemplary embodiments, the inhibitor can be selected from the group consisting of anti-ANPEP antibody, anti-ANPEP aptamer, ANPEP small interfering RNA, ANPEP small internally segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, ANPEP antisense oligonucleotides and small molecule ANPEP inhibitors. In a preferred embodiment, the inhibitor Is an anti-ANPEP antibody (i.e., an antibody that binds to ANPEP).

In a second aspect the invention provides a piuripotent ceil population, wherein the population does not express functional ANPEP. Functional ANPEP can be knocked out or inhibited by a method selected from generation of knock-in null mutant ANPEP ceil population using homologous recombination, transcription activator-like effector nuc!eases{TALE:Ns), clustered regulatory interspaced short pa!indromic repeat (CRiSPR) Cas-based RNA-guided DNA endonuc!eases technology, generation of ANPEP knockout cell line using homologous recombination, TALEN or CRISPR technology, generation of knock-in point mutation of ANPEP using homologous recombination, TALEN or CRISPR technology, anti-ANPEP antibody, anti-ANPEP aptamer, ANPEP small interfering RNA, ANPEP small internally segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, ANPEP antisense oligonucleotides and small molecule ANPEP inhibitors. In some embodiments, cells, tissues or organs derived from the piuripotent ceil population that does not express functional ANPEP can be transplanted into a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosed exemplary aspects have other advantages and features which will be more readily apparent from the detailed description, the appended claims, and the accompanying figures. A brief description of the figures is below.

Figure 1 shows that CD13-nuli skin grafts survive longer. (A) Gross images of wild type (WT) and CD13-nuil skin grafts in WT recipients at days 25 (top) and 100 (bottom). (B) Skin graft measurements over time demonstrate decreased diameter until comp!ete rejection in WT grafts compared to CD13-nul! grafts. (C) H&E staining of skin graft sections demonstrate higher numbers of infiltrating ceils in WT compared to CD13-null skin grafts. (D) Quantification of immunohistochemicai staining of skin graft sections demonstrate higher leve!s of CDS staining, indicative of T ceil infiltration, in WT grafts. WTWT= WT donor tissue, WT recipient; OWT= KO donor tissue, WT recipient; WTKO= WT donor tissue, KO recipient; KOKO= KO donor tissue, KO recipient. Figure 2 shows that mast ceil presence is higher in surviving grafts. {A) Toiuidine blue staining of mast cell granules in skin at baseline. (B) Quantification of toiuidine blue staining in skin graft tissue at days 5-7 post-transplant indicates ioss of mast eel! granule staining in WT graft tissue. {C) Quantification of toiuidine blue staining in draining lymph nodes of mice receiving minor histocompatibility (MiHC) mismatch skin grafts demonstrates higher levels of mast ceils that have not degrartulated in recipients receiving CD13-nuli donor tissue.

Figure 3 shows that cultured mast cells demonstrate different reactivity. (A) Bone marrow derived mast cells demonstrate 80-90% positivity for c-kit expression after 4- 6 weeks in culture as welt as high forward and side scatier profiles by flow cytometry. (B) Co-culture of bone marrow derived mast celts show loss of forward and side scatter profile upon stimulation (left panels} as well as stimulation of proliferation {right panels) demonstrated by ioss of CFSE dye. Interestingly, WT and KO mast ceils preserved more of their original ce!i morphology and displayed !ess proliferation when stimulated by CD13-nul! dermal fibroblasts. In addition, CD13-nu!S mast cells a!so maintained more of their original eel morphology and proliferated less than WT mast ceils when stimulated with WT dermal fibroblasts. Figure 4 shows SL13 treatment is sufficient to prolong WT graft survival. (A) WT skin grafts show prolonged survival in WT gender mismatched recipients treated for two weeks with SL13. Vehicle treated controls demonstrated signs of rejection as early as day 13 post-transplant white SL13 treated recipients maintained functional grafts past 3 weeks even after discontinuation of SL13 at time of surgery

Figure 5 shows that mES transplantation recapitulates skin graft studies. (A) Gross images of teratomas generated in immune-competent hosts at four weeks shows clear growth advantage cy CD13-nuii mES as well as apparent growth advantage in CD13-nu!i hosts. {B-C} Proliferation is enhanced in CD13-nuii teratomas as seen in quantification of Ki67 expression (B) as well as BrDU incorporation (C). (D) This growth advantage is not seen in immune-compromised nude mice. (E) There is less CD3+ T celt infiltration in CD13-nu!i teratomas, especia!iy in those teratomas generated in CD13-nuil hosts. (F) WT teratomas also demonstrate higher numbers (15% in WT versus 3.29% in CD13 KO) of CD1 1b+CD11 c+ dendritic cells and (G) F4/80+ macrophages by flow cytometry. (H) Toiuidine blue staining and

quantification of mast ceils within teratomas indicate higher levels in CD13-nu!i teratomas. Figure 6 shows a schematic for iHC mismatch skin graft. Donor male skin is harvested from the dorsal side of the ear and transplanted onto the shaved exposed backs of female recipient mice.

Figure 7 shows (A) Flow cytometry for immune ceS! populations in post- transplant Day 5 spleen.

Figure 8 shows that mixed lymphocyte reactions with WT and CD13-nuii MiHC mismatched spienocytes show that CD13-nufS spienocytes demonstrated reduced proliferation upon stimulation compared to WT spienocytes.

Figure 9 shows {A) a SL13 treatment Scheme. {B} Check for SL13 antibody in serum of treated and control mice

Figure 10 shows CD 13-nui! mES cell characterization. (A) CD13-nuil mESCs express the p!uripotency marker Oct4. |B) WT mES generate teratomas containing ee!i types of ail! three germ iayers. fC) CD13-nuii teratomas also display ceil types of all three germ iayers. (D) None of the teratomas display any significant apoptosis. (E) CD13-nui! teratomas contain more CD31 and aSMA positive staining cells, indicating higher levels of vascularity.

DETAILED DESCRIPTION OF THE INVENTION

Methods well known to those skilled in the art can be used to construct expression vectors and recombinant bacteria! ceils according to this invention. These methods include in viiiO recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and PCR techniques. See, for example, techniques as described in Maniatis et a ., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York; Ausubei ef a/., 1989, CURRENT

PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WiSey intersciersce. New York, and PCR Protocols: A Guide to Methods and Applications {Innis er a/., 1990, Academic Press, San Diego, CA).

Ail publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes. Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a", "an", and "the" include plural referents unless the context cleariy dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.

In a first aspect the invention provides a method of limiting development of transplant rejection, comprising administering to a subject in need of a transpiant and/or treating a donor transplant with an effective amount of an inhibitor of alanyi (membrane) a inopeptidase {ANPEP} to limit development of transpiant rejection in the subject.

The inventors have surprisingly discovered that the methods of the invention allow the ability of donor transplant to engraft and survive with little to no immune suppression required of the recipient. Furthermore, the ceils, tissues and organs of the invention in which ANPEP is inhibited or that do not express functional ANPEP can be transplanted universally without minor histocompatibility complex (MiHC) matching, human leukocyte antigen (HLA) matching or immune suppression. Thus, the inhibition of ANPEP expression, activity or function in a donor transplant or cells, tissues or organs to be transplanted can allow more successful transplantation by mitigating or eliminating the immune reactions triggered by transplantation.

For uses in this application, CD13 and ANPEP will be used synonymously. The approved HUGO Gene Nomenclature Committee (HGNC) Symbol for human CD13 is ANPEP, and the approved HGNC name is alanyi (membrane) aminopeptidase. Previous names include: CD13, PEPN; APN; LAP1 ; PI 50; GP150. Synonyms include: aminopeptidase , aminopeptidase N, microsomal aminopeptidase. Human ANPEP has the following identifiers: UniProtKB/Swiss-Prot, PI 5144 {SEQ ID NO: 01 ); NCBI Reference Sequence (mRNA), N _001 150.2 (SEQ ID NO: 02); and NCBI Reference Sequence (protein), IMP_0G1141.2 (SEQ ID NO: 03) and point mutations {SEQ ID NOs: 04-06). CD13 is a type II zinc-dependent metailopeptidase that is found on the surface of ail myeloid cells in addition to pericytes, activated endothelial cells, and subsets of organ-specific epithelial ceils. St is a multifunctional protein with both enzyme-dependent and independent functions that contribute to adhesion, ceil migration, angiogenesis, inflammatory trafficking, adhesion, antigen presentation, and endocytosis.

Table 1 : ANPEP Sequences

Human Aminope idase N (EC=3.4.11.2} ANPEP

>sp!P151 4!AMPM_HUMAN aminopeptidase N (SEQ ID HO: 01}

S ATTL DQS KAWf; R YR.L PU L P IS S YR V T LRP Y^"L T PNISRGL YV G S 3 TVE T GREAT DV I

X X H S KKLMYT LS Q GH R¥V LRGVGG S O ? F I> I D TE L VE P TE Y L S LKS S LVK DS Q ΥΞΜ D S EFEGELASDLAGFY^"RSEYMEGKVP,ΚΛ ' TTQ OAAEAR SFPCFDEPAMKAEENITLIHP KBLTALSK LPEGFSTPLPEBPKSKVTEΕΉΤTPE STY'LLAFI SEFDYVEKQASNGVLI RIw£EFSAIAAGHGDYAEKrr&PT^

TYRENSLLEDPLSSSSSNKERWTVTAHELAHQSFG!iLVTTEWS¾-5DLSLi;SGEASYVEYL GADYAEPTKilLEDLMVLifr^RV AVDAEASSHPLSTPASEINTPAQISELFDAISYS G ASVLF^LSSFLSEDVFEOGLASYLHTFAYQKTIYL L DHLQSA KRSIQLFTTVRDI KRSTLQ GEPVITVBTSTGTLSOEHFLLBPDS TRPSEFilYVWIVPITSIRDGRQOODY

IIS^'DAFHLASAHKVPVTEALiJ TLELTEEEQYMPSEAALSSLSYFRLMFDESEVYGP KN YL KQV PLFIKiT NNTiiK EXPE^^

SiSPNKNPIHPNLRSTVYCKAIAQGGEEEWDFAWEQFRSATL¾EAB LRAALAGS SL I LNRYLSYTLNPDLIRKQIATSTIISITIJ VI QGLVWDFVQS KK LFKBYGGGSFSESN LIQSVTRRFSTEYELQQLEQEKKDHEETGFGSGTRALEQALEKTKA IK VKEKKEYVLQ WETENSE

sapl&ns alanyl {membranes amlnopep ldase {ANPEP) , mRNA NCBI Reference Sequence: NM_001150.2 <SEQ ID NO: 02)

gggacggcgg cggcgcagct cggaacccgc cagggtccag ggtccaggtt ccagcgcccg gcggcccagg caccccccga gcccagctcc acacaccgtt cctggatct ctctccecag gcggagcgtg cc ctgccca gtccagtgac cttcgcctgt tggagccctg gttaattttt gcccagtctg cctgttgtgg gg tcctcc ctttggggat ataagcccgg cctggggctg ctccgttctc tgcctggcct gaggctccct gagccgcctc cccaccatca ccatggccaa gggcttctat atttccaagt ccctgggcat cctggggatc ct ctgggcg tggcagccgt gtgcacaatc atcgcactgt cagtggtgta ctcccaggag aagaacaaga acgccaacag ctcccc gtg gc tccacca ccccgtccgc ctcagccacc accaaccccg cctcggccac caccttqgac caaagtaaaq cgtggaatcg ttaccgcctc c caacacgc tgaaacccga ttcctaccgg gtgacgctga gaccgtacct cacccccaat gacaggggcc tgtacgtttt taagggctcc agcaccgtcc gtttcacctg caaggaggc.c actgacgtca tcatcatcca cagcaagaag ctcaactaca ccctcagcca ggggcacagg gtggtcctgc gtggtgtggg aggctcecag ccccccgaca ttgacaagac tgagctggtg gagcccaccg agtacctggt ggtgcacctc aagggctccc tggtgaagga cagccagtat gagatggaea gcgagttcga gggggagttg gcagatgacc tggcgggctt ctaccgcagc gagtacatgg agggcaatgt cagaaaggtg gtggccacta cacagatgca ggctgcagat gcccgqaagt ccttcccatg cttcgatgag ccggccatga aggccgagtt caacatcacg cttatccac ccaaggacct gacagc ctg tc aacatg ttcccaaagq tcccagcacc ccacttccag aagaccccaa ctggaatgtc actgagttcc acaccacgcc caagatgtcc acgtacttgc tggccttcat tgtcagtgag ttcgactacg tggagaagca ggcatccaat ggtgtcttga tccggatctg ggcccggccc agtgccattg cggcgggcca cggcgattat gccctqaacg tgacgggccc catccttaac ttctttgctg gtcattatga cacaccctac ceaetcccaa aatcagacca gattggcctg ccagacttca acgccggcgc catggagaac tqgggactqg tgacctaccg ggagaactcc ctgctgttcg accccctgtc ctcctccagc agcaacaagg agcgggtggt cactgtgatt gctcatqagc tggcccacca gtggtt ggg aacctggtga ccatagagtg gtggaatgac ctgtggctga acgagggctt cgcctcctac gtggagt cc tgggtgctga ctatgcggag cccacctgga acttgaaaga cctcatggtg ctgaatgatg tgtaccgcgt gatggcagtg gatgcactgg cctcctccca cccgctgtcc acacccgcct cggagatcaa cacgccggcc cagatcagtg agctgtttga cgccatctcc tacagcaagg gcgcctcagt cctcaggatg ctctcca ct tcctgtccga ggacgtattc aagcagggcc tggcgtccta cctccacacc tttgcctacc agaacaccat ctacctgaac ctgtgggacc acctgcagga ggctgtqaac aaccggtcca tccaactccc caccaccgtg cgggacatca tgaaccgctg gaccctgcag atgggcttcc cggtcatcac ggtggatacc agcacgggga ccctttccca ggagcacttc ctccttgacc ccgattccaa tgttacccgc ccctcagaat tcaactacgt gtggattgtg cccatc.acat ccatcagaga tggcagacag cagcaggact actggctgat agatgtaaga gcccagaacg atctcttcag cacatcaggc aatgagtggg tcctgctgaa cctcaatgtg acgggctatt accggqtgaa ctacgacgaa gagaactgga ggaagattca gactcagctg cagagagace actcggccat ccctgtcatc aatcgggcac agatcattaa tgacgccttc aacctggcca gtgcccataa ggtccctgtc actctqgcgc tqaacaacac cctcttcctg attgaagaga gacagtacat gccctg gag gccgccctga gcagcctgag ctacttcaag ctcatgtttg accgctccga ggtctatggc cccatgaaga act cctgaa gaagcaggtc acacccctct tcattcactt cagaaataat accaacaact ggagggagat cccagaaaac ctgatggacc agtacagcga ggttaatgcc atcagcaccg cctgctccaa cggagt.tcca gagtgtgagg agatggtctc tggccttttc aagcagtgga tggagaaccc caataataac ccgatccacc ccaacctgcg gt.ccac gt.c tactctcaacg ctatcg cca gggcggggag gaggagtggg acttcgcctg ggagcagttc cciaa-atgcca cactggtcaa tgaggctgac aagctccggg cagcc tggc ctgcagcaaa gagttgtgga tcctgaacag gtacctgagc tacaccctga ac cggactt a tccggaag caggacgc a cctctaccat catcag att accaacaacg tcattgggca aggtctggtc tgggactttg tccagagcaa. ctggaagaag ctttttaacg attatgqtgg tgqctcgttc tc ttctcca acetcatcca ggcagtgaca cgacgattct ccaccgagta tgagctgcag cagctggage. agttcaagaa ggacaacgag gaaacaggct tcggctcagg cacccgggcc ctggagcaag ccctggagaa. gacgaaagcc aac tcaagt gggtgaagga gaacaaggag gtggtgctcc agtggttcac agaaaacagc aaatagtccc cagcc ttga agtcacccgg cccccatgca aggtgcccac atgt ccat cccagcggct ggtgcaqggc Ct.CC-i;1.ccfc ggagcccgag geaccagtcit cctcccctca aggacaaagt ctcc¾gccca cgttc.tctct gcctgtgagc cagtctagtt cctqat.gacc caggctgcct gagca ctcc cagcccctgc ccctcatgcc aaccccgccc taggcetggc atggcacctg tcgcec g g ccctggggct gatctcaggg aagc cagct ccaggg cag atgagcagaa gctct gatg gaca tgaac ggccttgetg ggggccgccc tgtaccctct ttcacctttc taaaga ctaaatct.ga ggaatcaaca gqgcagcaqa tctqtatatt tttttctaag actaaaatgta aataaaggat ttctagat.ga aaaaaaaaaa aaaaaaaaaa aai-:a,saaaaa aaaaaaaaaa aaaaaaaaaa aaa aaaaa amiisopeptidase N precursor [Homo sapiens}

MCEI Reference Sequence; NP_001141.2 {SEQ ID NO: 03)

RKGFYISKS LGILGILLGV AAYCTIIALS WYSQEXNKH ANSSFVASTT FSASATTNPA SATTLDQSKA WNRYRLPKTL KFDSYRVTLR PYLTFKLRGL YVFEGSSTVR FTCKEAT^'CVI IIHSKELNYT LSQGHR LR GvGGSQPFDX D TELVEF E YLWfiLKGSL V I>S¾YE¾D3 EFEGELABBL AGFYP.SEYME SHVRKWATT QMQA DARES EFCFDEPAMK ASFMITLTHP KDLTALSN L PEGPSTPLPE BFKWNVTEFH TTPKMSTYLL AFIvSEFDYV EKQASEGYLI RIWAEFSAIA AGHGDYALNV TGPILNFFAG HYB PYPLFK SBOIGLPDFN AGAMENKGLV TYREESLLFD PLSSSSSKEE R TvIAHEL AHQKFGELYT IEWWNBLKLN EGFASYVEYL GABYAEPT8K LKUWVLKW YRVMAV^'DALA. SSKPLSTFAS EXETFAQTSE LFL-ATS SKG ASVLRMLSSF LSEDVFKQGL ASYLHTFAYO. HTIYLNLWBH LQEAVEERSI QLPTTVRBIM NKS5TLQ GFF VITVDTSTGT LSQSHFLLDF S VTRPSEF NYVWIVPITS lEBGRQQ-QBY LIBVRAQEC LFSTSGNEKV LLKLEVTGYY RVNYDEENSR EIOTQLQRDK SAIPVXNRAQ IINDAFHLAS AHKVPV LAL NKTLFLIEER QYMPivEAALS SLSYFKLMFD RSEVYGP KK Y^"LEEQV LF IHFEEETEIS REIPEKLMBQ YSEVBAIS7& CSEGVPECEE MVSGLFEQSM EEFNNEPIHF NLRSTVYCHA IAQGGEEEWD FAWEQFREAT LVEEADELEA ALACSKELWI LNRYLSYTLK PDLIRKQLAT STIISITKKV IGOGLVWDFV QSNWKKLFND YGGGSFSFSK LIQAVTRRFS TEYELQOLEQ FKKDNEETGF GSGTBALEQA LEKTKA I VEEEEEWLQ WFTENSE

AMP _HUMAN Aminopeptidase N enzymatically inactive mutant H392A (SEQ ID NO; 04}

MA GFYISKSLGILGILLGVAAVCTIIALSvVYSQEKEEEAS^

SATTLDQSEASKRYELFNTLEPBSYRvTLEFYLTPiiBRGLYvFEGSSTvRFTCREATBVI 11H3EELKYTLS Q GHRWLP.GVGGSQ;PFBIDKTELVEFTEYLWSLS GSLVKD3 QY'E DS SFEGELABDLAGFYRSEYKEGE^/EEWAT Q OAAIfAEESFFCFBEFAMEAEFKITLIHF DLT¾LSKMLPKGPSTPiPEDPKWNV EFHTT KMSTYL-J-FIVSEFDYVEKQ¾SHGVLI RIWAP.PSAIASGHGDYAL-^/TGPII^FFAGHYDTPYPLP SD8IGIJiiFNAGAIffiOTGLy TYEEKSLLFBFLSS3SSNKERWTVIAHELAAOSFGELV IEWWiBLSLEEGFASYVEYL GADYAEFT NLEDLMVLEBvYRVMAVBALASSHPLSTFAGEINTFAQISELFtaiSYSKG ASVLF^L3SFLSEDVFEOGLA3YLHTFAYQMTIYLEL BKLQEA KRSIQLFTTVRDI E TLQMGFFYTTVDTSTGTLSOEHFELDFBSNVTEFSEFNYV^IVFT SIRDGRQQOBY WLIDVRAQNBI>FSTSGBEIP/LL;IJ^

11isBAFKLASAHKVFVTLALKE LFLIEE RQY'KFSEAALS3;L3YFKLRiF ItRSEVYGP EU YLKEQvTFLFIHFE iTEE^EEIFEEL BOYSS AISTACSNGVFSCEEMVSGLFKOiv EEPNKNFIHPNLRSTVY'CKAIA.QG GEEEWDF_¾i'5EQFE!i ATLVEEADKL RAAL CSKEL I LERYLSYTLiiPLLIEEQDATSTIISITHEVIGQGLV^DFVQSESiiKKLFEDYGGGSFSFSN LIQSVTRRFS EYELQQLEQFKKBHEETGFGSGTRALECJALEKTEAEI VEEEEEVVLQ WFTEKSE AMPN_HUMAN Aiainopeptidase N enzymatically inactive mutant H388A

{SEQ ID NO: 05)

MAE FY^'13KSL ILGILL VAAVC 11 LSWYSQSKN NAN3SpVASTTPSASATT FA

SATTLE¾S A 5RYRL?i¾TL PBSYRvTLR?YLTPNBRGLYV KGS3T¥RFTCKEaTBVI

IIHSK LNYTLSQGSR LRGVGGSQ PDI ETELYEPTEYLVVHLKGSLV DSQYEMDS

EFEGELADBLAGFYESEYMEG EKWATTQMQAADAEKSFPCFBEFAIiKAEFN^

KDLTALSNMLPE FSTP'LP¾DF^¾^

RIWAR.?3AXSAGfiGDY'ALNVTGPILNFFAGHY'DTFY 'LPKSDQIGLPDFUAGAMENS?GLV TYRENSLLFDPLSSSSSNKERWTVTAAELAHQSEG!iLVTTEWSSiDLSLiiEGFAGYVEYL GADYAEFTKiJLEDLMVLiTDVYRVI4AVBALASSHFLSTPASEI TPA.QISΞLFDAIS^rSKG AS LR¾LSSFLSEDVFK¾GLASYLHTFAYQNTXYLif^

NR¥TLQMGFPVITVDTSTGTLSQEHFLLDPDS£J^RPSEFSY TVPITSIRDGRQQQDY LIBVRAQfΐBLF;T3GiJE VLLf;LI3VTGYYRVKYDSEMWR.KI¾TQLORDHS I P IUFA

IIKIAFJLASAHEVPVTLALIJKTLFLISERQYMPWEAALGSLSYF L FBRSEVYGPM K YL EQVTPLFIKFRNNTiiKW EXPE L^

E!iFNN PIHFNLRSTVYCNAIAOGGEEEKBFAKEQFEiiATLviiEADKLRAALACSKEL I LKR^"LSY'TLNPDLIEKQIA.TS 1131TN VIGQ L¾DFVQS KK LFNBYGGGSFSFSK LIQAVTRRFSTEYELQQLEQFSEBIJEETGFGSGTRALEgALEKTEAKI EEKKEWLg WFTSMS

AMPN_HD S_ Aiainopeptidase U non-phosphoryla able mutant Y6F {SEQ ID NOt OS)

MA GFFIS SLGTLGILLGVAJ¾VCTI ALS^^SQEir3¾NAKSSF¾STTPSASATTiiFA SATTLBC-SΚΑΕΉΐΕΥRLPJJ L PI>SYP.VTLRPYLTFNIR.GLYVFKGSSTVRFTGEEATI' X^' IIHSRKL YTLGQGHEVVLRGVGGSQPPDIBKTELVEFTEYLVVHLKGS ^EDSQYE BS EFEGELADBLAGFYRSEYMEGKVR ATTQ QAABAR 3FPCFDEPA KAEFKITLIHF KDLTAL3MMLP GF3XPLPEDPfTiKVTEFHTTPKM3TYLLAFIVSEFDYVEKCASNGVLI EI ARPSAIAAGHGDYALNvTGPILNFFAGHYi:TPYPLPKSDQIGLPDFiiAGA E.iJKGL

Yi^KSLLFDPLSSSSS i^VVTyiHE-J-H^ F NL TIEiTOIDIJiLIffiGFASYVEYL

BY"AEPTSS LKDL VLNDVYPVM VDAIiASSHPLSTFASEIKTFAQXSELEDAXSYS G ASVLR LSSFLSEBVF QGLASYLHTFAYQNTXYLNLSDHLQEAVNNP.SIQLFTTYEDXM KR TLQ GFPVITVIfTSTGTLGQEHFLLIfPBSKVTRPSEFNYVWIVPITSIRBGRQQQBY WLIDVFAQ!iBLFSTSGSE LL!iLNVTGYYRVNYDEESWR TQTQLQRDHSAIPvT^

II.ilDAE¾LASAHEVPVTLALNNTLFLIEERQYMPSE.AA,LSSLSYFELMFDRSEVYGPMKIJ YL KQVTPLFIHFRSUTNNSREIFENLMDOYSEVKSISTAGSiiGVPEGEE VSGLFEG¾a ENΡίΙΝϊίPIHFULRSTVYCMAIAOGGEESWDFAv E FRNATLVNEADKLEAALACSEELsvI LiSRYLSYTLMPBLIREQBATSTIISITK IGOGL BFVQSIJWEELFKDYGGGSFSFSIi LIQAVTREFSTEYELOQLEQFEEBNEETGFGSGTRALEQALEET ANTKWVEENKEWLQ

ft'FTEUS

Mus mwsculus alanyl {membrane} aminopeptidase {AlfPEF} , mRN

MCBI Reference Sequence; HM_008486,2 (SEQ ID HO: 07)

ggcagtgggg ctccaccccc tgtgaqgata taagctggcc ccggggctgc tgttctttcc tcttggcctg agctattccg agctccctgt ccaccqgcat catggccaag gggttctaca tttccaagac cctgggcatc ttgggcatcc tgttgggtgt ggcagctgtg tgtaccatca tagctctgtc ggtggtctac gctcaggaga agaataggaa tgcagagaac tctgccacag cccccacgct cccgggcagc acctcagcca ccaccgcaac caccacccct gctgtagatg aaagcaagcc ttggaaccag tatcgcttgc ctaagactct tatacctgac tcctaccggg tgatcctgag accctacctc acccc aaca atcagggcct gtacatcttc caaggcaaca gtactgttcg ctttacctgc aaccagacca cggatgtcat tatcatccac agcaaaaagc tcaactacac cctcaaagga aaccacaggg tggtgttgcg aaccctggac ggcactccgg cacctaacat tgacaaaacg gaactggtag agcgt ctga gtacctggtg gtgcacctgc aggggtccct ggtagagggc cgtcaqtacg agatggacag ccagttccag ggggaactgg ctgatgacct gg tggctt taccgcagcq agtacatgga aggagacgtc aagaaagtgg tggctacaac gcagatgcag gctgctgatg ctcggaaatc ctttccatgt tttgatgagc cagccatgaa ggccatgttc aacatcacac tcatctaccc caacaacctc atagctctgt ctaatatgct tcccaaagag tccaagccct atccggaaga cccttcctgc accatgactg agttccactc caccectaag atgtccacat acctgctggc ctaeategtg aqcgagttca aaaatataag ctccgtctca gccaatggtg tccagattgg aatctgggct cggcccagtg ccattgatga gggccagggt gattaegcac tgaacgttac aggccccatc ctaaatttct ttgcccaaca ttataataca tc taccct taccaaagtc tgaccagatt gccctgcctg acttcaacgc tggagccatg gagaaetggg gtctggtgac ctaccgtgag agctccctgg tctttgactc tcagtcctcc tccattagca acaaggagcg ggtggtcact gtgattgctc acgagctggc ccatcagtgg tttggcaacc tggtgactgt ggcttggtgg aatgatctgt ggctgaacga gggctttgcc tcctacgtgg aatatctggg tgctgactat gcagagccta cctggaatct gaaagacctc atggtactga atgatgtgta ccgtgtgatg gccgtggatg cccttgcctc ctcccaccca ctgtccagtc ctgctgacga gatcaaaaca ccagaccaga tcatggagct gtttgacagc atcacctaca gcaagggagc ctcagtcatc aggatgctgt ccagtttcct gacagaqqac ctgtttaaga agggcctttc at ttatct cacacct cc agtactegaa caccgtttat ctggacctgt gggaacacct gcaaaaggcc gtgaaccaac agacagctgt ccaacccccg gccacggtgc gcactatcat ggaccgctgg attctacaga tgggctttcc cgttatcact gtgaacacca atacaqgaga aatctcccag aaacacttcc tcctggattc caagtccaac gttacccgcc cctccgagtt taattacatc tggatcgcgc ccattccatt tctcaaaagt ggacaggagg atcactactg gctggatgtc gagaaaaacc agagtgeaaa gttccagaca tcctccaatg aatggatctt actgaacatt aacgtaaccg gctactacct ggttaactat gatgagaaca actggaagaa gcttcagaat cagctgcaaa cagacctttc tgttatccct gtcatcaacc gagcacagat tatccacgac tccttcaacc tggccagtgc taaaatgata cccatcaccc tggcgctgga caacaccctc ttcctggtca aagagqcgga gtacatgccc tggcaggctg ccctgagcag cctcaactac ttcacactca tgttcgaccg ctcggaggtc tacggcccca tgaagaggta tctgaagaag caagttacgc ccctcttctt ctacttccaa aatagaacca acaactgggt caaccgtcct ccaacgctga tggaqcagta caatgaaatt aacqccatca gcaccgcctg ttccagtggt ctcaaagagt qtagggacct ggtcgttgag ctctatagtc agtggatgaa aaac .ctaat aataacacga tccaccccaa ccttcggtct actgtctact gcaatgccat tgctttcggt ggcgaagaag agtggaactt tgcttgggaa cagttccgga atgcaactct ggtgaacgaa gcggacaaac tccggtcagc cttggcctgt ageaaagatg tgtggatttt gaacaggtac ctgagttaca ctctgaaccc ggactacatc cggaagcagg acaccacctc caccatcatc agcattgcca qcaacgtqgc tgggcaccct ctggtttggg actttgtccg aagcaactgg aagaaactgt ttgagaatta cggtggagga tctttctcct ttgccaatct catccaggga gtgacccggc gcttctcctc tgagttcgag ctgcagcagc tggagcagtt taaagcggat aactcagcca caggctttgg caccggcact cgggctctgg agcaagccct ggagaagaeg agagecaaca tcgactgggt gaaggagaac aaagatgcgg tattcaagtg gttcacagag aacagcagtt agtt ctggt tctqagaacc acttgtccca gtatgacacc tcttact te tcagcagcct gtgcagggtc tctgtcctca gagctccaga caccagcatc ctaetctcaa ggatgaagtc tccagcctgt ggagccagcc tagctcctaa ctgtcaggct gacggacacc tcccaggtct tgcaccctca tgccaactct gccccaggtc caggcctctg gggctgatct cagggaagee cagctctgaa gctagattta ctgqacaaag ggcagcctgg aaaqagactc ectgaatget ttactatccc tgccccctac ccccacccct accccccacg agatccagaa ccaaagaatc aacagggcac aagatctata tatattttta agagaaaatg taaataaaga atttetaaaa tgaa aaaaa aaaa a a

amlnopeptidase N IMus m scul s]

MCBI Reference Sequence; NP_032512,2 (SEQ ID HO: 08)

MAKGFYI KT LGILGTLLG ¾&TCTIIftLS WYSQE KSN AEKSATAFTL FGS SATTAT

TTPAVDESKP KNQYRLPKTL IPDSY VILR FYLTPNNQGL YIFQGUSTVE F CKQTTDVI

IIHSEELNYT LEGNHRWLR TLDGTPAPNI DKTELVSRTE YLWHLQGSL YEGRQYEMBS

QFQGELAPDL AGFYRSEYME DV KWA QMQA D R 3 FPCFDEPA K AMFMITLIYP

KELISLSKKL FKESEFYPED PSC MTEFHS TPKMSTYLLS YIVSEF ISIS SVSANGVQIG

IKAEPSAIDE GQGDYAiEVT GPILISFFAQH YNTSYPLPKS DQIALPDFBA GASEHWGLYT

YRESSLVFDS QSS3ISNKER WTVIAEELA HQWFGIJLVTV A WNDL LIJE GFASYVEYLG

ABYASPTWKL EDLMVLUPvY KVMAvBAEAS SHPLSSPADE TKTPDQIMEL FBSTTYSEGA

3VIRKLS3FL TEDLFEKGLS SYLETYQYSIJ TVYLDLWEHL QKAVNQQTAV QPPATVRTIM

DP. ILQ GFF VITVETNTGE ISQKHFLLBS KS VTRPSEF MYIKTAPIFF LESGQEΙΉY5*J

LBvEEKySAK FQTSSBEWIL LiJ IKVTGYYL VNY&EBBSfKK L¾K¾LQTDLS VIPVilTEAQI

BHDSFNLASA KMIPITLALD iSTLFLVKEAE Y P QAALS3 LIJYFTLMFBE SEVYGPMKRY

L KQVTPLFF YFQKRTSmV KRPPTLMEQY ITSINAI3TAC SSGLEECRDL WELY3Q M

NPffffEITIHPff LRSTVYCEIAI ¾FGGEEE¾KF AWSQFRIIATL HEABKLRSA LACSEDVWIL MRYLSYTLiSP DYIRKQBTTS Tl lS I&SfJVa GHPLYWD VR SEWKKLFEEY GGGSFSFAML

IQGVTRRFSS EFSLOQLSQF KADNSATGFG '1' I E TR3NIDWV ESK DSVFKW

The inhibitor can prevent the expression, activity and/or function of ANPEP; any suitable ANPE inhibitor can be used, as deemed most appropriate for an intended use. In exemplary embodiments, the inhibitor can be selected from the group consisting of anti-ANPEP antibody, anti-ANPEP aptamer, ANPEP smaii interfering RNA, ANPEP small internally segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, ANPEP antisense oligonucleotides and small mo!ecu!e ANPEP inhibitors, in a preferred embodiment, the inhibitor is an anti-ANPEP antibody (i.e., an antibody that binds to ANPEP).

As used herein, the term "subject" or "patient" is meant to include any subject for which a donor transplant or donor or treatment may be required. "Subjects" or "patients" can comprise both humans and non-humans and includes, but is not limited to, humans, monkeys, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, insects, horses, chickens, and so on. Most preferably, the subject is human. In some embodiments, the subject or patient may require a graft or transplant due to a congenitaf defect (e.g., congenita! anomaly of the heart, limbs or kidneys), traumatic injury, acquired allergic reactions or disease sequelae (e.g., chronic kidney disease can be a sequel of diabetes), in an exemplary embodiment, a patient born with a congenital heart defect can receive a new heart or heart tissue with no defect created with the piuripotent ceils that lack ANPEP expression, in another embodiment, tissues such as skin, muscle or nerve can be created and stored in hospitals for the immediate transplant into trauma patients. In yet another embodiment, therapeutic cells that produce insulin or mast cell stabilizers can be created from piuripotent cells Sacking ANPEP expression and transplanted into diabetes patients or patients suffering from allergic airway disease and asthma. In embodiments where the immune system should be enhanced instead of suppressed, such as in cancer patients, CD13 inhibition would not be used; rather, CD13 function will be enhanced to exert the corresponding effects on immunity.

Transplant rejection occurs when transplanted tissue is rejected by the recipient's immune system, which destroys the transplanted tissue. Transplant rejection can be iessened by determining the molecular similarity between donor and recipient and by use of immunosuppressant drugs before and after the transplant procedure, immune cells from the subject receiving the donor transplant or donor graft recognize the donor transplant or donor graft as "foreign". The recipient's immune cells then attack the transplanted donor transplant or donor graft which can result in transplant rejection . In addition, residual immune celis in the donor transplant or donor graft can mobilize to a recipient's peripheral lymphoid organs and initiate graft versus host disease, attacking host cells and causing extensive damage. The methods and compositions of the invention can prevent transplant rejection by mitigating or eliminating the immune reactions of the recipient to the donor transplant or donor graft.

As used herein, "treating transplant rejection" or "limiting development of transplant rejection" means accomplishing one or more of the following: 1 ) reducing the severity of an immune response to a donor transplant or donor graft; 2) inhibiting or preventing development of an immune response at the donor transplant or graft location; 3) inhibiting or preventing the worsening of an immune response at the donor transplant or graft location; or 4} inhibiting or preventing the development of immune-mediated com lications ultimately resulting in rejection of the donor transplant or graft.

In an embodiment, the subject receiving a donor transplant is treated with an ANPEP inhibitor before the transplant procedure occurs. The inhibiior can be administered to the subject one day, two days, three days, four days five days, six days, one week, two weeks, three weeks, four week or more before the transplant procedure; and continued to be administered for one week, two weeks, three weeks, one month, two months, three months or more after the transplant procedure as necessary in order to prevent transplant rejection. I another embodiment, the donor transplant is treated with an ANPEP in ibitor for the transplant procedure. The inhibitor can be administered to the donor transplant one day, two days, three days, four days five days, six days, one week, two weeks, three weeks, four week or more before the transplant procedure; and continued to be administered for one week, two weeks, three weeks, one month, two months, three months or more after the transplant procedure as necessary in order to prevent transplant rejection. In some embodiments, both the subject receiving the donor transplant and the donor transplant are treated with an ANPEP inhibitor. In such cases, the inhibitor can be administered to the subject and donor transplant one day, two days, three days, four days five days, six days, one week, two weeks, three weeks, four week or more before the transplant procedure; and continued to be administered for one week, two weeks, three weeks, one month, two months, three months or more after the transplant procedure as necessary in order to prevent transplant rejection. As used herein, the term "donor transplant" or "donor graft" refers to a population of ceils, or a tissue or an organ that is to be moved from one body to another or from a donor site to another location on the subject's own body, for the purpose of replacing the recipient's damaged or absent tissue or organ, in some embodiments, the donor transplant can be re-grown from the patient's own ceils (e.g., plurspotent ceils or stem ceils, or cells extracted from the failing organs). In other embodiments, the donor iranspiant or graft can be grown from a pfuripotent ceil population lacking ANPEP, as described herein.

The donor transplant or graft may be a ceil, tissue, or organ , as is suitable for an intended use. Exemplary donor transplants or grafts can be selected from, but are not limited to: skin ceils, beta cells (i.e., cells in the pancreas located in the islets of Langerhans), cardiac ceils, brain cells, kidney cells, iiver ceils, ceils of the digestive tract and accessory digestive organs, salivary gland cells, adrenal gland ceils, prostate ceils, lung cells, pancreatic cells, bone ceils, immune cells, hematopoietic ceils, vascular cells, cells of the eye, connective tissue cells, musculoskeletal ceils, bone tissue, musculoskeletal tissue, cornea tissue, skin tissue, heart valves, blood vessels, immune ceils, connective tissue, Sung tissue, skin, a cornea, a kidney, a iiver, a Sung, a pancreas, a heart, and intestine, in preferred embodiments, the donor graft is comprised of skin ceils, skin tissue or beta-cells.

The cell, tissue and/or organ to be transplanted can be syngeneic, allogenic or xenogenic to the subject receiving the transplant. As used herein, the term

"syngenic" or "syngeneic" refers to ceils, tissues or organs that are genetically identical or are derived from a genetically identical source to the transplant recipient {e.g., an Identical twin), especially with respect to antigens or immunological reactions. Such ceils, tissues or organs are called isografts. As used herein, the term "allogenic" or "allogeneic" refers to cells, tissues or organs that are not genetically identical or are derived from a non-geneticaliy identical source to the transplant recipient (e.g., a non-related donor), especially with respect to antigens or immunological reactions. Such ceils, tissues or organs are called allografts, allogeneic transplants, homografts or aliotransplants As used herein, the term

"xenogenic" or "xenogeneic" refers to ce!is, tissues or organs that are from a different species to the transplant recipient (e.g., a pig donor to a human recipient), especially with respect to antigens or immunological reactions. Such ceils, tissues or organs are called xenografts or xenotranspiants.

In another embodiment, the invention provides a method of reducing an immune response comprising administering to a subject in need thereof with an effective amount of an inhibitor of aianyi (membrane) aminopeptidase (ANPEP) to reduce an immune response. In an embodiment, the subject in need thereof is a subject with an autoimmune condition, an immune hyper-reactive condition, a chronic

inflammatory condition, or is in need of a transplant. An autoimmune condition can include, but is not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of Juvenile idiopaihic arthritis, glomerulonephritis. Graves' disease, Guii!ain-Barre syndrome, fdfopaihic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multip!e sclerosis, pemphigus pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis,

sc!eroderma/systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo or granulomatosis with polyangiitis (Wegener's), immune hyper-reactive conditions can include, but are not limited to, allergies, athma, eczema or chronic fatigue syndrome. Chronic inflammatory conditions can include, but are not limited to appendicitis, bursitis, colitis, cystitis, dermatitis, phlebitis, rhinitis, tendonitis, tonsillitis or vasculitis.

The subject or the donor transplant or graft may be treated with the inhibitor in any suitable manner (i.e., in vitro; ex vivo; in vivo) to inhibit expression, activity and/or function of ANPEP. As used herein, an "inhibitor' of expression, activity and/or function of ANPEP includes compounds that block the function, peptidase activity and/or signaling of ANPEP, compounds that reduce or prevent the transcription of ANPEP DMA into RNA, compounds that reduce or prevent the translation of ANPEP RNA into protein, and compounds that reduce or prevent the function of ANPEP protein. Such inhibiting can be complete inhibition or partial inhibition, such that the expression and/or activit of ANPEP is reduced, resulting in a reduced protease activity, adhesion or signaling, and prevention of limitation of receptor-mediated antigen uptake and presentation in dendritic cells and regulation of endocytosis and innate immune regulation in any cell type. Such inhibitors are selected from the group consisting of: antibodies that bind to ANPEP; aptamers that can interfere with ANPEP; antisense oligonucleotides directed against the ANPEP DNA or mRNA; smaii interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNA) or small internally segmented interfering RNAs (sisiRNA) directed against ANPEP protein, DNA, or mRNA, small molecule ANPEP inhibitors and any other chemical or biological compound that can interfere with ANPEP activity. The inhibitor can be used alone or together with other agents as an immunosuppressant to decrease the activity of the immune system, and may prevent transplant rejection or graft-versus- host disease.

When the inhibitor comprises an antibody, such antibodies can be polyclonal or monocionai. The antibodies can be humanized, fully human, or murine forms of the antibodies. Such antibodies can be made by well-known methods, such as described in Hariow and Lane, Antibodies; A Laboratory Manual, Coid Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). In some embodiments, additional amino acid residues may be added to either the N- or C-terminus of the antibody or antibody fragment. When the inhibitor comprises an aptamer, such aptamers can be oSigonucleic acid or peptide molecules that bind to a specific target molecule.

Methods of constructing and determining the binding characteristics of aptamers are well known in the art, and the aptamers can be isoiated from random iibraries or they can be previously identified peptides. When the inhibitor comprises antisense oligonucleotides, such antisense oligonucleotides can be small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNA) or small internally segmented interfering RNAs (sisiRNA). Methods of constructing and determining the binding characteristics of antisense oligonucleotides are well known in the art, and the antisense oligonucleotides can be designed using known programs or they can be previously identified antisense oligonucleotides.

Administering of ANPEP inhibitors to a subject in need can be via any suitable route as deemed appropriate for an intended use. In certain embodiments, the inhibitor, as described herein, can be administered a!one. in certain embodiments, the inhibitor can be administered prior to the administration of at least one other therapeutic agent, in certain embodiments, the inhibitor can be administered concurrent with the administration of at least one other therapeutic agent. In certain embodiments, the inhibitor can be administered subsequent to the administration of at least one other therapeutic agent, in other embodiments, the inhibitor can be administered prior to the administration of at least one other therapeutic agent. As will be appreciated by one of skill in the art, in some embodiments, the inhibitor can be combined with the other agent/compound. In some embodiments, the inhibitor and other agent can be administered concurrently. In some embodiments, the inhibitor and other agent are not administered simultaneously, with inhibitor being administered before or after the agent is administered. In some embodiments, the subject receives both the inhibitor and the other agent during a same period of prevention, occurrence of a disorder, and/or period of treatment. The methods and cells of the disclosure can be used for a wide variety of

pharmaceutical, cosmetic, and medicinal purposes that are known in the art.

in some embodiments, an inhibitor of ANPEP or pharmaceutical compositions comprising an inhibitor of ANPEP can be administered in combination therapy, i.e., combined with other agents, in certain embodiments, the combination therapy comprises the inhibitor, in combination with at least one other agent. Agents include other immunosuppressive agents, but are not limited to corticosteroids and

glucocorticoids {e.g., Cortisol, hydrocortisone, cortisone, prednisone, prednisolone, meihylprednisoione, dexamethasone, betamethasone, triamcinolone,

beciometasone, fludrocortisone acetate, deoxycorticosterone acetate or

aldosterone), calcineurin inhibitors and drugs acting on immunophiiins (e.g., cycSosporine or tacrolimus), mTOR inhibitors {e.g. , everolimus or siriumus),

antiproliferative drugs {e.g., azathioprine, cyclophosphamide, mycophenolic acid, mycophenolate mofeti!, mizoribine. Additional agents may also include, but are not limited to antibodies or biologies, such as anti-CD3 antibodies, anti-CD20 antibodies, anti-IL2antibodies, anti-PD-1 antibodies, anti-CTLA4 antibodies or other

immunosuppressive agents.

In certain embodiments, the invention provides for pharmaceutical compositions comprising the inhibitor and a therapeutically effective amount of at feast one additional therapeutic agent, together with a pharmaceutically acceptable diluent, carrier, solubiiizer, emulsifier, preservative and/or adjuvant.

in certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In some embodiments, the formulation materials) are for sub-cutaneous (s.c.) and/or intravenous (i.V.) administration. In certain embodiments, the inhibitor of ANPEP or pharmaceutical composition comprising an inhibitor of ANPEP can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmoiarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition, in certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, giutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents {such as mannitoi or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)}: comp!exing agents (such as caffeine, polyvinylpyrrolidone, beta- cyciodextrin or hydroxypropyl-beta-cycSodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophiiic polymers (such as

polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming countersons (such as sodium); preservatives {such as benza!konium chloride, benzoic acid, salicylic acid, thsmerosal, phenethyi aicohoS, methyiparaben, propylparaben, ch!orhexidine. sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as piuronics, PEG, sorbitan esters, poiysorbaies such as polysorbate 20, poiysorbate 80, triton, trornethamine, lecithin, cholesterol, tyioxapal); stability enhancing agents {such as sucrose or sorbitol:); tonicity enhancing agents (such as alkali metal haiides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles;

diluents; excipients and/or pharmaceutical adjuvants. {Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company {1995). In some embodiments, the formulation comprises PBS; 20 m NaOAC, pH 5.2, 50 mM Nad; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose.

in certain embodiments, the optimal pharmaceutical composition comprising an ANPEP inhibitor will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra, in certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.

In certain embodiments, the primar vehicle or carrier of an inhibitor of ANPEP or pharmaceutical compositions comprising an inhibitor of ANPEP can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial: cerebrospinal fluid, possibly supplemented with other materials common in

compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate-buffered saline, in certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles, fn certain embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-S.5, or acetate buffer of about pH 4.0-5.5, which can further inciude sorbsto! or a suitable substitute therefore.

fn certain embodiments, the effective amount of an inhibitor of ANPEP or pharmaceutical compositions comprising an inhibitor of ANPEP as described herein, with or without at ieasi one additional therapeutic agent, to be employed

therapeuticaiiy iil depend, for example, upon the therapeutic context and objectsves. One skilled in the art wiil appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vvii! thus vary depending, in part, upon the inhibitor delivered, the indication for which an inhibitor described herein, with or without at ieast one additional therapeutic agent, is being used, the route of administration, and the size {body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. In certain embodiments, a typicai dosage comprising an antibody that binds to and inhibits ANPEP can range from about 0.1 pg/kg to up to about 100 mg kg or more, depending on the factors mentioned above, in certain embodiments, the dosage can range from 0.1 pg/kg up to about 100 mg/kg; or 1 pg/kg up to about 100 mg/kg; or 5 pg/kg up to about 100 mg/kg.

in certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of the inhibitor described herein and/or any additional therapeutic agents in the formulation used. In certain embodiments, a clinician wiil administer the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose, or as two or more doses {which may or may not contain the same amount of the desired fusion protein) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routine!'/ performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of the inhibitor of ANPEP or pharmaceutical compositions comprising an inhibitor of ANPEP is in accord with known methods, e.g. oraiiy, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricufar, intramuscular,

subcutaneously, intra-ocu!ar, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.

In certain embodiments, the inhibitor of ANPEP or pharmaceutical compositions comprising an inhibitor of ANPEP can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired inhibitor has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired fusion protein can be via diffusion, timed-release bo!us. or continuous administration.

In a second aspect the invention provides a piuripotent ceii population, wherein the population does not express functional ANPEP. Functional ANPEP ca be knocked out or inhibited by a method selected from the group consisting of generation of knock-in null mutant ANPEP ceii population using homologous recombination, generation of knock-in nuli mutant ANPEP ceil population using transcription activator-iske effector nucleases ( TALENs), generation of knock-in null mutant ANPEP celS population using clustered regularly interspaced short palindromic repeats (CRISPR) technology generation of ANPEP knockout ceil Sine using homologous recombination, generation of ANPEP knockout ceil Sines using TALEN, generation of ANPEP knockout cell lines using CRISPR technology, generation of ANPEP mutant cell lines using homologous recombination, generation of ANPEP mutant ceil lines using TALEN, generation of ANPEP mutant ceil Sines using CRISPR technology,anti-ANPEP antibody, anti-ANPEP aptamer, ANPEP small interfering RNA, ANPEP smali internally segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, ANPEP antisense oligonucleotides and small molecule ANPEP inhibitors.

As used herein, the term "piuripotent eel! population" refers to animal, especially mammalian, preferably human, piuripotent cells. Piuripotent cells refer to unspeciaiized cells that have the ability to self-renew for long periods of time and differentiate into specialized ceils with specific functions. Piuripotent cells can refer to stem ceils that have the potential to differentiate into an of the three germ layers: endoderm (e.g., interior stomach fining, gastrointestinal tract, the lungs), mesoderm {e.g., muscle, bone, bSood, urogenital), or ectoderm (e.g., epidermal tissues and nervous system). Cell pfuripotency can be a continuum, ranging from the piuripotent cell that can form every cell of the embryo proper, e.g., embyronic stem cells and induced piuripotent stem cells, to the incompletely or partially piuripotent cell that can form cells of all three germ layers, but that may not exhibit ail the characteristics of completely piuripotent ceils. Piuripotent cells can refer to progenitor cells which have the gene activation potential to differentiate into multiple, but limited cell types. For example, a piuripotent blood stem cell is a hematopoietic ceii and this ceii type can differentiate itself into several types of blood ceil types like lymphocytes, monocytes, or neutrophils, but cannot differentiate into brain cells, bone ceils or other non-blood cell types. Piuripotent cells can be found in many, but not ail human eel! types (for example, piuripotent ceiis have been found in adipose tissue, cardiac ce!is. bone marrow, and mesenchymal stroma! cells).

As used herein, the term "functional ANPEP" refers to ANPEP protein with both enzyme-dependent and independent functions that contribute to adhesion, cell migration, angiogenesis, inflammatory trafficking, adhesion, antigen presentation, and endocytosis. in an embodiment, the ceiis, tissues and organs of the invention lacking functional ANPEP can have endogenous ANPEP knocked-out of the cell, tissue or organ or expression can be disrupted at transcription or translation step and therefore the cell, tissue or organ therefore does not express ANPEP. In another embodiment, the ceils, tissues or organs of the invention lacking functional ANPEP can have endogenous ANPEP replaced by knock-in of a mutant ANPEP that does not function properly and therefore ANPEP cannot contribute to adhesion, cei! migration, angiogenesis, inflammatory trafficking, adhesion, antigen presentation, and endocytosis. in yet another embodiment, the ceiis, tissues or organs of the invention lacking functional ANPEP can have endogenous ANPEP inhibited and therefore ANPEP cannot contribute to adhesion, eel migration, angiogenesis, inflammatory trafficking, adhesion, antigen presentation, and endocytosis.

in an embodiment, the invention provides a piuripotent cell population in which functional ANPEP has been knocked out or inhibited by a method selected from the group consisting of: generation of knock-in null mutant ANPEP eel! population using homologous recombination, generation of knock-in null mutant ANPEP ceil population using TALEN, generation of knock-in null mutant ANPEP cell population using CRISPR technology, generation of ANPEP knockout cell lines using homoiogous recombination, generation of ANPEP knockout ceil lines using TALEN, generation of ANPEP knockout cell lines using CRISPR technology, generation of ANPEP mutant cell lines using homologous recombination, generation of ANPEP mutant ceil lines using TALEN, generation of ANPEP mutant ceil lines using CRISPR technology, anti-ANPEP antibody, anti-ANPEP aptamer, ANPEP small interfering RNA, ANPEP small internally segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, smali molecule inhibitors of ANPEP and ANPEP antisense oligonucleotides. CRISPR/Cas-based RNA-guided DNA endonuc!eases are genome editing tools (Wang et ai, 2013 One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR Cas-Mediated Genome Engineering." Cell 153(4):910- 18). Transcription activator-like effector nucleases (TALENs) comprise chimeric nucleases that are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA c!eavage domain. TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homoiog -directed repair at specific genomic locations {Gaj ei a!., 2013 "ZFN, TALENI, and CRISPR/Cas-based methods for genome engineering." Trends Biotechnoi. 31(7};397-405).

In another embodiment, the invention provides a differentiated population of celis for transplanting into a subject in need thereof, wherein the cells are derived from the piuripotent ceils lacking functionai ANPEP. The differentiated population of cells can be selected from the group consisting of: skin ceils, beta ceils, cardiac celis, brain ceils, kidne cells, liver ceils, ceils of the digestive tract and accessory digestive organs, saiivary gland ceils, adrenal gland cells, prostate celis, lung ceils, pancreatic ceils, bone cells, immune cells, hematopoietic cells, vascular ceils, cells of the eye, connective tissue cells and musculoskeletal ceils. In a preferred embodiment, the ceils are skin cells or beta cells lacking functionai ANPEP.

The differentiated population of cells can be derived from the piuripotent cells lacking functional ANPEP by a number of protocols that are already developed or are currently being optimized by others in the field. These methods can include, but are not limited to, systematic induction of differentiation using special celi culture matrices, media and growth factor or small molecule supplements administered i specific time windows that mimic normal developmental timepoints, introduction of certain genes and growth factors or cytokines into cells to promote immediate switching of ceil types, or the in vivo introduction of certain genes, growth factors or cytokines into ceiis to promote endogenous differentiation of ceils.

In yet another embodiment, the invention provides a tissue for transplanting into a subject in need thereof, wherein the tissue is derived from the piuripotent ceil population lacking functional ANPEP. The tissue can be selected from the group consisting of: bone tissue, muscuioskeietaS tissue, cornea tissue, skin tissue, heart valves, and b!ood vessels, immune ceils, connective tissue, iung tissue. In a preferred embodiment, the tissue is skin tissue lacking functionai ANPEP.

The differentiated tissue can be derived from the piuripotent cells lacking functional ANPEP by a number of protocols that are already developed or are currently being optimized by others in the field. These methods can include, but are not limited to, systematic induction of differentiation using special ceil culture matrices, media and growth factor or smaii molecule supplements administered in specific time windows that mimic normal developmental timepoints, introduction of certain genes and grov h factors or cytokines into cel!s to promote immediate switching of ceil types, or the in vivo introduction of certain genes, growth factors or cytokines into ce!is to promote endogenous differentiation of ceiis. Ceiis generated in vitro can be seeded onto bioengineered scaffolds that can provide the three dimensional structure of the tissue of interest. These scaffolds can be created by three dimensional printing with biomateriais specifically developed for these purposes, including but not limited to, synthetic materials, protein based materials and polysaccharide based materials, such as polygiycoiic acid, poiyiactic acid, fibrin, giycosaminogiycans.

In a further embodiment, the invention provides an organ for transplanting into a subject in need thereof, wherein the organ is derived from the pluripotent celi population lacking functional ANPEP. The organ can be selected from, but is not limited to, the group consisting of: cornea, skin, kidney, fiver, lung, heart, pancreas and intestine. In a preferred embodiment, the organ is skin lacking functional ANPEP.

The differentiated organ can be derived from the pluripotent cells lacking functional ANPEP by a number of protocols that are already developed or are currently being optimized by others in the field. These methods can include, but are not limited to, systematic induction of differentiation using special cell culture matrices, media and growth factor or small molecule supplements administered in specific time windows that mimic normal developmental timepoints, introduction of certain genes and growth factors or cytokines into cells to promote immediate switching of cell types, or the in vivo introduction of certain genes, growth factors or cytokines into ceils to promote endogenous differentiation of cells. Differentiated ceiis or stem ceils can be seeded onto bioengineered scaffo!ds to promote further development of a complete organ with the various different cell types and organization that is associated with the particular organ of interest. Bioengineered scaffolds can be created through three dimensional printing with biomaterials or deceilufarization of existing organs according to protocols optimized b experts in that field. In an alternative method, pluripotent stem cells can be used to generate large multidimensional organoids according to culture methods currently being developed by other experts in the field.

In an another embodiment, the invention provides cells, tissues or organs for transplanting into a subject in need thereof, wherein the cells, tissues or organs are derived from the pluripotent celi population lacking functional ANPEP and also comprise at least one additional modification. I some embodiments, a additional modification can be the genetic engineering of the ceils, tissues or organs lacking functional ANPEP to express a gene or peptide. In such an embodiment, the donor transplant can also serve as a means for administering therapeutic proteins and peptides. The peptide is not limited to any particular peptide, but can include any peptide that can be used to treat any number of diseases, disorders or conditions. For example, the therapeutic peptide can include, but is not !imited to, nesiritide, ceruletide, bentiromide, exenatide, gonadoreiin, enfuvirtide, vancomycin, icatibant, secretin, leuprolide, glucagon recombinant, oxytocin, bivalirudin, sermoreiin, gramicidin D, insulin, capreomycin, calcitonin, vasopressin, cosyntropin, bacitracin, octreotide, abareiix, vapreotide, thymaifasin, mecasermin, cetrore!ix, teriparatide, corticotropin or pramlintide.

in an additional embodiment, the invention provides a method of transplanting celis, tissues and/or organs into a subject in need thereof comprising: pre-treating the ceils, tissues and/or organs with ANPEP inhibitors prior to transplanting the cells, tissues and/or organs lacking or blocking functional ANPEP into a subject in need thereof or treating the subject with ANPEP inhibitors prior to transplant. The cell, iissue and/or organ to be transpianied can be syngeneic, aliogeneic or xenogeneic to the subject receiving the transplant.

As used herein, the term "syngenic" or "syngeneic" refers to ceils, tissues or organs that are geneticaily identicai or are derived from a genetically identicai source to the graft recipient (e.g., an identicai twin), especially with respect to antigens or immunological reactions. Such ceils, tissues or organs are caiied isografts.

As used herein, the term "allogenic" or "allogeneic" refers to celis, tissues or organs that are not genetically identicai or are derived from a non-geneticaily identicai source to the graft recipient {e.g., a non-related donor), especially with respect to antigens or immunological reactions. Such cells, tissues or organs are caiied allografts, allogeneic transplants, homografts or a!iotransplants

As used herein, the term "xenogenic" or "xenogeneic" refers to cells, tissues or organs that are from a different species to the graft recipient (e.g., a pig donor to a human recipient), especially with respect to antigens or immunological reactions. Such ceils, tissues or organs are called xenografts or xenotransplants.

EXAMPLES

The Examples that follo are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention. Example 1 : ANPEP expression and the immune response

To determine the role of ANPEP in immune responses elicited by stem eel! transplantation, mouse embryonic stem {mES) ce!!s were derived from ANPEP WT and ANPEP KO mice. These eels proliferate at similar rates and express characteristic piuripotency markers «7 vitro. When implanted into syngeneic mice, mES of both genotypes form teratomas that contain cell types representing ait three embryonic germ layers, demonstrating piuripotency in vivo as well. Surprisingly, teratomas generated from ANPEP KO mES grow more rapidly, achieve a larger size, and show a higher degree of differentiation than those generated from ANPEP WT mES celis. Analysis of potential underlying causes showed that ANPEP KO teratomas contained fewer infiltrating T-iymphocytes and dendritic cells, indicating that lack of ANPEP expression may permit stem ceils to escape immune detection and allow implants to thrive. In agreement with this hypothesis, growth and differentiation of WT and ANPEP KO mES implanted into immunocompromised mice were comparable, consistent with an underlying immune mechanism. Consequently, modulation of ANPEP expression may alter immune responses toward implanted stem ceils and enhance their engraftment to ultimately facilitate repair.

Example 2: Assessment of ANPEP expression and immunomodu!ation

To study the immunomodulatory effects of ANPEP further, the inventors have been studying skin grafts between ANPEP WT and ANPEP KO mice with gender or major histocompatibiiity-mismatches. Surprisingly, the skin graft studies confirm the observations with the teratomas. ANPEP KO female skin engrafts successfully on both ANPEP WT and ANPEP KO male mice, while ANPEP WT female skin is rejected from both ANPEP WT and ANPEP KO male mice after 14 days.

Comparison of histological sections at sites of engraftment shows that there is significant inflammation and disruption of normal skin architecture in ANPEP WT skin grafts. There is oniy mild inflammation in ANPEP KO skin engrafted in ANPEP WT mice, while there is virtually no disruption of ANPEP KO skin engrafted in ANPEP KO mice. These studies provide surprising and important evidence that immunological toierance can be engendered through ANPEP blockade.

Example 3: Skin grafts lacking CD13 survive longer with diminished

inflammation.

Initial characterization of mice lacking CD 13 expression on a global level demonstrated no notable developmental deficiencies or defects in homeostatic processes (Winnieka ef ai. 2010). However, while site-specific mechanisms influenced specific outcomes, the inflammatory responses of CD13-nuii animals were universally compromised upon challenge in various injury models (Pereira ef al. 2013, Rahman ef al. 2013, Subramani et al. 20 3, Ghosh ef al. 2014), Based on these observations, it was determined sf the diminished inflammatory responses seen in CD13-nuii mice could potentially extend to the inflammatory reactions mediating transplant rejection. Using wild type and global CD13-nu!i mice in a standard mode! of minor histocompatibility (MiHC) mismatched allograft rejection (Figure 6), long- term survival of full-thickness GD13-nuif male donor skin grafts was observed on female recipients that survived for over 100 days {Figure 1A). The CD13-nu!i grafts maintained their original size as compared to VVT skin grafts that wither and gradually shrink in diameter until fully rejected between two to three weeks post-transplant (Figure 1B). Furthermore, the CD13-nui! grafts retained the characteristic thin, vascularized skin of the donor dorsal ear and did not adopt the thick fur of the host dorsal skin, suggesting that CD13 can be a potential instigator of allograft rejection. Indeed, while there is evidence of inflammatory cell infiltration into CD13-nulS graft tissue after one week, the number of cells and the extent of tissue damage was significantly less than that observed in VVT skin grafts (Figure 1C).

Immunohistochemica! quantification of CDS positive cells in grafts at five days post- transplant indicated that significantly more T cells infiltrated into WT grafts as compared in GDI 3-nufS grafts in both WT and CD13-nui! recipients (Figure D). Flow cytometric analyses of host spleens receiving grafts of either genotype at days 5 and 1 GO post-transplant indicated that there were no significant differences in various splenic immune populations (Figure 7} implying that the differences in graft acceptance observed were most likely due to local events in the graft and draining lymph nodes.

Example 4. Mast cell-mediated inflammation is reduced in CD13-nuli grafts

Wast cells are traditionally considered pro-inflammatory mediators of the acute immune response; however, recent studies have elucidated novel anti-inflammatory roles for these cells particularly in the setting of aliograft survival (Gaiii er ai. 2005, Lu ef a/. 2006), where mast cells have been shown to be critical for graft survival and their degranulation reverses tolerance to skin allografts (Lu ef al. 2006, de Vries ef ai. 2009). Pertinent to this study, mast cells are known to express high levels of CD 13, but the functional ro!e of CD13 in mast ceil activity has yet to be demonstrated. To determine the contribution of mast cells to this model of allograft survival, WT and CD13-nu!i skin grafts were examined for the presence of mast cells and their degranulation status using to!uidine blue to stain mast cell-specific granular contents. Interestingly, higher levels of toluidine blue staining was observed in CD13-nu!i graft tissue, potentially due to reduced mast cell degranuiation in the absence of GDI 3 (Figure 2A and 2B). This increase in granule staining was a!so seen in the draining iymph nodes, but not the spleens of mice that received CD 13-nuli skin grafts (Figure 2C). Orthotopic applications of gender mismatched dermai fibroblasts survive ionger when mixed with CD13-nuli mast ceils. These results confirm the histological observations that inflammation is reduced in the absence of CD13. Furthermore, given that mast cells are known to produce IL-4, a known stimulator of reparative M2 macrophages, and 1L-10, a potent immunosuppressor, it is possible that the diminished inflammation and elevated number of alternatively activated macrophages in CD13-nuil grafts (Figure 1F) can be attributed to a degranulation-independent antiinflammatory function of mast ceils.

Example 5. Absence of CD13 on mast ceiis corresponds to diminished mast cell activation and degranuiation In vitro

To confirm the in vivo observations of effects of CD13 on mast ceil activity, primary mast ceils were derived from bone marrow of WT and CD13-nufS mice according to previously published protocols (Kafesnikoff and Ja!ii 2011 ). After 4-6 weeks of culture, cultures consisted of 80-90% ckit+Fc£iR1a+ mast cells with a high degree of forward and side scatter, indicating these cells are large and complex (Figure 3A). To test the response of mast ceils to MiHC determinants, isolated WT or CD13-nuil mast ceils were co-cultured with gender-mismatched dermal fibrobiasts and changes were assessed by flow cytometry. The forward and side scatter profiles of both WT and CD'S 3-nuil mast cells were decreased indicating loss of granularity and size, presumably the result of degranuiation; however, the changes in GDI 3-nuli mast cell size and granularity were significantly iess upon stimulation with either WT or CD 13- nuli dermal fibrobiasts than WT mast cells (Figure 3B), This is in accord with results from mixed lymphocyte reactions between gender mismatched splenocytes where the response of CD13-nuil splenocytes was considerably weaker (Figure 8).

However, MiHC mismatched CD13-nuli stimulators invoked more robust responses from WT splenocytes than WT mast cells, suggesting a lack of aliorecognition.

Additionally, the supernatant collected from CD^'13-nuii mast cells cultured with dermal fibrobiasts contained higher levels of !L-4. To determine if CD13-nuSi mast cells could be exerting their anti-inflammatory role at least in part by skewing macrophage differentiation towards the reparative 2 phenotype at the expense of a pro-inflammatory Ml phenotype, WT and CD13-nuil mast cells are co-cultured with WT and CD13-nuii immature macrophages for 1-3 days before determining relative numbers of M1 and M2 macrophages by flow cytometry. As a whole, these data show that GDI 3-nuli mast ce!i degranulation and pro-inflammatory responses are impaired while a more anti-inflammatory or immunosuppressive microenvironment is enhanced, thus promoting transplant survival.

Example 6. Transient blockade of CD13 prolongs graft survival

To confirm the effects on transplant acceptance were due to CD13, a CD13 biocking antibody, SL13, was used in transient treatment studies (Figure 9A}. Following two weeks of intraperitoneal SL13-mAb administration, SL13 was detectable in the serum of treated mice, but not in the serum of mice treated with vehicle alone or isotype control (Figure 9B}. All of the mice tolerated the treatment and after two weeks there were no significant pathological changes in any major organ indicating that SL13 can be used safely in vivo. After the two week treatment period, SL13 treatment was halted and WT female recipients of both groups received gender-mismatched skin grafts from WT and CD13-nu!i male donors. Similar to studies in the global CD13- null animals, CD13-nuii grafts universally survived until time of harvest in both groups (Figure 4A left panels: KO male skin graft). Importantly, rejection of WT male skin was delayed by 3 weeks in treated WT recipients, well after WT skin grafts had been rejected by the untreated mice (Figure 4A right panels; WT male skin graft).

Reversal of graft acceptance following the withdrawal of SL13 treatment indicated that CD13 blockade using a monoclonal antibody prolongs graft survival. SL13 treatment for 1 week prior to skin transplant can prolong graft survival when SL13 is routinely given. Once SL13 treatment is discontinued, WT skin grafts show signs of failing. Toluidine biue staining of WT skin that survives in SL13 treated mice indicates an increase in mast ceil staining as compared to that seen in CD13-nuSl grafts, suggesting that a similar mechanism of locally induced immune acceptance.

Example 7. Tissues derived from mESC Sacking CD13 are accepted by immune- competent mice.

Pluripotent stem cell derived therapies are the ultimate goai of regenerative medicine; however, their utility in the clinic is hampered by the same immunologic reactions that limit current transplantation therapies (Tang and Drukker 2011 }. To determine if CD13 blockade could be applied in a stem cell-derived therapeutic setting, mouse embryonic stem cells (mES) were generated from WT and CD13-null mice. These cells displayed characteristic mES ceil morphology and expressed traditional piuripotency markers Oct4 and Nanog {Figure 10A). Both WT and CD13- null mESCs proliferated at similar rates in vitro and generated ail three germ layers when differentiated in teratoma assays (Figure 1GB and 10C). Interestingly, CD 13- nui! mES generated substantially larger teratomas than did WT mES even though starting ceil numbers, host mice and duration of growth are matched (Figure 5A). These CD13-nuii teratomas also proliferated at higher levels as indicated by increased Ki87 staining and BrDU incorporation {Figure 5B and 5C), but did not display any significant apoptosis by Tunef staining (Figure 1 QD, arrows indicate apoptotic cells). Furthermore, CD13-nul! teratomas are also more vascularized as detected by endothelial CD31 and pericyte aSMA staining (Figure 10E).

Overall, similar to the observations with CD13-nui! skin grafts these data indicate that CD13-null mES possess a survival advantage over WT mES. To ascertain the potential: immunological processes underlying this apparent growth advantage, WT and CD13-nu!l mES were injected subcutaneously into immunocompromised mice lacking T, B and NK cells. Both WT and CD 3-null mES generated teratomas of equal: size and at similar rates (Figure 5D) confirming that the difference in growth was not attributable to inherent differences in the embryonic stern ceils themselves, but rather to events occurring at the donor-host interface. Analysis of infiltrating immune ceils in teratomas generated in immunocompromised mice shows no significant differences; while numbers of infiltrating CD3+ T ceils in immune competent mice are higher in WT in CD13-null teratomas. Furthermore, highly significant differences in numbers of infiltrating T ceils were observed in teratomas generated in CD13-nuil hosts (Figure 5E). This would imply that WT mES are more capable of stimulating an immune reaction against the transplanted ceils and that the response of CD 3-nuli mice to immunogenic donor ceils is impaired and is in agreement with the immune cell analyses in the skin graft studies discussed above. Additionally, higher levels of dendritic cells and macrophages were detected in WT teratomas {Figure 5F). Toiuidine blue staining of teratoma sections also indicated higher ievels of mast cell staining within and around CD13-nuif teratomas, consistent with reduced mast cell degranulation in the skin graft model (Figure 5G).

Taken together, these data demonstrate that the CD 13 blockade-dependent transplant of cells and tissues is most likely due to the absence CD13-mediated immunological rejection. To test the applicability of CD 3-b!ockade in promoting ceil therapy, skin and lung ceils were generated from male WT and CD13-null mES and transplanted into WT female recipients. The invention wou!d significantly increase the success of organ and skin transplant as wei! as eliminate the time transplant recipients must wait for a suitable donor transplant or graft to become available. Furthermore, this invention can minimize the necessity of systemic immune suppression, thereby significantly improvsng quality of life in terms of avoiding infection and taking medication with deleterious side effects. The method would provide universal transpiants or grafts and obviate the need and cost of creating personalized pluripotent cell lines and differentiated tissues that would only foe applicable to one or a few patients.

Furthermore, the invention could potentially be used by transplant and reconstructive surgeons for patients who require any type of graft or organ. For example, skin grafts could be used for severely burned patients without the current limitations of time, access to viable skin_: or immunogenicity. Neurons or neuronal support cells such as oligodendrocytes could also be produced for transplant into patients suffering from neuromuscular diseases. Cardiac ceils could be transplanted into patients who suffered myocardial infarctio or congestive heart failure.

Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended ciaims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.

Claims

WHAT IS CLAIMED IS:

1. An isolated piuripotent cell population, wherein the population does not

express functional ANPEP.

2. The isolated piuripotent ceil popuiation of claim 1 , wherein functional ANPEP has been knocked out or inhibited by a method selected from the group consisting of: generation of knock-in nuii mutant ANPEP ceil popuiation using homologous recombination, generation of knock-in null mutant ANPEP ceil population using TALEN, generation of knock-in null mutant ANPEP ceil population using CRiSP technology, generation of ANPEP knockout ceil lines using homologous recombination, generation of ANPEP knockout cell lines using TALEN, generation of ANPEP knockout cell lines using CRISPR technology, generation of ANPEP mutant cell Sines using homologous recombination, generation of ANPEP mutant cell lines using TALEN, generation of ANPEP mutant cell lines using CRISPR technology, anti- ANPEP antibody, anti-ANPEP aptamer, ANPEP small interfering RNA, ANPEP small internally segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, smaii molecule inhibitors of ANPEP and ANPEP antisense oligonucleotides .

3. The isolated piuripotent ceil popuiation of any of claims 1 -2, wherein the ceiis are cultured into a differentiated population of cells.

4. The differentiated population of cells of claim 3, wherein the cells are selected from the group consisting of; skin ceils, beta ceils, cardiac cells, brain celis, kidney cells, liver cells, ceils of the digestive tract and accessor digestive organs, salivary gland ceils, adrenal gland cells, prostate ceiis, lung cells, pancreatic celis, bone cells, immune celis, hematopoietic cells, vascular ceils, cells of the eye, connective tissue ceils and muscu!oskeietal ceils.

5. The differentiated population of cells of claim 4, wherein ceiis comprise a tissue selected from the group consisting of: bone tissue, musculoskeletal: tissue, cornea tissue, skin tissue, kidney tissue, liver tissue, pancreatic tissue, heart valves, blood vessels, immune ceils, connective tissue and lung tissue.

6. The differentiated popuiation of cells of claim 4, wherein cells comprise an organ selected from the group consisting of: cornea, skin, kidney, liver, lung, pancreas and intestine. The differentiated population of eels of any of claims 4-6, wherein the ceils are genetically engineered to express a therapeutic peptide.

A method of transpiantation comprising: transplanting the ceils, tissues and/or organs of any of claims 1 -7 into a subject in need thereof.

The method of claim 8, wherein the subject in need thereof is pre-ireaied with an effective amount of an inhibitor of aiany! (membrane) aminopeptidase (ANPEP).

The method of claim 9, wherein the inhibitor is selected from the group consisting of: anti-ANPEP antibody, anti-ANPEP aptamer, ANPEP small interfering RNA, ANPEP small intemaliy segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, ANPEP antisense oligonucleotides, and smal! molecule inhibitors of ANPEP.

The method of claim 10, wherein the inhibitor is an anti-ANPEP antibody.

The method of claim 8, wherein the ceil tissue and/or organ is selected from the group consisting of: skin ceils and skin tissue.

The method of any of claims 8-12, wherein the ceil tissue and/or organ is syngeneic, allogeneic or xenogeneic to the subject.

The use of the ceils, tissues and/or organs of any of claims 1-7 for transplanting into a subject in need thereof.

A method of iimiting deve!opment of transplant rejection, comprising:

a) administering to a subject in need of a transplant, and/or

b) treating a donor transplant with,

an effective amount of an inhibitor of alanyi {membrane} aminopeptidase (ANPEP) to limit development of transplant rejection in the subject.

The method of claim 15, wherein the subject receives a donor transplant and wherein the method includes treating the donor transplant: wherein treating the donor transplant comprises pre-treatment with an inhibitor of alanyf (membrane) aminopeptidase (ANPEP).

A method of any of claims 15 or 16, wherein the subject's immune system is rejecting the donor transplant, comprising administering to the subject an effective amount of an inhibitor of afanyl (membrane) aminopeptidase (ANPEP). The method of any of claims 15- 17. wherein the subject is receiving a donor transplant or has received a donor transplant and the donor transplant is selected from the group consisting of: skin ceils, beta ceiis, cardiac cel!s, brain cel!s, kidney ceils, liver cells, cells of the digestive tract and accessory digestive organs, salivary gland ce!!s, adrenal giand cells, prostate cells, Sung ceils, pancreatic cells, bone ceils, immune ceils, hematopoietsc ceils, vascular ceils, cells of the eye, connective tissue ceiis, musculoskeletal ceils, bone tissue, musculoskeletal tissue, cornea tissue, skin tissue, heart valves, blood vessels, immune cells, connective tissue, lung tissue, skin, a cornea, a kidney, a liver, a iung, a pancreas, a heart, and intestine.

The method of claim 18, wherein the donor transplant is selected from the group consisting of: beta cells, skin ceiis and skin tissue.

The method of any of claims 15- 19. wherein the subject is receiving a donor transplant or has received a donor transplant and the donor transplant is syngeneic, allogeneic or xenogeneic to the subject.

A method of reducing an immune response comprising administering to a subject i need thereof with an effective amount of an inhibitor of a!anyl {membrane} aminopeptidase (ANPEP) to reduce an immune response.

The method of claim 21 , wherein the subject in need thereof is a subject with an autoimmune condition, an immune hyper-reactive condition, a chronic inflammatory condition, or is in need of a transplant.

The method of any of claims 15-22, wherein the inhibitor is selected from the group consisting of anti-ANPEP antibody, anti-ANPEP aptamer, ANPEP small interfering RNA, ANPEP small internally segmented interfering RNA, ANPEP short hairpin RNA, ANPEP micro RNA, ANPEP antisense oligonucleotides, and small molecule inhibitors of ANPEP.

The method of any of claims 15-23, wherein the inhibitor comprises an anti- ANPEP antibody.