AU2015201974B2 - Antibodies against a proliferating inducing ligand (APRIL) - Google Patents

Abstract

The present invention relates to a binding compound which binds to human APRIL. More specifically the invention provides, compositions of anti-APRIL specific antibodies and methods to use such antibodies in modulating the biological activity APRIL, particularly in inflammatory diseases, inhibition of cell proliferation and cancer.

Description

Antibodies against a proliferating inducing ligand (APRIL)

The present application is a divisional application of Australian Application No. 2010220421, which is incorporated in its entirety herein by reference.

The present invention relates to isolated antibodies or fragments thereof which binds to human APRIL., polynucleotides encoding such antibodies and host cells producing said antibodies. The antibodies can be used to inhibit immune cell proliferation and/or survival, to treat cancer and to treat an inflammatory disease. APRIL is expressed as a type-II transmembrane protein, but unlike most other TNF family members it is mainly processed as a secreted protein and cleaved in the Golgi apparatus where it is cleaved by a furin convertase to release a soluble active form (Lopez-Fraga et ah, 2001, EMBO Rep 2, 945-51 ,). APRIL assembles as a noncovalently linked homo-trimer with similar structural homology in protein fold to a number of other TNF family ligands (Wall weber et ai., 2004, Mol Bio/343,283-90). APRIL binds two I NF receptors: B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TAG) (reviewed in Kimberley et ai., 2009,1 Ceil Physiol. 218(1):1-8). in addition, APRIL has recently been shown to bind heparan sulphate proteoglycans (HSPGs) (Hendriks et a!., 2005, Cell Death Differ 12, 637-48). APRIL· shows high homology (30%) to another member of the TNF superfamily, B cell activating factor belonging to the INF family (BAFF or B Lymphocyte stimulator, BLyS), with which it shares binding to its receptors, BCMA and TAG. BAFF is also known to bind a unique receptor, BAFF-Receptor, and through this mediates crucial survival signals during B cell development (reviewed in Kimberley et ah, 2009, J Cell Physiol. 218(1):1-8). APRIL and BAFF have been suggested to form mixed trimers (Roschke et ai., 2002, J Immunol. 169(8):4314-21). Such mixed trimers were found to occur at a higher prevalence in rheumatoid arthritis (RA) patients. APRIL is predominantly expressed by immune cell subsets such as monocytes, macrophages, dendritic cells, neutrophils, B-cells, and T -cells, many of which also express BAFF. In addition, AP.R1L can be expressed by non-iminune cells such as_ osteodasts, epithelial ceils mi a variety of tumour tissues (reviewed In Klnihedey et at, 2m%J Cell Physiol 218(1):1-8).

The fimctifon of APRIL was established using mouse genetic models. MFJRIL tmnsgeulo mice develop normally, bat showed enhanced T cell survival sad elevated levels of IgM antibodies (Stein at si, 2002. J Clin invest 109,1587-98). la addition, T cell independent type II responses were enhanced. Aged hAPRIL transgenic mice displayed extreme ealargemeat and re-organisatioa of the lymph system and enlarged spleen doe to infiltmtien of Q>5 positive B cells, a phenotype closely resembling human B-CLL {Planelles et ah, 2004, Corner (Ml, 399-408). APRIL deficient mice were Iband to have decreased levels of IgA m circulation and open challenge with a T~ cell dependent antigen (Castigti et ah, 2004. Pmc Not! Acad Sci USA: Ml, 3903-8; Varfolomeev et ah, 2604, Moi Cell Bia! 24,997-1006), Next, APRIL, along with BAFF, was demonstrated to inaction la ekxss-s witch recombination (CSR) of antibodies to both IgG and IgA, mdependently of CB40-CB40Lsignaling (Litinskiy et si., 2002, Nat Immumsi 3, 822-9). hr addition, APRIL was deuiosstrated to be less critical than BAFF in B eel! niaintonanee, tad was shown to have a role in B cell signalling and drive both proliferation and survival of human and murine B cells in-vitro (reviewed in Kimberley et at,, 2009, JCeUPkjmel 218(1.):1-8).. APRIL was originally identified based on Its expression 1» cancer cells (Bahne et al«, 1998, J Exp Med 188,1185-90). High expression levels of APRIL mRNA were found in a pane! of tumour eel linos as well as human primal tuffiotns sack as colon, and a lymphoid carcinoma. In addition, APRIL feaasfected murine ibrohlast MH-3T3 cells were shown to grow more rapidly in immtmodeicient mice. Mote importantly, blocking APRIL using a soluble APRIL receptor was shown to inhibit tumour growth of long and colon carcinomas (Rennert et ah, 20QQ^ JExp Med 192,1677-84).

Chronic Lymphocytic Leukaemia (CLL) B calls express both APRIL sod APRIL-reeeptors. In addition, it was shown that APRIL protected CLL cells against spontmeons and dmg4ndueed apoptosis and stimulated MF^xB acrivaiion (reviewed in Kimberley et &L, 20(19, J CM Physiol 218(1):1-8). A retmspective study under 95 CLL patients showed Increased levels of APRIL In serum* which correlated with disease progression and overall patient survival, with a poorer prognosis for patients Wath high APRIL serosa levels (Blanelles et al., 2dOI, lfaspo/d/£?g?ssr ^2s 1284-5),

Similarly, (mo-eased levels of) APRIL was shorn to fee expressed m Hodgkin’s lymphoma* Non-Hodgkin’s lymphoma (NHL) and Multiple Myeloma (MM) (reviewed la Kimfeeiiey et Μ.* 2009, JCeS Physiol. 218(1):1-¾ A retrospective study in DLBCL patients (NHL) showed that high APRIL expression in cancer lesions correlated with a poor survival rate (Sehwalkr etal* 2(102* B!md 109,331-8), Using NHL and MM cell-lines It was shown that treatment with APRIL or BA® Increased survi val viaNF-κΒ activation and up-regulation of prmsirvival proteins (reviewed in Kimberley et aL, 28^JC$IIP%0&L 218(1):1-8). In accordance with tills pro-sttrvival role of APRIL, MM celts were shown to undergo apoptosis when enittoed in the presence of T.ACI~Fe. Since BAFF- receptor was less oSeeive In enhancing apoptosis, this indicates that APRS/, and not BAEP is prhnariy responsible for enhanced survival in these cells (Abe et ai, 2006, Leukemia 2(6 1313-5). APRIL was also found to he over-expressed in a number of cell lines derived from solid tumours. Indeed* APRIL was able to stimulate ίη-vitro proliferation of a number of these oeiliines (royiewed in Kimbedey et at* 280%JGeM Physiol 21.8(1):1-8),

Boo to Its role m B cell biology APRIL also plays a role m many autolmtrmne diseases. Indeed, amelcept (apommerdhl 1A€I«Fe propa^ in numerous clinical trials for treatment of several aidoimmune diseases (reviewed in Gallo et si, 2008*

Carr Qpinldvmdg Xh*®$ 9(11):1216-22). Increased semrn levels of APRIL and BAFF have been reported in many SLE patient (Royama et al* 2005, Am Ekgum Dm 64* 1065-7). A retrosptwtiye analysis revealed that APRIL serum levels tended to correlate with a&tbdsDNA antibody litres, Evidence that APRIL may play a functional role la. 8LE was obtained by testing the effect of TACLFc fission protein Into lupus prone mice (Gross et ah, 2000, Nature 484* 995-¾ which prevented disease development and prolonged survival ία addition, inhibition of APRIL and RAFF with TACI-Fo is the CIA mouse mode! of .rheumatoid 'arthritis was also found to pieveat disease progression and lower disease scores» compared with controls (Gross et al,. 2001, Immunity IS» 289-302; Wang et al, 2001, Nat immumt 2» 632-7). Also in another arthritis .medfel, synovirnn-SClB mouse cMmeras, TACI-Fc showed a beneficial effect (Seller et si,» 2005, J din Invest 115, 3083-92), Treatment with TA€I-Fe resulted in the disappsaran.ce of Germinal Centers m the synovial tissue, decreased Ig production and decreased production of IFH-gamma.

It was later reported Fiat tire synovial fluid of patients with inflammatory arlMtis had sigaiiciroliy increased APRIL levels compared with, those with patients suffering from mm-snflmmm&ny arthritis such as osttmaifhritls (Stohl et at, 2006» EmimrMetab Immune Disord Drug Targets 6, 351-8; Tan et at, 2003,. Arthritis Rheum 48,982-92).

Several studies focused on the presence of APRIL I» the sera of patients suffering from a wider range of systemic ioimrmc-based rheumatic diseases (now also including Sjogren’s syndrome, Reiter’s syndrome, psarialie arthfitis, polymyositis, and ankylosing ^wadylitis) and found sigaMcastly Increased APRIL levels in these patients, suggesting an Important role for APRIL In these diseases as well (lonsson et al.» 1986» ScandJ Rheumatol Su.ppl 61,1.66-9; Rosehke et ai, 2002, J Immunol 169, 4314-21).

Finally, Increased APRIL expression has also bees linked to Multiple Sclerosis (MS), APRIL expression was found to be increased in die astrocytes of MS sufferers compared with normal controls, lids is in line with, the described APRIL expression In. glioblastomas and m the serum of glioblastoma patients (Deshayes et at, 2004, One&gem 23» 3005-12; Roth et al., 2001» Cell Death IMJjfer 8,403-10), APRIL plays a crucial role in the survival and proliferative capacity of several B~eeil malignancies, and potentially also some solid tumours. APRIL is also emerging as a key player in mRannualoty diseases or autoimmunity, Thns»: strategies to antagonise APRIL are a therapeutic goal for a somber of these diseases, Indeed cinlcal studies targeting APRIL with TACLFc (Ataeieept) are currently ongoiug tor treatment of several autoimmune diseases, However, TACI-Fe also targets BAFF, a factor involved in normal B-cell maintenance. Antibodies directed against APRIL have been described in W09614328, W02001/60397, WO2002/94192, W09912965, WO2001/196528 and W09900518. This invention describes antibodies targeting APRIL specifically. The antibodies in this invention fully block the binding of APRIL to TACI and at least partially to BCMA. Some antibodies according to the invention fully block the binding to both BCMA and TACI. Such molecules are useful in a therapy for a number of conditions in which circulating soluble APRIL correlates with disease activity and progression. Since expression levels of APRIL can be used as diagnostic and prognostic markers for different diseases, these antibodies can also be applied in such tests.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

The invention provides binding compounds such as isolated antibodies or antibody fragments which bind to human APRIL.

According to a first aspect, the present invention provides an isolated or recombinant antibody or antibody fragment which binds to human A Proliferating Inducing Ligand (APRIL) comprising: a. an antibody heavy chain variable region comprising CDRs SEQ ID NOs: 15, 16, and 17; and b. an antibody light chain variable region comprising CDRs SEQ ID NOs: 18, 19, and 20.

According to a second aspect, the present invention provides an isolated or recombinant antibody or antibody fragment which binds to human APRIL wherein the antibody or antibody fragment binds to an epitope having the amino acid sequence of SEQ ID NO: 38 or EGQ.

According to a third aspect, the present invention provides an isolated or recombinant antibody or antibody fragment which competes for a binding epitope on human APRIL with the antibody or antibody fragment of the invention, and: a. binds to human APRIL with a KD of about 10~8 to ΙΟ'11 M; b. binds to human APRIL with about the same Kd as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8; or c. blocks binding of human TACI to human APRIL with an IC50 of about 2 nM or lower.

According to a fourth aspect, the present invention provides a composition comprising the isolated or recombinant antibody or antibody fragment of the invention in combination with a pharmaceutically acceptable carrier or diluents.

According to a fifth aspect, the present invention provides a method of treating an APRIL-dependent condition comprising administering an effective amount of the isolated or recombinant antibody or antibody fragment of the invention or the composition of the invention wherein the APRIL-dependent condition is selected from the group consisting of APRIL-dependent cancers, APRIL-dependent inflammatory diseases or APRIL-dependent auto-immunity.

According to a sixth aspect, the present invention provides a method of inhibiting immune cell proliferation and/or survival comprising administering an effective amount of the isolated or recombinant antibody or antibody fragment of the invention or the composition of the invention.

According to a seventh aspect, the present invention provides a diagnostic assay for detecting expression of human APRIL in a cell, tissue or serum by labelling the isolated or recombinant antibody or antibody fragment of the invention and detecting binding of the labelled isolated or recombinant antibody or antibody fragment in the cell, tissue or serum.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

In some embodiments the binding compound blocks binding to TACI and BCMA. In some embodiments, the APRIL binding compound of the invention includes one or more of the antibody CDRs (Complementarity Determining Regions) selected from SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; and in further embodiments, includes one or more antibody light chain CDRs of SEQ ID NOs: 12, 13, 14, 18, 19 and 20 and/or antibody heavy chain CDRs of SEQ ID NOs: 9, 10, 11, 15, 16 and 17. In some embodiments, the binding compound is a chimeric antibody, human antibody, humanized antibody or a fragment thereof.

In one embodiment, the invention provides a binding compound which binds to human APRIL comprising antibody heavy chain CDRs SEQ ID NOs: 9, 10 and 11, or variants of any said sequences; and antibody light chain CDRs SEQ ID NOs: 12, 13 and 14, or variants of any said sequences.

In another embodiment, the invention provides a binding compound which binds to human APRIL comprising antibody heavy chain CDRs SEQ ID NOs: 15, 16 and 17 or variants of any said sequences; and antibody light chain CDRs SEQ ID NOs: 18, 19 and 20 or variants of any said sequences. . M another embodiment, the kvenikm comprises a binding eomponnd which bind to team APRIL comprising as antibody heavy chain vt«fahle region comprising the amino add sequence of SEQ ID MO: 5 and a antibody light chain variable region comprising the ammo add sequence selected from die group of SEQ ID NO: 6,

In yet another embodiment, die invention comprises a binding compound which bind to human APRIL comprising a antibody heavy ebain variable region comprising the amino acid sequence of SEQ IB NO: 7 and a antibody fight chain variable region comprising the amino acid sequence of SEQ ID NO: 8«

In another mbodimmt the invention comprises an antibody, wherein the heavy chain has the variable region sequence of SEQ ID MO: 5 and Is joked to a IgGI constant region and the light chain has the sequence of SEQ ID NO: 6 and is joked to the ,¾ constant region. In particular,. the coastest region k fora mouse or human origin. More in pardeukr, the antibody is hAPRIL.O! A.

In another embodiment lire invention comprises m aitibody, wherein the heavy chain has hie variable region sequence of SEQ ID NO: 7 and is joined to a Ig<31 constant regkn and the light chsk has the sequence of SEQ ID NO: 8 and is joined to tbe at constant region. In particular, the constant region is from mouse or human origin. More hrparticnlar, the antibody is hAPRIL.03 A.

In another embodiment the invention comprises a variant of a Hading compound which hind to human APRIL, wherein any of said variaatfs) may comprise up to three amino acid modifications in the previous ideniliiad CDRs of each the autihody heavy and light chain variable regions.

In another embodiment the invention comprises a variant of a binding compound whleh binds to human APRIL, wherein any of said variaotfs) may comprise up to three amiao acid modificarions m each of the pre vio us identified CDRs in each of the antibody heavy aid light chain variable regions, hr another embodiment the invention comprises a. variant of a binding compound which binds to human APRIL, wherein any of said variant(s) may comprise up to three amino acid modifications in the previous identified CDR sequences In each of the antibody heavy and light chain variable regions.

The invention also comprises a binding compound that fully blocks the binding of APRIL with human TACI and at least partially blocks the binding with human J3CMA, M anufeer effi1&amp;>diment the invention comprises a binding compound feat felly blocks fee fending of APRIL wife human TACI Mid wife human BCMA. hi another embodiment the mvention. comprises a binding compound which Mad to human APRIL* wherein tits binding command Mods human APRIL with a Κ» of about 10 nM or lower; and blocks bmdiag of human TACI aud/or human BCMA to hamau APRIL with m 10.¾¾ of about 2 obi or lower,

The Invention also comprises a binding compound which fends to human APRIL wherein the binding compound has the same epitope specificity as the antibodies described above i&amp; competes for fee binding epitope of fee antibodies described above. ht some embodiments fee invention comprises a brndisg compomid which competes for a binding epitope on human APRIL wife any of fee mfebodies described above, aid binds human APRIL with a Kd of about 10 nM or lower. In particular, fee epitope on. human APRIL is fee epitope winch bind to the antibodies hAPEiL.01A and hAPRII..-,03A3 prcfeaMy hAPRILjlA,

In another embodiment the invention comprises a binding compound which competes for a hinding epitope on human APRIL wife any of the antibodies described above and binds to human APRIL wife about fee same Κ» as an antibody having a hsmy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising fee amino acid sequence of SEQ ID NO: &amp;

In another embodiment the invention composes a binding compound which competes for a hinding epitope on human. APRIL wife any of the compounds described above and binds to human APRIL wife about the same 1¾ as an antibody having a.heavy chain comprising fee amino acid sequence of SEQ ID NO: 7 and a light chain comprising fee amino acid sequence of SEQ ID NO: 8,

In another embodiment the invention comprises a binding compound which competes tor a binding epitope on human APRIL wife any of fee audhodles described above and blocks binding of humanTACI and/or human BCMA to human APRIL wife m ICsg of about 2 nM or lower..

In another embodiment the invention comprises a binding compound which hinds to the conformational human APRIL epitope SMP8HP (pmferahiy IRSMPSHPDRA) optionally supported by TLFE. and/or QDVTFTMOQ. &amp; yet a&amp;other embodiment the invention comprises a binding compound which. binds Μ some embodiments the binding compound of the im?ention Is a chimeric antibody or a fragment thereof

Isa another embodiment the binding compound of the invention is a human antibody or a fragment thereof.

In anotber embodtiaesrt &amp;e binding compound of the Invention Is a hutnarazed antibody or a fragment thereof. la. another embodiment the invention comprises a binding compound, preferably a hunmized antibody, with the above identified CDlTs and a human heavy chain constant region variant and a human fight chain constant legion variant, wherein each constant region variant comprises up to 20 conservatively modified amino acid

The Invention also comprises the 'binding compound as described above which inhibits the proliferation and survival of B~eei!s. lie invention also comprises onelelc adds eneodingthe anti-APRIL binding compound of the Invention. Included in the invention are nucleic acids encoding any one of the amino acid sequences enclosed In SEQ ID NOS: 5 to 20. Also Included vrithin the invention are nucleic acids comprising SEQ ID NOS 1,2,3 or 4. lit addition, the invention also comprises the nucleic acids encoding the variants of the amino add

The invention also comprises cells and expression vectors comprising: nucleic- acids

Further, the invention comprises a method of producing a binding compound of the invention comprising; (a) cultoring the host cell comprising a nucleic acid encoding an antibody or antibody fragment of the-invention in culture medium raider conditions comprising fee light and heavy cfeam variable regions; and <b) recovering the polypeptides from tie host cell or culture medium.

The invention also comprises compositions eomprMng a binding compound of file invention la combination wife a pbamaeeatieafiy acceptable earner or diluent The invention also comprise® a method of inhibiting the proliferation and/or survival of an Immune cell, comprising administering to a subject in need thereof a therapeatieally effective amount of a binding compound of the invention. la one embodiment, the method .may be used to treat cancer. In another embodiment, the method may be nse to teat an autoimniane or inflammatory disease.

In some embodiments, the invention comprises a method of inhibMng the pnhferaiion and/or survival of an immune cell, comprising administering to a subject In need thereof a fiiempeuficaliy effective amount of a binding compound of the invention, and further comprising measnring B eel proEfcmtion and/or survival &amp;x two ia a sample derived from fee sabj eef, wherein an. iafeibffion of fee prollferatioa and/or survival of

In other embodiments, the in vention comprises a method of inhibiting fee proliferation and/or survival of as Immune cell, comprising administering to a subject in need thereof a feempetstieally effective amount of a Imiding compound of the Invention,, and further comprising measuring B cell proliferation and/or survival ex vivo in a sample derived front fee subject, wherein an increase in B eel proliferation and/or survival predicts fee likelihood fete fee treateieai will he successful.

Thu invention also comprises m immunoeonpgate comprising an and-APRIL binding compound of the im'e&amp;tkm, linked to a feempeatie agent snob as a bacterial toxin or a mdiotoxKt Mondiiniting eaamples of cytotoxic agents include texol, cydoeh&amp;lasm B* mitomycin, etoposide and vincristine or other antimclabolites, alkylating agents, antibiotics and antirmtoties.

The invention also comprises a method of snMMting fee proilfertelon and/or sarvlval.ef an immmte cell, comprising eemtaeting an immune cell wife, a binding compound of fee present Invention. Ιη some embodiments,. anri-APRIL binding compounds cm be combined with a treatment thatis eonsid eredto besttmdard of care in cancer ortmioimmime or in^annaatoQcdiaease. Rationale for such eoittferoaiions Is i&amp;d concarmot increased ant»si»ii5M|>ii|oH, by aail~APElL will Induce or tkdtifefe initial clinical response to standard of cate ireMmeni, induce durable elmicd response aud long-term. immune control of disease. M anotlier emfeodiment the brnding compounds of the presmt ie%^ittoa are used diagnostically. embodiment the broding eompouuds of the invention m&amp; used to nseasare B cell proliferation and/or survival ex vivo in a sample derived fern the si^^ivhstpfo. tht;jb9|^i|N|l# c€the proliferation aai/orsurviwl of the B eel Indicates that the treatment with the binding compound as described here above should be continued..

In mother embodiment the binding compounds aceotriing to the mvmti on are isolated antibodiesM anhhody^ which bind to human APRIL,

The term kmilhody” mfers to any form of antibody that exhibits the desired biological activity, such as nihibitiug binding of a ligand to its receptor, or by kdnhMag ligand-induced signaling of &amp; receptor. Thus, “anybody* is used in the broadest sense and Speeifically covers, but is not limited to, roonockmal antiixfeies (including foil length monoclonal mitibodies), polyulonal antibodies, andmultispeciflc antihpdies (e.g, MspeciSo anybodies). tsA«tifcody fragmeuf’ and “antibody binding fragment” mem an%en-bindiBg fragments and analogues of an antibody, typically mdudlng at least a portion of the antigen binding or variab le regions (e.g„ one or more CDRs) of the parental antibody . An antibody Bsgmcnt retains at least some bfthe binding spedfeity of the patonfel antibody. Typically, an antibody fragment retains at least 10% of the parental binding activity when that: activity is expensed on a molar basis. Preferably, an antibody fragment rotaim at least 20%; 50%, ?0%, 80%, 90%, 95% or 100% or more of the parental antibody'’s binding affinity for the target. Examples of antibody fragments include, bat am not limited to, Fab, Fab*» F(ab% and Fv fragments; dlabodies; linear antibodies; siagle-cbam antibody molecules, e,g., se-Fv* nnibodies (technology front Cknmah); nanobodies (technology front Domairds); domain antibodies (technology front AWym); and miilii^jeoiSo antibodies formed from antibody fragments. Engmeered antibody variants me reviewed in Bolliger and Hudson* 2005, Nat A ηψφ fragment” is comprised of one light chain and the ChI sad variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

An wFc* region contains two heavy chain fragments comprising the €«1 and €h2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. A ”Fab* fmgntenf* contains one light chain and a portion of one heavy chant that contains the Ye domain, and the C domain, and also the region between tie ChI and C hs domains, such that an Interchain disulfide bond can be fomted between the two heavy chains of two Fab* fragments to fonn a.F(ab*) amolecole. A fragment*' contains two light chains and two heavy chains containing a portion of the constant region between the Cat and Cm domains, such that m Merchant disulfide bond is formed between the two heavy chains. A F(abs) 2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond . between the two heavy chains,

Tire "Fv region” comprises the variable regions from both the heavy and light chains, hut lacks die constant regions. A "smgte-^kain Fv antibody** (or "sePv antibody”) refers to antibody fragments comprising the Vh and % domains of an antibody, wherein these domains are present in a single polypeptide chain, Oeneraliy, the Fv polypeptide fbrlher comprises a polypeptide Baker between foe V« sad Vl domains which ©sables the se-Fv to form foe desired structure for antigen binding. For a review of scFv, see I%u:fohimJ994, The Phaiumao£MUXJY of Monocuxnal AmTBODras, voi 113, Itosenburg and Moose eds. Springer-Verlag, New York, pp. 269-315. See also, Jmemationa! Patent Application Publication No, WO 88/01649 sod US. Pat Nos. 4,946,778 and 5,260,203. A “foabody” is a small antibody fragment with two antigen-bmdmg sites. The fotgments comprises a heavy chain variable domain (Vs*) connected to a light chain variable domain (Vt) in the same polypeptide chain (Vh~Vl or VT -Ye). By using a linker that is too short to allow pairing between the two domains on toe same chain, the domains am forced to pair with die complementary domains of mother chain and create two antigeo-binding sites. Diabodles am described more folly In, e.g,, BP 404,097; WO 93/11161; and Holliger et ai, 1993, Proc. Nail lead Set DM 96,6444-6448. A Momain antibody fiagment” is an immimologisally factional immtmoglobulm fragment containing only the variable region of a heavy ebain or foe variable region of a light chain. In some Instances, two or mors Vh regions am covalently j oined with a peptide linker to create a bivalent domain antibody fragment The two YH regions of a bivalent domain antibody fragment may target the same or different antigens.

As used herein antibody hAPRlL.01 A is a mouse antibody wherein tire heavy chain has the variable region sequence of SEQ H) NO: 5 and Is joined to a IgGI constant region and the light chain has the variable region sequence of SEQ ID NO: 6 and Is joined to the κ constant region, Antibody hAPB.IL.03A is a mouse antibody, wherein the heavy chain has foe variable region sequence of SEQ ID NO: 7 and is joined to a IgGI constant region and the light chain has foe variable region sequence of SEQ ID NO: 8 and is joined to foe a constant region,

An antibody fragment of foe invention may comprise a suffici ent portion of foe constant region to permit dimerization (or multimeiisslion) of heavy chai ns that have seduced disulfide linkage capability, for example where alleast one of the hinge cysteines notimliy Involved in Iker-heavy chain disulfide linkage is altered as described Iwiuu la another embodiment, m. antibody fragment, for example ose that comprises the Fc region* retains'at least oae of the biological functions normally associated with the Fe region whoa present la m intact antibody, sock as FoRn binding, aatibody half life modulation, ADCC (antibody dependent cellular cytotoxicity) function, and/or complement binding (for example, where the antibody has a glycosylation profile necessary for AJDCC fenetion or complement binding).

The tern* ^cMmeric'* antibody refers to antibodies in which a portion of the hea vy .and/or light chain is identical with or homologous to corresponding sequences In aitibodies derived from a particular species or belonging to a particular antibody class or subclass, while the rerttamdcr of the cbaln(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological acti vity (See, for example, U.S, Pat, Ho. 4,816,567 and Morrison el el., 1984, Prmx Nmi Acad Set USA 81* 6S51-6855).

As used herein* the term %umanked antibody” refers to forms of antibodies that contain sequences from nondtnman (c.g,, murine) antibodies as well m human mnibodies. Sucli antiJhodies contain minimal sequence derived from nondmman immrmoglobulB, In general, Ihe humanked antibody will comprise substantially all of at least one, and typically two, variable domains, la which all or substantially all of the hj'pervariable loops correspond to those of a non-hinn&amp;n hnmmioglohnlia and all or substantially all of the FE regions are those of a human hamunoglobnlia sequence.

The humanked antibody optionally also will comprise at least a portion of an immimoglohulm constant region (Fc), typically that: of a human hmnunoglobidm. The tananked forms of rodent antibodies will essentially comprise the sane COR sequences of the pamuM rodent antibodies, although certain amino acid substitutions may be included to increase affinity. Increase stability of the humanked antibody* o? for other reasons, However» as €BR loop exchanges do not uniformly result in an antibody with the same binding properties as the antibody of origin* changes In framework residues (FR), residues involved to CDR loop siippc&amp;t. might also be mtrodueed in humanised antibodies to preserve antigen binding affinity (Kabat eiaL 1991,,/. Immunol. 147,1709).

Hie term “antibody'' also Includes “fully human” antibodies, uu, antibodies that comprise human inimunoglobohnprotem sequences only, A folly human antibody may contain, murine carbohydrate chains if podueed in a mouse. In a mo« cell, or in a hybridoina derived front a mouse cell Similarly * “mouse antibody” or “rat antibody” refer to an antibody that eoniprlses only moose or rat immunoglobulin, sequences, respectively. A fully human antibody may he generated In a human being, in a transgenic animal having human immunoglobulin germline sequences, by phage display or other molecular fetologies! methods. Also, moomhiuaut immuno^ofenlius may also be made to transgenic mice. 8m M«ds2 et at, 1997, Nature Genetics 15,146-156, See As Abgenli. and Medarex technologies.

The antibodies of the present invention also Include aatihodles with modified (or Mocked) Fc regions to provide altered effector functions. See, e.g., U.S« Fat. Mo. 5,624,821; ¥02003/086310; W02005/I20571: ¥02006/0057702; Frusta, 2006, Αώκ Drug Delivery Rev, 58:640-656. Such modffication can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (subsfituiions, deletions and insertions), glycosy'lation or deglyeosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antihodies in therapeutic antibodies, and a longer half-fife would .result in less frequent dosing, with the concomitant increased convenience and decreased use of material . -See firesta, 201)5, J. Allergy Clin. Immimim, 731 at. 734-35.

The antibodies of the pmsmit invention also include antibodies with intact Fe regions that provide fid! effector functions, e.g. antihodies of Isotype IgGL which induce compiement-dependent cytotoiddty (GDC) or antibody dependent eolMar cytotoxicity (ABCC) hi the a targeted cel. -

The antibodies may also fee conf ugatod (&amp;&amp;* co valeatly linked) to molecules that Improve stability'' of die antibody during storage or increase the halt-life of the antibody in vim, -Examples of molecules tiiat increase the hall-life ate albumin (e.gf3, human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylaled derivfeives of antibodies can fee prepared using techniques well team® in the art. See, e.g,a Chapman, 2902,zMv, DmgDelm Rev, 54,531-545; Anderson andTomasi, 1988, X Immunol Mejk&amp;ds 189» 37-42; Suzuki ei aLs 1984, BmcMm, Biophys, Acta 7§8, 248255; aid Brekke aid Sandlie, 2003, Nature Rev, 2,52-62,

Antibodies used is the present mveuiion will usually bind with at feast a Kjj of about 1CT3 M, more usually at least 10**M, typically at least 19*7 M, more typically at least 10“ SMS preferably at feast: about 10*®M, and more preferably at feast 104e M, and .most preferably at least 1041 M. See, e.g,t Presta, etol, 2801, Ifiromk Hmmast 85,379» 389; Yang, eiaL 2001, Crk Rev, Oncol, fkmatol 38, 17-23; Carnahan, eta!., 2003, Clin. Cancer Rex. (Suppl.) 9 3982s-3990s.

Aatsfoody affinities may be determined using standard analysis.

The term %ypervariable region)8 as used herein, refers to fee amine acid residues of an antibody which are responsible for antlgen-hindfeg. The hypervariaMe region comprises amino acid .residues from a ^complementarity detennlning region*’ or ^CDR)5 defined, by sequence atignment, for example residues 24-34 (LI)* 50-55 (L2) and 89-97 (L3> .1» the light chafe variable domain and 31-35 (HI), 50-65 <B2) and 95102 (H3) in fee heavy chain variable domain; see Rabat et al, 1991, Sequences of pmtems of Immanologfeal Merest, Stb Ed. Public Health Sendee, National festltntes of Health, Befeesda, Md< and/or those residues from a feypervari&amp;blc loop* (H¥L), as defined structurally, tor example, residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in fee light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (M3) in the heavy chain variable domain; see Chofeia and Leak), '1987, J. Mai Bial. 196, 901-917, "Fjramewoxk* or ”FRW .residues are those variable domain residues other than fee hypervariable region residues as herein defined:,

An ^isolated” antibody is one that has been identified and separated and/or recovered horn a component of its natural environment Contaminant components of Its natural eaviroranent are materials that wonld interfere wife diagnostic or thempcBtic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or aonpmielnaceona solutes, In gome embodiments, the antibody will be'psiSed-0) to pester ftaa 95% fey weight oi aMifeo# ss dshmm»ed fey the Lowry method, and most l^^rabty mom than 99% fey weight, (2) to a degree saffidept to ofehda at least 15 residues ofN-iemhaa! or internal amino acid sequence by use of a spuming cop s^etnhaiv or (3) to homogeneity by SDS-fAGE under reducing ornonredueing conditions using Coomassle bins op, preffrably, silver staiu, isolated m&amp;bodg-mjfadm the antibody msitu within mconthinani cells since at least one component of the Mtibody*s aamtat environment willnot fee pmsepb Grdmaiily, however, isolated a^body will fee fHeptued fey at least one purification step.

An "isolated* nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic add Molecule with, which it is ordinarily associated M the natural source of dm antibody nucleic add. An isolated auclde acid molecule is otfear tfeau in the form or setting is wMch ft is found in nature, Isolated nucleic add molecules therefore are distisgaisfeed fern the nucleic add nteleeule as it exists in natural cells. Ilowever, an Isolated nucleic add moiecule inehides a sucldc acidmolecule contained ia-.edtts that ordinarily express the andfeody where, ibr example^ t&amp;e bueleic acid molecule is in a chromosomal location different fern that of natural cells.

The antibody" as used herdn refers to an antibody obtained tom a impulsion of smfestaniM antibodies,theindhddtsal mtihodies comprising the population are identical except for possible iMarally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly speeiffe, being directed against a single antigenic site. Furthcrnime, in contrast to conventional (polydosal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is dhrooted against a single determinant on the antigen, The modifier "monoekupr Indicates the character of the antibody as being obtained Iroroa substantially homogeneous popnlatlos. of antibodies, and is not to be construed as tempting production of ibc antibody by any particular method. For example, the monoclonal mtibodies fe be nsed in accordance with the present Invention may be made by the hybridoma method fiM- described by Kohler et al„ 1975« Mame 256, 495* or may be made by reeomlnnani BNA methods (see, for example, U.S. Fat No. 4,816.,567). The feiOBoekmsI aatlbodles^ may also be isolated Bom phage aotlfeody libraries «sing the techniques described in Clacksou et ais 1991, Nature 352,624-621 and Marks et ah, 1991,1 Mol Biol 222,581-597* for example. The .monoclonal antibodies harem SfsedfiotUy include fehimerie” antibodies.

As used herein, tire term ‘Immune eel” Includes cells that are ofhematopofetie origm and that play a role in the hmntme rospotm Immune cells include Iwnphoeytos, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, feasopMls* and granulocytes.

As used herein, an ^immunoeonjugate58 refers to an miti-APRIL antihody, or a feagrnent tbeTeof. eonjugated to a therapeutic moiety, such m a bacterial toxin, a cytotoxic drag or a radiotoxin- Toxic moieties can be conjugated to antibodies of the invention using methods available in the art

As used herein, a sequence **vadaaf* refers to a sequence that differs from file disclosed sequence at one or mom anilno add .residues bnt which retains the biological acti vity of the resulting molecule. “Conservatively modified variants85 or “conservadve amino acid refers to substitutions of amino acids are kaowato those of skill in this art and may be made generally without altering the biological activity of fire resdtfeg molecule, Those of skill in this atl recognise that, in general, single amino acid substimdons in nonessential regions of a polypeptide do not substantially alter biological activity (see, e,g;, Watson, et ul, Molecular Biology of the Gene, He Beniamin/Cummisgs Pub. Co,». p< 224 (4th Edition 1987)). Snob exemplary suhsfltutieos are preferably made in. accordance with those set forth below as fellows:

Exemplary Cmison^ative Amino Acid Substitutions

As nsed herein, the testa ‘‘about** refers to a value that is within m acceptable mm range for the particular value as determined by one of ordinary skill is the art» which will depend In patten hew the value Is measured or determined» i*, the limitations of the measurement system. For example, "about* can mean within i or more than 1 standard deviation per the practice In the art Altematlvely, ’’about" or "comprising essentially of" can mean a range of up to 20%. Furthermore, particularly' with respect to biological systems or processes, the terms am mean up to an order of magnitude or up to Mold of a value. When particular values are provided In the application and claims, unless otherwise stated, ibe meaning of‘’about" or “comprising essentially of* should he assumed to be within m acceptable error mage lor that particular value. “Specitically” binds, when .referring to a lipmdAeeeptef, aadhody/antigea, or other binding pair. Indicates a binding reaction which Is determinative of the presence of the protein, e.jp, APRIL, in a heterogeneous population of proteins and/or other biologies, Thus, under designated conditions, a specified iigand/antigeh binds to a particular reeepmr/aBtibody and does not bind in a significant amount to other proteins present in the sample. ‘hMmMstipom® tod %eap8M” a« n applies to m animal, human, experimental subject, cell, tissue, organ, or Mological: laid, «e^«^c<mtaK^:pi8R exopnous pharmacepeal, therajtoife, diagnostic agent, or composition to the animal, human, subject, cell, issue, Μ|βη» or Mologieal Brad. iSA<pimstj;ati0a,i aad iftreatmeat” caa refer, e.g,, to therapeutic,. pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell meom|sgpos contact of a reagent to the cell, as well as contact of a raageot to a fluid, where the tide! is in ecmtact with the cell ‘^dpnlstmtiaa4* and also pptB in vitro and ex vivo treaaeeats, e,g,, of a cell, by a reagent, diagnostic, binding composition, or % another cell Μο»ό«ϊ«ή»1 Anifepdtes

Monoclonal antibodies to fcumao APRIL can be made according to knowledge and skill in the art of peeiteg test subjects with human APRIL antigen and then isolating hybridomas expressing antibodies having the desired sequence or fpetional Mamramsics. DMA ^codiagihe maaoclmtsl: aaibodtes is readily isolated and sequenced «sing conventional procedures (e,g., by using oligonucleotide probes that are capable of binding specliioaliy to genes encoding the heavy and Hght chains of the monoclonal antibodies), The hybridoxna cells serve as a preferred source of s uch DMA, Once Isolated, the DMA may be placed into expression vectors, which Me then transfected Mo host cells such as E, cell cells, simian COS cells, Chinese hamster ovary (CM0) «ells, or myeloma cells that do not otherwise produce imaiimoglobdm protep to obtain the synthesis of monoclonal antibodies imthe raeomfelnarahosi cells, ReeomMnant pmdoetion of antibodies will he described in more detail below, &amp; a fethesr embodiment, antibodies or antipdy fragments cm he isolated fena anybody phage libraries generated eskg tbe teMinqnes desMbM k MeCafferty et af, 1990, Nature., 348,552-554, CMkson et al,s 199if Nature 352, €14*628, and Mrafe et at, 1991, J, Mol Biol 222, $81-59? desMM tire isolation of mmme and human antibodies, respectively, using phage libraries, Subsequent publications describe the production of high affinity {«M range) hwsm antibodies by chain shuffling (Marks et al„ 1992, Ekt/Techming?* 18» 779-713), as wall m combinatorial mfection and in vivo tecomhimtlm as a strategy for constructing very large phage libraries (Waterhouse et at, 199% Nuc, Acids, Km, 11,2263-2266). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridonm techniques for isolation of monoclonal antibodies. CMraerie Antibodies

The antibody DNA also may be modified, for example, by substituting the coding sequence for human heavy- and iigbbehaln. constant domains in place of the homologous murine sequences (IIS, Fat, No. 4,816,567; Morrison* et al, 1984, Proc. NadAe&amp;d jSet USA, 81,6851), or by covalently joining to tie ImmimoglobiiM» coding sequence ail or part of the coding sequence for noo~hnmrmogloholln material (e.g., protein domains). Typically such non-immunoglobulin material is substituted for the constant domains of an antibody, or is substituted tor the variable domains of one antigen-combining si te of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigenmombMag site having specificity for a different antigen,

HuMMbsed and Human Antibodies A humauteed antibody bas one or mom amino acid residues from a. source that is nonhuman. The noo-human amino acid residues are often referred to os “import” residues, and are typically taken feom an “import* variable domain, Humanimtion can be performed generally following the method of Winter and co-wofkers (Jones et al, 1986, Nefure 321, 522-525; lleohmann et al., 1988, NaUm, 332» 323-327; Yerhoeyen et al,, 1988, Science 239,1534-1536), by subsrituling rodent CDEs or CJPR sequences lor the corresponding sequences of a human antibody. Accordingly, such ffiumaulaed* antibodies are antibodies wherein substantially less than as intact human variable domain Ms been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some 1¾ residues are substituted by residues from analogous sites k non-human, for example, rodent antibodies.

The «choice of human variable domains, both light sad heavy, to he aged la oinking the feinaanfeed antibodies is very impoftant to reduce antigenicity, According to the so-called "best-fit* method, the sequence of the vari able domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. lire human sequence which is closest to that of the rodent is then accepted as the human Ihsaework (PR) for the iinmaniEed antibody (Sims et at* 1987, J. Immm&amp;l 151» 2296; Choihia et a!,, 1987*«/ Mol, Biol 196* 901), Another method uses a parhcular hmnework derived lost foe consensus sequence of all human antibodies of a particular subgroup of fight or heavy chains. The same framework may he used for several different humanized antibodies (Carter et at.» 1992* Pme. Natl Acad Sci USA 89* 4285; Presto et al„ 1993» J. Immnol 151,2623),

It is further important that antibodies he humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a proterred method, humanized antibodies are prepared fey a process of analysis of the parental sequences and various conceptual bumanized products using threedimensional models of the pateniai and h umanized sequences, lluee-dlmessional immunoglobulin models are commonly available and are familiar to those skilled in the art, Computer programs are available which illustrate and display probable threedimensional eonformational slmeturos of selected candidate immunoglobulin sequence. Inspection of these displays penults analysis of the likely role of the residues in the iunotioniag of the candidate inummoglobulfe sequence, be.» the analysis of residues that influence tire ability of the candidate ImmunoglobulM to hind its antigen. In this way, FR residues can be selected and combined horn the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for tire target auhgcH(s),is achieved. Irt general, the CDR residues are directly and most substantially involved in influencing antigen binding.

Humanization of antibodies is a straightforward pote&amp;i engineering task, Nearly all murine antibodies can be humanized by CDR grafting, resulting in the retention of antigen binding, See» Lo, Benny, K.C., editor, in Antibody Engineering: Methods ami Protocols, volume 24S, Humana Press, New Jersey, 2004.

Alternatively, It Is now possible to produce transgenic animals (e,g>, mice) that arc capable), upon Ixmnmuzaiion, of producing a fell repertoire of human antibodies in fee absence of endogenous feimxinoglobidia production. for example. It has been described diet the homozygous deletion of fee antibody heavy-chain joining region (JH) gene la cMmeric and, getm-llne mutant mice results in complete inhibition of endogenous anti body production,. Transfer of the human germ-line immtmogloballa gene array la such germ-hne mutaui mice will result la fee prediction of human antibodies upon antigen challenge. See, e.g., Jakobovits et a!., 1993, Free. Nmi, Acad Sci USA 90,2551; Jakobovits et a!., 1993, Nafom 302,255-25$; Bmggenaaaa et al, 1993, Year iniMmmoiogp 7,33; and Dueliosal et at, 1092, Nature 355,258. Human antibodies can also be derived from phage-display libraries (Hoogenhooni et at«, 1991, d Mol MM. 227,381; Matte et at, d Mol MM 1991,212,581-597; Vaughan et 1996, Nature Biotech 14,3 09),

Amino acid sequence variants of humanized imi-APRIL antibodies are prepared by mboduciag appropriate nucleotide changes into fee humanized aufeAPRlL antibodies* DMAs, or by peptide synthesis. Such variants include, for example, deletions from, and/or Insertions into, and/or subshteiions of, residues within fee amino add sequences shown for fee humanized and-APRIL antibodies. Any combination of deletion, insedioB, and substitution is made to arrive at fee final construct, provided feat fee final construct possesses fee desired characteristics. The amino add changes also may alter post-translatioaai processes of fee humanized and-APRIL antibodies, such as changing fee number or position of giycosyMion sites, A oseJM method for identification of certain residues or regions of the humanized and-APRIL antibodies polypeptides feat am preferred locations for .mutagenesis Is called *alanme soanmog mutagenesis," as described by Gttojimgham and Wells, 1989,

Science 244,1081 -1085, Here, a residue or group of target residues are identified (e.g., charged, residues such as Atg, Asp, His, Ly s, and Gla) and mpfeced by a neutral or negatively charged amino add (most preferably alanine or polyaiaalne) to affect the mtetactibn of fee amino acids wife APRIL antigen, The amino add residues dmnonstmting fimcdonal smritivity to fee sobshtufems fees am refined by iferodoelog further or other variants at or for, fee sites of substitution, Thus, while the site for miroduciog an. amino acid sequence variation is predeteimned., fee nature of the mutation, ρβτ s&amp; need not be p-edeierrmned. For «sample, to analyse fee performance of a mutation at a given site, Ala scanning ox mndom mutagenesis is conducted at the target codon or region and the expressed humaufeed anthAPRIL antibodies' variants are screened lor the desired activity.

Ordinarily, ammo'acid-sequence variants of fee humanized anii-APRIL antihodies will have an amino acid sequence -having at least 75% amino acid sequence identity wife fee original humanized antibody amino acid sequences of either fee heavy or the light chain mote preferably at least 80%, mom preferably at least 85%, mom preferably at least 90%, and most preferably at least 95%, 98% or 99%; Identity or homology wife respect to this sequence is defined herein as fee percentage of ammo add .residues In the candidate sequence feat a-e identical wife fee humanized residues, after aligning fee sequences and introducing gups, If necessary, to achieve fee maximum percent sequence identity, aid not considering any conservative snhsiitntions as pari of tie sequence identity, Hone of N-tcrariual, €4emaoat, or internal extensions, deletions, or Insertions iMo fee antibody sequence shall be construed as aSncting sequence identity or homology.

Antibodies having fee characteristics identified herein as being desirable in humanized anti-APElL antibodies can be screened for inhibitory biologic activity in v&amp;m or suitable binding affinity. To screen fer antibodies feat bind to feu B€MA or TACI epitopes on human APRIL bound by an antibody of interest (e.g,, those feat-block binding of APRIL), a routine cross-blocking assay such as feat described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be pcrlomicd, Antibodies feat bind to fee same epitope are likely to crossblock In such assays, bat not all cross-blocking antibodies mil necessarily bind at precisely fee .same epitope since cross-blocking may result from sterie Mndrsnee of . antibody binding fey antibodies bind at overlapping epitopes, or e%a nearby nonAlternatively* epitope mapping, eg,, as described. In Clampe et ols 1995, </ Biol Chem. 27% 1388-1394, cm be performed io determine whether tie antibody binds an epitope of interest "Alanine scanning mutagenesis»* as described by Cmminghaor and Wells, I §89, Science 244,1081-1085, or some otber form of point mutagenesis of amino acid residues in human APRIL may also be used to determine the fencifona! epitope for mifi-APRIL antibodies of foe present invention.

Additional antibodies binding to the same epitope as m antibody of the present Invention may be obtained, for example, by screening of antibodies raised against APRIL for binding to foe epitope, or by mnnunMaiion of an animal with a peptide comprising a fragment of human APRIL comprising foe epitope sequences (eg,, SCMA or TACi), Antibodies font bind to foe same fenotionai epitope might be expected to exMMt stellar biological activities, sad» as Mocking receptor binding, and such activities can he eonlirmed by fonetional assays of lb© antibodies.

Antibody afisltiea may be determined «sing standard analysis, Preferred binding eomponnds sticb as e,g< bnnianlzcd antibodies are those tint bind brnnan APRIL with a K4 value of no more than about l x !0"f; preferably no more than about 1x10“^ mom preferably no more than about lxtO*8; aid most preferably no more than about IxICT18 or even Ixl0''u M,

The himianfoed antibody can be selected from my ©lass of imnnmoglobalms, including Igbl IgG, IgD, IgA, and IgB. Preferably, tire antibody is an IgG antibody. Any isotype of IgG can be Used, including IgGn IgCri, Ig%5 and IgG*. Variants of the IgG isotypes are also amtemplaied, The humanized antibody may comprise sequences from, more than one class or isotype, Opfruiizaiion of foe necessary constant domain sequences to generate the desired biologic activity is readily achieved by screening foe antibodies in the biological assays described In foe Examples,

Likewise* either class of light chain can he used in the compositions and methods herein. Specifically»· kappa lambda, or variants tbereof are useful in foe present compositions and methods. lie ^ίκκίΐβδ and mrtifeody fragments of the Mvention may also be conjugated with cytotoxic payloads such as cytotoxic agents ot tadiomtcleotides such as mTc^Y, ml% 2i\ uC,m$,me% l% l% 3% SiCt, S5Tof s%.a> &amp;>Co, 59Fei 57Se, mBi\, mm>mm47Sc, ^1¾aod4% 3% and ^Fe.Snch antibody eenfogates may be used in femnuinotheiapy to selectively target and Mil cells ejqptsmng a target (the antigen for that antibody) on their surface. Exemplary cytotoxic agents incladeiicta., vinca^alkaloid*^meihotreocaie, Psuedomonas exotoxin, aaporin, di^di^atoxk, cispiatAn, &amp;xorubicm, abris toxin, gelonin and pokew^ antiviral protein.

The aiiibodics and antibody fragments of the mvc.ni.km may also be conjugated with frnemseentor ehentilhiminexeeni labels, including iooropbores such as tare earth cbelates, fluorescein and its derivatives, rliodamine and its derivatives, Isothiocyanste, phyeoer>frimy phycoey&amp;nin, allopbycocyanm, o~phti.*aladehyde, fiuorescamhie, *s2Eu, dansyb sntbeliferone, loMfein, lunkml label, isolunnoal label an. aromatic acridb.uxrm ·> * ester label, as Imidaxoie label, anactidlmmm salt laid, an oxalate ester label, an. aeqnotin MofeFavidin, spin labels and stable free radicals,

Any .method known m the art for conjugating the antibody molecules or protein offileeoles.-of theiaveniots to the various moieties oiay be employed, ioelnding those methods described by Hunter et ah, 1962, Nature 144,945; David ei at,. 197% Bioehemistry 13,1614; Fain 1981, J. Immunol Meth. 40,219; ^d Nypen, 1., 1982, Hlsteshem. and Oytochent. 30,407. Methods for aanjogating antibodies and proteins are conventional and well known in &amp;g it.

Antibody ftedioatinn

When using recombinant teclnaqiies, the antibody can be podaeed ktr^llnlarly. In feeperipiasmic space, or dimedyseereted Into the medium. If the antibody is produced inttaediitiarly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for «sample* by cenhd&amp;pdos or nlWSltmdm Carter et aL, 1992, Bio/Techmhgy 10,163-167 describe a procedure for isolating antibodies winch arc secretedto the peripla^aic ^ce rfB. cofr. Badly, cell paste is tlmwed m tire presence of sodlom acetate (pH 3.5), BDTA, and phenyhnetbyisidfonylinoride (PMSF) over abottt 30 min. Ceil debris east be removed by eenMfegaiion. Where die antibody is secreted into the medium, snperaatants from such expression, systems are generally first concentrated using a commercially available· protein coacentratio» filter; for example, an Amicon or Millipore Pelllcon uhrafilfration unit A protease Mnbstor such as FMSF may be Included I» any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from toe cells can he pudfied using, for example, feydtmylapaiito ohromatogmphy, gel electrophoresis, dialysis, and affinity cinematography, with affiuiiy oliromatopaphy Mug the preferred purification teckiique. The saitahility of protein A as an affinity ligand depends on toe species and isotype of any inmiunoglohulln Fe region that is present in the antibody . Protein A can fee used to purity antibodies that are based on human .gamma.!, .gamma,!, or .gnmma.4 heavy chains (landmark at nf, 19S3, X Jmsmtol Meik 62,1-13). Protein Θ is recommended lor ail mouse isotypes and for human .garnmaJ (Guss e£alt 1986, , BIMBO /5,1567“ ! 575). The matrix to which the affinity ligand is attached is most often agarose, hut other matrices are available. Mechanically stable matrices such as controlled pom glass or poIy(slyTOiiedlvinyi)benr«n.e allow for faster .flow rates and shorter processing times than can be achieved with agarose, Where the antibody comprises a CH3 domain, the Bafcerbond ABX™ msin. (1, T, Baker, PMllipsbnrg, NX) is useful for pimfication. Other techniques tor protein purffieatlon such as finotionation on an iomexchange column, ethanol precipitation, Reverse Phase HPLC, ckomntogtapby on silica, chromatography on heparin SEPIMR0SBTM chronmiography on an anion or nation exchange resin (such as a polyaspartie acid column), chwxmMofocusmg, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to he recovered, in one einbodlment, the glycoprotein may be puttied using adsorption onto a lectin substrate (e,g. a lectin affinity eolarnn) to remove fueose-coniainmg glycoprotein from the preparation and thereby emidi lor fitcosefrree glyeopmtein.

Pharmaceutical Foimmlatitms 1¾¾ invention comprises pharmaceutical foundations of a» APRIL binding compound. To prepare phtrraaceatical or sterile compositions, tire antibody or ffisgment thereof Is admixed with a phammceixficaliy acceptable earner or excipient, see, e.gk, Remington** PharmiceutiCiOi Smemm and U.S. Fharmampeia: Naiiami Farmdmyt Mack Publishing Company, Easton, PA (1984). Fonnnlaiions of tierapeotie arid diagnostic agents may ho prepared by mixing with physiologically acceptable carriers, excipients* or stabilisers in the form of, e.g., lyopMIixed powders, slurries, aqueous solutions or stspensions (see, e,g,, Hardman, et &amp;ls 2001, Goodman md Giiman '$ The Pharmacal&amp;gimi Basis ofTkempentkst McGrawvHill, Now York, NY; Chamaio, 2000* Remmgkm; Pie Sewme and.Practice of Pharmacy* Lipphieott, Williams, and Wilkins, New York, NY; Avis, et aL (eds,), 1903, Pkarmammieoi Dosage Form: Parmmmi Medheotiam* Marcel Mte, MY; Llebermao, et at (eds,), 1,990, Pharmaceutical Bmage Forms; Tablets,. Marcel Bekker, NY; Liebcrman, et &amp;L (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Siyetems* Marcel Dekker, NY; 'Weiner and Kotkoskie, 2000, Excipient Pm&amp;iy am18qfefy% Marcel Bekker, Inc., New York, NY).

Toxicity and therapeutic efficacy of the antibody composMons, administered alone or in combination with an immunosuppressive agent, can be determined by standard pharmaceutical procedures In cell cultures or experimental animals, for detemiining the LD&amp; (the dose letbal to 50% of the population) aid tbe £1½ (the dose thempeihlcaily effective in 50% of the population), The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LBso sod EDsg. The data obtained tot these cell culture assays and animal studies can be used in formulating a .range of dosage for use in humans. The dosage of sack compounds lies preferably within a range of circulating concentrations that include the ED$8 with little or no toxicity- The dosage may vary within rids range depending upon Ere dosage form employed and die route of adminlsiration ntilked.

Suitable routes of admiaistfation iaclnde parenteral administration, such as Intramuscnlar, intravenous, or subeutansous achtniiisltatlon and oral adnihitstration.

AfeMsMkm ^ asiibefdy Med inthe pJtesmas^^ to practice 1½ method of the present invention can he corned oat in a variety of conventional ways, sadt as otst iopshoa* MtalahoBj. topical applieaiioner cutaneous, subcutaneous, ktraperitone&amp;l, pamntemL intmarterial or intravenous injection. Ια one embodiment, the tedlsg compound of the inventio» is admMstered inhrtvenously. In soother embodiment, the binding compound of the invention is mbtuiusie^d subcutaneously,

Albnuatlvely, one may administer the antibody M a local rather than systemic manner, ibr example, via injection of the aaiibody directly into the site erf action, often in a depot or sustained release formniatlom Fiathsmose, one^a^'':adminis^¾e;.8at*bo^ in a targeted drug delivery system.

Gmdauce in selecting appropiate doses of aahtedtea* cytokines, mtd snmll molecules are available (see, e.g„, Wawrzynczak, 1996, Jnhbody Jlwrapp^ Bios Scientific Pub, Ltd, OxfedshimfUK; Kresina (ed-), 1991 ^MϋnmhmίAmihύdm'f CyiiMmsmtd Arthritis? M&amp;raei Dekker, Hew York, HY; Bach (ed,), 1993, M&amp;n&amp;clamii Antibodies andFepfide XHsewss? Marcel Bekker, Hew York, NY; Baefi,

Med 34%601-60%Milgrora, etaL? m%NewBngl I MM 34J, 1966-19^; Sl^non, &amp;αΙ*,2ΜΙ> New Engl J, Med 344,783-792; Bcmaminovitz, etal, 2m,mrnMngl <1 Med 342,613-619: Ghosh, etat., 2W\New Engl d Med S4S, 24-32; Lipsky, ei ok, 2000, AW Engld Med 343,1594-1602).

Deiemmadion of the appropriate dose is made by fire clinician, e„g., using pammeters or factors known or suspected in the art to affect treatment or predicted to affect Ocatment Generally, the dose begins opdmam dose and It Ik increased by small mcremeste ihetusfier until the deshed or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of e.g., the inffaamtaiion or level of inflammatory c>roMnes piodttced. A p.mferred dose potoeol is one involving fie mavhnal dose or dose frequency that avoids significant ondeshable side effects. A total weekly dose is geueraSy at least 0.05 pg4g body weight, mote generally at least 0.2 pg/kg, most generally at tost 0,5 pg/kg, typically at feast 1 pg/kg, mm typically at feast JO pg/fcg, most typically at least 100 pg/'kg, preferably at least 0.2 mg/kg, mom preferably at feast 1.0 mg/ig, most preferably at least 2.0 mg/kg, optinsaliy at tost 10 mg/kg, more optimally at least 25 mg/kg, md most optimally at least 50 mg/kg (see, e.g,, Yang* et ®ls 2003, New Engl J. Med M% 427-434; Herold, ei <£, 2002, Nm BngL J. Med 34€, 16924698; Lm, et alf 1999., J. .Nettwl Neurasttfg, Psydr 67,451 »456; Fortielji, et ol., 2003, Comer Imnmml Immmaiker. 52,133444). The desired dose of a small moleenle therapeutic, e.g,, a peptide mimetic, natural product, or organic chemical, is about die same as for an antibody or polypeptide, on a mofcs/kg basis.

As used herein, 4nhibiff or Mtreaf* or %eatmenf5 includes a postponement of development of the symptoms associated with disease and/or a induction in die severity of such. symptoms that will or are expected to develop with said disease. The terms ferfber include amdioratisg odstlng symptoais, preveating additional symptoms, and ameliorating or pmvenfing the underlying causes of such symptoms* Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject rath a disease,

As used herein, the toon isfeempeudea!Iy elective amouofi’ or "elfeetwe amount* refers to an amount of m anti-APRIL antibody or fragment thereof, that when administered alone or in combination with an additional therapeutie agent to a cell, tissue, or subject is effective to peeve»* or ameliorate the disease or condition to be treated. A. therapeutically clfectlvc dose furdter refers to that, amount of the compound sufficient to result hi amelioration of symptoms, eg., treatment, healing, prevention or amelioration of the relevant medical, condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an mdividual active ingredient admhnsteed alone, a therapeutically etYeotlve dose refers to that ingredient alone. When applied to a comhination, a therapeutically eflnetive dose refers to combined amounts of the active mgrediente that result in die therapeutic effect, whether admimstemd in combination, serially or shnnltaneonsiy, An efieetlve amount of therapeutic wall decrease the symptoms typically by at least 10%; usually by at least 29%; preferably at least about 30%; more preferably at least 40%, and moat preferably by at least 50%.

Methods for eo-admimstmtloB or tnsatoeat wife a second therapeutic ageat are well tow is fee art see, e.g,, Berdmms* ei&amp;L (eds.), 2001, Goodman emdGUmem ’$.The Pharmacological Masts ofTheropeMks, 10&amp;cd,5 Mo€Raw411I, New York, NY; Poole and Peterson (eds,), 2001^ Pharmacothcri^euticsforAdwmced Practice: Λ Practical Approach, Lippineott, Williams <k Wilkins, Phils,, PA; Chahner mid Longo (eds,). 2001, Cancer Chemotherapy and Bwtherapy, Lippincoir, Williams &amp; WIlMns, Phi la., PA.

The pharmaceutical composMon of the invention may also contain other agent, including but not limited to a eyfetsodc, eheinofe«mpefeie, cytostatic, aafemrgiogemc or mdmtefeholtte agent, a tumor targeted agent an immune sfenutafeig or immune modolatiog agent or an antibody mpfugatcd to a cytotoxic, cytostatic, or otherwise toxic agent The pharmaceutical composition can also be employed with other therapeutic modalities such as surgery, chemotherapy and radiation*

Therapeutic Uses for fee Antibody and Antibody Fragments of the Invention The antibodies and antigen binding ingaieuts of the invention, which spedlically bhid to human APRIL, can be used to treat several diseases in which fee activity of APRIL is central to pathology. Broadly speaking this includes cancer, autoAmmimity, hiSammatniy diseases and potentially multiple sclerosis, a CMS disease.

Cmmr

The antibody or aatigeo binding fragments of the invention which specifically hind APRIL can he used to treat cancer. ihufemed eaneem whose growth and survival may be inhibited by fee invention include any cancers known to express APRIL- and depend on this for proliferati ve signals, Mon»limiting examples of snch cancers rnctude several B cell malignancies, such as Clitome Lynaphocytic Lctfeaernia (CLL), Multiple Myeloma, Hodgkin’s lymphoma and Norn Hodgkin's lymphoma including Burkitfis lymphoma and diffuse large B-cell lymphoma, and also potentially several solid tumors such as glioblastomas, where APRIL expression has been reported.

The binding compounds of the invention may be used alone or in combination with other anti-cancer agents, such as chemotherapeutic reagents or other biological agents. Additionally the invention includes refractory or recurrent malignancies or treatment of metastases derived from any of these malignancies.

Autoimmune Disease

The binding compounds of the invention may be used to treat several autoimmune diseases, where the expression of APRIL has been shown to play a role in pathology. Examples of such diseases are Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Sjogren's syndrome. In addition, higher than normal titres of APRIL were found in the serum of multiple sclerosis patients and also increased levels were found in their astrocytes. Thus, APRIL is a contributing factor to disease pathology and therapeutic blockage of APRIL in MS may be beneficial.

Non-Therapeutic Uses for the Antibody and Antibody Fragments of the Invention

The non-therapeutic uses for these antibodies include flow cytometry, western blotting, enzyme linked immunosorbent assay (ELISA), immunohistochmistry.

The antibodies of this invention may also be used as an affinity purification reagent via immobilization to a sepharose column.

The antibody may also be useful in diagnostic assays, e.g., for detecting expression of APRIL in specific cells, tissues, or serum. For diagnostic applications, the antibody typically will be labelled (either directly or indirectly) with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories: biotin, fluorochromes, radionucleotides, enzymes, iodine, and biosynthetic labels.

The antibodies of the present invention may be employed in any known assay method, snob as competitive binding assays, direct and indirect sandwich assays a«d mmsimopiedpitaticm assays, ZoArnfibadim, A Manual QfTeckmqms> ppj 47-158 (CRC Press, Inc. 1987),

The antibody may also fee used for in vim diagnostic· assays. Generally, the antibody is labeled, with a radionuclide so that the antigen or cells expressing it can be localized, using iinmunoscintiogtaphy or positron emission tomography.

Legends in An figures

Ptgnru 1

Figure 1. shows APRIL reactivity and BCMA-blocking activity of hAPRtL.Ol A and hAPlilL.OSA hybridoma supernatants, Figure 1A .shows liAPRtL.OlA mid MPR1L.03A biudiug to FLAO-MFRIL eaftaml by m anti-FLAG antibody, ApdIy-5 antibody was used as a positive control Figure IB dsmosstndes that RAPRlLdll A and MPRIL.03A hybridoma snpemateits, and not Aprily-5 block the binding of FLAG-hAPRIL to BCMA-Fc,

Figure l

Figure 2 shows distinct binding mid reeeptorfelocldrig cbaracteristles of purified hAPEDLOlA and hAPRIL,03 A antibodies, Figure 2A confirms binding of purified MPML.Q1A and h.APRILJBA to FLAO«hAPML, captured by an anti-FLAG antibody, Figure 2B shows that only liAPRIL.03 A hinds FLACFhAPRlL that is captured by BCMA-Fc. Figure .20 shows that MPMLM A felly Mocks FLAG-hAPRIL binding to BCMA~Fc, wMle hAPRfL.03A partially blocks this interaction. Figure 2D demonstrates that hAPRIL.01A and MFRIL.03A both felly block FLAG-hAPRIL with TAO-Fc,

Figure 3

Figure 3 shows the receptor-blocking ELISAs for hAPRIL.OlA, MFRIL.OM, and 12 known conmiereiafly available monoclosal anti.~.APRIL snfikjdies, This illustrates that hAFRIL.OIA and MPR1L.03A are rmique m their ability to Mock APRIL binding to BCMA (Figure 3 A) and TACT (Figure 3B).

Figure 4

Figure 4 shows that hAPRIL.01 A and hAPRIL.OSA Mock APRB.<~drIvea B«eeM proliferation and Isotype elass-swifeMng but do not affect BAPF-reediaied processes. Figure 4A Is an In-vitro B»eeII assay vMck demonstrates that the described monoclonal antibodies. Mock kiown APRIL fenotions such as the somvsl and prolifemdoB of B ceils and production of class-switched IgA antibodies. Of significance is the demonstration that both monoclonal antibodies block APRIL aethity more effectively than TACLFe, which was administered at cquterolar concentration. Figure 4B shows that the antibodies do not affect BAFP~dnven B cells responses* while TACLFc blocks these processes,

Figures .

Figure 5 shows the results of targeting APRIL with bAPRIL.OIA and bAPRlLJBA (panel. A) or TA€I-Fc (panel B) fywiw, in a TAudependcM B eel response,

Transgenic mice were challenged with MP-FteoS., and treated with hAPRIL.01A, IiAPRIL,03A md TACLFc twice per week, PBS and mouse IgGl were used as negative controls. The imranooglohulm litres (IgA* IgM and IgCi) were measured by ELISA, hAFRILill 4 MPRIL*03A and to a lesser extent TACI-Fe are able to inhibit APRIL mediated B cell responses ia the hAPRIL transgenic mice Md reduce immimoglobuiln levels to that of the WT,

Figure b

Figure 6 shows the effect of targeting APRIL with hAPRXL.GI A* hAFR.IL.03A and TACI-Fe on B-cell popMafems in the spleen (panel A) or peritoneal cavity (panel B). Transonic mice were challenged with NP-Ficoll, and treated with hAPRIL .01 A* hAPRlL.03A, TAd-Fc twice per week PBS and mouse IgGl were used as negative controls, Alter 30 days of treatment spleens and cells fern die peritoneal cavity were harvested and analyzed by Bow eymmeiry, Treatment with hAFRIL.OI A or hAPRILJBA did not allbet fee (s«h)popn!ation of B-cehs in the spleen. In contrast, TACLFc strongly reduced the total B-ceU poimlation mid mature and T2 softpopalaiions, In fee puitonesl cavity, TACI-Fo affected fee ratio of B1 m B2~eells, while hAPRILOlA and kAFRILilS A did not affect these subpopuknlons.

Figure 7

Flgare 7 shows fee variable region sequences ofhAFRIL.OlA and hAFRIL-jM, Figures 7A and ?B show the amino acid seqaesees of the heavy and light dak variable sequence of hAPML.OiA, respectively, Figures 7€ and 7D shows die amkio add sequences of the heavy and light chain variable sequence ofhAPML.03A» respectively, .

Examples

Iiepfe h ImmwMxaiion and setetfen of aMi-APRIL antibodies Immuuls&amp;fioa. of Mice with APRIL cBMA

To'generate antibodies agsmsi die human APRIL protein, a eDNA eticoding the Ml length open reading frame of APRIL was snbdoned Into the pCI-neo vector (Promega, Madison, Wl)> Expression of the obtained vector was cheeked by transient transfection of pCRnawbAPRIL In 293 cells (,American Type Cfetee Collection, Manassas, VA) aid imnmnoblotting wife moose anidiAPML IgG! Aprily-5 (1:5,000) {Alexis, San Diego, CA), followed by goat anti-mouse IgGI TIRP (1:2,000) (Southern Biotechnology, Bimiingham, AL),

Mice were innnunfred by gene gun intmoskation using a Helios Gene gun (BioRad, Hercules, CA) and DNA coated gold ballets (BioRad) following mamfeetererk imimetions. Briefly, 1 pin gold particles ware coated widipCl-neo»hiiPML cBNA and commercial expression vectors for mouse f ItSL and mouse 0M-CSF in a 2:1:1 ratio (both from AMevron, Fargo, MB), A total of 1 pg of plasmid IMA was used to coat 5001.¾ of gold bullets,

Speciilealiy, 7-8 weeks old female BALB/C mice were itnmuniged in the ears with a gene gun, receiving 4 or 5 cycles of a shot la both earn. Approximately, a 1:3,200 anti-itAPEIL titer was detected by ELISA m mouse serum after three DMA IcmuMsstions In the ELISA, all incubation steps were followed by a wash step wife PBST (PBS wife 0.1% Tween 20) 3 times, Maxiserp 96-¾¾¾ Immunoplates (Nunc, Rochester, NY) were coated with rabbit and-FLAGpolyclonal antibody (50 ng/well ia PBS) (Sigma, St Louis, MO) overnight at 4 *C and Mocked with 10% Goat semm/PBST tm 1 boor at RT. Plates wre incubated with supernatant (1. :4 in PBS) from 293T culls transiently transfected with CMV promoter driven secreted form ofFJLAG-MPRIL (pCR3~hAPRIL) for 1 h at SX followed fey ineabatioas wMi mouse sera dilutions and 1:2,000 BRPwnnjngated goat aaikaoiise IgG (Soihbern Bioteelmology) for I hour each at RT, After the final PBST wash, at$4iAPBXL immnnomactlvhy was'visualized with 100 μ! OptlKLA TMB substrate (BD Biosdences, Franklin Lake, NJ), Reactions wem stopped with 100 μΙ 0,5 M BaS04 aid absorbances were read at 460 aad 620 urn. Mice that demonstrated rcacdvity against hAPRIL were immtnilzed fora final, fourth, time and sacrificed four days later, Brythroo>1e~clepleied spleen cell populations were prepared as described previously (Steeubakkers et«/., 1992, J- Immuml Metfa 152: 69-77; Steenbaktas et at, 1994, Mol Ehl Rep. 19:125434) and teen at 44{rC,

Selection ef ant WML antibody producing B cells To select R celt donee producing anfi~APRlL antibodies* 1,5 x ID7 erythrocyte-depleted splenoeytes were subjected to two rounds of negative panning on 2.3 x 107 Dynabeads# M»450 tosylmcfivaied beads (Ihvitrogen, Carlsbad, CA) coated wife and-FLAG M2 antibody (Sigma), 50 pg anti-FLAG M2 antibody was coated per 1x10s beads in 5Ο0μί according to manufacturer’s instructions. Beads and splenocyte suspension were incubated for 30 minutes on ice and resuspended in cold DMEM F12/P/S/I0%BCS, Unbound splenocyles were separated Born the beads using the Dynal MFC (Magnetic Particle Concentrator) (Invitrogen), For the positive paimlng, splenoeytes were incubatedwith 23 x 107 beads coated with anti-PLAG M2 bound to FIACKhAPRIL for 30 minutes on ice. Beads and unbound splenoeytes were separated as described above with a total of 12 washes.

Antigen-specifk B-cells were enltnred as described by Steenbakkers et aL, 1994, Mol, Biot Rep, 19:125434. Briefly, selected B-ceils were mixed with 7,5% (v/v) T~celi supematant and 50,000 itmdtalod (2,500 RAD) BL-4 B5 nursing cells hi a final volume of200 pi DMEM F12,T/S/i0%RCS in a 96~wel! fiat-bottom tissue culture plates. On day eight, supernatants were screened for bAPRIL reactivity by ELISA as described above, 21 APRIL-^eactive supernatants were Identified and tested, for their ability to inhibit the interaction of APRIL with BCMA-Fc. In the ELISA* all incubation steps were followed by a wash step with PBST (PBS with 0J% Tween 20) 3 times, A Maxisorp 96-weU immuneplate was coated with BCMA-Fc ($0 ng/well in PBS) (E&amp;D Systems, Minneapolis, MM) overnight at 4 °C and blocked with 10% Goat serum/FBST for l boor at RT. FIJkG-hAPRIL containing supernatants wea» pre~ incnbated with antibody-containing B-eeil supernatants for l hour at RT and then added to the BCMA-Fc coated plate for 1 hour at RT. Bound PLAG-teAPRGL was detected by incubation with. ,1 pg/m! anti-PL AG BioM2~Mofm antibody (Sigma) and 1:2,000 Sireptavidin-fiRP (Southern Biotechnology) for I hour each at RT. After the final PBST wash, APRIL-bound BCMA-Fc was visualized with 100 μί OptlBIA TMB substrate (BB Blosdences). Reactions were stopped with 100 μί 0,5 M H3SO4, and absorbances were read at 460 and 620 nai.

Subsequently, S B~eeII clones were immortalized by mini~elmftTofiision following , published procedures (Steenbakkers et aL, 1992, / Immunol MetL· 152,60»77; Steenhakkers et a!., 1994, Mol Biol Rep, 19,125-34). Specifically, B-cells were mixed with 106 NS-1 myeloma cells, and serum was removed by washing with DMEM F12 media. Ceils were treated with pronase solution for three minutes and washed with fission medium, Rieeirolhsions were performed in a 50 μ! fusion chamber by an alternating electric field of 30s, 2 MHz, 400 V/om followed by a square, high field pulse of 10 qs, 3 kY/cm and again by an alternating electric field of 30s, 2 MB2,400 V/crn . Contents of the chamber were transferred to hybrldonm selective medium and plated in a 96-well plate under limiting dilution conditions. On day 14 following the fusions* hyhridoma supernatants were screened for APRIL reactivity and ΒΟΜΆ-Mocking activity', as described above. Two distinct anti-hAI^RIL hybxidomas, named hAPRIL.OlA and MPRIL.03A wore Isolated and subdoned by limited dilution to safeguard their integrity. hAPRIL reaeftvify and BCMA-MocMng activity' of MPRIL.OIA and hAPRIL»03A antibodies were confirmed with hyhridoma supernatants (see Figure 1), -37-

Example 2: Purification and characterization of anti-APRIL antibodies Stabilization of anti-APRIL producing hybridomas and purification of anti-APRIL antibodies

Clonal cell populations were obtained for each hybridoma by multiple rounds of limiting dilutions (six for hAPRIL.Ol A and four for hAPRIL.03A). Stable hybridomas were cultured in serum-free media using CELLine® bioreactors (Integra-Biosciences, Chur, Switzerland) according to manufacturer's instructions. Following 7-10 days in culture, supernatants were harvested and fdtered through a 0.22 μΜ nitrocellulose membrane. Supernatants were diluted 1:1 in high salt binding buffer (1M Glycine/2M NaCl, pH 9.0), and antibodies were purified with Protein G HiTrap 5 ml columns (GE Healthcare, Piscataway, NJ). After PBS wash of the column, antibodies were eluted with 0.1 M Glycine pH 2.7 and neutralized with 3 M Tris. Buffer was exchanged for PBS using PD-10 gel-filtration columns (GE Healthcare). Antibodies were concentrated with Amicon Ultra-15 centrifugal filter units (Millipore, Billerica, MA) and quantified using spectrophotometry.

Using a mouse monoclonal antibody isotyping test kit (Serotec, Raleigh, NC), the (sub)-isotype of both hAPRIL.OIA and hAPRIL.03 A antibodies was determined to be IgGl, Kappa.

Binding Analysis

Protein-based ELISA experiments using purified hAPRIL.OIA and hAPRIL.03A antibodies were performed to determine apparent binding affinities (reported as EC50 values). Binding was compared to mouse anti-h APRIL IgGl Aprily-5 (Alexis).

Maxisorp 96-well immunoplates (Nunc) were coated with either rabbit anti-FLAG polyclonal antibody (Sigma) or BCMA-Fc (R&amp;D Systems) at 50 ng/well in PBS overnight at 4 C and blocked with 10% Goat serum/PBST for 1 hour at RT. Plates were washed with PBST 3 times and incubated with supernatant (1:4 in PBS) containing FLAG-hAPRIL for 1 hour at RT. Plates were again washed with PBST 3 times and incubated with hAPRIL.OIA, hAPRIL.03 A, and Aprily-5 antibodies (10 pg/ml high test with 10-fold dilutions in triplicates) for 1 h at RT. After three washes with PBST, bound antibodies were detected with goat anti-mouse IgG-HRP (1:2,000) (Southern Biotechnology) for 1 hour at RT. Plate was washed three times with PBST, - and AFML-reaedvfty was visualized with OptiEIA TMB substrate (Becton Dickinson), The <mneemratlon for kfof-maxinial binding is reported as a meam of relative binding affinity, Wlm FLAGfoAPRIL· was captured by the aati-FLAG aatibody (Figuso 2A)S ECso values for hAPEIL.OlA, hAFML03A and Aprily-S were calculated as 2,2,1.4, and 1 JnM, respeefotefy. When FLAG~hAPR)I, was captured by BCMA-Fc (Figure 2B)S hAPRELOlA antibody binding was not observed, suggesting that the APRIL-BCMA mieractien bitted theMPRlL.OlA epitope. I» 'contrast, binding of bAfiRIL.CBA to the AFRJL«BCMA complex, was observed. Antibody detection of fee receptor-ligand complex may prove useful k diagnostic assays wd for research purposes to .Mow the clearance of soluble APRIL,

Kiaetie amlysfe by fefe~SgM iuterferometry (FertdWe)

To feather characterize the binding diaMcfefisties of tbe antibodies, each was profiled using bkvdlgM laterferometry on the Octet system (ForteBlo, Menlo Park. CA) to elucidate binding kinetics and calculate equilibrium binding constants. TMs assay was performed by coupling purified liAPMLJlA and hAPRIL,C6A isitibodies to amine-reactive biosensors (Fortehio) using standard amine chemistry. Recombinant human APRIL (RM3 Systems) binding to and dissociation fern the biosensors was then observed at two concentrations, I and 2 pg/ml. Specifically, aminemaactive biosensors were pre-wetted by immersing them in wells containing 0.ΙΜ MES pH88 5 for 2 minutes. The biosensors were then activated using a 0.1M MBS / Θ.4Μ EDO mbfosre for S minutes, hAPRIL ,01A and MPRILiBA antibodies were coupled by Immersing the biosensors in a solution of 5 pg/mL of foe antibody for 18 minutes, The biosensor surface was quenched using a solution of 1M e&amp;anolamme pH JL5 for 7 minutes. Biosensors were eqoilihm&amp;d in PBS for 5 minutes, Association of recombinant APRIL was observed by placing the biosensors in wells containing either 1 or 2 pgbrd APRIL and monitoring .interferometry for 20 minutes. Dissociation was measured after transfer of foe biosensors into RBS and monitoring of foe interferometry signal for 2(1 minutes, The observed on and off rates (¾¾ and 1¾) were fit using ah! binding global fit model» and the cqmMMom binding constant Kb was calculated (sec Table !),

Table L Binding characteristics of humanized anbdiAPRIL anbixaiies of the invention

Receptor Blockade

Blocking abilities dfhAPRIL.OIA and hAPEBLOSAwere confirmed «sing purified antibodies, Maxisorp Sfowell plates were coated with either BCMA-Fc (Ε&amp;Ϊ) Systems) or TACBFc (RM> Systems) at 50 nglwell overnight at 4 *C and Mocked with 10% Goat «aan/PBST for 1 boor at RT. PIJkG~hAFRIL containing mpermtants were prerincobated with hAPEHLOIA* MFRJOL.03A* and AptllyRS Mhkxlies (10 pghnl high test with ICLfold dilations la triplicates) for 1 h at ET, Plates were washed with PBST 3 times, and hound FLAGdiAPRIL was detected by incubation with 1 gghni a&amp;tl-FL AG BIoMSfoiohn antibody (Sigma) and 1:2,000 SbcpiavidhtdiEP (Soother» Biotechnology) for 1 hour each at RT, After die Sad PBST wash* APRIL-botmd BCMA»Fc was visualized with OptiEiA TMB substrate (BD Bioselenees). As shown in Figures 2C and 2D, hAPRILJB A folly blocks FLAG-hAPRIL binding to BCMA«Fc and TAOt-Pc, whereas fcAPRIL,03A folly blocks FLAG-hAPRIL binding to TACJ-Fe, while only partially blocking the ItAPRJL-BCMA~Fc istemetioa, Aprily-5 does not Mock F LAG-h APRIL binding to either BCMA-Fe or TACI-Fe, The eonceatondon of half-maxirausi inhibition (ICso) was determined for hAPRSuOIA as 1.2 and 0.4 nM for BCMA-Fc and FACIAL respectively. The IC53 for hAPRIL J3A to TACLFc \vas determined as 1.3 nM.

Commensal Antibodies

Cotnmerciaily available anti-APRIL antibodies were obtained as described in Table 2, Table 2. Connnercially avatlaMe ani-hisnan APRIL monoclonal aitlbodies

To study whether the blocking chamoieristles ofhAPRIL«QlA and hAPRILJBA are imiqm all known commercially available anti-APRIL antibodies were tested for their ability to block the interaction of FLA0-hA.FRIL to BCMA-Fc and TACI-Fe (Kgutes 3 A and 3®), Blockade of receptor binding was studied using m EOS A. Ait ELISA plate was coated with SO with 1 ΟΟμΙ of BCMA-Fe at 1 pghnl or with ΙΟΟμΙ of TACI-Fe at a concentration of 2 gg/ml la coating bailer Mid tuenbaied overnight at 4 *C, The pkte was the® waslied with PBS/0»2%Tween and then incubated with for I hoar at 37 ”C with 100 μΐ PBS/5% BSA per well The plate was then washed tour times with PBS/02%Tweera In a separate piste APRIL monoclonal antibodies were pre-mixed with APRIL siipematant and incubated ibr 30 minntes on Ice, Conditioned medium containing soluble APRIL was diluted 1 k 4 and inked witli an ecpal volume of PBS eonimuiug the antibodies titrated in doubling dintions starting with. 5 pgfmL 100 μΐ of tiie pro-kcttbakd mix was transferred to the ELIS A plate and incubated for 2 hours at 37 *C. The plate was then waslied four times Ά PBSAO%Twseii« And-Flag-HRP antibody was then diluted in PBS at a eoncenhmtlon of! :1000 and then 1.(10 pi of this added to each well and Incubated for 1 hour at 37 The plate was then 'washed fear times with PBS/(i2%Twe«a and then 100 μΐ of ABTS added- to each well (the ABTS was dilated to the ratio 10 inf of reagent plus 5 μ! of 1¾¾ made Immediately before addition), life colour was allowed to develop and thm the OD at 405 tm read cm an ELISA plate reader, Human IgGl was used as a control protein to coat die plate as Ibis is the same kotype as the Fc«fcsion proteins and controlled for APRIL sticking to the plate non-speclfieidly, As is apparent from Figure d, nose of the commerelally amiable antibodies was able to block fee binding of FLAG-APK1L- to either TACI-Fc or B€MA»Fg* whereas bAPlIL.OIA and LAPRILilB A do inlijfcit (pariialy) the binding to TACI-Fe and BCMA-Fc,

Species Cross-Reactivity

Binding ofbAPRIL,01A and hAPHJOL.(BA to moose APRIL was also examined by BLAcore, hot no binding of either antibody was observed. The antibodies appear only to bind human APRIL. ,

Example $t Fimefmnal ProSlIng of Marine ani-Haiaan APRIL AMlbedies Mouse 11 cell response to APRIL

In order to show that the antibodies of this invention ean functionally block APRIL In-vitro a mouse B cell assays was used to examine two APRIL driven responses In B cells - proliferation and IgA production. AH cell lines were maintained at 3?*€ with 5% C€fe, Mouse splenocytos and purified B cells were grown In RPMI~ 1640 (Gibco) supplemented with 8% FC$> 2 mM Glutamine-and Beta-mereapioethanol at 50 μΜ» and supplemented with penicillin and streptomycin at a concentration of 1 Cigghnl, Splenic mouse B cells were isolated from. wiM-type mice using magnetic activated eel separation (MACS) columns with CB45R/B220 MACS beads (Miltmyi Biotee, Utteebt* The Netherlands). The cells were cultured in 96-well, round-bottomed nferoiiter plates at a density of 2 x Kf /well In a fetal volume of200 pi, For all assays conditioned medium eontalsisg fee various forms of soluble APRIL were normalised for expession levels prior to-use. To .measure proMfemtkug cells were treated with and-IgM (Jackson hmnusoReseardt) and soluble APRIL meondi^ or as purified protein at a final eoHcenimtkmofl |ig/ml. Crossdinfcmg anli-Flag monoclonal antibody was added to the well at a M eoneentmdon of 1 ug/ffil The cells were incubated at 37 *€ and after 48 hours poised witLBJ μ€Ι {0.011 Μ&amp;|>'®ί£ trMatod thymidine, GE Healthcare, 7% bfeiirfaod^ &amp;r 18 hoots, before harvesting* To measure IgA production, moose B cells were eidtwed andtoled with APRIL, as above. Fallowing incubation for 6 dap, supernatant was collected sad assayed for IgA content by ELISA. Briefly, ELISA plates were ooateft with 2 ggfed amb^nse-Ig (Southern Biotech^ Mocked wife PBS/1% BSA and incubated with, the collected supernatant Bound IgA was then detected with HR? labelled aoti^mope-lgA (Southern Biotech, IJliboem, the Kditerlaad^c# a control, cells were treated with IS pg/ml LBS (lavi vogen) pins ! ng/ml of human TG.Fp (Sigsia-Aldrich). As shown in Figaro 4A, hAPRIL.OlA and to a lesser extent bAFRILi)3 A are able to JnMhft AP1IL induced class^swiich recombination as was determined by the reduced IgA seorotion from mouse splenic B -cells. TACRFe m a control Inhibited the IgA secretion, while mouse IgGJ and human Ig did not affect the APML-mdneed IgA secretion drum splenic R-eells. in addition, hAFBIL,0i A pd hAPRILLBA were demonstrated to Inhibit APKil-kidoced mouse splente B~cell prolfomtiom To establish tjte pemfiei^ of the antibodies, tfe efihetof hAPML.01 A and hAPRIL.03A on BAPF-indueed IgA secretion mid proliferation was studied. As shown in Figure 4B, neither hAPRIL.OiA norhAfRIL,03 A inhibited BAFF induced IgA secretion and prolifemtkm, while TACI-Fc as a eosirol inhibited both processes.

Experiment to Mock APML function

To demonstrate Miin-vivo MocMng e^ct of &amp;e antibodies on APRIL thnctlon, we eaammedthe ability of the Mdbodies to bkK4;ise'HP~FicoU induced hnmotaLresponse in mice. The mice used were 8%lft week old API^L iratsgenie (TO) mice and wiidtype (Wl) litfermates, k)tb on a CS7BL/i background, The APML toansgemc mice express human APRIL under the Lck~dislal promoter, which directs transgene exppsic® to maftrm thymocytes and peripheral T lymphocytes (Stein et ah, 2002, J Clin Invest M% 1587-98). The mice were bred to the anhnal facility o f the Academic Medical Center m&amp; the exi^hnont w'p g|?proved by the msdtoddnal ctMual committee. The mice were divided into several grasps and treated as follows: five APRIL WT mice were treated with PBS (2Ο0μ1) and 5 groups of five APRIL transgenic mice were treated with, the Mowing molecules: hAPRILOIA or h APRIL. 03 A or TACI-Fc or suMsotype-mMehed control antibody msigGi_k (200 pg/monse is 200 μ! PBS) or PBS, Treatment of Sic mice was started 3 days before the NP-Fieel imnnmimiion (day 0; 100 pi ip. with 250 m of the itmrrmnogm) - mjectioos were continued twice a week for 28 days. Blood was collected via tail vela at day -1* 3* 7,14 imd 28. An&amp;(4^dtwxy~mtToplie»aceiyl) (NP>8peeffie aBtihodtes (IgM, IgG aid IgA) were assayed in 6 independent ELIS A nstng diluted sera (1:100 for IgA; 1:500 for IgG and 1:2,000 for IgM) as previously described Pardenbetg et al ,s immune! Ceil Biol M(6% 530-4, (2008)). Briefly 96~we!l ELISA plates (Greiner) were coated with. NP-BSA at 5 pg/nil (Bioseareh Teehncrfogies) in sodium carbonate buffer (pH 9,6) overnight at 4°C. The wells were Mocked with 1% BSA tor 1 hr at 37 XI and incubated with diluted sera for '2 hrs at room temperature. HRP^oupgaied isotype specific antibodies (Goat anti-mouse IgG, IgA and IgM - Bom Southern Biotech) were used as revealing antibodies. All dilutions were made in PBS/BSA 1.%/Tweea 20 0.05¾. One way ANOVA test was used to check statistical sigaificancc between the groups TG (PBS) vs TO (hAPRiL.Oi A) and TG (PBS) vs TO (hAPML JSA). As apparent front Figure 5, both hAFRIL.Oi A and liAPRiLiBA inhiMted the T-cell independent B~oeli responses in vivo. TA€I~Fc InhiMted this response less efficient PBS and. moose IgOl as an rsotype-matched control, did not affect the IgA, IgM and IgG aori-NP response, To examine the long-term effect of hAPRIL.OI A and hAPRIIABA on B cell Imputations mice were treated as described above, tin day 30, mice were sacrificed arid the spleen and· peritoneal exudate cavity (PEC) analysed for B cell expression by flow cytometric Briefly, splenoeytes and iyu^djoeytess from the PEC were separated Bom red blood cells by one wash with erythrocyte lysis buffer and then counted, Cells w« washed and resuspended la PBS/1% BSA and seeded in 96-weti round-bottomed plates at a density of 5 x l(f par well. Next, cells were stained with the following antibodies at the recommended coneenhations: B220-PITC (BD hioseiaoee) and CD3-AK5 (ehloscience); IgD-PITC (BD bioscienee) and IgM-PE (BD bioscienee); IgD-PiTC (BD biosdence), CD3-APC (ebioseiense) and CB43-PE (BD bioscienee). Antibodies were incubated for 40 mimrtes, washed three dines with PBS/134 BSA usd then analysed by Sow cytometry using the FACSGalifew (Becton Lfickcimm). B220* B-eelk, mature B~cel h (i'gD'lgM'"5) and T2 B-eetls (tgl.f ig<Vf ) in spleen were quantified (see Figure 6A). hi addition, B! (CDdJ’IgD^) and B2 (CD43I§D'!) suhpopulations were quantised in PEC (see Figure 6B}> The decease in B eells in response to TACI-Fc treatment is evident Bom both the spleen and fee PEC,, fecle-ating that long term administration of TACI-Fe may have a detrimental effect oa normal B cell populations. This is not seen wife bAPRIL.Ql A and hAPKILJ3 A antibodies, suggesting feat m eases where APRIL bin not BARF is the primary cause of pathology, the antibodies of this inYention may show less side-eiYeets than TACI~Fe<

Example 4: Anri-APRIL antibodies sequences Cloning oflmmonoglobuRn eDNAs

Degenerate primer PCR-based methods were used to determine die DMA sequences encoding the variable regions for the mouse antibodies that are expressed fey hvbridomas liAPE.IL.0i A and hAPEXL.03A. Total ENA was Mated from Sxl O6 v hyferidonm cells using TRXZOL Clmdirogem), and gene specific eBNAs for die heavy and light chains were synthesised using the iSeript Select cBMA synthesis Mt (Biorad) according to fee manufacturers instructions. The Yh sad Yl genes were P€R-amplified using a Novagen -based Ig-primar set (Novagen, Sas Diego, €A) and Taq polymerase (lovitrogen), AO PCR products feat matched fee expected amplieon m® of 500 bp were cloned into pCR4 TOPO vector (Invifrogsn}, and the cousimcis wet» transformed in BH5« £. caii Chwitrogen) according ίο the manufacturer’s Instructions. Clones were screened fey colony PCR using uuiYersal MIS forward and reverse primers, and two clones from each reaction were seleetod &amp;r DMA sequencing analysis. Sequences we*» searched against databases of gmalk© and rearranged igV variable region sequences using NCBI Ig-BIast BLASTN 22 J 6

Blast results tor hAPRlLOIA and hAPRIL.03A showed one in-feme Vk sequence and one In frame Yf sequence for each antibody, The amino acid sequences were confirmed by mass spectromehy. Hie sequences are disclosed in fee attached Sequence Listing, Figure 7 and listed in Table 3,

Table 3: Setp&amp;nee ID numbers for murine tmti-hnmaa APRIL antibodies of this invention

Example Si Epitope mapping «stag Pepscan method Synthesis of peptides and pepsean screening

The synthetic linear and CLIPS peptides were synAesiaed and screened using credit» card format mini PHPSCAM cards (455-well plats whh 3 ol wells) as described by Slootstra et aL (Slootstra at at, 1996» Mol Diversity 1,87-96) and Timmerman et at (Timmerman et al» 2007, X Mol Recognit. 2% 283-299), The binding of antibodies (hAPRILOI A and hAPRIL.03A) to each peptide was tested in a PEPSCAN-based enKyme»linked imrmmo assay (ELISA). The 455-wdl creditcard-fonnat polypropylene cards, conteimng the covalently linked peptides, were menbaled with sample (for «sample 1 tigtoi tmitbedy dilated in&amp;PBS solution containing 5% horse serum (vol/voi) and 5% ovalbumin (waaghttool)} and 1% Tween 80 (4®€, overnight). After washing the peptides were incubated with m anti-antibody peroxidase (dilition 1/1000» for example rabbit anti-mouse peroxidase, Southern Biotech) (1 hour, 25σ€), and subsequently* alter washing the peroxidase substrate 2?2!»akn(MS-3“ ediyltmMMaadme sulfonate (ABTS) and 2» id/ml 3% H202 were added. After 1. boor the color development was measured. The enter development of the ELISA was quantised wife a CCD-camem and as image processing system. The setup consists of a €CD»caniera and a 55 nan lens (Sony CCD Video Camara XC-77RE, Mfcoa micro» niMror 55 mm fO J lens), &amp; camera adaptor (Sony Camam adaptor DC-77RM) and Image Processing Software,

Synthesis Peptides

A total of4225, primarily, CLIPS peptides were synfeesiaed. The target sequence used, 147 amino acids, with loops according to alignment with lXU2.pdb underlined: RAVLTOKQg;KOHSVLBI.yPINA,rSKDDSDVrEVMWOMLRRGRGLOAOGYQV MODAGVYLIABQVIJ'OIiyiFrMC^VYSRBGOgRjOETLPECffiSMPSHPllSAV

Loops on “top* side of protein; QK&amp;QHSVLHL (SEQ13140:22¾ ALRRGRGL (SEQ- ID 140:23¾ QAQGYOVRI (SEQ ID 140:24), QDAGWEL (SEQ ID MOt25), SREGQGRQETV· (SEQ ID KO:26), PHLHQ0D1LSV (SEQ; IB NO:27) and loops on “bottom* side ofprotek: MATSKDDSDVTE (SEQ ID 140:28¾ YLFQDVTFTMG (SEQ m 140:29), IRSMPSHPDRAYMSC (SEQ ID N0:3§)s UPRARAKL (SEQ ID N0:31), NLSPHGXFLOF (SEQ ID NO'32). The inferaotmeetkg regions are mostly sheets. Note tlmt fee “top5* and "bottom*5 side are chosen arbitrarily.

The following CLIPS topologies were used: T2 CUPS couples to the side-chain of two cysteines to form a single loop topology, while T3 CLIPS couples to the side-chak of three cysteines to form double loop topology, while I2T2 CLIPS fest T2 couple to two cysteines (labeled C)* and second 11 couples to two cysteines and fatally T2T3 CLIPS T2 couples to two cysteines and T3 couples to three eystemes.

In total 20 different sets of peptides were synthesized: 1914 (set-1): Allowdappiag 35~mer se^aees cowkg file 147 AA target sepeme were synthesized. la this set the different loops, when present m the sequence* as deSaedabove were m doable loop or sfaecWiks topology through two T2 CLIPS. 191-2 (set-2) A total offfmesheels were identified, AO 9x9 combinations were synthesized to tmmie doahfe sheet eonfornmtions, IS» seqnence OSQ -was used as a linker, 191-3 (set-3) lire same as set-2 as explained above but with a shorter sheet length* 191-6 (seM) All overlapping linear 35~mer sequences covering the complete 147 A A target sequence were syotlwsiEed. 191-7 (set-5) All overlapping linear 15-mer sequences covedng the complete 147 AA target segapice were synthesized, 191-8 (seffda) Short linear sequences (of varying length) only euvermg the loop regions of the complete 147 AA target sequence warn synthesized, 191-16 (set-6h) Different peptides were selected ffom the five bottom*5 loops, These worn twmmbined In a 9x9 matrix onto the T3 COPS taihim double looped topologies whh “hottpmf loops of two different lengths, 191-17 (set-7) All overlapping 135 different 15-nter segnences ww synthesized wi® a cysteine at position 1, &amp; and 15, the three cysteine were coupled to a T3 CLIPS, 191-18 (set-9) Long yersiom of the six *%ψ* loops and long versions of the four ^hoterf’1 loops were recombined with each other on the T3 CLIPS, 191-19 (sefflO) Six^SisAFdur different sized loops of the 4opw loop region. were all recombined with each other on. the T3 CLIPS. 191-20 (set-1Iff 7,184¾¾)) 33 different sequences broadly covering &amp;e %jpn or “bottom5'’ loops were recombined with other on the T3 CUPS , ttese sets of peptides ate m sets .11, 17, IB, 19 and 20. Reason for this “scattering5* is the card layout, 191-22 (set-12) Diflerent sized loops of all “top5* and “bottom” loops were synthesized as single loops oh T2 CLIPS, 191-23 (set-13) Ail overlapping single looped I5~mer sequences covering the complete target pmteiit synthesized on Ί2 CUPS, 191-24 (set-14) Sixdifferent 9-mer sequences eovemtg the "‘top5* loops were mcomMnedv^oaehmahxfeStt^eloopedinatrix onl'213 COPS cothhlimllon, 191-25 (set-15) The same set of overlapping peptides as set-1, All overlapping 35-mer sequences covering the complete I47AA target sequence were synthesized, la this· set the different loops, when present in the sequence, as deimec! above were consdamed late triple loop topology through T3T2 CLIPS. 191-26 (set-16) Six different 9-mer sequences covering the ^bottom” loops ware recombined with each 'in a 6x6x6 triple topped matrix <m T2T3 CLIPS combination.

Bata analysis and epitope deferMiaaticM

Each antibody was tested on. all 4225 peptides and their binding values were ranked. Clearly· re-occurring sequences m most the top hinders (-top 1%) wars considered as epitope candidates. Two additional snpporting analyses were clone. Firstly, it was investigated if multiple identified parts can. t®a one dlsconfinpons epitope. This was done through the homologous structure l XIJ2,pdh. Secondly, It was investigated if each of multiple Identified binding parts was recognised without support of the other part. These two parameters, co-loeatkation. on the 3D structure and independent recognition, were used- to support that a eonfornmiional and discontinuous epitope was identified. IferbAFRIL.OlA it was determined that it binds to 1RSMPSHFDRA(SEQ ID NO-33), wife the core region being SMFSHP (B1Q ID MO:34). The TLFR (SEQ ID NO:35) and/or QDYIFTMGQ (SEQ ID NO:36) (core region is YTFTM (SEQ ID NOD?)) motifs were shown to support fee binding of hAPRIL.OIA, hAFRIL.OSA was shown to hkd YSRBGQGRQ (SEQ ID NODE) motif with core region being EGQ. The TFFMGQ (SEQ ID NO:39) motif was shovm to support biuding of hAFSIL.OM.

Claims (17)

1. Claims

   1. An isolated or recombinant antibody or antibody fragment which binds to human A Proliferating Inducing Ligand (APRIL) comprising: a. an antibody heavy chain variable region comprising CDRs SEQ ID NOs: 15, 16, and 17; and b. an antibody light chain variable region comprising CDRs SEQ ID NOs: 18, 19, and 20.
2. 2. The isolated or recombinant antibody of claim 1 wherein the antibody or antibody fragment fully blocks the binding of APRIL with human transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and partially blocks the binding of APRIL to human B-cell maturation antigen (BCMA).
3. 3. The isolated or recombinant antibody or antibody fragment of claim 1 or claim 2 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
4. 4. The isolated or recombinant antibody or antibody fragment of any one of the preceding claims, wherein the antibody or antibody fragment: a. binds human APRIL with a Kd of about 10 nM or about 10~8 to 10'11 M; and b. blocks binding of human TACI to human APRIL with an IC50 of about 2 nM or lower.
5. 5. An isolated or recombinant antibody or antibody fragment which binds to human APRIL wherein the antibody or antibody fragment binds to an epitope having the amino acid sequence of SEQ ID NO: 38 or EGQ.
6. 6. An isolated or recombinant antibody or antibody fragment which competes for a binding epitope on human APRIL with the antibody or antibody fragment of any one of claims 1 to 3, and: a. binds to human APRIL with a Kd of about lOnM or about 10"8 to 10"11 M; b. binds to human APRIL· with about the same KD as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8; or c. blocks binding of human TACI to human APRIL with an IC50 of about 2 nM or lower.
7. 7. The isolated or recombinant antibody or antibody fragment of any one of claims 1 to 6, wherein the antibody or antibody fragment is: a. a chimeric antibody or an antibody fragment thereof; b. a human antibody or an antibody fragment thereof; c. a humanized antibody or an antibody fragment thereof; or d. an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, bispecific mAb and a diabody.
8. 8. The isolated or recombinant antibody or antibody fragment of any one of claims 1 to 6 wherein the antibody or antibody fragment inhibits the proliferation and survival of B-cells.
9. 9. A composition comprising the isolated or recombinant antibody or antibody fragment of any one of claims 1 to 8 in combination with a pharmaceutically acceptable carrier or diluent.
10. 10. A method of treating an APRIL-dependent condition comprising administering an effective amount of the isolated or recombinant antibody or antibody fragment of any one of claims 1 to 8 or the composition of claim 9 wherein the APRIL-dependent condition is selected from the group consisting of APRIL-dependent cancers, APRIL-dependent inflammatory diseases or APRIL-dependent auto-immunity.
11. 11. A method of inhibiting immune cell proliferation and/or survival comprising administering an effective amount of the isolated or recombinant antibody or antibody fragment of any one of claims 1 to 8 or the composition of claim 9.
12. 12. A diagnostic assay for detecting expression of human APRIL in a cell, tissue or serum by labelling the isolated or recombinant antibody or antibody fragment of any one of claims 1 to 8 and detecting binding of the labelled isolated or recombinant antibody or antibody fragment in the cell, tissue or serum.
13. 13. The method of claim 10 wherein the APRIL-dependent cancer is a B-cell malignancy, a Non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, or an APRIL-dependent solid tumor.
14. 14. The method of claim 10, wherein the cancer is selected from the group consisting of Chronic Lymphocytic Leukemia (CLL), Multiple Myeloma, Hodgkin’s lymphoma, Burkitt’s lymphoma, diffuse large B-cell lymphoma and a glioblastoma.
15. 15. The method of claim 10 wherein the inflammatory disease is selected from the group consisting of Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Sjogren’s syndrome or Multiple Sclerosis.
16. 16. Use of the isolated or recombinant antibody or antibody fragment of any one of claims 1 to 8 in the manufacture of a medicament for treating an APRIL-dependent condition wherein the APRIL-dependent condition is selected from the group consisting of APRIL-dependent cancers, APRIL-dependent inflammatory diseases or APRIL-dependent auto-immunity.
17. 17. Use of the isolated or recombinant antibody or antibody fragment of any one of claims 1 to 8 in the manufacture of a medicament for inhibiting immune cell proliferation and/or survival.