AU2015201974A1 - 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

- 1 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. 5 The present invention relates to isolated antibodies or fragments thereof which binds to human APRIL, pol ynucleotides encoding such antibodies and host cells producing said antibodies. The antibodies can be used to inhibit inunune cell proliferation and/or survival, to treat cancer and to treat an inflammatory disease. 10 APRIL is expressed as a type-Il 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 al., 2001, EMIBO Rep 2 945-5 1 ,). APR IL assembles as a noncovalently 15 linked horno-trimer with similar structural homology in protein fold to a number of other TNF family ligands (Wall weber et al., 2004, Mol Bio/343, 283-90). APRIL binds two TNF receptors: B cell maturation antigen (BCMA) and transintmnbra ne activator and calcium modulator and cyclophilin ligand interactor (TACI) (reviewed in Kimberley et al., 2009, J Cell Physiol. 218(1):1-8). In addition, 20 APRIL has recently been shown to bind heparan sulphate proteoglycans (HSPGs) (Hendriks et al., 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, 25 BLyS), with which it shares binding to its receptors. BCMA and TACI. 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 al., 2009, J Cell Physiol. 218(1):1-8). APRIL and BAFT have been suggested to form mixed trimers (Roschke et al., 2002, J Immunol. 169(8):4314-21). Such mixed trimers were found to 30 occur at a higher prevalence in rheumatoid arthritis (RA) patients.

- 1 a 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.RIl. can be expressed by non-immune cells such as osteociastsepithelial cells and a variety of tumour tismues (reviewed in Kimberley et al, 2009, JCell PhysiaL 218(11-8), The function of APiUL was established using mouse genetic models. hAPRIL 5 trasgenic mice develop normally, but showed enhanced T cell survival and elevated levels of IgM antibodies (Stein et at, 2002, J Clin nest 109, 15857-98). In addition, T ell independent type I responses were enhanced. Aged hAPRIL transgenic mice displayed extreme enlargement and re-organisation of the lymph system and enlarged spleen due to infilration of CD5 positive B cells, a phenotypc closely resembling o human B-CLL (Planelles et al 2004, Caner Cel6, 399-408),. APRIL deficient mice were found to have decreased levels of IgA in circulation and upon challenge with a T cell dependent antigen (Castigli et al, 2004, Proc NaAcadSi U SA 101, 3903-8; Varfolomeev et al., 2004, Mo Cell BioX 24, 9971006), Next APRIL, along with BAFF, was demonstrated to function in elss-switch reconination (CSR) of is antibodies to both IgG and IgA, independently of CD40-CD4OL signaling (Litinskiy et al 2002, Nat hnmunol 3, 822-9), in addition, APRIL was demonstrated to be less critical than BAFF in B cell maintenance, but was shown to have a role in B cell signalling and drive both proliferation and survival of human and marine B cells In vitro(reviewed in Kimbeley et at, 2009, J CellPhysio 218(t):1 -8), 20 APIUL vas originally identified based on its expression in cancer cells (Hahne et at, 1998, JEg Aed 188, 1185-90). High expression levels of APRIL niNA were found in a panel of tumour cell lines as well as human primary tumours such as colon, and a lymphoid carcinomad.In addition, APRIL tmasfected marine fibroblast NFII3T3 cells 25 were shown to grow more rapidly in imnumodeficient mice More importantly, blocking APRIL using a soluble APRIL receptor was shown to inhibit tumour growth of hmg and colon carcinomas (Rennert et at, 2000, ]Psp Med 192, 1677-84). Chronic Lyiphocytic Leukaemia (CLL) B cells express both APRIL and APRIL 3o receptors. In addition, it was shown that APRIL protected CILL cells against spontaneous and drg-induced apoptosis and stAimulated NF-iB activation (reviewed in Kimberley et a., 2009, J Cell Pkvsio, 218(1):14). A retrospective study under 95 CLL patients shoed increased levels f APRIL in serum, which correlated with disease progression and overall patient survival, with a poorer prognosis for patients with high APRIL serum levels (Planelles et al, 2007, Haematologica 92, 1284-5). 5 Similady, (increased levels oi) APRIL was shown to be expressed in Hodgkin's lymphoma, Nonodgkifs lymphoma (NHL) and Multiple Myeloma (MM) (reviewed in Kimberley et al, 2009, JCel PhysioL 218(1):1-8) A retrospective study in DLBCL patients (NHL) showed that high APRIL expression in cancer lesions correlated with a poor survival rate (Schwaller et aL, 2007, Blood 109,331-8) Using NHL and MM el i lines it was shown that treatment with APRIL or BAFF increased survival via NF-rB activation and up-regulation of pro-survival proteins (reviewed in Kimberley et at, 2009 JCe PhysioL 218(1):14)In accordance with this pro-survival role of APRIL, MM Cells were shown to undergo apoptosis when cultured in the presence of TACi-c, Since BAF- receptor was less effective in enhancing apoptosis, this indicates that is APR1L. and not BAFF is primarily responsible for enhanced survival in these cells (Abe et al. 2006, Leukemia 20, 1313-5), APRIL was also found to be over-expressed in a number of ceil lines derived from solid tumours Indeed, APRIL was able to stimulate in-vitro proliferation ofa number 20 of these cell lines (reviewed in Kimbeey et at, 2009:J Ce01 Physiot 218(l):18), Due to itsrole in B cell biology APRIL also plays a role in many autoimmune diseases, Indeed, atacicept (a commercial TAC-Fc preparation) is already in numerous clinical trials for treatment of several autoimmune diseases (reviewed in Gatto et a, 2008, 25 Curr Opin fweshig Drug. 9(11 ):126-27) Increased serwn levels of APRIL and BAFF have been reported in many SLE patients (Koyama i at, 200$, Ann Rheum Dis 64, 1065-7). A retrospective analysis revealed that APRIL serum levels tended to correlate with anti-dsDNA antibody tires. Evidence that APRIL may play a functional role in SLE was obtained by testing the effect of TACI-F fusion protein into lupus 30 prone mice (Goss et at,2000, Nture 404, 995-9), which prevented disease development and prolonged survival.

-4 In addition, inhibition of APRIL and BAFF with TACI-Po in the CIA mouse model of rheumatoid arthritis was also found to prevent disease progression and lower disease scores, compared with controls (Gross et al., 2001, Immunity 15, 289-302; Wang et al., 200 MtaIinnol 2,6324) Also in another arthritis model, synovium-SCIL)mouse s chimeras, TACI-e showed a beneficial effect (Seyler et al 2005, JClin Invest 115, 3083-92). Treatment with TACI-Fe rested in the disappearance of Germinal Centers in the synovial tissue, decreased Ig production and decreased production of FN gamma It was later reported that the synovial fluid of patients with inflammatory arthritis had 1 signifcantly increased APRIL levels compared with those with patients suffering from non-inflammatory arditis such as osteoarthritis (Stohl et al., 2006; Endocr Metab 1rnwme Disord Drug Targets 6, 351-8; Tan et al, 2003,Arthrits Rhewn 48, 982-92). Several studies focused on the presence of APRIL in the sera of patients suffering from 1s a wider range of systemic immune-based rheumatic diseases (now also including Sjdgren's syndrome, Reiters syndrome, psoiatic arthritis, polymyositis, and ankylosing spondylitis) and found significantly increased APRIL levels in these patients, suggesting an important role for APRIL in these diseases as well (Jonsson et at,1986,candlJ Rhewmatdl %pp 61, 166-9; Roscbke et aL, 2002, J emmunol 169, 2u 4314-21). Finally, increased APRIL expression has also been linked to Multiple Sclerosis (MS). APRIL expression was found to be increased in the astrocytes of MS sufferers compared with normal controls. This is in line with the described APRIL expression in glioblasto mas and in the serum of glioblastoima patients (Deshayes et at 2004, 25 Oncogene 23,3005-42; Roth et al, 2001, Cel Death Dfer8 403-10), APRIL plays a crucial role in the survival and proliferative capacity of several B-cell malignancies and potentially also some solid tumours. APRIL is also emerging as a key player in inflammatory diseases or autoinmunity, Thus, strategies to antagonise 30 APRIL are a therapeutic goal for a number of these diseases. Indeed clinical studies targeting APRIL with TACI-c (Atacicept) are currently ongoing for treatment of several autoimmune diseases. However, TACI-Fc also targets BAFF, a factor involved in normal B-cell maintenance. Antibodies directed against APRIL have been described inWO9614328W, VO2001/60397, WO2002/94192, W09912965, VO2001/196528 and W0990051 & This invention describes antibodies targeting APRIL specifically The antibodies in this invention fully block the binding of APRIL to TACI and at least s partially to BCMA. Some antibodies according to the invention fully block the binding to both BCMA and TACI Suich molecules are useful in a therapy for a number of conditions in which circulaiting soluble APRIL correlates with disease activity and progression, Since expression levels of APRIL can be used as diagnosic and prognostic markers for different diseases, these antibodies can also be applied in such I0 tests. The invention provides binding compounds such as isolated antibodies or antibody fragments which bind to human APRIL Is 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 (Complementary 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 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 bind to human 25 APRIL comprising antibody heavy chain CDRs SEQ ID Ns: 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 bind to human APRIL comprising antibody heavy chain CDRs SEQ I) NOs: 15, 16 and 17 or 30 variants of any said sequences; and antibody light chain CDRs SEQ ID NOs: 18, 19 and 20 or variants of any said sequences.

In another embodiment the invention comprises a binding compound which bind to human APRIL comprising an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a antibody light chain variable region compdsrng the amino acid sequence selected from the group of SEQ ID NO: 6, In yet another embodiment, the invention comprises a binding compound which bind to human APRIL comprising a antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a antibody light chain variable region composing the amino acid sequence of SEQ UD NO:8S In another embodiment the invention comprises an antibody, wherein the heavy chain to has the variable region sequence of SEQ ID NO: 5 and is joined to a IgG I constant region and the light chain has the sequence of SEQ I) NO: 6 and is joined to the s constant regKionn particular, the constant region is fom mouse or human origin. More in particular, the antibody is hAPRILO 1A. In another embodiment the invention comprses an antibody wherein the heavy chain ia has the varable region sequence of SEQ ID NO: 7 and is joined to a IgOl constant region and the light chain has the sequence of SEQ ID NO: 8 and is joined to the K constant region. In particular, the constant region is from mouse or human origin More in particdar, the antibody is hAPRILS)3A. n another embodiment the invention comprises a variant of a binding compound which 20 bind to human APRIL, wherein any of said variant(s) may comprise up to three amino acid modifications in the previous identified CDRs of each the antibody heavy and light chain variable regions. In 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 2 acid modifications in each of the previous identified CDRs in each of the antibody heavy and light chain variable regions, i 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 modifcations in the previous identified CDR sequences in each of the antibody 3o heavy and light chain variable region$. 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 BCMA, In another embodiment the invention comprises a binding compound that fully blocks the binding of APRIL with human TACI and with hmnan BCMA, In another embodiment the invention comprises a binding compound which bind to human APRIL, whewin the binding compound binds human APRIL with a K,,) of about s 10 nM or lower; and blocks binding of human TACI and/or human BCMA to human APRIL with an ICQ of about 2 nM or lowest The invention also comprises a binding compound which binds to human APRIL wherein the binding compound has the same epitope specificity as the antibodies described above ie, competes for the binding epitope of the antibodies described io above. In some embodiments the invention comprises a binding compound which competes for a binding epitope on human APRIL with any of the antibodies described above, and binds human APRIL with a K{ of about 10M n or lower In particular, the epitope on. human APRIL is the epitope vich bind to the antibodies hAP RIL.01A and 1s hAPRL03A preferably hAPRILOI A, In another embodiment the invention comprises a binding compound which competes for a binding epitope on human APRIL with any of the antibodies described above and binds to human APIL with about the same Kr as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the 20 amino acid sequence of SEQ ID NO: 6; In another embodiment the invention comprises a binding compound which competes for a binding epitope on human APRIL with any of the compounds described above and binds to human APRIL with about the same K 1 as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain 25 comprising the amino acid sequence of SEQ I) NO 8 In another embodiment the invention composes a binding compound which competes for a binding epitope on human APRIL with any ofthe antibodies described above and blocks binding of human TACT and/or human BCMA to human APRIL with an ICo of about 2 nrM or lower. 30 In another emnbodiment the invention comprises a finding compound which binds to the confornational human APRIL epitope SMPSHP (preferably IRSMPSHPDRA) optionally supported by TLFR and/or QDVTFTMQ- -8 In yet another embodiment the invention composes a binding compound lich binds to the confirmation human APRIL epitope VSREQQGRQ opdonally supported by TFT4GQ In some embodiments the binding compound of the invention is a chimerie antibody or 5 a fiagment thereofE In another embodiment the binding compound of the invention is a human antibody or a fragment thereof In another embodiment the binding compound of? the invention is a humanized antibody or a fragment thereof 1o In another embodiment the invention composes a binding compound, preferably a humanized antibody, with the above identified CDR's and a human heavy chain constant region variant and a human light chain constant region vacant, wherein each constant region variant comprises up to 20 conservatively modified amino acid substitutions. 15 In another embodiment the binding compound of the invention is a antibody fragment selected from Fab, Fabt FabtSH, Fv scFv, F(ab) 5 bispecifc mAb or a diabody fragment 'The invention also comprises the binding compound as described above which inhibits the proliferation and survival of B-cells. 20 The invention also comprises nucleic acids encoding the anti-APRIL binding compound of the invention Included in the mention are nucleic acids encoding any one ofthe amino acid sequences enclosed in SEQ ID NOS 5 to 20, Also included within the invention are nucleic acids comprising SEQ ID NOS 1,2, 3 or 4. In addition, the invention also comprises the nucleic acids encoding the variants of the amino acid 5 sequences as described hereinabove. The invention also comprises cells ad expression vectors comprising nucleic acids encoding the binding compound of the invention, Further, the invention comprises a method of producing a binding compound of the invention comprising: (a) culturing the host cell co mpiising anucleic acid encoding an 3o antibody or antibody fragment of the invention in culree medium under conditions wherein the nucleic acid sequence is expressed, thereby producing polypeptides -9 comprising the light and heavy chain variable regions; and (b) recovering the polypeptides from the host cell or culture medium. The invention also comprises compositions comprising a binding compound of the invention in combination with a pharmaceutically acceptable carrier or diluent 5 The invention also composes a method of inhibiting the proliferation and/or survival of an immune cell, comprising administering to a subject in need thereof a therapeutically effective amount of a binding compound of the invention. In one embodiment, the method may be used to treat cancer. In another embodiment, the method may be use to treat an autoinimune or inflammatory disease, 0) In some embodiments, the invention comprises a method of inhibiting the proliferation and/or survival of an immune cell, comprising administering to a suject in need thereof a therapeutically effective amount of a binding compound of the invention, and further comprising measuring B cell proliferation and/or survival ex vivo in a sample derived from the subject, wherein an inhibition of the proliferation and/or survival of ii the B cell indicates that the treatment should be continued. In other embodiments, the invention comprises a method of inhibiting the proliferation and/or survival of an immune cell, comprising administering to a suiect in need thereof a therapeutically effective amount of a binding compound of the invention, and further comprising measuring B cell proliferation and/or survival ex vivo in a sample 2P derived from the subject, wherein an increase in B cci proliferation and/or survival predicts the likelihood that the treahnent will he successfdL The invention also comprises an imniunoconjugate comprising an anti-APRIL binding compound of the invention, linked to a therapeutic agent such as a bacterial toxin or a radiotoxin. Non-limiting examples of cytotoxie agents include taxol, cytochalasin B, as niitomycin, etoposide and vincristine or other antimetabolites alkylating agents, antibiotics and antimitotics. The invention also comprises a method of inhibitinge t proliferation and/or survive of an imrune cell, comprising contacting an immune cell with a binding compound of the present invention. in sInome embodiments the method comprises further administering a second therapeutic agent or treatment modality.

- 10 In sonic embodiments, antifAPRIL binding compounds can be combined with a treatment that is considered to be standard of care in cancer or autoimmune or inflammatory disease. Rationale for such combinations is that concurrent increased inmmne inhibition by anti~APRIL will induce or facilitate iitial clinical response to s standard of care treatment, induce double clinical response and long-term immune control of disease. In another embodiment the binding compounds of the present invention are used diagnostically. in yet another embodiment the binding compounds of the invention are used to to mes UreB cell proliferation and/or survival ex vivo in a sample derived from the subject, wherein an inhibition of the proliferatia and/or survival of the B cell indicates that the treatment with the binding compound as described here above should be continued In another embodiment the binding compounds according to the invention are isolated 1s antibodies or antibody fragments which bind to human APRIL, The tenn "antibody" refers to any form of antibody that exhibits the desired biological activity, such as inhibiting binding of a ligand to its receptor, or by inhibiting ligand 20 induced signaling of a receptor Thus "antibody" is used in the broadest sense and specifically covers 4 but is not limited tononoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and nultispecitle antibodies (e. g bispecitic antibodies) 2s "Antibody fragment" and "aibody binding fragment mean antigen-binding fragments and analogues of an antibodytypically including at least a portion ofthe antigen binding or variable regions (e.g. one or more CDRs) of the parental antibody. An antibody fragment retains at least some of the binding speciefiity of the parental antibody. Typically, an antibody fragment retains at least 10% of the parental binding 3o activity when that activity is expressed on amolar basis. Preferably, an antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the parental antibody's binding affmity for the target, Examples of antibody fragments - 11 include, but are not limited to, Fab Fab, F(ab*) and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules e.g. sc-Fvunibodies (technology from Gennab); nanobodies (technology fron Domxantis); domain antibodies (technology from Ablynx); and muhispeciftic antibodies formed from antibody fragments. s Engineered antibody variants are reviewed in flolliger and Hudson, 2005, Nat BiotechnoL 23, 112641136. A Fab fragmet" is comprised of one light chain and the CHI and variable regions of one heavy chai. The heavy chain of a Fab molectle cannot foran a disulfide bond with lo another heavy chain molecule. An "Fe" region contains two heavy chain fragments comprising the Cul and Ca2 domains of an antibody, The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of te CH3 domains. 15 A TaW fragnen" contains one light chain and a portion of one heavy chain that contains the V1 domain and the C nil domain and also the region between the C44 and C V domains, such that an interchain disulfide band can be fomied between the two heavy chains of two Fab' fragments to fann a F(ab) molecule. 20 A TF(ah'bfragment" contains two light chains and two heavy chains containing a portion of the constant region between the Cal and CQ domains, such that an interchain disulfide bond is fom-ed between the two heavy chains. A F(ab')z fragment thus is composed of two Fabfragments that are held together by a disuffide bond. 5 between the two heavy chains. The "Tv region' comprises the variable regions from both the heavy and light chains, but lacks the constant regions, 30 A "inglesbain Fv antibody" (or "scFv antibody") refers to antibody fragments comprising the V 8 and V 1 domains of an antibody, wherein these domains are present in a single polypeptide chain. GeneraLy, the Fv polypeptide further comprises a - 12 polypeptide linker between the Vn and VI domains which enables the scFv to fonn the desired structure for antigen binding, For a review of scfv, see Pluekthun, 1994;THE PHARMACOLOGY OF MoNOCLONu ANTiBODIES, voL 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, International Patent Application 5 Publication No. WO 88/01649 and U.S. Pat. Nos, 4,946, 778 and 5,260,203, A "diabody" is a small antibody fragment with two antigen-binding sites. The fragments comprises a heavy chain variable domain (Vn) connected to a light chain variable domain (Vt) in the same polypeptide chain (Vn-Vi. or VT-V). By using a 10 linker that is too shod to allow pairing between the two domains on the same enain, the domains arc forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., 1P 404,097; WO 93/11161; and Holliger et al, 1993, Proc. Nal Acad Sc UISA 90, 6444 6448, 15 A "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more Varegions are covalently joined with a peptide linker to create a bivalent domain antibody fragment. The two VI regions of a 20 bivalent domain antibody fragment may target the same or different antigens. As used herein antibody hAPRIL0IA is a mouse antihody wherein the heavy chain has the variable region sequence of SEQ ID 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 25 the K constant region. Antibody hAPRIL.03A is a mouse antibody, wherein the heavy chain has the variable region sequence of SEQ ID NO: 7 and is joined to a IgGI constant region and the light chain has the variable region sequence of SEQ ID NO: 8 and is joined to the K constant region. 30 An antibody fragment of the invention may comprise a sufficient portion of the constant region to pennit dimerization (or multimerization) of heavy chains that have 13 reduced disulfide linkage capability, for example where at least one of the hinge cysteines nonnally involved in iner-heavy chain disulfide linkage is altered as described herein. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally s associated with the Fe region when present in an intact antibody, such as FoRn binding, antibody half life modulation, ADCC (antibody dependent cellar cytotoxicity) fuoncion and/or complement binding (for example, where the antibody has a glycosylation profile necessary for ADCC function or complement binding). i The term "chimeric" antibody refers to antibodies in which a portion of the heavy mid/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to s another antibody class or subelassas well as fragments of such antibodies, so long as they exhibit the desired biological activity (See, for example, U.S. Pat.No. 4,816,567 and Morrison et aL, 1984, Proc, NatL Acad Set USA St 685 1-6855) As used herein, the term "humanized antibody" refer to forms of antibodies that 20 contain sequences from nonrhuman (eg, urine) antibodies as Well as human antibodies. Such antibodies contain minimal sequence derived from non-hunman inmnmoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops corrspond to those of a non-human immuinoglobulin and all or 25 substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an mnoglobulin constant region (Fe), typically that of a human immiunoglobulin. The humanized fonns of rodent antibodies will essentially comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions so may be included to increase affinity, increase stability of the humanized antibody, or for other reasons. However, as CDR loop exchanges do not unifornly result in an antibody with the same binding properties as the antibody of origin, changes in - 14 framework residues (FR), residues involved in CDR loop suppoit might also be introduced in humanized antibodies to preserve antigen binding affinity (Kahat et al, 1991, Immun 147, 1709) s 'The term "antibody" also includes "fully human" antibodies, i -, antibodies that comprise human innunoglobulin protein sequences only. A fully human antibody may contain mine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, "mouse antibody" or "rat antibody" refer to an antibody that comprises only mouse or rat immunoglobulin sequences, o respectively. A fully human antibody may be generated in a human being, in a transgenic animal having human immunoglobulin gennline sequences, by phage display or other molecular biological methods, Also, recombinant immunoglobul ins may also be made in transgenie mice, See Mendez et at 1997, Nature Gene tics 15,146-156, See aav Abgenix and Medarex technologies. 15 The antibodies of the present invention also include antibodies with modified (or blocked) Fe regions to provide altered effector functions. See, e:g., U1S. Pat. No 5,624,821; W02003/0863 10; W02005/120571; W02006/005 7702; Presta, 2006, Adk Drug Dehvepy Rev, 58:640-656. Such modification can be used to enhance or suppress 20 various reactions of the immune system, with possible beneficial effects in diagnosis and therapy Alterations of the Fe region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation and adding multiple Fe, Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent dosing, with the concomitant 25 increased convenience and decreased use of material. See Presta, 2005,] Alergy Chn. hntmwaL116, 731 at 734-35, The antibodies of the present invention also include antibodies with intact Fe regions that provide fU effector functionseeg. antibodies of isotype igG1 which induce complement-dependent cytotoxicity (CDC) or antibody dependent celludar cytotoxicity 3o (ADCC) in the a targeted cell. The antibodies may also be conjugated (e.g., covalently linked) to molecules that improve stability of the antibody during storage or increase the half-ife of the antibody - 15 in vWo Examples of molecules that increase the half4ife are albumin (e.g.. human sern albumin) and polyethylene glycol (PEG). Albumin-linked and PElylated derivatives of antibodies can be prepared using techniques well known in the art. See, g. Chapman, 2002, Adi. Drug Deliv. Rem 54, 531-545; Anderson and Tomasi, 1988, s .1 knmunot Method i109, 37-42; Suzuki et al, 1984, Biochim Biophys Acta 78, 248 255; and Breke and Sandlie,2003, Mature Rev, 2, 52-62. Antibodies used in the present invention will usually bind with at least a KD) of about I0M, more usually at least 10 M, typically at least 10' 7 M, more typically at least 10 8M, preferably at least about .10 4, and more preferably at least IW 14, and most w preferably at least 10- M. &e e g., Presta, e at, 2001, Thromb, Haemost 85, 379 389; Yang, et aL 2001, 0Cri Rev. OncoL tematot 38, 17-23; Camahan, et al, 2003, (Cin Cancer Res (Supph) 9 3982s-3990s, Antibody aflinities may be determined using standard analysis. i The term "hypervariahle region" as used herein, refers to the amino acid residues of an antibody which are responsible for antigen-binding The hypervariable region comprises amino acid residues from a complementarity detennining region" or TDR," defined by sequence alignment, for example residues 24-34 (LI) 50-56 (L2) and 8997 (L3) in the light chain variable domain and 31-35 (111) 50-65 (12) and 95 2o 102 (B3) in the heavy chain variable domain; see Kabat t aL, 1991, Sequences of proteins of munological Interest, 5th Ed Public Health Senice, National Institutes of Health, Bethesda;Md and/or those residues from a "hypervariable loop" (HVL), as defined strueturally, for example, residues 26-32 (L) 50-52 (L2) and 91-96(13) in the light chain variable domain and 26-32 (111), 53-55 (Ff) and 96-101 (H3) in the 25 heavy chain variable domai; see Chothia and Lesk 1987,] MWt Bi 19 901-917, Traneworki or "FI residues are those variable domain residues other than the hypervariable region residues as herein defMined. An "isolated" antibody is one that has been identified and separated and/or recovered 30 from a component of its natural environment; Contaminant components of its natural enviromnent are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or - 16 nonproteinaceous salutes. In some embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as detenmined by the Lowry method, and most preferably more than 99% by weight, (2)to a degree sufficient to obtain at least 15 residues of Ntemrinal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDSPAGiunder reducing ornonreducing conditions using Coonassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibodys natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one puificatdon step. An "isoIated" nuclei acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which i is ordinarily associated in the natural source of the antibody nuclei acid. An isolated nucleic acid molecule is other than in the form or setting in vich it is found in nature is Isolated nucleic acid molecules therefore are distinguished from the nucleic acid moleule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different -from that of natural cells. 20 The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly 25 specific being directed against a single antigenic site. Furthennore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoconal" indicates the character of the antibody as being obtained from a substantially no homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the 17 hybridoma method first described by Kohler et al, 1975,,Ntaure 256,495, or may be made by recombinant DNA methods (see, for example, US. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, 1991, Nature 352, 624-628 and Marks et al, 5 1991, J MoZ Biot 222. 581-597, for example. The monoclonal antibodies herein specifically include "chimeric" antibodies. As used herein, the tern "immune cell" includes cells that are of hematopoietic origin and that play a role in the mnuine response. Immune cells include lymphocytes, such i) as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, cosinophils, mast cells, basophils, and granulocytes. As used herein an "imrnlunoconjugate" refers to an anti-APRIL antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a bacterial toxin, a cytotoxic drug 13 or a radiotoxin. Toxic moieties can be conjugated to antibodies of the invention using methods available in the art. As used herein, a sequence "variant"refers to a sequence that differs from the disclosed sequence at one or more amino acid residues but which retains the biological 20 activity of the resulting molecule. "Conservatively modified variants" or "conservative amino acid substitution" refers to substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of 25 skill in this art recognize that, in generaL, single amino acid substitutions in non essential regions of a polypeptide do not substantially alter biological activity (see, e.g, Watson, et at, Molecular Biology qf the Gene,Th Bejamii/Cminngs Pub Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are preferably made in accordance with those set forth below as follows: Exemplary Conservative Amino Acid Substitutions Orio'nal residue eConservative substitution| Ala (A) | Gy;Z Ser 18 Original residue Conservative substitution Arg (R) Lys, His Asn (N) Gin; His Asp () Glu; Asn ys (C) | Ser; Ala SIn (Q Asn G Lv (GA Ala le (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M)Leu;. Ile; Tyr Phe (F) Tyr; Met; Leu Pr.P Ala 8cr jS) 'hr Trp (W) Tr Phe * Tyr (Y) Trp; Phet Val (Y) - 11e; Leu As used herein, the term "about"refers to a value that is within an acceptable enror range for the particular value as determined by one of ordinary skill in the arts which will depend in part on how the value is measured or determined, Le,, the lImitations of s the measurement system. For example, "about" can mean within I or more than 1 standard deviation per the practice in the art Alternatively, "about" or "comprising essentialV of" can mean a range ofup to 20%Furthermore particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value, When particular values are provided in the application and wo claims unless otherwise stated, the meaning of "about" or "comprising essentially of" should be assumed to be within an acceptable error range for that particular value "Specifically" binds, when referring to a ligandreceptor$ antibody/antigen, or other binding pair, indicates a binding reaction which is determinative of the presence of the 1s protein, eg. APRIL, in a heterogeneous population of proteins and/or other biologies, Thus, under designated conditions, a specified ligand/antigen binds to a particdar receptor/antibody and does not bind in a significant amount to other proteins present in the sample, 19 "Administration" and "teatnent," as it applies to an animal human, experimental subject, cell, tissue, organic or biological fluid, refers to contact of an exogenous phanautical, therapeutic, diagnostic agent or composition to the animal, hunamn zs subject, cell, tissue, orgat, or biological fluid. "Administation" and treatment" can refer, e~g., to therapeuticphannacokinetic, diagnostic, research, and experimental methods. Treatment of a cel encompasses contact of a reagent to the cell, s well as contact of a reagent to a fluid, where the ftid is in contact with the cell "Administration" and "treatment" also means in vitro and ex viva treatmentse.g ofa w cell, by a reagent, diagnostic, binding composition, orby another cell. Monoelonal Antibodies Monoxlonal antibodies to human APRIL can be made according to knowledge and skil in the art of injecting test subjects with human APRIL antigen and then isolating hybridonas expressing antibodies having the desired sequence or functional characteristics, DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of 2 binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies), The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E, coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to z5 obtain the synthesis of nvnoelonal antibodies in the recombinant host cells Recombinant production of antibodies will be described in more detail below. In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in Mctafferty et al, 30 1990, Nature, 348, 552-554. Clackson et al, 1991.Niature, 352, 624-628, and Marks e aL, 1991,J. Mot Bio1 222, 581-597 describe the isolation of marine and human antibodies, respectively, using phage libraries. Subsequent publications descrtibe the -20 production of high affinity (nM range)human antibodies by chain shuffling (Marks et aL, 1992, Bio/1hhnology 0, 779483), as well as combinatoral infetion and in vivo recombination as a strategy for constmting very large phage libraries (Waterhouse et at 1993, Nw Acids, Res 21, 2265-2266). Thus, these techniques are viable s alternatives to traditional monoclonal antibody hybridona techniques for isolation of monoclonal antibodies. Chimerie Andbodies The antibody DNA also may be modified, for example, by substituting the coding do sequence for human heavy- and lighh-cain constant domains in pae ofthe homologous urine sequences (US, Pat No, 4,816,567; Morrison, et at, 1984, Proc Nad A cad Set USA, 81, 6851), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for non-immunoglobulin material (eg., protein domains). Typically suchnon-immunoglobulin material is substituted for the y constant domains lof an antibody, or is substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity fbr a different antigen 2o Humanized and Human Antibodies A humanized antibody has one or more amino acid residues from a source that is non human Tie non-human amino acid residues arc often referred to as "Import" residues, and are typically taken from an import " variable domain HiUnianization can be perbrmed generally following the method of Winter and co-workers (Jones et aL, 2s 1986, Nature 321, 522-525; Riechmann C at, 1988,Nature,332; 323-327; Verhoeyen et at, 1988, Science 239, 1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species 30 In practice.humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in non-hwnan, for example, rodent antibodies.

21 The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so called "best-fit" method, the sequence of the variable domain of a rodent antibody is s screened against the entire library of known human variable-domain sequences. The hunan sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al, 1981,J liphmmnal 151,, 2296; Chothia et at, 1987,1MoL BioL 196 901). Another method uses a particular framework derived from the cosensus sequence of al human antibodies of a particular io subgroup of light or heavy chains. The same framework may be used for seve ra diferent humanized antibodies (Carter et aL, 1992, Proc. Nar! Acad ScL USA 89, 42851 Presta metal, 1993', bnal 151,2623). It is further important that antibodies be humanized with retention of high affinity for 1s the antigen and other favorable biological properties, To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and hunanized sequences. Three-dimensiond innmunoglobtin models are commonly available and are familiar to those skilled in the 2n art. Computer progrns are available which illustrate and display probable three dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immuoglobulin sequence, i&e, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its 25 antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved, In general, the CDR residues are directly and most substantially involved in influencing antigen binding as HMunanization of antibodies is a straightforward protein engineering task, Nearly all mune antibodies can be humanized by CDR, grafting, resulting in the retention of -22 antigen binding, See, Lo, Benny, KC, editor, in Antibod Engineering: Methods and Protocols, volume 248, humana Press New Jersey, 2004. Alternatively, it is now possible to produce transgenic animals (eOg,' mice) that are s capable, upon hnnmnkzation of producing a fWll repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described dat the homozygous deletion of the antibody heavy-chain joining region (MH) gene in chimeric and germ-ine mutant Mice results in complete inhibition of endogenous antibody production Tnsfer ofthe hmian gernline immunoglobulin 1o gene army in such gem-line mutant mice will result in the production of human antibodies upon antigen challenge, See, e.g., Jakobovits et at 1993, Proc Nad. Acad Sci USA 90, 2551; Jakobovits et al, 1993, atwure 362, 255-258; Bgruggermann et al 1993, Year in Immunology 7 33; and Duchosal et al, 1992, Ntvre 355,258 hanan antibodies can also be derived fromn phage-display libraries (Hoogenboom et at, 1991, is J )I Biot 227,381; Marks et aL, £ Mo Bio. 1991, 222, 581-597; Vaughan et al, 1996, Nature Biotech 14, 309) Anno acid sequence variants of humanized anti-APRIL antibodies are prepared by introducing appropriate nucleo tide changes into the humanized anti- APRL antibodies' 20 DNAs, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences shown for the humanized anti-APRIL antibodies Any combination of deletion, insertion, and substitution is made to arrive at the final constuct, provided that te final constmct possesses the desired characteisties. The amino acid changes also may alter 25 post-translational processes of the hnumanized anti-APRIL antibodies, such as changing the number or position of glycosylation sites. A useful method for identification of certain residues or regions of the humanized anti APRIL antibodies polypeptides that are preferred locations for mutagenesis is called ahine scanning mutagenesis," as described by Gurighim and Wells, 1989, 3m Science 244, 10811 085, Here, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Gl) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with APRIL antigen. The amino acid residues demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for,the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the 5 nmtationperse need not be predetermined For example, to analyze the performance of a mutation at a given site, Ala scanning or random mutagenesis is conducted at the target codon or region and the expressed humanized anti-APRIL antibodies' variants are screened for the desired activity. 0 Ordinarily, amino acid sequence variants of the humanized anti-APRIL antibodies will have an amnino acid sequence having at least 75% amino acid sequence identity with the original humnanized antibody amino acid sequences of either the heavy or the light chain more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, 98% or 99% Identity or homology with is respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the humanized residues, after aligning the sequences and introducing gaps, if necesmry, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence idendty. None of N-terminal, C-terminal, or internal extensions, deletions, or 20 insertions into the antibody sequence shall be construed as affecting sequence identity or homology. Antibodies having the characteristics identified herein as being desirable in humanized anti-APRIL antibodies can be screened for inhibitoxy biologic activity in vitro or 23 suitable binding affinity To screen for antibodies that bind to the BCMA or TACI epitopes on human APRIL bound by an antdbody of interest (eg< those that block binding of APRIL), a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be perfonned, Antibodies that bind to the same epitope are likely to cross so block in such assays, but not all cross-blodking antibodies will necessarily bind at precisely the sameepitope since cross-blocidng may result from steric hindrance of -24 antibody binding by antibodies bind at overapping epitopes or even nearby non overlapping epitopes Altenatively, epitope mapping, e~g., as described in Champe et al, 1995, MiL Chem 270, 1388-1394, can be perfonned to determine whether the antibody binds an i epitope of interest. "Alaninc scanning rnutagenesisi as described by Cunningham and Wells, 1.989, Science 244, 1081-1085, or some other form of point mutagenesis of amino acid residues in human APRIL may also be used to determine the functional epitope for anti-APRIL antibodies of the present invention. Additional antibodies binding to the same epitope as an antibody of the present 10 invention may be obtained, for example, by screening of antibodies raised against APRIL for binding to the epitope, or by imnumnization of an animal with a peptide comprising a fragment of human APRIL comprising the epitope sequences (e.g. BCMA or TACI). Antibodies that bind to the same functional epitope might be expected to exhibit similar biological activities, such as blocking receptor binding, and is such activities can be confirmed by functional assays of the antibodies. Antibody aflinities may be determineed using standard analysis. Preferred binding compounds such as e.g. humanized antibodies are those that bind hunan APRIL with a Ka value ofno more than about lx10, preferably no more than about Dxl more preferably no more than about lxi0t and most preferably no more than about lxI 010 20 or even 1x 101 M The humanized antibody can be selected from any class of inmmunoglobulins including Ig A IgG, l), i gA, and FgBE Preferably, the antibody is an IgG antibody, Any isotype of IgG can be used, including IgGj, lg&3. IgCt, and IgG4. Variants of the IgG 25 isotypes are also contemplated. The humanized antibody may comprise sequences from. more than one class or isotype; Optimization of the necessary constant domain sequences to generate the desired biologic activity is readily acheed by screening the antibodies in the biological assays described in the Examples, Likewise, either class of light chain can be used in the compositions and methods 3o herein. Specificallykappa, lambda, or variants thereof are usefulin the present compositions and methods.

25 The antibodies and antibody fragments of the invention may also be conjugated with eytotoxic payloads such as cytotoxic agents or radtionucleotides such as eh',fY, "in, 1 4 C 1251, 1s 31 C 1 C, kN, X S Cr, To, "tRa Co, 5 9 Fe. 57 5e "tYu, ,,ICU, 2 ij A Ph, Se Pd Th, and )KC 4 1Mn1 r and Fe. Such antibody conjugates may be used in immunotherapy to selectively target and kill cells expressing a target (the antigen for that antibody) on their surface, Exemplary cytotoxdc agents include ricin, vinca alkaloid, methotrexate, Psuedomonas exo toxin, saporin, diphtheria toxin, cisplatin, doxorubicin, abrin toxingelonin and pokeweed antiviral protein, t The antibodies and antibody fragments of the invention may also be conjugated with fluorescent or chemilluninescent labels, including fluorophores such as rae earth chelates, fluorescein and its derivatives, rhodamine and its derivatives isothiocyanate, phyeoerythru, phycocyanin, allophycocyanin, o-pthaladehyde, fluorescamine, Eu, dansyl, unibelliferone, luciferin, lurninal label, isoluminal label, an aromatic acridinium i5 ester label, an imidazole label, an aeddimium salt labeL, an oxalate ester label, an aequarin label, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals Any method known in the at for conjugating the antibody molecules or protein molecules of the invention to the various moieties may be employed, including those o methods described by Hunter et aL, 1962, Nature 144, 945; David et at, 1974, Biochemistry 13,1014; Pain et at, 1981, J. immunol. Meth. 46, 219; and Nygren, J. 1982. Histochen and Cytochem, 30, 407 Methods for conjugating antibodies and proteins are conventional and well known in the art, 25 Antibody Purification When using recombinant techniques, the antibody can be produced intracellulary, in the peripiasmic space, or directly secreted into the medium If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centdifugation or ultrafiltration. Carter ct aL, 1992, so Bi/Tchnodgy 10, 163-167 describe a procedure for isolating antibodies which are secreted to the periplasmic space ofE. cal. Briefly, cell paste is thawed in the presence of sodium acetate (pH 35), EDTA, and phenyhnethylsalfonylfluoride (PMSF) over -26 about 30 minn Cell debris can be removed by centrifligation, Where the antibody is secreted into the medium, supematants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit, A protease inhibitor such as PMSF 5 may be included in 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 the ells can be puified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity 10 chromatography, with affmity chromatography being the preferred purification technique The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fe region that is present in the antibody Protein A can be used to purify antibodies that are based on human gamma,1, .gana2, or -gamma4 heavy chains (Lindmark t a, 1983, Jnnmnot Meth 62, 1-13), Protein G 15 is recommended for all mouse isotypes and for human sgamma3 (Guss et at, 1986, EMBO J5, 1567-1575), The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable mattces such as controlled pore glass or poly(styrenedivinyl)benzne allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody 20 comprises a CH3 domain, the Bakerbond ABXrM resin (3, Baker, Phillipsburg, NI) is useful for purification, Other techniques for protein purification such as fractionation on an ion-exchange cohunn, ethanol precipitation, Reverse Phase HPLC, chromatography on siica, chromatography on heparin SEPHIAROSETM chromatogmphy on an anion or cation exchange resin (such as a polyaspartic acid 5 column),chromatofocusing, SDS PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. In one embodiment, the glycoprotein may be purified using adsorption onto a lectin substrate (eg. a lectin affinity column) to reniove fuasecentairang glycoprotein frn 30 the preparation and thereby enrich for facose-free glycoprotein.

-27 Pharmaceutical Formulations The invention comprises phannaceutical fonnulations of an APRIL binding compound. To prepare pharnaceutical or sterile compositions, the antibody or fragment thereof is admixed with a phannaceutically acceptable carrier or excipientsee, ezg, Remingtoni 5 Pharmoceutical Seiences and US Piarnwcopeia: National Formtdary, Mack Publishing Company, Easton, PA (1984). Fonnulations of therapeutic and diagnostic agents may be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of., lyophilized powders, slurries, aqueous sohions or suspensions (see, eg Hardman, etal, 2001, Goodman and Gihnan's The m1 Pharmacological Basis of Therapeutics, Mcsraw-Hill, New York, NY- Gennaro, 2000,Remington The Science and Practice of Pharmacy;LippincottWilliams, and Wiikins, New York, NY; Axis, et aL (eds., 1993, Pharmaceutical Dosage Forms: Parenteral Medications, Marel Dekker, NY; Lieberman, et aL (eds), 1990, Phamaceutical Dosage Forms: Tablts, Marcel Dekker, NY; Liebeman, et at (eds.), 1s 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Evipient Toxicity and Sfety, Marcel Dekker, Inc., New York, NY). Toxicity and therapeutic efficacy of the antibody compositions, administered alone or 2- in combination with an immunosuppressive agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eag for determining the LD 5 (the dose lethal to 50% of the population) and the E3)so (the dose therapeutically 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 2- LDao and EDA). The data obtained from these cell cu'titure assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED j with little or no toxicity, The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. 30 Suitable routes of administration include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration and oral adniistration.

28 Administration of antibody used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subeutaneous, intraperitoneal, parenterla intraarterial or intravenous injection In one embodiment, 5 the binding compound of the invention is administered intravenously, In another embodiment, the binding compound of the invention is administered subcutaneously. Alternatively, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into the site of action, often in a i0 depot or sustained release fonnulation. Furthermore, one may administer the antibody in a targeted drug delivery system Guidance in selecting appropriate doses of antiboldies, cytokines, and small molecules are available (see'g e, Wawrzynczak, 1996, Antibody Therapy. Bios Scientific Pub. an Ltd, Oxfordshire, UK Kresina (ed), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed), 1993, Monocdonal Antibodies andPeptide Therapy in Autohnmune Diseases, Marcel Dekker, New York NY; Baert, et al, 2003, New Engl J Med 348, 601-608, Milgrin, et at, I99,New Ehgl i Med. 341. 19661973; Slamon, eta!,, 2001, New EngL I. Med 344,783-792; Beniaminovitz, 2a et al, 2000, New Engl J; Med 342, 613-619; Ghosh. et at, 2003, New EngJ di Med 348, 24-32; Lipsky, et at, 2000, New Dg. i Med 343, 1594-1602). Detennination of the appropriate dose is made by the clinician, eg., using parameters or factors known or suspected in the art to affet treatment or predicted to affect 25 treatment, Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small inTrements thereafter until the desired or optimum effect is achieved relative to any negative side effects important diagnostic measures include those of symptoms of e.g, the inflammation or level of inflanmatory cytokines produced. s0 A preferred dose protocol is one involving the maxinal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose is generally at least - 29 0,05 pg~kg body weight, more generally at least 0.2 pg/kg, most generally at least 0,5 pg/gtypically at least I pgkg, more typically at least 10 pg/kg, most typically at least 100 pg/kg, preferably at least 0,2 mg/kg, more preferably at least 1.0 mg/g, most preferably at least 2.0 mg/kg, optimally at least 10 mg/kg, more optimally at least 25 , mg&g, and most optimally at least 50 mg/kg (seeg Yang, aL 2003, New Eng. J Med. 349, 427434; Herold, er at 2002, Mw Eng J, Med 346, 1692-1698; Liu, et all 1999,] Nezwot Neurosurg. Psyck 67,451-456; Pordelji, et al, 2003, Cancer AnmunoL mmunoher. 52, 133-144). The desired dose of a small molecule therapeutic, e.g,, a peptide mimetic, natural product, or organic chemical, is about the wo same as for an antibody or polypeptide, on a moles/kg basis. As used herein, "inhibit" or "treat" or "treatment" includes a postponement of development of the symptoms associated with disease and/or a reduction in the severity of such symptoms that will or are expected to develop with said disease. The terms 1 further include ameliorating existing symptoms preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms Thus, the temns denote that a beneficial result has been conferred on a vertebrate subject with a disease As used herein, the tenn "therapeutically effie!ive amount" or "effective amount" 20 refers to an amount of an anti-APUL antbody or fragment thereof, that when adnistered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or amliorate the disease or condition to be treated. A, therapeutically effective dose further refers to that amount of the compound sufficient to result in ameliortion of symptoms, eg., treatment, healing, prevention or 2- amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient admnistered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic 30 effect, whether administered in combination, serially or siaultaneously An effetive amount of therapeutic will decrease the symptoms typically by at least 10%; usually by -30 at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. Methods for co-administration or treatment with a second theraeutic agent are wel s known in the art, see, Hardman, et aL (eds4, 2001, Goodman and Gilman 's The Pharnacologcal Basis of Therapeutics, le edt McGrawIHill, New York, NY; Poole and Peterson (eds4 200 Phaumacotherapeuticsfor Advanced Pactice: A Practical Approach, Lippincott, Williams & Wilkins, Pbila, PA; Chabner and Longo (eds) 2001, Cancer Chemotherapy and Biotherapy, iippineott Williams & Wilkins, Phila., in PA. The pharmaceutical composidin of the invention may also contain other agent, including but not limited to a cytotoxic, cherothempeutic cytostatic, anti-angiogenic or antimetabolite agent, a tumor targeted agent an immune stimulating or inunune is modulating agent or an antibody canjigated 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 the Antibody a-nd Antibcdy Fragments of the Invention 20 The antibodies and antigen binding fragments of the invention, which specifically bind to human APRiL, can be used to treat several diseases in which the activity of APRIL is central to pathology. Broadly speaking this includes cancer, auto-immunity, inflammatory diseases and potentially multiple sclerosis, a CNS disease. 25 Cancer The antibody or antigen finding fragments of the invention which specifically bind APRIL can be used to treat cancer. Prefen-ed cancers whose growth and survival may be inhibited by the invention include any cancers known to express APRIL and depend on this for proliferative signals. Non-limiting examples of such cancers include several auo B cell malignancies, such as Chronic Lyiphocytic Leukaeinia (CLL);Maltiple Myeloma, Hodgkin's lymphoma and Non- Hodgkin's lymphoma including Burkitt's 31 lumphoma and diffuse large B cell lympboma 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 hi combination with 5 other anti-cancer agents, such as chemothempeutic reagents other biological agents. Additionally the invention includes reframtory or remurent malignancies or treatment of netastases derived from any of these malignancies. Autoimmune Disease io The binding compounds of the invention may be used to treat several autoinmune diseases, where the expression of APRIL has been sown to play a role in pathology. Examples of such diseases are Rheumatoid Arthritis (RA), Systemic Lupus Erythemtosus (SLE) and Sjogren's syndrome, In addition, higher then normal tires of APRIL were found in the serum ofmultiple sclerosis patients and also increased levels found in their astrocytes, Thus APRIL is a contributing factor to disease pathology and therapeutic blockage of APIL in MS may be beneficial Non-Therapetic Uses for the Antibody and Antibody Fragments of the Invention The non-therapeutic uses for these antibodies include flow cytometry, westem blotting, m enzyme linked inmunosorbant assay (ELISA), inmnnobistochmistry. The antibodies of this inventionmay also be used as an affinity purification reagent via immobilization to a sepharose column. 2- The antibody may also be useful in diagnostic assaye~g for detecting expression of APRIL in specific cells, tissues, or sermm For diagnostic applications, the antibody typically will be labeled (either directly or indirectly) with a detectable moiety. Numerous labels are available which can be generally grouped into the flowing categories: biotin, fluorochromes, radionucleotides, enzymes, iodine, and biosynthetic so labels.

32 The antibodies of the present invention may be employed in any known assay method, sudh as competitive binding assays, direct and indirect sandwich assays and imnunoprecipitation assays. Zola, MonocionalAnibadiss A Manual qf Techniques pp 147-158 (CRC Press Inc. 1987). The antibody may also be used for in vivo diagnostic assays. General, the antibody is labeled with a radionuclidel so that the antigen or cels expressing it can be localized using irntmnuoseintiography or positron emission tomography, o Legends to the figures Figure I Figure 1 shows APRIL reactivity and BCMA-blocking activity of hAPRILOIA and hAPRIL.03A hybridoma supematants, Figure IA shows hAPRILOIA and hAPRILA3A binding to FLAG-hAPIRiL catured by an anti-F LAG antibody. Aprily-5 is antibody was used as a positive control. Figure 1B demonstrates that hAPRILJ A and IAPRIL.03A hybridoma supematants, and not Aprily-5 block the binding of FLAG hAPRILto BCMA-Fc, Figure 2 Figure 2 shows distinct binding and receptor-blocking characteristics of purified 20 hAPRtL01A and hAPRILO3A antibodies Figure 2A confirms binding of purified hAPRLI0A and hAPRILO3A to FLAG-hAPRIL, captured by an anti-FLAG antibody, Figure 2B shows that only iAPRILJI3A binds FLAG-hAPRIL that is captured by BCMA-Fc. Figure 2C shows that heAPRIL;01A AiMly blocks FLAG bAPRIL binding to BCMA-Fe. while hAPRILS}3A partially blocks this interaction, 5 Figure 2D demonstrates that hAPRJL) I A and hAPRIL03A both fully block FLAG hAPRIL with '[ACI-Fe, Figure 3 Figure 3 shows the receptor-blocking ELISAs for hIAPRIL01A hAPRIL)3A and 12 known commercially available monoclonal anti-,APRIL antibodies. This illustrates that IAPPUL.01A and hAPRILO3A ar unique in their ability to block APRIL binding to BCM4A (Figure 3A) and TACI (Figure 3B). Figure 4 Figure 4 shows that hAPRILGIA and IAPRILS03A block APRILdriven B-cell s proliferation and isotope classv-sithhing but do not affect BAFF-mediated processes. Figure 4A is an in-vitro B-cell assay which demonsrates that the described smonoclonal antibodies block known APRIL funictions such as the survival and proliferation of B cells and production of class-switched IgA antibodies. Of significance is the demonstration that both monoclonal antibodies block APRIL activity more effectively o than TACl-Fc, which was administered at equimolar concentration Figure 4B shows that the antibodies do not affect BAFF-driven B cells responses, while TACTFe blocks these processes, Figure 5 Figure 5 shows the results of targeting APRIL wit hAPRILOQA and hAPRL3A s(panel A) or 'IAC-Fo (panel B) in-vivo in a T-independent B cell response. Transgenic mice were challenged with NP-Ficoll, and treated with hAPRILO1A, hAPRILO3A and TACT-Fe twice per week PBS and mouse IgGI were used as negative controls The inmunokglobulin itres (IgA. IgM and IgG) were measured by ELISA.. hAPRIL01 A, hAPRILO3A and to a lesser extent TACt-Ft are able to inhibit 20 APRIL mediated B cell responses in the hAPRIL transgenic rice and reduce imunuoglobulin levels to that of the WT. Figure 6 Figure 6 shows the effect of targeting APRIL with IiAPRIL01An IAPRILI03A and TACT-F on B-cell populations in the spleen (panel A) or peritoneal cavity (panel B). 2s Tranusgeic mice were challenged with NP-Ficoll, and treated with lAPRIE0iA, hAPRIL043A, TACI-Fe twice per week. PBS and mouse IgGI were used as negative controls. After 30 days of treatment spleens and cells from the peritoneal cavity were harvested and analyzed by flow cytometry, Treatment with hAPRILGI A or hAPRILQ3A did not affect the (sub)popuiation of B-cells in de spleen. In contrast, so TACT-Fe strongly reduced the total B-cell population and mature and T2 34 subpopulations. In the peritoneal cavityTAClFE affected the ratio of B 1 vs B2cehs, wYhiie hAPRIL.OIA and h-APRILO3A did not affect these subpopulations. Figure 7 5 Figure 7 shows the variable region sequences of hAPRIL01A and hAPRIL03A. Figures 7A and 713 show the amino acid sequences of the heavy and light chain variable sequence of hAPRIL,01A, respectively Figures 7C and 7D shows the amino acid sequences of the heavy and light chain variable sequence of hAPRILO3A, respectively. i Examples Example 1: Imumnization and selection of anti-APRIL antibodies Imauaization of Mice with APRIL eDNA To genetrae antibodies against the human APRIL protein, a cDNA encoding the full length open reading frame of APRIL was subloned into the pCl-neo vector (Promega, s Madison, WI, Expression of the obtained vector was checked by transient transfection of pl-neo-hAPRIL in 293 cells (American Type Culture Collection, Manassas, VA) and inmmunoblotting with mouse anti-hAPRIL IgG1 Aprily-.5 (1:53.000) (Alexis, San Diego, CA), followed by goat antinouse IgG1HRP (1:2,000) (Southern Bioteelmology, BirminghamAL 20 Mice were immunized by gene gun immunization using a Helios Gene gun (BioRad, Hercules, CA) and DNA coated gold bullets (BioRad) following manufacturers instructions Briefly, I Rm gold particles were coated with pCbneo-hAPRIL cDNA and commercial expression vectors for mouse 131, and mouse 0M CSF in a 2:1 25 ratio (both from Aldevron, Fargo, ND). A total of I pxg of plasmid DNA was used to coat 500 pg of gold bullets, Specifically, 7-8 weeks old female BALMC mice were immunized in the ears with a gene gun, receiving 4 or 5 cycles of a shot in both earsApproximately, a 1:3,200 anti o hAPRIL titer was detected by ELISA in mouse serum after three DNA inumunizations In the ELISA, all incubation steps were followed by a wash step with PBST (PBS with - 35 0.1% Tween 20) 3 times. Maxisorp 96-well immunoplates (Ntn4 Rochester, NY) were coated with rbbit anti-FLAG polyclonal antibody (50 ng/well in PBS)(Sigma, St Louis, MO) overnight at 4 *C and blocked with 10% Goat serunVPBST for 1 hour at RT. Plates were incubated with supernatant (1-4 in PBS) from 293T cells 5 transiently transfected with CMV promoter driven secreted four of FLAG-hAPRIL (pCR3-hAPRIL) for I h at RT2 followed by incubations with mouse seta dilutions and I :2,000 -RP-conjugated goat anti-mouse IgG (Southem Biotechnology) for 1 hour each at RT. After the final PBST wash, anti-hAPRML immunoreactivity was visualized with 100 pl OptiEA TMIB substrate (BD) Biosciences, Franklin Lake, NJ). Reactions 1o wverestopped with 100 p1 05 M Fi 2

SO

4 and absorbances were read at 460 and 620 nm. Mice that demonstrated reactidvity against hAPRIL were immunized for a final, fourth time and sacrificed four days later, Erythrocyte-depled spleen cell populations were prepared as described previously (Steenbakkers et al, 1992,] Immunol Meth, 152; 69-77; Steenbakkers et al. 1994, Mat Blot Rep. 19: 125-134) and frozen at -140'C Selection of anti-APRIL antibody producing B cells To select B cell clones producing anti-APRIL antibodies, L5 x 107 erythrocyte depleted splenocytes were subjected to two rounds of negative panning on 2,3 x 10I Dynabeads@t M-450 tosylactivated beads (nvitrogen, Carlsbad, CA) coated with anti 20 FLAG M2 antibody (Sigma). 50 pg anti-FLAG M2 antibody was coated per Ix 1Os beads in 500sI according to nanufacturer's instmetions. Beads and splenocyte suspension were incubated for 30 minutes on ice and resuspended in cold DMIEM F12!P!S/l0%BCS Unbound splenocytes were separated from the beads using the Dynal MPC (Magnetic Particle Concentrator) (Inviirogen). For the positive panning, 25 splenocytes were incubated with 23 x 10 beads coated with anti-FLAG M2 bound to FLAGhAPRIL for 30 minutes on ice, Beads and unbound splenocytes were separated as described above with a total of 12 washes. Antigen-specific B-cells were cultured as described by Steenbakkers et al., 1994, Mol, BioL, Rep. 19: 125-134, Briefly, selected B-cells were mixed with 75% (viv) T-cell o supernatant and 50,000 irradialed (2,500 RAD) EL-4 B5 during cells in a final volume of 200 1 DMEM F12/P/S/i0%BCS in a 96-well flat-bottom tissue culture plates. On day eight, superatants were screened for hAPRIL reactivity by ELISA as described - 36 above, 21 APRIL-reactive supematants were identified and tested for their ability to inhibit the interaction of APRIL with BCMA-F. In the EISA, all incubation steps were followed by a wtash step with PBST (PBS with 0.1% Tween 20) 3 times. A Maxisorp 96-well immunoplate was coated with BCMA-Ec (50 ng/well in PBS) (R&D 5 Systems, Minneapolis, MN) overnight at 4 *C and blocked with 10% Goat serumi/PBST for 1 hour at RT. FLAG-hAPRIL containing supernatants were pre incubated with antibody-containing B-cell supematants for 1 hour at RT and then added to the BCMA-Fc coated plate for 1 hour at RT. Bound FLAG-IiAPKTL was detected by incubation with I pg/ml anti-FLAG BioM2-biotin antibody (Sigma) and io 1.:2,000 Sireptavidin-HRP (Southem Biotechnology) for I hour each at RT. After the final PBST wh, AP'RIL bound BCMA-Fc was visualized with 100 pl OptiBlA TMB substrate (BD Biosciences). Reactions were stopped with 100 10.5 M H2504, and absorbances were read at 460 and 620 nm, Subsequently, 8 B-cell clones were immortalized by mini-ectroftsion following is published procedures (Steenbakkers et al., 1992, J limunal Meth. 152, 69-77; Steenbakkers et al, 1994, MoL Biol Rep. 19, 125-34). Specifically B-Ceils were mixed with 106 NS-1 myeloma cells, and serum was removed by washing with DMEM F1 2 media. Cells were treated with pronase solution for three minutes and washed with fusion medium, Electrofusions were performed in a 50 pl fusion chamber by an 20 alternating electric field of 30s, 2 Mlz, 400 V/em followed by a square, high field pulse of 10 pts, 3 kV/cm and again by an alternating electric field of 30s, 2 MHz, 400 V/cm. Contents of the chamber were transferred to hybridoma selective medium and plated in a 96-well plate under limiting dilution conditions. On day 14 following the fasions, hybridoma supernatants were screened for APRIL reactivity and BICMA 25 blocking activity, as described above. Two distinct anti-hAPRIL hybridomas, named hAPRIL01A and hAPRIL13A were isolated and subaloned by limited dilution to safeguard their integrity. hAPRIL reactivity and BCMA.-blocking activity of hAPRIL}OIA and IAPRJL,03A antibodies were confirmed with hvbridoma 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 runds of i limiting dilutions (six tor hAPRL 1IA and four for hAPRIL.03A). Stable hybridomas were cultured in serum-free media using CELLine bioreactors (lntegra-iosciences. Chr, Switzerland) according to manufacturers instructions Following 7-10 days in cultreN supernatants were harvested and filtered through a 0.22 pM nitreellulose membrane Superatants were diluted l in high salt binding buffer (I M Giycine/2M 1 NaCl 3 pH 9,0), and antibodies were purified with Protein G HliTrap 5 ml cohnns (GE H-eahare, Piscataway N) After PBS wash of the column, antibodies were eluted with 0,1 M Glycine pH 23 and neutralized with 3 MFris, Bufferwas exchanged for PBS using PD10 gelfltration columns (GE Healthcare) Antibodies were concentrated with Amnicon Ultra-i5 ceniriihgaifilter units (Millipore, Billerica, MA) 1 and quantified using spectrophotometry. Using a mouse monoclonal antibody isotyping test kit (Serotee Raleigh, NC), the (sub)isotype of both hAPRIL.01A and hAPRIL.03A antibodies was detennined to be IgG 1.Kappa 20 Binding Analysis Protein-based ELISA experiments using p fied hAPRIL101 A and hAPRIL.03A antibodies were perfonned to determine apparent binding affinities (reported as EQ 0 values) Binding was compard to mouse anti-hAPRIL IgG1 Aprily-5 (Alexis). Maxisorp 96-well imninoplates (Nano) were coated with either rabbit anti-FLAG v5 polyclonal antibody (Sigma) or BCMA-Fc (R&D Systems) at 50 ug/well in PBS ovemight t 4 *C and blocked with 10% Coat serum/PBST for I hour at RT. Plates were washed with PBST 3 times and incubated with supernatant (I4 in PBS) containing FLAG-hAPRIL for 1 hour at RT. Plates were again washed with PEST 3 times and incubated with IAPRRIL0A, tAPRIL.03A, and Aprily-5 antibodies (10 30 pg/m high test with 10-fold dilutions in triplicates) for I h at RT. After three washes wih PBST. bound antibodies were detected with goat anti-mouse IgG-HRP (12,000) (Southern Biotechnology)for 1 hour at RT. Plate was washed three times with PST, 38 and APRIL-reactivity was visualized with OptiEiA TMB substrate (Becton Dickinson)X The concentation for half-maximal binding is reported as a measure of relative binding affinity When FLAG-hAPRIL was captured by the anti-FLAG antibody (Figure 2A), F{s values for hAPRLIL01A, hAPRIL03A and Aprily-5 were calculated as 2. 2L4, s and 11 nM, respectively. When FLAG-hAPRIL was captured by BCMA-Fc (Figtre 2B), hAPRILG1A antibody binding was not observedsuggesting that the APRlL BCMA interaction blocked the hAPRILSsA epitope. In contract binding of hAPRIL.03A to the APRIL-BCMA complex was observed. Antibody detection of the receptordigand complex may prove useful in diagnostic assays and for search io purposes to folow the clearance of soluble APIUL Kinetic anaysis by biodight interferometry (ForteBio) To further characterize the binding characteristics of the antibodies, each was profiled using biolight interferometry on the Octet system (ForteBio, Menlo Park, CA) to is elucidate binding kinetics and calculate equilibrium binding constants. This assay was performed by coupling purified hAPRILOIA and hAPRIL B3A antibodies to amine reactive biosensors (Forvebio) using standard amine chemistry. Recombinant human APRIL (R&D Systems) binding to and dissociation from the biosensors was then observed at two concentrations, I and 2 pg/t. Specifically, amine-reactive biosensors 2o were pr-wetted by immersing them in wells containing 0-M IfES pH= 5 for 2 minutes. The biosensors were then actdvated using a N 1M NHS / 0.4M EDC mixture for 5 minutes hAPRIL01A and hAPRILO3A antibodies were coupled by immersing the biosensors in a solution of 5 gg/mL of the antibody fbr 18 minutes. The biosensor surface was quenched using a solution of IM ethanolamine pH 8,5 for 7 minutes. z Biosensors were equilibrated in PBS for 5 minutes Association of recorrbinant APRIL was observed by placing the biosensors in wells containing either I or 2 pg/ml APRIL and monitoring interferometry for 20 minutes. Dissociation was measured afler transfer of the biosensors into PBS and monitoring of the interferometry signal for 20 minutes. The observed on and off rates (k<b and kd) were fit using a 1:1 binding global 30 fit model, and the equilibrium hindina constant K 0 was calculated (see Table 1).

- 39 Table I. Binding characteristics of humanized anti-hAPRIL antibodies of the invention mt A h --------- ------------- .APRTLOI A 4,9E+,04 3.69E-05 hAPRRLOA 7,-4E§44 4,21E- 05 5,5SF~It0 Receptor Blockade Blocking abilities ofhAPRILA01A and hAPRiL,03A were confined using purified s antibodies, Maxisorp 96-well plates weA coated with either BCMA-e (R&D Systems) or TACI-Fe (R&D Systems) at 50 ng/well ovvrght at 4 NC and blocked with 10% Goat semmfPBST for 1 hour at RT. FLAGAhtAPRIL containing supematants were pie-icubated with hAPR1I 1A, IAPRILO03A and Aprily-5 antibodies (10 pg/mI high test with 10-fold dilutions in triplicates) for I h at RT. Plates were washed to with PBST 3 times and bound FLAGh-APRIL was detected by incubation with I pg/nl antiELAi BioM2-biotin antibody (Sigma) and 1:2,000 StreptavidinlHRP (Southern Biotechnology)for 1 hour each at RI. After the final PBST wash, APRIL bound BCMA-Fe was visualized with OptiEilA TMB substrate (11) Biosciences), As shown in Figures 2C and 2D hAPRIL.0l A fully blocks FLAG-hAPRIL binding to i5 BCMA-Fc and TACITPc, whereas hAPRIL:03A fully blocks FLAG-hAPRIL binding to TACI-Fe, while only partially blocking the hAPRIL-BCMA-Fc interaction. Aprily 5 does not block FLAG-hAPRiL finding to either BCMA-Fe or TACI-Fe. The concentration of half-maxiun inhibition (ICjo) was determined for bAPRIL.OIA as 12 and . rnM for BCMA-Fe and TACT-Fe, respectively. The IC5j for hAPRILO3A to 2{ TACt-Fe was detenined as 13 nM Comercial Antibodies Commercially available anti-APRIL antibodies were obtained as described in Table 2 25 Table 2, Conunercially available anti-human APRIL mnonoclanal antibodies Antibody Company Cat no. Alexis ALX-804148-C100 pPL-2 Alexis ALX-804-844-C100 40 Aprily-5 AlexIs ALX-804 8 C100 Apnly-S Alex is ALX-804449-C100 Sacha-1 Alexis ALX404-141-C00 Sacha-2 Alexis ALX-804804-C1O ani-CD256. clone T3-6 BioLegend 318502 ifetSpan mouse ant human APRIL Biosciences LS-Ci8658 LifeSpan mouse ani human APRIL Biosciences LS-CI8659 LifeSpan house anti-human APRIL Biosciences L8-C18687 TNFSFi3 monoclonal antibody (MO I), clone 114-E8 Tebu-bio H000087414 M01 TNFSF13 monoclonal antibodyy (M02), clone G3 (ABNOVA) H000087414M02 Human APRIL/TNFSF13 MAb (Clone 101115) R and D MAB84 To study whether the blocking characteristics of hAPRILiA and hAPRIL .03A are unique. all known commerially available anti-APRIL antibodies were tested for their ability to block the interaction ofFLAG-hAPRiL to BCMA-Fe and TACJ-F (Figures 3A end 3B), Blockade of receptor binding was studied using an ELSA An ELSA plate was coated with 50 with II00d of BCMA-FE at I pg/il or with 1 00pl of TACI Fe at a concentration of 2 ptg/ml in coating bufItr and incubated ovemight at 4 0C. The plate was then washed with PBS/02%Tween and then incubated with for I hour at 37 *C with 100 pl PBS/5% B1SA per well The plate was then washed four times with 10 PBS/02%Tween In a separate plate APRIL monoclonal antibodies were pre-mixed with APRIL supematant and incubated for 30 minutes on ice, Conditioned medium containing soluble APRIL was diluted I in 4 and mixed with an equal volume of PBS containing the antibodies titrated in doubling dilutions starting with 5 pg/ml. 100 P1 of the pre-incubated nix was transferred to the ELISA plate and incubated for 2 hours at us 37 *C, 'he plate was then washed four times with PBS/0.2%Tween. AntiFlag-HRP 41 antibody was then dihted in PBS at a concentration of 111000 and then 100 p of this added to each well and incubated for 1 hour at 37 C The plate was then washed four times with PBS/0;2%TIween and then 100 pl of ABTS added to each well (the ABTS was diluted to the ratio 10 ml of reagent plus S p of 1-120z made immediately before addition) The colour was allowed to develop and then the OD at 405 mnr read on an ELISA plate reader, Human IgGI was used as a control protein to coat the plate as this is the same isotype as the Focfusion proteins and controlled for APRIL sticking to the plate non-specifically, As is apparent from Figure 3, none of the commercially available antibodies was able to block the binding of FLAG-APRIL to either TACJ-Fc io or BOMA-RE, whereas hAPRILA)IA and hAPRIL.03A do inhibit (partially) the binding to TACT-Fe and BCMA-Fe. Species Cross-Reactivity Biding ofhAPRIL01A and IAPRIL03A to mouse APRIL was also examined by 1s BLAcore, bot no binding of either antibody was observed, The antibodies appear only to bind human APRIL Example 3: Funetional Profling of Murine anti-Human APRIL Anibodies Mouse H cell response to APRIL In order to show that the antibodies of this invention can functionally block APRIL in 20 vitro a mouse B cell assays was used to examine two APRIL driven responses in 1 cells proliferation and IgA production, All cell lines were maintained at 37C with 5% CO 2 Mouse splenocytes and purified B cells were grown in RPMII- 1640(Gibc)supplemented with 8% FCS, 2 mM Glutanilne 25 and Beta-mercaptoethanol at 50 pM, and supplemented with penicillin and streptomycin at a concentration of logg/ml Splenic mouse B cells were isolated from wild-type nice using magnetic activated cell separation (MACS) columns with CD45R/B220 MACS beads {Miltenyi Biotec, Utrecit, rhe Neftherlands). The cells were cultured in 96-well round-bottomed nicrotiter plates at a density of 2 x 10'?/well 30 in a final volume of 200 pl For all assays conditioned medium containing the various forms of soluble APRIL were normalised for expression levels prior to use, To measure proliferation, cells were treated with anti-lgM (Jackson hm noResearch) and soluble 42 APRIL in conditioned medium or as purified protein at a final concentration of I ggint Cross-linking antiaPlag monoclonal antibody was added to the well at a final concentration of I pg/mit The cells were incubated at 37 'C and after 48 hours pulsed with 0.3 pCi (Ot I0 MBq) of tritiated thymidine ([6 11] Thymidine, GE Healthcare, s The Netherlands) for 18 hours, before h arvesting. To measure IgA production, mouse B cells were cultured and treated with APRIL, as above. Following incubation for 6 days, supernatant was collected and assayed for IgA content by ELISA. Briefly, ELISA plates were coated with 2 jig/mI anti-mousedg (Southern Bionech), blocked with PBS/1% BSA and incubated with the collected supernatant. Bound IgA was then it detected with HRP labelled anti-mouseigA (Southem Biotech; Uithoon. the Nethelands). As a control, oells were treated with 10 pg/ml LPS (Invivogen) plus I ng/mi of human TGFp (Sigm-Aldrich). As shown in Figure 4A, hAPRIL.01A and to a lesser extent hAPRIL.03A are able to inhibit APRIL induced class-switch recombination as was determined by the reduced IgA secretion onom mouse splenic B is cells. TACI-Fc as a control inhibited the IgA secretion, while mouse IgG1 and human Ig did not affect the APRILinduced IgA secretion from splenic B-cells. In addition, hiAPRILOI A and hAPRIL 03A were demonstrated to inhibit APRilinduced mouse splenic B-cell proliferation To establish the specificity of the antibodies, the effect of hAPRILOIA and hAPRIL03A on BAFF-induced IgA secretion and proliferation was 20 studied, As shown in Figure 4B, neither hAPRILL01A nor hAPRJLA}3A inhibited BAFF induced IgA secretion and proliferation, while TACIFe as a control inhibited both processes, In-vivo Experiment to block APRIL function 25 To demonstrate an in-vivo blocking effect of the antibodies on APRIL function, we examined the ability of the antibodies to block the NP-Ficoll induced humoral response in mice. The mice used were 810 week old APRIL transgenic (TG)mice and widtype (WT)littermates, both on a C57BL16 background. The APRIL transgenic mice express human APRIL under the Lek-distal promoter, which directs transgene 30 expression to nature thymocytes and peripheral T lymphocytes (Stein et at, 2002,2 Clin Invest 109, 1587-98). The mice were bred in the animal facility of the Academic Medical Center and the experiment was approved by the institutional ethical - 43 committee. The mice were divided into several groups and treated as follows: five APRIL WT mice were treated with PBS (200pd) and 5 groups of tve APRIL transgenic mice were treated with the following molecules: hAPRILOI A or hiAPRILO)3A or TACt-lc or subisotypeumtched control antibody rasigGt k (200 5 pg/mouse in 200 pl PBS) or PBS, Treatment of the mice was started 3 days before the NP-FitoH inmnnzation (day 0; 100 td isp. with 250 pg of the immunogen) -'injections were continued twice a week for 28 days. Blood was collected via tai vein at day -1,3, 7,14 and 28. Anti-44-hydroxy-nitrophenacetyl) (NP)-specifie antibodies (IgM, IgG and IgA) were assayed in 6 independent EOLISA using diluted semi (1:100 for igA; 1:500 io for IgG and 1 :2,000 for IgM) as previously described (Hardenberg et al., Immunol Cell Biol 86(6), 5304. (2008)). Briefly 96-well ELISA plates (Greiner) were coated with NP-BSA at 5 pg/nd (Biosearcb Technologies) in sodium earbonate buffer (pH 9,6) overnight at 4*C The wells were blocked with 1% BSA for I hr at 37*C and incubated with diluted sera for 2 hra at room temperatum HRP-conjugated isotype specific is antibodies (Goat anti-mouse IgG, IgA and 1gM - from Southem Biotech) were used as revealing antibodies. All dilutions were made in PBS/BSA I1%/Tween 20 0.05%. One way ANOVA test was used to check statistical significance between the groups TO (PBS) vs TO (hAPRILf01A) and TG (PBS) vs TO (bAPRIL03A). As apparent from Figure 5, both hAPRIL01A and hAPRILO3A inhibited the T-cell independent B-eeR 20 responses in vivo, TACI-Fc inhibited this response less efficient. PBS and mouse IgGI as an isotype-matched control, did not affect the IgA, IgM and IgG anti-NP response To examine the long-term effect of hAPRIL.0IA and hAPRILO3A on B cell populations mice were treated as described above, On day 30 mice were sacrificed and the spleen and peritoneal exudate cavity (PEG) analysed for 8 cell expression by flow 21 cytometry Briefly, splenocytes and lymphocytes from the PEC were separated from red blood cells by one wash with erythrocyte lysis buffer and then counted Cels were washed and resuspended in PBS/I % BSA and seeded in 96-well round-ottomed plates at a density of 5 x I? per welL Next, cells were stained with the following antibodies at the commended concentrations: B220-FITC (BUD bioscience) and CD3-APC 30 biosciencece; IgD-FITC (BD bioscience) and 1gM-PB (BD bioscience); IgD-FITC (I bioscience), CD3-APC (ebioscience) and CD43PE D(13D bioscience). Antibodies were incubated for 40 minuteswashed three times with PBS/1% BSA and then -44 analysed by flow cytometry using the FACSCalibur (Becton Dickenson). B220 t

B

ceIls, mature 1-cells (igDigM) and T2 B-eeis (gIYIgM5 in spleen were quantified (see Figure 6A) In addition B1 (CD43'I gD and B2 (CD43TgDY) subpopulations were quantified in PEC (see Figure 6B). The decrease in B cells in s response to TACL-c treatment is evident from both the spleen and the PBC, indicating that long term administration of TACI-Fc may have a detrimental e~ct on norma B cell populations. This is not seen with hAPRIL.01A and hAPRILO3A antibodies, suggesting that in cases where APRIL but not BAFF is the primary cause of pathology the antibodies of this invention may show less side-effects than TACI-e. 10 Example 4 Anti-APIL antibodies sequences Cloning of Immanoglobvlin MDNAs Degenerate primer PCR-based methods were used to determine the DNA sequences encoding the variable regions for the mouse antibodies that are expressed by s hybridomas hAPILG01A and hAPRI03A, Total RNA was isolated from SxO hybridomas cells using TRIZOL (Invitrogenj, and gene specific cDNAs for the heavy and light chains were synthesized using the iScript Select eDNA synthesis kit (Biorad) according to the manufacturer's instructions. The V 1 4 and V genes were PCR ampliiied using a Novagen -based Ig-primer set (NovagenSan Diego. CA) and 'Faq 20 polymerase (Invitrogen). All PCR products that matched the expected amplicon size of 500 bp were cloned into pCR4 'OPO vector (Invitrogen4 and the constructs were transformed in DH5a K coli (Invitrogen) according to the manufacturer' s instructions. Clones were screened by colony PCR using universal M13 forward and reverse primers and two clones from each reaction were selected for DNA sequencing 25 analysis. Sequences were searched against databases of germine and rearranged IgV variable region sequences using NCBI Ig-Blast BLASTN 2.16 (httotwwnab.mn rni 'ovfprosbast Blast results for hAPRfL01A and hAPRIL03A showed one in-fane Vg sequence and one in [bane V{ sequence for each antibody. The amino acid sequences were confirmed by mass spectrometry, The 30 sequences are disclosed in the attached Sequence Listing, Figure 7 and listed in Table

I.

-45 Table 3: Sequence I) numbers for marine anti-human APRIL antibodies of this invention SEQ ID NO Description 3 hAPRILOIA heavy chain variable region (DNA) 2 hAPRIL3OIA light chain variable region (DNA) 5 hIAPRILOA heavy chain variable region (DNA) 4 hAPRILO01A light chain variable region (DNA) 5 hAPRILGIA heavy chain variable region (AA) 9 hAPRILOIA light chain variable region (AK) IIAbain variable Leis A) 10 hAPRIL03A light chain viable region (A ) 9 hAPRIL0IA heavy chain CDRI (AA) 10 APRIL0 A lihavy chain CDR2 (AA) 13 hAPRIL0 1 A havgy chain CDR3 (AA) 12 hAPRILOIA light chain CDRI (AA) 13 hAPRIL.,O IA. light chain C20R2 (A) '14 hAPRILOTA light chain CIR3 (X&A) 16 hAPRIL03A heavy chain CDR2 (PA) 16 hAPRHO3.A heavy chain CDRZ (AtA) 17 IIAPRL.03Aeavy chain CDR3 (AA) 18 hAP1ULO3A light chain CDR1 (AA) 2 hAPRLO3A light chain CDR3 (AA) Example 5: Epitope mapping using Pepsean method 5 Synthesis of peptides and pepstan screening The synthetic linear and CLIPS peptides were synthesized and screened using credit card format mini PEPSCAN cards (455-well plate with 3 ul wells) as described by Slootstra et at (Slootstra et al, 1996, Mol Diversity 1, 87-96) and Timmemian et at (Timmerman ct al, 2007,J Mol Recognit. 20,283-299). The binding of antibodies o (APRIL01A and hAPRIL03SA) to each peptide was tested in a PEPSC AN-based enzyme-linked imnuno assay (ELISA). The 455-well creditcard-fonrat polypropylene cards, containing the covalently linked peptides, were incubated with sample (for - 46 example I ughul antibody diluted in a PBS solution containing 5% horse serum (vol/vol) and 5% ovalbumin (weightvol)) and 1% Tween 80 (40C, overnight), After washing the peptides were incubated with an anti-antibody peroxidase (dilution 1/1000, for example rabbit anti-mouse peroxidase, Southem Biotech) (I hour, 251C), and 5 subsequently, after washing the peroxidase substrate 2,2

T

-azino-di-3 ethylbenzthiazoline sufonate (ABTIS) and 2, ul; 3% 1202 were added, After I hour the color development was measured. The color development of the ELISA was quantified with a LCD-camera and an image processing system. The setup consists of a CCD-camera and a 55 nun lens (Sony CCD Video Camara XC-77RR, Nikon micro wo nikkor 55 2n8n /1 lens), a camera adaptor (Sony Camar adaptor DC-77RR) and Image Processing Software, Synthesia Peptides A total of 4225, primarily, CLIPS peptides were synthesized The target sequence used, 1s 147 amino acids, with loops according to alignment with 1XU2 pdb underlined: RAVLTKQKKQ'ISLHNPINAISK1DDSDVEVMWQPALRRGRGLQAQCYGY RIQQA0V YLLY QVLT-AQVD L)(MGQVVSRE UQQQQBLFRCIRSMPSI{l)RAY NSCYSAGVFHLHOCDILSVIIPRARAKLNLSPHGTFLGFYKL (SEQ ID N0:21). Loops on topsidee of protein: QKKQHSVLHL (SEQ ID NO:22) ALRRGRGL (S:EQ 20 U) NO:23), QAQGYGVRI (SEQ ID NO24), QDAOVYLL (SEQ ID NO:25), SREGQGRQETV (SEQ ID NO:26), PHLHQGDILTSV (SEQ ID NO:27) and loops on "bottom" side of protein: 1NATSKDDSDVTE (SEQ ID NO:28x VLEQDVTFTMG (SEQ I D NO:29), RSMPSHPDRAYNSC (SEQ ID N0:30), 1 PRARAKL (SEQ ID NO31), NLSPHiGTFLGF (SEQ ID NO32). The intercnnecting regions are mostly as sheets Note thal the "top" and "bottom" side are chosen arbitrarily. The following CLIPS topologies were used: T2 CLIPS couples to the side-chain of two cysteines to form a single loop topology, while T3 CLIPS couples to the side-chain of three cysteines to form double loop topology, while 1212 CLIPS first T2 couples to two cysteines (labeled C) and second 12 couples to two cysteines and fmally T2T3 3o CLIPS T2 couples to two cysteines and T3 couples to three cysteines. In total 20 different sets of peptides were synthesized: -47 191-I (set-): All overlapping 35-Mer sequences covering the complete 147 AA target sequence were synthesized. In this set the different loops, when present in the sequence as defined above were constrained in double loop or sheet-like topology through two T2 CLIPS 191-2 (set-2) A total of nine sheets were identified, All 9x9 combinations were synthesized to mimic double sheet conformations. The sequence SG was used as a linked 191-3 (set-3) The same as set-2 as explained above but with a shorter sheet length. 191-6 (set-4) All overlapping linear 35-mer sequences covering the complete 147 AA 1o target sequence were synthesized, 191-7 (set-5) All overlapping linear 15-mer sequences covering the complete 147 AA target sequence were synthesized. 191-8 (set-6a) Short linear sequences (of varying length) only coveting the loop regions of the complete 147 AA target sequence were synthesized. 1s 191-16 (set-6b) different peptides were selected from the five "'ottom"loops These wee Mrecombined in a 9x9 matrik onto the T3 CLPS to form double looped topologies with "bottom" loops of two different lengths, 191-i7 (set-7) All overdapping 135 different 15-mer sequences were synthesized with a cysteine at position 1/8 and 15 The three cysteine were coupled to a T3 CLIPS. 20 19148 (set-9) Long versions of the six "top"loops and long versions of the four "bottom" loops were recombined with each other on the '3 CUPS. 19149 (set-0) Six+Six+Four different sized loops of the "top"loop region were all recombined with each other on the T3 CLIPS, 191-20 (set- Ui 17,18,1%20)33 different sequences broadly covering the "top" or .2s "bottom" loops were recombined with other on the T3 CLIPS, These sets of peptides are in sets .i, 17, 18, 19 and 20. Reason for this "scattering" is the card layout 191-22 (set- 12) Diftfrent sized loops of all "top" and "bottom" loops were synthesized as single liops on T2 CLIPS. 191-23 (set13) All overlapping single looped 15-mer sequences covering the complete 30 target protein were synthesized on T2 CUPS. 191-24 (set44) Six different 9-mer sequences covering the "top" loops were recombined with each in a 6x6x6 triple looped matrix on T2T3 CLIPS combination.

- 48 19125 (set-i5) The same set of overlapping peptides as set- All overlapping 35-mer sequences coverng the complete 147AA target sequence were synthesized In this set the different loops, when present in the sequence, as defined above were constrained into triple loop topology through T312 CLIPS. 5 191 26 (set- 16) Six different 9-mer sequences covering the "bottom" loops were recombined with each in a 6\6x6 tiple looped inatrix on 72T73 CLIPS combination. Data analyis and epitope deterdnation Each antibody was tested on all 4225 peptides and their binding values were ranked. 1e Clearly re-occurring sequences in most the top binders (~top I%) were considered as epitope candidates, Two additional supporting analyses were done. Firstly, it was investigated if multiple identified parts car form one discontinuous epitope This was done through the homologous structure 1XU2.pdb. Secondly, it was investigated if each of multiple identified binding parts was recognized without support of the other is part. These two paramneter, co-localization on the 3D structure and independent recognition, were used to support that a conformational and discontinuous epitope was idenitied For IAPRILIA it was determined that it binds to IRSMPSHPDRA (SEQ ID NO:33, with the core region being SMPSHP (SEQ ID NO:34). The 1,FR (SEQ II) NO:35) and/or QI)VTEFTMGQ (SEQ ID NO36) (core region is VTFTI (SEQ ID 2o NO:37)) motifs were shown to support the binding of APRILJ01A. hAPR[L03A was shown to bind VSREGQGRQ (SEQ ID NO:38) motif, with core region being EGQ. The TFTMGQ (SEQ ID N0:39) motif was shown to support binding of hAPRIL.03A.

Claims (7)

1. 5. 'The binding compound of any of the above claims, wherein the binding compound fully blocks the binding of APRIL with human TACI and at least partially blocks the binding with human BCM. 6 The binding compound of claim 5, wherein the binding canpound fully blocks 30 the binding of human April with hmnan 3CMA, 7 The binding compound of any of the above claims, wherein the binding compound: -50 a. binds human APRIL with a Ko of about 10 nM or lower; and b blocks bining of human TACI and/or human BCMA to human APRIL with an ICD of about 2 nM or lower;
2. 8. A binding compound wich binds to human APRIL wherein the binding i compound has the same epitope specificity as the compound selected from claim 3.
3. 9. A binding compound which competes for a binding epitope on human APRIL with any of the binding compounds of claims 3, and has one of the following characteristic: 10 a. binds human APRIL with a K 0 of about 10 nM or lower; bl binds to human APRIL with about the same K1 as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 6; e binds to human APRIL with about the same KD as an antibody having a 15 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; d. blocks finding of' human TAC land/or human BCMA to human APRIL with an ICso of about 2 nMT or lower. 10 The binding compound of any of the above claims.wherein the binding 20 compound is: a, a chimeric antibody or a fragment thereof, b a human antibody or a fragment thereof; c, a humanized antibody or a fragment thereof; or d. an antiody fragment selected fonm the group consisting of Fab. Fabt Fab' 5S1, Fv, scFv; F(ab} bispecifie nAb and a diabody. IL The binding compound of any of the above claims, wherein the binding compound inhibits the proliferation and survival of B-cells
4. 12. An isolated polynucleotide encoding the binding compound of any one of claims I to 11, 30 13 An expression vector comprising the isolated polynucleotide of claim 12.
5. 14. A host cell comprising the expression vector of claim 13. 51
6. 15. A method of producing a binding compound according to any one of claims I to II comprising: a, cuturing the host cell of claim 14 in culture medium under conditions wherein the polynueleotide is expressed, thereby producing polypeptides 5 comprising the light and heavy chain variable regions; and b, recovering the polypeptides ftron the host cell or culture medium.
7. 16. A composition comprising the binding compound of any one of claims 1 to 11 in combination with a pharmaceuticaly acceptable carter or diluent, 17, Binding compound of any one of claims I to 11 for use in therapy 10 18. The use of the binding compound of any one of claims I to 11 for a. inhibition of immune cell proliferation and/or survival; b, treatment of cancer; C. treatment of an autoimmune disease or d, treatment of an inflammatory disease. is 9. The use of the binding compound of any one of claims I to I I in a diagnostic method,