Anti-CD269 antibody-drug conjugate (ADC), anti-CD269 antibody and use thereof

Abstract

The present invention relates to anti-CD269 antibody-drug conjugates (ADCs), humanized antibodies or antibody fragments that bind to CD269 (BCMA), and their use in treating medical disorders associated with the presence of pathogenic B cells. The anti-CD269 antibody-drug conjugates (ADCs) bind to CD269 (BCMA), thereby disrupting the interaction between CD269 and its natural ligands (BAFF and APRIL).

Description

[0001] This application is a divisional application of an invention patent application with an application date of April 30, 2015, an international application number of PCT / EP2015 / 059562, a national application number of 201580021693.5, and an invention name of “Anti-CD269 (BCMA) Humanized Antibody”. Technical Field

[0002] The present invention relates to humanized antibodies or antibody fragments that bind to CD269 (BCMA), thereby disrupting the interaction between CD269 and its natural ligands (BAFF and APRIL), and their use in the treatment of plasma cell-mediated diseases such as multiple myeloma and autoimmune diseases. Background Art

[0003] B cell maturation antigen (BCMA) is the 17th member of the tumor necrosis factor receptor superfamily (TNFRSF). Its natural ligand is the B cell activating factor (BAFF; also known as BLyS or TALL-1, TNFSF13B) and proliferation-inducing ligand involved (through interaction with further ligands) in regulating different aspects of humoral immunity, B cell development and homeostasis.

[0004] BCMA is highly expressed on malignant plasma cells, such as multiple myeloma (MM), a non-Hodgkin lymphoma of myeloid B cells, and malignant plasma cells in plasma cell leukemia (PCL), which is more aggressive than MM and constitutes about 4% of all cases of plasma cell disorders. In addition to MM and PLC, BCMA is also detected on Hodgkin and Reed-Sternberg cells in patients with Hodgkin lymphoma (Chiu et al. (2007) Blood 109: 729-739). Similar to its function on plasma cells, ligands that bind to BCMA have been shown to regulate the growth and survival of multiple myeloma cells expressing BCMA (Novak et al. (2004) Blood 103: 689–694). BAFF and APRIL signaling through BCMA is considered a prosurvival factor for malignant plasma cells; therefore, elimination of BCMA-positive tumor cells and / or disruption of ligand-receptor interactions should improve treatment outcomes in multiple myeloma and autoantibody-dependent autoimmune diseases.

[0005] There are currently a variety of treatments available for multiple myeloma (Raab et al. (2009) Lancet 374:324–339). Chemotherapy results in only partial control of multiple myeloma in most subjects; only a very few undergo chemotherapy resulting in complete regression. Therefore, a combination approach is often used, usually involving the additional administration of corticosteroids, such as dexamethasone or prednisone. However, corticosteroids are plagued by side effects, such as reduced bone density. Stem cell transplantation has also been proposed, using one's own stem cells (autologous) or using cells from a close relative or matched unrelated donor (allogeneic). In multiple myeloma, most transplants performed are of the autologous type. Although not curative, such transplants have been shown to prolong life in selected patients (Suzuki (2013) Jpn J Clin Oncol 43:116–124). Alternatively, thalidomide and its derivatives have recently been used for treatment but are also associated with suboptimal success rates and high costs. Recently, the protease inhibitor bortezomib (PS-341) has been approved for the treatment of relapsed and refractory MM and has been used alone or in combination with established drugs in many clinical trials, resulting in encouraging clinical results (Richardson et al. (2003) New Engl J Med 348: 2609-2617; Kapoor et al. (2012) Semin Hematol 49: 228-242). The cost of combined therapy is relatively high, and the success rate still has significant room for improvement. Due to the accumulation of side effects when multiple drugs are used simultaneously, the combination of treatment options is not ideal. New methods for treating plasma cell diseases, particularly multiple myeloma, are needed.

[0006] The ability to specifically target plasma cells also has great benefits for treating autoimmune diseases. Mild forms of autoimmune diseases are usually treated with nonsteroidal anti-inflammatory drugs (NSAIDs) or disease-modifying antirheumatic drugs (DMARDs). Steroids and strong immunosuppressants such as cyclophosphamide (a cytotoxic agent) targeting cells in the cycle are usually used to treat more severe forms of systemic lupus erythematosus (SLE) involving organ dysfunction due to active disease. Only recently, the antibody belimumab targeting cytokine BAFF present at high levels in the serum of patients with autoimmune diseases has been approved by the Food and Drug Administration (FDA) for use in SLE. However, only newly formed B cells in the human body rely on BAFF to survive, while memory B cells and plasma cells are less sensitive to selective BAFF inhibition (Jacobi et al. (2010) Arthritis Rheum 62:201–210). For rheumatoid arthritis (RA), TNF inhibitors were the first biologics to be licensed, followed by abatacept, rituximab, and tocilizumab, among others: they inhibit key inflammatory pathways involved in joint inflammation and destruction, however at the expense of increased infection risk due to relative immunosuppression (Chan et al. (2010) Nat Rev Immunol 10:301–316, Keyser (2011) Curr Rheumatol Rev 7:77–87). Despite the approval of these biologics, patients with SR and SLE often show persistence of autoimmune markers, most likely related to the presence of long-lived, sessile plasma cells in the bone marrow that resist CD20-mediated ablation, e.g., by rituximab and high-dose glucocorticoid and cyclophosphamide therapy.

[0007] Antibodies that bind to CD269 (BCMA) and their use in treating various B cell-related medical disorders have been described in the art. Ryan et al. (Molecular Cancer Therapeutics, 2007 6(11), 3009) describe anti-BCMA antibodies obtained by immunizing rats with peptides of amino acids 5 to 54 of the BCMA protein. WO 2012 / 163805 describes BCMA binding proteins, such as chimeric antibodies and humanized antibodies, their use for blocking the interaction of BAFF and / or APRIL with BCMA and their potential use in treating plasma cell malignancies such as multiple myeloma. The antibodies disclosed therein are obtained by immunizing mice with recombinant peptides of amino acids 4 to 53 of the BCMA protein. WO 2010 / 104949 also discloses various antibodies that preferentially bind to the extracellular domain of BCMA and their use in treating B cell-mediated medical conditions and disorders. WO 2002 / 066516 and WO 2012 / 066058 disclose bivalent antibodies that bind both BCMA and an additional target and their potential use in treating B cell related medical disorders. No details on the binding properties and specific epitopes of the bivalent antibodies are provided in either disclosure. Summary of the invention

[0008] In view of the prior art, the technical problem underlying the present invention is to provide a preparation suitable for treating human diseases associated with pathogenic plasma cells, such as multiple myeloma and autoimmune diseases. This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0009] Therefore, the object of the present invention is to provide a humanized antibody or antibody fragment that binds to CD269 (BCMA), specifically to an epitope in the extracellular domain of CD269 (BCMA).

[0010] The antibodies disclosed herein include humanized sequences, particularly preferably humanized sequences of the VL and VH binding regions, which retain appropriate ligand affinity as described for the chimeric antibody J22.9-xi.

[0011] In different embodiments of the present invention, the amino acid sequence modifications used to obtain the humanized sequence can occur in the CDR region or in the framework region of the original chimeric antibody J22.9-xi, wherein the framework region should be understood as the region of the variable domain of the protein belonging to the immunoglobulin superfamily and being less "variable" than the CDR.

[0012] It was completely surprising that the specific humanized sequences provided herein, preferably involving the CDR regions of the combined VL and VH regions, showed specific and strong binding as demonstrated in the experimental examples, and maintained the binding characteristics of the original chimeric antibody J22.9-xi to the extent that their desired therapeutic effects were retained.

[0013] Therefore, the present invention relates to an antibody or antibody fragment comprising a VH domain comprising the following CDR sequences:

[0014] -RYWX1S (H-CDR1; SEQ ID NO. 15, wherein X1: I, F, L, V, YC, G, A, S, T);

[0015] -EINPX2X3STINYAPSLKDK (H-CDR2; SEQ ID No.16), where X2X3: SS, NS, TS, GS, KS, RS, SD, SN, DE;

[0016] -SLYX4DYGDAX5DYW (H-CDR3; SEQ ID NO. 17), wherein X4: Y, L, A, V, F, I, W and / or X5: Y, L, F, I, V, A, C,

[0017] The antibody or fragment thereof specifically binds to an epitope of the extracellular domain of CD269 (BCMA).

[0018] It is particularly surprising that the humanized antibodies described herein display sequence changes compared to the original chimeric antibody, particularly in the CDRs of said chimeric antibody, which humanized antibodies retain sufficient binding properties to their targets for therapeutic effect.

[0019] The technical staff will not anticipate that the binding characteristics of humanized variants will be similar to original chimeric antibodies or mouse antibodies. Considering the sequence changes of variable domains, especially CDR, the useful binding characteristics of the humanized sequences confirmed herein are considered to astonishing technical effects. The comparison of the binding characteristics between partially humanized antibodies and fully humanized antibodies has also shown the improvement of fully humanized sequences. This shows the result that is completely unexpected. The initial modification of chimera (partially humanized) causes some losses in binding affinity. However, further humanized introduction then causes enhanced combination, wherein "fully humanized" sequence shows a binding characteristic similar to the original chimera, thereby carrying out such significant sequence modification without serious loss of binding affinity, demonstrating astonishing technical effects.

[0020] In a preferred embodiment, the antibody or antibody fragment as described herein is characterized in that the VH domain comprises the CDR sequence RYWIS (SEQ ID NO. 18) or RYWFS (SEQ ID NO. 19).

[0021] In a preferred embodiment, the antibody or antibody fragment as described herein is characterized in that the VH domain comprises the CDR sequence EINPNSSTINYAPSLKDK (SEQ ID No. 20) or EINPSSSTINYAPSLKDK (SEQ ID No. 21).

[0022] In a further embodiment of the present invention, the 54th amino acid of the VH domain may be related to any given amino acid or modified amino acid. As shown in the examples below, potential glycosylation of the N amino acid at this residue does not significantly destroy the antibody specificity and strong binding to the target epitope. In view of this information, the present invention relates to an antibody or antibody fragment comprising a CDR2 sequence as described herein, wherein any given amino acid or modified amino acid may appear at the 54th amino acid of the VH domain in the CDR2 sequence.

[0023] In a preferred embodiment, the antibody or antibody fragment as described herein is characterized in that said VH domain comprises the CDR sequence SLYYDYGDAYDYW (SEQ ID NO. 22).

[0024] The present invention further relates to an antibody or antibody fragment comprising a VL domain comprising the following CDR sequences:

[0025] -KASQSVX1X2NVA (L-CDR1; SEQ ID NO. 23), wherein X1X2: ES, SS, TS, QS, HS, DH;

[0026] - SASLRFS (L-CDR2; SEQ ID NO 24); and / or

[0027] -QQYNNYPLTFG(L-CDR3; SEQ ID NO.25),

[0028] Wherein, the antibody or fragment thereof specifically binds to an epitope of the extracellular domain of CD269 (BCMA).

[0029] Additionally, for the LC sequence, surprisingly, modifications of the CDR3 sequence did not have a significant deleterious effect on binding to the BCMA target.

[0030] Furthermore, the antibodies of the invention also display unexpected and beneficial stability characteristics following administration when in solution, whether isolated or purified, or in vitro, and in vivo, which would not be expected from the sequence changes made to the original chimeric antibody.

[0031] In a preferred embodiment, the antibody or antibody fragment as described herein is characterized in that the VL domain comprises the CDR sequence KASQSVDSNVA (SEQ ID NO. 26).

[0032] In a preferred embodiment, an antibody or antibody fragment as described herein comprises a VH domain comprising the following sequence:

[0033] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWX1SWVRQAPGKGLVWVGEINPX2X3STINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYX4DYGDAX5DYWGQGTLVTVSS(SEQ ID NO.4), wherein X1: I, F, L, V, YC, G, A, S, T; X2X3: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS; X4: Y, L, A, V, F, I, W; and

[0034] In a preferred embodiment, the antibody or antibody fragment as described herein is characterized in that it comprises a VH domain comprising a sequence according to SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0035] In a preferred embodiment, an antibody or antibody fragment as described herein comprises a VH domain comprising the sequence

[0036] EIVMTQSPATLSVSPGERATLSCKASQSVX1X2NVAWYQQKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR (SEQ ID NO. 12), where X1X2: ES, SS, TS, QS, HS, DH.

[0037] In a preferred embodiment, the antibody or antibody fragment as described herein comprises a VH domain, said VL domain comprising the sequence according to SEQ ID NO.14.

[0038] In a preferred embodiment, the antibody or antibody fragment as described herein comprises a VH domain comprising a sequence according to SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9 and a VH domain comprising a sequence according to SEQ ID NO.14.

[0039] The present invention further relates to an antibody or antibody fragment as described herein, comprising a VH domain, wherein the VH domain comprises a sequence according to

[0040] X1VQLX2X3SGGGLVQPGGSLX4LSCAASGX5X6FX7X8YWZ1SWVRX9APGKGLEWX 10 GEINPZ2SSTINYAPSLKX 11 X 12 FX 13 ISRDNAKNTLYLQMX 14 X 15 X 16 RX 17 EDTAX 18 YYCASLYYDYGDAZ3DYWGQGTX 19 VTVSS(SEQ ID No.41), where X1: Q, E; X2: Q, V; X3: Q, E; X4: K, R; X5: I, F; I, V; X11: D, G; X12: K, R; X13: I, T;

[0041] and wherein at least one of Z1: I, F, L, V, YC, G, A, S, T, preferably I or F; Z2: S, N, T, G, K, R, D, preferably S and / or Z3: Y, L, F, I, V, A, C, preferably Y;

[0042] And wherein the antibody or fragment thereof specifically binds to an epitope of the extracellular domain of CD269 (BCMA).

[0043] This embodiment encompasses different humanized antibodies, in particular their VH sequences, all variants defined by advantageous humanization performed in the CDRs as described herein.

[0044] The present invention further relates to an antibody or antibody fragment as described herein, comprising a VL domain, wherein the VL domain comprises a sequence according to

[0045] DIVMTQSX1X2X3X4X5X6SVGDX7VX8X9TCKASQSVESNVAWYQQKPX 10 QX 11 PKX 12 LIX 13 SX 14 X 15 LRFSGVPARFX 16 GSGSGTDFTLTISX 17 LqCy 18 AX 19 YX 20 CQQYNNYPLTFGAGTKLELKR(SEQ ID No.42), where X1: Q, P; X2: R, A; X3: F, T; X4: M, L; X5: T, S; X11: S, A; X12: A, L; X13: F, Y;

[0046] And wherein the antibody or fragment thereof specifically binds to an epitope of the extracellular domain of CD269 (BCMA).This embodiment encompasses different humanized antibodies, in particular their VL sequences, all variants defined by advantageous humanization performed in the CDRs as described herein.

[0047] Preferred embodiments of humanized antibody variants

[0048] As disclosed in detail herein, the sequences of preferred embodiments of the present invention according to J22.9-xi are humanized to provide a more compatible reagent for administration to human subjects. Different humanized sequence variants of J22.9-xi were generated and tested for binding affinity and specificity to both human and macaque CD269 (BCMA). The results from the binding assays confirmed that the humanized sequences maintained the desired binding properties of the chimeric reagent J22.9-xi. In the sequences below, the underlined regions represent CDRs or putative CDRs.

[0049] Preferred embodiments regarding humanized VH variants

[0050] Additional information on the sequences of the humanized antibodies of the invention is provided below.

[0051] Chimeric sequence:

[0052] HC mouse (SEQ ID No.1):

[0053] QVQLQQSGGGLVQPGGSLKLSCAASGIDFS RYWMS WVRRAPGKGLEWIG EINPDSSTINYAPSLKDK FIISRDNAKNTLYLQMSKVRSEDTALYYCA SLYYDYGDAMDYW GQGTSVTVSS

[0054] The HC mouse sequence represents the heavy chain variable region (VH) originally developed for the chimeric antibody J22.9-xi, which includes VL and VH domains obtained from a mouse antibody, capable of binding to an epitope in the extracellular domain of CD269 (BCMA), and the VL and VH domains are fused to human CL and CH domains, respectively.

[0055] Partially humanized sequences:

[0056] HC partial humanization (SEQ ID No.2):

[0057] EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYWMS WVRQAPGKGLEWVG EINPDSSTINYAPSLKGR FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAMDYW GQGTLVTVSS

[0058] The HC portion humanized sequence refers to a modified amino acid sequence (by amino acid substitution) compared to the chimeric antibodies disclosed herein, wherein the VL and VH binding regions are modified with respect to their sequence to make them more suitable for administration to humans.

[0059] Humanized VH sequence:

[0060] hHC01 (SEQ ID No. 3):

[0061] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWMS WVRQAPGKGLVWVG EINPDSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAMDYW GQGTLVTVSS

[0062] Humanized VH sequence with post-translational modification motifs removed:

[0063] hHC02 (SEQ ID No. 4):

[0064] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWX1S WVRQAPGKGLVWVG EINPX2X3STINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYX4DYGDAX5DYW GQGTLVTVSS

[0065] in:

[0066] X1: I, F, L, V, YC, G, A, S, T, preferably I or F;

[0067] X2X3: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS;

[0068] X4: Y, L, A, V, F, I, W, preferably Y; and / or

[0069] X5: Y, L, F, I, V, A, C, preferably Y;

[0070] The "hHC01" and "hHC02" humanized sequences refer to new amino acid sequences that include sequence changes compared to the original chimeric sequences and partially humanized sequences described herein.

[0071] PTM mutations are intended to remove potentially harmful post-translational modification motifs from the protein while maintaining favorable binding properties. Positions 1, 5, 6, 19, 27, 28, 34, 39, 46, 48, 54, 69, 84, 85, 86, 88, 93, 107 and / or 115 of hHC01 and hHC02 are preferably mutated (substituted) compared to the original chimeric antibody sequence. The importance of the substitutions is primarily related to the resulting amino acid rather than the originating amino acid. Therefore, the changes can also be made from the original chimeric amino acids or other variants such as the corresponding amino acids of the partially humanized sequence.

[0072] The following substitutions are novel compared to the chimeric (SEQ ID No 1) sequence:

[0073] - amino acid M34 of the HC (VH) sequence is replaced by any amino acid, preferably I, L, F, V, YC, G, A, S, T;

[0074] -Amino acid E46 of the HC (VH) sequence was replaced by V;

[0075] - amino acids D54 and S55 of the HC (VH) sequence are replaced by any combination of amino acids, preferably SS, TS, GS, KS, RS, SD, SN, DE;

[0076] - amino acid Y101 of the HC (VH) sequence is replaced by any amino acid, preferably L, A, V, F, I, W; and / or

[0077] - Amino acid M107 of the HC (VH) sequence is replaced by any amino acid, preferably L, Y, F, I, V, A, C.

[0078] Sequences that can be modified at those residues required for direct interaction with BCMA:

[0079] hHC03 - modified amino acids involved in interaction with BCMA (SEQ ID No 5):

[0080] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYX1MX2 WVRQAPGKGLVX3VGX4 INPDSSTINYAPSLK DK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SX5X6X7DYGDX8MDYW GQGTLVTVSS

[0081] Among them, the preferred amino acids are

[0082] X1: W, F, Y, preferably W;

[0083] X2: S, T, N, Q, D, E, preferably S;

[0084] X3: W, F, Y, preferably W;

[0085] X4: E, Q, preferably E;

[0086] X5: L, I, V, G, A, preferably L;

[0087] X6: Y, X, preferably Y;

[0088] X7: Y, F, L, I, V, M, preferably Y; and / or

[0089] X8: A, G, V, preferably A.

[0090] "hHC03" humanized sequence refers to a new amino acid sequence that includes amino acid sequence changes compared to both the original chimeric sequence and the partially humanized sequence. These sequence changes are intended to reflect potential changes in amino acids that can be replaced for binding to the BCMA target while maintaining favorable binding properties. The importance of the replacement mainly relates to the resulting amino acid rather than the originating amino acid. Therefore, changes can also be made from the corresponding amino acids of the original chimeric amino acids or other variants.

[0091] For example:

[0092] - amino acid W33 of the HC (VH) sequence is W, F, Y;

[0093] - amino acid S35 of the HC (VH) sequence is S, T, N, Q, D, E;

[0094] - amino acid W47 of the HC (VH) sequence is W, F, Y;

[0095] -Amino acid E50 of the HC (VH) sequence is E, Q;

[0096] - Amino acid L99 of the HC (VH) sequence is L, I, V, G, A;

[0097] - amino acid Y100 of the HC (VH) sequence is Y, X;

[0098] - amino acid Y101 of the HC (VH) sequence is Y, F, L, I, V, M; and / or

[0099] -Amino acids A106 A, G, V of the HC (VH) sequence.

[0100] In general, any changes made to a CDR region during the humanization process may also be considered a feature of the CDR sequence when considered independently of the framework sequence as a whole. Such modified CDR sequences may be considered a defining feature of the invention within the context of the modified CDR sequence in the overall framework region described herein, or independently of the context of the modified CDR sequence in the overall framework region described herein. For example, the CDR sequences identified by underlining in hHC01 to hHC03 may be considered a defining feature of the invention independent of the surrounding framework sequence.

[0101] Specific examples of humanized HC (VH) sequences:

[0102] hHC04 (SEQ ID NO 6):

[0103] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWIS WVRQAPGKGLVWVG EINPNSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0104] hHC05 (SEQ ID NO 7):

[0105] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWF WVRQAPGKGLVWVG EINPNSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0106] hHC06 (SEQ ID NO 8):

[0107] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWIS WVRQAPGKGLVWVG EINPSSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0108] hHC07 (SEQ ID NO 9):

[0109] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWF WVRQAPGKGLVWVG EINPSSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0110] Comparison:

[0111] The CLUSTAL W (1.83) multiple sequence alignment of different substitution positions in the HC sequence was Figure 8 Appropriate sequence comparisons are provided in . "Generalized sequence" refers to a HC sequence in which each X represents a potential amino acid change for any given amino acid. Preferred amino acid substitutions are those described above for each potential mutation position.

[0112] Preferred embodiments of humanized VL variants

[0113] Chimeric sequence:

[0114] LC mouse (SEQ ID No.43):

[0115]

[0116] The LC mouse sequence represents the variable region (VL) of the light chain originally developed for the chimeric antibody J22.9-xi, which includes VL and VH domains obtained from a mouse antibody, capable of binding to an epitope in the extracellular domain of CD269 (BCMA), and the VL and VH domains are fused to human CL and CH domains, respectively.

[0117] Partially humanized sequences:

[0118] LC partial humanization (SEQ ID NO 10):

[0119]

[0120] The LC part humanized sequence refers to a modified sequence (by amino acid substitution) compared to the chimeric antibody disclosed in the examples of the invention herein, wherein the VL and VH binding regions are modified with respect to their sequence to make them more suitable for administration to humans.

[0121] Humanized VL sequence:

[0122] hLC01 (SEQ ID NO11):

[0123]

[0124] Humanized VL sequence with post-translational modification motifs removed:

[0125] hLC02 (SEQ ID NO12):

[0126]

[0127] in:

[0128] X1X2: ES, SS, TS, QS, HS, DH, ES preferred.

[0129] "hLC01" and "hLC02" humanized sequences refer to new amino acid sequences that include sequence changes compared to both the original chimeric sequences and the partially humanized sequences described herein.

[0130] PTM mutations are intended to remove potentially harmful post-translational modification motifs from the protein while maintaining favorable binding properties. Compared to the original chimeric antibody sequence, positions 1, 8, 9, 10, 13, 15, 17, 19, 20, 21, 22, 30, 41, 43, 45, 49, 58, 63, 70, 77, 83, 85 and / or 87 of hLC01 and hLC02 are preferably mutated (substituted). The importance of the substitutions is primarily related to the resulting amino acid rather than the originating amino acid. Therefore, changes may also be made from the corresponding amino acids of the original chimeric amino acids or other variants.

[0131] The following substitutions are novel with respect to the chimeric and partially humanized sequences:

[0132] - Amino acid D1 of the LC (VL) sequence was replaced by E;

[0133] - Amino acid V15 of the LC (VL) sequence was replaced by P;

[0134] - Amino acid D17 of the LC (VL) sequence was replaced by E;

[0135] - Amino acid V19 of the LC (VL) sequence was replaced by A;

[0136] - Amino acid T22 of the LC (VL) sequence was replaced by S;

[0137] - amino acids D30 and S31 of the LC (VL) sequence are replaced by any combination of amino acids, preferably ES, SS, TS, QS, HS, DH;

[0138] - amino acid V58 of the LC (VL) sequence is replaced by I; and / or

[0139] - Amino acid D70 of the LC (VL) sequence was replaced by E.

[0140] Sequences that can be modified in their CDR binding regions at those residues required for direct interaction with BCMA:

[0141] hLC03 - modified amino acids involved in interaction with BCMA (SEQ ID NO 13)：

[0142] EIVMTQSPATLSVSPGERATLSC KASQSVDX1X2VX3 WX4QQKPGQAPRALIX5 X6AX7X8RX9S GIPARFSGSX 10 X 11 GTEFTLTISSLQSEDFAVYYC X 12 QX 13 NNX 14 PX 15 TFG AGTKLELKR

[0143] The preferred amino acids are:

[0144] X1: S, H, T, N, D, Q;

[0145] X2: N, E, Q;

[0146] X3: A, G, V, S, T, L, I;

[0147] X4: Y, F, L, I, V, A, G;

[0148] X5: Y, F, L;

[0149] X6: S, T;

[0150] X7: S, T, D, N, H, E, Q;

[0151] X8: L, V, I, M;

[0152] X9: F, L, I, V, Y, M;

[0153] X 10 : G, X;

[0154] X 11 : S, X;

[0155] X 12: Q, V, L, I, M;

[0156] X 13 : Y, F, L, I, Q;

[0157] X 14 : Y, F, R, Q, K; and / or

[0158] X 15 :L,I,V,F.

[0159] "hLC03 humanized sequence" means a new amino acid sequence that includes amino acid sequence changes compared to both the original chimeric sequence and the partially humanized sequence. These sequence changes are intended to reflect potential changes in amino acids that can be substituted for binding to the BCMA target while maintaining favorable binding properties. The importance of the substitutions primarily relates to the resulting amino acids rather than the originating amino acids. Therefore, changes can also be made from the corresponding amino acids of the original chimeric amino acids or other variants.

[0160] For example:

[0161] - Amino acid S31 of the LC (VL) sequence is S, H, T, N, D, Q;

[0162] - Amino acid N32 of the LC (VL) sequence is N, E, Q;

[0163] - Amino acids A34 of the LC (VL) sequence are A, G, V, S, T, L, I;

[0164] - Amino acid Y36 of the LC (VL) sequence is Y, F, L, I, V, A, G;

[0165] - Amino acid Y49 of the LC (VL) sequence is Y, F, L;

[0166] - Amino acid S50 of the LC (VL) sequence is S, T;

[0167] - Amino acid S52 of the LC (VL) sequence is S, T, D, N, H, E, Q;

[0168] - Amino acid L53 of the LC (VL) sequence is L, V, I, M;

[0169] - Amino acid F55 of the LC (VL) sequence is F, L, I, V, Y, M;

[0170] - Amino acid G66 of the LC (VL) sequence is G, X;

[0171] - Amino acid S67 of the LC (VL) sequence is S, X;

[0172] - Amino acid Q89 of the LC (VL) sequence is Q, V, L, I, M;

[0173] - Amino acid Y91 of the LC (VL) sequence is Y, F, L, I, Q;

[0174] - amino acid Y94 of the LC (VL) sequence is Y, F, R, Q, K; and / or

[0175] -Amino acid L96 of the LC (VL) sequence is L, I, V, F.

[0176] In general, any changes made to a CDR region may also be considered a feature of the CDR sequence when viewed independently of the framework sequence as a whole. Such modified CDR sequences may be considered a defining feature of the invention either within the context of the modified CDR sequence in the overall framework region described herein, or independently of the context of the modified CDR sequence in the overall framework region described herein. For example, the CDR sequences identified by underlining in hLC01 to hLC03 may - in unmodified or substituted form - be considered a defining feature of the invention independently of the surrounding framework sequence.

[0177] Examples of humanized LC sequences:

[0178] hLC04 (SEQ ID NO 14):

[0179] EIVMTQSPATLSVSPGERATLSC KASQSVESNVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR

[0180] Comparison:

[0181] The CLUSTAL W (1.83) multiple sequence alignment of each potential change site in the LC sequence was Fig. 9 Appropriate sequence comparisons are provided in. "Generalized sequence" refers to a LC sequence in which each X represents a potential amino acid change. Preferred amino acid substitutions are those described above for each potential mutation position.

[0182] Thus, the present invention relates to humanized sequences according to hHC01, hHC02, hHC03, hHC04, hHC05, hHC06, hHC07, hLC01, hLC02, hLC03 and / or hLC04, or any given combination thereof.

[0183] The present invention encompasses all possible combinations of potential modifications to any given potential variant residue (identified as X in the "broad" sequence) set forth herein. By combining one or more of these different substitutions, humanized variants that exhibit the desired binding properties of the chimeric antibodies originally developed and confirmed herein can be generated. The antibodies described herein, or portions thereof, also encompass sequences having at least 80%, preferably 90%, sequence identity with those humanized sequences explicitly disclosed or disclosed by sequence formula.

[0184] Preferred embodiments of antibody epitopes

[0185] The present invention thus relates to an isolated antibody or antibody fragment that binds to CD269 (BCMA), wherein the antibody binds to an epitope that includes one or more amino acids from residues 13 to 32 of CD269 (BCMA).

[0186] The amino acid sequence of residues 13 to 32 of CD269 is shown in SEQ ID No. 40. The N-terminal sequence of CD269 is provided in SEQ ID No. 39. The extracellular domain of CD269 is provided as SEQ ID No. 38.

[0187] Antigens including the extracellular domain of CD269 according to SEQ ID No. 38 are used for immunization to generate the binding specificity of the mouse antibodies and chimeric antibodies described herein. Using the entire CD269 protein or fragments thereof including membrane-bound or intracellular domains as antigens during antibody generation, antibodies can be generated that bind to hidden or intracellular domains of CD269, thereby making such preparations unsuitable or unfavorable for therapeutic applications. The antibodies of the present invention are therefore defined by their binding to the extracellular domain of CD269. Specific epitopes within the extracellular domain also represent a preferred new and unexpected feature of the present invention.

[0188] Fab fragments prepared from mouse antibodies or chimeric antibodies were crystallized in complex with purified BCMA extracellular domains, and the complex structure was solved. Structural analysis revealed detailed information about the epitopes of the antibodies of the present invention and their biological relevance. It is an advantageous property that the antibodies of the present invention bind to an epitope of one or more amino acids from residues 13 to 32 of CD269 (BCMA) including the extracellular domain, because this region shows significant overlap with the binding sites of two natural ligands of CD269, BAFF and APRIL. No anti-CD269 antibodies described in the art so far show such a comprehensive overlap with BAFF and APRIL binding sites.

[0189] In one embodiment, the isolated antibody or antibody fragment of the present invention is characterized in that the antibody binds to an epitope comprising one or more of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27 or 32 of CD269 (BCMA). In another embodiment, the isolated antibody or antibody fragment of the present invention is characterized in that the antibody binds to an epitope consisting of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27 and 32 of CD269 (BCMA). These residues represent amino acids that directly interact with the antibodies of the present invention, as identified by the crystal structure data provided herein. These residues are numbered with respect to SEQ ID No. 39, which provides the N-terminal sequence of CD269.

[0190] In one embodiment of the invention, the isolated antibody or antibody fragment is characterized in that the antibody binding to CD269 (BCMA) disrupts BAFF-CD269 and / or APRIL-CD269 interaction.

[0191] The binding of the antibody of the present invention to the extracellular domain of CD269 disrupts the BAFF-CD269 interaction. Since the binding sites of APRIL and BAFF are located at sites similar to the antibody epitope, the binding of the antibody to CD269 also blocks the APRIL-CD269 interaction.

[0192] Comparison of the specific epitopes of the antibodies of the invention with the binding sites of APRIL and BAFF, whose crystal structures have been solved and their interactions mapped, reveals a comprehensive overlap in the binding sites of the natural ligands and the antibodies described herein. This represents an advantageous and unexpected aspect of the invention and enables reliable and efficient disruption of the BAFF-CD269 and / or APRIL-CD269 interaction.

[0193] The present invention thus relates to an isolated antibody or antibody fragment as described herein, wherein the antibody disrupts the APRIL-CD269 interaction by binding to an epitope comprising one or more of the amino acids at residues 13, 15, 17, 18, 19, 22, 26, 27, 30, 31, 32, 33, 34, 35 of CD269 (BCMA), in particular consisting of amino acids at residues 13, 15, 17, 18, 19, 22, 26, 27, 32. These amino acids correspond to the binding sites of APRIL on CD269, respectively, as well as overlapping residues of CD269 that bind both the antibodies as described herein and APRIL.

[0194] In another embodiment, the invention thus relates to an isolated antibody or antibody fragment as described herein, wherein the antibody disrupts BAFF-CD269 interaction by binding to an epitope comprising one or more of amino acids at residues 13, 15, 16, 17, 18, 19, 22, 25, 26, 27, 29, 30, 31, 32, 34, 35 of CD269 (BCMA), in particular consisting of amino acids at residues 13, 15, 16, 17, 18, 19, 22, 26, 27, 32. These amino acids correspond to the binding sites of BAFF on CD269, respectively, as well as overlapping residues of CD269 that bind both the antibodies as described herein and BAFF.

[0195] Although antibodies binding to CD269 have been described in the art, which also potentially disrupt the interaction of APRIL or BAFF with CD269, no relevant disclosure has been provided concerning the specific epitopes of such antibodies. It cannot be assumed that the antibodies described previously also bind to epitopes with such comprehensive overlap as the antibodies of the present invention. Even if the interaction of APRIL or BAFF with CD269 has been shown to be disrupted, this could potentially occur due to binding to a rather different epitope and subsequent steric hindrance of the APRIL or BAFF anchor. The extent of disruption of the interaction of APRIL or BAFF with CD269 caused by existing antibodies has not been previously documented.

[0196] The antibodies of the invention enable efficient and reliable disruption, which potentially represents an improved technical effect compared to those described in the art. In vitro blocking assays can be performed to determine and compare BAFF and / or APRIL disruption, for example using the extracellular domain of human BCMA and recombinant BAFF or APRIL.

[0197] In preferred embodiments, the epitope specificity exhibited by the antibodies described herein with high affinity binding represents a novel and unexpected technical effect. In essence, the exceptionally high affinity of the J22.9 antibody and its humanized variants not only provides "disruption" or "blocking" of the binding of the natural ligand; but the ultra-high affinity of the antibodies of the present invention ensures that when the antibodies are present, the natural ligands are essentially completely or almost completely excluded from binding to their BCMA targets.

[0198] As disclosed in the examples below, the affinities of the humanized antibodies as described herein are surprisingly high and relatively better than similar approaches attempted in the prior art. A Kd in the pM range is generally regarded as an excellent affinity that cannot be foreseen in normal practice.

[0199] In another aspect, the humanized antibodies or antibody fragments of the invention bind to CD269 with high affinity, for example, the antibody binds to human CD269 with an affinity of 100 nM, 90, 80, 70, 60, 50, 40, 30 nM or less, with an affinity of 20 nM or less, or with an affinity of 15 nM or less, or with an affinity of 5 nM or less, or with an affinity of 1000 pM or less, or with an affinity of 500 pM or less, or with an affinity of 100 pM or less, or with an affinity of 80 pM or less, or for example, with an affinity of about 50 pM, when measured by surface plasmon resonance such as Biacore.

[0200] In further embodiments, the antibody binds to human CD269 at about 1 pM to about 100 nM, or about 100 pM to about 50 nM, or about 200 pM to about 20 nM when measured by surface plasmon resonance such as Biacore.

[0201] Further preferred embodiments of the present invention

[0202] In one embodiment, the invention relates to antibodies or antibody fragments comprising an amino acid sequence defined by one or more amino acids that interact directly with the CD269 target and / or by one or more amino acids that interact via water interactions (see Tables 1 to 6). The large number of water interactions involved in the binding of the antibodies to the epitopes as described herein represent an unusual and surprising aspect of the binding. In particular, the high affinity of the antibodies directed to the particular epitopes described herein, combined with the large number of water interactions involving the binding surface between the antibodies and the epitopes, represent a surprising and unexpected aspect of the invention.

[0203] The present invention therefore relates to an antibody or antibody fragment comprising an amino acid sequence as described herein, wherein the sequence is characterized in that, with respect to the chimeras disclosed herein or with respect to the corresponding residues of the humanized sequence variants disclosed herein, there are specific amino acid residues involved in the interaction surface with the target epitope via a water bridge according to Table 5, said specific amino acid residues being selected from the group comprising Ser31, Asn32, Tyr36, Ser50, Ser52, Gly66, Gln89, Tyr91 and / or Tyr94 of the light chain, and / or Trp33, Ser35, Trp47, Glu50, Leu99 and / or Tyr101 of the heavy chain.

[0204] Although the example of water bridge formation for antibodies was performed with J22.9-xi chimeras, the inventors claim that the technical effect is maintained in the humanized variants of the present invention because the binding characteristics are maintained in the humanized variants compared to the original chimeric antibodies tested. In the heavy chain, the only mutated water bridge residue is Y101, but its water interactions involve main chain (i.e., backbone) atoms and can therefore be reasonably assumed to be unchanged by mutating the side chains; in the light chain, there are no mutations in residues involved in water bridges.

[0205] The antibodies of the present invention can be further characterized as amino acid residues involved in epitopes that interact with the antibodies described herein via water bridges. Relevant features are provided in Table 5. For example, in one embodiment, the present invention is therefore characterized in that residue Ser31 of the light chain interacts with Thr32 of CD269 via a water molecule. Such descriptions of the binding properties of the antibodies of the present invention are intended for each interaction as provided in Table 5.

[0206] In addition, the present invention includes sequence variants of the antibodies described herein, wherein one or more residues involved in the "water bridge" interaction are modified so that the direct side chain interaction is "replaced" into the sequence at the expense of the water "bridge". For example, a mutation or change can be made in the amino acid sequence to replace water from the interaction surface without significantly affecting the affinity of the interaction. The present invention therefore relates to antibodies or antibody fragments comprising an amino acid sequence as described herein, wherein the sequence is characterized as, with respect to the chimeras disclosed herein or with respect to the corresponding residues of the humanized sequence variants disclosed herein, sequence variations involving those amino acid residues on the interaction surface with the target epitope through a water bridge according to Table 5, the amino acid residues being selected from the group comprising Ser31, Ser31, Asn32, Tyr36, Ser50, Ser52, Gly66, Gln89, Tyr91 and / or Tyr94 of the light chain, and / or Trp33, Ser35, Trp47, Glu50, Leu99 and / or Tyr101 of the heavy chain. Variations at corresponding positions in the humanized antibodies described herein may involve any given amino acid substitution, preferably, one that will effectively displace water from the interaction while retaining similar binding properties with respect to epitope affinity and specificity.

[0207] In one embodiment of the invention, the isolated antibody or antibody fragment is characterized in that the antibody is glycosylated, preferably comprising N-linked oligosaccharide chains, preferably at Asn297 of the heavy chain.

[0208] Glycosylation of antibodies refers to the attachment of carbohydrates or glycans to antibodies. N-linked glycans are attached to the nitrogen of asparagine or arginine side chains. The carbohydrate chains attached to the target protein serve different functions. For example, certain proteins do not fold properly unless they are first glycosylated. Furthermore, polysaccharides attached to the amide nitrogen of arginine in proteins impart stability to certain secreted glycoproteins. Glycosylation in this case is not a limiting requirement for proper folding, but rather the non-glycosylated protein can be degraded more quickly.

[0209] As demonstrated in the examples of the present invention, deglycosylation of the antibodies disclosed therein results in a decrease in therapeutic efficacy compared to the glycosylated form of the antibody. Surprisingly, glycosylation plays a significant role in maintaining the activity of the antibody. Glycosylation therefore represents a preferred embodiment of the present invention associated with unexpected technical advantages.

[0210] As demonstrated in the Examples herein, although the overall tumor burden of animals treated with J22.9-xi-N-glycan (deglycosylated) was not significantly different from that of animals receiving an isotype control antibody, the lifespan of these mice was significantly improved compared to the isoAb treated group. Since J22.9-xi-N-glycan was shown to be unable to induce ADCC or CDC, this result suggests that the binding of J22.9-xi to BCMA alone impedes tumor growth. It is reasonable to consider that this is due to the blockade of the interaction between the receptor and its natural ligands (APRIL and BAFF). This aspect of the invention and the antibodies described herein represent a surprising technical effect that cannot be obtained from antibodies of the prior art. J22.9-xi-N-glycan (deglycosylated) can be considered a control sample for evaluating potential therapeutic effects in the described experiments, which control sample enables the antibody to bind to its target epitope without the downstream effects of ADCC or CDC. The antibodies of the present invention, preferably those with glycosylation, thus demonstrate such effective site binding that can prevent (or significantly disrupt) cytotoxicity caused by binding of natural ligands. This feature of the antibodies described herein is not described in similar antibodies described in the art.

[0211] Although the examples on glycosylation of antibodies were performed with J22.9-xi chimeras, the inventors claim that the technical effect is maintained in the humanized variants of the present invention because the binding characteristics of the humanized variants are maintained compared to the original chimeric antibodies tested. The preferred position for glycosylation (Asn297 of the heavy chain) is not directly linked to any mutated residue and is located in the constant region of human intact IgG. Therefore, it is reasonable to assume that there is no difference in the glycosylation pattern at this position present in any J22.9 variant compared to the chimeric antibodies.

[0212] Use and functional aspects of the invention

[0213] The antibodies of the present invention are capable of binding to the epitopes described herein, blocking the interaction of the natural ligand of the epitope and inducing CDC and ADCC.

[0214] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a factor induced by the antibodies of the present invention that produce the desired therapeutic effect. ADCC is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antigens. After cells expressing CD269 bind to the antibodies of the present invention, ADCC can be induced. Classical ADCC is mediated by natural killer (NK) cells; macrophages, neutrophils and eosinophils can also mediate ADCC. Since ADCC depends on a prior antibody response, ADCC is part of the adaptive immune response. Mouse experiments can show that ADCC is an important mechanism of action of therapeutic antibodies as described herein.

[0215] A preferred embodiment of the invention relates to an isolated antibody or antibody fragment as described herein for use as a medicament for the treatment of a medical disorder associated with the presence of pathogenic B cells.

[0216] In one embodiment of the invention, the medical disorder is a CD269-related disorder, preferably associated with pathogenic B cells, preferably a disease of plasma cells and / or memory B cells.

[0217] The plasma cell disorder may be a cancer of the plasma cells, such as multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, or plasma cell leukemia. The plasma cell disorder may be a cancer of the B lymphocytes, such as Hodgkin's disease.

[0218] In one embodiment of the invention, the medical disorder is an autoimmune disease associated with autoreactive plasma cells and / or autoreactive memory B cells, such as an inflammatory autoimmune disease, for example systemic lupus erythematosus or rheumatoid arthritis.

[0219] Thus, the present invention also encompasses methods of treatment for the medical disorders as disclosed herein, preferably comprising administering to a subject in need of such treatment a therapeutically effective amount of the antibody.

[0220] A further aspect of the invention relates to an antibody drug conjugate (ADC) comprising an antibody or antibody fragment as described herein.Anti-CD269 antibody drug conjugate "anti-CD269 ADC" can be described as an anti-CD269 antibody or fragment thereof conjugated to a therapeutic agent.In certain embodiments, the ADC comprises an anti-CD269 antibody (e.g., a humanized variant of J22.9-xi described herein).

[0221] When administered to a subject with a CD269 expressing medical condition such as cancer or an autoimmune disorder, an ADC or ADC derivative as described herein produces a clinically beneficial effect on cells expressing CD269. In one embodiment, an anti-CD269 antibody or derivative thereof is conjugated to a cytotoxic agent such that the resulting ADC or ADC derivative exhibits a cytotoxic effect on cancer cells expressing CD269, preferably when occupied or internalized by said cancer cells expressing CD269.

[0222] The anti-CD269 ADC or ADC derivative is preferably internalized and accumulated in cells expressing CD269, in which the ADC or ADC derivative exerts a therapeutic effect (e.g., a cytotoxic effect). Specifically, a particularly suitable part for conjugation to an antibody or antibody derivative is a chemotherapeutic agent, a prodrug converting enzyme, a radioisotope or compound, or a toxin. For example, an anti-CD269 antibody or its derivative can be conjugated to a cytotoxic agent such as a chemotherapeutic agent or a toxin (e.g., a cytostatic agent or a cytocidal agent).

[0223] Another aspect of the present invention relates to a preferably isolated nucleic acid molecule selected from the group consisting of:

[0224] a) A nucleic acid molecule comprising the following nucleotide sequence

[0225] - which encodes an isolated antibody or antibody fragment according to any one of the preceding claims,

[0226] - which encodes an amino acid sequence selected from the group consisting of those according to SEQ ID 1 to 31 and 41 to 42,

[0227] - comprising a sequence or a sequence fragment of SEQ ID No. 32 to 36,

[0228] b) a nucleic acid molecule complementary to the nucleotide sequence according to a);

[0229] c) a nucleic acid molecule comprising a nucleotide sequence having a sequence identity sufficient to be functionally similar / equivalent to the nucleotide sequence according to a) or b), preferably having a sequence identity of at least 80%, preferably 90%, more preferably 95% to the nucleotide sequence according to a) or b);

[0230] d) a nucleic acid molecule which, as a result of the genetic code, is degenerate to a nucleotide sequence according to a) to c); and

[0231] e) A nucleic acid molecule in which the nucleotide sequence according to a) to d) is modified by deletion, addition, substitution, translocation, inversion and / or insertion and which is functionally similar / equivalent to the nucleotide sequence according to a) to d).

[0232] A further aspect of the invention relates to a host cell, such as a bacterial cell or a mammalian cell, preferably a hybridoma cell or cell line, capable of producing an antibody or antibody fragment as described herein and / or comprising a nucleic acid molecule as described herein.

[0233] A further aspect of the invention relates to a pharmaceutical composition comprising an isolated antibody or antibody fragment as described herein, a nucleic acid molecule as described herein or a host cell as described herein, and a pharmaceutically acceptable carrier.

[0234] A further and surprising aspect of the invention is the improved stability of the antibodies as disclosed herein. The antibodies can be easily stored for extended periods of time under appropriate conditions without any loss of binding affinity. Appropriate testing has been performed with respect to the stability of activity after storage at -80°C or 4°C, which demonstrated the unexpectedly good stability and retention of activity of the antibodies after storage at the aforementioned two temperatures ( Figure 3c ). This improved stability is obvious for chimeric antibodies and is also unexpected for humanized variants thereof. Surprisingly, under long-term storage conditions, the stability of humanized variants is higher than that of chimeric antibodies.

[0235] A further advantage of the antibodies as described herein is the systemic depletion of myeloma cells as demonstrated in the examples. Antibodies previously disclosed in the prior art have not been shown to exhibit the desired anti-plasma cell effects in a systemic manner. Studies conducted on antibodies of the prior art have only disclosed subcutaneous injection with myeloma cells and subsequent treatment of isolated cell populations. The present invention provides antibodies capable of systemic depletion of cancerous multiple myeloma cells after their intravenous injection, as demonstrated in the examples. Effective depletion of target cells represents a technical effect not previously described in the prior art, in addition to the effective properties of the antibodies of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0236] The present invention is described by way of example with the embodiments and drawings disclosed herein. The drawings provided herein represent specific embodiments of the present invention and are not intended to limit the scope of the present invention. The drawings are considered to provide further descriptions of possible and potential preferred embodiments that enhance the technical support of one or more non-limiting embodiments. Description of the drawings:

[0238] Figure 1a to Figure 1d : In vitro characterization of J22.9-xi.

[0239] FIG. 2A to FIG. 2B : Structure of CD269 (BCMA) and J22.9-xi Fab:CD269 complex.

[0240] Figure 3a to Figure 3c: In vitro cytotoxicity of J22.9-xi.

[0241] Figure 4a to Figure 4g : Effects of J22.9-xi in xenografted NSG mice.

[0242] Figure 5a to Figure 5d : Treatment of established tumors.

[0243] Figures 6a to 6d : Tumor treatment in the early stages of the disease.

[0244] Figure 7 : Instability of hybridoma J22.9.

[0245] Figure 8 : Sequence of the humanized sequence of HC compared with J22.9-xi.

[0246] Fig. 9 : Sequence of the humanized sequence of LC compared with J22.9-xi.

[0247] Fig.10 : Sequence optimized variants of J22.9-xi showed similar binding in ELISA.

[0248] Fig.11 : Sequence-optimized variants of J22.9-xi showed similar binding in flow cytometry.

[0249] Figure 12: SPR raw data.

[0250] Fig.13 : Gel electrophoresis of antibody variants.

[0251] Detailed description of the accompanying drawings:

[0252] Figures 1a to 1d :In vitro characterization of J22.9-xi. ELISA ( Figure 1a ) or by flow cytometry using CD269-positive MM.1S cells ( Figure 1b ), J22.9-xi binds to BCMA in a concentration-dependent manner. ( Figure 1c ) The binding affinity of J22.9-xi to BCMA was determined by surface plasmon resonance measurement using the indicated concentrations of BCMA. Figure 1d )J22.9-xi blocks the interaction between BAFF and BCMA adsorbed to microtiter plates.

[0253] FIG. 2A to FIG. 2B :( Figure 2A)CD269 (BCMA) recognition surface. Three views of the CD269 (BCMA) extracellular domain showing the binding epitope residues of BAFF / APRIL and J22.9-xi. In the upper figure, a direct view of the binding surface of CD269 (BCMA): the light gray shading indicates all residues of the binding epitopes including BAFF and APRIL as discerned from their crystal structures, and the black residues (shown as spheres) do not contact any BAFF, APRIL or J22.9-xi; a subset of epitope residues involved in J22.9-xi binding is shown in a surface representation; the remaining light gray residues shown as spheres are part of both BAFF and APRIL epitopes but do not directly contact J22.9-xi. The middle and lower figures are shown in the same representation as the upper figure, but are rotated 90° toward and away from the observer, respectively. ( Figure 2B ) Two views of the J22.9-xi Fab:CD269 complex. J22.9-xi is shown in surface representation with the heavy chain in light grey and the light chain in dark grey. CD269 (BCMA) is shown in ribbon representation bound to the J22.9-xi antigenic cavity region. On the left is a full view of the Fab:CD269 complex; on the right is an oblique view to show the complex bound to the cavity.

[0254] Figure 3a to Figure 3c :In vitro cytotoxicity of J22.9-xi. ( Figure 3a ) CD269-positive MM.1S-Luc cells were mixed with human PBMCs at an effector to target ratio of 20:1 and incubated with the indicated concentrations of J22.9-xi for 4 hours. Open symbols indicate the cytotoxic activity of J22.9-xi without-N-glycans when incubated with PBMCs from donor 1 and donor 2. Error bars indicate SEM. ( Figure 3b ) Deglycosylation does not affect the binding of J22.9-xi to MM.1S cells. ( Figure 3c ) Storage of J22.9-xi at 4°C or -80°C for 3 weeks did not affect cytotoxicity.

[0255] Figure 4a to Figure 4g : Effects of J22.9-xi in xenografted NSG mice. Figure 4a ) Tumor development over time in mice administered 200 μg of J22.9-xi or control antibody twice weekly and in untreated control mice. Figure 4b ) Total tumor burden between days 6 and 41 (( Figure 4a ) area under the curve (AUC). Plotted are mean values ​​with SEM (**P<0.01, ***P<0.001, t-test). ( Figure 4c ) Overall survival of J22.9-xi and isotype control mice. P values ​​were calculated using the log-rank (Mantel-Cox) test. Figure 4d-1 ) Detection of MM.1S-Luc cells in the indicated groups that were not administered therapeutic antibodies. Below the rightmost image, the numbers (41, 41, 44, 40) indicate the days after tumor cell injection when a particular mouse died. ( Figure 4d-2 ) Detection of MM.1S-Luc cells in the indicated groups at days 21 and 28. Dorsal view. ( Figure 4e )Relationship between J22.9-xi concentration and tumor development. Figure 4f ) Total tumor burden between days 6 and 42 (( Figure 4e ) AUC). Mean values ​​with SEM (**P<0.01, ***P<0.001, t-test). ( Figure 4g ) Overview of the experiment timeline.

[0256] Figure 5a to Figure 5d : Treatment of established tumors. ( Figure 5a ) Tumor development over time in mice administered 200 μg of J22.9-xi or control antibody twice weekly and in untreated control mice. Figure 5b ) Total tumor burden between days 8 and 48 (( Figure 5a ) AUC). Plotted are mean values ​​with SEM (**P<0.01, ***P<0.001, t-test). ( Figure 5c ) Total survival of J22.9-xi and isotype control mice. P values ​​were calculated using the log-rank (Mantel-Cox) test. Figure 5d An overview of the experimental timeline is provided in .

[0257] Figures 6a to 6d :Tumor treatment in the early stages of the disease. ( Figure 6a ) Tumor growth when treated with 2 μg, 20 μg, or 200 μg of J22.9-xi, or 200 μg of isotype control antibody or J22.9-xi without N-glycans, and tumor growth in the absence of tumors. Figure 6b Total tumor burden from day 9 to day 44 (( Figure 6a )). Shown are the mean values ​​with SEM (**P<0.01, ***P<0.001, t-test). ( Figure 6c Survival of mice treated with ) antibody and control xenograft SCID-Beige mice. P values ​​were calculated using the log-rank (Mantel-Cox) test. Figure 6d An overview of the experimental timeline is provided in .

[0258] Figure 7: Instability of hybridoma J22.9. Supernatant of hybridoma J22.9 tested in ELISA on BCMA-coated microtiter plates on day 1 was positive for binding to BCMA. Subsequent analysis at the indicated time points revealed a decrease in the binding capacity of the supernatant. The culture medium was changed on days 7, 14 and 21.

[0259] Figure 8 : Summary of humanized antibody sequences compared to J22.9-xi. Sequence comparisons were performed using standard alignment software. X: variable amino acids according to humanized sequence; HCg: general heavy chain variable sequence; HCm: mouse heavy chain variable sequence; HCpH: partially humanized heavy chain variable sequence; CDRs are underlined in general and mouse sequences.

[0260] Fig. 9 : Summary of humanized antibody sequences compared to J22.9-xi. Sequence comparisons were performed using standard alignment software. X: variable amino acids according to mouse or humanized sequence; HCg: general heavy chain variable sequence; HCm: mouse heavy chain variable sequence; HCpH: partially humanized heavy chain variable sequence; CDRs are underlined in general and mouse sequences.

[0261] Fig.10 : The binding of chimeric J22.9-xi and humanized variants (J22.9-H corresponds to the humanized sequence SEQ ID No.27; J22.9-FSY corresponds to the humanized and PTM-modified SEQ ID No.28; J22.9-ISY corresponds to the humanized and PTM-modified SEQ ID No.29) was tested by using microtiter plates coated with human BCMA (hBCMA) or cynomolgus monkey BCMA (cyBCMA).

[0262] Fig.11 : The binding of chimeric J22.9-xi and humanized variants (J22.9-FSY corresponds to humanized and PTM-modified SEQ ID No. 28; J22.9-ISY corresponds to humanized and PTM-modified SEQ ID No. 29) was tested by flow cytometry using the human MM cell line RPMI-8226.

[0263] Figure 12: SPR raw data: Chimeric J22.9-xi and humanized variants were measured by surface plasmon resonance (SPR) spectroscopy with human BCMA ( Fig. 12A ) and macaque BCMA ( Fig. 12B ) binding affinity. IgG was immobilized on a Proteon GLH sensor chip by amine chemistry and binding was measured with BCMA in the mobile phase. The order of the raw data in the figures follows the order of the samples listed in the figure captions.

[0264] Fig.13 : Gel electrophoresis of antibody variants. Antibody variants were run in non-reducing SDS-PAGE and the antibody variants were stained to show protein migration. DETAILED DESCRIPTION

[0265] As used herein, antibody generally refers to a protein consisting of one or more polypeptides encoded by a fragment of an immunoglobulin gene or immunoglobulin gene. When the term "antibody" is used, the term "antibody fragment" is also considered to be associated therewith. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as countless immunoglobulin variable region genes. Light chains are classified as κ and λ. Heavy chains are divided into γ, μ, α, δ or ε, which in turn define immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. Basic immunoglobulin (antibody) structural units are known to include tetramers or dimers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" (L) chain (about 25kD) and a "heavy" (H) chain (about 50-70kD). The N-terminal of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antibody recognition. The terms "variable light chain" and "variable heavy chain" refer to the variable regions of the light chain and heavy chain, respectively. Optionally, the antibody or immunoglobulin portion of an antibody can be chemically conjugated to or expressed as a fusion protein with other proteins.

[0266] The antibodies of the invention are intended to bind to mammalian, especially human, protein targets. The use of protein names may correspond to either the mouse or human form of the protein.

[0267] "Specific binding" should be as understood by those skilled in the art, wherein the skilled person is well aware of the various experimental procedures that can be used to test binding and binding specificity. Some cross-reaction or background binding may be unavoidable in many protein-protein interactions; this is not to detract from the "specificity" of the binding between the antibody and the epitope. When considering the term "specificity" in understanding the interaction between the antibody and the epitope, the term "directed against" is also applicable.

[0268] The antibodies of the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single variable region fragments (ssFv), single domain antibodies (such as VHH fragments from nanobodies), single chain fragments (scFv), Fab fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, anti-idiotypic antibodies and epitope binding fragments or any combination of the above, provided that they maintain the original binding properties. Mini antibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptibodies can also be used in the methods of the present invention. The immunoglobulin molecules of the present invention can be any class (i.e., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules. Therefore, the term antibody used herein also includes antibodies and antibody fragments synthesized de novo by modification of the entire antibody or using recombinant DNA methods.

[0269] Humanized antibodies comprising one or more CDRs of an antibody of the invention or derived from said antibody can be prepared by any method known in the art. For example, four general steps can be used to humanize a monoclonal antibody. These steps are: (1) determining the nucleotide sequence and expected amino acid sequence of the starting antibody light chain variable domain and heavy chain variable domain, (2) designing the humanized antibody, i.e., determining which antibody framework region to use in the humanization process, (3) actual humanization methods / techniques, and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 5,225,539; 6,548,640.

[0270] The term humanized antibody means at least a portion of the framework region of an immunoglobulin, and optionally a portion of the CDR region or other regions that are related to the combination is derived from or adjusted to a human immunoglobulin sequence. For example, it is possible to prepare a humanized, chimeric or partially humanized form of a mouse monoclonal antibody by recombinant DNA technology, for example, not according to the mouse and / or human genomic DNA sequence encoding H and L chains or the cDNA clone encoding H and L chains. The mouse antibody of humanized form can be produced by connecting the CDR region of a non-human antibody to a human constant region by recombinant DNA technology (Queen et al., 1989; WO 90 / 07861). Alternatively, the monoclonal antibody used in the method of the present invention can be a human monoclonal antibody. For example, a phage display method can be used to obtain human antibodies (WO 91 / 17271; WO 92 / 01047).

[0271] As used herein, humanized antibodies also refer to forms of non-human (e.g., murine, camel, llama, shark) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as antibody Fv, Fab, Fab', F(ab')2 or other antigen binding sequences) that contain minimal sequence derived from non-human immunoglobulin.

[0272] As used herein, human antibody or humanized antibody means an antibody with an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a person and / or using any technique known in the art or disclosed herein for preparing human antibodies to prepare human antibodies or humanized antibodies. Human antibodies with the same epitope specificity as a particular mouse antibody can be selected by competitive binding experiments or other means. Humanized antibodies of the present invention surprisingly share the useful functional properties of mouse antibodies to a great extent. Human polyclonal antibodies in the form of human serum immunized with an immunogenic agent can also be provided. Optionally, such polyclonal antibodies can be concentrated by affinity purification using amyloid fibrillar and / or non-fibrillar polypeptides or their fragments as affinity reagents. Monoclonal antibodies can be obtained from serum according to the technology described in WO 99 / 60846.

[0273] The present invention further relates to the use of antibodies or fragments thereof (e.g., variable regions) as described herein in identifying molecules or affinity reagents suitable for selectively binding to a target. Affinity reagents, antibodies or fragments thereof according to the present invention may be pegylated, wherein pegylation refers to the covalent attachment of a polyethylene glycol (PEG) polymer chain to an antibody of the present invention. Pegylation may be routinely achieved by incubation of a reactive derivative of PEG with a target molecule. Pegylation of antibodies may potentially shield the reagent from the host immune system, resulting in reduced immunogenicity and antigenicity of the reagent or increased hydrodynamic size of the reagent, which may extend the circulation time of the reagent by reducing renal clearance.

[0274] The variable region of an antibody refers to the variable region of a single antibody light chain or the variable region of an antibody heavy chain, or a combination. The variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also known as hypervariable regions). The CDRs of each chain are held together in close proximity by the FRs and together with the CDRs from the other chains form the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (Fifth Edition, 1991, National Institutes of Health, Bethesda, Maryland); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273: 927-948). As used herein, CDRs may refer to CDRs defined by either method or by a combination of both methods.

[0275] In some embodiments, the invention provides antibodies, which include at least one CDR, at least two, at least three or more CDRs substantially identical to at least one CDR, at least two, at least three or more CDRs of the antibodies of the invention. Other embodiments include antibodies with at least two, three, four, five or six CDRs substantially identical to at least two, three, four, five or six CDRs of the antibodies of the invention or derived from the antibodies of the invention. In some embodiments, at least one, two, three, four, five or six CDRs are at least about 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98% or 99% consistency with at least one, two or three CDRs of the antibodies of the invention. It is understood that for the purposes of the present invention, binding specificity and / or overall activity are generally retained, although the degree of activity compared to the antibody may vary (may be larger or smaller).

[0276] The half-life and cytotoxic potential of antibodies mainly depend on the interaction of the Fc domain with different Fcγ receptors. In the case of antibody half-life, the newly born Fc receptor (FcRn) plays a major role. The receptor is expressed in several cell types and tissues that can absorb serum proteins into its recycling endosome, such as monocytes and vascular endothelial cells. In some endosomes, the pH is reduced to close to 6, and under these conditions, antibodies can be bound to FcRn. This interaction protects antibodies from degradation until the antibodies are released again into the blood that destroys the binding with the receptor at physiological pH (Roopenian and Akilesh (2007) Nat Rev Immunol 7: 715-725). The higher the affinity of the antibody to FcRn at pH 6, the longer the half-life of the antibody. The known Fc fragment mutations for stabilizing this interaction are summarized in Presta (2008, Curr Opin Immunol 20: 460-470).

[0277] Therapeutic antibodies can work by several mechanisms when bound to their targets. Binding itself can trigger signal transduction, which can lead to programmed cell death (Chavez-Galan et al. (2009) Cell Mol Immunol 6:15-25). It can also block the interaction between a receptor and its ligand by binding to a receptor or ligand. If the signal important for survival is affected, this interference can lead to apoptosis (Chiu et al. (2007) Blood 109:729-739). Regarding cell depletion, there are two known major effector mechanisms. The first is complement-dependent cytotoxicity (CDC) to target cells. There are three known different pathways. However, in the case of antibodies, an important pathway for CDC is the classical pathway, which starts by binding to the constant region of IgG or IgM through C1q (Wang and Weiner (2008) Expert Opin Biol Ther 8:759-768).

[0278] The second mechanism is called antibody-dependent cell-mediated cytotoxicity (ADCC). The effector function is characterized by recruiting immune cells expressing the Fc receptors of the respective isotypes for antibodies. ADCC is mainly mediated by Fcγ receptors (FcγR) that can be bound to IgG molecules alone or as an immune complex by activation. Mice show three kinds of activated Fcγ receptors (FcγRI, FcγRIII and FcγRIV), and people show five kinds of activated Fcγ receptors (FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA and FcγRIIIB). These receptors are expressed in natural immune cells such as granulocytes, monocytes, macrophages, dendritic cells and natural killer cells, and therefore natural immune cells are connected to the adaptive immune system. According to cell type, when identifying the target cells of antibody markers, there are several modes of action of cells carrying FcgR. Granulocytes usually release vasoactive and cytotoxic substances or chemoattractants, but can also carry out phagocytosis. Monocytes and macrophages respond with phagocytosis, oxidative burst, cytotoxicity, or release of proinflammatory cytokines, while natural killer cells release granzymes and perforins and can also trigger cell death through the interaction of FAS on target cells with their Fas ligands (Nimmerjahn and Ravetch (2008) Nat Rev Immunol 8:34-47; Wang and Weiner (2008) Expert Opin Biol Ther 8:759-768; Chavez-Galan et al. (2009) Cell Mol Immunol 6:15-25).

[0279] Antibody-dependent cell-mediated cytotoxicity (ADCC) can also be improved by strengthening the binding of the Fc domain to activating Fcγ receptors (FcγRs). This can also be achieved by mutations in the Fcγ domain as summarized in Presta (2008, Curr Opin Immunol 20:460-470).

[0280] Another approach to altering ADCC is to manipulate the carbohydrate moiety present in every IgG at Asn 297. Defucosylation and removal of sialic acid from the termini of the carbohydrate molecule are known to increase the cytotoxic potential of antibodies (Anthony and Ravetch (2010) J Clin Immunol 30 Suppl 1: S9-14).

[0281] Sequence variants of the claimed nucleic acids, proteins and antibodies that maintain the properties of the invention, such as defined by the claimed % sequence identity, are also included within the scope of the invention. Such variants show alternative sequences, but substantially maintain the same binding properties, such as target specificity, because the specific sequences provided are referred to as functional analogs or as functionally similar. Sequence identity refers to the percentage of identical nucleotides or amino acids when performing a sequence alignment.

[0282] It will be appreciated by those of ordinary skill in the art that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any native gene. Nevertheless, the present invention particularly contemplates polynucleotides that vary due to differences in codon usage. The present invention also encompasses deletions, substitutions and other changes that fall within the sequence identity described.

[0283] Modification of protein sequences that can occur by replacement is also included in the scope of the present invention. Replacement as defined herein is a modification of the amino acid sequence of a protein, wherein one or more amino acids are replaced with the same number of (different) amino acids, producing a protein containing an amino acid sequence different from the primary protein, preferably without significantly changing the function of the protein. As added, the replacement can be natural or artificial. It is well known in the art that amino acid replacement can be performed without significantly changing the function of the protein. This is particularly true when the modification involves a "conservative" amino acid replacement, which is an amino acid replaced with another amino acid of similar properties. Such a "conservative" amino acid can be a natural amino acid or a synthetic amino acid, which can be replaced without significantly affecting the structure and function of the protein due to size, charge, polarity and conformation. Frequently, many amino acids can be replaced by conservative amino acids without adversely affecting the function of the protein.

[0284] In summary, the nonpolar amino acids Gly, Ala, Val, Ile and Leu; the nonpolar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can be substituted for each other even if they belong to different groups.

[0285] Substitution variants have at least one amino acid residue and the different residues inserted in its position in the antibody molecule removed. The most interesting mutation sites for replacement include hypervariable regions, but the change of FR is also considered. If such replacement causes a change in biological activity, then be named as "exemplary replacement" in the following table, or the larger change further described below with reference to amino acid class can be introduced and screened product.

[0286] Potential amino acid substitutions:

[0287] Original residue Preferred conservative substitutions Example of illustrative substitution Ala(A) Val Val; Leu; Ile Asg(R) Lys Lys; Gln; Asn Asn(N) Gln Gln; His; Asp, Lys; Arg Asp(D) Glu Glu; Asn Cys(C) Ser Ser; Ala Gln(Q) Asn Asn、Glu Glu(E) Asp Asp; Gln Gly(G) Ala Ala His(H) Arg Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu(L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met(M) Leu Leu; Phe; Ile Phe(F) Tyr Leu; Val; Ile; Ala; Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr Tyr; Phe Tyr(Y) Phe Trp; Phe; Thr; Ser Val(V) Leu Ile; Leu; Met; Phe; Ala; norleucine

[0288] Substantial modifications of the biological properties of the antibody are achieved by selecting substitutions that differ significantly in their effects on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution (e.g., sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the majority of side chains.

[0289] Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids are ω amino acids and cyclohexylalanine of different chain lengths as neutral non-polar analogs; citrulline and methionine sulfoxide as neutral non-polar analogs, phenylglycine as an aromatic neutral analog; cysteic acid as a negatively charged analog and ornithine as a positively charged amino acid analog. As with naturally occurring amino acids, this list is not exhaustive, but merely an example of substitutions well known in the art.

[0290] Antibodies of the present invention can be produced by transfecting host cells with an expression vector comprising the encoding sequence of antibodies of the present invention. Expression vectors or recombinant plasmids are produced by combining these encoding sequences of antibodies with conventional control sequences that can be controlled to replicate and express in host cells and / or can be secreted from host cells. Control sequences include promoter sequences such as CMV promoters and signal sequences that can be derived from other known antibodies. Similarly, a second expression vector can be produced that has a DNA sequence dna encoding complementary antibody light chain or heavy chain. In some embodiments, this second expression vector is identical to the first, except within the scope of relevant encoding sequences and selectable markers, so as to ensure that each polypeptide chain is functionally expressed as far as possible. Alternatively, the heavy chain and light chain encoding sequences of the antibody can be located on a single vector.

[0291] The host cell of the present invention is co-transfected with the first and second vectors by conventional techniques (or simply transfected by a single vector) to produce a transfected host cell of the present invention, the host cell comprising both a recombinant or synthetic light chain and a heavy chain. The transfected cells are then cultured by conventional techniques to produce engineered antibodies of the present invention. The antibodies comprising the combination of the recombinant heavy chain and / or the light chain are screened from the culture by appropriate assays such as ELISA or RIA. Similar conventional techniques can be used to construct other antibodies.

[0292] Those skilled in the art can select vectors suitable for cloning and subcloning steps used in the construction of the method and composition of the present invention. For example, conventional pUC series cloning vectors can be used. A vector pUC19 is commercially available. The components of such vectors, such as replicons, selection genes, enhancers, promoters, signal sequences, etc., can be obtained from commercial or natural sources, or synthesized by known programs for instructing the expression and / or secretion of the product of the recombinant DNA in the selected host. For this purpose, other appropriate expression vectors can also be selected, and many types of them are known in the art for mammalian, bacterial, insect, yeast and fungal expression.

[0293] The present invention also encompasses cell lines transfected with recombinant plasmids containing the coding sequences of the antibodies of the present invention.Host cells used for cloning and other manipulations of these cloning vectors are also conventional.

[0294] Host cells or cell lines suitable for expression of antibodies of the present invention include mammalian cells such as NS0, Sp2 / 0, CHO (e.g., DG44), COS, HEK, fibroblasts (e.g., 3T3) and myeloma cells, for example, expressed in CHO or myeloma cells. Human cells can be used, thus enabling the molecules to be modified by human glycosylation patterns. Alternatively, other prokaryotic or eukaryotic cell lines can be used. Selection of suitable mammalian host cells and methods for transformation, cultivation, amplification, screening and product generation and purification are known in the art.

[0295] According to the present invention, a method for producing an anti-CD269 antibody of the present invention that binds to human CD269 and neutralizes human CD269 activity is provided, the method comprising the following steps: providing a first vector encoding the heavy chain of the antibody; providing a second vector encoding the light chain of the antibody; transforming a mammalian host cell (e.g., CHO) with the first vector and the second vector; culturing the host cell of step (c) under conditions that direct the antibody to be secreted from the host cell into the culture medium; recovering the secreted antibody in step (d). Once expressed, the desired binding properties of the antibody can be evaluated using the methods described herein.

[0296] The present invention encompasses immunoconjugates (interchangeably referred to as "antibody drug conjugates" or "ADCs") comprising an antibody according to the present invention as described herein, including but not limited to an antibody conjugated to one or more cytotoxic agents such as chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes (i.e., radioconjugates). Techniques for conjugating therapeutic agents to proteins, especially antibodies, such as the anti-CD269 antibody drug conjugates of the present invention, are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., ed., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (Robinson et al., ed., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological And Clinical Applications). Applications (Pinchera et al., eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., eds., Academic Press, 1995); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58.See also, e.g., PCT Publication WO 89 / 12624.).

[0297] Typically, ADC or ADC derivatives include a connector region between a therapeutic agent and an anti-CD269 antibody or derivative thereof. As described above, in typical embodiments, the connector is cleavable under intracellular conditions, so that the connector is cut under the intracellular environment to release the therapeutic agent from the antibody. For example, in some embodiments, the connector is cut by a cutting agent present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The connector can be, for example, a peptidyl connector cut by an intracellular peptidase or protease (including but not limited to a lysosomal protease or an endosomal protease). In other embodiments, the cleavable connector is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, a pH-sensitive connector is hydrolyzable under acidic conditions. In another embodiment, the connector is cleavable under reducing conditions (e.g., a disulfide connector). Various disulfide linkers are known in the art (see, e.g., Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.).

[0298] Typically, the linker is substantially insensitive to the extracellular environment. In other non-mutually exclusive embodiments, the linker promotes cell internalization. In certain embodiments, when conjugated to a therapeutic agent (i.e., in the environment of a linker-therapeutic agent portion of an ADC or ADC derivative as described herein), the linker promotes cell internalization. In yet another embodiment, when conjugated to a therapeutic agent and an anti-CD269 antibody or derivative thereof (i.e., in the environment of an ADC or ADC derivative as described herein), the linker promotes cell internalization. A variety of linkers that can be used with the present compositions and methods are described in WO 2004010957, filed on July 31, 2003, entitled “Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease,” and U.S. Provisional Application No. 60 / 400,403, filed on July 31, 2002, entitled “Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease,” the disclosures of which are incorporated herein by reference.

[0299] In certain embodiments, the immunoconjugate comprises an antibody as described herein, including but not limited to an antibody and a chemotherapeutic agent or other toxin. Enzymatically active toxins and fragments thereof that can be used include diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin IIA chain, alpha-sarcin, Aleurites fordii proteins, dianthus proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies.

[0300] The antibodies or fragments thereof of the present invention may also be conjugated to one or more toxins, including but not limited to calicheamicin, maytansinoids, dolastatins, aurostatins, trichothecenes and CC1065, and derivatives of these toxins having toxin activity. Suitable cytotoxic agents include but are not limited to dolavaline-valine-dolaisoleucine-dolaproline-phenylalanine (MMAF) and monomethyl auristatin E (monomethyl auristatin E) (MMAE), and MMAE in ester form, DNA minor groove binders, DNA minor groove alkylating agents, enediynes, lexitropsins, duocarmycins, taxanes including paclitaxel and docetaxel, puromycin, dolastatins, maytansinoids, and vinca alkaloids. Specific cytotoxic agents include topotecan, morpholino adriamycin, rhizoxin, cyanomorpholino adriamycin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1, DM-4, ​​fusobacterin. Other suitable cytotoxic agents include anti-tubulin agents such as auristatins, vinca alkaloids, podophyllotoxin, taxanes, baccatin derivatives, cryptophysin, maytansinoid alkaloids, combretastatin or dolastatin. Anti-tubulin agents include dimethylvaline-valine-dolaisoleucine-dolaproline-phenylalanine-p-phenylenediamine (AFP), MMAF, MMAE, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, colcimid, estramustine, cemadotin, scutellarin, maytansine, DM-1, DM-4 or eleutherobin.

[0301] In some embodiments, the immunoconjugate includes an antibody conjugated to dolastatin or a dolastatin peptide analog and derivative auristatin (U.S. Pat. Nos. 5,635,483; 5,780,588), which have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents and Chemother. 42:2961-2965). Dorastatin and auristatin (which is a pentapeptide derivative of dorastatin) drug moieties can be linked to antibodies via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172). Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF, which are disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Pat. No. 7,498,298. As used herein, the abbreviation "MMAE" refers to monomethyl auristatin E. As used herein, the abbreviation "MMAF" refers to dolavaline-valine-dolarisoleucine-dolarproline-phenylalanine.

[0302] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods known in the field of peptide chemistry (see Schroder and K.Lubke, "Peptides," Vol. 1, pp. 76-136, 1965, Academic Press).

[0303] Maytansinoids can be used as an active agent coupled to an antibody or its fragment according to the present invention. Maytansinoids are mitotic inhibitors that work by inhibiting tubulin polymerization. Maytansine (U.S. Patent No. 3,896,111) was first isolated from the East African shrub Maytansin (Maytenus serrata). Subsequently, it was found that some microorganisms can also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). High cytotoxic maytansinoid drugs can be prepared from ansamitocin precursors produced by fermentation of microorganisms such as Actinosynnema. Antibodies-maytansinoid conjugates are prepared by chemically connecting antibodies to maytansinoid molecules without significantly reducing the biological activity of antibodies or maytansinoid molecules. See, for example, U.S. Patent No. 5,208,020. An average of 3-4 maytansinoid molecules are conjugated per antibody molecule, showing effectiveness in increasing target cell cytotoxicity without negatively affecting the function or solubility of the antibody, although even one molecule of toxin / antibody is expected to increase cytotoxicity above that of using unmodified antibodies. Maytansinoids are well known in the art and can be synthesized or isolated from natural sources by known techniques.

[0304] Examples of selected calicheamicin family of antibiotics can be used as active agents coupled to antibodies or fragments thereof according to the present invention. The calicheamicin family of antibiotics can produce double-stranded DNA damage at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296. Another anti-tumor drug that can be conjugated to antibodies is the antifolate QFA. Both calicheamicin and QFA have intracellular sites of action and are not easy to pass through the plasma membrane. Therefore, the cellular uptake of these agents by antibody-mediated internalization greatly improves their cytotoxic effects.

[0305] Other anti-tumor agents that can be conjugated to antibodies include BCNU, streptozoicin, vincristine and 5-fluorouracil, a family of agents collectively referred to as LL-E33288 complexes described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicins (U.S. Pat. No. 5,877,296). The present invention further contemplates the formation of immunoconjugates between antibodies and compounds having nucleolytic activity (e.g., ribonucleases or DNA endonucleases such as deoxyribonucleases; DNA enzymes). For selective destruction of tumors, antibodies can include highly radioactive atoms.

[0306] In the sense of the present invention, a pharmaceutically acceptable carrier can be any non-toxic substance that does not significantly interfere with the effectiveness of the biological activity of the antibody of the present invention in a harmful sense. Obviously, the characteristics of the carrier will depend on the route of administration. Such a composition may contain, in addition to the active substance and the carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers and other substances well known in the art. The preparation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as in a variety of treatment regimens such as oral, parenteral, intravenous, intranasal and intramuscular administration and formulations, for the development of appropriate administration and treatment regimens using the specific compositions described herein.

[0307] The drug containing the active ingredient (antibody or antibody fragment), or the pharmaceutical composition, can be in a form suitable for oral administration, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. The composition for oral use can be prepared according to any method known in the art of manufacturing pharmaceutical compositions, and such a composition can contain one or more agents selected from the group consisting of sweeteners, flavoring agents, colorants and preservatives to provide pharmaceutically beautiful and palatable preparations. Tablets contain active ingredients mixed with non-toxic pharmaceutically acceptable excipients suitable for making tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. Tablets can be uncoated or they can be coated by known techniques to delay decomposition and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time. For example, time-delay substances such as glyceryl monostearate or glyceryl distearate can be used. They can also be coated. The present invention also refers to pharmaceutical compositions for topical application, oral administration, inhalation, or cutaneous, subcutaneous or intravenous injection. The technician knows the carriers and additives required for the specific application form.

[0308] When a therapeutically effective amount of the active substance (antibody or antibody fragment) of the present invention is administered by intravenous injection, cutaneous injection or subcutaneous injection, the active substance may be in the form of a pyrogen-free, parenterally acceptable aqueous solution.

[0309] The present invention also relates to administering a therapeutically relevant amount of an antibody described herein in the treatment of a subject having a medical disorder as disclosed herein. As used herein, the term "therapeutically effective amount" means the total amount of each active ingredient of a pharmaceutical composition or method sufficient to show a meaningful patient benefit. The amount of active substance in the pharmaceutical composition of the present invention depends on the nature and severity of the condition to be treated, as well as the nature of the prior treatment the patient has received. Larger doses may be administered until the patient obtains the optimal therapeutic effect, and at that point, the dose is no longer increased further.

[0310] The preparation of such non-gastrointestinal acceptable solutions should pay attention to pH, isotonicity and stability, etc., and the preparation is within the technology of the art. Preferred pharmaceutical compositions for intravenous, cutaneous or subcutaneous injection should contain isotonic solvents, such as sodium chloride injection, Ringer's injection, glucose injection, glucose and sodium chloride injection, lactated Ringer's injection or other solvents known in the art in addition to the active substance. The pharmaceutical composition of the present invention also contains stabilizers, preservatives, buffers, antioxidants or other additives known to those skilled in the art.

[0311] The dosage of the antibody administered obviously depends on many factors well known in the art, such as the chemical nature of the antibody and the pharmaceutical formulation, and the patient's weight, body surface, age and gender, as well as the time and route of administration. For adults, the dosage can be exemplarily 0.001 μg to 1 g per day, preferably 0.1 μg to 100 mg per day, more preferably 1 μg to 100 mg per day, and even more preferably 5 μg to 10 mg per day. In continuous infusion, the dosage is exemplarily 0.01 μg to 100 mg per kilogram of body weight per minute, preferably 1 μg to 10 mg.

[0312] In another aspect of the invention, an antibody according to the invention as described herein is provided for use in the treatment of a B cell-mediated or plasma cell-mediated disease or antibody-mediated disease or disorder selected from the group consisting of: multiple myeloma (MM), chronic lymphocytic leukemia (CLL), non-secretory multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), primary macroglobulinemia (Waldenstrom's Macroglobulinemia), plasma cell leukemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, type I and II cryoglobulinemia, light chain deposition disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders, and Epidermolysis bullosa acquisita; or any non-Hodgkin lymphoma B cell leukemia or Hodgkin lymphoma (HL) with BCMA expression, or any disease in which the patient develops neutralizing antibodies to a recombinant protein replacement therapy, wherein the method comprises the step of administering to the patient a therapeutically effective amount of an antibody described herein.

[0313] B cell disorders can be divided into defects in B cell development / immunoglobulin production (immunodeficiency) and excessive / uncontrolled proliferation (lymphoma, leukemia). As used herein, B cell disorders refer to both types of diseases, and methods of treating B cell disorders with antibodies are provided.

[0314] In one aspect of the invention, the disease is multiple myeloma

[0315] Also provided is the use of an antibody as described herein in the manufacture of a medicament for treating a disease or disorder as described herein.

[0316] For example, in one aspect of the invention, there is provided use of an antibody as described herein for treating or preventing diseases and disorders that respond to modulation (such as inhibition or blocking) of the interaction between BCMA and the ligands BAFF and APRIL.

[0317] In one embodiment of the invention, the isolated antibody or antibody fragment is used to treat a B lymphocyte cancer, such as Hodgkin lymphoma.

[0318] In one embodiment of the invention, the isolated antibody or antibody fragment is used to treat an autoimmune disease, such as a medical disorder associated with inflammation, preferably an autoimmune disease with an inflammatory component, wherein the autoimmune disease is selected from the group consisting of Takayasu arteritis, giant cell arteritis, familial Mediterranean fever, Kawasaki disease, polyarteritis nodosa, cutaneous polyarteritis nodosa, hepatitis-associated arteritis, Behcet's syndrome, Wegener's granulomatosis, ANCA-vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, connective tissue disease vasculitis, Hennoch- Purpura, cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, tropical aortitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, leprosy nodular arteritis, primary vasculitis of the CNS, thromboangiitis obliterans, paraneoplastic vasculitis, urticaria, Dego's disease, myelodysplastic syndrome, erythema protuberans, hyperimmunoglobulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Crohn's disease, ulcerative colitis, autoimmune myositis, diabetes, multiple sclerosis, Guillain-Barré syndrome Syndrome), histiocytosis, osteoarthritis, atopic dermatitis, periodontitis, chronic sinusitis, psoriasis, psoriatic arthritis, microscopic colitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis, cryoglobulinemia, Sjogren's syndrome syndrome), lupus erythematosus, aplastic anemia, myelofibroma, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, idiopathic achalasia, bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, autoimmune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphocytopenia, Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atrophic thyroiditis, Graves' disase, autoimmune polyglandular syndrome, autoimmune Addison's syndrome Syndrome, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, antiphospholipid syndrome, myasthenia gravis, stiff-man syndrome, Goodpasture's syndrome, sympathetic ophthalmia, folliculitis, Sharp syndrome and / or Evans syndrome, in particular hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, preferably rheumatoid arthritis or multiple sclerosis.

[0319] sequence

[0320] Preferred antibody sequences of the present invention are:

[0321]

[0322]

[0323]

[0324]

[0325]

[0326] Preferred nucleotide sequences

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333] Preferred sequences of the present invention belonging to CD269 (BCMA):

[0334]

[0335] Preferred general amino acid sequences including humanized sequence modifications:

[0336]

[0337]

[0338] Example

[0339] The present invention is demonstrated by way of the embodiments disclosed herein. The embodiments provided herein represent only specific embodiments of the present invention and are not intended to limit the scope of the present invention. The embodiments are considered to provide further descriptions of possible and potential preferred embodiments that enhance the technical support of one or more non-limiting embodiments.

[0340] Although the examples concerning the crystallization of the antibody-epitope complex and the in vivo and in vitro antitumor effects were performed using the original chimeric antibody J22.9-xi, the inventors claim that these technical effects are maintained in the humanized variants of the present invention, since the binding characteristics are maintained in the humanized variants compared to the original chimeric antibody tested. The data from the chimeric antibodies are therefore provided as reference material and indication of the industrial applicability and usefulness of the claimed human variants. Initial biological data indicate comparable effects between J22.9-xi and the humanized variants.

[0341] Binding and blocking characteristics of the interaction between J22.9-xi and BCMA

[0342] The new chimeric antibody (J22.9-xi) binds to the extracellular domain of human CD269 (BCMA, TNFRSF17). This was initially determined by ELISA and flow cytometry based on the human multiple myeloma cell line MM.1S ( Figure 1a , Figure b). Surface plasmon resonance (SPR) was used to determine the affinity of J22.9-xi for BCMA. Figure 1c The average Kd shown was 54 pM.

[0343] It is known that BCMA triggers signals important for the survival of multiple myeloma and plasma cells in vivo through interaction with its ligands BAFF and / or APRIL (Mackay F et al. (2003) Annu Rev Immunol 21:231-264). Therefore, in vitro blocking assays were performed using the extracellular domain of human BCMA and recombinant BAFF. Binding of J22.9-xi to BCMA significantly blocked the interaction between the receptor and its ligand BAFF. Using an isotype control antibody instead of J22.9-xi, the binding of recombinant BAFF to BCMA was not affected ( Figure 1d ).

[0344] The crystal structure of the J22.9-xi-Fab-BCMA complex reveals an extensive binding interface with BCMA

[0345] The Fab fragment prepared from J22.9-xi was crystallized in complex with the purified 46-amino acid residue BCMA extracellular domain, and the complex structure was solved at a resolution of 1.9 angstroms. High-quality electron density was observed for residues 6 to 41 of BCMA, indicating extensive interactions with J22.9-xi, primarily with the light chain of the antibody ( Figure 2BThis interface, which occupies 740.4 square angstroms and involves one-third of the BCMA residues, covers 12 of the 16 residues of the same epitope observed in the crystal structure of BMCA complexes with APRIL and sTALL1 (also known as BAFF), including the conserved DxL motif (Gordon NC et al. (2003), BAFF / BlysS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site. Biochemistry 42(20):5977-83 and Patel DR et al. (2004), Engineering an APRIL-specific B Cell Maturation Antigen. JBC 279(16):16727-35), providing a clear rationale for the blocking effect seen in in vitro assays with BAFF ( Figure 2A ). Interactions with J22.9-xi additionally include direct side chain contacts with Ala20 and Pro23 in BCMA, residues that are not part of the binding epitope covered by BAFF and APRIL, and several water-mediated hydrogen bonds. The overall conformation of BCMA is very similar in all three structures, with a C-α rmsd of 1.4 Å between the J22.9-xi and APRIL complex and 1.5 Å between the J22.9-xi and sTALL1 complex; the respective C-α rmsds of the J22.9-xi BCMA binding epitope (residues 13 to 30) are 0.98 and 0.88 Å. Although it is recognized that the same BCMA epitope has a DxL motif at its core, the binding site of J22.9-xi is very different from that of sTALL1 and APRIL, as is the binding of interactions that include the interface.

[0346] If you can Figure 2B As seen in Tables 1 and 2, 19 amino acids from J22.9-xi (6 from the heavy chain (Table 1) and 13 from the light chain (Table 2)) form a direct connection with 12 residues from the extracellular domain of CD269.

[0347] Table 1: List of amino acid interactions between the heavy chain of J22.9-xi and BCMA. These lists of interactions were generated using the software PDBsum (Laskowski RA (2009)).

[0348]

[0349] Table 2: List of amino acid interactions between the light chain of J22.9-xi and BCMA. These interaction lists were generated using the software PDBsum (Laskowski RA (2009)).

[0350]

[0351] Table 3: Interaction list of residues involved in CD269:APRIL and CD269:BAFF binding (residues not directly contacted by J22.9 are Underlined These interaction lists were generated using the software PDBsum (Laskowski RA (2009)).

[0352]

[0353]

[0354]

[0355] Table 4: Residues of the CD269 target bound by direct contacts of J22.9, APRIL and / or BAFF. The residues of the CD269 target directly contacted only by J22.9 are Underlined (20, 23). Residues of the CD269 target that are not directly contacted by J22.9 are in bold (30, 31, 33, 34, 35 for APRIL; 25, 29, 30, 31, 34, 35 for BAFF).

[0356]

[0357] Table 5: J22.9 water interactions (J22.9-xi:H2O:CD269). The data in Table 5 were generated using the software LigPlot (Wallace and Laskowski, European Bioinformatics Institute).

[0358] (sc = side chain H bond; mc = main chain H bond)

[0359]

[0360] The potent cytotoxic efficacy of J22.9-xi is strongly reduced after deglycosylation

[0361] A luciferase-based cytotoxicity assay was established using the MM.1S-Luc cell line transduced with luciferase. In this assay, bioluminescence was detected only from live cells because the luciferase released by dead cells due to the lack of ATP in the culture medium could not function. PBMCs from healthy donors were isolated and mixed with MM.1S-Luc cells at a ratio of 20:1. Bioluminescence was measured after 4 hours.

[0362] The in vitro cytotoxicity of J22.9-xi was determined in PBMC preparations from 4 unstimulated donors. The cytotoxic potential varied slightly between PBMCs from different donors. Within 4 hours of incubation, cell lysis reached 18 to 35% at a J22.9-xi concentration of 125 ng / ml. Increasing the J22.9-xi concentration to 1 μg / ml increased cell lysis by up to 56% ( Figure 3a ).

[0363] After deglycosylation of J22.9-xi with PNGase F (J22.9-xi-N-glycan), the cytotoxic activity dropped to less than 8%, while the binding of J22.9-xi-N-glycan to BCMA-positive MM.1S cells remained unchanged ( Figure 3a , Figure 3b ).

[0364] J22.9-xi reduces tumor burden and prolongs survival in xenograft mice

[0365] We used NOD scid common gamma chain knockout (NSG) mice lacking functional B, T, and NK cell populations. These were cultured with 1 × 10 7 Mice injected intravenously with MM.1S-Luc cells developed hind limb paralysis within 6 weeks ( Figure 4d-1 ). The date of first symptom onset defined the date of sacrifice.

[0366] Inject 1 × 10 7 After MM.1S-Luc, mice were randomly divided into 3 groups. The first group (n=2) did not receive treatment until the end of the experiment, while the second group (n=5) and the third group (n=6) received injections of 200μg isotype control or J22.9-xi antibody twice a week, respectively. The antibody was administered intraperitoneally (ip) for 6 weeks from the date of tumor cell injection. Tumor growth was monitored using IVIS spectrum once a week. Bioluminescence was measured 3 minutes after intraperitoneal injection of luciferin.

[0367] A similar course of tumor development was found in both the untreated group and the group receiving the control antibody, whereas the group treated with J22.9-xi showed a significantly lower tumor burden starting from the first measurement point on day 6 ( Figure 4aIn addition, this group showed a smaller overall tumor burden during the entire monitoring period ( Figure 4b ). Isotype control treated animals had a median survival of 46 days after cell injection. Mice receiving J22.9-xi survived an average of 26 days longer. This corresponds to a 55% increased survival rate ( Figure 4c ). On day 28 after cell injection, extensive infiltration of tumor cells into the spinal and inguinal lymph nodes was seen in untreated mice and mice receiving isotype control antibodies ( Figure 4d-2 ).

[0368] 200 μg of antibody was administered to mice, corresponding to approximately 10 mg / kg body weight. To test the efficacy of J22.9-xi at lower doses, we divided MM.1S-Luc xenografted mice into 4 groups. The first group (n=7) received 200 μg of control antibody twice a week, and the second, third (n=3 each) and fourth groups (n=9) were injected with 2 μg, 20 μg or 200 μg twice a week, respectively. Injections and monitoring were performed as described above.

[0369] Although tumors developed as expected in the control group mice, a significant restriction of tumor growth was observed in the groups receiving 20 μg or 200 μg of J22.9-xi ( Figure 4e , Figure 4f ).exist Figure 4g An overview of the experimental timeline is provided in .

[0370] Growth of established tumors was arrested for 5 weeks during J22.9-xi treatment

[0371] Therapeutic administration was mimicked by delaying the start of antibody treatment until 5 days after tumor cell injection. Xenograft mice were divided into two groups (n=6). Animals received 200 μg of isotype control or J22.9-xi antibody twice a week. The first treatment was completed on day 8 after cell injection. Although there was no measurable tumor-derived bioluminescence in the group receiving J22.9-xi (n=5) by day 35, a steady increase in tumor load was observed in animals receiving isotype control antibodies (n=6). Figure 5a , Figure 5b Mice from the isotype control group survived an average of 56 days after cell injection, whereas all mice that received J22.9-xi were still alive at day 77 ( Figure 5c ).exist Figure 5d An overview of the experimental timeline is provided in .

[0372] Enhanced early treatment with J22.9-xi prevented tumor growth for 7 weeks

[0373] To further evaluate the effect of treatment timing on tumor growth, different antibodies were administered over 5 consecutive days starting from the day of tumor cell injection. After intravenous injection of cells, animals were randomly divided into 5 groups. The first group (n=5) was treated with 200μg isotype control antibody / injection (intraperitoneal injection), while the second group (n=6) received 200μg / injection of J22.9-xi-N= polysaccharide antibody. Mice in the third group (n=4), the fourth group (n=5) and the fifth group (n=5) received 200μg, 20μg and 2μg / injection of J22.9-xi antibody, respectively. Bioluminescence measurements began on the 9th day after cell injection. By day 44, no bioluminescence from tumors was seen in any group that received the complete J22.9-xi antibody. Although tumor growth was slowed in animals treated with J22.9-xi-N-polysaccharides, the overall tumor load was not significantly different from those animals that received the isotype control antibody ( Figure 6a , Figure 6b Although the overall tumor burden in animals treated with J22.9-xi-N-glycan (deglycosylated) was not significantly different ( Figure 6b ), but the lifespan of these mice was significantly increased compared to the isoAb-treated group ( Figure 6c ). Because J22.9-xi-N-glycans were shown to be unable to induce ADCC or CDC, this result suggests that the binding of J22.9-xi alone to BCMA impedes tumor growth. It is reasonable to assume that this is due to blocking the interaction between the receptor and its natural ligands (APRIL and BAFF). Figure 6d An overview of the experimental timeline is provided in .

[0374] Humanization of J22.9-xi

[0375] The J22.9-xi antibody was humanized based on sequence alignment and data obtained from the crystal structure. The variable region sequences were aligned to their respective human homologs using IgBLAST (NCBI) or Clustal (EBI). Each proposed mutation was evaluated by visual inspection of the structure before alteration.

[0376] Binding of humanized variants to the BCMA target

[0377] The mutants were tested for binding to BCMA using flow cytometry, ELISA, and SPR. TMXPR36; Bio-Rad) measures the affinity of the humanized antibodies. The binding data show surprising results about the specificity and affinity of the humanized antibody variants to the same epitope as tested J22.9-xi. As shown in the table below, it is completely surprising that the humanized antibodies described herein exhibit comparable binding characteristics to the original chimeric antibodies. The SPR data reveal that the affinity of the humanized variants is similar to that of the chimera, and is sufficient to assume their clinical relevance based on the data of the original chimeric antibodies provided herein. The technician would not expect to maintain the binding characteristics to such an extent by modifying the CDRs during the humanization of the chimera.

[0378] ELISA was performed as described herein using BCMA-coated microtiter plates (1 μg / ml). Fig.10 As observed in Figure 3, all humanized variants had comparable binding to human and macaque BCMA compared with J22.9-xi.

[0379] Flow cytometry was also performed using the humanized variants described herein and showed equivalent binding of all humanized variants to both human and macaque BCMA (ref. Fig.11 )

[0380] SPR analysis was also performed and the affinity of the humanized antibody variants was measured. As can be observed in the table below (Table 6), the affinity of the humanized variants (J22.9-H corresponds to the humanized sequence SEQ ID No. 27; J22.9-FSY corresponds to the humanized and PTM-modified SEQ ID No. 28; J22.9-ISY corresponds to the humanized and PTM-modified SEQ ID No. 29).

[0381] Table 6. SPR data

[0382]

[0383] Amino acid 54 in CDR2 of J22.9 heavy chain:

[0384] To remove a potential post-translational modification site in humanized J22.9, residue D54 of the heavy chain CDR2 was mutated to asparagine (N), inadvertently creating a new potential modification site for N-linked glycosylation. The heavy chain containing the mutation at N54 migrated slower on SDS gels ( Fig.13 ), indicating a larger size, and that the CDRs are glycosylated.

[0385] However, the corresponding IgG, J22.9-FNY, binds BCMA in FACS and ELISA, and crystallizes in complex with BCMA. Although not completely accurate, the 2.7 angstrom resolution structure shows clear electron density extending from the N54 side chain - consistent with sugar modification of the residue. Surprisingly, such a large extension of the side chain does not disrupt binding to BCMA, and from these observations it can be expected that multiple and different amino acid substitutions can be tolerated at this position, potentially also derivatizations other than sugars.

[0386] method

[0387] Cell lines and culture

[0388] The human multiple myeloma cell line MM.1S (Greenstein et al. (2003) Exp Hematol 31:271-282) was obtained from Prof.B. (MDC, Berlin, Germany). To monitor tumor cell growth in vivo, luciferase and GFP were cloned into the pFU vector of the lentiviral vector system ViraPower (Invitrogen). Monoclonal cell lines were isolated by fluorescence-activated single cell sorting via GFP expression in transduced cells. Cell lines (all from PAA) were cultured in RPMI-1640 without phenol red, containing 10% fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin.

[0389] HEK293-6E cells purchased from the National Research Council of Canada were maintained in Freestyle F17 medium (Invitrogen) supplemented with 7.5 mM L-glutamine (PAA), 0.1% Pluronic F-68 (Invitrogen) and 25 μg / ml G418 (Invitrogen). Cells were grown in conical flasks (Corning) at 110 rpm and 37° C. in a 5% CO2 atmosphere.

[0390] Antibody production and purification

[0391] To obtain antibodies that bind to BCMA, standard hybridoma technology was used. Four BL / 6 wild-type mice were immunized six times with incomplete Freund's adjuvant and 30 μg of the extracellular domain of human BCMA fused to glutathione S-transferase (GST) at the C-terminus. After cell fusion, the following screening period, J22.9 hybridomas were shown to secrete anti-BCMA antibodies.

[0392] Due to the instability of hybridomas, the variable regions of the light and heavy chains of hybridoma J22.9 were amplified and cloned upstream of the human κ or IgG1 constant domain genes. The chimeric J22.9-xi antibody was produced by transiently co-transfecting 293-6E cells with a 1:2 mixture of DNA plasmids encoding the light and heavy chains, respectively. Briefly, 293-6E cells were resuspended in serum-free Freestyle F17 medium at 1.7 × 10 6 Cells / ml, and polyethyleneimine was used to transfect with a final concentration of 1 μg / ml culture medium. On the 2nd day after transfection, Freestyle F17 containing 100% transfection volume of 1% tryptone N1 (Organo Technie) was supplied to the cells. On the 7th day, the cells were harvested by centrifugation, and the filtered (0.45 μm) culture medium was passed through a 3.5 ml protein A agarose column (Bio-Rad). The column was washed with 10 ml phosphate buffered saline (PBS), and the antibody was eluted by adding 20 mM sodium acetate, 150 mM NaCl at pH 3.5. 2 ml of fractions were directly collected in a tube containing 100 μl 1M HEPES at pH 7.5 to neutralize. The final yield of full-length IgG is close to 40 mg / l culture medium

[0393] Because hybridoma J22.9 lost the ability to produce / secrete anti-BCMA antibodies ( Figure 8 ), therefore the variable regions of the heavy and light chains were amplified using PCR and then cloned at the 5' end of the human constant IgG1 and kappa light chain genes, respectively. The chimeric J22.9-xi antibody was produced by co-transfecting 293-6E cells with these two plasmids. The production of the antibodies of the present invention is therefore inherently difficult and cannot be achieved by straightforward conventional methods.

[0394] An isotype control antibody consisting of the J22.9-xi heavy chain and a random chimeric kappa light chain was generated in parallel with J22.9-xi. This antibody was shown to be unable to bind to BCMA by ELISA and flow cytometry.

[0395] N-linked oligosaccharide chains at Asn297 of the J22.9-xi heavy chain were enzymatically removed using N-glycosidase F (PNGase F) (NEB). 10 mg of J22.9-xi was incubated with 15,000 units of PNGase F in 500 μl PBS (pH 7.4) at 37°C for 36 hours, then the buffer was exchanged to sterile PBS.

[0396] Binding and blocking abilities of J22.9-xi determined by enzyme-linked immunosorbent assay (ELISA)

[0397] Microtiter plates were coated with the extracellular domain of human BCMA at 10 μg / ml. The coated BCMA was detected with serially diluted J22.9-xi and isotype controls ranging from 1 to 1000 ng. Binding of J22.9-xi or isotype control antibodies to coated BCMA was detected with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibodies (Jackson ImmunoResearch, 109-035-098, 1:5,000 dilution).

[0398] Microtiter plates were coated with 1 μg / ml of the extracellular domain of human or macaque BCMA (hBCMA or cyBCMA, respectively). The coated BCMA was detected with serial dilutions of J22.9-xi, J22.9-H, J22.9-ISY, and J22.9-FSY ranging from 0.26 pM to 500 nM. Binding of antibodies to coated BCMA was detected with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibodies (Jackson ImmunoResearch, 109-035-098, 1:5,000 dilution).

[0399] For blocking experiments, 1 mg / ml human recombinant BAFF fused to a His tag (Biomol) was applied after antibody and washing, and detected using mouse anti-His tag (AbD Serotec, AD1.1.10, 1:5,000 dilution, HRP conjugated) antibody. All ELISAs were developed using BD OptEIA reagents A and B (BD Bioscience) and measured at 450 nm and 570 nm using a microplate spectrophotometer (BioTek).

[0400] Flow cytometric analysis

[0401] For cell surface antigen detection experiments, homemade antibodies (J22.9-xi, J22.9-H, J22.9-ISY, J22.9-FSY and isotype control) and commercially available mouse anti-His tag (AbD Serotec, AD1.1.10, 1:100 dilution, Alexa Fluor 488 conjugated) and goat anti-human IgG1 (Jackson ImmunoResearch, 109-116-098, 1:400, PE conjugated) antibodies and human recombinant BAFF fused to His tag (Biomol) were used. Experiments were performed on FACSCalibur or FACSCanto II flow cytometers (BD Bioscience). Data were analyzed using Flowjo software version 7.6 (TreeStar Inc.).

[0402] Generation of Fab and Fab:BCMA complexes

[0403] (Fab) 2 fragments were generated from full-length J22.9-xi IgG by incubation with pepsin. J22.9-xi was passed through a PD-10 buffer exchange column into 50mM sodium acetate at pH 3.5, and pepsin was added at 30μg per mg of J22.9-xi. Incubation for 2.5 hours at 37°C was sufficient to completely digest the crystallizable fragment (Fc) region, and inactivated by exchanging pepsin to PBS (pH 7.2) on a PD-10 column. (Fab) 2 fragments were reduced to individual Fabs by adding 2-mercaptoethylamine (50mM) in the presence of 5mM ethylenediaminetetraacetic acid (EDTA). Incubated at 37°C for 90 minutes, reduced cysteine ​​was blocked by alkylation with 500μM iodoacetamide for 30 minutes, and then buffer exchanged to fresh PBS. The Fab fragments were combined with 1.5 molar equivalents of purified BCMA, and the complex was separated by size exclusion chromatography on a Superdex 75 16 / 60 column. Fractions were analyzed on 4 to 12% SDS polyacrylamide gels and fractions containing both Fab and BCMA were pooled and concentrated for crystallization experiments.

[0404] Crystallization of Fab:BCMA complex

[0405] 0.5 molar equivalents of pure BCMA were added to the concentrated complex to ensure saturation, and the concentrated complex was subjected to crystallization screening. Initial Fab:BCMA crystallization conditions were identified in 96-well sitting drop plates using a Gryphon pipetting robot (200nl drop) by commercial screening (Qiagen), and optimized with hanging drops (2-3 μl) in 24-well plates. The complex was concentrated to 8 mg / ml and crystallized at 20°C in 21% PEG 3350, 0.1M BisTris pH 6.5 and 5mM CuCl2. After 3 days, the crystals appeared as clusters of thin disks, and their final size (0.2-0.3 mm) was obtained in nearly 7 days. The clusters were separated and the individual disks were rapidly frozen with liquid nitrogen in a mother liquor with 20% glycerol as a protective agent. Complete diffraction data were collected from a single crystal at the BESSY synchrotron at the Helmholtz Zentrum Berlin. The structure was solved by molecular replacement at a resolution of 1.9 angstroms using the experimental phase of the structure from efalizumab (3EO9) as a search model. Data processing was performed using the ccp4 suite of programs, structure refinement was performed using Phenix (Adams PD, et al. (2010), Acta Cryst. D 66: 213-221), and modeling and evaluation was performed using Coot. (Emsley et al., Acta Crystallographica Section D-Biological Crystallography, 2010, 66: 486-501). The structure was analyzed using PyMOL (PyMOL Molecular Imaging System, Version 1.5.0.4 LLC) imaging.

[0406] In vitro cytotoxicity assay

[0407] In this assay, the cytotoxic effect of J22.9-xi was determined by measuring the luminescence of the remaining viable cells in a bioluminescence reader. Briefly: Freshly obtained human filter buffy coat (FBC) was backwashed by gravity with 160 ml elution buffer (PBS (pH 7.4) containing 5 mM Na2-EDTA and 2.5 [w / v] sucrose). Monocytes were separated from the eluted cells by Ficoll gradient centrifugation. Monocytes were taken from the interphase and washed twice with elution buffer. After erythrocyte lysis, PBMCs were washed again, counted and adjusted to 1×10 by dilution with RPMI / 10% FCS w / o phenol red. 7 5×10 cells / ml. 50 μl RPMI 4MM.1S-Luc cells were plated in microtiter plates. 10 minutes before adding 100 μl PBMC, MM.1S-Luc cells were incubated with 200 μl sample volumes of J22.9-xi or isotype control antibody serial dilutions. After adding target cells, antibodies and effector cells, the microtiter plates were centrifuged (300 × g) for 2 minutes at room temperature (RT) and stored at 37 ° C, 5% CO2. The control wells were treated with 1% Triton X instead of antibody to completely lyse. After incubation for 4 hours, 25 μl PBS containing fluorescein (250 ng / ml) was applied to each well, and the bioluminescence of live cells was measured in a bioluminescence reader (Tecan). Specific cytotoxicity was calculated according to the following formula:

[0408] 100−[value(J22.9-xi)−value(total lysis)] / [value(isotype control)−value(total lysis)]×100.

[0409] In vivo studies

[0410] NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CSF2)2Ygy Tg(IL3)1Ygy Tg(KITLG)3YgyJGckRolyJ mice (NSG) from Jackson Laboratory and CB17.Cg-Prkdcscid Lystbg / Crl mice from Charles River Germany (Sulzfeld, Germany) were used. Experiments were performed with mice aged 8-14 weeks. All animal studies were performed under specific pathogen-free conditions according to institutional and national guidelines. In experimental cases involving treatment of established tumors and tumor therapy in the early stages of the disease, CB17.Cg-Prkdcscid Lystbg / Crl mice were used. The phenotypes of the two mouse strains mentioned herein are very similar. These animals do not have functional B cells, T cells, and NK cells. Slightly slower tumor growth was observed in CB17.Cg mice, indicating an even more promising effect of the therapeutic antibodies of the present invention.

[0411] On day 0, 1 × 10 7 MM.1S-Luc cells induce a xenograft model of multiple myeloma. In this model, untreated animals develop hind limb paralysis within 6 weeks. The occurrence of such symptoms indicates the end point of the experiment.

[0412] For effectiveness studies, antibodies were administered intraperitoneally (ip) twice a week or for 5 consecutive days starting from day 0. J22.9-xi antibodies were provided at doses of 2 μg, 20 μg, or 200 μg per injection; for isotype control antibodies, 200 μg / injection was used. After intraperitoneal injection of 150 μg of luciferin, the bioluminescence of MM.1S-Luc cells was measured using the IVIS spectrum (Caliper Life Sciences). Measurements were performed once a week. At each time point, 3 untreated mice were also administered luciferin. The total flow values ​​of these animals were subtracted from each measurement or displayed in the figure.

[0413] For treatment of established tumors, antibody treatment was initiated 5 days after injection of MM.1S-Luc cells. 200 μg of J22.9-xi or isotype control antibody was administered twice weekly over a period of 6 weeks.

[0414] Humanization of J22.9-xi

[0415] The variable region sequences of the heavy and light chains (mouse) were aligned with human sequences from the corresponding heavy and light chain subtypes to determine the residue changes required to generate fully humanized sequence variants. Using the crystal structure of the J22.9-xi:hBCMA complex, each modification was first evaluated in silico to identify mutations that could potentially disrupt the binding of the antibody to BCMA. Two complete J22.9 variable region genes for each chain were synthesized, one with the original mouse sequence and one with the fully humanized sequence (i.e., containing all necessary humanized mutations), each with two added restriction enzyme sites to divide the gene into three boxes. After marking potential problem mutations, different combinations of the original mouse and fully humanized gene boxes were generated, and their corresponding IgGs were expressed and purified, and FACS analysis was performed with BCMA-positive cells to evaluate binding. The marked problem residues were mutated individually using PCR to demonstrate their effect on affinity for BCMA, and the binding of the final optimized construct to both human and macaque BCMA was quantitatively evaluated by SPR.

[0416] SPR

[0417] SPR was performed on Proteon XPR36 using phosphate buffered saline (PBST) supplemented with 0.005% Tween 20. Whole IgG at a concentration of 15 μg / ml was immobilized on a Proteon GLH sensor chip using standard amine chemistry according to the manufacturer's instructions. For binding experiments, human or macaque BCMA in PBST was used as the mobile phase. The association constant (k) was determined in parallel from multiple concentrations of BCMA (0.4 to 800 nM for hBCMA and 2.7 to 1 μM for cynoBCMA). on ) and dissociation constant (koff ) to calculate the binding affinity (K d ), assuming a single-site binding model.

[0418] Furthermore, further experiments revealed that preferred embodiments of the present invention provide surprising and unexpected effects, thereby solving the problems of the present invention in non-obvious ways.

[0419] References

[0420] Adams, PD, Afonine, PV, Bunkoczi, G., Chen, VB, Davis, IW, Echols, N., Headd, JJ, Hung, LW, Kapral, GJ, Grosse-Kunstleve, RW, et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221.

[0421] Al-Lazikani, B., Lesk, AM Chothia, C. (1997). Standard conformations for the canonical structures of immunoglobulins. J Mol Biol 273, 927-948.

[0422] Anthony, RM Ravetch, JV (2010). A novel role for the IgG Fc glycan: the anti-inflammatory activity of sialylated IgG Fcs. J Clin Immunol 30 Suppl 1, S9-14.

[0423] Chan et al. (2010) Nat Rev Immunol 10:301–316.

[0424] Chavez-Galan, L., Arenas-Del Angel, M.C., Zenteno, E., Chavez, R. Lascurain, R. (2009). Cell death mechanisms induced by cytotoxic lymphocytes. Cell Mol Immunol 6, 15-25.

[0425] Chiu, A., Xu, W., He, B., Dillon, SR, Gross, JA, Sievers, E., Qiao, X., Santini, P., Hyjek, E., Lee, JW, et al. (2007). Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL. Blood 109, 729-739.

[0426] Gordon, NC, Pan, B., Hymowitz, SG, Yin, J., Kelley, RF, Cochran, AG, Yan, M., Dixit, VM, Fairbrother, WJ Starovasnik, MA (2003). BAFF / BLyS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site. Biochemistry 42, 5977-5983.

[0427] Greenstein, S., Krett, NL, Kurosawa, Y., Ma, C., Chauhan, D., Hideshima, T. and Eerson, K.C. Rosen, ST. (2003). Characterization of the MM.1 human multiple myeloma (MM) cell lines: a model system to elucidate the characteristics, behavior, and signaling of steroid-sensitive and -resistant MM cells. Exp Hematol 31, 271-282.

[0428] Jacobi, A. M., Huang, W., Wang, T., Freimuth, W., Sanz, I., Furie, R., Mackay, M., Aranow, C., Diamond, B. Davidson, A. (2010). Effect of long-term belimumab treatment on B cells in systemic lupus erythematosus: extension of a phase II, double-blind, placebo-controlled, dose-ranging study. Arthritis Rheum 62, 201-210.

[0429] Kapoor, P., Ramakrishnan, V. and Rajkumar, SV (2012). Bortezomib combination therapy in multiple myeloma. Semin Hematol 49, 228-242.

[0430] Keyser, FD (2011). Choice of Biologic Therapy for Patients with Rheumatoid Arthritis: The Infection Perspective. Curr Rheumatol Rev 7, 77-87.

[0431] Laskowski, RA (2009). PDBsum new things. Nucleic Acids Res 37, D355-359.

[0432] Mackay, F., Schneider, P., Rennert, P. Browning, J. (2003). BAFF AND APRIL: a tutorial on B cell survival. Annu Rev Immunol 21, 231-264.

[0433] Nimmerjahn, F. Ravetch, JV (2008). Fc gamma receptors as regulators of immune responses. Nat Rev Immunol 8, 34-47.

[0434] Novak, AJ, Darce, JR, Arendt, BK, Harder, B., Henderson, K., Kindsvogel, W., Gross, JA, Greipp, PR Jelinek, DF (2004). Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival. Blood 103, 689-694.

[0435] Queen et al., 1989; WO 90 / 07861.

[0436] Patel, DR, Wallweber, HJ, Yin, J., Shriver, SK, Marsters, SA, Gordon, NC, Starovasnik, MA Kelley, RF (2004). Engineering an APRIL-specific B cell maturation antigen. J Biol Chem 279, 16727-16735.

[0437] Presta, LG (2008). Molecular engineering and design of therapeutic antibodies. Curr Opin Immunol 20, 460-470.

[0438] Raab, MS, Podar, K., Breitkreutz, I., Richardson, PG Anderson, KC (2009). Multiple myeloma. Lancet 374, 324-339.

[0439] Richardson et al. (2003) New Engl J Med 348:2609-2617.

[0440] Ryan et al. (Molecular Cancer Therapeutics, 2007 6(11), 3009).

[0441] Roopenian, DC Akilesh, S. (2007). FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol 7, 715-725.

[0442] Suzuki, K. (2013). Current therapeutic strategy for multiple myeloma. Jpn J Clin Oncol 43, 116-124.

[0443] Thorpe et al., 1982, Immunol. Rev. 62: 119-58.

[0444] Wang, SY and Weiner, G. (2008). Complement and cellular cytotoxicity in antibody therapy of cancer. Expert Opin Biol Ther 8, 759-768.

[0445] Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584. Sequence Listing <110> Max-Debruk-Helmholtz Association for Molecular Medicine <120> Anti-CD269 antibody-drug conjugate (ADC), anti-CD269 antibody and use thereof <130> XII 2136 / 14WO <160> 43 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ile Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 2 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (34)..(34) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (54)..(55) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (101)..(101) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (107)..(107) <223> Xaa can be any naturally occurring amino acid <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Xaa Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Xaa Xaa Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Xaa Asp Tyr Gly Asp Ala Xaa Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (33)..(33) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (35)..(35) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (47)..(47) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (50)..(50) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (99)..(101) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (106)..(106) <223> Xaa can be any naturally occurring amino acid <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Xaa Met Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Xaa Val 35 40 45 Gly Xaa Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Xaa Xaa Xaa Asp Tyr Gly Asp Xaa Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Asn Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 7 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Phe Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Asn Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Ser Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Phe Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Ser Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 10 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Val Gly 1 5 10 15 Asp Glu Val Thr Leu Thr Cys Lys Ala Ser Gln Ser Val Asp Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Asp Asp Leu Arg Phe Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 11 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 11 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Leu Arg Phe Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 12 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (30)..(31) <223> Xaa can be any naturally occurring amino acid <400> 12 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Xaa Xaa Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Leu Arg Phe Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 13 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (31)..(32) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (34)..(34) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (36)..(36) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (49)..(50) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (52)..(53) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (55)..(55) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (66)..(67) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (89)..(89) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (91)..(91) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (94)..(94) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (96)..(96) <223> Xaa can be any naturally occurring amino acid <400> 13 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Xaa Xaa 20 25 30 Val Xaa Trp Xaa Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile 35 40 45 Xaa Xaa Ala Xaa Xaa Arg Xaa Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Xaa Xaa Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Xaa Gln Xaa Asn Asn Xaa Pro Xaa 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 14 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 14 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Glu Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Leu Arg Phe Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (4) <223> Xaa can be any naturally occurring amino acid <400> 15 Arg Tyr Trp Xaa Ser 1 5 <210> 16 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any naturally occurring amino acid <400> 16 Glu Ile Asn Pro Xaa Xaa Ser Thr Ile Asn Tyr Ala Pro Ser Leu Lys 1 5 10 15 Asp Lys <210> 17 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (4) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (10)..(10) <223> Xaa can be any naturally occurring amino acid <400> 17 Ser Leu Tyr Xaa Asp Tyr Gly Asp Ala Xaa Asp Tyr Trp 1 5 10 <210> 18 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 18 Arg Tyr Trp Ile Ser 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 19 Arg Tyr Trp Phe Ser 1 5 <210> 20 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 20 Glu Ile Asn Pro Asn Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu Lys 1 5 10 15 Asp Lys <210> twenty one <211> 18 <212> PRT <213> Artificial sequence <220> <223> Ab <400> twenty one Glu Ile Asn Pro Ser Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu Lys 1 5 10 15 Asp Lys <210> twenty two <211> 13 <212> PRT <213> Artificial sequence <220> <223> Ab <400> twenty two Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp 1 5 10 <210> twenty three <211> 11 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (7)..(8) <223> Xaa can be any naturally occurring amino acid <400> twenty three Lys Ala Ser Gln Ser Val Xaa Xaa Asn Val Ala 1 5 10 <210> twenty four <211> 7 <212> PRT <213> Artificial sequence <220> <223> Ab <400> twenty four Ser Ala Ser Leu Arg Phe Ser 1 5 <210> 25 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 25 Gln Gln Tyr Asn Asn Tyr Pro Leu Thr Phe Gly 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 26 Lys Ala Ser Gln Ser Val Asp Ser Asn Val Ala 1 5 10 <210> 27 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 28 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Phe Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Ser Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 29 <211> 450 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45 Gly Glu Ile Asn Pro Ser Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Asp Lys Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 30 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 30 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Leu Arg Phe Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 31 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 31 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Glu Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Leu Arg Phe Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 32 <211> 1450 <212> DNA <213> Artificial sequence <220> <223> Ab coding <400> 32 gaattccacc atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt 60 ccacagtgaa gtgcagctgg tcgaatctgg aggaggcctg gttcagcctg gtggcagcct 120 taggctctct tgtgcagcct ctggctttac cttctcacgg tattggatga gctgggtgag 180 acaggctcca gggaaaggtc tggtgtgggt aggggagata aaccccgata gcagcacgat 240 caactatgct ccgtcactga aagacaagtt caccatttcc cgcgataatg ccaagaacac 300 tctctacttg cagatgaatt cccttcgagc cgaggataca gcggtgtact actgcgccag 360 tctgtactac gactatgggg acgcaatgga ctattgggga caaggcacac tggtgactgt 420 tagctccgcg tcgaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac 480 ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aacctgtgac 540 ggtctcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca 600 gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac 660 ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt 720 tgagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct 780 ggggggaccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg 840 gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt 900 caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca 960 gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 1020 tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac 1080 catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg 1140 ggaggagatg accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag 1200 cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc 1260 tcccgtgctg gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag 1320 caggtggcag caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca 1380 ctacacgcag aagagcctct ccctgtcccc gggtaaatga gtgcgacggc cgggcggcgg 1440 cggcggatcc 1450 <210> 33 <211> 1450 <212> DNA <213> Artificial sequence <220> <223> Ab coding <400> 33 gaattccacc atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt 60 ccacagtgaa gtgcagctgg tcgaatctgg aggaggcctg gttcagcctg gtggcagcct 120 taggctctct tgtgcagcct ctggctttac cttctcacgg tattggttca gctgggtgag 180 acaggctcca gggaaaggtc tggtgtgggt aggggagata aaccccagca gcagcacgat 240 caactatgct ccgtcactga aagacaagtt caccatttcc cgcgataatg ccaagaacac 300 tctctacttg cagatgaatt cccttcgagc cgaggataca gcggtgtact actgcgccag 360 tctgtactac gactatgggg acgcatacga ctattgggga caaggcacac tggtgactgt 420 tagctccgcg tcgaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac 480 ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aacctgtgac 540 ggtctcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca 600 gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac 660 ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt 720 tgagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct 780 ggggggaccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg 840 gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt 900 caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca 960 gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 1020 tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac 1080 catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg 1140 ggaggagatg accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag 1200 cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc 1260 tcccgtgctg gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag 1320 caggtggcag caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca 1380 ctacacgcag aagagcctct ccctgtcccc gggtaaatga gtgcgacggc cgggcggcgg 1440 cggcggatcc 1450 <210> 34 <211> 1450 <212> DNA <213> Artificial sequence <220> <223> Ab coding <400> 34 60. gaattccacc atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt ccacagtgaa gtgcagctgg tcgaatctgg aggaggcctg gttcagcctg gtggcagcct 180. taggctctct tgtgcagcct ctggctttac cttctcacgg tattggatca gctgggtgag acaggctcca gggaaaggtc tggtgtgggt aggggagata aaccccagca gcagcacgat 240 caactatgct ccgtcactga aagacaagtt caccattcc cgcgataatg ccaagaacac tctctacttg cagatgaatt cccttcgagc cgaggataca gcggtgtact actgcgccag 360 420. tctgtactac gactatgggg acgcatacga ctattgggga caaggcacac tggtgactgt tagctccgcg tcgaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac 480 ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aacctgtgac 540 ggtctcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca 600 gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac 660 ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acagagagt 720 tgagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccacc ctgaactcct 780 ggggggaccg tcagcttcc tctcccccc aaaacccaag vakaccctca tgatctcccg 840 gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt 900 caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca 960 gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 1020 tggcaggag tacaagtgca aggctccaa caaagccctc ccagccccca tcgagaaaac 1080 catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc cccatcccg 1140 ggaggagatg accagaacc aggtcagcct gacctgcctg gtcaaggct tctatcccag 1200 cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc 1260 tcccgtgctg gactccgacg gctcctttt cctctatagc aagctcaccg tggacagag 1320 caggtggcag cagggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca 1380 ctacacgcag aagagcctct ccctgtcccc gggtaaatga gtgcgacggc cgggcggcgg 1440 cggcggatcc 1450 <210> 35 <211> 718 <212> DNA <213> Artificial sequence <220> <223> Ab <400> 35 gaattccacc atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt 60 acactccgag atcgtgatga cccagtctcc tgctaccctg agcgtttctc ccggtgaaag 120 ggccacactc agctgcaaag cctctcaaag cgtggacagc aatgtcgcct ggtatcagca 180 gaaacctggc caagctccga gagcactgat ctattccgcg tcattgcgct tttccggcat 240 accagcacgg tttagtggct cagggagtgg gactgagttc actctgacga ttagctccct 300 tcagtcagag gatttcgccg tgtactactg tcagcagtac aacaactatc ccctcacatt 360 cggagctgga accaagctgg aactgaagcg tacggtggct gcaccatctg tcttcatctt 420 cccgccatct gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa 480 cttctatccc agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa 540 ctcccaggag agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac 600 cctgacgctg agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca 660 tcagggcctg agctcgcccg tcacaaagag cttcaacagg ggagagtgtt agggatcc 718 <210> 36 <211> 718 <212> DNA <213> Artificial sequence <220> <223> AB <400> 36 gaattccacc atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt 60 acactccgag atcgtgatga cccagtctcc tgctaccctg agcgtttctc ccggtgaaag 120 ggccacactc agctgcaaag cctctcaaag cgtggagagc aatgtcgcct ggtatcagca 180 gaaacctggc caagctccga gagcactgat ctattccgcg tcattgcgct tttccggcat 240 accagcacgg tttagtggct cagggagtgg gactgagttc actctgacga ttagctccct 300 tcagtcagag gatttcgccg tgtactactg tcagcagtac aacaactatc ccctcacatt 360 cggagctgga accaagctgg aactgaagcg tacggtggct gcaccatctg tcttcatctt 420 cccgccatct gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa 480 cttctatccc agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa 540 ctcccaggag agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac 600 cctgacgctg agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca 660 tcagggcctg agctcgcccg tcacaaagag cttcaacagg ggagagtgtt agggatcc 718 <210> 37 <211> 285 <212> PRT <213> Artificial sequence <220> <223> HIS tag <400> 37 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Three Gln Ser Met With Arg Tyr With Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Val Leu Asp With Arg Tyr Gly Val Ser Arg With Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe was synthesized by Arg and Glu synthesized by Pro Gln and Asp synthesized by Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg 210 215 220 Gly Ser Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser Leu 225 230 235 240 Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr Pro 245 250 255 Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser Val 260 265 270 Lys Gly Thr Asn Ala Leu Glu His His His His His His 275 280 285 <210> 38 <211> 53 <212> PRT <213> Artificial sequence <220> <223> Extracellular domain <400> 38 Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser Leu Leu His 1 5 10 15 Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr Pro Pro Leu 20 25 30 Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser Val Lys Gly 35 40 45 Thr Asn Ala Leu Glu 50 <210> 39 <211> 56 <212> PRT <213> Homo sapiens <400> 39 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala Leu Glu 50 55 <210> 40 <211> 20 <212> PRT <213> Homo sapiens <400> 40 Tyr Phe Asp Ser Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys 1 5 10 15 Ser Ser Asn Thr 20 <210> 41 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (1) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (19)..(19) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (27)..(28) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (30)..(31) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (39)..(39) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (48)..(48) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (66)..(67) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (69)..(69) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (84)..(86) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (88)..(88) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (93)..(93) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (115)..(115) <223> Xaa can be any naturally occurring amino acid <400> 41 Xaa Val Gln Leu Xaa Xaa Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Xaa Leu Ser Cys Ala Ala Ser Gly Xaa Xaa Phe Xaa Xaa Tyr 20 25 30 Trp Glx Ser Trp Val Arg Xaa Ala Pro Gly Lys Gly Leu Glu Trp Xaa 35 40 45 Gly Glu Ile Asn Pro Glx Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60 Lys Xaa Xaa Phe Xaa Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Xaa Xaa Xaa Arg Xaa Glu Asp Thr Ala Xaa Tyr Tyr Cys 85 90 95 Ala Ser Leu Tyr Tyr Asp Tyr Gly Asp Ala Glx Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Xaa Val Thr Val Ser Ser 115 120 <210> 42 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Ab <220> <221> misc_feature <222> (8)..(13) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (18) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (20)..(21) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (41)..(41) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (43)..(43) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (46)..(46) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (49)..(49) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (51)..(52) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (63)..(63) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (77)..(77) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (83)..(83) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (85)..(85) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (87)..(87) <223> Xaa can be any naturally occurring amino acid <400> 42 Asp Ile Val Met Thr Gln Ser Xaa Xaa Xaa Xaa Xaa Xaa Ser Val Gly 1 5 10 15 Asp Xaa Val Xaa Xaa Thr Cys Lys Ala Ser Gln Ser Val Glu Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Xaa Gln Xaa Pro Lys Xaa Leu Ile 35 40 45 Xaa Ser Xaa Xaa Leu Arg Phe Ser Gly Val Pro Ala Arg Phe Xaa Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Xaa Leu Gln Ser 65 70 75 80 Glu Asp Xaa Ala Xaa Tyr Xaa Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 43 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Ab <400> 43 Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Thr Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Ser Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Arg Gln Ser Pro Lys Ala Leu Ile 35 40 45 Phe Ser Ala Ser Leu Arg Phe Ser Gly Val Pro Ala Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Rec 100 105

Claims

1. An anti-CD269 antibody-drug conjugate for delivering a therapeutic agent to a CD269-expressing cell, comprising an antibody or an antibody fragment, wherein: The antibody or antibody fragment thereof comprises a VH domain and a VL domain, wherein the VH domain comprises: - CDR1 sequence: RYWIS (SEQ ID NO. 18) or RYWFS (SEQ ID NO. 19); - CDR2 sequence: EINPNSSTINYAPSLKDK (SEQ ID No. 20) or EINPSSSTINYAPSLKDK (SEQ ID No. 21); and -CDR3 sequence: SLYYDYGDAYDYW (SEQ ID NO.22), The VL domain comprises: - CDR1 sequence: KASQSVESNVA; - CDR2 sequence: SASLRFS (SEQ ID NO 24); and - CDR3 sequence: QQYNNYPLTFG (SEQ ID NO. 25), wherein the antibody or fragment thereof specifically binds to an epitope of the extracellular domain of CD269, and wherein the antibody-drug conjugate is conjugated to a therapeutic agent, whereby the antibody-drug conjugate comprises a linker located between the therapeutic agent and the antibody or antibody fragment.

2. The anti-CD269 antibody-drug conjugate according to claim 1, wherein The antibody or antibody fragment thereof comprises a VH domain and a VL domain, The VH domain comprises the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWX1SWVRQAPGKGLV WVGEINPX2X3STINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYC ASLYX4DYGDAX5DYWGQGTLVTVSS (SEQ ID NO.4), where X1: I or F; X2X3: SS or NS; X4: Y; and X5: Y, The VL domain comprises the following sequence: EIVMTQSPATLSVSPGERATLSCKASQSVX1X2NVAWYQQKPGQAPRAL IYSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAG TKLELKR (SEQ ID NO. 12), where X1X2: ES.

3. The anti-CD269 antibody-drug conjugate according to claim 1, wherein The antibody or antibody fragment thereof comprises a VH domain comprising a sequence according to SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.

9.

4. The anti-CD269 antibody-drug conjugate according to claim 1, wherein The antibody or antibody fragment thereof comprises a VL domain comprising a sequence according to SEQ ID NO.

14.

5. The anti-CD269 antibody-drug conjugate according to claim 1, wherein The antibody or antibody fragment thereof comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence according to SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9, and the VL domain comprises a sequence according to SEQ ID NO.

14.

6. The anti-CD269 antibody-drug conjugate according to claim 5, wherein The VH domain has at least 90% sequence identity with the sequence according to SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9, and the VL domain has at least 90% sequence identity with the sequence according to SEQ ID NO.

14.

7. The anti-CD269 antibody-drug conjugate according to any one of claims 1 to 6, wherein The linker is a peptidyl linker that is cleaved by an intracellular peptidase or protease.

8. The anti-CD269 antibody-drug conjugate according to any one of claims 1 to 6, wherein The linker is a pH sensitive cleavable linker.

9. The anti-CD269 antibody-drug conjugate according to any one of claims 1 to 6, wherein The linker is cleavable under reducing conditions.

10. The anti-CD269 antibody-drug conjugate according to any one of claims 1 to 6, wherein The therapeutic agent is selected from topotecan, morpholino doxorubicin, rhizoxin, cyanomorpholino doxorubicin, dolastatin-10, echinomycin, combretastatin, chalicheamicin, maytansine, DM-1, DM-4, ​​fusobacterin, auristatin, vinca alkaloids, podophyllotoxin, taxane, baccatin, dimethylvaline-valine-dolaisoleucine-dolaproline-phenylalanine-p-phenylenediamine, MMAF, MMAE, vincristine, vinblastine, vindesine, vinorelbine, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicine, colchicine, estramustine, cemadotin, spondylolipids or acanthosides.

11. Use of the anti-CD269 antibody-drug conjugate according to claim 1 in the preparation of a medicament for treating a medical disorder associated with the presence of pathogenic B cells, wherein: The medical disorder associated with pathogenic B cells is a cancer of the plasma cells, such as multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia or plasma cell leukemia, or a cancer of the B lymphocytes, such as Hodgkin's disease.

12. Use of the anti-CD269 antibody-drug conjugate according to claim 1 in the preparation of a medicament for treating a medical disorder associated with the presence of pathogenic B cells, wherein: The medical disorder associated with pathogenic B cells is an autoimmune disease associated with autoreactive plasma cells and / or autoreactive memory B cells, wherein the autoimmune disease associated with autoreactive plasma cells and / or autoreactive memory B cells is systemic lupus erythematosus or rheumatoid arthritis.

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