RU2787629C2 - Methods and materials for assessment of response to therapy against plasma-blasts and plasma cells - Google Patents

Abstract

FIELD: pharmaceutics; medicine.

SUBSTANCE: invention relates to the field of pharmaceutics and medicine, namely to a method for the treatment of a disease in a patient, including: a) measurement of a level of CD38-expressing cells in a patient compared to a reference individual without a disease, where CD38-expressing cells are plasma-blasts and plasmacytes; and b) administration to the patient of therapeutically effective amount of an anti-CD38 antibody, where the disease is an autoimmune disease, and, after treatment with an anti-CD20 antibody, an increased level of CD38-expressing cells was detected in the patient compared to the reference individual, and where, in a biological sample obtained in the patient after treatment with the anti-CD20 antibody, the following was detected: i) an increased level of CD38-expressing plasma-blasts and plasmacytes compared to the reference subject, when analyzing free light chains of Ig, and ii) the presence of an increased level of at least one gene increased in CD38-expressing cells compared to the reference individual.

EFFECT: use of an anti-CD38 antibody for the treatment of patients, when measuring plasma-blasts and plasma cells before the treatment of patients suffering from an autoimmune disease, which cannot reach remission of the disease, using therapy based on an anti-CD20 antibody.

17 cl, 20 dwg, 6 tbl, 21 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application pursuant to 119(e) 35 USC claims priority to U.S. Provisional Application 62/362963, filed July 15, 2016, the entire disclosure of which is incorporated herein by reference.

INCLUSION BY SENDING MATERIAL SUBMITTED IN ELECTRONIC FORM

[0002] By reference, the machine-readable list of nucleotide/amino acid sequences filed simultaneously with this application and designated as follows: file "266608SeqListing.txt" in ACII (text) format, 56 kilobytes in size, created on July 14, 2017, is fully included.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0003] The present invention relates to anti-CD38 antibodies and their use as therapeutic and diagnostic agents. The present invention also relates to methods for the treatment of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. The present invention also relates to diagnostic testing methods for determining patients having autoimmune diseases to be treated.

BACKGROUND OF THE INVENTION

[0004] Plasma cells and plasmablasts are antibody-secreting cells (ASCs) that are important in pathogenic processes associated with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). They are implicated in the pathogenesis of many antibody-mediated autoimmune diseases, including myasthenia gravis, Sjögren's syndrome, multiple sclerosis (MS), and autoimmune thyroiditis. See Jacobi AM, Mei H, Hoyer BF, et al., Ann Rheum Dis (2010) 69 (1): 305-8; Dorner T, Isenberg D, Jayne D, et al., International Roundtable on B cells as Therapeutic Target for Intervention, Autoimmun Rev (2009) 9 (2): 82-9; Tipton CM, Fucile CF, Darce J, et al., Nat Immunol (2015) 16 (7): 755-65; and Cepok S, Rosche B, Grummel V, et al., Brain (2005) 128 (Pt 7): 1667-76. Plasmablasts are terminally differentiated B cells that form rapidly, with most becoming short-lived effector cells and a minority becoming long-lived plasma cells.

[0005] The majority of B cells express CD20 and are effectively and sustainably eliminated by antibody therapeutics directed against this cell surface protein. See Silverman GJ, Arthritis Rheum (2006) 54 (8): 2356-67. However, some cells of the B-cell lineage, such as plasmablasts and plasma cells, do not express significant amounts of CD20 and are not amenable to effective reduction by direct CD20-targeted elimination. See Silverman 2006. Thus, the ability of CD20-targeted drugs to influence this population of cells is limited to the elimination of the CD20+ B cell population, which can eventually differentiate into plasmablasts, with little effect on established plasmablasts and plasma cells. See Silverman GJ and Boyle DL, Immunol Rev (2008) 223: 175-85.

[0006] Clinical studies involving SLE patients have shown that high levels of plasmablasts before treatment with anti-CD20 drugs correlate with inefficient elimination of B-cells in the blood. In addition, incomplete elimination of plasmablasts from the blood may have clinical consequences, including accelerated recurrence and reduced survival. See Vital EM, Rawstron AC, Dass S, et al., Arthritis Rheum (2011) 63 (3): 603-8. Similar results have been demonstrated in RA patients treated with anti-CD20 monoclonal antibodies. See Dass S, Rawstron AC, Vital EM, et al., Arthritis Rheum (2008) 58 (10): 2993-9; and Owczarczyk K, Lal P, Abbas AR, et al., Sci Transl Med (2011) 3 (101): 101ra92.

[0007] To solve these problems, methods have been developed to indirectly monitor the levels of plasmablasts and plasma cells in the blood by assessing the mRNA transcripts expressed primarily in these cell types. See Vital EM, Dass S, Buch MH, et al., Arthritis Rheum (2011) 63 (10): 3038-47; Streicher K, Morehouse CA, Groves CJ, et al., Arthritis Rheumatol (2014) 66 (1): 173-84; and Owczarczyk, 2011. Using this method, CD20 non-responders were retrospectively identified as patients with high levels of IgJ-rich plasmablasts and plasma cells and low levels of FCRL5 (Fc receptor-like protein 5), which is expressed in CD20+ non-plasmoblasts.

[0008] This indirect approach is simple and can be used in most clinical situations. Blood samples collected using conventional sampling tubes (eg, PAXgene tubes) have long-term stability when properly stored. However, transcript assays in whole blood do not readily allow direct quantification of plasmablasts and plasma cells, and, depending on the uniqueness of the transcripts assayed in a given cell population, changes in other cell types may influence the results.

[0009] Direct measurement of the levels of plasmablasts and plasma cells before or in response to therapy that affects B-cells should help solve this problem. However, the viability of plasmablasts rapidly declines after vein sampling. With modern techniques, monitoring of plasmablast levels requires processing on the day of collection, which is difficult to standardize in different clinical centers.

[0010] Although plasmablasts and plasma cells do not express significant amounts of CD20, these cells, along with NK cells, constitutively express high levels of CD38, like B cells and T cells, in which CD38 expression is upregulated upon activation. See Malavasi F, Deaglio S, Funaro A, et al., Physiol Rev (2008) 88 (3): 841-86. CD38, also known as cyclic ADP-ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal endoplasmic domain. CD38 is a member of a group of related membrane-bound or soluble enzymes, including CD157 and Aplysia ADFR cyclase. This family of enzymes has the unique ability to convert NAD to cyclic ADP-ribose or nicotinamide adenine dinucleotide phosphate.

[0011] Recent studies in humans show that anti-CD38 mAbs can eliminate many types of blood cells, including plasmablasts and plasma cells.

SUMMARY OF THE INVENTION

[0012] Anti-CD38 antibodies in combination with measurement of plasmablasts and/or plasma cells before or during treatment can be used to treat patients suffering from rheumatoid arthritis, systemic lupus erythematosus or other autoimmune disease who cannot achieve disease remission with therapy based on CD20 or other therapies that do not provide persistent elimination of plasmablasts and/or plasma cells.

[0013] In one aspect, the invention provides a method of treating a disease in a patient, comprising administering a therapeutically effective amount of an anti-CD38 antibody to the patient, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease , and the patient was shown to have elevated levels of CD38-expressing cells prior to treatment with the anti-CD38 antibody compared to the control individual.

[0014] In another aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering a therapeutically effective amount of an anti-CD38 antibody to the patient, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and the patient after treatment with an anti-CD20 antibody showed an increased level of CD38-expressing cells compared to a control individual.

[0015] In a further aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and in a biological sample obtained from a patient prior to treatment with an anti-CD38 antibody, an increased level of CD38-expressing cells was shown compared to a control individual.

[0016] In another aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and in a biological sample obtained from a patient after treatment with an anti-CD20 antibody, an increased level of CD38-expressing cells was shown compared to a control individual.

[0017] In yet another aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease , and in a biological sample obtained from a patient prior to treatment with an anti-CD38 antibody, the presence of an increased level of plasma cells and plasmablasts was shown compared to a control individual in the analysis of free light chains of Ig.

[0018] In a further aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and in a biological sample obtained from a patient after treatment with an anti-CD20 antibody, the presence of an increased level of plasma cells and plasmablasts was shown compared to a control individual when analyzing free Ig light chains.

[0019] In yet another aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease , and in a biological sample obtained from a patient prior to treatment with an anti-CD38 antibody, the presence of an increased level of a specific mRNA or mRNA group of CD38-expressing cells was shown compared to a control individual.

[0020] In another aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and in a biological sample obtained from a patient after treatment with an anti-CD20 antibody, the presence of an increased level of total mRNA of CD38-expressing cells was shown compared to a control individual.

[0021] In a further aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of a first anti-CD38 antibody, wherein the first anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is autoimmune disease, and a biological sample obtained from a patient prior to treatment with an anti-CD38 antibody was shown to have an increased level of CD38-expressing cells compared to a control individual by flow cytometry.

[0022] In yet another aspect of the invention, there is provided a method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of a first anti-CD38 antibody, wherein the first anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is autoimmune disease, and a biological sample obtained from a patient after treatment with an anti-CD20 antibody was shown to have an increased level of CD38-expressing cells compared to a control individual using flow cytometry.

[0023] In another aspect of the invention, a method is provided for assaying CD38-expressing cells in whole blood, comprising:

a) obtaining a whole blood sample from an individual, where the sample includes erythrocytes, leukocytes and an anticoagulant;

b) lysis of erythrocytes to obtain a processed sample;

c) freezing the processed sample at a temperature of approximately -20°C or below for approximately 24 hours after receiving the sample, obtaining a frozen sample;

d) measuring the number of CD38-expressing cells in the sample for approximately 72 hours after sample acquisition, where the frozen sample is warmed to room temperature before measuring the volume of CD38-expressing cells.

[0024] Provided herein are reagents and methods for binding to CD38, as well as methods for treating CD38-associated diseases and detecting CD38 using CD38-specific binders, including anti-CD38 antibodies.

[0025] Thus, in some embodiments, an isolated antibody specific for human CD38 (SEQ ID NO: 1) and cynomolgus monkey CD38 (SEQ ID NO: 2) is described for use in conjunction with various aspects of the invention. In some embodiments, the isolated antibodies described herein may consist of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDR regions) described herein as HCDR1, HCDR2, and HCDR3, and wherein the light chain variable region includes the three CDR regions described herein as LCDR1, LCDR2 and LCDR3. The sequences of the CDR regions are as follows: HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4), HCDR3 (SEQ ID NO: 5), LCDR1 (SEQ ID NO: 6), LCDR2 (SEQ ID NO: 7 ) and LCDR3 (SEQ ID NO: 8).

[0026] In other embodiments, the isolated antibody may include a heavy chain variable region, wherein the heavy chain variable region sequence comprises SEQ ID NO: 9. In other embodiments, the isolated antibody may comprise a light chain variable region, wherein the light chain variable region sequence comprises SEQ ID NO: 10. In other embodiments, the heavy chain variable region comprises SEQ ID NO: 9 and the light chain variable region comprises SEQ ID NO: 10.

[0027] In some embodiments, the isolated antibody may include a heavy chain variable region, wherein the heavy chain variable region sequence comprises SEQ ID NO: 21. In other embodiments, the isolated antibody may comprise a light chain variable region, wherein the light chain variable region sequence comprises SEQ ID NO: 22. In other embodiments, the heavy chain variable region comprises SEQ ID NO: 21 and the light chain variable region comprises SEQ ID NO: 22. Such a combination of heavy chain variable region and light chain variable region is referred to as Ab79. In some embodiments, the isolated antibody comprises an Fc domain. In other embodiments, the Fc domain is a human Fc domain. In other embodiments, the Fc domain is a variant Fc domain.

[0028] In some embodiments, an isolated antibody specific for human CD38 (SEQ ID NO: 1) and cynomolgus monkey CD38 (SEQ ID NO: 2) is described for use in conjunction with various aspects of the invention. In some embodiments, isolated antibodies described herein may be composed of six CDR regions, where each CDR region of that antibody may be different from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 with 0, 1 or 2 amino acid substitutions.

[0029] In other embodiments, an isolated antibody specific for human CD38 (SEQ ID NO: 1) and cynomolgus CD38 (SEQ ID NO: 2) is described for use in conjunction with various aspects of the invention. The isolated antibodies described herein may be composed of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDR regions) described herein as HCDR1, HCDR2, and HCDR3, and where the light chain variable region includes three CDR regions, described herein as LCDR1, LCDR2 and LCDR3. The sequences of the CDR regions are as follows: HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14), HCDR3 (SEQ ID NO: 15), LCDR1 (SEQ ID NO: 16), LCDR2 (SEQ ID NO: 17 ) and LCDR3 (SEQ ID NO: 18).

[0030] In some embodiments, the isolated antibody may include a heavy chain variable region, wherein the heavy chain variable region sequence comprises SEQ ID NO: 11. In other embodiments, the isolated antibody may comprise a light chain variable region, wherein the light chain variable region sequence comprises SEQ ID NO: 12. In some embodiments, the isolated antibody may include a heavy chain and a light chain, where the heavy chain sequence comprises SEQ ID NO: 11 and the light chain comprises SEQ ID NO: 12.

[0031] In other embodiments, the isolated antibody may include a heavy chain variable region, wherein the heavy chain variable region sequence comprises SEQ ID NO: 19. In other embodiments, the isolated antibody may comprise a light chain variable region, wherein the light chain variable region sequence comprises SEQ ID NO: 20. In other embodiments, the heavy chain variable region comprises SEQ ID NO: 19 and the light chain variable region comprises SEQ ID NO: 20. This combination of heavy chain variable region and light chain variable region is referred to as Ab19.

[0032] In other embodiments, an isolated antibody specific for human CD38 (SEQ ID NO: 1) and cynomolgus monkey CD38 (SEQ ID NO: 2) is described for use in conjunction with various aspects of the invention. The isolated antibodies described herein may be composed of six CDR regions, where each CDR region of a given antibody may be different from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 with 0, 1 or 2 amino acid substitutions.

[0033] In some embodiments, an isolated anti-CD38 antibody is provided that specifically binds to human CD38 (SEQ ID NO: 1) and cynomolgus monkey CD38 (SEQ ID NO: 2), wherein the antibody binds to human CD38 with a KD of approximately 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 or greater, and links cynomolgus monkey CD38 to a KD of approximately 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 or greater.

[0034] In some embodiments, antibodies are provided that compete with Ab79 and/or Ab19 for binding to human CD38 and/or cynomolgus CD38.

[0035] The methods of the present invention may be described as embodiments in any of the following listed paragraphs. It should be understood that any of the embodiments described herein may be used in combination with any other embodiment(s) described herein, to the extent that the combined embodiments do not conflict with each other.

[0036] 1. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and the patient prior to treatment with an anti-CD38 antibody, an increased level of CD38-expressing cells was shown compared to a control individual.

[0037] 2. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and the patient after treatment with an anti-CD20 antibody, an increased level of CD38-expressing cells was shown compared to the control individual.

[0038] 3. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and biologically a sample obtained from a patient prior to treatment with an anti-CD38 antibody showed an increased level of CD38-expressing cells compared to a control individual.

[0039] 4. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and biologically a sample obtained from a patient after treatment with an anti-CD20 antibody showed an increased level of CD38-expressing cells compared to a control individual.

[0040] 5. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and biologically a sample obtained from a patient prior to treatment with an anti-CD38 antibody was shown to have an increased level of CD38-expressing plasmablasts and plasma cells compared to a control individual in the analysis of free Ig light chains.

[0041] 6. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and biologically a sample obtained from a patient after treatment with an anti-CD20 antibody was shown to have an increased level of CD38-expressing cells compared to a control individual in the analysis of free Ig light chains.

[0042] 7. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and biologically a sample obtained from a patient prior to treatment with an anti-CD38 antibody was shown to have elevated levels of at least one gene upregulated in CD38-expressing cells compared to a control individual.

[0043] 8. A method of treating a disease in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD38 antibody, wherein the anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and biologically a sample obtained from a patient after treatment with an anti-CD20 antibody was shown to have elevated levels of at least one gene upregulated in CD38-expressing cells compared to a control individual.

[0044] 9. The method according to any of the preceding claims, wherein the anti-CD38 antibody comprises:

[0045] a) a heavy chain variable region, comprising:

[0046] i) a first CDR region comprising SEQ ID NO: 3;

[0047] ii) a second CDR region comprising SEQ ID NO: 4;

[0048] iii) a third CDR region comprising SEQ ID NO: 5; and

[0049] b) a light chain variable region, comprising:

[0050] i) a first CDR region comprising SEQ ID NO: 6;

[0051] ii) a second CDR region comprising SEQ ID NO: 7;

[0052] iii) a third CDR region comprising SEQ ID NO: 8.

[0053] 10. The method of claim 9, wherein the heavy chain variable region comprises SEQ ID NO: 9.

[0054] 11. The method of claim 9 wherein the light chain variable region comprises SEQ ID NO: 10.

[0055] 12. The method according to any one of items 9-11, wherein the heavy chain variable region comprises SEQ ID NO: 9 and the light chain variable region comprises SEQ ID NO: 10.

[0056] 13. The method according to any one of items 9-11, wherein the heavy chain comprises SEQ ID NO: 21 and the light chain comprises SEQ ID NO: 22.

[0057] 14. The method according to any one of items 1-8, wherein the anti-CD38 antibody comprises:

[0058] a) a heavy chain variable region, comprising:

[0059] i) a first CDR region comprising SEQ ID NO: 13;

[0060] ii) a second CDR region comprising SEQ ID NO: 14;

[0061] iii) a third CDR region comprising SEQ ID NO: 15; and

[0062] b) a light chain variable region, comprising:

[0063] i) a first CDR region comprising SEQ ID NO: 16;

[0064] ii) a second CDR region comprising SEQ ID NO: 17;

[0065] iii) a third CDR region comprising SEQ ID NO: 18.

[0066] 15. The method of claim 14, wherein the heavy chain variable region comprises SEQ ID NO: 11.

[0067] 16. The method of claim 14, wherein the light chain variable region comprises SEQ ID NO: 12.

[0068] 17. The method according to any one of items 14-16, wherein the heavy chain variable region comprises SEQ ID NO: 19 and the light chain variable region comprises SEQ ID NO: 20.

[0069] 18. The method according to any one of items 14-16, wherein the heavy chain comprises SEQ ID NO: 34 and the light chain comprises SEQ ID NO: 35.

[0070] 19. The method of any one of 9 and 14, wherein the anti-CD38 antibody further comprises an Fc domain.

[0071] 20. The method of claim 19, wherein the Fc domain is human.

[0072] 21. The method of claim 19, wherein the Fc domain is a variant of the Fc domain.

[0073] 22. The method according to any one of items 1-8, wherein the CD38 antibody interacts with at least K121, F135, Q139, D141, E239, W241, C275, K276, F284, P291, and E292 SEQ ID NO: 1.

[0074] 23. A method of treating a disease in a patient, comprising administering a therapeutically effective amount of a first anti-CD38 antibody to the patient, wherein the first anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and a biological sample obtained from a patient prior to treatment with an anti-CD38 antibody was shown to have an increased level of CD38-expressing cells compared to a control individual by flow cytometry.

[0075] 24. A method of treating a disease in a patient, comprising administering a therapeutically effective amount of a first anti-CD38 antibody to the patient, wherein the first anti-CD38 antibody is an isolated antibody that specifically binds human CD38 (SEQ ID NO: 1), the disease is an autoimmune disease, and a biological sample obtained from a patient after treatment with an anti-CD20 antibody was shown to have an increased level of CD38-expressing cells compared to a control individual by flow cytometry.

[0076] 25. The method according to any one of items 23 and 24, wherein the CD38-expressing cells were stained with a second fluorochrome-conjugated anti-CD38 antibody.

[0077] 26. The method according to item 25, wherein the second anti-CD38 antibody is an antibody as defined in any one of items 14-18.

[0078] 27. The method according to any one of items 21-24, wherein the first anti-CD38 antibody is an antibody as defined in any one of items 9-13.

[0079] 28. The method according to any one of items 1-27, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, ulcerative colitis, graft versus host disease, myasthenia gravis, Sjögren's syndrome, multiple sclerosis and autoimmune thyroiditis.

[0080] 29. The method according to any one of items 1-27, wherein the autoimmune disease is rheumatoid arthritis.

[0081] 30. The method according to any one of items 1-27, wherein the autoimmune disease is systemic lupus erythematosus.

[0082] 31. A method for analyzing CD38-expressing cells in whole blood, comprising:

[0083] a) obtaining a whole blood sample from an individual, where the sample includes erythrocytes, leukocytes and an anticoagulant;

[0084] b) lysis of erythrocytes to obtain a processed sample;

[0085] c) freezing the processed sample to obtain a frozen sample, where the freezing occurs within about 24 hours after receiving the whole blood sample from the individual;

[0086] d) measuring the number of CD38-expressing cells in the sample for approximately 72 hours after sample acquisition, where the frozen sample is warmed to room temperature before measuring the number of CD38-expressing cells.

[0087] 32. The method according to item 31, wherein the frozen sample is kept at a temperature of approximately -20°C or less before measuring the number of CD38-expressing cells.

[0088] 33. The method according to any one of items 1-32, wherein the CD38-expressing cells are plasma cells and/or plasmablasts.

[0089] 34. The method according to any one of items 1-32, wherein the CD38-expressing cells are plasma cells.

[0090] 35. The method according to any one of items 1-32, wherein the CD38-expressing blood cells are plasmablasts.

[0091] These and other embodiments, features, and potential advantages will become apparent with reference to the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0092] FIG. 1 shows the expression profile of CD38 on cells of the lymphoid lineage, an asterisk indicates high expression of CD38. CD38 expression was determined on pro-B cells (CD34 ⁺ CD19 ⁺ CD20 ^- ), activated B cells (CD19 ⁺ CD20 ⁺ ), plasma cells (CD138 ⁺ CD19 ^- CD20 ^- ), activated CD4 ⁺ and CD8 ⁺ T cells, NKT cells (CD3 ⁺ CD56 ⁺ ) and NK cells (CD56 ⁺ CD16 ⁺ ). In addition, CD38 expression was detected on lymphoid progenitor cells (CD34 ⁺ CD45RA ⁺ CD10 ⁺ CD19 ^- ), but not on lymphoid stem cells. In addition, increased expression of CD38 was observed on mature DCs and activated monocytes.

[0093] FIG. 2 shows the heavy and light chain sequences of Ab79 and Ab19.

[0094] FIG. 3 shows human and cynomolgus monkey CD38 sequences.

[0095] FIG. 4 shows the human CD38 epitopes that each of Control 1 and 2, Ab19 and Ab79 antibodies binds.

[0096] FIG. 5 shows increased expression of CD38 in PBMCs from SLE patients using a commercial anti-human CD38 antibody.

[0097] FIG. 6 shows the percentage change in cell count in cynomolgus monkeys 24 hours post-dose.

[0098] FIG. 7 shows recovery from elimination following a single dose of Ab79.

[0099] FIG. 8 shows the results in one HuSCID mouse for a significant reduction in all Ig isotypes after a single dose of Ab79.

[00100] FIG. 9 is similar to FIG. 8 for Ab79 elimination activity in HuSCID mice as described in the Examples.

[00101] FIG. 10 shows a significant reduction in tetanus response in the HuSCID model after treatment with Ab79.

[00102] FIG. 11, again in the HuSCID model, shows a significant increase in survival after treatment with Ab79, in essentially one type of graft versus host model.

[00103] FIG. 12 shows differences in CD38 antigen expression between human and mouse PBMCs using commercial antibodies to each.

[00104] FIG. 13 shows the therapeutic effect under conditions of inflammation when immune cells are eliminated from peripheral blood with a surrogate mouse anti-CD38 antibody.

[00105] FIG. 14 shows that Ab79 eliminates blood plasmablasts (CD38+, CD27+), plasma cells (CD138+, CD27+) and (CD138+, IRF4+).

[00106] FIG. 15 shows that Ab79 eliminates bone marrow long-lived plasma cells (CD19-, CD38+, CD138+).

[00107] FIG. 16 shows sample collection, culturing, and experimental endpoints.

[00108] FIG. 17 shows the sensitivity of ASA to Ab79 in the blood of healthy volunteers (FIG. 17A) and individuals with SLE (FIG. 17B) as measured by the ELISpot.

[00109] FIG. 18 shows that Ab79 reduces ACK, which produces autoantibodies 9G4 (FIG. 18A) and Ro (FIG. 18B).

[00110] FIG. 19 shows that plasmablasts and plasma cells (CD45+, CD3-, CD19+, CD27+, CD38+) can be detected in whole blood by flow cytometry using the methods described herein.

) Ab79 (0,1 мг/кг); (

) Ab79 (0,3 мг/кг); (

) Ab79 (0,6 мг/кг).[00111] FIG. 20 shows flow cytometry of blood cells preserved by the fix/freeze method described herein after administration of Ab79 to healthy volunteers and shows that Ab79 eliminates plasmablasts and plasma cells. (o) Placebo; (•) Ab79 (0.03 mg/kg; (

) Ab79 (0.1 mg/kg); (

) Ab79 (0.3 mg/kg); (

) Ab79 (0.6 mg/kg).

DETAILED DESCRIPTION OF THE INVENTION

[00112] OVERVIEW

[00113] The extracellular domain of CD38 has been shown to have bifunctional enzymatic activity as it has both ADP-ribosyl cyclase and ADP-ribosyl hydrolase activities. Thus, CD38 can catalyze the conversion of NAD ⁺ to cADFR (cyclase) and can then hydrolyze it into ADP-ribose (hydrolase). cADFR is involved in the mobilization of calcium from the intracellular depot and is the second mediator important for cell proliferation, differentiation, and apoptosis.

[00114] Increased expression of CD38 has been reported in various diseases of hematopoietic origin and has been described as a negative prognostic marker in chronic lymphoblastic leukemia. Such diseases include, but are not limited to, multiple myeloma (Jackson et al. (1988)), chronic lymphoblastic leukemia (Moribito et al. (2001), Jelinek et al. (2001), Chevalier et al. (2002), Durig et al. (2002)), B-cell chronic lymphocytic leukemia, acute lymphoblastic leukemia (Keyhani et al. (2000)), including B-cell acute lymphocytic leukemia, Waldenström macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, pro -lymphocytic/myelocytic leukemia, acute myeloid leukemia (Keyhani et al. (1993)), chronic myeloid leukemia (Marinov et al. (1993)), follicular lymphoma, NK cell leukemia and plasma cell leukemia. Thus, CD38 represents a useful target in the treatment of diseases of the hematopoietic system.

[00115] Several anti-CD38 antibodies are in clinical trials for the treatment of CD38-associated cancers. Thus, anti-CD38 antibodies with therapeutic effect and/or diagnostic applications are useful. The invention provides two different sets of anti-CD38 CDR regions that bind to different CD38 epitopes and that bind both human CD38 and cynomolgus CD38, as well as antibodies that contain these CDR regions.

[00116] In addition, the present invention shows that antibodies against CD38 find use in the diagnosis and/or treatment of inflammation and/or immunological disorders associated with activated lymphocytes, including, in particular, autoimmune diseases. As shown herein, CD38 is expressed in immature hematopoietic cells, downregulated in mature cells, and re-expressed at a high level in activated lymphocytes and plasma cells. For example, CD38 is highly expressed in activated B cells, plasma cells, activated CD4+ T cells, activated CD8+ T cells, NK cells, NKT cells, mature dendritic cells (DCs), and activated monocytes.

[00117] The results presented herein are surprising in that the presence of anti-CD38 autoantibodies has been associated with diabetes, chronic autoimmune thyroiditis, and Graves' disease (see Antonelli et al, Clin. Exp. Immunol. 2001 126: 426-431; Mallone et al., Diabetes 50: 752 (2001) and Antonelli et al., J. Endocrinol Invest 27: 695-707 (2004), which are incorporated by reference.

[00118] Thus, the antibodies of the invention find use in the diagnosis and/or treatment of many diseases, including, but not limited to, autoimmune diseases as discussed below, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA ), inflammatory bowel disease (IBD), and ulcerative colitis.

[00119] Thus, for example, patients with high plasma cell counts can be selected, such as SLE patients who exhibit high plasma cell levels, as well as RA patients who have been shown to be refractory to treatment with CD20-based therapy. .

[00120] The anti-CD38 therapeutic antibodies of the present invention bind to CD38-positive cells, resulting in the depletion of these cells, such as activated lymphocytes, through a variety of mechanisms of action, including, but not limited to, CDC, ADCC, and apoptotic pathways, as provided in herein, leading to the treatment and/or reduction of the severity of autoimmune diseases.

[00121] One benefit not seen with some anti-CD38 antibodies in cancer clinical trials is the ability to bind to cynomolgus monkey CD38 since these primates are used in preclinical studies, which could lead to early dose assessment, toxicity, efficiency, etc.

[00122] CD38 PROTEINS

[00123] Thus, the present invention provides isolated anti-CD38 antibodies that specifically bind human CD38 protein (and, as described below, additionally and preferably specifically bind primate CD38 protein). As is known in the art, CD38 proteins are found in organisms of many species. Of particular use in the present invention are antibodies that bind to both the human CD38 protein and the CD38 proteins of primates, especially primates used in clinical research, such as cynomolgus monkeys (Macaca fascicularis, cynomolgus monkeys, sometimes referred to herein as monkeys). "cynomolgus"). "Human CD38" or "human CD38 antigen" refers to a protein of SEQ ID NO: 1 or a functional fragment thereof, such as an epitope, as defined herein. Typically, CD38 has a short intracytoplasmic tail, a transmembrane domain and an extracellular domain, in some embodiments, the antibodies of the invention bind to the extracellular portion of the CD38 protein. By "cynomolgus CD38" is meant herein SEQ ID NO: 2, which is 92% identical to human CD38.

[00124] Synonyms for CD38 include ADP-ribosylcyclase 1, cADFR hydrolase 1, Cd38-rs1, cyclic ADP-ribose hydrolase 1, 1-19, and NIM-R5 antigen.

[00125] In some embodiments, the anti-CD38 Ab79 antibodies of the invention interact with CD38 at a number of amino acid residues including K121, F135, Q139, D141, M142, D202, V203, H205, Q236, E239, W241, S274, C275, K276, F284, C287, V288, K289, N290, P291, E292, D293. As provided herein, other antibodies that interact with these residues also find use in therapeutic and diagnostic purposes.

[00126] In some embodiments, anti-CD38 antibodies of the present invention do not necessarily (and in some cases preferentially) bind to other members of the CD38 family, such as CD157. For example, preferred embodiments herein do not bind to human CD157 SEQ ID NO: 23 (GenBank accession number NP_004325).

[00127] ANTIBODIES

[00128] The present invention provides anti-CD38 antibodies, typically therapeutic and/or diagnostic antibodies, as described herein. Antibodies that find use in the present invention may take a variety of formats as described herein, including traditional antibodies, as well as derivatives, fragments and mimetics of antibodies, described below. As such, the invention provides antibody structures that contain a set of 6 CDR regions as defined herein (comprising a small number of amino acid changes as described below).

[00129] Traditional structural units of antibodies usually include a tetramer. Each tetramer usually consists of two identical pairs of polypeptide chains, with each pair having one "light" (typically having a molecular weight of approximately 25 kDa) and one "heavy" chain (typically having a molecular weight of approximately 50-70 kDa) . Human light chains are classified into kappa and lambda light chains. Heavy chains are subdivided into mu, delta, gamma, alpha, or epsilon and define the isotype of the antibody: IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2a, IgG3 and IgG4. IgM has subclasses including, without limitation, IGM1 and IgM2. Thus, "isotype" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions. Known isotypes of human immunoglobulin are IgG1, IgG2a, IgG3, IgG4, IgA1, IgA2, IGM1, IgM2, IgD and IgE. It should be understood that therapeutic antibodies may also include hybrids of isotypes and/or subclasses.

[00130] The N-terminal portion of each chain includes a variable region of approximately 100-110 or more amino acids that is primarily responsible for antigen recognition. In the variable region, three loops assemble in each of the heavy chain and light chain V domains to form an antigen binding site. Each of the loops is referred to as a complementarity determining region (hereinafter referred to as "CDR region") in which the amino acid sequence variation is most significant. "Variable" refers to the fact that some segments of the variable region differ greatly in sequence in different antibodies. The variability within the variable region is unevenly distributed. Instead, the V regions consist of relatively constant fragments called framework regions (FRs) of 15-30 amino acids separated by shorter regions of high variability called "hypervariable regions", each of which is 9-15 amino acids or more in length.

[00131] Each VH and VL consists of three hypervariable regions ("complementarity determining regions", "CDR regions") and four FR regions, arranged from the N-terminus to the C-terminus in the following order: FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4.

[00132] The hypervariable region typically spans amino acid residues from about amino acid residues 24-34 (LCDR1; "L" stands for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and residues around about 31 -35B (HCDR1; "H" stands for heavy chain), 50-65 (HCDR2) and 95-102 (HCDR3) in the variable region of the heavy chain; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and/or residues forming a hypervariable loop (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53 -55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region Chothia and Lesk (1987) J Mol Biol 196:901-917 Specific CDR regions of the invention are described below.

[00133] Throughout this specification, the Kabat numbering system is commonly used to denote a residue in the variable domain (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) (e.g., Kabat et al., supra (1991 )), with the EU numbering system used for the Fc region.

[00134] CDR regions contribute to the formation of an antigen-binding, or more specifically, epitope-binding site of antibodies. "Epitope" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. Epitopes are groups of molecules, such as amino acids or sugar side chains, and usually also have specific structural properties as well as specific charge properties. . One antigen may have more than one epitope. For example, as shown herein, two different antibodies, referred to herein as "Ab19" and "Ab79", bind to different epitopes on the CD38 molecule.

[00135] An epitope may include amino acid residues directly involved in binding (also referred to as the immunodominant component of an epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide; in other words, the amino acid residue is present in the recognizable region of the specific antigen-binding peptide.

[00136] Epitopes can be conformational or linear. A conformational epitope is formed as a result of spatial convergence of amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished due to the fact that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[00137] An epitope typically includes at least 3, and more often at least 5 or 8-10 amino acids, in a unique spatial pattern. Antibodies that recognize the same epitope can be tested in a simple immunoassay that shows the ability of one antibody to block another antibody from binding to a target antigen, such as "binning" as described in the Examples. X-ray crystallography studies as shown in the Examples determined the amino acid residues that bind both the antibodies of the invention (including Ab19 and Ab79) and the prior art (Control 1 and Control 2) as shown in FIG. four.

[00138] In the present invention, Ab79, as described in the Examples, interacts with certain amino acid residues of CD38, including K121, F135, Q139, D141, M142, E239, W241, S274, C275, K276, F284, V288, K289, N290, P291, E292 and D293. It should be noted that these residues are identical in humans and cynomolgus monkeys, except that S274 is actually F274 in cynomolgus. These residues may represent an immunodominant epitope and/or residues within a recognizable region of a specific antigen-binding peptide.

[00139] In the present invention, Ab19 binds to another epitope including G91, E103, E1034, D105, Q107, M110, K111, T114, Q115, T148, V192, R194, R195, F196, A199, H228, N229, Q231, E233 and K234. It should be noted that these residues are identical in humans and cynomolgus monkeys, except that M110 is V110 in cynomolgus and A199 is T199 in cynomolgus.

[00140] Thus, in some embodiments, antibodies that compete with Ab79 and Ab19 when bound at any of these epitopes can be used to treat autoimmune diseases. It should be noted that there is some overlap between Ab79 and Control 1 (BM1); thus, antibodies that compete with Ab79 and are not BM1 find use in the present invention.

[00141] Thus, the present invention provides antibodies that bind to human and cynomolgus CD38 and interact with at least 80%, 90%, 95%, or 98% of these residues. In other words, the area of the interaction zone does not exceed the area of these residues.

[00142] The C-terminal portion of each strand defines a constant region that is primarily responsible for effector function. Kabat et al. assembled a plurality of primary sequences of heavy chain and light chain variable regions. Based on the degree of sequence conservation, they classified individual primary sequences in the CDR and framework region and made a list of them (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al., incorporated by reference in full) .

[00143] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig) domain", as used herein, is meant a region of an immunoglobulin having a defined tertiary structure. Within the scope of the present invention, heavy chain domains are of interest, including heavy chain constant domains (CH) and hinge domains. In relation to IgG antibodies, each IgG isotype contains three CH regions. Thus, the "CH" domains within IgG are as follows: "CH1" refers to positions 118-220 according to the Kabat EU index. "CH2" refers to provisions 237-340 according to the EU index according to Kabat, and "CH3" refers to provisions 341-447 according to the EU index according to Kabat.

[00144] Another type of heavy chain Ig domain is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" is meant herein a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at position 220 EU, and the IgG CH2 domain begins at position 237 EU. Thus, in the case of IgG, the antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbering corresponds to the Kabat EU index. In some embodiments, such as in the case of the Fc region, a lower hinge is included, where "lower hinge" typically corresponds to positions 226 or 230.

[00145] Fc regions are of particular interest within the scope of the present invention. By "Fc" or "Fc region" or "Fc domain" as used herein is meant a polypeptide comprising the antibody constant region excluding the first domain of the immunoglobulin constant region and, in some cases, the hinge portion. Thus, Fc refers to the last two domains of the constant region of IgA, IgD and IgG, the last three domains of the constant region of immunoglobulins IgE and IgM and the flexible hinge at the N-terminus with respect to these domains. In the case of IgA and IgM, Fc may include a J chain. In the case of IgG, the Fc domain includes the Cγ2 and Cγ3 immunoglobulin domains (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the Fc region of a human IgG heavy chain is generally defined as comprising C226 or P230 residues at its C-terminus, where the numbering corresponds to Kabat's EU index. In some embodiments, as described in more detail below, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcγR receptors or to an FcRn receptor.

[00146] In some embodiments, the antibodies are full length. By "full-length antibody" is meant herein a structure that constitutes the natural biological form of an antibody, including variable and constant regions, including one or more modifications, as described herein.

[00147] In the alternative, antibodies can be a variety of structures including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies , antibody fusion constructs (sometimes referred to as "antibody conjugates"), and fragments of each, respectively. Structures that are also based

[00148] In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to: (i) Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) Fd fragment consisting of VH and CH1 domains, (iii) Fv fragment consisting of VL and VH domains of one antibody; (iv) a dAb fragment (Ward et al., 1989, Nature 341: 544-546, incorporated by reference in its entirety), which consists of a single variable, (v) isolated CDR regions, (vi) F(ab') 2 fragments, a bivalent fragment comprising two linked Fab fragments, (vii) single chain Fv (scFv) molecules, where the VH domain and VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al ., 1988, Science 242: 423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 5879-5883, incorporated by reference in their entirety), (viii) bispecific single chain Fv (WO 03/11161 , hereby incorporated by reference) and (ix) "diabodies" or "triatebodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326: 461-479; WO 94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 6444-6448, incorporated by reference in full).

[00149] CHIMERIC AND HUMANIZED ANTIBODIES

[00150] In some embodiments, the antibody may be a mixture of different species, such as a chimeric antibody and/or a humanized antibody. Thus, in the present invention, sets of CDR regions can be used with framework and constant regions that differ from those specifically described by the sequence herein.

et al., 1998, Protein Eng 11: 321-8, полностью включенные посредством отсылки. Гуманизация или другие способы снижения иммуногенности вариабельных участков нечеловеческих антител могут включать способы переукладки, как описано, например, в публикации Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91: 969-973, полностью включенной посредством отсылки. В одном варианте осуществления исходное антитело было подвергнуто созреванию аффинности, как известено в уровне техники. Способы на основе структуры могут использоваться для гуманизации и созревания аффинности, например, как описано в USSN 11/004,590. Способы на основе отбора могут использоваться для гуманизации и/или созревания аффинности вариабельных участков антитела, в том числе, без ограничения перечисленными, способы, описанные в Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272 (16): 10678-10684; Rosok et al., 1996, J. Biol. Chem. 271 (37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16 (10): 753-759, полностью включенные посредством отсылки. Другие способы гуманизации могут включать перевивание только частей CDR-областей, в том числе, без ограничения перечисленными, способы, описанные в USSN 09/810,510; Tan et al., 2002, J. Immunol. 169: 1119-1125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084, полностью включенных посредством отсылки.[00151] Generally, "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from organisms of more than one species. For example, "chimeric antibodies" typically include mouse (or rat, in some cases) variable region(s) and human constant region(s). "Humanized antibodies" generally refers to non-human antibodies in which the variable domain framework regions have been replaced with sequences present in human antibodies. Typically, in a humanized antibody, all but the CDR regions of the antibody are encoded by a polynucleotide of human origin, or identical to such an antibody, except for its CDR regions. CDR regions, some or all of which are encoded by nucleic acids derived from a non-human organism, are grafted onto the beta-fold framework of a human antibody variable region to produce an antibody whose specificity is determined by the grafted CDR regions. The production of such antibodies is described, for example, in WO 92/11018, Jones, 1986, Nature 321: 522-525, Verhoeyen et al., 1988, Science 239: 1534-1536, which are incorporated by reference in their entirety. "Backmutation" of selected acceptor framework residues to the appropriate donor residues is often required to restore affinity that is lost in the original grafted construct (US5530101; US5585089; US5693761; US5693762; US6180370; US5859205; US5821337; US 6,054,297; US 6,407,213, which are incorporated by reference in their entirety). The humanized antibody will also optimally include at least a portion of an immunoglobulin constant region, typically from a human immunoglobulin, and thus will typically include a human Fc region. Humanized antibodies can also be generated using mice with genetically modified immune systems. See Roque et al., 2004, Biotechnol. Prog. 20:639-654, incorporated by reference in its entirety. Various techniques and methods for humanizing and reengineering non-human antibodies are known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA) and references cited therein. , which are incorporated by reference in their entirety). Humanization methods include, but are not limited to, those described in Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988; Nature 332: 323-329; Verhoeyen et al., 1988, Science, 239: 1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86: 10029-33; He et al., 1998, J. Immunol. 160:1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89: 4285-9, Presta et al., 1997, Cancer Res. 57 (20): 4593-9; Gorman et al., 1991, Proc. Natl. Acad. sci. USA 88: 4181-4185;

et al., 1998, Protein Eng 11: 321-8, incorporated by reference in their entirety. Humanization or other means of reducing the immunogenicity of non-human antibody variable regions may include refolding techniques as described, for example, in Roguska et al., 1994, Proc. Natl. Acad. sci. USA 91: 969-973, incorporated by reference in its entirety. In one embodiment, the parent antibody has been subjected to affinity maturation as is known in the art. Structure-based methods can be used for humanization and affinity maturation, for example, as described in USSN 11/004,590. Selection-based methods can be used to humanize and/or affinity mature the variable regions of an antibody, including, but not limited to, the methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272 (16): 10678-10684; Rosok et al., 1996, J. Biol. Chem. 271 (37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16 (10): 753-759, incorporated by reference in their entirety. Other humanization methods may include grafting only portions of the CDRs, including, but not limited to, the methods described in USSN 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084, incorporated by reference in their entirety.

[00152] In one embodiment, the antibodies of the invention may be multispecific antibodies, and in particular bispecific antibodies, also sometimes referred to as "diabodies". These antibodies bind to two (or more) different antigens or different epitopes on the same antigen. Diabodies can be obtained by various methods known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4: 446-449, incorporated by reference in its entirety), for example, obtained chemically or from hybrid hybridomas.

[00153] In one embodiment, the antibody is a minibody. Minibodies are minimal antibody-like proteins, including scFv linked to a CH3 domain. See Hu et al., 1996, Cancer Res. 56: 3055-3061, incorporated by reference in its entirety. In some cases, the scFv may be connected to the Fc region and may include part or all of the hinge region.

[00154] Antibodies of the present invention are typically isolated or recombinant. "Isolated" when used to describe the various polypeptides disclosed herein means a polypeptide that has been identified and separated from and/or recovered from the cell or cell culture in which it was expressed. Typically, an isolated polypeptide is obtained in at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificity. For example, an isolated antibody that specifically binds to CD38 contains essentially no antibodies that specifically bind to antigens other than CD38.

[00155] An isolated antibody that specifically binds to an epitope, isoform, or variant of human CD38 or cynomolgus CD38 may, however, be cross-reactive with other related antigens, for example from other species, such as CD38 species homologues. In addition, the isolated antibody may be substantially free of other cellular material and/or chemicals.

[00156] Isolated monoclonal antibodies having different specificities can be combined in a well-defined composition. Thus, for example, Ab79 and Ab19, if necessary, can be combined in one composition.

[00157] Anti-CD38 antibodies of the present invention specifically bind CD38 ligands (e.g., human and cynomolgus monkey CD38 proteins of SEQ ID NOs: 1 and 2. "Specific binding" or "specifically binds to" or is "specific for" a particular antigen or epitope means a binding that is measurably different from a non-specific interaction.Specific binding can be measured, for example, by determining the binding of a molecule in comparison with the binding of a control molecule, which is usually a molecule with a similar structure that has no binding activity.For example, specific binding can be determined in competition with a control molecule that is similar to the target.

[00158] Specific binding for a particular antigen or epitope may exhibit, for example, an antibody having a KD for the antigen or epitope of at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^-6 M, at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^-11 M, according to at least about 10 ^-12 M or more, where KD refers to the rate of dissociation of the interaction of a particular antibody-antigen. Typically, an antibody that specifically binds an antigen will have a KD that is 20, 50, 100, 500, 1000, 5000, 10,000 or more times greater relative to the control molecule compared to the antigen or epitope.

[0100] In addition, specific binding for a particular antigen or epitope may exhibit, for example, an antibody having KA or Ka for the antigen or epitope at least 20, 50, 100, 500, 1000, 5000, 10,000 or more times more in relation to the epitope compared to the control, where KA or Ka refer to the rate of association of the interaction of a particular antibody-antigen.

[0101] ANTIBODY MODIFICATIONS

[0102] The present invention also provides various antibodies. Thus, there are many modifications that can be introduced into the antibodies of the invention, including, but not limited to, amino acid modifications in the CDR regions (affinity maturation), amino acid modifications in the Fc region, glycosylation variants, other types of covalent modifications, etc. d.

[0103] By "variant", as used herein, is meant a polypeptide sequence that differs from the original polypeptide sequence by modifying at least one amino acid. Amino acid modifications may include substitutions, insertions and deletions, the former being the most preferred in many cases.

[0104] As a rule, variants may include any number of modifications, provided that the function of the protein is preserved, as described herein. Thus, in the case of amino acid variants derived from, for example, Ab79 or Ab19 CDR regions, the antibody should still specifically bind to human and cynomolgus CD38. Similarly, if the amino acid variants are derived from an Fc region, for example, the antibody variants must retain the necessary receptor binding functions for the particular application or indication of the antibody.

[0105] However, as a rule, from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions are usually used, since often the goal is to change the function with a minimum number of modifications. In some cases, there are from 1 to 5 modifications, while in many embodiments, 1-2, 1-3 and 1-4 are also used.

[0106] It should be noted that such a number of amino acid modifications may be present in functional domains: for example, it may be desirable to have from 1-5 modifications in the Fc region of wild-type proteins or recombinant proteins, as well as from 1 to 5 modifications in the Fv region , for example. A variant polypeptide sequence will preferably have at least about 80%, 85%, 90%, 95%, or up to 98% or 99% identity with respect to the original sequences (e.g., variable regions, constant regions, and/or Ab79 heavy and light chain sequences). and/or Ab19). It should be noted that depending on the size of the sequence, the percent identity will depend on the number of amino acids.

[0107] By "amino acid substitution" or "substitution", as used herein, is meant the replacement of an amino acid at a specific position in the parent polypeptide sequence with another amino acid. For example, substitution S100A refers to a polypeptide variant in which serine at position 100 is replaced by alanine. By "amino acid insert" or "insert" as used herein is meant the introduction of an amino acid at a specific position in the parent polypeptide sequence. By "amino acid deletion" or "deletion" as used herein is meant the removal of an amino acid at a specific position in the parent polypeptide sequence.

[0108] By "original polypeptide", "original protein", "precursor polypeptide", or "precursor protein", as used herein, is meant an unmodified polypeptide that is subsequently modified to form a variant. As a rule, the original polypeptides in this document are Ab79 and Ab19. The parent polypeptide may refer to the polypeptide itself, compositions that include the parent polypeptide, or the amino acid sequence that encodes it. Thus, "original Fc polypeptide" as used herein means an Fc polypeptide that has been modified to produce a variant, and "original antibody" as used herein means an antibody that has been modified to produce a variant antibody.

[0109] By "wild type" or "WT" or "native", as used herein, is meant an amino acid sequence or nucleotide sequence that exists in nature, including allelic variants. Protein, polypeptide, antibody, immunoglobulin, IgG WT, etc. has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0110] By "variant Fc region", as used herein, is meant an Fc sequence that differs from such wild-type Fc sequence by modification of at least one amino acid. An Fc variant may refer to the Fc polypeptide itself, compositions comprising an Fc variant polypeptide, or an amino acid sequence.

[0111] In some embodiments, one or more amino acid modifications are made to one or more CDR regions of an antibody (Ab79 or Ab19). Typically, only 1 or 2 or 3 amino acids are changed in any one CDR region, and typically no more than 4, 5, 6, 7, 8, 9, or 10 changes are made in a set of CDR regions. However, it should be understood that any combination of 0, 1, 2 or 3 substitutions in any CDR region can be independently and optionally combined with any other substitution.

[0112] In some cases, amino acid modifications in CDR regions are referred to as "affinity maturation". An affinity-matured antibody is an antibody that has one or more changes in one or more CDR regions that results in an improvement in the affinity of the antibody for the antigen compared to the parent antibody that does not have the change(s). In some cases, although rare, it may be desirable to reduce the affinity of an antibody for its antigen, but this is generally not preferred.

[0113] Affinity maturation can be performed to increase the binding affinity of an antibody for an antigen by at least about 10% to 50-100-150% or more, or 1 to 5 times, compared to the "parent" antibody. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are prepared using known techniques. See, for example, Marks et al., 1992, Biotechnology 10: 779-783, which describes affinity maturation by heavy chain (VH) and light chain (VL) variable domain shuffling. Random mutagenesis of residues in the CDR region and/or framework region is described, for example, in Barbas, et al. 1994 Proc. Nat. Acad. Sci, USA 91: 3809-3813; Shier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, J. Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154(7): 3310-9; and Hawkins et al, 1992, J. Mol. Biol. 226:889-896.

[0114] Alternatively, amino acid modifications can be made in one or more of the CDR regions of the antibodies of the invention that are "silent", eg, that do not significantly change the affinity of the antibody for the antigen. They may be made for a number of reasons, including optimization of expression (as may be done for nucleic acids encoding antibodies of the invention).

[0115] Thus, within the scope of the definition of CDR regions and antibodies according to the invention, variants of CDR regions and antibodies are included; that is, the antibodies of the invention may include amino acid modifications in one or more of the Ab79 and Ab19 CDR regions. In addition, as provided below, amino acid modifications can also be independently and optionally made in any region outside the CDR regions, including framework and constant regions.

[0116] In some embodiments, Ab79 and Ab19 antibody variants specific for human CD38 (SEQ ID NO: 1) and cynomolgus CD38 (SEQ ID NO: 2) are described. This antibody consists of six CDRs, where each CDR of this antibody may be different from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 0, 1, or 2 amino acid substitutions. In other embodiments, the CD38 antibody variant consists of six CDRs, where each CDR of the antibody may be different from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 with 0, 1, or 2 amino acid substitutions.

[0117] In some embodiments, the anti-CD38 antibodies of the invention consist of a variant Fc domain. As is known in the art, the Fc region of an antibody interacts with a number of Fc receptors and ligands, conferring a number of important functional properties referred to as effector functions. These Fc receptors include, but are not limited to (in humans) Fc-RI (CD64), including Fc-RIA, Fc-RIB, and Fc-RIC isoforms; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158, correlated with antibody-dependent cellular cytotoxicity (ADCC)) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), FcRn (neonatal receptor), C1q (complement protein involved in complement-dependent cytotoxicity (CDC)) and FcRn (neonatal receptor affecting serum half-life). Suitable modifications can be made in one or more positions as generally described, for example, in US patent application 11/841,654 and references cited therein, US 2004/013210, US 2005/0054832, US 2006/0024298, US 2006 /0121032, US 2006/0235208, US 2007/0148170, USSN 12/341769, US Pat. No. 6,737,056, US Pat. No. 7,670,600, US Pat. binding to Fc receptors.

[0118] In addition to the modifications described above, other modifications may be made. For example, molecules can be stabilized by including disulfide bridges connecting the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14: 1239-1245, incorporated by reference in its entirety). In addition, there are many covalent modifications of antibodies that can be made, as described below.

[0119] Covalent modifications of antibodies are included in the scope of the present invention, and usually, but not always, they are carried out post-translationally. For example, several types of covalent modifications of an antibody are introduced into a molecule by reacting certain amino acid residues of the antibody with an organic derivatizing agent that is capable of interacting with selected side chains or N- or C-terminal residues.

[0120] Cysteinyl residues are usually reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercur benzoate , 2-chloromercur-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

[0121] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide can also be used; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

[0122] Lysinyl and N-terminal residues are reacted with anhydrides of succinic or other carboxylic acids. Derivatization with these agents causes a change in the charge of lysinyl residues. Other suitable reagents for derivatizing alpha-amino residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione and transaminase-catalyzed reaction with glyoxylate.

[0123] Arginyl residues are modified by reaction with one or more standard reagents, which include phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under alkaline conditions due to the high pKa value of the guanidine functional group. In addition, these reagents can interact with lysine groups, as well as the arginine epsilon-amino group.

[0124] Specific modification of tyrosyl residues can be performed, with particular interest in introducing spectral labels on tyrosyl residues, by reaction with aromatic diazonium compounds or tetranitromethane. Most often, N-acetylimidazole and tetranitromethane are used to obtain O-acetyltyrosyl compounds and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125I or 131I to produce labeled proteins for use in radioimmunoassay, with the chloramine T method described above being suitable.

[0125] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N-R'), where R and R' are optionally different alkyl groups such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonium-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues are converted into asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0126] Derivatization with bifunctional agents is suitable for crosslinking antibodies to a water-insoluble carrier matrix or surface for use in various methods in addition to the methods described below. Commonly used crosslinkers include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example 4-azidosalicylic acid esters, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidyl propionate), and difunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate give photoactivated intermediates that can crosslink in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates are described in US Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440, which are incorporated herein by reference in their entirety, are used to immobilize proteins.

[0127] Glutaminyl and asparaginyl residues are often deamidated to form the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated in a moderately acidic environment. Any form of these residues is within the scope of the present invention.

[0128] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983] incorporated herein by reference in its entirety), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0129] In addition, as will be apparent to those skilled in the art, labels (including fluorescent, enzymatic, magnetic, radioactive, etc., can be introduced into antibodies (as well as other compositions according to the invention)).

[0130] GLYCOSYLATION

[0131] Another type of covalent modification are glycosylation changes. In another embodiment, the antibodies disclosed herein may be modified to include one or more modified glycoforms. By "modified glycoform" as used herein is meant a carbohydrate composition that is covalently attached to an antibody, wherein said carbohydrate composition is chemically different from the carbohydrate composition of the parent antibody. The preferred form of the modified glycoform is afucosylation, which has been shown to correlate with increased ADCC function, presumably as a result of tighter binding to the FcγRIIIa receptor.In this context, "afucosylation" means that most of the antibody produced in cells -hosts, is essentially free of fucose, e.g., 90-95-98% of antibodies produced do not have a significant amount of fucose as a component of the carbohydrate group of the antibody (usually attached to N297 in the Fc region). These antibodies typically exhibit at least 50% or greater affinity for the FcγRIIIa receptor.

[0132] Modified glycoforms can be prepared by various methods known in the art (Umana et al., 1999, Nat Biotechnol 17: 176-180; Davies et al., 2001, Biotechnol Bioeng 74: 288-294; Shields et al. , 2002, J Biol Chem 277: 26733-26740; Shinkawa et al., 2003, J Biol Chem 278: 3466-3473; US 6.602.684; USSN 10/277.370; USSN 10/113.929; /29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1, incorporated herein by reference in its entirety; (Potelligent® technology [Biowa, Inc., Princeton, NJ]; GlycoMAb® glycosylation engineering technology [Glycart Biotechnology AG, Zurich, Switzerland]).Many of these technologies are based on the control of the level of fucosylated and/or branched oligosaccharides covalently attached to the Fc region, for example, by expression of IgG in various organisms or cell lines, created or otherwise obtained (for example, CHO Lec- 13 or YB2/0 rat hybridoma cells), by regulation of enzymes, in involved in the glycosylation pathway (eg, FUT8 [α1,6-fucosyltransferase] and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying the carbohydrate(s) after IgG expression. For example, the "modified sugar antibody" or "SEA technology" developed by Seattle Genetics is to add modified saccharides that inhibit fucosylation during synthesis; see, for example, 20090317869 incorporated by reference in its entirety. The modified glycoform generally refers to another carbohydrate or oligosaccharide; thus, the antibody may include a modified glycoform.

[0133] Alternatively, a modified glycoform may refer to an IgG variant that includes a different carbohydrate or oligosaccharide. As is known in the art, glycosylation profiles may depend on the sequence of the protein (eg, the presence or absence of specific glycosylated amino acid residues discussed below) or the host cell or organism in which the protein is synthesized. Specific expression systems are discussed below.

[0134] Peptide glycosylation is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate group to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognition sequences for the enzymatic attachment of a carbohydrate molecule to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the addition of one of the sugars, N-acetylgalactosamine, galactose or xylose, to a hydroxyamino acid, usually serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0135] The addition of glycosylation sites to an antibody is conveniently accomplished by changing the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Said change can also be made by adding or replacing one or more serine or threonine residues in the original sequence (for O-linked glycosylation sites). For convenience, the amino acid sequence of an antibody is preferably altered by changes at the DNA level, in particular by mutating the DNA encoding the target polypeptide at preselected bases, resulting in the formation of codons that will be translated into the desired amino acids.

[0136] Another way to increase the number of carbohydrate groups of an antibody is to chemically or enzymatically combine glycosides with a protein. These procedures have the advantage that they do not require the production of a protein in the host cell that is capable of N- and O-linked glycosylation. Depending on the coupling method used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine sulfhydryl groups, (d) free hydroxyl groups such as as hydroxyl groups of serine, threonine or hydroxyproline, (e) aromatic residues such as aromatic residues of phenylalanine, tyrosine or tryptophan, or (f) glutamine amide group. These methods are described in WO 87/05330 and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, which are incorporated by reference in their entirety.

[0137] Removal of carbohydrate groups present in the parent antibody (eg, post-translationally) can be done chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to a trifluoromethanesulfonic acid compound or equivalent. This action leads to the elimination of most or all of the sugars except for the binding sugar (N-acetylglucosamine or N-acetylgalactosamine), while the polypeptide remains intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131, which are incorporated by reference in their entirety. Enzymatic cleavage of carbohydrate groups in polypeptides can be performed using various endo- and exoglycosidases, as described in Thotakura et al., 1987, Meth. Enzymol. 138:350, which is incorporated by reference in its entirety. Glycosylation at potential glycosylation sites can be prevented by using the tunicamycin compound as described in Duskin et al., 1982, J. Biol. Chem. 257:3105, which is incorporated by reference in its entirety. Tunikamicin blocks the formation of protein-N-glycosidic bonds.

[0138] Another type of covalent modification of an antibody involves linking the antibody to various non-protein polymers, including, without limitation, various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, as described, for example, in Nektar Therapeutics PEG Catalog 2005-2006 (available on the web). Nektar website), US Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, which are incorporated by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made at various positions in the antibody to facilitate attachment of polymers such as PEG. See, for example, U.S. 2005/0114037 A1, which is incorporated by reference in its entirety.

[0139] CERTAIN CDR AND VARIABLE REGION EMBODIMENTS

[0140] The present invention proposes a number of antibodies, each of which has a specific set of CDR regions (including, as described above, some amino acid substitutions). As described above, antibodies can be defined as sets of 6 CDRs, variable regions, or full length heavy and light chains including constant regions. In addition, as described above, amino acid substitutions can also be made. Generally, in the context of changes in CDR regions, due to the relatively short length of CDR regions, amino acid modifications are usually described by the number of amino acid modifications that can be made. While this also applies to the discussion of the number of amino acid modifications that can be introduced into variable, constant, or full length sequences, in addition to the number of changes, these changes can also be defined in "% identity". Thus, as described herein, the antibodies included in the invention are 80%, 85%, 90%, 95%, 98%, or 99% identical to the SEQ ID NOs listed herein.

[0141] With respect to the Ab79 antibody, the set of CDR regions is as follows: the three heavy chain CDR regions include SEQ ID NO: 3 (HCDR1) HCDR1, SEQ ID NO: 4 (HCDR2), and SEQ ID NO: 5 (HCDR3), and the three light chain CDR regions include SEQ ID NO: 6 (LCDR1), SEQ ID NO: 7 (LCDR2), and SEQ ID NO: 8 (LCDR3).

[0142] With respect to Ab19, the set of CDR regions is as follows: HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14) and HCDR3 (SEQ ID NO: 15), and LCDR1 (SEQ ID NO: 16) , LCDR2 (SEQ ID NO: 17) and LCDR3 (SEQ ID NO: 18).

[0143] Explicitly excluded from the present invention are the antibodies SEQ ID NOs: 24 and 25 (Control 1 heavy and light chains) and SEQ ID NOs: 26 and 27 (Control 2 heavy and light chains). It should be noted that these antibodies are not cross-reactive with cynomolgus CD38, as discussed below.

[0144] The antibodies of the invention are cross-reactive with human and cynomolgus CD38 and thus are cross-species cross-reactive antibodies. A "cross-species-cross-reactive antibody" is an antibody that has a binding affinity for a first species mammalian antigen that is nearly the same as the binding affinity for the second species mammalian homologue of that antigen. Cross-species cross-reactivity can be expressed, for example, as the ratio of the KD of an antibody to a mammalian antigen of the first species to the KD of the same antibody to a mammalian homologue of that antigen of the second species, where the ratio is 1.1, 1.2, 1.3, 1.4 , 1.5, 2, 5, 10, 15, up to 20. Alternatively or additionally, an antibody has "interspecies cross-reactivity" when it exhibits therapeutic or diagnostic efficacy when administered to a second species. Thus, in this case, the antibodies of the invention are cross-reactive with cynomolgus CD38, exhibit preclinical efficacy when administered to cynomolgus primates, and are thus considered to be cross-reactive.

[0145] In some embodiments, antibodies are provided that compete with antibodies of the invention (eg, Ab79 and/or Ab19) for binding to human CD38 and/or cynomolgus CD38, but do not include either BM1 or BM2. Competition for binding to CD38 or a portion of CD38 between two or more anti-CD38 antibodies can be determined using any suitable technique as is known in the art.

[0146] Competition as used herein refers to any detectably significant reduction in the ability of an antibody of the invention (eg, Ab79 or Ab19) to bind to its specific binding partner, eg, CD38, in the presence of a test compound. Typically, competition means at least about 10-100% reduction in the binding of an antibody of the invention to CD38 in the presence of a competitor, as measured by standard methods such as ELISA or Biacore® assay. Thus, for example, criteria for competitiveness can be established that show at least about 10% relative inhibition; at least about 15% relative inhibition; or at least about 20% relative inhibition before the antibody is considered sufficiently competitive. In cases where epitopes belonging to competing antibodies are closely spaced on the antigen, competition may be marked by greater than about 40% relative inhibition of CD38 binding (e.g., at least about 45% inhibition, such as at least about 50% inhibition, e.g. at least about 55% inhibition, such as at least about 60% inhibition, such as at least about 65% inhibition, such as at least about 70% inhibition, such as at least about 75% inhibition, such as at least about 80% inhibition, such as at least about 85% inhibition, such as at least about 90% inhibition, such as at least about 95% inhibition or higher relative inhibition).

[0147] In some cases, one or more components of competitive binding assays are labeled as discussed below in the context of diagnostic applications.

[0148] There may also be competition between CD38 antibodies for more than one CD38 epitope and/or part of CD38, for example, in conditions where the binding properties of the antibody with respect to a particular region of CD38 are retained in its fragments, for example, in the case of a well-presented a linear epitope located in the various fragments tested, or a conformational epitope that is present in sufficiently large fragments of CD38, as in CD38.

[0149] Evaluation of competition typically includes evaluation of relative inhibitory binding using an antibody of the invention, CD38 (human and/or cynomolgus) and test molecule. Test molecules can include any molecule, including other antibodies, small molecules, peptides, and so on. The compounds are mixed in amounts that are sufficient to allow a comparison to be made that provides information on the selectivity and/or specificity of the molecules under study compared to other molecules present.

[0150] The amounts of test compound, CD38, and antibodies of the invention may vary. For example, ELISA scores require approximately 5-50 μg (e.g., approximately 10-50 μg, approximately 20-50 μg, approximately 5-20 μg, approximately 10-20 μg, etc.) of anti-CD38 antibody and/or CD38 targets to assess if competition exists. The conditions must also be suitable for linking. Generally, physiological or near physiological conditions (eg, temperatures of about 20-40° C., pH of about 7-8, etc.) are suitable for binding an anti-CD38:CD38 antibody.

[0151] Often the competition is characterized by a significantly higher relative inhibition than about 5%, as determined using ELISA and/or FACS analysis. It may be desirable to establish a higher relative inhibition threshold as a criterion/determinant of what is an appropriate level of competition in a particular context (e.g., when a competitive assay is used to select or screen for new antibodies designed with a putative function of blocking the binding of another peptide or molecule that binds CD38 (eg, natural binding partners of CD38, such as CD31, also called CD31 antigen, EndoCAM, GPIIA, PECAM-1, platelet/endothelial cell adhesion molecule, or naturally occurring anti-CD38 antibodies).

[0152] In some embodiments, an anti-CD38 antibody of the present invention specifically binds to one or more residues or regions in CD38, but also does not cross-react with other proteins with homology to CD38, such as BST-1 (bone marrow stromal cell antigen). 1) and Mo5, also called CD157.

[0153] Generally, no cross-reactivity means less than about 5% relative competitive inhibition between molecules as assessed by ELISA and/or FACS analysis using sufficient numbers of molecules under appropriate assay conditions.

[0154] INHIBITION OF CD38 ACTIVITY

[0155] The disclosed antibodies may find use in blocking ligand-receptor interaction or inhibiting receptor component interaction. Anti-CD38 antibodies of the invention may be "blocking" or "neutralizing". "Neutralizing antibody" refers to an antibody whose binding to CD38 results in inhibition of the biological activity of CD38, such as its ability to interact with ligands, enzymatic activity, signaling ability and, in particular, the ability to cause activation of lymphocytes. Inhibition of CD38 biological activity can be assessed using one or more of several standard in vitro or in vivo assays known in the art (see Examples below).

[0156] "Inhibits binding" or "blocks binding" (eg, when referring to inhibiting/blocking the binding of a CD38 binding partner to CD38) includes both partial and complete inhibition/blocking. Inhibiting/blocking the binding of a CD38 binding partner to CD38 can reduce or alter the normal level or type of cell signaling that is present when a CD38 binding partner binds to CD38 without inhibition or blocking. Inhibition and blocking are also expected to include any measurable reduction in the binding affinity of a CD38 binding partner to CD38 in contact with an anti-CD38 antibody compared to a ligand not in contact with an anti-CD38 antibody, such as blocking the binding of a CD38 binding partner to CD38. by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0157] The disclosed anti-CD38 antibodies can also inhibit cell growth. "Inhibits growth" includes any measurable reduction in growth of cells in contact with an anti-CD38 antibody compared to the growth of the same cells not in contact with an anti-CD38 antibody, such as inhibition of cell culture growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.

[0158] In some embodiments, the disclosed anti-CD38 antibodies are capable of eliminating activated lymphocytes, plasmablasts, and plasma cells. "Elimination" as used herein means a measurable reduction in blood levels (eg, as tested in cynomolgus monkeys) of activated lymphocytes and/or plasma cells compared to untreated animals. Typically, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% elimination is observed. As shown in the Examples below, in addition, one particular advantage that the antibodies of the present invention exhibit is the resilience of these cells after dosing; that is, as is known for some therapies (eg, using anti-CD20 antibodies), cell depletion can continue for long periods of time, causing undesirable side effects. As shown herein, the effect on activated lymphocytes and/or plasma cells is reversible.

[0159] METHODS FOR PRODUCING ANTIBODIES OF THE INVENTION

[0160] The present invention also provides methods for producing disclosed anti-CD38 antibodies. These methods include culturing a host cell containing the isolated nucleic acid(s) encoding the antibodies of the invention. As will be appreciated by those skilled in the art, this can be done in a variety of ways, depending on the nature of the antibody. In some embodiments, when the antibodies of the invention are full-length conventional antibodies, for example, a heavy chain variable region and a light chain variable region under conditions under which the antibody is produced and can be isolated.

[0161] In general, nucleic acids are provided that encode antibodies of the invention. Such polynucleotides encode the variable and constant regions of each heavy and light chain, although the present invention also contemplates other combinations in accordance with the compositions described herein. The present invention also provides oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to those polynucleotides.

[0162] Polynucleotides can be in the form of RNA or DNA. Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogues and synthetic DNA are included within the scope of the present invention. The DNA may be double-stranded or single-stranded, and in case the DNA is single-stranded, may be a coding (sense) strand or a non-coding (antisense) strand. The coding sequence that encodes a polypeptide may be identical to the coding sequence provided herein, or may be a different coding sequence which, due to redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA provided herein.

[0163] In some embodiments, the nucleic acid(s) encoding the antibodies of the invention are included in expression vectors, which may be extrachromosomal or designed to integrate into the genome of the host cell into which they are introduced. The expression vectors may contain any number of suitable regulatory sequences (including, but not limited to, transcription and translation control sequences, promoters, ribosome binding sites, enhancers, origins of replication, etc.) or other components (selection genes, etc.) that are known to be operably linked in the state of the art. In some cases, two nucleic acids are used and each is placed in a separate expression vector (eg, heavy chain in the first expression vector, light chain in the second expression vector) or, alternatively, they can be placed in a single expression vector. It will be apparent to those skilled in the art that the design of the expression vector(s), including the choice of regulatory sequences, may depend on such factors as the choice of host cell, the level of expression of the target protein, and so on.

[0164] In general, nucleic acids and/or expression can be introduced into a suitable host cell to produce a recombinant host cell using any method appropriate to the selected host cell (e.g., transformation, transfection, electroporation, infection), when In this, the nucleic acid molecule(s) is operably linked to one or more expression control elements (eg, in a vector, in a construct created by cellular processes, integrated into the genome of the host cell). The resulting recombinant host cell can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a suitable culture medium supplemented with suitable salts, growth factors, antibiotics, nutritional supplements, etc.) resulting in the production of the polypeptide(s). In some cases, heavy chains are produced in one cell and light chains in another.

[0165] Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, VA, including, but not limited to, Chinese Hamster Ovary cells ( CHO), HEK 293 cells, NSO cells, HeLa cells, newborn hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg Hep G2), as well as many other cell lines. Non-mammalian-derived cells, including but not limited to bacterial, yeast, insect, and plant cells, can also be used to express recombinant antibodies. In some embodiments, the antibodies can be produced in transgenic animals such as cows or chickens.

[0166] General molecular biology techniques for antibody expression, purification, and screening are described, for example, in Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001 and 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5: 683-689 ; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2: 339-76; and Morrison, S. (1985) Science 229: 1202.

[0167] USES AND INDICATIONS

[0168] Once produced, the antibodies of the invention find use in a variety of fields, including the diagnosis of CD38-associated diseases and their treatment.

[0169] CD38 RELATED STATES

[0170] In one aspect of the invention, methods are provided for diagnosing and treating a condition associated with inflammation and immunopathological diseases, in particular, diseases associated with activated lymphocytes. As shown herein, CD38 is expressed in immature hematopoietic cells, downregulated in mature cells, and re-expressed at a high level in activated lymphocytes and plasma cells. For example, CD38 is highly expressed in activated B cells, plasma cells, activated CD4+ T cells, activated CD8+ T cells, NK cells, NKT cells, mature dendritic cells (DCs), and activated monocytes.

[0171] The anti-CD38 therapeutic antibodies of the present invention bind to CD38-positive cells, resulting in the elimination of these cells, such as activated lymphocytes, through various mechanisms of action, including CDC and ADCC pathways.

[0172] Thus, any autoimmune disease in which there is either an increased expression of CD38, or an increased number of cells expressing CD38, as a component of the disease, can be treated using antibodies according to the invention. These include, but are not limited to, allogeneic islet transplant rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune adrenal disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune immune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac disease dermatitis, chronic fatigue syndrome with immune dysfunction, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Goodpasture's syndrome, transpl reaction anti-host antate (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, parenchymal organ transplant rejection, stiffness syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis , uveitis, vasculitis such as vasculitis in dermatitis herpetiformis, vitiligo and Wegener's granulomatosis.

[0173] In some embodiments, a specific use of the present antibodies is in the diagnosis and/or treatment of a number of diseases, including, without limitation, autoimmune diseases, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) , inflammatory bowel disease (IBD), ulcerative colitis, and graft-versus-host disease.

[0174] Thus, for example, patients with high plasma cell levels, such as SLE patients who have high plasma cell levels, as well as RA patients who have been shown to be refractory to treatment using therapy based on CD20.

[0175] It is known in the art that certain conditions are associated with cells that express CD38, and that some conditions are associated with overexpression, high expression density, or increased expression of CD38 on cell surfaces. Whether or not a cell population expresses CD38 can be determined using methods known in the art, such as flow cytometry to determine the percentage of cells in a given population that are labeled with an antibody that specifically binds CD38, or immunohistochemical assays, as described generally below, for diagnostic applications. For example, a population of cells in which CD38 expression is found in approximately 10-30% of cells may be considered weakly positive for CD38; and a population of cells in which CD38 expression is found in more than about 30% of cells can be considered positive for CD38 (as in Jackson et al. (1988), Clin. Exp. Immunol. 72: 351-356), although other criteria can be used to determine if a cell population expresses CD38. Cell surface expression density can be determined using methods known in the art, such as, for example, flow cytometry measurement of the average fluorescence intensity of cells that have been fluorescently labeled using antibodies that specifically bind CD38.

[0176] ANTIBODY COMPOSITIONS FOR IN VIVO ADMINISTRATION

[0177] Antibody preparations used in accordance with the present invention are prepared for storage by mixing an antibody having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980 ]), in the form of lyophilized preparations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0178] The dosage form herein may also contain more than one active compound that is necessary for the particular indication being treated, preferably those that have additional activities that do not adversely affect each other. For example, it may be desirable to obtain antibodies with other specificities. Alternatively or additionally, the composition may contain a cytotoxic agent, a cytokine, a growth inhibitor, and/or a small molecule antagonist. Such molecules are preferably present in combination in amounts that are effective for the intended purpose.

[0179] The active substances can also be enclosed in microcapsules made, for example, by coacervation or interfacial polymerization methods, for example, hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0180] Dosage forms to be used for in vivo administration must be sterile or nearly sterile. This is easily achieved by filtration through sterilizing filter membranes.

[0181] Sustained release formulations can be made. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of molded articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactides (US Patent 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of a copolymer of lactic acid-glycolic acid and leuprolide acetate), and poly-D-(-)-3-hydroxy-butyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules for over 100 days, some hydrogels release proteins for shorter periods of time.

[0182] When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be developed for stabilization, depending on the mechanism used. For example, if it is found that the mechanism of aggregation is the formation of intermolecular S-S bonds as a result of thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, controlling moisture content, using appropriate additives, and developing special polymer matrix compositions.

[0183] METHODS OF ADMINISTRATION

[0184] Antibodies of the invention are administered to an individual according to known methods, such as intravenous administration as a bolus or continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation way. Intravenous or subcutaneous administration of the antibody is preferred.

[0185] TREATMENT METHODS

[0186] In the methods of the invention, therapy is used to provide a positive therapeutic response to a disease or condition. By "positive therapeutic response" is meant an improvement in a disease or condition and/or a reduction in symptoms associated with the disease or condition. For example, a positive therapeutic response may refer to one or more of the following improvements in a disease: (1) reduction in the number of neoplastic cells; (2) increased neoplastic cell death; (3) inhibition of neoplastic cell survival; (5) inhibiting (ie, slowing down to some extent, preferably stopping) tumor growth; (6) increase patient survival; and (7) some relief from one or more symptoms associated with the disease or condition.

[0187] Positive therapeutic responses for any disease or condition can be determined according to standardized response criteria established for that disease or condition. Tumor response can be assessed by changes in tumor morphology (i.e. total tumor mass, tumor size, etc.) using screening methods such as magnetic resonance imaging (MRI), X-ray imaging, computed tomography ( CT), bone scintigraphy, endoscopy, and tumor biopsy sampling, including bone marrow aspiration (BMA), and circulating tumor cell counts.

[0188] In addition to these positive therapeutic responses, the individual undergoing therapy may experience a positive effect in improving the symptoms associated with the disease.

[0189] Thus, in the case of B-cell tumors, the individual may experience a decrease in so-called B-symptoms, i.e. night sweats, fever, weight loss and/or urticaria. In the case of precancerous conditions, therapy with an anti-CD38 therapeutic agent can block the development and/or increase the time to development of an associated malignant condition, for example, the development of multiple myeloma, in individuals suffering from undetermined monoclonal gammopathy (MGUS).

[0190] Improvement in disease can be characterized as a complete response. By "complete response" is meant the absence of clinically detectable disease with normalization of any previously abnormal radiographic findings, bone marrow and cerebrospinal fluid (CSF), or abnormal monoclonal protein in the case of myeloma .

[0191] Such a response may persist for at least 4-8 weeks, or sometimes 6-8 weeks, after treatment in accordance with the methods of the invention. Alternatively, improvement in disease may be categorized as partial response. By "partial response" is meant at least approximately 50% reduction in all measurable tumor mass (i.e., the number of malignant cells present in an individual's body, or the measured mass of tumors, or the amount of abnormal monoclonal protein) in the absence of new lesions that may persist within 4-8 weeks or 6-8 weeks.

[0192] Treatment according to the present invention includes a "therapeutically effective amount" of drugs used. "Therapeutically effective amount" refers to an effective amount, in doses and for periods of time required to achieve the desired therapeutic result.

[0193] A therapeutically effective amount may vary depending on such factors as the pathological condition, age, sex and weight of the patient, as well as the ability of drugs to cause the desired response in the patient. A therapeutically effective amount is also one in which any toxic or unwanted effect of the antibody or antibody fragment is offset by a therapeutically beneficial effect.

[0194] A "therapeutically effective amount" in an anticancer therapy can also be measured by its ability to stabilize the progression of a disease. The ability of a compound to eliminate B cells can be assessed in an animal model system to predict efficacy in human autoimmune diseases.

[0195] Alternatively, this property of the composition can be assessed by testing the ability of the compound to deplete B cell populations using in vitro assays known to one of skill in the art. A therapeutically effective amount of a therapeutic compound can deplete populations of B cells or otherwise relieve symptoms in an individual. One of skill in the art will be able to determine such amounts based on such factors as the size of the individual, the severity of the individual's symptoms, and the particular composition or route of administration chosen.

[0196] Application regimens are selected to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over a period of time, or the dose may be proportionally reduced or increased as indicated, depending on the therapeutic situation. Compositions for parenteral administration may be formulated in unit dosage form for ease of administration and uniformity of dose. Unit dosage form as used herein refers to physically discrete units suitable as unit doses for individuals to be treated; each unit contains a predetermined amount of active compound, calculated to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier.

[0197] The specification for unit dosage forms of the present invention is dictated by and directly dependent on: (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations common in the art of formulating active compound formulations for the treatment of sensitivity in patients.

[0198] Effective doses and administration schedules of the anti-CD38 antibodies used in the present invention depend on the disease or condition being treated and can be determined by those skilled in the art.

[0199] An exemplary, non-limiting range for a therapeutically effective amount of an anti-CD38 antibody used in the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example, about 0.1- 20 mg/kg, such as about 0.1-10 mg/kg, such as about 0.5, such as about 0.3, about 1, or about 3 mg/kg. In another embodiment, the antibody is administered at a dose of 1 mg /kg or more, such as a dose of 1 to 20 mg/kg, for example, a dose of 5 to 20 mg/kg, for example, a dose of 8 mg/kg.

[0200] A medical professional of average skill can easily determine and prescribe the required effective amount of the pharmaceutical composition. For example, a physician or veterinarian may begin administering doses of the drug used in a pharmaceutical composition at levels below those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0201] In one embodiment, the CD38 antibody is administered by infusion at a weekly dose of 10 to 500 mg/kg, such as 200 to 400 mg/kg. 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours.

[0202] In one embodiment, the CD38 antibody is administered by slow continuous infusion over an extended period, such as more than 24 hours, as needed to reduce side effects, including toxicity.

[0203] In one embodiment, the CD38 antibody is administered at a weekly dose of 250 mg to 2000 mg, such as, for example, 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg, or 2000 mg, up to 8 times, for example, 4 to 6 times. Administration may be by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours. Such a regimen may be repeated one or more times as needed, for example after 6 months or 12 months. The dose can be determined or adjusted by measuring the amount of a compound of the present invention in the blood upon administration, for example by taking a biological sample and using anti-idiotypic antibodies that target the antigen-binding region of an anti-CD38 antibody.

[0204] In another embodiment, the anti-CD38 antibody is administered once a week for 2-12 weeks, eg for 3-10 weeks, eg for 4-8 weeks.

[0205] In one embodiment, the CD38 antibody is administered with maintenance therapy, such as, for example, once a week for a period of 6 months or more.

[0206] In one embodiment, the anti-CD38 antibody is administered according to a schedule comprising one infusion of the anti-CD38 antibody followed by an infusion of the radioisotope-conjugated anti-CD38 antibody. The scheme can be repeated, for example, after 7-9 days.

[0207] As non-limiting examples, treatment according to this invention can be provided as a daily dose of antibody in an amount of approximately 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg/kg per day on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or alternatively at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 after the start of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination of them.

[0208] DIAGNOSTIC USE

[0209] The proposed antibodies against CD38 also find use in in vitro or in vivo imaging of tumors or autoimmune pathological conditions associated with CD38. In some embodiments, the antibodies described herein are used for both diagnosis and treatment, or only for diagnosis. In the case where anti-CD38 antibodies are used for diagnosis and for treatment, some embodiments are based on two different anti-CD38 antibodies for two different epitopes, with the diagnostic antibody not competing with the therapeutic antibody for binding, although in some cases the same antibody can be used for both. For example, in some instances, Ab19 is used diagnostically (typically labeled as discussed below) while Ab79 is used therapeutically, or vice versa. the antibody is labeled as described herein. In addition, the therapeutic and diagnostic antibody composition can also be co-administered with other drugs as provided herein.

[0210] In many embodiments, the diagnostic antibody is labeled. By "labeled" in the present description means that one or more elements, isotopes or chemical compounds are attached to the antibodies disclosed herein to ensure detection during a screening or diagnostic procedure. Generally, labels fall into several classes: a) immune labels, which can be an epitope included as a fusion partner that is recognized by an antibody, b) isotopic labels, which can be radioactive or heavy isotopes, c) small molecule labels, which may include fluorescent and colorimetric dyes (such as fluorescein isothiocyanate, also known as FITC) or molecules such as biotin which provide other labeling methods, and d) labels such as particles (including vials for ultrasonic labeling) or paramagnetic labels which provide visualization of the body. Labels can be introduced into antibodies at any position and can be introduced in vitro or in vivo during protein expression, as is known in the art.

[0211] Diagnosis can be performed either in vivo, by administering a diagnostic antibody that allows whole body imaging as described below, or in vitro, on a sample or biological sample obtained from a patient. "Sample" or "biological sample" as used herein includes any number of materials including, but not limited to, bodily fluids (including, but not limited to, whole blood, plasma, urine, serum, lymph, saliva, anal and vaginal secretions). , sweat and semen), as well as tissue samples obtained from biopsies of the relevant tissues. In some embodiments, the biological sample is a whole blood sample.

[0212] In some embodiments, in vivo imaging is performed, including, but not limited to, ultrasound, CT scans, X-ray, MRI, and PET scans, as well as optical techniques that use optical labels for tumors near the body surface.

[0213] In vivo imaging of diseases associated with CD38 can be performed using any suitable technique. For example, ⁹⁹ Tc labeling or labeling with another β-emitting isotope can be used to label anti-CD38 antibodies. Variations of this technique may include the use of magnetic resonance imaging (MRI) to improve imaging compared to methods that use a gamma camera. Similar methods and principles of immunoscintigraphy are described, for example, in Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum Press 1988), Chase, "Medical Applications of Radioisotopes", in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro et al. , (eds.), pp. 624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies", in Biotechnology And Pharmacy 227-49, Pezzuto et al., (eds.) (Chapman & Hall 1993).

[0214] In one embodiment, the present invention provides an in vivo imaging method wherein an anti-CD38 antibody is conjugated to a detection aid, the conjugated antibody is administered to a recipient, e.g., by injection into the blood stream, and the presence and location of the labeled antibody in the recipient's body is determined. Using this technique and any other diagnostic method provided herein, the present invention provides a method for screening for the presence of disease-associated cells in a patient or in a biological sample obtained from a patient.

[0215] For diagnostic imaging, radioisotopes can be linked to an anti-CD38 antibody either directly or indirectly via an intermediate functional group. Useful functional intermediates include chelating agents such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example, US Pat. No. 5,057,313). In such diagnostic assays involving radioisotope-conjugated anti-CD38 antibodies, the dose of anti-CD38 conjugated antibody delivered to the patient is generally kept as low as possible by selecting the isotope with the best combination of minimum half-life, minimum retention time in the body, and minimum amount of isotope, which provides detection and accurate measurement.

[0216] In addition to radioisotopes and radiopaque agents, diagnostic methods can be performed using anti-CD38 antibodies that are conjugated with dyes (such as the biotin-streptavidin complex), contrast agents, fluorescent compounds (such as fluorescein isothiocyanate, also known as FITC) or molecules and contrast agents (eg, paramagnetic ions) for magnetic resonance imaging (MRI) (see, for example, US patent 6,331,175, which describes MRI methods and the production of antibodies conjugated to MRI contrast agents). Such diagnostic/detecting agents may be selected from those used in magnetic resonance imaging and fluorescent compounds.

[0217] To load an anti-CD38 antibody with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which various chelating groups are attached to bind the ions. Such a tail may be a polymer such as polylysine, a polysaccharide or other derivatized or derivatized chain having side groups to which chelating groups can be attached, such as, for example, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes and the like, known to be useful for this purpose.

[0218] Chelates can be coupled to anti-CD38 antibodies using standard chemistry methods. The chelate is typically coupled to the CD38 antibody with a moiety that allows for bonding to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal crosslinking.

[0219] Examples of potentially useful metal chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs used with diagnostic isotopes in the general energy range of 60-4000 keV such as ¹²⁵ I, ¹²³ I, ¹²⁴ I, ⁶² Cu, ⁶⁴ Cu, ¹⁸ F, ¹¹¹ In, ⁶⁷ Ga, ⁹⁹ Tc, ⁹⁴ Tc, ¹¹ C, ¹³ N, ¹⁵ O and ⁷⁶ Br, for radioisotope imaging.

[0220] The labels include a radionuclide, a radiopaque agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an echo contrast agent, and a photosensitive agent. Such diagnostic agents are known and any such known diagnostic agent may be used. Non-limiting examples of diagnostic substances may include a radionuclide such as ¹¹⁰ In, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Zr, ⁹⁴ mTc, ⁹⁴ Tc, ⁹⁹ mTc, ¹²⁰ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ^154-158 Gd, ³² P, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁵¹ Mn, ⁵² mMn, ⁵⁵ Co, ⁷² As, ⁷⁵ Br, ⁷⁶ Br, ⁸² mRb, ⁸³ Sr or other sources of γ-, β-radiation or positrons.

[0221] Suitable paramagnetic ions may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium ( III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III). Metal-based contrast agents may include lanthanum (III), gold (III), lead (II), or bismuth (III).

[0222] Echo contrast agents may include liposomes, such as gas-filled liposomes. Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds and thallium compounds.

[0223] These and similar chelates, in combination with non-radioactive metals such as manganese, iron and gadolinium, can be used in MRI diagnostic methods in combination with antibodies against CD38. Macrocyclic chelates such as NOTA, DOTA and TETA can be used with various metals and radiometals, most preferably gallium, yttrium and copper radionuclides, respectively. Such metal chelate complexes can be made very stable by selecting the ring size for the target metal. Other ring type chelates, such as macrocyclic polyesters, which are of interest for stable binding of radionuclides such as ²²³ Ra, can also be used in diagnostic methods.

[0224] Anti-CD38 antibodies can also be used, for example, to detect the expression of an antigen of interest in specific CD38-expressing cells, such as plasmablasts and plasma cells, tissues, or serum. In some embodiments, the anti-CD38 antibodies described herein can be used when expression of an antigen of interest is detected in CD38-expressing plasmablasts and plasma cells. For diagnostic applications, the antibody will typically be labeled with a detectable group for in vitro assays. As will be appreciated by those skilled in the art, there are a number of labels suitable for use in in vitro studies. Suitable dyes for use in this aspect of the invention include, but are not limited to, fluorescent lanthanide complexes, including europium and terbium complexes, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl coumarins, quantum dots (also referred to as "nanocrystals" ; see US Patent Application 09/315,584 incorporated herein by reference), pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue™, Texas red, Cy dyes (Cy3, Cy5, etc.), dyes alexa (including Alexa, phycoerythin, BODIPY, and others described in the 6th edition of the Molecular Probes Handbook, Richard P. Haugland, hereby expressly incorporated by reference.

[0225] Thus, the present invention provides diagnostic anti-CD38 antibody conjugates, wherein the anti-CD38 antibody conjugate is conjugated to a contrast agent (such as for magnetic resonance imaging, computed tomography, or an echo contrast agent) or a radionuclide, which can be, for example, emitting γ-, β-, α-, Auger electrons or positrons by an isotope or detectable molecule (such as fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, etc.). In some embodiments, the diagnostic anti-CD38 antibody conjugate of the present invention may be an anti-CD38 antibody conjugated to fluorescein isothiocyanate, also known as FITC. In some embodiments, the anti-CD38 conjugated antibody may be referred to as a fluorescein-anti-CD38 antibody conjugate. It will be apparent to one of skill in the art that other reagents known in the art may be used to conjugate fluorescein to an anti-CD38 antibody, and that in each such case the resulting conjugate may be referred to as a fluorescein-anti-CD38 antibody conjugate. Such fluorescein-anti-CD38 antibody conjugates as described herein can be used in in vitro assays such as flow cytometry.

[0226] METHODS OF ANALYSIS

[0227] Suitable assay methods for use in connection with the present invention include, but are not limited to, flow cytometry, "Ig" free light chain assays, analysis of mRNA transcripts, and the like. It should be understood that any standard methods known in the art carrying out such methods of analysis can be used in connection with the present invention.

[00159] In some embodiments, the methods described herein can be performed using flow cytometry as a means to determine the number of CD38-expressing cells in a biological sample obtained from a patient. It should be understood that, as used herein, a biological sample obtained from a patient may be a whole blood sample, a serum sample, or a processed blood sample. In some embodiments, the biological sample obtained from the patient is a whole blood sample. It should be understood that a biological sample obtained from a patient may include one or more cell types, including, but not limited to, erythrocytes and any of the cell types shown in FIG. 1. As used herein, "CD38-expressing cells" means any cell that has been shown to express CD38, including, but not limited to, pro-B cells (CD34 ⁺ CD19 ⁺ CD20 ^- ), activated B cells ( CD19 ⁺ CD20 ⁺ ), plasmablasts (CD19 ⁺ CD27 ⁺ ), long-lived plasma cells (CD138 ⁺ CD19 ^- CD20 ^- ), short-lived plasma cells (CD138 ⁺ CD19 ⁺ CD20 ^- ), activated CD4 ⁺ and CD8 ⁺ T cells, NKT- cells (CD3 ⁺ CD56 ⁺ ) and NK cells (CD56 ⁺ CD16 ⁺ ). In addition, CD38 expression was found on lymphoid progenitor cells (CD34 ⁺ CD45RA ⁺ CD10 ⁺ CD19 ^- ), but not on lymphoid stem cells. In addition, increased CD38 expression was noted on mature DCs and activated monocytes.

[0228] It should be understood that flow cytometry can be performed by any method known in the art using any suitable flow cytometry instrument. Such methods include standard sample preparation methods well known in the art in connection with flow cytometry. In some embodiments, the flow cytometry methods are performed using staining methods that facilitate cell sorting in a biological sample. It should be understood that any staining method generally known in the art for use in connection with flow cytometry can be used. In some embodiments, one or more labeled antibodies or antibody conjugates are contacted with a whole blood sample as described herein. It is well known in the art that multiple cellular targets can be counted using multicolor flow cytometry. See, for example, Baumgarth, N and Roeder, M, J Immun Methods (2000) 243: 77-97. In some embodiments, at least one labeled antibody or antibody conjugate can be used in combination with the flow cytometry methods described herein to determine the cells contained in a biological sample obtained from a patient. In some embodiments, at least one labeled antibody or antibody conjugate for use in combination with the flow cytometry methods described herein is a fluorescein-anti-CD38 antibody conjugate. In some embodiments, the fluorescein-anti-CD38 antibody conjugate is Ab19-fluorescein or Ab79-fluorescein.

[0229] In some embodiments, a method for performing a flow cytometry analysis is provided, comprising contacting a biological sample obtained from a patient with a lyse/fix solution (also referred to as a resuspension solution), such as a FACS™ Lyse solution, comprising at least one component, capable of causing lysis of certain cells, such as erythrocytes; and at least one component capable of fixing the sample, such as an aldehyde or an alcohol. alcohols such as methanol or ethanol, which are known to those skilled in the art as precipitating or denaturing fixatives capable of coagulating proteins, or aldehydes such as formaldehyde or glutaraldehyde. It should be understood that formaldehyde may polymerize to paraformaldehyde (PFA), and in the presence of methanol, formaldehyde may not polymerize to paraformaldehyde. It should also be understood that for applications in which alcohol-free formaldehyde is used as a component in the lyse/fix solution, the lyse/fix solution may optionally contain a buffer, such as a PBS buffer. In some embodiments, the biological sample is whole blood. In some embodiments, a patient's whole blood sample may be contacted with a lyse/fix solution, such as FACS™ Lyse solution, to lyse the red blood cells contained in the whole blood sample (see Einwallner, E, Subbasic, A, Strasser, A, et al., J Immun Methods (2013) 390: 127-132 and Tiirikainen, M. Cytometry (1995) 20: 341-348, which are incorporated herein by reference). In some embodiments, a biological sample obtained from a patient is contacted with a FACS™ Lyse solution to form a processed sample prior to freezing the sample for storage. In some embodiments, a biological sample obtained from a patient is contacted with a FACS™ Lyse solution to form a processed sample immediately prior to flow cytometry analysis, without freezing the processed sample for storage. In some embodiments, a biological sample obtained from a patient may be contacted with a FACS™ Lyse solution for about 2 minutes to about 30 minutes. In some embodiments, a biological sample may be contacted with a FACS™ Lyse solution for about 4 minutes. up to about 20 minutes, or from about 5 minutes to about 10 minutes.

[0230] In some embodiments, a processed sample obtained after contacting a biological sample obtained from a patient with a lyse/fix solution, such as a FACS™ Lyse solution, is cooled to a temperature of approximately -20°C or less to obtain a frozen sample. In some embodiments, the implementation of the processed sample is cooled to approximately -80°C. In some embodiments, the processed sample is cooled to approximately -78°C. In some embodiments, the implementation of the processed sample is cooled to approximately -70°C. In some embodiments, the implementation of the processed sample is cooled to approximately -60°C. In some embodiments, the implementation of the processed sample is cooled to approximately -50°C. In some embodiments, the implementation of the processed sample is cooled to approximately -40°C. In some embodiments, the processed sample is cooled to approximately -30°C. In some embodiments, the implementation of the processed sample is cooled to approximately -20°C. In some embodiments, the implementation of the processed sample is cooled to a temperature from approximately -80°C to -20°C. In some embodiments, a treated sample obtained after contacting a biological sample obtained from a patient with a lyse/fix solution, such as FACS™ Lyse solution, is refrigerated for approximately 24 hours or less after contact. In some embodiments, the treated sample is refrigerated for about 12 hours or less after lysis. In some embodiments, the processed sample is cooled for about 6 hours or less.

[0231] After freezing the processed sample, in some embodiments, the implementation of the frozen sample can be heated to approximately room temperature before counting the cells in the biological sample using flow cytometry. In some embodiments, the frozen sample may be warmed to approximately room temperature for approximately 78 hours or less after cooling. In some embodiments, the frozen sample may be warmed to approximately room temperature for approximately 72 hours or less after cooling. In some embodiments, the frozen sample may be warmed to approximately room temperature for approximately 54 hours or less after cooling. In some embodiments, the frozen sample may be warmed to approximately room temperature for approximately 48 hours or less after cooling. In some embodiments, the frozen sample may be warmed to approximately room temperature for approximately 30 hours or less after cooling. In some embodiments, the frozen sample may be warmed to approximately room temperature for approximately 24 hours or less after cooling. In some embodiments, the frozen sample may be warmed to about room temperature for about 72 hours to about 78 hours after cooling. In some embodiments, the frozen sample may be warmed to about room temperature for about 48 hours to about 54 hours after cooling. In some embodiments, the frozen sample may be warmed to about room temperature for about 24 hours to about 30 hours after cooling.

[0232] In some embodiments, the invention provides a method for analyzing CD38-expressing cells in whole blood, comprising: obtaining a sample of whole blood from an individual, where the sample includes red blood cells and an anticoagulant; lysis of erythrocytes to obtain a processed sample; cooling the treated sample to a temperature of about -20°C or less within about 24 hours after receiving the sample, obtaining a frozen sample; and measuring the number of CD38-expressing cells in the sample within about 72 hours after obtaining the sample, where the frozen sample is warmed to room temperature before measuring the number of CD38-expressing cells.

[0233] It should be understood that changes in the number of plasmablasts or plasma cells present in a biological sample obtained from a patient can be monitored using an Ig free light chain (FLC) assay. Any free Ig light chain assay known to those skilled in the art, such as the FREELIGHT® assay (BindingSite, UK), can be used to measure free Ig light chains. See, for example, Fassbinder T, Saunders U, Mickholz E, et al., Arthritis Res Ther (2015); Draborg AH, Lydolph MC, Westergaard M, et al., PLoS One (2015) 10 (9): e0138753. doi: 10.1371/journal.pone.0138753. eCollection 2015. SLE leads to polyclonal activation of plasma cells, patients have increased levels of kappa and lambda light chains compared to healthy individuals, and this increase in antibodies and plasma cells is considered part of the pathogenesis of the disease (Draborg, 2015). The presence of FLC in serum was indicated as a marker of SLE, and FLC in urine as a marker of class III/IV lupus nephritis. Serum FLC levels are altered by treatment with rituximab or mycophenolate mofetil (MMF) and correlate with other blood plasmablast and plasma cell markers (Fassbinder, 2015; Draborg, 2015).

[0234] In some embodiments, the invention relates to a method for measuring a biomarker of increased expression of at least one gene enriched in CD38-expressing cells in a biological sample obtained from a patient, compared with the level of expression of a gene enriched in CD38-expressing cells , in a biological sample obtained from a control individual. In some embodiments, the invention relates to a method for measuring a biomarker of increased expression of a gene enriched in CD38-expressing cells in a biological sample obtained from a patient compared to a threshold value of a gene enriched in CD38-expressing cells. In some embodiments, the gene enriched in CD38-expressing cells is IgJ. In some embodiments, the gene enriched in CD38-expressing cells is CD38. In some embodiments, the implementation of the biomarker includes mRNA. In some embodiments, the biological sample is whole blood. In some embodiments, the patient has received at least one previous treatment. In some embodiments, at least one previous treatment comprises treatment with an anti-CD20 antibody.

[0235] In some embodiments, the invention provides a method for predicting a patient's response to therapy, comprising measuring in a biological sample obtained from the patient, the expression of a biomarker of increased expression of at least one one gene enriched in CD38-expressing cells, and comparing the expression of at least at least one gene enriched in CD38-expressing cells in a biological sample of a patient with the expression of the same at least one gene enriched in CD38-expressing cells in a biological sample obtained from a control individual or with a threshold value of at least one gene, enriched in CD38-expressing cells, where increased expression of at least one gene enriched in CD38-expressing cells in a biological sample of a patient compared to expression in a biological sample of a control individual or with a threshold value predicts the patient's response to therapy. In some embodiments, the plasma cell/plasmoblast enriched genome is IgJ. In some embodiments, measuring expression of at least one gene includes measuring mRNA. In another embodiment, the measurement of mRNA includes a PCR method, RNA sequencing (RNAseq), or a microarray chip. In some embodiments, the implementation of the biological sample includes whole blood. In some embodiments, the patient has received at least one previous treatment. In some embodiments, at least one previous treatment comprises treatment with an anti-CD20 antibody.

[0236] It should be understood that the measurement of the level of expression of a gene enriched in CD38-expressing cells can be performed using any standard methods known in the art. For example, the level of mRNA can be measured using a microarray. In another aspect, gene expression is measured by polymerase chain reaction (PCR), RNAseq, or quantitative real-time polymerase chain reaction (qPCR). In another aspect, gene expression is measured by multiplex PCR. In another embodiment, expression of a gene of interest in a biological sample of a patient is considered elevated when compared to expression of the gene of interest in a biological sample of a control individual if the relative mRNA level of the gene of interest in the patient sample is greater than 2-fold the mRNA level of the control. In another embodiment, the relative level of mRNA of the gene of interest in the patient sample is greater than 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, or 30-fold higher than the level in the control sample. In another embodiment, the expression of a gene of interest in a biological sample of a patient is considered low relative to the expression of that gene in a biological sample of a control subject, if the relative level of mRNA of the gene of interest in the patient sample is less than 2 times the mRNA level of the control subject. In another embodiment, the expression of a gene of interest in a biological sample of a patient is considered low compared to the expression of a given gene in a biological sample of a control subject, if the relative level of mRNA of the gene of interest in the patient sample is less than 3 times, 5 times, 10 times, 15 times , 20 times, 25 times or 30 times higher than the level in the control sample.

[0237] mRNA levels can be measured and quantified using various methods known to those skilled in the art, including the use of commercial kits and reagents. One such method is the polymerase chain reaction (PCR). Another method for use in quantitative analysis is quantitative real-time PCR or qPCR. See, for example, Innis MA et al., eds., Academic Press, Inc. (1990); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987, and periodic updates); and "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994). Another method for use in quantitative analysis is RNAseq. See Wang, Zhong; Gerstein, Mark; Snyder, Michael. "RNA-Seq: a revolutionary tool for transcriptomics". Nature Reviews Genetics 10(1): 57-63.

[0238] In another aspect, the level of gene expression is measured using a PCR method or a microarray method. In one embodiment, the microarray method involves the use of a microarray chip with one or more nucleic acid molecules that can hybridize under stringent conditions to a nucleic acid molecule encoding a gene as defined above. In one embodiment, the PCR method is qPCR. In one embodiment, the PCR method is multiplex PCR. Microarray analysis is a multiplex technology that typically uses an array of thousands of nucleotide probes arrayed on a chip to hybridize to, for example, a cDNA or cRNA sample under highly stringent conditions. Target probe hybridization is typically detected and quantified by detecting fluorophore-, silver- or chemiluminescent-labeled targets to determine the relative abundance of nucleic acid sequences in the target. In conventional microchips, probes are attached to a solid surface by covalent bonding to a chemical matrix (using epoxysilane, aminosilane, lysine, polyacrylamide, or others). The solid surface is, for example, glass, a silicon chip, or microscopic spheres. Various microchips are available for sale, including those from, for example, Affymetrix and Illumina.

[0239] PRODUCTS

[0240] In other embodiments, an article of manufacture is provided that contains materials suitable for the treatment of the disorders described above. The product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. Containers can be made from various materials such as glass or plastic. The container contains a composition that is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a hypodermic stopper). The active substance in the composition is an antibody. The label on or associated with the container indicates that the composition is being used to treat the selected condition. The product may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. It may additionally include other materials required by the commercial and user position, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

EXAMPLES

[0241] The following examples are provided to illustrate, but not limit, the invention.

[0242] EXAMPLE 1: Construction of expression vectors comprising polynucleotides encoding human, cynomolgus and mouse CD38

[0243] To construct a vector expressing human CD38 (huCD38), a polynucleotide encoding huCD38 was isolated from cDNA obtained from the Origene Technologies Trueclone® human cDNA collection. The isolated huCD38 was cloned into a stable expression vector (XOMA, Inc.) containing a neomycin resistance (neoR) gene that allowed selection of G418 (geneticin) resistant transfectants. The huCD38 gene present in the selected transfectants was sequenced to detect any possible sequence errors. Sequence errors that did not match the GenBank sample NM_001775 were corrected by site-directed PCR mutagenesis. The final vector DNA was confirmed by 5' sequencing.

[0244] To construct a vector expressing cynomolgus monkey CD38 (cyCD38), a polynucleotide encoding cyCD38 was isolated from DNA obtained from BioCain-Institute cDNA from normal monkey (cynomolgus) spleen tissue. The isolated cyCD38 was cloned into a stable expression vector (XOMA, Inc.) containing the neoR gene, which allowed selection of G418 (geneticin) resistant transfectants. The cyCD38 gene present in the selected transfectants was sequenced to detect any possible sequence errors. Sequence errors that did not match the template in Genbank AY555148 were corrected using site-directed PCR mutagenesis. The final DNA vector was confirmed by sequencing.

[0245] To construct a vector expressing mouse CD38 (moCD38), a polynucleotide encoding moCD38 was isolated from DNA obtained from Origen's TrueORF collection. The isolated moCD38 was cloned into a stable expression vector (XOMA, Inc.) containing the neoR gene, which allowed selection of G418 (geneticin) resistant transfectants. The moCD38 gene present in the selected transfectants was sequenced to determine any possible sequence errors. Sequence errors that did not match the GenBank sample NM_007646 were corrected by site-directed PCR mutagenesis. The final vector DNA was confirmed by sequencing.

[0246] EXAMPLE 2: Generation of CD38 Expressing Chinese Hamster Ovary (CHO) Cells

[0247] To create CHO cells expressing huCD38, muCD38 and cyCD38, CHO cells were transfected with linearized DNA. After one week under selection conditions, cells were sorted by flow cytometry and cells with the highest expression of huCD38, muCD38 or cyCD38 (top 15%) were plated in 96-well plates to obtain single colonies. The rest of the cells were also sown with selection to obtain reserve colonies. Approximately 12-14 days after seeding, single colonies were identified and transferred to 96-well deep well plates. Clones were subjected to screening using FACS analysis after the second passage. The best producer clones were subcultured and propagated in shake flasks. The top 2 clones were frozen and/or cultured for testing for the introduced mycoplasma agent and for scaling up.

[0248] To construct a luciferase reporter for disseminated xenograft models, a commercial vector containing the CMV promoter/luciferase gene/neomycin resistance selectable marker (Promega, Madison, WI) was used to generate a stable line of transfectants in Daudi Burkitt's lymphoma cells.

[0249] EXAMPLE 3: Phage display libraries and screening for agents that bind CD38

[0250] The selection of target-specific antibodies from the phage display library was performed according to the methods described in Marks et al. (2004, Methods Mol. Biol. 248: 161-76). Briefly, the phage display library was incubated with 100 pmol of biotinylated CD38 at room temperature for 1 h, and then the resulting complex was captured using 100 μl of streptavidin bead suspension (DYNABEADS® M-280 Streptavidin, Invitrogen). Non-specific phages were removed by washing the beads with wash buffer (5% milk in PBS). Bound phages were eluted with 0.5 ml of 100 nM triethylamine (TEA) and immediately neutralized by adding an equal volume of 1M Tris-Cl, pH 7.4. The eluted phage pool was used to infect E. coli TG1 cells in logarithmic growth phase and the phagemid was isolated as described in Marks et al., Id. The selection was repeated for a total of three rounds.

and R. Aitken, Humana Press; "Panning of Antibody Phage-Display Libraries", Coomber, D.W.J., pp. 133-145, и "Selection of Antibodies Against Biotinylated Antigens", Chames et al., pp. 147-157). Коротко, три лунки планшета NUNC(MAXISORP покрывали 50 мкл рекомбинантного CD38 (R&D systems) при концентрации 10 мкг/мл в PBS. После инкубирования в течение ночи при 4°C, свободные связывающие сайты блокировали 5% молоком в PBS в течение одного часа при комнатной температуре. Затем приблизительно 200 мкл фаговой библиотеки в 5% молоке/PBS добавляли в блокированные лунки и инкубировали при комнатной температуре в течение приблизительно одного-двух часов. Лунки промывали, и связанный фаг элюировали при использовании стандартных методов (см., например, Sambrook and Russell, Molecule Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001). Элюированный фаг амплифицировали путем заражения клеток-хозяев E.coli TG 1 в логарифмической фазе роста. Зараженные клетки TG 1 собирали с помощью центрифугирования при 2500 об/мин в течение пяти минут, сеяли на 15 см чашки с агаром с 2YT-ампициллин-2% глюкозы и инкубировали при 30°C в течение ночи. Процесс пэннинга затем повторяли при использовании амплифицированного фага. Цикл пэннинга, элюции и амплификации повторяли три раунда.[0251] Alternatively, phage display libraries were panned with immobilized CD38 (R&D systems) to determine a panel of antibody fragments with the ability to bind CD38. Panning was performed using standard techniques (see, for example, Methods in Molecular Biology, vol. 178: Antibody Phage Display: Methods and Protocols Edited by: PM

and R. Aitken, Humana Press; "Panning of Antibody Phage-Display Libraries", Coomber, DWJ, pp. 133-145, and "Selection of Antibodies Against Biotinylated Antigens", Chames et al., pp. 147-157). Briefly, three wells of a NUNC(MAXISORP plate) were coated with 50 µl of recombinant CD38 (R&D systems) at 10 µg/ml in PBS. After overnight incubation at 4°C, free binding sites were blocked with 5% milk in PBS for one hour at Approximately 200 µl of the phage library in 5% milk/PBS was then added to blocked wells and incubated at room temperature for approximately one to two hours. and Russell, Molecule Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001. Eluted phage was amplified by infecting E. coli TG 1 host cells in logarithmic growth phase. Infected TG 1 cells were harvested by centrifugation at 2500 rpm for five minutes, plated on 15 cm 2YT-ampicillin-2% glucose agar plates and incubated at 30° C. overnight. was repeated using amplified phage. The cycle of panning, elution and amplification was repeated for three rounds.

[0252] After completion of panning, single colonies of seeded TG1 cells were used to inoculate media in 96-well plates. Microcultures were grown to an OD600 of 0.6 and at this point soluble scFv expression was induced by adding 1 mM IPTG followed by overnight incubation on a shaker at 30°C. Bacteria were pelleted by centrifugation and the periplasmic extract was used to test scFv binding to immobilized CD38 using standard ELISA and FACS binding assay.

[0253] For FACS binding screening, CHO cells stably expressing CD38 were used to screen scFv fragments in periplasmic extract (PPE) for their ability to bind native membrane-bound CD38. Stock and CHO transfectants (cell lines expressing human CD38 or Cynomolgus CD38 or mouse CD38) were resuspended separately to 2×10 ⁶ cells/ml in PBS (Life Technologies), 0.5% BSA (Sigma-Aldrich), and 0.1 % NaN ₃ (Sigma-Aldrich) (FACS buffer). Original CHO cells not expressing CD38 were used as a negative control. Twenty-five µl aliquots of cells were seeded into 96-well V-bottom plates (cat.Costar 3897) and 25 µl of periplasmic extract containing myc-labeled scFv antibody fragment was added to the cells, after which the mixture was incubated at 4°C for within 30 minutes. The cells were then washed twice, after which the pellet was resuspended in 25 μl mouse anti-c-myc antibody (1/1000 in FACS buffer) (Roche) and incubated again at 4°C for 30 minutes. The cells were then washed twice and resuspended in 25 µl 1/200 dilution of anti-mouse IgG-PE in FACS buffer (Jackson labs) and incubated again at 4°C for 30 minutes. Cells were then washed twice to remove excess unbound antibody, resuspended in 70 µl FACS buffer and examined on BD FACScan®. The obtained data were evaluated using the program FlowJo (TreeStar, Inc.). Positive samples were determined by comparing the average CD38 fluorescence intensity of the transfected CHO cell and the median fluorescence intensity of the original CHO cell line (CD38-).

[0254] The antibody clones that bound human CD38 were sequenced to identify unique clones. The unique scFV clones were then assessed by dissociation rate as determined by the Biacore® assay. 200RU to 500RU human recombinant CD38 (Cat. R&D systems 2404-AC or equivalent) were immobilized using standard amine coupling chemistry (Biacore®) on a CM5 or equivalent chip. A checkpoint was also prepared, which was activated and then blocked without protein immobilization. This was done by diluting the antigen to 1-3 μg/ml in acetate buffer, pH 5.0, and passing it over the activated surface until the desired level of immobilization (3-5) minutes was obtained. The surface was then blocked with ethanolamine. Periplasmic extracts were diluted one to one with assay buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% polysorbate 20, pH 7.4, with 2 mg/ml BSA (bovine serum albumin) ). The diluted periplasmic extract was passed over Surface Plasmon Resonance (SPR) surfaces at 30 minutes for 300 seconds with an additional 900 seconds to monitor dissociation time. Regeneration was performed with a single 8 second injection of 100 mM HCl. The control point data were subtracted from the active surface data, after which the dissociation curves were fitted using a 1:1 dissociation model in the Biacore® T100 software.

[0255] The best scFV clones were converted into IgG1 antibodies. FACS binding screening was repeated on reformatted IgG1 clones using original CHO cells and CHO cells expressing human, mouse, and cynomolgus CD38 to ensure retention of binding properties and to assess interspecies cross-reactivity. FACS analysis of reformatted IgG clones was performed as described above, but the steps consisting of adding anti-c-myc antibody and anti-mouse IgG-PE were replaced by a single step in which full-length human IgG binding was detected by adding phycoerythrin-conjugated anti-human IgG (Jackson Labs).

[0256] EXAMPLE 4: Cellular in vitro assays of reformatted IgG clones

[0257] Approximately 150 clones were reformatted into human IgG1 antibodies and five of them (Ab19, Ab43, Ab72, Ab79 and Ab110) were fully evaluated using a panel of assays as described below. The performance of reformatted IgG clones in in vitro and in vivo assays was compared with two antibodies, BMTK4-1 (also referred to as control-1, BM-1 or BMTK-1) (SEQ ID NOS: 24 and 25; heavy and light chain variable regions) and BMTK4-2 (also referred to as control-2, BM-2 or BMTK-2) (SEQ ID NOS: 26 and 27; heavy and light chain variable regions), whose amino acid sequences were derived from those of the known anti-CD38 antibody, daratumumab ( also referred to as HuMax-CD38, disclosed in International Application Publication WO 06/099875) and SAR650984 (disclosed in International Application Publication WO 08/047242), respectively. Palivizumab (Synagis®) (MedImmune), a clinically approved antibody that recognizes respiratory syncytial virus, served as a negative control for CD38 binding.

[0258] EXAMPLE 5: Ab79 Binding Detection by Immunofluorescence

[0259] Ab79 labeled with Alexa Fluor®-488 was applied to frozen sections of normal human colorectal tissue, prostate, and lymph node. Alexa Fluor®-488 dye labeled palivizumab (Synagis®) served as a negative staining control. The resulting immunofluorescent staining images are shown in FIG. 4. The pattern of staining observed for Ab79 was identical to that observed using a commercial anti-CD38 polyclonal antibody on normal human colorectal tissue, prostate, and lymph node (data not shown).

[0260] Alexa Fluor®-488 dye-labeled Ab79 was also applied to normal and multiple myeloma bone marrow samples (data not shown). Since Ab79 bound to ~10% of normal bone marrow cells, >90% of bone marrow cells affected by multiple myeloma, Ab79 binding was shown in 4 out of 4 samples tested.

[0261] The ability of Ab79 to bind to various cell lines (MOLP-8, DAUDI, RPMI and MCF7) was also investigated. All cells of MOLP-8 (human multiple myeloma), DAUDI (lymphoblast derived from a patient with Burkitt's lymphoma) and RPMI (cell line derived from a patient with chronic myelogenous leukemia) showed Ab79 binding. The breast cancer line, MCF7, was generally negative for binding to Ab79 (data not shown).

[0262] Antibodies conjugated with Alexa Fluor® 488 were stained on 8 µm sections frozen in a cryostat, which were fixed in a mixture of ethanol/acetone for 5 min, after which they were incubated with antibodies for 1 hour at room temperature in a controlled humidity chamber . Sections were then washed, DAPI-containing section embedding medium (Vector Laboratories, cat. H1500) was added, and a coverslip was placed.

[0263] EXAMPLE 6: Evaluation of Ab79 Expression in Multiple Myeloma (MM) and Chronic Lymphocytic Leukemia (CLL)

[0264] Binding of Ab79 to bone marrow samples from multiple myeloma patients was examined by flow cytometry either after enrichment in CD138+ cells or by gating on CD138+CD45-/1o cells (FIG. 7A). Ab79 was found to be expressed in >95% of cells from four of six multiple myeloma samples. The binding profile of Ab79 was generally similar to that of the anti-CD38 antibody used in clinical laboratories. In addition, Ab79 bound cells from patients with chronic lymphocytic leukemia (FIG. 7B).

[0265] To measure the binding of Ab79 to MM and CLL using FACS, patient samples were processed for 24 hours. Peripheral blood mononuclear cells were isolated in Ficoll-Paque™ (GE Healthcare) according to the manufacturer's instructions. Expression analysis was performed using the following panels of antibodies, the clones of which are indicated in brackets. Panel MM: Ab79-Alexa Fluor®-488, CD45-PerCP (2D1), CD138-APC (MI15). CLL panel: Ab79-Alexa Fluor®-488; CD5-PE (UCHT2), CD45-PerCP (2D1), CD19-APC (SJ25C1). 5 µl of PE, PerCP or APC labeled antibody or 10 µl of Alexa Fluor®-488 labeled antibody or isotype control was added to each well or tube containing 100 µl of 0.2 x 10 ⁶ PBMC or CD138 enriched cells from bone marrow aspirate. Samples were incubated for 30 min at room temperature, after which the erythrocytes were lysed using BD Pharmlyse according to the manufacturer's instructions. All samples were washed three times in FACS buffer. Samples were fixed in 1% paraformaldehyde and tested on BD FACSCanto™ II or BD FACSCalibur™.

[0266] EXAMPLE 7: Anti-CD38 antibody-induced CDC assays

[0267] Cross-reactive clones of cynomolgus were tested for the ability to induce complement-dependent cytotoxicity (CDC). MOLP-8 cells were seeded at a density of 10,000 cells/well in a black 96-well flat-bottomed tissue culture plate in 50 μl of complete medium (RPMI supplemented with 10% fetal bovine serum). 50 µl of 2x anti-CD38 antibody, control IgG antibody, or medium alone was added to each well and left to incubate at room temperature for 10 minutes. Varying amounts (2-15 µl) of purified rabbit complement (Cat. #CL 3441 Cedarlane Laboratories, Canada), depending on the cell line, were added to each well except control wells. After one hour of incubation at 37°C, the plates were brought to room temperature, 100 μl of CytoTox Glo™ cell titer calculation reagent (Promega G7571/G7573) was added to the wells, the plate was shaken for 5-7 minutes and the luminescence was read on a plate analyzer EnVision® (Perkin Elmer). Test conditions: cells only; cells + complement; cells+IgG control+complement; cells+antibody+complement% CDC was calculated using the following equation:

100-(RLU _T /RLU _C )×100),

[0268] where RLU _T are the relative luminescence units of the test sample, and RLU _C are the relative luminescence units of the sample with complement only. Statistical analysis was performed using the PRISM program. EC ₅₀ values determined from % CDC versus antibody concentration curves are shown in Table 1.

[0269] EXAMPLE 8: Anti-CD38 antibody-induced ADCC assays

[0270] Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated using Daudi, MOLP-8 and RPMI-8226 cell lines as target cells. PBMCs were isolated as effector cells by Ficoll-Plaque™ separation from the leukocyte layer or by LRS obtained from Stanford Blood Center (Palo Alto, CA). Samples were diluted 1:3 with 2% FBS in PBS, 15 ml Ficoll-Plaque™ (GE Healthcare) was carefully layered onto 35 ml of the diluted sample and centrifuged at 1800 rpm (no quick stop) for 25 minutes. The cloudy intermediate phase containing the PBMCs was collected, washed 3 times with 2% FBS in PBS and frozen in aliquots of 50×10 ⁶ cells/ml in 10% DMSO/FBS. Frozen aliquots of PBMCs were thawed and cultured overnight in 10% FBS/RPMI+5 ng/ml recombinant human IL2 (R&D systems #202-IL) at 2×10 ⁶ per ml, if needed.

[0271] When analyzing ADCC, all steps were performed in complete environments. 5,000 target cells were seeded per well of a 96-well plate, to which 50 µl of 3x anti-CD38 antibody, IgG control, or medium alone was added, followed by 50 µl of human effector PBMCs at a target:effector cell ratio (T:E) of 1 :25-1:50. The plates were rapidly centrifuged for ~30 seconds at 800 rpm to pool all cells in close proximity. After 4 hours at 37°C, the plates were centrifuged at 1100 rpm for 5 minutes and 100 μl of the supernatant was transferred to a white plate. 100 µl of CytoTox Glo™ reagent (cat. Promega #G9292) was added to the supernatant and the plates were left shaking for 20-30 min at RT. Luminescence was read on an EnVision® plate analyzer (Perkin Elmer) and the percent specific lysis was calculated using the following equation:

(RLU _T /RLU _E/T )/(RLU _L /RLU _E/T )×100

[0272] where RLU _T are the relative luminescence units of the test sample, and RLU _E/T are the relative luminescence units of the sample containing target cells and only effector cells, and RLU _L are the relative luminescence units for cells after Triton X-100 lysis. Statistical analysis was performed using the PRISM program. EC50 values determined from % specific lysis versus antibody concentration curves are shown in Table 1.

TABLE 1. CDC, ADCC, and agonist activity of IgG reformatted antibodies

Antibody CDC EC50 nM (MOLP-8) ADCC EC50 nM (DAUDI) ADCC EC50 nM (MOLP-8) ADCC EC50 nM (RPMI-8226) Apoptosis EC50 nM (DAUDI) BM-1 0.48±0.16 0.03±0.02 0.036±0.013 0.13±0.03 0.057 BM-2 0.65±0.18 0.04±0.02 0.024±0.005 0.15±0.04 0.062 Ab19 0.98±0.26 0.08±0.03 0.038±0.008 0.46±0.15 0.032 Ab43 2.2 0.12±0.09 0.027±0.018 3.84+1.34 1.56 Ab72 0.66±0.49 0.14±0.12 0.193±0.037 2.35±0.99 0.35 Ab79 1.1±0.39 0.03+0.02 0.047±0.012 0.46±0.19 0.048 Ab110 1.99±0.71 0.24±0.17 0.874±0.804 2.98±0.91 0.40 Ab164 2.00±0.83 ND 0.165±0.154 1.2±0.24 0.31

[0273] EXAMPLE 9: Determination of affinity using FACS

[0274] MOLP-8 cells expressing CD38 were suspended in 1% FBS buffer at a viable cell concentration of approximately 2 million cells/ml. Tabs to be tested were serially diluted (2-fold) in wells of two 96-well plates in 1(PBS. The last well of each titration contained only buffer. Additional PBS and cell suspension were added to each well so that the final volume was 300 µl/well, with each well containing approximately 100,000 cells.The mAbs are listed below with the corresponding range of final mAb binding site concentrations (2×molecular concentration) used for titration:

Control 1, [mAb] binding site=50.8 nM -49.7 pM;

Control 2, [mAb] binding site=49.5 nM -48.3 pM;

Ab 43, [mAb] binding site=49.3 nm-48.2 pM;

Ab 110, [mAb] binding site=204 nm -49.9 pM;

Ab 79, [mAb] binding site=103 nm -25.3 pM;

Ab 72, [mAb] binding site=103 nm -25.2 pM;

Ab 19, [mAb] binding site=100 nm -12.2 pM;

[0275] The plates were placed in a plate shaker for 5 hours at 4°C, after which the plates were washed 3 times at 4°C with 1x PBS. Then, 200 μl of 99 nM specific goat anti-human Fc IgG polyclonal Cy5 antibody (Jackson ImmunoResearch Laboratories, 109-175-008) was added to each well and the plates were shaken for 30 minutes at 4°C. The plates were washed again 2× at 4° C. 1(PBS, then a FACSCanto™ II HTS flow cytometer was used to record the mean fluorescence intensity (MFI) of 5000 events for each well containing a unique concentration of mAb binding site. The antibody region was non-linearly approximated by Scientist 3.0 using the equation below to estimate KD:

F=p[(KD+LT+n(M))-{(KD+LT+n(M))2-4n(M)(LT)}1/2]/2+B

[0276] where F (mean fluorescence intensity), LT (total mAb binding site concentration), p (proportional constant that relates fluorescence units to bound mAb), M (cell concentration in moles; 0.553 fM based on 100,000 cells in 300 µl), n (number of receptors per cell), B (background signal) and KD=equilibrium dissociation constant.

[0277] For each antibody titration curve, a KD score was obtained by free-running P, n, B, and KD in a non-linear analysis. For a detailed derivation of the above equation, see Drake and Klakamp (2007), "A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants", J. Immunol. Methods 318: 157-62, which is incorporated herein by reference. Table 3 shows the obtained KD values for all antibodies in descending order of affinity, as well as the 95% confidence interval for each approximation in brackets. The antibody binding site concentration (2×molecular concentration) was used for non-linear curve fitting.

[0278] EXAMPLE 10: Determination of affinity using Biacore®

[0279] The affinity of IgG antibodies to the soluble CD38 ectodomain (ECD) was determined using Biacore™ A100 surface plasmon resonance (SPR) assay at 22°C. A goat anti-human IgG polyclonal antibody (Caltag H10500) was immobilized on the CM5 biosensor chip using a standard combination of amines for points 1, 2, 4 and 5 in all four flow cells of the chip. Immobilization levels at each point ranged from 5865 RU to 6899 RU. Human CD38 was purchased from R&D systems (cat. 2404-AC, lot #PEH020812A). Stock CD38 was determined using the methods detailed in Pace et al. (1995) "How to measure and predict molar absorption coefficient of a protein", Protein Science 4 (11): 2411-23 and Pace and Grimsley (2004) "Spectrophotometric determination of protein concentration", in Current Protocols in Protein Science, Chapter 3: Unit 3.1, the contents of which are incorporated herein by reference.

[0280] Working buffer was prepared by degassing HEPES buffered saline, 0.005% polysorbate 20, and adding filtered BSA to a final concentration of 100 μg/mL. All eight purified mAbs were diluted to approximately 2 μg/ml with running buffer. Preliminary experiments assessing the amount of each mAb taken to maintain surface capacity (Rmax) showed no more than ~100 RU. For each mAb capture/antigen injection cycle, mAbs were captured at points 1 and 5 in each flow cell, with adjacent points 2 and 4 serving as the respective reference surfaces. Each diluted mAb was captured for 1 minute at a flow rate of 10 μl/min, after which the working buffer was passed for three minutes to stabilize the surface. HuCD38 was passed over all four flow cells for 120 seconds at 30 μl/min over a concentration range of 193.7 nM-3.0 nM (2x serial dilution) followed by a dissociation phase of 15 minutes. All samples were prepared in running buffer and randomly administered in triplicate with seven buffer injections in between for dual control. Surfaces were regenerated with two 20 second pulses of 10 mM glycine, pH 1.7.

[0281] All sensorgram data was processed using the Scrubber 2.0c program with a global fit using the Scrubber 2.0c 1:1 interaction model. The resulting binding constants are shown in Table 2.

TABLE 2

Antibody FACS KD (nM)
MOLP-8 FACS KD (pM)
RPMI-8226 Biacore Ka
(M ^{-1 s-1} ) Biacore Kd (with ^-1 ) Biacore KD (nM) BM-1 1.1 (0.9) 802 4.49×10 ⁴ 2.46×10 ^-3 54.8 BM-2 1.6 (0.6) 428 4.24×10 ⁵ 2.27×10 ^-3 5.4 Ab19 0.4 (0.3) - 1.54×10 ⁵ 8.10×10 ^-4 5.3 Ab79 1.2 (1.1) 508 1.22×10 ⁵ 6.75×10 ^-4 5.5 Ab72 0.6 (0.4) - 1.44×10 ⁴ 1.82×10 ^-3 126 Ab110 1.0 (0.1) - 1.22×10 ⁵ 1.71×10 ^-1 1400 Ab43 1.1 (0.3) - 2.72×10 ⁵ 1.46×10 ^-1 537 Ab164 1.4 (0.7) - 1.99×10 ⁵ 7.15×10 ^-2 359

[0282] EXAMPLE 11: immunofluorescence internalization probes

[0283] Immunofluorescence techniques were used to assess the internalization of anti-CD38 antibodies into MOLP-8 cells. MOLP-8 cells were harvested and 5×10 ⁶ cells were stained for 10 min at 4°C in RPMI-1640 using 1 μg of each anti-CD38 antibody directly conjugated to Alexa Fluor® 488. Cells were washed in PBS containing 1% BSA , and 1×10 ⁶ cells were incubated for 3 or 6 hours at 4°C or 37°C. Surface staining was quenched for 30 min at 4°C using 2 μg of rabbit anti-Alexa Fluor®-488 (Invitrogen). Cells were washed and fixed in PBS with 1% PFC, transferred to a 96-well microassay plate (BD Biosciences) and evaluated by flow cytometry using a FACSCanto™ II flow cytometer (BD Biosciences) or visualized using ImageXpress (Micro (Molecular Devices) ) at 20(increase.

[0284] EXAMPLE 12: Epitope binning with Biacore®

[0285] The Biacore® A100 system was used to binn the two control antibodies, as well as Ab 19 and Ab 79. Antibodies were first immobilized at high and low density on a CM5 chip using NHS/EDC coupling chemistry. For each cycle of the epitope binning experiment, CD38 was first passed over these surfaces. Similar to the ELISA sandwich assay, the unique antibody (taken from a set of immobilized antibodies) was then passed over surfaces containing CD38/antibody complexes. Surfaces were regenerated using pulses of phosphoric acid at the end of each cycle. Data was recorded at 22°C using HBS-P (10 mM 10 HEPES, pH 7.4, 150 mM NaCl, 0.005% P-20) supplemented with BSA. The resulting sensograms were processed using the Epitope Mapping module in the Biacore(A100 Evaluation) software package, as well as the trial Scrubber for A100 datasets. Table 3.

TABLE 3

Ab79 BM1 Ab19 BM2 Ab79 0 0 1 1 BM1 0 0 1 0 Ab19 1 1 0 0 BM2 1 0 0 0

[0286] EXAMPLE 13: In vivo assay in oncology

[0287] The in vivo efficacy of Ab19 and Ab79 was studied in a disseminated Daudi-luciferase model of human lymphoma. Female 6-8 week old CB.17 SCID mice from Taconic Laboratories were intravenously injected with 1×10 ⁶ Daudi-Luc tumor cells. On study day, 7 mice were intraperitoneally injected with: palivizumab, Ab 79, Ab19, Control 1 and Control 2. Bioluminescent imaging was performed weekly starting on day 21 using the Xenogen IVIS system (Caliper Life Sciences) to monitor tumor mass. For imaging, the animals were ip injected with luciferase substrate (150 mg/kg) 10 min before imaging, after which the animals were anesthetized with isoflurane and imaged. The results are shown in FIG. 8 and FIG. nine.

[0288] EXAMPLE 14: Correlation of inflammatory and immunological disease

[0289] CD38 has been shown to be highly elevated in PBMC cells after activation. In resting PBMCs derived from healthy donors, less than 20% of resting cells express CD38, with the estimated number of receptors being approximately 10,000-20,000 per cell. Activated PBMCs, again obtained from healthy donors, showed 60-75% of CD38-expressing cells with 110,000-160,000 receptors per cell.

[0290] In addition, as shown in FIG. 5, CD38 showed increased expression in PBMCs of SLE patients.

[0291] Samples were immunohistologically examined for CD38 overexpression. We studied 19 samples obtained from patients with SLE, which showed increased expression of CD38 on B and T memory cells and pDCs. Analysis in 3 patients with RA showed infiltration of CD38 plasma cells into the synovial tissue of all three patients, as well as strong staining of the synovial tissues with the Ab79 antibody. Analysis in 7 patients with Crohn's disease and 6 patients with ulcerative colitis showed infiltration of CD38-expressing plasma cells into the smooth muscle of the colon (the study was performed on two samples of primary cells from patients). It should be noted that the same number of healthy patients were studied, the results of which were not shown.

[0292] EXAMPLE 15 Elimination Using Anti-CD38 Antibodies

[0293] Administration of doses of Ab79 to cynomolgus monkeys shows a significant reduction in lymphocytes, B and T cells, and NK cells. The antibody was administered by 30-minute intravenous infusion at the doses shown in FIG. 6, data were recorded after 24 hours. Similar data was shown on days 4, 7, 11 and 18.

[0294] However, pK/pD data show recovery of animals after dosing. As shown in FIG. 7, the lymphocyte count returned to similar control levels a few days after the single dose by 30-minute IV infusion, even after the highest dose, indicating that there was no problem with the lymphoid progenitor cells.

[0295] EXAMPLE 16: Autoimmune disease models

[0296] USED THREE DIFFERENT MODELS

[0297] The HuScid mouse model served as a model for pseudo-graft-versus-host disease as well as a model for tetanus toxoid, IgG depletion, and overall survival. CB17/HuSCID mice were injected with ASGM1 to eliminate NK cells and then injected with human PBMC the next day. Engraftment was carried out for 7-10 days with serum sampling for randomization of human Ig. The next day, mice were treated with Ab79 at 10 mg/kg followed by TTd, with a second dose 4 days later, a third dose 3 days thereafter, and serum was collected for human Ig counts and detection of tetanus toxoid antibodies 3 days thereafter ( only 10 days after the first dose). FIG. 8 shows the results of a single donor mouse indicating a significant reduction in all Ig isotypes with treatment. FIG. 9 shows the results of the average Ig levels for each group of recipients, with each data point corresponding to the average value in each group (from n=5 to n=10). FIG. 10 shows a significant reduction in tetanus response with Ab79 treatment. Finally, in FIG. 11 shows overall significant survival with Ab79 treatment.

[0298] MICE SURROGATE EXPERIMENTS

[0299] Since Ab79 does not cross-react with rodent CD38, a surrogate antibody was created. As a preliminary measure, using a commercial anti-human CD38 antibody and a commercial anti-mouse CD38 antibody showed significant differences in CD38 expression levels in different cell types in human and mouse (see Figure 12), demonstrating that the biology of the disease is different. Thus, the use of antibodies that cross-react with monkey CD38 in monkey models can give significantly better results.

[0300] A surrogate antibody was generated showing a similar depletion of CD38+ cells derived from spleen, blood and bone marrow (data not shown), as well as from peripheral blood (see FIG. 13). In addition, in FIG. 14 shows the kinetics of wasting in spleen, blood, and bone marrow collected on days 1, 2, or 3 following a single dose of 10 mg/kg to a surrogate mouse. Rapid depletion of B cells in the blood has been shown within 24 hours, with slower depletion shown in the spleen and bone marrow.

[0301] MOUSE SLE MODEL

[0302] A surrogate mouse anti-CD38 antibody was tested in the MRL/1pr model using the following intermediate assays (e.g., every two weeks) in blood: dsDNA autoantibodies, CBC, FACS for T/B cell depletion, urinary albumin, and skin inflammation. Blood endpoints included, but were not limited to: dsDNA autoantibodies, CBC, FACS for T/B cell depletion, in spleen, lymph nodes, and bone marrow: organ weights, immune cell counts, FACS for T/B lymphocytes, in kidney: organ weight, histopathology (H/E and PAS), cellular arrangement, inflammatory cells and skin inflammation (histopathology).

[0303] COLLAGEN-INDUCED ARTHRITIS (CIA) MODEL

[0304] A murine surrogate antibody model of CIA was used, with 7 groups of mice to evaluate pro-prophylactic, prophylactic, and therapeutic efficacy. All mice were immunized with CII/CFA on day 0, with a booster injection of CII/CFA on day 21, typically resulting in disease progression from day 21 to 42 (end of study). Group 1 (10 mice) received 10 mg/kg surrogate antibody ip twice a week starting on day 0 (pro-prophylactically). Group 2 (n=10) was given the same dose starting on day 21. Group 3 (n=10) was given the same dose from disease onset (day 25-28). Group 4 (n=10) received the same dose but with hIgG1 (e.g. isotype) on day 0. Group 5 (n=10) received the same dose of hIgG1 but starting on day 21. Group 6 (n=10) on day 21, 0.5 mg/l dexamethasone in water was administered and Group 7 (n=5) was not treated.

[0304] A study of CIA in cynomolgus monkeys was performed using n=3 in each group, group 1 - naive animals, vehicle only. Group 2 received a single dose of Ab79 at 3 mg/kg or 10 mg/kg, q1w (prophylactic treatment started on day 7 after collagen immunization). Group 3 was given Ab79 at 3mg/kg or 10mg/kg q1w, for therapeutic treatment, starting at disease onset, on day 21 or day 28. Group 4 was given dexamethasone 0.1mg/kg q1d, also at disease onset.

[0306] EXAMPLE 17: Ab79 eliminates human plasma cells/plasmoblasts in vitro

[0307] In an in vitro culture system using human peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMCs), the addition of Ab79 depletes PB/PC populations that are characterized by surface expression of CD27, CD38 and CD138 and intracellular plasma cell markers IRF4 (FIG. 14). Ab79 depletes both short lived plasma cells (CD19+plasma cells) and long lived plasma cells (CD19 plasma cells) from the bone marrow (FIG. 15). Plasma cells and plasmablasts are the main antibody-secreting cells (ASCs) in the body. See Hibi T and Dosch HM. Eur J Immunol (1986) 16(2): 139-145. ASC measured directly with the ELISpot (FIG. 16) is reduced in healthy and SLE patient samples (FIG. 17). In healthy patient samples in the upper panel, treatment with Ab79 results in a 70% reduction in IgG-producing cells from the blood and bone marrow. Similar depletion is seen in samples from SLE patients. In addition, the number of cells producing autoantigen-specific VH4-34 9G4+ antibodies and anti-Ro antibodies is reduced (FIG. 18). These results highlight the potential of the Ab79 monoclonal antibody as a therapeutic agent for the treatment of SLE through PB/PC elimination. By targeting CD38, a molecule highly expressed on all PB/PC cells, Ab79 effectively depletes both short-lived and long-lived ACKs.

[0308] EXAMPLE 18 Detection of PB/PC in Whole Blood Using a Method Appropriate for Clinical Use and Ab79 Treated Individuals

[0309] Flow cytometric analyzes of whole blood from a healthy volunteer show that CD38 is highly expressed on PB/PC and that staining using the fix/freeze method described below with Ab19 mAbs clearly sorts CD45+, CD3-, CD19+ , CD38+, CD27+ B cell subset that contains PB and PC (FIGS. 19A-D).

[0310] Whole blood collected from a healthy volunteer was stained with CD3-PE); CD27-APC; CD45-AF700 CD19-V450; CD38-FITC. For flow cytometry, cells were gated to detect single cells, then forward (FSC) and side scatter (SSC) light scatter was used for comparison regarding blood cell size and granularity (A), and based on this, CD45+ staining of mononuclear cell lymphocytes was determined (B). B cells in this population were identified as CD3-CD19+ cells (C). Plasmablasts and plasma cells were identified as CD27+CD38 bright cells (panel D).

[0311] To stain whole blood samples, tubes containing aliquots of donor samples were centrifuged at 500 RCF for 5 minutes, the supernatant was removed, and the tubes were tilted in a rack to disperse the cell pellet. The cells were washed with 2 ml of PBS with 1% BSA, centrifuged at 500 RCF for 5 minutes, the supernatant was removed and the tubes were tilted in a rack to disperse the cell pellet. The number of cells was determined for each sample and brought the number of cells to a level of 20×10 ⁶ cells/ml to 40×10 ⁶ cells/ml PBS with 1% BSA if necessary. 200 μl were taken with a pipette into a tube with the appropriate marking. The required amount of human serum was added to each tube, shaken and placed in the dark to incubate for 10 minutes. An appropriate amount of antibody was added to each tube. All samples were thoroughly shaken and incubated for 30 minutes at room temperature in the dark. After incubation, the cells were washed with 2 ml of PBS with 1% BSA. The tubes were centrifuged at 500 RCF for 5 minutes, the supernatant was removed and the tubes were tilted in a rack to disperse the cell pellet. After centrifugation, the cells were reconstituted in 500 μl of 1% paraformaldehyde. Samples were analyzed on a BD FACSCanto II flow cytometer. Registration was performed up to approximately 1,000,000 events or until 240 seconds elapsed. The following reagents were used for staining:

[0312] CD3 PE (Clone: SK7) -BD, catalog number 347347;

CD27 APC (Clone: M-T271) -BD Pharmingen, part number 558664;

CD45 AF700 (Clone: HI30) -Biolegend, p/n 304024;

CD19 V450 (Clone: HIB19) -BD Horizon, P/N 560353;

CD38 (Ab19);

1X FACSLyse;

PBS with 1% BSA;

1% paraformaldehyde solution;

Quantum MESF FITC Calibration Beads (LabCorp Analytical Systems);

Quantum MESF APC Calibration Beads (LabCorp Analytical Systems);

[0313] EXAMPLE 19: Stability comparison of PB/PC in whole blood without fixative or after FACSlyse treatment and freezing

[0314] Standard Storage Method and Flow Cytometry

[0315] Five samples of whole blood from healthy donors were collected in tubes containing the anticoagulant heparin sodium. Samples were tested in triplicate by two independent operators on Day 0 (<2 hours post-collection). The samples were then kept at ambient temperature and examined one at a time on Day 1 (24-30 hours), Day 2 (48-54 hours) and Day 3 (72-48) hours after collection. This was followed by staining as described in Example 18.

[0316] The results are shown in Table 4.

Table 4

Donor Days after collection Total number of registered events % CD19+B cells that are CD38++/CD27++ Donor 1 0 1104419 0.85 1 226287 0.14 2 1066353 0.13 3 1094968 0.17 Donor 2 0 885060 0.64 1 232424 0.40 2 615546 0.16 3 500499 0.20 Donor 3 0 241812 0.58 1 830574 0.14 2 853470 0.15 3 528522 0.07 Donor 4 0 754839 2.23 1 1046385 0.64 2 964089 0.29 3 577233 0.27 Donor 5 0 1112831 3.80 1 1098387 0.99 2 1129494 0.61 3 625239 0.42

[0317] c. Fix/Freeze Storage Method and Flow Cytometry

[0318] Collected five samples of whole blood from healthy donors (anticoagulant heparin sodium) and divided the samples into four aliquots. The first aliquot was examined fresh according to the FACSLyse method without a freezing step to establish the initial number of plasmablasts. Freshly collected samples were tested in triplicate by two independent operators on Day 0 (<2 hours post-collection). The remaining three aliquots were frozen at -80°C after incubation with FACSLyse for 10 minutes. The second aliquot was thawed at 38°C in a water bath and processed on Day 1 (24-30 hours). The third aliquot was thawed on Day 2 (48-54 hours) and the fourth aliquot on Day 3 (72-78 hours) after collection. Treatments on Days 1-3 were performed one at a time.

[0319] Frozen aliquots were thawed in a water bath at 38°C until the sample became liquid in the conical part, then placed in a shaker to complete the thawing process. The tubes were centrifuged at 500 RCF for 5 minutes, the supernatant was removed and the tubes were tilted in a rack to disperse the cell pellet. The cells were washed with 2 ml of PBS with 1% BSA, centrifuged at 500 RCF for 5 minutes, the supernatant was removed and the tubes were tilted in a rack to disperse the cell pellet. The number of cells was determined for each sample and brought the number of cells to a level of 20×10 ⁶ cells/ml to 40×10 ⁶ cells/ml PBS with 1% BSA if necessary. 200 μl were taken with a pipette into a tube with the appropriate marking. The required amount of human serum was added to each tube, shaken and placed in the dark to incubate for 10 minutes. An appropriate amount of antibody was added to each tube. All samples were thoroughly shaken and incubated for 30 minutes at room temperature in the dark. After incubation, the cells were washed with 2 ml of PBS with 1% BSA. The tubes were centrifuged at 500 RCF for 5 minutes, the supernatant was removed and the tubes were tilted in a rack to disperse the cell pellet. After centrifugation, the cells were reconstituted in 500 μl of 1% paraformaldehyde. Samples were examined on a BD FACSCanto II flow cytometer. In total, approximately 1,000,000 events were recorded or analysis continued until 240 seconds elapsed. The results are shown in Table 5.

Table 5

Donor Days after collection Total number of registered events % CD19+ B cells that are CD38++/CD27++ Donor 1 0 1000000 0.14 1 830650 0.10 2 908750 0.09 3 737475 0.12 Donor 2 0 230400 0.94 1 794125 1.03 2 662925 1.05 3 714550 1.08 Donor 3 0 248500 0.17 1 934225 0.21 2 315400 0.24 3 842975 0.20 Donor 4 0 892050 0.32 1 1000000 0.49 2 528175 0.32 3 600675 0.24 Donor 5 0 257450 0.48 1 1000000 0.49 2 962875 0.43 3 1000000 0.47

EXAMPLE 20: Ab79 does not inhibit staining with CD38-specific mAb Ab19

[0320] Five whole blood samples from healthy donors (anticoagulant heparin sodium) were collected and Ab79 (10 μg/ml and zero) was administered for one hour at 37°C. After incubation, the samples were divided into four aliquots. The first aliquot was examined fresh by the FACSlyse method without the freezing step to establish the baseline plasmablast level. Freshly collected samples were tested in triplicate by two independent operators on Day 0 (<2 hours post-collection). The other three aliquots were frozen at -80°C after incubation with FACSLyse for 10 minutes. The second aliquot was thawed in a water bath at 38°C and processed on Day 1 (24-30 hours). The third aliquot was thawed on Day 2 (48-54 hours) and the fourth aliquot on Day 3 (72-78 hours) after collection. Treatments on Days 1-3 were performed one at a time.

[0321] Frozen aliquots were thawed in a water bath at 38°C until the sample became liquid in the conical part, then placed in a shaker to complete the thawing process. The tubes were centrifuged at 500 RCF for 5 minutes, the supernatant was removed and the tubes were tilted in a rack to disperse the cell pellet. The cells were washed with 2 ml of PBS with 1% BSA, centrifuged at 500 RCF for 5 minutes, the supernatant was removed and the tubes were tilted in a rack to disperse the cell pellet. The number of cells was determined for each sample and brought the number of cells to a level of 20×10 ⁶ cells/ml and 40×10 ⁶ cells/ml PBS with 1% BSA if necessary. 200 μl were taken with a pipette into a tube with the appropriate marking. The required amount of human serum was added to each tube, vortexed and placed in the dark to incubate for 10 minutes. An appropriate amount of antibody was added to each tube. All samples were thoroughly vortexed and incubated for 30 minutes at room temperature in the dark. After incubation, the cells were washed with 2 ml of PBS with 1% BSA. Centrifuge tubes at 500 RCF for 5 minutes, remove the supernatant and tilt the tubes in a rack to disperse the cell pellet. After centrifugation, the cells were reconstituted in 500 μl of 1% paraformaldehyde. Samples were examined on a BD FACSCanto II flow cytometer. A total of approximately 1,000,000 events were recorded or the analysis continued until 240 seconds elapsed. The results are shown in Table 6.

Table 6

Healthy blood donors Total number of registered events % CD19+B cells that are CD38++/CD27++ Total number of registered events % CD19+ B cells that are CD38++/CD27++ In the presence of 10
micrograms/ml Ab79 Without adding Ab79 Donor 1 1000000 0.14 1000000 0.14 Donor 2 818625 0.86 230400 0.94 Donor 3 247425 0.18 248500 0.17 Donor 4 255675 0.28 892050 0.32 Donor 5 255400 0.48 257450 0.48

[0322] EXAMPLE 21: Healthy Ab79 Treated Individuals Demonstrate a Dose-Dependent Reduction in PB/PC as Measured by Fix/Freeze Method and Flow Cytometry Assays

[0323] PB/PC levels were assessed in the highest dose cohorts in a randomized, double-blind, placebo-controlled study of Ab79 with single doses in healthy individuals. Ab79 or placebo was administered either by 2-hour intravenous infusion or by subcutaneous administration in doses of various sizes. Whole blood was collected at the clinical center. Pre-treatment samples (2 screenings, Day -1 and Day 1 pre-dose) were used to determine baseline. Samples after treatment were collected at 14 time points over a period of time from 1 hour to 78 days depending on the route of administration. Most of the PB/PC data were collected from individuals treated with sc. All samples were preserved using the fixation/freeze method described above and examined by multicolor flow cytometry assays.

[0324] A comparison of the median number of PB/PC cells in the blood of individuals who received SC treatment showed that after the administration of placebo there were differences in the levels of PB/PC over time, however, Ab79 treatment led to a consistent decrease in PB/PC in the Ab79 treatment group which persisted for up to 20 days. The results are shown in FIG. 20.

SEQUENCE LIST

SEQ ID NO: 1 (CD38 Homo sapiens; NP_001766.2)

MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI

SEQ ID NO: 2 (CD38 Macaca fascicularis; AAT36330.1)

MANCEFSPVSGDKPCCRLSRRAQVCLGVCLLVLLILVVVVAVVLPRWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI

SEQ ID NO: 3 (HCDR1 Ab79)

GFTFDDYG

SEQ ID NO: 4 (HCDR2 Ab79)

ISWNGGKT

SEQ ID NO: 5 (HCDR3 Ab79)

ARGSLFHDSSGFYFGH

SEQ ID NO: 6 (LCDR1 Ab79)

SSNIGDNY

SEQ ID NO: 7 (LCDR2 Ab79)

RDS

SEQ ID NO: 8 (LCDR3 Ab79)

QSYDSSLGS

SEQ ID NO: 9 (Heavy chain Ab79)

EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLA

SEQ ID NO: 10 (Light chain Ab79)

QSVLTQPPSASGTPGQRVTISCSGSSSNIGDNYVSWYQQLPGTAPKLLIYRDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSVFGGGTKLTVLGQPKANPTVTLFPPSSEEL

SEQ ID NO: 11 (Heavy chain Ab19)

EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYDMTWVRQAPGKGLEWVAVISYDGSDKDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYGFSGPSMDVWGQGTLVTVSSASTKGPSVFPLA

SEQ ID NO: 12 (Light chain Ab19)

QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYQQLPGTAPKLLIYSDSNRPSGVPDRFSSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSRVFGGGTKLTVLGQPKANPTVTLFPPSSEEL

SEQ ID NO: 13 (HCDR1 Ab19)

GFTFNNYD

SEQ ID NO: 14 (HCDR2 Ab19)

ISYDGSDK

SEQ ID NO: 15 (HCDR3 Ab19)

ARVYYYGFSGPSMDV

SEQ ID NO: 16 (LCDR1 Ab19)

NSNIGSNT

SEQ ID NO: 17 (LCDR2 Ab19)

SDS

SEQ ID NO: 18 (LCDR3 Ab19)

QSYDSSLSGSR

SEQ ID NO: 19 (Heavy chain Ab19) with constant region

EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYDMTWVRQAPGKGLEWVAVISYDGSDKDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYGFSGPSMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 20 (Light chain Ab19) with constant region

QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYQQLPGTAPKLLIYSDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSRVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK

SEQ ID NO: 21 (Heavy chain Ab79)

EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 22 (Light chain Ab79)

QSVLTQPPSASGTPGQRVTISCSGSSSNIGDNYVSWYQQLPGTAPKLLIYRDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYTVSCQVTHEGS

SEQ ID NO: 23 (CD157 Homo sapiens; NP_004325)

MAAQGCAASRLLQLLLQLLLLLLLLAAGGARARWRGEGTSAHLRDIFLGRCAEYRALLSPEQRNKNCTAIWEAFKVALDKDPCSVLPSDYDLFINLSRHSIPRDKSLFWENSHLLVNSFADNTRRFMPLSDVLYGRVADFLSWCRQKNDSGLDYQSCPTS EDCENNPVDSFWKRASIQYSKDSSGVIHVMLNGSEPTGAYPIKGFFADYEIPNLQKEKITRIEIWVMHEIGGPNVESCGEGSMKVLEKRLKDMGFQYSCINDYRPVKLLQCVDHSTHPDCALKSAAAATQRKAPSLYTEQRAGLIIPLFLVLASRTQL

SEQ ID NO: 24 (Control 1; Heavy chain variable region)

EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

SEQ ID NO: 25 (Control 1; Light chain variable region)

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKR

SEQ ID NO: 26 (Control 2; Heavy chain variable region)

QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYYGSNSLDYWGQGTSVTVSS

SEQ ID NO: 27 (Control 2; Light chain variable region)

DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIKR

SEQ ID NO: 28 (Ab43 heavy chain)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSRINSDGSSTSYADSMKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYYYYAMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 29 (Ab43 light chain)

QSVLTQPPSASGTPGQRVTISCSGGSSNIGYKTVNWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGLVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWCSHREKTVAPTEGSTV

SEQ ID NO: 30 (Heavy chain Ab72)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMNWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSNYDFWSGYYYGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 31 (Light chain Ab72)

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSKTVSWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAARSTNIIFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWCSHRSYSCQVTHEGSTVEKAP

SEQ ID NO: 32 (Heavy chain Ab110)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSIIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRATWGGATHDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 33 (Light chain Ab110)

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLNGVLFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWCSHREKTVAPTEGSTV

SEQ ID NO: 34 (Heavy chain Ab19) with constant region

EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYDMTWVRQAPGKGLEWVAVISYDGSDKDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYGFSGPSMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 35 (Light chain Ab19) with constant region

QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYQQLPGTAPKLLIYSDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSRVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK

[0325] After a detailed description of the invention and with reference to specific variants of its implementation, it will be obvious that modifications and variations are possible without departing from the scope of the invention defined in the attached claims. More specifically, although certain aspects of the present invention are defined herein as being particularly advantageous, it is intended that the present invention should not necessarily be limited to these specific aspects of the invention.

--->

SEQUENCE LIST

<110> TAKEDA PHARMACEUTICAL COMPANY LIMITED

SMITSON, Glennda

ESTEVAM, Jose

JONES, Nicholas

<120> METHODS AND MATERIALS FOR ASSESSING THE RESPONSE TO THERAPY AGAINST PLASMOBLASTS

AND PLASMA CELLS

<130> 67376-266608

<140> PCT patent application number

<141> 2017-07-14

<150> 62/362.963

<151> 2016-07-15

<160> 35

<170>PatentIn version 3.5

<210> 1

<211> 300

<212> Protein

<213> Homo sapiens

<400> 1

Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys

1 5 10 15

Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val

20 25 30

Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln

35 40 45

Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu

50 55 60

Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val

65 70 75 80

Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys

85 90 95

His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu

100 105 110

Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser Arg Ile

115 120 125

Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr

130 135 140

Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys

145 150 155 160

Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp

165 170 175

Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val

180 185 190

Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu

195 200 205

Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser

210 215 220

Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu Glu Ala

225 230 235 240

Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu Cys Gln Asp

245 250 255

Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile Gln

260 265 270

Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val

275 280 285

Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile

290 295 300

<210> 2

<211> 301

<212> Protein

<213> Macaca fascicularis

<400> 2

Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys

1 5 10 15

Arg Leu Ser Arg Arg Ala Gln Val Cys Leu Gly Val Cys Leu Leu Val

20 25 30

Leu Leu Ile Leu Val Val Val Val Val Val Val Leu Pro Arg Trp Arg

35 40 45

Gln Gln Trp Ser Gly Ser Gly Thr Thr Ser Arg Phe Pro Glu Thr Val

50 55 60

Leu Ala Arg Cys Val Lys Tyr Thr Glu Val His Pro Glu Met Arg His

65 70 75 80

Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser

85 90 95

Lys Tyr Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Val Lys

100 105 110

Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Thr Leu Leu Trp Ser Arg

115 120 125

Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe

130 135 140

Thr Leu Glu Asp Met Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp

145 150 155 160

Cys Gly Glu Phe Asn Thr Phe Glu Ile Asn Tyr Gln Ser Cys Pro Asp

165 170 175

Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr

180 185 190

Val Ser Arg Arg Phe Ala Glu Thr Ala Cys Gly Val Val His Val Met

195 200 205

Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly

210 215 220

Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Ala Leu Glu

225 230 235 240

Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu Cys Gln

245 250 255

Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile

260 265 270

Arg Phe Phe Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys

275 280 285

Val Lys Asn Pro Glu Asp Ser Ser Cys Leu Ser Gly Ile

290 295 300

<210> 3

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain CDR1 Ab79

<400> 3

Gly Phe Thr Phe Asp Asp Tyr Gly

15

<210> 4

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain CDR2 Ab79

<400> 4

Ile Ser Trp Asn Gly Gly Lys Thr

15

<210> 5

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain CDR3 Ab79

<400> 5

Ala Arg Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His

1 5 10 15

<210> 6

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain CDR1 Ab79

<400> 6

Ser Ser Asn Ile Gly Asp Asn Tyr

15

<210> 7

<211> 3

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain CDR2 Ab79

<400> 7

Arg Asp Ser

1

<210> 8

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain CDR3 Ab79

<400> 8

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser

1 5 10

<210> 9

<211> 135

<212> Protein

<213> Artificial sequence

<220>

<223> Ab79 heavy chain variable region

<400> 9

Glu Val Gln Leu Leu 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

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Trp Asn Gly Gly Lys Thr His Tyr Val Asp Ser Val

50 55 60

Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ser 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 Arg Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala

130 135

<210> 10

<211> 129

<212> Protein

<213> Artificial sequence

<220>

<223> Ab79 light chain variable region

<400> 10

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu

<210> 11

<211> 134

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain Ab19

<400> 11

Glu Val Gln Leu Leu 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 Asn Asn Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asp Lys Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 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 Arg Val Tyr Tyr Tyr Gly Phe Ser Gly Pro Ser Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala

130

<210> 12

<211> 130

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain Ab19

<400> 12

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Gly Ser Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu

130

<210> 13

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Ab19 heavy chain CDR1

<400> 13

Gly Phe Thr Phe Asn Asn Tyr Asp

15

<210> 14

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Ab19 heavy chain CDR2

<400> 14

Ile Ser Tyr Asp Gly Ser Asp Lys

15

<210> 15

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Ab19 heavy chain CDR3

<400> 15

Ala Arg Val Tyr Tyr Tyr Gly Phe Ser Gly Pro Ser Met Asp Val

1 5 10 15

<210> 16

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain CDR1 Ab19

<400> 16

Asn Ser Asn Ile Gly Ser Asn Thr

15

<210> 17

<211> 3

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain CDR2 Ab19

<400> 17

Ser Asp Ser

1

<210> 18

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain CDR3 Ab19

<400> 18

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Arg

1 5 10

<210> 19

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain Ab19

<400> 19

Glu Val Gln Leu Leu 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 Asn Asn Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asp Lys Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 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 Arg Val Tyr Tyr Tyr Gly Phe Ser Gly Pro Ser Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 20

<211> 217

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain Ab19

<400> 20

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Gly Ser Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 21

<211> 453

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain Ab79

<400> 21

Glu Val Gln Leu Leu 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

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Ser Trp Asn Gly Gly Lys Thr His Tyr Val Asp Ser Val

50 55 60

Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ser 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 Arg Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 22

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain Ab79

<400> 22

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 23

<211> 318

<212> Protein

<213> Homo sapiens

<400> 23

Met Ala Ala Gln Gly Cys Ala Ala Ser Arg Leu Leu Gln Leu Leu Leu

1 5 10 15

Gln Leu Leu Leu Leu Leu Leu Leu Leu Ala Ala Gly Gly Ala Arg Ala

20 25 30

Arg Trp Arg Gly Glu Gly Thr Ser Ala His Leu Arg Asp Ile Phe Leu

35 40 45

Gly Arg Cys Ala Glu Tyr Arg Ala Leu Leu Ser Pro Glu Gln Arg Asn

50 55 60

Lys Asn Cys Thr Ala Ile Trp Glu Ala Phe Lys Val Ala Leu Asp Lys

65 70 75 80

Asp Pro Cys Ser Val Leu Pro Ser Asp Tyr Asp Leu Phe Ile Asn Leu

85 90 95

Ser Arg His Ser Ile Pro Arg Asp Lys Ser Leu Phe Trp Glu Asn Ser

100 105 110

His Leu Leu Val Asn Ser Phe Ala Asp Asn Thr Arg Arg Phe Met Pro

115 120 125

Leu Ser Asp Val Leu Tyr Gly Arg Val Ala Asp Phe Leu Ser Trp Cys

130 135 140

Arg Gln Lys Asn Asp Ser Gly Leu Asp Tyr Gln Ser Cys Pro Thr Ser

145 150 155 160

Glu Asp Cys Glu Asn Asn Pro Val Asp Ser Phe Trp Lys Arg Ala Ser

165 170 175

Ile Gln Tyr Ser Lys Asp Ser Ser Gly Val Ile His Val Met Leu Asn

180 185 190

Gly Ser Glu Pro Thr Gly Ala Tyr Pro Ile Lys Gly Phe Phe Ala Asp

195 200 205

Tyr Glu Ile Pro Asn Leu Gln Lys Glu Lys Ile Thr Arg Ile Glu Ile

210 215 220

Trp Val Met His Glu Ile Gly Gly Pro Asn Val Glu Ser Cys Gly Glu

225 230 235 240

Gly Ser Met Lys Val Leu Glu Lys Arg Leu Lys Asp Met Gly Phe Gln

245 250 255

Tyr Ser Cys Ile Asn Asp Tyr Arg Pro Val Lys Leu Leu Gln Cys Val

260 265 270

Asp His Ser Thr His Pro Asp Cys Ala Leu Lys Ser Ala Ala Ala Ala

275 280 285

Thr Gln Arg Lys Ala Pro Ser Leu Tyr Thr Glu Gln Arg Ala Gly Leu

290 295 300

Ile Ile Pro Leu Phe Leu Val Leu Ala Ser Arg Thr Gln Leu

305 310 315

<210> 24

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> Control Heavy Chain 1

<400> 24

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 25

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Control light chain 1

<400> 25

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 26

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Control Heavy Chain 2

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr

65 70 75 80

Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 27

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Control light chain 2

<400> 27

Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly

1 5 10 15

Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 28

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain Ab43

<400> 28

Glu Val Gln Leu Leu 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 Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Met

50 55 60

Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ser 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 Arg Gly Gly Tyr Tyr Tyr Tyr Ala Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 29

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain Ab43

<400> 29

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Tyr Lys

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 30

<211> 455

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain Ab72

<400> 30

Glu Val Gln Leu Leu 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 Ser Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 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 Lys Asp Ser Asn Tyr Asp Phe Trp Ser Gly Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 31

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain Ab72

<400> 31

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys

20 25 30

Thr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Ala Arg Ser

85 90 95

Thr Asn Ile Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 32

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain Ab110

<400> 32

Glu Val Gln Leu Leu 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 Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg Ala Thr Trp Gly Gly Ala Thr His Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 33

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain Ab110

<400> 33

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu

85 90 95

Asn Gly Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 34

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain Ab119

<400> 34

Glu Val Gln Leu Leu 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 Asn Asn Tyr

20 25 30

Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asp Lys Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 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 Arg Val Tyr Tyr Tyr Gly Phe Ser Gly Pro Ser Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 35

<211> 217

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain Ab19

<400> 35

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

Ser Gly Ser Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<---

Claims (35)

1. A method of treating a disease in a patient, wherein the method includes: a) measuring the level of CD38-expressing cells in a patient compared to a control individual without disease, where CD38-expressing cells are plasmablasts and plasmacytes; and b) administering to the patient a therapeutically effective amount of an anti-CD38 antibody, where the disease is an autoimmune disease and after treatment with an anti-CD20 antibody, the patient was found to have an increased level of CD38-expressing cells compared to a control individual, and where the anti-CD38 antibody is an isolated antibody that specifically binds to human CD38 (SEQ ID NO:1) and contains: a) a heavy chain variable region comprising: i) a first CDR containing SEQ ID NO:3; ii) a second CDR containing SEQ ID NO:4; and iii) a third CDR containing SEQ ID NO:5; and b) a light chain variable region comprising: i) a first CDR containing SEQ ID NO:6; ii) a second CDR containing SEQ ID NO:7; and iii) a third CDR containing SEQ ID NO:8, where in a biological sample obtained from a patient after treatment with an anti-CD20 antibody, it was found: i) an increased level of CD38-expressing plasmablasts and plasma cells compared to a control subject in the analysis of free Ig light chains, and ii) having an elevated level of at least one gene upregulated in CD38-expressing cells compared to a control individual, where said at least one gene is CD38 or IgJ, and where the anti-CD38 antibody depletes plasmablasts and plasma cells after administration, and where the patient treated with anti-CD38 antibody showed a dose-dependent decrease in plasmablasts and/or plasma cells. 2. The method of claim 1 wherein the heavy chain variable region comprises SEQ ID NO:9. 3. The method of claim 1 wherein the light chain variable region comprises SEQ ID NO:10. 4. The method of claim 1 wherein the heavy chain variable region comprises SEQ ID NO:9 and the light chain variable region comprises SEQ ID NO:10. 5. The method of claim 1 wherein the heavy chain comprises SEQ ID NO:21 and the light chain comprises SEQ ID NO:22. 6. The method of any one of claims 1 to 5, wherein the anti-CD38 antibody further comprises an Fc domain. 7. The method of claim 6 wherein the Fc domain is a human Fc domain. 8. The method of claim 6 wherein the Fc domain is a variant of an Fc domain. 9. The method according to any one of claims 1 to 3, wherein the anti-CD38 antibody interacts with at least K121, F135, Q139, D141, E239, W241, C275, K276, F284, P291 and E292 of SEQ ID NO:1. 10. The method of claim 1, wherein a biological sample obtained from a patient after treatment with an anti-CD20 antibody was found to have an increased level of CD38-expressing cells compared to a control individual as measured by flow cytometry. 11. The method of claim 10, wherein the CD38-expressing cells are stained with a second fluorochrome-conjugated anti-CD38 antibody. 12. The method according to any one of claims 1 to 11, wherein the autoimmune disease is selected from the group consisting of autoimmune thrombocytopenia, immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, ulcerative colitis, graft-versus-host disease, myasthenia gravis, Sjögren's syndrome, multiple sclerosis, and autoimmune thyroiditis. 13. The method of claim 12 wherein the autoimmune disease is rheumatoid arthritis. 14. The method of claim 12 wherein the autoimmune disease is systemic lupus erythematosus. 15. The method of claim 12 wherein the autoimmune disease is myasthenia gravis. 16. The method of claim 12, wherein the autoimmune disease is selected from the group consisting of autoimmune thrombocytopenia, immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura. 17. The method of claim 1, wherein the patient is not responding to treatment with CD20-based therapy.

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