RU2782950C2 - Subcutaneous injection of anti-cd38 antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for the treatment of a disease with increased CD38 expression in an individual is proposed. The method includes subcutaneous injection to the individual of a standard dosage form of an isolated human anti-CD38 antibody, where the standard dosage form is in a volume of 3 ml or less at a dose from 0.03 to 0.6 mg per 1 kg of the body weight.

EFFECT: invention provides effective therapy of autoimmune and cancer diseases with low doses and volumes of anti-CD38 antibodies, preventing side effects and inconveniences related to injection of high doses of preparations for systemic therapy with anti-CD38 antibodies.

15 cl, 108 dwg, 15 tbl, 7 ex

Description

Cross-reference to related applications

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 62/617146, filed January 12, 2018, the full disclosure of which is hereby incorporated by reference.

Incorporation by reference of material submitted electronically

[0002] Included herein by reference is a machine-readable nucleotide/amino acid sequence listing submitted concurrently with this application and identified as follows: A 20 kb ASCII (text) file named "101588-5009-WO-Sequence-Listing.txt ”, created on December 4, 2018.

FIELD OF THE INVENTION

[0003] Disclosed are methods of administering isolated anti-CD38 antibodies at low doses and in small volumes via subcutaneous (SC) administration.

BACKGROUND OF THE INVENTION

[0004] 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 cytoplasmic domain. CD38 is a member of a group of related membrane-bound or soluble enzymes that includes CD157 and the sea snail ADFR cyclase ( Aplysia ). This family of enzymes has the unique ability to convert NAD to cyclic ADP ribose or nicotinic acid adenine dinucleotide phosphate. CD38 is involved in Ca ²⁺ mobilization and signal transduction through tyrosine phosphorylation of numerous signaling molecules including phospholipase Cγ, ZAP-70, syk and c-cbl. Based on these observations, CD38 is an important signaling molecule in the maturation and activation of lymphoid cells during their normal development. Among hematopoietic cells, a set of functional effects have been attributed to CD38-mediated signaling, including lymphocyte proliferation, cytokine release, regulation of B-lymphocyte and myeloid cell development and survival, and induction of dendritic cell (DC) maturation.

[0005] CD38 is expressed in immature hematopoietic cells, downregulated in mature hematopoietic cells, and re-expressed in large numbers in activated lymphocytes and plasma cells. For example, high expression of CD38 is observed in activated B cells, plasma cells, activated CD4+ T cells, activated CD8+ T cells, NK cells, NKT cells, mature DCs, and activated monocytes (US Patent No. 8362211). CD38 deficiency in mice has been associated with reduced levels of peripheral T-regulatory and invariant NKT cells, defects in humoral B-cell responses, and directed DC migration, as well as an attenuated form of collagen-induced arthritis (CIA) (Chiba et al . (2005) Arthrit Rheum 52: 1941-48).

[0006] The presence of anti-CD38 autoantibodies has been associated with a number of diseases, including diabetes, chronic autoimmune thyroiditis, and Graves' disease (Antonelli et al. (2001) Clin. Exp. Immunol. 126: 426-431; Mallone et al. (2001) Diabetes 50: 752 and Antonelli et al (2004) J Endocrinol Invest 27: 695-707).

[0007] Increased expression of CD38 has been documented in various diseases, including autoimmune diseases and cancer. Such diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), myasthenia gravis (MG) (Yilmaz et al. (2018) Ann. Clin. Transl. Neurol, 5 (11): 1408-1414), optic neuromyelitis (ONN) (Chihara et al. (2011) Proc. Natl. Acad. Sci. USA108 (9): 3701-6) immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TPP) (Behzad et al. (2018) APMIS126 (6): 523-532) antiphospholipid syndrome (APS) (Alvarez-Rodriguez et al. (2018) Int. J. Mol. Sci, 19 (2): pii ), pemphigus vulgaris (PE), pemphigus foliaceus (PL), anti-NMDA receptor encephalitis (ANRE), autoimmune hemolytic anemia (AHA), Graves disease, membranous nephropathy, Sjogren's syndrome (SS), ANCA-associated vasculitis, acquired bullous epidermolysis (PBE), Hashimoto's thyroiditis, scleroderma and IgG ₄ -associated disease. In patients with RA, the number of plasma cells in the articular tissue is increased compared to controls. In SLE, plasma blasts are elevated in peripheral blood in patients with more active disease. However, current therapies based on CD20-associated depletion of the B cell population, such as rituximab, effectively deplete the CD20+ B lymphocyte population, but cannot directly and effectively deplete plasma cell populations or plasma blasts because they do not express CD20. Thus, RA or SLE patients with high levels of plasma cells or plasma blasts are unlikely to benefit significantly from CD20-based therapy.

[0008] Therapeutics targeting CD38, which is highly expressed in plasma cells, plasma blasts, NK cells, activated B cells, plasmacytoid dendritic cells, and activated T cells, can provide effective treatment for RA and SLE, as well as other diseases characterized by the expression of CD38. The level of CD38-expressing plasma blasts in the peripheral blood of adult SLE patients treated with rituximab and oral steroids was the best predictor of relapse time. In addition, circulating immunoglobulin (Ig)-secreting cells that express high levels of CD38 have been identified in the peripheral blood of SLE patients with active disease, and the level of this subpopulation has been associated with treatment-induced reductions in anti-double-stranded DNA (anti-dsDNA) levels. antibodies, proteinuria, and disease activity (Grammer et al . (2003) J. Clin. Invest. 112: 1506-1520). In addition, plasma cells are sensitive to proteasome inhibition and are downregulated in the peripheral blood of refractory SLE patients exposed to bortezomib (Alexander et al . (2015) Ann. Rheum. Dis. 74 (7): 1474-1478). This decrease was consistent with a decrease in anti-dsDNA antibodies and a corresponding improvement in disease activity in each patient. Unfortunately, therapy has been associated with treatment-related adverse events (AELI; eg, neuropathy, diarrhea) that may result from proteasome inhibition in non-lymphoid cells (eg, neurons, epithelium). Taken together, these data indicate that a specific reduction in the number of CD38-expressing plasma cells may provide a significant improvement in the risk profile for patients with refractory SLE. Because current treatments for RA and SLE induce good clinical response and sustained remission in only a small number of patients, there is an urgent need to explore additional treatment mechanisms.

[0009] Increased expression of CD38 has been documented in various diseases of hematopoietic origin, as well as in derived cell lines, and has been described as a negative prognostic marker in hematological cancers. Such diseases include, but are not limited to, multiple myeloma (MM), chronic lymphocytic leukemia, B-cell chronic lymphocytic leukemia (B-CLL), including B-cell acute lymphocytic leukemia, B- and T-cell acute lymphocytic leukemia (ALL ), acute lymphoblastic leukemia, Waldenström macroglobulinemia, mantle cell lymphoma, prolymphocytic/myelocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), follicular lymphoma, NK cell leukemia, plasma cell leukemia, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (LB), T-cell lymphoma (TKL), hairy cell leukemia (VCL), and Hodgkin's lymphoma (HL). In addition, CD38 expression is predictive of patients with conditions such as B-CLL (Dürig et al . (2002) Leukemia 16:30-35; and Morabito et al . (2001) Leukemia Res. 25:927 -932) and acute myelogenous leukemia (Keyhani et al . (1999) Leukemia Res. 24: 153-159). Therefore, CD38 provides a useful target for the treatment of diseases of the hematopoietic system.

[0010] Several anti-CD38 antibodies are in clinical trials for the treatment of CD38-associated cancers. However, these prior art therapeutic anti-CD38 antibodies bind to erythrocytes and platelets, which may explain why a higher dosage requirement is needed to overcome such a deficiency caused by erythrocyte binding. For example, treatment with daratumumab (Darzalex® - an anti-CD38 IgG1 isotype monoclonal antibody approved by the FDA and commercially available from Janssen Oncology) requires a very high dose (≥16 mg/kg) and an intensive regimen (8x weekly, 8x every other week, then monthly) for optimal antitumor activity (Xu et al. (2017) Clin. Pharmacol. Ther. 101 (6): 721-724). Binding of daratumumab to CD38 on erythrocytes and platelets results in a positive antiglobulin test (indirect Coombs test), which can persist up to 6 months after the last infusion of daratumumab (Sullivan et al. (2017) Blood 129 (22): 3033-3037). This is an important property of daratumumab and other antibodies that bind red blood cells. Although CD38 is expressed on erythrocytes at a level approximately 1000 times lower than on myeloma cells (deWeers et al. (2011) J. Immunol. 186 (3): 1840-1848), in the blood of a patient with MM with active disease, there are approximately 36,000 red blood cells for every myeloma cell (Witzig et al. (1993) Cancer 72 (1): 108-113). Therefore, there are 36 times more CD38 molecules expressed by the erythrocyte population compared to the tumor cell population. Thus, it has been hypothesized that erythrocytes will bind a significant amount of any anti-CD38 antibody administered, resulting in the need to administer large doses in order to obtain sufficient levels of anti-CD38 antibody to achieve a therapeutic effect on tumor cells.

[0011] Accordingly, treatment with anti-CD38 antibodies is currently focused on intravenous (IV) administration due to the large volume of antibody required to achieve therapeutic efficacy, because such large volumes are not suitable for subcutaneous administration, and there are a limit to how concentrated antibodies can be in dosage form. For example, a dose of 1200 mg of daratumumab given intravenously over at least 2 hours is approved for the treatment of relapsed and refractory multiple myeloma (RRMM) and new-onset multiple myeloma (MMMM). Subcutaneous (SC) daratumumab dosage form consists of 1800mg in 15mL to be mixed with Enhance™ (containing Halozyme to promote absorption) and administered 8X weekly, 8X every other week, and then once a month , is currently in phase 3 clinical trials for PPMM.

[0012] Another anti-CD38 antibody, isatuximab (commercially available from Sanofi Genzyme and currently in phase 3 clinical trials), is administered at 10 mg/kg and 20 mg/kg, corresponding to 700-1400 mg per patient 70 kg, with a predicted injected volume of 3.5-14 ml.

[0013] The higher doses and volumes required of prior art anti-CD38 antibodies in current clinical practice can also cause serious side effects, such as, for example, hemolytic anemia, a condition in which red blood cells are destroyed faster than they can be. replaced. In an open, uncontrolled study, isatuximab was administered intravenously to 97 patients at a dose of 3 mg/kg every 2 weeks (n=23); at a dose of 10 mg/kg every 2 weeks for 2 cycles, followed by every 4 weeks (n=25); at a dose of 10 mg/kg every 2 weeks (n=24); and 20 mg/kg once a week for 4 doses (1 cycle), followed by every 2 weeks (n=25). The most common severe (grade 3/4) side effect was anemia, which affected 24% of patients (see http://www.onclive.com/conference-coverage/asco-2016/isatuximab-monotherapy-effective- for-heavily-pretreated-myeloma, the 2016 ASCO Annual Meeting Richter et al (2016) J Clin Oncol 34 (suppl): abstr 8005). In one study of daratumumab, 45% of all patients had anemia (19% of which were grade 3) and 48% of patients had thrombocytopenia (10% of which were grade 3 and 8% of which were grade 4) (See, for example, https://www.rxlist.com/darzalex-side-effects-drug-center.htm; Costello (2017) Ther. Adv. Hematol. 8 (1): 28-37). Therefore, patients treated with isatuximab or daratumumab should be closely monitored for these life-threatening and other serious side effects.

[0014] The physical problems in administering large amounts of monoclonal anti-CD38 antibodies to patients subcutaneously illustrate the need in the art for stronger anti-CD38 antibodies, since a stronger antibody could achieve the desired pharmacological effects in a smaller amount/volume and thus allow more effective forms of administration. A stronger antibody could result in smaller volume formulations that are more effective than daratumumab (Darzalex) in patients in whom depletion of the CD38-expressing cell population is warranted, such as for the treatment of autoimmune diseases and hematologic cancers. The advantages of administering a smaller amount of drug include an administration that takes seconds compared to minutes for SC daratumumab and hours for IV daratumumab, isatuximab, or MOR202, as well as fewer infusion reactions (as was demonstrated for subcutaneous administration of daratumumab). Reducing the amount of time a patient has to spend in an infusion center will allow for home-based treatment options, and increase the efficiency of infusion and the productivity of infusion centers; reduce health care costs per patient as a result of increased institutional efficiency; and expand use if the drug can be used by patients without access to an infusion center.

[0015] AB79 is a fully human IgG1 immunoglobulin monoclonal antibody that specifically binds to CD38 with high affinity (Kd=3.5 nM) (U.S. Patent No. US 8,362,211, the contents of which are hereby incorporated by reference in their entirety). AB79 inhibits the growth of CD38-expressing tumor cells by depleting the cell population through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). AB79 also reduces the level of plasma cells and plasma blasts in blood taken from healthy subjects and patients with systemic lupus erythematosus (SLE). In the case of SLE, 80% of the plasma cell population, including short-lived and long-lived plasma cells, is reduced. In addition, the number of cells producing pathogenic autoantibodies was also reduced, including VH4-34 9G4+ antibodies (70% reduction), anti-Ro antibody (70% reduction), and anti-dsDNA antibody (80% reduction). Daratumumab, an anti-human CD38 monoclonal antibody, also depletes CD38-expressing plasma blasts and plasma cells in samples from patients with SLE and RA in a dose-dependent manner in vitro . Unlike daratumumab, AB79 cross-reacts with cynomolgus monkey-expressed CD38, providing a unique opportunity to determine whether a decrease in CD38-expressing cells would affect inflammation and tissue damage in a non-human primate model of autoimmune disease. In healthy cynomolgus monkeys, depletion efficiency for B and T cells as well as NK cells was positively correlated with CD38 expression level and AB79 dose level (PCT Application No. PCT/US2017/042128; US Patent No. US 8362211).

[0016] Given that many CD38 antibodies in clinical practice are not suitable for subcutaneous administration at low doses or in small volumes and have dangerous side effects, there remains a need in the art for antibody formulations that are safer, more convenient, and more effective in the treatment of diseases for which CD38 binding is prescribed, such as autoimmune diseases and hematological cancers.

BRIEF DESCRIPTION OF THE INVENTION

[0017] Provided herein are methods of treating diseases in which CD38 binding is prescribed, such as, for example, autoimmune diseases and hematologic cancers, comprising subcutaneous administration of isolated anti-CD38 antibodies at surprisingly low doses and/or small volumes.

[0018] In one aspect, the invention relates to a method of treating a disease wherein CD38 binding is administered to a subject, the method comprising the step of subcutaneously administering to a subject suffering from CD38 binding a therapeutically effective amount of isolated human anti-CD38 an antibody sufficient to treat a disease, wherein the anti-CD38 antibody comprises a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence SEQ ID NO: 5; and a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 6, a CDR2 having the amino acid sequence of SEQ ID NO: 7, and a CDR3 having the amino acid sequence of SEQ ID NO: 8.

[0019] In another aspect, the invention relates to a method of treating a disease wherein CD38 binding is administered to a subject, the method comprising the step of subcutaneously administering to a subject suffering from CD38 binding a therapeutically effective amount of isolated human anti-CD38 an antibody sufficient to treat a disease, wherein the anti-CD38 antibody comprises a VH chain region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence of SEQ ID NO: 5; and a VL chain region containing a CDR1 having the amino acid sequence of SEQ ID NO: 6, a CDR2 having the amino acid sequence of SEQ ID NO: 7, and a CDR3 having the amino acid sequence of SEQ ID NO: 8, where the anti-CD38 antibody is administered in a volume of 3 milliliters or less.

[0020] In another aspect, the invention relates to a method of treating a disease wherein CD38 binding is administered to a subject, the method comprising the step of subcutaneously administering to a subject suffering from CD38 binding a therapeutically effective amount of isolated human anti-CD38 an antibody sufficient to treat a disease, wherein the anti-CD38 antibody comprises a VH chain region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence of SEQ ID NO: 5; and a portion of the VL chain containing CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 7, and CDR3 having the amino acid sequence of SEQ ID NO: 8, the anti-CD38 antibody is administered at a dose of 0, 03 to 0.6 milligrams per kilogram of body weight.

[0021] In one aspect, the anti-CD38 antibody does not cause hemolytic anemia or thrombocytopenia.

[0022] In one aspect, administration of an anti-CD38 antibody results in less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% of the occurrence of adverse events that occurred during the course of treatment (AELI), grade 3 or 4, selected from the group, consisting of anemia, hemolytic anemia, thrombocytopenia, fatigue, infusion reactions (IR), leukopenia, and lymphopenia.

[0023] In one aspect, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% depletion of the red blood cell population.

[0024] In one aspect, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% platelet depletion.

[0025] In one aspect, the disease is selected from the group consisting of autoimmune disease and cancer.

[0026] In one aspect, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis, myasthenia gravis (MG), optic neuromyelitis (ONN), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), antiphospholipid syndrome (APS), pemphigus vulgaris (PO), pemphigus foliaceus (PL), anti-NMDA receptor encephalitis (ANRE), autoimmune hemolytic anemia (AHA), diseases Graves, membranous nephropathy, Sjögren's syndrome (SS), ANCA-associated vasculitis, acquired epidermolysis bullosa (PBE), bullous pemphigoid (BP), Hashimoto's thyroiditis, scleroderma, IgG ₄ -associated disease, and graft versus host disease. (Yilmaz V, et. al. , Ann Clin Transl Neurol. 2018 Sep 22; 5 (11): 1408-1414; Chihara N, Aranami T, Sato W, Miyazaki Y, Miyake S, Okamoto T, Ogawa M, Toda T , Yamamura T. Proc Natl Acad Sci US A. 2011 Mar 1; 108 (9): 3701-6; Behzad MM, et al. , APMIS. 2018 Jun; 126 (6): 523-532; or Alvarez-Rodriguez L ., et al., Int J Mol Sci. 2018 Feb 16;19(2).

[0027] In one aspect, the hematologic cancer is selected from the group consisting of multiple myeloma, NK/T cell lymphoma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, B cell lymphoma, and lymphoma Burkitt.

[0028] In one aspect, the hematologic cancer is multiple myeloma. In some embodiments, the multiple myeloma is selected from the group consisting of PPMM and WWMM.

[0029] In one aspect, the VH region of the chain has the amino acid sequence of SEQ ID NO: 9, and the VL region of the chain has the amino acid sequence of SEQ ID NO: 10.

[0030] In one aspect, the anti-CD38 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12.

[0031] In one aspect, a therapeutically effective amount is from 0.03 to 0.6 milligrams per kilogram of body weight.

[0032] In one aspect, the therapeutically effective amount is in a volume of 3 milliliters or less.

[0033] In one aspect, the therapeutically effective amount is in a volume of 2 milliliters or less.

[0034] In one aspect, the therapeutically effective amount is in a volume of 1 milliliter or less.

[0035] In one aspect, the human anti-CD38 antibody is administered in the form of a pharmaceutically acceptable composition.

[0036] In another aspect, the invention provides a method for treating hematologic cancer in a subject, the method comprising the step of subcutaneously administering to a subject having hematologic cancer a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the hematologic cancer, wherein the anti- The CD38 antibody contains a VH chain region containing a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence of SEQ ID NO: 5; and a VL chain region containing a CDR1 having the amino acid sequence of SEQ ID NO: 6, a CDR2 having the amino acid sequence of SEQ ID NO: 7, and a CDR3 having the amino acid sequence of SEQ ID NO: 8.

[0037] In another aspect, the invention provides a method for treating hematologic cancer in a subject, the method comprising the step of administering to a subject having hematologic cancer a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the hematologic cancer, wherein the anti-CD38 the antibody contains a VH chain region containing a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence of SEQ ID NO: 5; and a VL chain region containing a CDR1 having the amino acid sequence of SEQ ID NO: 6, a CDR2 having the amino acid sequence of SEQ ID NO: 7, and a CDR3 having the amino acid sequence of SEQ ID NO: 8, where the anti-CD38 antibody is administered in a volume of 3 ml or less, 2 ml or less, or 1 ml or less.

[0038] In another aspect, the invention provides a method for treating hematologic cancer in a subject, the method comprising the step of subcutaneously administering to a subject having hematologic cancer a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the hematologic cancer, wherein the anti- The CD38 antibody contains a VH chain region containing a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence of SEQ ID NO: 5; and a VL chain region containing a CDR1 having the amino acid sequence of SEQ ID NO: 6, a CDR2 having the amino acid sequence of SEQ ID NO: 7, and a CDR3 having the amino acid sequence of SEQ ID NO: 8, wherein the anti-CD38 antibody is administered at a dosage of 0 .03 to 0.6 milligrams per kilogram of body weight.

[0039] In one aspect, the anti-CD38 antibody does not cause hemolytic anemia or thrombocytopenia.

[0040] In one aspect, administration of an anti-CD38 antibody results in less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% occurrence of one or more Grade 3 or 4 AEs selected from the group consisting of anemia, including hemolytic anemia , thrombocytopenia, fatigue, infusion reactions (IR), leukopenia, and lymphopenia.

[0041] In one aspect, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% red blood cell depletion.

[0042] In one aspect, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% platelet depletion.

[0043] In one aspect, the hematologic cancer is selected from the group consisting of multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-cell lymphoma, NK/T-cell lymphoma, and lymphoma Burkitt.

[0044] In one aspect, the hematologic cancer is multiple myeloma. In some embodiments, the multiple myeloma is selected from the group consisting of PPMM and WWMM.

[0045] In one aspect, the VH portion of the chain has the amino acid sequence of SEQ ID NO: 9 and the VL portion of the chain has the amino acid sequence of SEQ ID NO: 10.

[0046] In one aspect, the anti-CD38 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 11 and the light chain amino acid sequence of SEQ ID NO: 12.

[0047] In one aspect, a therapeutically effective amount is from 0.03 to 0.6 milligrams per kilogram of body weight.

[0048] In one aspect, the therapeutically effective amount is in a volume of 3 milliliters or less.

[0049] In one aspect, the therapeutically effective amount is in a volume of 2 milliliters or less.

[0050] In one aspect, the therapeutically effective amount is in a volume of 1 milliliter or less.

[0051] In one aspect, the human anti-CD38 antibody is administered in the form of a pharmaceutically acceptable composition.

[0052] In another aspect, the invention provides a unit dosage form comprising an isolated antibody which comprises the amino acid sequence of the heavy chain variable region of SEQ ID NO: 9 and the amino acid sequence of the light chain variable region of SEQ ID NO: 10, wherein the isolated antibody binds to CD38 where the unit dosage form is formulated for subcutaneous administration of the antibody at a dose of 0.03 to 0.6 milligrams per kilogram of body weight.

[0053] In one aspect, the heavy chain contains the amino acid sequence of SEQ ID NO: 11 and the light chain contains the amino acid sequence of SEQ ID NO: 12.

[0054] In one aspect, the unit dosage form has a volume of 3 milliliters or less.

[0055] In one aspect, the unit dosage form has a volume of 2 milliliters or less.

[0056] In one aspect, the unit dosage form has a volume of 1 milliliter or less.

[0057] In one aspect, the unit dosage form is formulated for subcutaneous administration of an antibody in the treatment of hematologic cancer selected from the group consisting of multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-cell lymphomas and Burkitt's lymphomas.

[0058] In one aspect, the hematologic cancer is multiple myeloma. In some embodiments, the multiple myeloma is selected from the group consisting of PPMM and WWMM.

[0059] In one aspect, the anti-CD38 antibody does not cause hemolytic anemia or thrombocytopenia.

[0060] In one aspect, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% red blood cell depletion.

[0061] In one aspect, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% platelet depletion.

[0062] These and other embodiments, features, and potential benefits will become apparent with reference to the following description and drawings.

BRIEF DESCRIPTION OF GRAPHICS

[0063] The objects and features of the present invention may be better understood in view of the drawings described below, in which:

[0064] In FIG. Table 1 shows FA data for the cynomolgus macaque (Javanese) for the s.c. dose groups described in Table 2. Anti-drug antibodies (ADA) were detected using a verified qualitative electrochemiluminescent (ECL) assay . The frequency increased over time and affected PK when a certain threshold titer was reached, around 1000 (~log(7)).

[0065] In FIG. 2 shows the FC data for the Javanese and the FC model for AB79. Panels A and B show primary iv PK data from 8 macaque studies; panel A - the first 7 days after the first dose, and panel B - the entire period of observation. S/C data have been omitted (see Figure 1 for S/C data). Panel C displays the final structure of the PK model, including the target-mediated drug distribution (DDD) marked with a blue box. _Vc denotes the volume of the central chamber of the model in which AB79 concentrations are observed (labeled "Conc"). V _P denotes the volume of the peripheral chamber of the model. _Rtot denotes the model chamber for antibody-bound and unbound CD38 receptor. K _SYN and K _DEG denote the rate constants of formation and degradation of the receptor, and K _INT is the rate constant of internalization (complex elimination rate constant). K _SS is the equilibrium state constant, defined as K _SS = (K _OFF +K _INT ) / K _ON , where K _OFF is the dissociation rate constant and K _ON is the binding rate constant. The DF panels show overlays of the linear two-chamber model predictions (median, 95% predictive interval) without the MRPM component and observational data for the 3 lowest doses (Study 8). Notice the different timelines between panels D , E , and F.

[0066] In FIG. 3 shows the effects of AALP and FA in a 13-week toxicology study on a Javanese. This estimate is based on the final PK population model (Figure 2, Table 2). The following model fit criteria (KAM) plots are presented, stratified by dose and route of administration. (Keizer et al. (2013) CPT Pharmacometrics Syst. Pharmacol. 2: e50): (1) conditional weighted residuals (SVR) versus time; (2) predicted concentration depending on the population model; (3) SVR depending on the population model; and (4) observed concentration versus individual model prediction.

[0067] In FIG. 4 shows the KAM plots for the final population model of FC, stratified by dose and route of administration (i.v. - crosses, s.c. - triangles). Fig. 4A - summary data; Fig. 4B - dose 0.03 mg/kg; Fig. 4C - dose 0.1 mg/kg; Fig. 4D - dose 0.3 mg/kg; Fig. 4E - dose 1.0 mg/kg; Fig. 4F - dose 2.0 mg/kg; Fig. 4G - dose 3.0 mg/kg; Fig. 4H - dose 30 mg/kg; Fig. 4I - dose 80 mg/kg; and Fig. 4J - dose 100 mg/kg.

[0068] In FIG. 5 shows a comparison of CD38 expression on the surface of human and macaque NK-, B- and T-cells. The parameters measured by flow cytometry were standardized, and the signals were presented as equivalent soluble fluorochrome (MEF) molecules. Human and macaque blood lymphocytes bind similar levels of AB79. Direct comparison of CD38 expression levels on macaque NK cells (CD3-, CD159a+), macaque B cells (CD3-, CD20+) and macaque T cells (CD3+), and human NK cells (CD3-, CD16/CD56+), Human B cells (CD3-, CD19+) and human T cells (CD3+) analyzed by flow cytometry. Mean Fluorescence Intensity (MFI) for AB79 staining for each cell population was converted to MERF units using a standard curve generated using Rainbow Beads reagent (Spherotech; Lake Forest, IL). The data shown are from 3 individuals of each species and demonstrate MERF ± SD for each cell type. There are differences in CD38 expression between blood lymphocytes, with higher levels of AB79 binding on NK cells > B cells > T cells. The pattern of AB79 binding is similar in monkey blood cells, but the level of AB79 binding/CD38 expression is lower.

[0069] In FIG. Figure 6 shows the inter- and intra-subject variability of data for T cells, B cells, and NK cells in placebo-treated animals from the study indicated in Figure 6. 5. FIG. 6A - NK cells; Fig. 6B - B cells; and Fig. 6C - T cells.

[0070] In FIG. 7 shows pre-dose numbers of NK-, B-, and T-cells (cells per µl), stratified by study (top row) or sex (bottom row). Fig. 7A - NK cells relative to studies; Fig. 7B - B cells relative to studies; Fig. 7C - T cells relative to studies; Fig. 7D - NK cells relative to male/female; Fig. 7E - B cells relative to male/female; and Fig. 7F - T cells relative to male/female.

[0071] In FIG. 8 shows AB79-dependent depletion of NK cell, B cell and T cell populations. The graphs focus on changes that occurred during the first 7 days after treatment with the first dose of AB79. It was possible to combine data from single and multi-dose studies with weekly or biweekly dosing regimens. Fig. 8A - minimal depletion of the NK cell population; Fig. 8B - 1 week after ^1st dose for NK cells; Fig. 8C is the average profile per dose group for NK cells; 8D - minimal depletion of the B cell population; 8E - 1 week after ^1st dose for B cells; Fig. 8F is the average profile per dose group for B cells; 8G - minimal depletion of the T cell population; Fig. 8H - 1 week after ^1st dose for T cells; and Fig. 8I is the average profile per dose group for T cells. AC plots show individual minimum cell numbers (i.e., maximum PD effect), individual cell numbers 7 days after the first dose, mean cell population depletion profiles per dose, and the structure of the PK-PD model for NK cells, respectively. DF plots show the same information for B cells and GI plots show the same information for T cells.

[0072] In FIG. 9 shows the effect of AB79 antibody treatment on erythrocytes two days post-dose (FIG. 9A) and total lymphocyte count on the first post-dose day in Study 7 (Table 2) (FIG. 9B).

[0073] In FIG. 10 shows human simulated PK and depletion profiles of NK cell, B cell and T cell populations for AB79. Based on scaled macaque PK and PK-PD models, PK was modeled for 5 single i.v. and s.c. doses and depletion profiles of cell populations (0.0003 to 1 mg/kg). The graphs on the left show data after iv injection, and the graphs on the right show data after s/c injection. The first row of graphs displays FC profiles. The lower limit of quantitation (LQQ) of 0.05 μg/mL is indicated by the horizontal dotted line. The lowest dose FC was completely covered by the noise, and only at doses of 0.03 mg/kg did FC reach levels above the LEL. Fig. 9A - IV administration of AB79; Fig. 9B - subcutaneous injection of AB79; Fig. 9C - IV administration for NK cells; Fig. 9D - s / c injection for NK cells; Fig. 9E - IV administration for B cells; Fig. 9F - sc injection for B cells; Fig. 9G - iv administration for T cells; Fig. 9H - sc injection for T cells.

[0074] In FIG. 11 shows the design of a single dose escalation toxicity study of AB79 in healthy volunteers. A total of 74 subjects were randomized into 6 IV and 4 SC cohorts and received a single dose of AB79. Extensive blinded data on safety, PK and PD were analyzed after each cohort prior to dose escalation. Termination criteria included depletion of the target cell population to avoid potential immunosuppression in healthy volunteers. Each subject was followed up for 92 days post-dose.

[0075] In FIG. 12 shows KAM plots for PK-PD models stratified by route of administration (i.v. red; s.c. blue); Fig. 12A - NK cells; Fig. 12B - B cells; and Fig. 12C - T cells.

[0076] FIG. 13 demonstrates that AB79 depletes the macaque lymphocyte population. AB79 caused a dose-dependent depletion of the blood NK-cell > B-cell > T-cell population in female cynomolgus monkeys (n=4 per dose group) after a single IV dose of AB79; quantitation was performed using Flow-Count ^™ fluorospheres (Beckman-Coulter) using flow cytometry. Samples were collected during pre-treatment (week -1), day 1: pre-dose, 15, 30 minutes, 1, 4, 8, 24, 48, 96 and 168 hours post-dose, at 10, 15, 22 , 29, 36 43, 50 and 57 days. For clarity, only 2 weeks of data are shown. Mean cell numbers were calculated at each time point and used to calculate % of baseline. Fig. 13A - T cells; Fig. 13B - B cells; and Fig. 13C - NK cells.

[0077] FIG. 14 demonstrates that secondary immunity responses to human tetanus toxoid (TA) are reduced by administration of the AB79 antibody. CB17/SCID mice were injected with anti-asialo-GM1 to eliminate NK cells and then injected with 25 x 10 ⁶ human peripheral blood lymphocytes. After 7-10 days, serum samples were collected for human Ig evaluation, and the Ig level was the basis for randomization. Mice were given AS to elicit a secondary immune response and treated with these antibodies twice a week for 10 days. Three days after the last treatment, serum was collected and analyzed for anti-AS antibodies. The AB79 antibody caused a dose-dependent suppression of the secondary immunity response to AS, to the same extent as Rituxan (Rtx) isotype control (Iso), Rtx and AB79, all at 10 mg/kg.

[0078] FIG. 15 demonstrates that AB79 does not enhance cytokine induction. The AB79 antibody did not cause an increase in IL-6 levels in PBMC isolated from the blood of 4 different subjects after a 24 hour incubation compared to the IgG1 isotype control. Positive controls for PHA and anti-CD3 increased cytokine levels in all subjects, demonstrating that the cells had the ability to produce IL-6. Similar results were obtained with PBMC stimulated for 48 hours when levels of IL-2, IL-4, IL-10, GM-CSF, IFN-γ, and TNF-α were analyzed (data not shown). The bars display the following conditions for each subject: (i) no AT; (ii) isotype control; (iii) AB79; (iv) anti-CD3; (v) FGA. Each value is the average of triplicate wells ± SD measured 24 hours after incubation.

[0079] In FIG. 16A is a schematic of the experiment of FIG. 16B under dry binding, liquid binding and solution binding conditions (modified from Stebbings et al. (2007) J. Immunol. 179: 3325-3331).

[0080] FIG. 16B demonstrates that AB79 has no agonist activity. The AB79 antibody was highly concentrated when its solution was added to the wells and the liquid was allowed to evaporate (dry binding), compared to when AB79 was allowed to bind to the wells in solution (liquid binding) or added directly to the PBMC (solution binding). The AB79 antibody did not stimulate IL-6 or IL-2, IL-4, IL-8, IL-10, GM-CSF, IFN-γ, or TNF-α in any of the conditions analyzed after 24 hours. IL-8 was constitutively produced by PBMC and was not altered by any of the treatments (data not shown). Each measurement is the average of triplicate wells measured 24 hours after incubation for one subject.

[0081] In FIG. 17 shows an assessment of AB79 binding to human erythrocytes. In this study, no binding of AB79 (solid line histogram) or isotype control (dashed line histogram) to erythrocytes in whole blood of thirty human volunteers was observed in this study. Representative data for 15 out of 30 human volunteers are presented.

[0082] In FIG. 18 shows an assessment of AB79 binding to cynomolgus macaque erythrocytes. In this study, no binding of AB79 (solid line histogram) or isotype control (dashed line histogram) to erythrocytes in whole blood of thirty cynomolgus monkeys was observed in this study. Representative data for 15 out of 30 cynomolgus monkeys are presented.

[0083] In FIG. 19 shows an assessment of AB79 binding to human platelets. In this study, no binding of AB79 (solid line histogram) or isotype control (dashed line histogram) to CD61+ platelets was observed in whole blood of thirty human volunteers. Representative data for 15 out of 30 human volunteers are presented.

[0084] In FIG. 20 shows an assessment of AB79 binding to cynomolgus macaque platelets. In this study, no binding of AB79 (solid line histogram) or isotype control (dashed line histogram) to CD61+ platelets was observed in whole blood of thirty cynomolgus monkeys. Representative data for 15 out of 30 cynomolgus monkeys are presented.

[0085] In FIG. 21 shows an assessment of AB79 binding to CD45+ cynomolgus monkey lymphocytes. Binding of AB79 to CD45+ lymphocytes in unlysed whole blood of cynomolgus monkey. CD45+ lymphocytes were noted and then assessed for binding to AB79 (solid line histogram) or to isotype control (dashed line histogram). Binding of AB79 to a subpopulation of lymphocytes was found, as demonstrated in the fraction of cells to the right of the vertical dotted line. There is practically no binding of isotype control to lymphocytes.

[0086] In FIG. 22 shows the hierarchy for marked cells with (A) identification of erythrocytes and (B) identification of lymphocytes among human erythrocytes.

[0087] In FIG. 23 shows the binding capacity of the antibody for the biotin-streptavidin-BV421 AB79 complex and biotin-streptavidin-BV421 daratumumab. Bars represent (i) blank microbeads, (ii) streptavidin-BV421 only (negative control), (iii) AB79-biotin/Cav-BV421 (5 µg/mL), (iv) daratumumab-biotin/Cav-BV421 (5 µg /ml), (v) AB79-biotin/Cav-BV421 (10 μg/ml), (vi) daratumumab-biotin/Cav-BV421 (10 μg/ml). MIF=mean fluorescence intensity, Sav=streptavidin. A photomultiplier tube (PMT) voltage of 275 volts was used.

[0088] In FIG. 24 shows the binding of the biotin-streptavidin-BV421 AB79 and biotin-streptavidin-BV421 daratumumab complexes to CD38+ lymphocytes. Bars represent (i) AB79-biotin-strep-BV421 (0 µg/ml), (ii) unlabeled AB79 and AB79-biotin-strep-BV421 (10 µg/ml), (iii) AB79-biotin-strep-BV421 ( 10 µg/ml), (iv) daratumumab-biotin-strep-BV421 (0 µg/ml), (v) unlabeled daratumumab and daratumumab-biotin-strep-BV421 (10 µg/ml), (vi) daratumumab-biotin- strep-BV421 (10 μg/ml). The vertical lines represent the standard deviation; n=4 donors (3 donors for unlabeled conditions). MIF=mean fluorescence intensity.

AB79-биотин-стреп-BV421;

холодный (немеченный) AB79 и AB79-биотин-стреп-BV421;

даратумумаб-биотин-стреп-BV421;

холодный даратумумаб и даратумумаб-биотин-стрептавидин BV421.[0089] In FIG. 25 shows the binding of AB79 and daratumumab to human erythrocytes (% positivity per donor). Peripheral blood from four healthy volunteers incubated with biotin-streptavidin-BV421 AB79 complex (0; 0.1; 10; 100 µg/mL) or biotin-streptavidin-BV421 daratumumab (0; 0.1; 1; 10; 100 µg /ml) for 3 hours at room temperature on a soft shaker in the presence or absence of unlabeled AB79 (500 µg/ml) or unlabeled daratumumab (500 µg/ml). Legend:

AB79-biotin-strep-BV421;

cold (unlabeled) AB79 and AB79-biotin-strep-BV421;

daratumumab-biotin-strep-BV421;

cold daratumumab and daratumumab-biotin-streptavidin BV421.

AB79-биотин-стреп-BV421;

холодный AB79 и AB79-биотин-стреп-BV421;

даратумумаб-биотин-стреп-BV421;

холодный даратумумаб и даратумумаб-биотин-стреп-BV421.[0090] In FIG. 26 shows the binding of AB79 and daratumumab to human erythrocytes (total % positive data). Peripheral blood from four healthy volunteers incubated with biotin-streptavidin-BV421 AB79 complex (0; 0.1; 10; 100 µg/mL) or biotin-streptavidin-BV421 daratumumab (0; 0.1; 1; 10; 100 µg /ml) for 3 hours at room temperature on a soft shaker in the presence or absence of unlabeled AB79 (500 µg/ml) or unlabeled daratumumab (500 µg/ml). Legend:

AB79-biotin-strep-BV421;

cold AB79 and AB79-biotin-strep-BV421;

daratumumab-biotin-strep-BV421;

cold daratumumab and daratumumab-biotin-strep-BV421.

AB79-биотин-стреп-BV421;

холодный AB79 и AB79-биотин-стреп-BV421;

даратумумаб-биотин-стреп-BV421;

холодный даратумумаб и даратумумаб-биотин-стрептавидин BV421.[0091] In FIG. 27 shows the binding of AB79 and daratumumab to human erythrocytes (average fluorescence intensity for each donor). Peripheral blood from four healthy volunteers incubated with biotin-streptavidin-BV421 AB79 complex (0; 0.1; 10; 100 µg/mL) or biotin-streptavidin-BV421 daratumumab (0; 0.1; 1; 10; 100 µg /ml) for 3 hours at room temperature on a soft shaker in the presence or absence of unlabeled AB79 (500 µg/ml) or unlabeled daratumumab (500 µg/ml). Legend:

AB79-biotin-strep-BV421;

cold AB79 and AB79-biotin-strep-BV421;

daratumumab-biotin-strep-BV421;

cold daratumumab and daratumumab-biotin-streptavidin BV421.

AB79-биотин-стреп-BV421;

холодный AB79 и AB79-биотин-стреп-BV421;

даратумумаб-биотин-стреп-BV421;

холодный даратумумаб и даратумумаб-биотин-стреп-BV421.[0092] In FIG. 28 shows the binding of AB79 and daratumumab to human erythrocytes (total average fluorescence intensity). Peripheral blood from four healthy volunteers incubated with biotin-streptavidin-BV421 AB79 complex (0; 0.1; 10; 100 µg/mL) or biotin-streptavidin-BV421 daratumumab (0; 0.1; 1; 10; 100 µg /ml) for 3 hours at room temperature on a soft shaker in the presence or absence of unlabeled AB79 (500 µg/ml) or unlabeled daratumumab (500 µg/ml). Legend:

AB79-biotin-strep-BV421;

cold AB79 and AB79-biotin-strep-BV421;

daratumumab-biotin-strep-BV421;

cold daratumumab and daratumumab-biotin-strep-BV421.

изотип-контролем человеческого IgG1,

даратумумабом,

AB79 и

сапонин-контролем в мкг/мл. Каждый символ отображает среднее из 3-х репликат для человека (n=5 доноров).[0093] In FIG. 29 shows hemolysis (normalized %) of human erythrocytes caused by:

isotype control of human IgG1,

daratumumab,

AB79 and

saponin control in µg/ml. Each symbol represents the average of 3 human replicates (n=5 donors).

изотип-контролем человеческого IgG1,

даратумумабом,

AB79 и

сапонин-контролем в мкг/мл. Каждый символ отображает среднее из 3-х репликат для яванской макаки (n=5 доноров).[0094] In FIG. 30 shows hemolysis (normalized %) of cynomolgus macaque erythrocytes caused by:

isotype control of human IgG1,

daratumumab,

AB79 and

saponin control in µg/ml. Each symbol represents the average of 3 cynomolgus macaque replicates (n=5 donors).

[0095] In FIG. 31 shows (A) the mean serum concentration of AB79 in i.v. AB79-treated monkeys and the percentage change from pre-dose baseline in the mean absolute number of (B) CD20 ^- /CD3 ^- /CD16 ⁺ NK cells; (C) CD3 ^- /CD20 ⁺ B cells; and (D) CD3 ⁺ T cells in the peripheral blood of monkeys following an IV infusion of AB79. Control - drug carrier (open squares); 0.1 mg/kg (open diamonds); 0.3 mg/kg (open triangles); 1.0 mg/kg (open circles); 3.0 mg/kg (solid squares); 30.0 mg/kg (filled diamonds) and 80.0 mg/kg (filled triangles) of AB79. Animals from the open symbol cohorts (n=7 animals) were dosed weekly, and the solid symbol cohorts (n=5 animals) were dosed biweekly for 3 months. Error bars indicate standard deviation.

[0096] FIG. 32 shows concentration dependent binding of AB79 (A) to CHO cells expressing recombinant cynomolgus CD38; and (B) endogenous cynomolgus monkey CD38 expressed on CD3+ T lymphocytes, CD3-CD20+ B lymphocytes, and CD3-/CD20-/CD16+ NK cells. NK cell levels in whole blood of cynomolgus monkeys (n=3) after incubation with AB79 for 48 hours in culture are shown in FIG. 22C.

[0097] In FIG. 33 is a plot of the dosing strategy for Example 7.

[0098] In FIG. 34 shows (A) body weight measurements normalized to percent change since study entry: non-arthritis control animal (open circles); intact animals with arthritis (solid circles); animals treated prophylactically with AB79 antibody (open squares); animals receiving therapeutic treatment with AB79 antibody (solid squares); animals receiving therapeutic treatment with dexamethasone (open triangles); (B) Mean clinical arthritis scores for 16 joints: intact animals with arthritis (solid circles); animals treated prophylactically with AB79 antibody (open squares); animals receiving therapeutic treatment with AB79 antibody (solid squares); animals receiving therapeutic treatment with dexamethasone (open triangles); (C) number of swollen proximal interphalangeal (PIP) joints; and (D) mean oval area of 16 PIP joints, calculated and reported as the mean joint swelling score for each animal. Radiographic examination was performed by radiography of each PMF joint (E) and PF joint (F). Data are mean ± standard error for each group. * p < 0.05 and ** p < 0.01 compared with the intact group (one-way ANOVA/Dunn's multiple comparison test).

[0099] In FIG. 35 shows body weight measurements ( A ) normalized to percent change since study entry: non-arthritis control animal (open circles); intact animals with arthritis (solid circles); animals treated prophylactically with AB79 antibody (open squares); animals receiving therapeutic treatment with AB79 antibody (solid squares); animals receiving therapeutic treatment with dexamethasone (open triangles). Serum chemistry scores: CRP (B) and ALP (C) over time: data are mean ± standard error for each group. * p < 0.05 and ** p < 0.01 compared with the intact group (one-way ANOVA/Dunn's multiple comparison test).

[00100] In Fig. 36 shows quantitative histomorphometry of articular area and DMF joint surface, blinded by a bone histopathologist using toluidine blue-stained slides (32 x 22 animals = 704 slides). Individual results for each animal are plotted as mean ± standard error for each group. Statistical analysis was carried out as indicated for the graph above. (A) total area of articular cartilage; (B) the thickness of the damaged articular cartilage; (C) percentage of damaged articular surface; and (D) osteophyte area. Data are mean ± standard error for each group. * p < 0.05 and ** p < 0.01 compared with the intact group (one-way ANOVA/Dunn's multiple comparison test).

[00101] In Fig. 37 shows the indicators of biochemical analysis of serum: CRP (A) and alkaline phosphatase (B) over time. Control animal without arthritis (open circles); intact animals with arthritis (solid circles); animals treated prophylactically with AB79 antibody (open squares); animals receiving therapeutic treatment with AB79 antibody (solid squares); animals receiving therapeutic treatment with dexamethasone (open triangles). Data are mean ± standard error for each group. * p < 0.05 and ** p < 0.01 compared with the intact group (one-way ANOVA/Dunn's multiple comparison test).

[00102] In Fig. 38 shows the levels (A) of erythrocytes; (B) hematocrit; (C) reticulocytes, (D) platelets, (E) neutrophils, and (F) lymphocytes in animals over time: control animal without arthritis (open circles); intact animals with arthritis (solid circles); animals treated prophylactically with AB79 antibody (open squares); animals receiving therapeutic treatment with AB79 antibody (solid squares); animals receiving therapeutic treatment with dexamethasone (open triangles). Data are mean ± standard error for each group. * p < 0.05 and ** p < 0.01 compared with the intact group (one-way ANOVA/Dunn's multiple comparison test).

[00103] In Fig. 39 shows levels (A) of NK cells; (B) B cells; (C) T cells and (D) monocytes in peripheral blood over time: control animal without arthritis (open circles); intact animals with arthritis (solid circles); animals treated prophylactically with AB79 antibody (open squares); animals receiving therapeutic treatment with AB79 antibody (solid squares); animals receiving therapeutic treatment with dexamethasone (open triangles). Data are mean ± standard error for each group. * p < 0.05 and ** p < 0.01 compared with the intact group (one-way ANOVA/Dunn's multiple comparison test).

[00104] In Fig. 40 shows serum concentrations of AB79 in selected monkeys when administered prophylactically (A) or therapeutically (B). Serum concentrations of anti-AB79 antibodies in individual macaque monkeys at prophylactic (C) or therapeutic (D) administration.

[00105] In FIG. 41 shows mean serum concentration-time profiles for AB79 after a single 2-hour IV infusion of AB79 antibody at doses of 0.03 (square) and 0.06 (triangles) mg or a single s.c. injection of AB79 compound at a dose of 0.6 mg·kg ^-1 (circles) to healthy subjects. Error bars indicate standard deviation (n=6). in / in - intravenously; s / c - subcutaneously.

[00106] In Fig. 42A-42B show the levels of NK cells in the peripheral blood of healthy subjects after a single IV or SC administration of the AB79 antibody. in / in - intravenously; s / c - subcutaneously.

Абсолютное количество моноцитов (клетки/мкл),

NK-клеток (клетки/мкл),

Общее количество Т-клеток (клетки/мкл),

В-клеток (клетки/мкл),

плазмобластов (клетки/мкл). Центрированные кривые обозначают медиану. NK - естественный киллер (клетка); п/к - подкожно.[00107] In FIG. 43 shows the levels of plasmablasts, monocytes, B-, T- and NK- cells in the peripheral blood of healthy subjects after a single sc injection of placebo control, 0.1, 0.3 or 0.6 mg·kg ^-1 of AB79 antibody.

Absolute number of monocytes (cells/µl),

NK cells (cells/µl),

Total number of T cells (cells/µl),

B-cells (cells/µl),

plasmablasts (cells/µl). The centered curves denote the median. NK - natural killer (cell); s / c - subcutaneously.

плацебо,

AB79 0,0003 мг/кг

AB79 0,001 мг/кг

AB79 0,003 мг/кг

AB79 0,01 мг/кг

AB79 0,03 мг/кг

AB79 0,06 мг/кг

AB79 0,1 мг/кг

AB79 0,3 мг/кг

AB79 0,6 мг/кг. Ig - иммуноглобулин; в/в - внутривенно; п/к - подкожно.[00108] In Fig. 44 shows the change from baseline in total IgA, IgG and IgM in serum of healthy subjects after a single intravenous injection of placebo or 0.003 to 0.06 mg kg ^-1 of AB79 antibody, or a single s.c. injection of placebo or from 0, 03 to 0.6 mg·kg ^-1 compound AB79. Symbols denote the cohort mean, and error bars denote the standard error of the mean.

placebo

AB79 0.0003 mg/kg

AB79 0.001 mg/kg

AB79 0.003 mg/kg

AB79 0.01 mg/kg

AB79 0.03 mg/kg

AB79 0.06 mg/kg

AB79 0.1 mg/kg

AB79 0.3 mg/kg

AB79 0.6 mg/kg. Ig - immunoglobulin; in / in - intravenously; s / c - subcutaneously.

DETAILED DESCRIPTION OF THE INVENTION

[00109] This invention relates to methods for treating diseases associated with CD38, by subcutaneous administration of low doses (≤600 mg) and volumes (≤3 ml) of anti-CD38 antibodies.

[00110] AB79, daratumumab, isatuximab, and MOR202 are IgG1s that primarily kill tumors through antibody-dependent cellular cytotoxicity (ADCC). This mechanism requires that effector cells, such as NK cells, bind antibodies to target cells and form a lytic synapse to release cytotoxic agents in a targeted manner. The number of these effector cells in the blood is orders of magnitude lower than the number of red blood cells and platelets. For example, the ratio of red blood cells to NK cells in the blood is 20,000:1. In addition, approximately 36 times more CD38 molecules are expressed on erythrocytes than on myeloma cells in patients with active disease. It has been suggested that the effector activity of daratumumab, isatuximab, and MOR202 deviates from tumors, since effector cells are mainly associated with anti-CD38 antibodies associated with erythrocytes and platelets, preventing the formation of a lytic synapse with tumors, which leads to low efficiency of ADCC. In contrast, reduced or shorter-term erythrocyte and platelet binding to AB79 compared to daratumumab may allow effector cells to focus on the tumor, leading to more effective ADCC, higher antitumor activity, and a lower effective dose.

[00111] Treating patients with anti-CD38 antibodies that bind to red blood cells and platelets can also lead to life-threatening side effects. For example, treatment of PPMM with MOR202 has resulted in several treatment-related dangerous adverse events (AEAEs) (see, for example, Raab et al. (2015) Blood 126: 3035). The most common AEs of any grade were anemia (15 patients, 34%), fatigue (14 patients, 32%), infusion reactions (IR) and leukopenia (13 patients, 30% each), lymphopenia and nausea (11 patients, 25% each). AELI grade 3 or higher were reported in 28 patients (64%); the most common were lymphopenia (8 patients, 18%), leukopenia (5 patients, 11%), and hypertension (4 patients, 9%). IR occurred mainly during the first infusion; all were grades 1-2 except for one patient (grade 3). Infections were frequently reported (26 patients, 59%), but in most cases they were not considered treatment related. MOR202 is used in clinical practice only by intravenous infusion. This is in contrast to the present invention, which allows AB79 to be administered subcutaneously at low doses and in small volumes as described herein.

[00112] Other antibodies are known from Morphosys that target CD38 (see, for example, WO 2006/125640 which discloses four human antibodies: MOR03077, MOR03079, MOR03080 and MOR03100, and two mouse antibodies: OKT10 and IB4). These prior art antibodies are inferior to AB79 for a number of reasons. MOR03080 binds to human CD38 and cynomolgus monkey CD38, but with low affinity for human CD38 (K _D = 27.5 nM; determined by Biacore technology). OKT10 binds to human CD38 and cynomolgus monkey CD38, but with low/moderate affinity for human CD38 (K _D = 8.28 nM; determined by Biacore technology). MOR03079 binds to human CD38 with high affinity (K _D = 2.4 nM; determined by Biacore technology), but does not bind to cynomolgus monkey CD38. MOR03100 and MOR03077 bind to human CD38 with moderate or low affinity (K _D =10 nM and 56 nM, respectively; determined by Biacore technology). In comparison, AB79 binds to human CD38 and cynomolgus macaque with high affinity (for human CD38 K _D =5.4 nm; determined by Biacore technology). In addition, prior art antibodies have low ADCC as well as CDC activity.

[00113] An advantage of the more effective ADCC is the ability to deliver the CD38 therapeutic as a low volume injection. If AB79 is formulated at a concentration of 135 mg/mL, an effective dose for an 80 kg myeloma patient can be given as a single s.c. injection of <2.5 ml. In contrast, the s.c. dose of daratumumab in phase 3 clinical trials is 1800 mg suspended in 15 ml along with Enhance™ (Halozyme).

[00114] The safety, tolerability, pharmacokinetics, and pharmacodynamics of IV and SC AB79 were initially characterized in macaques. AB79 was administered as a single dose iv bolus or s.c. injection to cynomolgus monkeys in sterile saline (i.v.) or dilution solution (s.c.) at doses of 0.03, 0.1, and 0.3 mg/kg (4 females/group). For the IV route of administration, Cmax was approximately dose proportional from 0.03 to 0.3 mg/kg AB79, and AUC(0-t) was greater than dose proportional from 0.03 to 0.1 mg/kg AB79, but was approximately dose proportional from 0.1 to 0.3 mg/kg AB79. For the s.c. route of administration, Cmax and AUC(0-t) increased with dose, but AUC(0-t) increased more than dose proportionally after s.c. over the range of 0.03 to 0.3 mg/kg. According to calculations, T _1/2 ranged from 120 to 144 hours. The mean bioavailability for the SC route of administration was approximately 100% (120%, 73% and 120% for 0.03, 0.1 and 0.3 mg/kg, respectively). Administration of AB79 at 0.03, 0.1 or 0.3 mg/kg by single intravenous or subcutaneous injection to female cynomolgus monkeys was well tolerated. The putative pharmacological effects of mild to moderate reduction in lymphocytes (T-cells, B-cells) and dose-dependent reduction in NK cell populations were observed at all dose levels and for both routes of administration; the maximal depletion effects of cell populations after s.c. dosing were similar or slightly less than those observed after i.v. dosing at the same dose level (Roepcke et al. (2018) Pharmacol. Res. Perspect. 6 (3): e00402 ). Based on the results of this study, the no adverse effect level (NEAEL) was considered to be 0.3 mg/kg for both intravenous and subcutaneous routes of administration. Altered lymphocyte and NK cell counts returned to normal in the absence of AB79 after a 56 day period. AUC and Cmax in serum associated with UPCONE were 574 h * μg / ml and 7.94 μg / ml for IV administration, and 698 h * μg / ml and 2.15 μg / ml for SC administration, respectively.

[00115] The safety, tolerability, pharmacokinetics, and pharmacodynamics of AB79 were then clinically characterized in a randomized, double-blind, placebo-controlled study of a single intravenous (IV) infusion or subcutaneous (SC) injection of 1 ml in cohorts of healthy people with increasing dosage. AB79 was well tolerated, with all adverse events (AEs) being mild or moderate, and there were no early withdrawals due to AEs or infusion reactions (Fedyk et al. (2018) Blood 132: 3249). In higher dose cohorts, a transient, mild, or moderate increase in cytokines coincided with a decrease in CD38-expressing cells; clinical symptoms primarily included pyrexia, headache, and postural hypotension. No identified abnormalities in laboratory tests, electrocardiograms, vital signs, or physical examinations have been reported associated with AB79 antibody treatment. The AB79 antibody caused a decrease in plasmablast and natural killer (NK) levels at similar doses, with a semi-effective dose (ED ₅₀ ) of 0.003 mg/kg for the IV route and 0.1 mg/kg for the OC route. The decrease in the total number of immunoglobulins (Ig) M and A occurred without comparable changes in IgG. The total number of leukocytes, granulocytes, lymphocytes, erythrocytes and platelets remained within the normal range for all dose levels. Overall, AB79 selectively reduced the levels of plasmablasts and NK cells in the peripheral blood of healthy subjects when given i.v. or s.c. and was generally safe and well tolerated. This plasmacytolytic profile may be useful in the treatment of disorders caused by plasma cells or NK cells, malignant counterparts (eg, multiple myeloma and NK cell leukemia), and pathogenic antibodies or immunoglobulins.

[00116] AB79 can induce a therapeutic response (e.g., disease remission) in immunoglobulin- or antibody-mediated diseases relatively quickly because it targets plasma cells directly, in contrast to therapeutic agents that target B-cell progenitors of plasma cells (e.g., , anti-BAFF mAbs (eg, belimumab)), anti-CD20 mAbs (eg, rituximab), and Bruton's tyrosine kinase (TKB) inhibitors (eg, baricitinib)). These latter strategies target plasma cells indirectly, essentially abolishing de novo plasma cell formation by inhibiting progenitor differentiation. Existing pools of plasma cells remain relatively unaffected and continue to produce pathogenic antibodies/immunoglobulins throughout their lives. Thus, the lifespan of pre-existing plasma cells, some of which survive for decades, may lead to a potential reduction in pathogenic antibodies/immunoglobulins, so indirect strategies may exhibit a slower onset of activity and efficacy compared to AB79. The only other therapeutics that have shown a direct reduction in plasma cell counts are proteasome inhibitors (eg, bortezomib), and this class of drugs is relatively poorly tolerated and includes dose-limiting side effects (eg, neuropathy, diarrhea) that are associated with inhibition of the proteasome in cells. , other than plasma (eg, neurons, epithelial cells), since the proteasome is expressed ubiquitously in these tissues. Thus, the specificity of AB79 for CD38, combined with the limited expression profile of CD38, creates a mechanism of action that directly targets plasma cells while minimizing effects on non-plasma cells and has the potential to provide rapid efficacy in diseases caused by plasma cells. transformed analogs and/or pathogenic antibodies/immunoglobulins.

[00117] Anti-CD38 methods and dosage units of the present invention provide for the first time subcutaneous administration of therapeutically effective low doses and volumes of anti-CD38 antibodies, thereby providing unexpected benefits and avoiding the side effects, inconvenience, and costs associated with high dose administration of drugs. for systemic therapy with anti-CD38 antibodies.

[00118] The present invention provides methods and unit dosage forms for subcutaneously administering a therapeutically effective amount of an isolated anti-CD38 antibody to a patient in need thereof for the treatment of diseases in which CD38 binding is prescribed, including hematologic cancers. In some embodiments, the subcutaneous antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 9 and a light chain variable region comprising the sequence of SEQ ID NO: 10. An anti-CD38 antibody provided herein is capable of being therapeutically effective when administered at unexpectedly low doses and as such can be administered in an unexpectedly small volume, facilitating subcutaneous administration.

[00119] Another advantage of the anti-CD38 antibodies of this invention is that, unlike some other anti-CD38 antibodies in clinical practice, the anti-CD38 antibodies of this invention (e.g., AB79) are able to bind to CD38 of the cynomolgus macaque (Javanese ), providing a useful animal model for preclinical evaluation of tolerability, toxicity and efficacy, etc.

[00120] Another advantage of the anti-CD38 antibodies of this invention is that they can be used to screen for other antibodies that compete for binding to CD38 on the same epitope and may be useful in the methods and dosage units of this invention.

[00121] Another advantage of the anti-CD38 antibodies of this invention is that they can be used to screen for other antibodies with reduced or alternative (e.g., shorter duration) binding to erythrocytes and/or platelets compared to daratumumab, and can be useful in the methods and unit dosage forms of this invention, for example an antibody that competes with or binds to the same epitope as AB79.

[00122] Unless otherwise defined herein, scientific and technical terms used in connection with this invention will have the meanings generally understood by those of ordinary skill in the art. The meaning and scope of terms should be clear. However, in the event of any latent ambiguity, the definitions provided in this document take precedence over any dictionary or out-of-document definition. Moreover, unless the context otherwise requires, singular terms will include the plural, and plural terms will include the singular. The term "or" includes "and/or" unless otherwise indicated. In addition, the use of the term "including", "includes" or "included" is not intended to be limiting. Terms such as "element" and "component" include both elements and components containing a single unit, and elements and components that contain more than one subunit, unless specifically indicated otherwise.

[00123] The methods and techniques of this invention are generally performed in accordance with conventional methods well known in the art and described in various general and more specific references, which are cited and discussed in this description, unless otherwise indicated. Nomenclature used in connection with laboratory methods and methods, and proper laboratory methods and methods of cell and tissue culture, molecular biology, immunology, microbiology, genetics, chemistry and hybridization of proteins and nucleic acids, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry described in this document are well known and widely used in the art. For chemical synthesis, chemical analysis, preparation, formulation, delivery of pharmaceuticals and treatment of patients, standard techniques are used. Commercial enzymatic reactions and purification procedures are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein.

[00124] All section headings and designations are used for clarity and reference purposes only, and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects of the disclosure from different headings and sections, as appropriate, in accordance with the spirit and scope of the invention described herein.

[00125] Certain terms are defined below in order to make this invention more understandable.

[00126] The terms "human CD38" and "human CD38 antigen" refer to the amino acid sequence of SEQ ID NO: 1 or a functional fraction thereof, such as an epitope, as defined herein (Table 1). In general, CD38 has a short intracytoplasmic tail, a transmembrane domain, and an extracellular domain. The terms "cynomolgus CD38" and "cynomolgus monkey CD38 antigen" refer to the amino acid sequence of SEQ ID NO: 2, which is 92% identical to the amino acid sequence of human CD38 (Table 1). Synonyms for CD38 include cyclic ADP ribose hydrolase; cyclic ADP ribose hydrolase 1; ADP ribosylcyclase; ADP-ribosylcyclase 1; cADFR hydrolase 1; CD38-rs1; I-19; NIM-R5 antigen; 2'-phosphocyclic-ADP-ribose transferase; 2'-phospho-ADP-ribosyl cyclase; 2'-phosphocyclic-ADP-ribose transferase; 2'-phospho-ADP-ribosyl cyclase; and T10.

Table 1. Amino acid sequence of human CD38 and cynomolgus macaque. View Amino acid sequence
1234567890123456789012345678901234567890 SEQID NO human CD38 MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI one CD38 cynomolgus macaque MANCEFSPVSGDKPCCRLSRRAQVCLGVCLLVLLILVVVVAVVLPRWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI 2 human CD157 MAAQGCAASRLLQLLLQLLLLLLLLAAGGARARWRGEGTSAHLRDIFLGRCAEYRALLSPEQRNKNCTAIWEAFKVALDKDPCSVLPSDYDLFINLSRHSIPRDKSLFWENSHLLVNSFADNTRRFMPLSDVLYGRVADFLSWCRQKNDSGLDYQSCPTSEDCENNPVDSFWKRASIQYSKDSSGVIHVMLNGSEPTGAYPIKGFFADYEIPNLQKEKITRIEIWVMHEIGGPNVESCGEGSMKVLEKRLKDMGFQYSCINDYRPVKLLQCVDHSTHPDCALKSAAAATQRKAPSLYTEQRAGLIIPLFLVLASRTQL 13

[00127] The terms "therapeutically effective amount" and "therapeutically effective dosage" means an amount of a therapeutic agent that is sufficient to reduce or alleviate the severity and/or duration of a disorder or one or more of its symptoms; preventing the progression of the disorder; ensuring the reverse development of the violation; preventing the recurrence, development, manifestation or progression of one or more symptoms associated with the disorder; or enhancing or improving the prophylactic(s) or therapeutic(s) effect(s) of another therapy (eg, prophylactic or therapeutic agent), in dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on such factors as the course of the disease, the age, sex and body weight of the individual, and the ability of the drugs to elicit the desired response in the individual. A therapeutically effective amount of an antibody is that amount such that any toxic or detrimental effects of the antibody or antibody portion are offset by therapeutically beneficial effects. A therapeutically effective amount of an antibody for tumor therapy can be measured by its ability to stabilize the progression of the disease. The ability of a compound to inhibit cancer can be assessed in an animal model system predictive of efficacy in human cancers.

[00128] The terms "patient" and "subject" include both humans and other animals. Thus, the compositions, dosages, and methods disclosed herein are applicable to both human and veterinary medicine. In one embodiment, the patient is a mammal, such as a human.

[00129] The term "disease in which CD38 binding is prescribed" means a disease in which binding of a binding partner (e.g., an anti-CD38 antibody of this invention) to CD38 provides a prophylactic or therapeutic effect, including the reduction of one or more symptoms of the disease. Such binding may result in blocking of other factors or binding partners for CD38, neutralization of CD38, ADCC, CDC, complement activation, or some other mechanism by which the disease is prevented or treated. Factors and binding partners for CD38 include anti-CD38 autoantibodies that are blocked by the anti-CD38 antibodies of the invention. Such binding may be referred to as a consequence of the expression of CD38 by cells or a subset of cells, such as MM cells, whereby providing the subject with a binding partner for CD38 results in removal, such as lysis, of the cells, through, for example, hemolysis or apoptosis. Such expression of CD38 may be, for example, normal, overexpressed, inappropriately expressed, or due to activation of CD38 relative to normal cells or relative to other cell types, either in a disease-free state or in a diseased state.

[00130] The term "hematological cancer" refers to malignant neoplasms of hematopoietic tissues and includes leukemias, lymphomas, and multiple myelomas. Non-limiting examples of conditions associated with aberrant CD38 expression include, but are not limited to: multiple myeloma (MM) (Jackson et al . (1988) Clin. Exp. Immunol. 72: 351-356) including relapsed refractory MM (RRMM) or newly identified MM (WWMM); B-cell chronic lymphocytic leukemia (B-CLL) (Dürig et al . (2002) Leukemia 16: 30-35; Morabito et al. (2001) Leukemia Res. 25: 927-932; Marinov et al . (1993) Neoplasma 40(6): 355-358 and Jelinek et al (2001) Br J Haematol 115 : 854-861); acute lymphoblastic leukemia (Keyhani et al . (1999) Leukemia Res. 24: 153-159; and Marinov et al . (1993) Neoplasma 40(6): 355-358); chronic myeloid leukemia (Marinov et al . (1993) Neoplasma 40(6): 355-358); acute myeloid leukemia (Keyhani et al . (1999) Leukemia Res. 24: 153-159); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia or chronic myeloid leukemia (CML); acute myelogenous leukemia or acute myeloid leukemia (AML); acute lymphocytic leukemia (ALL); hairy cell leukemia (VCL); NK/T cell lymphoma, myelodysplastic syndromes (MDS) (Nurulhuda et al. (2017) Blood 130: 2814); and all subtypes and stages (for example, for CML: blast phase (BP), chronic phase (CP), or accelerated phase (UV)) of these leukemias and other hematological diseases, which are determined by morphological, histochemical and immunological methods that are well known to specialists in this field of technology.

[00131] The terms "neoplasia" and "neoplastic condition" refer to a condition associated with cell proliferation characterized by a loss of normal control resulting in one or more symptoms, including unregulated growth, lack of differentiation, dedifferentiation, local tissue invasion, and metastasis.

[00132] The term "isolated antibody" means an antibody that is essentially 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. An isolated antibody that specifically binds to an epitope, isoform, or variant of human CD38 or cynomolgus macaque CD38 may, however, be cross-reactive with other related antigens, eg from other species, such as CD38 species homologues. Moreover, the isolated antibody may be substantially free of other cellular materials and/or chemicals.

[00133] The term "erythrocytes" refers to hemoglobin-containing blood cells derived from the bone marrow, which carry oxygen to cells and tissues, and carry carbon dioxide back to the respiratory organs. Red blood cells are also referred to as red cells, red blood cells, hematids, and erythroid cells.

[00134] The terms "specific binding,""specifically binds to," and "specifically to," in relation to the interaction of a particular antibody, protein, or peptide with an antigen, epitope, or other chemical, means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of the molecule compared to the binding of a control molecule, which is usually a molecule of similar structure, while not possessing binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target. The anti-CD38 antibodies of this invention specifically bind CD38 ligands. The terms "specific binding", "specifically binds to" and "specific to" also mean that the interaction depends on the presence of a particular structure (eg, antigenic determinant or epitope) in the chemicals; for example, an antibody recognizes and binds to a specific structure of a protein rather than to proteins in general. If the antibody is specific for the "A" epitope, then the presence of a molecule containing the "A" epitope (or free, unlabeled "A") in a reaction that includes the labeled "A" and the antibody will reduce the amount of the labeled "A". associated with the antibody. Specific binding for a particular antigen or epitope may be manifested, for example, by a KD of an antibody relative to the antigen or epitope of at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^-6 M, according to at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 ^{- 12} M, where KD is the rate of dissociation of a particular antibody-antigen interaction. 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 higher for the control molecule relative to the antigen or epitope. In addition, specific binding for a particular antigen or epitope may be manifested, for example, by an antibody's KA or Ka score relative to the antigen or epitope being at least 20, 50, 100, 500, 1000, 5000, 10,000 or more times greater for the epitope. compared to control, where KA or Ka means the rate of association of a particular antibody-antigen interaction.

[00135] The term "over a period of time" refers to any period of time, such as minutes, hours, days, months, or years. For example, "within a certain period of time" may refer to at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least at least 105 minutes, at least 120 minutes, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least one day, at least two days, at least three days, at least 4 days, at least 5 days, at least 6 days, at least a week, at least a month, at least one year, or any period of time between them. In other words, the antibody from the composition can be absorbed by the individual to whom it is administered over a period of time of at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, at least 75 minutes, at least at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least one day, at least two days, at least three days, at least 4 days, at least 5 days, at least 6 days, at least a week, at least a month, at least one year, or the time interval between them.

[00136] A composition that "primarily" consists of a component means that the composition contains more than about 80%, in some embodiments more than about 90%, in some embodiments more than about 95%, in some embodiments more than about 97%, in some embodiments, more than about 98%, in some embodiments, more than about 99% of the component by weight.

[00137] The term "about" refers to a value close to the number, degree, volume, time, etc., with only minor variations in size up to 10%.

[00138] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier suitable for administering the compounds of this invention to mammals. Carriers include a liquid or solid excipient, diluent, excipient, solvent, or encapsulating material involved in the transfer or transport of the compound in question from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not causing harm to the patient. In one embodiment, the pharmaceutically acceptable carrier is suitable for intravenous administration. In another embodiment, the pharmaceutically acceptable carrier is suitable for local-regional injection. In another embodiment, the pharmaceutically acceptable carrier is suitable for subcutaneous administration. In another embodiment, the pharmaceutically acceptable carrier is suitable for subcutaneous injection.

[00139] The term "pharmaceutical composition" refers to drugs suitable for administration to a subject and treatment of a disease. When the anti-CD38 antibodies of this invention are administered as medicaments to a mammal, such as a human, they may be administered "as is" or as a pharmaceutical composition comprising the anti-CD38 antibody in combination with a pharmaceutically acceptable carrier and/or other excipients. The pharmaceutical composition may be in unit dosage form for administering a specific dose of the anti-CD38 antibody at a specific concentration, a specific amount, or a specific volume. Provided herein are pharmaceutical compositions comprising anti-CD38 antibodies, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers.

[00140] As a rule, the traditional structural units of the antibody contain a tetramer. Typically, each tetramer consists of two identical pairs of polypeptide chains, each pair has one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa) . Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha or epsilon and define the isotype of an antibody as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. Thus, "isotype" refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. Known isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE. Therapeutic antibodies may also contain hybrids of isotypes and/or subclasses.

[00141] Each variable heavy (VH) and variable light (VL) region (100 to 110 amino acids in length) consists of three hypervariable regions called "complementarity determining regions" (CDRs) and four framework regions (FR) (length about 15-30 amino acids) located from the amino-terminus to the carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. "Variable" refers to the fact that the KLOs vary greatly in sequence among antibodies, and thus defines a unique antigen binding site.

[00142] 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 from about 31- 35B (HCDR1; "H" stands for heavy chain), 50-65 (HCDR2) and 95-102 ( ^HCDR3 ) in the heavy chain variable region (Kabat et al. (1991) Sequences Of Proteins Of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD) and/or from those amino acid residues that form a hypervariable loop, such as residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the variable light chain regions and residues 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).

[00143] The Kabat numbering system is commonly used to denote a residue in a 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. (1991) Sequences Of Proteins Of Immunological Interest, 5th ^Ed . Public Health Service, National Institutes of Health, Bethesda, MD) with the EU numbering system used for the Fc fragment.

[00144] The term "domain of an immunoglobulin (Ig)" refers to a region of an immunoglobulin having a distinct tertiary structure. In addition to variable domains, each heavy and light chain has constant domains: constant heavy (CH) domains; constant light (CL) domains; and hinge domains. In relation to IgG antibodies, each IgG isotype has three CH regions. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Accordingly, "CH" domains in relation to IgG are as follows: "CH1" refers to positions 118-220 in accordance with the EU index, respectively Kabat. "CH2" refers to provisions 237-340 according to the EU index, respectively to Kabat, and "CH3" refers to provisions 341-447 according to the EU index, respectively to Kabat.

[00145] Another type of heavy chain Ig domain is the hinge region. The term "hinge region" refers to a flexible polypeptide containing amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EC position 220 and the IgG CH2 domain begins at a residue at EC position 237. Thus, for IgG, the antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1 ), while the numbering corresponds to the EC index, respectively Kabat. In some embodiments, such as the Fc moiety, a bottom hinge is included, with "bottom hinge" typically referring to positions 226 or 230.

[00146] The term "Fc fragment" refers to a polypeptide containing the constant region of an antibody, excluding the first constant region of the immunoglobulin domain and, in some cases, part of the hinge. Thus, Fc refers to the last two constant regions of the IgA immunoglobulin domains, IgD and IgG, to the last three constant regions of the IgE and IgM immunoglobulin domains, and the flexible hinge N-terminus of these domains. In IgA and IgM, Fc may include a J chain. In IgG, the Fc domain contains 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 fragment may vary, the Fc fragment of the human IgG heavy chain is usually defined as including residues C226 or P230 towards its carboxyl terminus, with the numbering corresponding to the EC index, respectively Kabat. In some embodiments, as described more fully below, amino acid modifications are made to the Fc fragment, eg, to change binding to one or more FcγRs or to an FcRn receptor.

CD38 antibodies

[00147] Accordingly, this invention relates to isolated anti-CD38 antibodies that specifically bind human and primate CD38 protein, and which have a reduced or less than 10%, less than 20%, less than 30%, less than 40%, less than 50% binding to human erythrocytes compared to daratumumab and thus find use in subcutaneous routes and unit dosage forms. Of particular use in this invention are antibodies that bind to both human and primate CD38 proteins, especially primates used in clinical trials such as the cynomolgus macaque ( Macaca flavicularis, cynomolgus monkey, also referred to herein as "Javanese") .

[00148] In some embodiments, anti-CD38 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 and D293 - AB79 epitope. Any antibody that interacts with these residues also finds use in therapeutic methods and dosage units according to this invention.

[00149] In some embodiments, anti-CD38 antibodies of the invention interact with CD38 at a number of amino acid residues 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 both humans and cynomolgus monkeys, except that S274 is essentially F274 in cynomolgus monkey. These residues may be an immunodominant epitope and/or residues in the region of action of a specific antigen-binding peptide.

[00150] In some embodiments, the anti-CD38 antibody comprises a heavy chain containing the following CDR amino acid sequences: GFTFDDYG (SEQ ID NO: 3; HCDR1 AB79), ISWNGGKT (SEQ ID NO: 4; HCDR2 AB79), and ARGSLFHDSSGFYFGH (SEQ ID NO: 5; HCDR3 AB79). In some embodiments, the antibody comprises a light chain containing the following CDR amino acid sequences: SSNIGDNY (SEQ ID NO: 6; LCDR1 AB79), RDS (SEQ ID NO: 7; LCDR2 AB79), and QSYDSSLSGS (SEQ ID NO: 8; LCDR3 AB79 ). In some embodiments, the antibody comprises a heavy chain containing the following CDR amino acid sequences: GFTFDDYG (SEQ ID NO: 3; HCDR1 AB79), ISWNGGKT (SEQ ID NO: 4; HCDR2 AB79), ARGSLFHDSSGFYFGH (SEQ ID NO: 5; HCDR3 AB79 ) and a light chain containing the following CDR amino acid sequences: SSNIGDNY (SEQ ID NO: 6; LCDR1 AB79), RDS (SEQ ID NO: 7; LCDR2 AB79) and QSYDSSLSGS (SEQ ID NO: 8; LCDR3 AB79). In some embodiments, the antibody comprises a heavy chain comprising the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 9.

EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLA (SEQ ID NO: 9).

[00151] In some embodiments, the antibody comprises a light chain comprising the light chain variable region (VL) amino acid sequence of SEQ ID NO: 10.

(SEQ ID NO: 10).

[00152] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of the VH region of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of the VL region of SEQ ID NO: 10.

[00153] As will be appreciated by those skilled in the art, variable heavy and light chains can be fused to human IgG constant domain sequences, typically IgG1, IgG2, or IgG4.

[00154] In some embodiments, the antibody comprises the heavy chain (HC) amino acid sequence of SEQ ID NO: 11.

EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

[00155] In some embodiments, the antibody comprises the light chain (LC) amino acid sequence of SEQ ID NO: 12.

QSVLTQPPSASGTPGQRVTISCSGSSSNIGDNYVSWYQQLPGTAPKLLIYRDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYTVSCAPHEGSQ2.

In some embodiments, the antibody comprises the HC amino acid sequence of SEQ ID NO: 11 and the LC amino acid sequence of SEQ ID NO: 12.

[00156] This invention encompasses antibodies that bind to both human and Javanese CD38 and interact with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% of these amino acid residues.

[00157] In some embodiments, these 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.

[00158] Alternatively, antibodies can be of a variety of structures, including but not limited to: antibody fragments, monoclonal antibodies, bispecific antibodies, mini-antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies , humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each, respectively. Specific antibody fragments include, but are not limited to: (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment (Ward et al. (1989) Nature 341: 544-546) which consists of a single variable domain; (v) isolated CDR regions; (vi) F(ab')2 fragments - a bivalent fragment containing two linked Fab fragments; (vii) single chain Fv molecules (scFv), where the VH domain and the 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. USA 85: 5879-5883); (viii) bispecific single chain Fvs (WO 03/11161) and (ix) "diabodies" or "tribodies" - multivalent or polyspecific fragments constructed by gene fusion (Tomlinson et al. (2000) Methods Enzymol. 326: 461-479; WO94 /13804 Holliger et al (1993) Proc Natl Acad Sci USA 90: 6444-6448).

Antibody modifications

[00159] This invention also relates to variants of anti-CD38 antibodies. That is, there are a number of modifications that can be made to the antibodies of this invention, including, but not limited to, amino acid modifications in the CDR (affinity maturation), amino acid modifications in the Fc fragment, glycosylation variants, other types of covalent modifications, etc. P.

[00160] The term "variant" means a polypeptide that differs from the parent polypeptide. Amino acid variants may include substitutions, insertions and deletions of amino acids. In general, variants may include any number of modifications as long as the function of the protein is still present, as described herein. That is, in the case of amino acid variants generated by a CDR from, for example, AB79, the antibody should still specifically bind to human and cynomolgus CD38, and not bind to erythrocytes, or be reduced or less than 10%, less than 20%, less than 30%, less than 40%, less than 50% erythrocyte binding ability compared to daratumumab. The term "variant Fc fragment" means an Fc sequence that differs from the wild-type Fc sequence or the parental Fc sequence due to at least one amino acid modification. An Fc variant may refer to the Fc polypeptide itself, compositions containing the Fc polypeptide variant, or an amino acid sequence. If amino acid variants are generated, for example, with an Fc fragment, the variant antibodies must retain the required functions for the specific application or indication of the antibody. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions can be used, such as substitutions 1-10, 1-5, 1-4, 1-3, and 1-2. Suitable modifications can be made in one or more positions, as generally described in, for example, US Pat. Nos. 6,086,875; 6737056; 7317091; 7670600; 8084582; 8188231; 8367805; 8937158; and also 9040041; all of which are expressly incorporated herein by reference in their entirety and, in particular, relate to specific amino acid substitutions that increase binding to Fc receptors.

[00161] For example, it may be appropriate to have 1 to 5 modifications in the Fc fragment of wild-type or engineered proteins, as well as 1 to 5 modifications in the Fv fragment. The polypeptide sequence variant will preferably have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the parental sequences ( for example, variable regions, constant regions and/or heavy and light chain sequences for AB79).

[00162] The term "amino acid substitution" means 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. The term "amino acid insertion" means the addition of an amino acid at a specific position in the parent polypeptide sequence. The term "deletion of an amino acid" means the removal of an amino acid at a specific position in the parent polypeptide sequence.

[00163] The terms "parent antibody" and "progenitor antibody" mean an unmodified antibody that is subsequently modified to produce a variant. In one embodiment, the parent antibody herein is AB79. The parent polypeptide can mean the polypeptide itself, compositions that contain the parent polypeptide, or the amino acid sequence that encodes it. Accordingly, the term "parental Fc polypeptide" means an Fc polypeptide that has been modified to produce a variant.

[00164] The terms "wild type", "WT", and "native" mean an amino acid sequence or nucleotide sequence that occurs in nature, including allelic variations. Protein, polypeptide, antibody, immunoglobulin, IgG, etc. wild type (WT) has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[00165] In some embodiments, one or more amino acid modifications are made to one or more CDRs of anti-CD38 antibodies. Typically, only 1, 2, or 3 amino acids are substituted in any single CDR, and typically no more than 4, 5, 6, 7, 8, 9, or 10 substitutions are made in a set of CDRs. However, it should be understood that any combination of no substitutions, with 1, 2, or 3 substitutions in any CDR may independently and optionally be combined with any other substitution.

[00166] Amino acid modifications in CDRs are sometimes referred to as "affinity maturation". An "affinity matured" antibody is one that has one or more changes in one or more CDRs that result in an improvement in the affinity of the antibody for the antigen compared to a parent antibody that does not have those changes. In some cases, it may be desirable to reduce the affinity of an antibody for its antigen.

[00167] Affinity maturation can be performed to increase the binding affinity of an antibody to an antigen by at least about 10% to 50%, 100%, 150%, or more, or 1 to 5-fold relative to the "parent" antibody. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the antigen. Affinity matured antibodies are prepared by known methods (e.g., Marks et al. (1992) Biotechnol. 10: 779-783; 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).

[00168] In another embodiment, amino acid modifications can be made to one or more of the CDRs in antibodies of this invention that are "silent"—for example, that do not significantly change the affinity of the antibody for the antigen. This may be done for a number of reasons, including optimization of expression (as may be done for nucleic acids encoding antibodies of the present invention).

[00169] Thus, CDR and antibody variants are included in the definition of CDRs and antibodies according to this invention; that is, the antibodies of this invention may include amino acid modifications in one or more of the CDRs in AB79. In addition, as discussed below, amino acid modifications can also be independently and optionally made in any region outside of the CDR, including the framework and constant regions.

[00170] In some embodiments, AB79 antibody variants are described that are specific for human CD38 (SEQ ID NO: 1) and cynomolgus monkey CD38 (SEQ ID NO: 2). 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/or SEQ ID NO: 8 substitution of 0, 1, 2 or more amino acids without significant change or inhibition of function.

[00171] In addition to the modifications described above, other modifications may be made. For example, molecules can be stabilized by inserting disulfide bridges connecting the VH and VL domains (Reiter et al. (1996) Nature Biotech. 14: 1239-1245). In addition, there are many covalent modifications of antibodies that can be made, as described below.

[00172] Covalent modifications of antibodies are included within the scope of this invention and are usually, but not always, carried out post-translationally. For example, several types of covalent modifications of an antibody are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or N- or C-terminal residues.

[00173] In some embodiments, an anti-CD38 antibody of the invention specifically binds to one or more residues or regions in CD38, but also does not cross-react with other proteins homologous to CD38, such as BST-1 (bone marrow stromal cell antigen). 1) and Mo5, also referred to as CD157.

[00174] Typically, no cross-reactivity means less than about 5% relative competitive inhibition between molecules as assessed by ELISA and/or flow cytometric analysis using sufficient numbers of molecules under appropriate assay conditions.

Inhibition of CD38 activity and reduced side effects

[00175] The disclosed antibodies may find use in blocking ligand-receptor interaction or inhibiting receptor component interaction. Anti-CD38 antibodies according to this invention can be "blocking" or "neutralizing". The term "neutralizing antibody" refers to an antibody whose binding to CD38 results in inhibition of the biological activity of CD38, for example, its ability to interact with ligands, enzymatic activity, signaling ability, and, in particular, its ability to cause activation of lymphocytes. Inhibition of CD38 biological activity can be assessed using one or more standard in vitro or in vivo assays known in the art.

[00176] The terms "inhibits binding" and "blocks binding" (eg, in relation to inhibition/blocking of the binding of CD38 antibodies to CD38) encompass both partial and complete inhibition/blocking. Inhibition/blocking of CD38 antibody binding to CD38 can reduce or alter the normal level or type of cellular signaling that occurs when a CD38 antibody binds to CD38 without inhibition or blocking. Inhibition and blocking is also contemplated to include any measurable reduction in the binding affinity of an antibody's CD38 binding to CD38 when contacted by an anti-CD38 antibody compared to a ligand not contacting an anti-CD38 antibody, such as blocking the binding of an antibody's CD38 to CD38 by at least about 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.

[00177] The disclosed anti-CD38 antibodies can also inhibit cell growth. The term "growth inhibiting" refers to any measurable reduction in cell growth upon contact with an anti-CD38 antibody compared to the growth of the same cells not contacted with an anti-CD38 antibody, e.g., inhibition of cell culture growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.

[00178] In some embodiments, the disclosed anti-CD38 antibodies are capable of depleting populations of activated lymphocytes and plasma cells. The term "population depletion" as used herein means a measurable reduction in serum levels of activated lymphocytes and/or plasma cells in a subject compared to intact subjects. Typically, population depletions of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% are observed. As demonstrated in the Examples below, one particular advantage that the antibodies of this invention have is the resilience of these cells after dosing; that is, as is known for some treatments (eg, using anti-CD20 antibodies), depletion of a population of cells can continue for long periods of time, causing undesirable side effects. As demonstrated herein, the effects on activated lymphocytes and/or plasma cells are reversible.

[00179] The anti-CD38 antibodies of the present invention can reduce side effects compared to prior art anti-CD38 antibodies. In some embodiments, AB79 does not cause AELV. In some embodiments, AB79 reduces AELV compared to other anti-CD38 antibodies such as MOR202. NEVL are usually labeled as grades 1, 2, 3, 4, and 5, with grade 1 being the least severe and grade 5 being the most severe. Based on FDA guidelines and other recommendations regarding the Common Terminology Criteria for Adverse Events (CTCAE) standards for oncology drugs (see e.g. https://evs.nci.nih.gov/ftp1/CTCAE/ CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf; also https://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm; and Nilsson and Koke (2001) Drug Inform. J. 35: 1289-1299) , such degrees are usually defined as follows. Grade 1 - mild: asymptomatic or with mild symptoms; only clinical or diagnostic observations; intervention has not been demonstrated. Grade 2 - moderate: minimal, local or non-invasive intervention demonstrated; age-appropriate limitations in instrumental daily activities. Grade 3 - Severe or medically significant but not life threatening: Demonstrated hospitalization or prolongation of hospitalization; disability; restrictions in daily self-care activities. Grade 4 - life-threatening consequence: urgent intervention is demonstrated. Grade 5 - AE-related death.

[00180] In some embodiments, AB79 reduces the rate of AEs compared to other anti-CD38 antibodies, such as MOR202. In some embodiments, AB79 reduces the severity of AEs compared to other anti-CD38 antibodies from grade 5 to grade 4. In some embodiments, AB79 reduces the grade of AEs compared to other anti-CD38 antibodies from grade 4 to grade 3. In some embodiments, AB79 reduces the severity of AEs compared to other anti-CD38 antibodies from grade 3 to grade 2. In some embodiments, AB79 reduces the severity of AEs compared to other anti-CD38 antibodies from grade 2 to grade 1.

[00181] In some embodiments, AB79 reduces the severity of one or more AEs selected from the group consisting of anemia (including hemolytic anemia), thrombocytopenia, fatigue, infusion reactions (IR), leukopenia, lymphopenia, and nausea. In some embodiments, AB79 reduces the incidence of one or more AEs selected from the group consisting of anemia (including hemolytic anemia), thrombocytopenia, fatigue, infusion reactions (IR), leukopenia, lymphopenia, and nausea.

[00182] In some embodiments, a diagnostic test is used to determine the presence and/or extent of anemia, including hemolytic anemia. Diagnostic tests for anemia, including hemolytic anemia, include measuring hemoglobin levels. Typically, hemoglobin levels are interpreted as follows: (i) very mild/no anemia: ≥12.0 g/dl; (ii) mild: 10-12 g/dl; (iii) moderate: 8-10 g/dl; (iv) severe: 6-8 g/dl; and (v) very severe: ≤6 g/dl. Other diagnostic tests for anemia, including hemolytic anemia, include measuring haptoglobin levels. Typically, a haptoglobin level of ≤25 mg/dl is indicative of anemia, including hemolytic anemia. Other diagnostic tests include the direct antiglobulin test (DAT) (also called the direct Coombs test), which is used to determine if red blood cells are coated in vivo with immunoglobulin, complement, or both.

[00183] In some embodiments, a diagnostic test is used to determine the presence and/or degree of thrombocytopenia. Typically, a diagnostic test for thrombocytopenia involves measuring the number of platelets per microliter (µL) of blood. Typically, there are 150×10 ³ -450×10 ³ platelets per µl of blood. As a rule, thrombocytopenia is diagnosed when <150×10 ³ platelets per µl of blood. Mild thrombocytopenia is usually diagnosed at 70-150×10 ³ platelets per µl of blood. Moderate thrombocytopenia is usually diagnosed with 20-70×10 ³ platelets per µl. Severe thrombocytopenia is usually diagnosed with <20×10 ³ platelets per µl of blood.

Indications for diseases

[00184] The antibodies, methods, and dosage units of this invention find use in a variety of applications, including the treatment or amelioration of CD38-associated diseases.

[00185] 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. Certain pathological conditions are associated with cells that express CD38, and certain pathological conditions are associated with overexpression, high density expression or overexpression of CD38 on cell surfaces. Whether or not a cell population expresses CD38 can be determined by methods known in the art, for example, flow cytometric determination of the percentage in a given population of cells labeled with an antibody that specifically binds CD38, or immunohistochemical assays, as generally described below for diagnostic applications. . For example, a population of cells in which CD38 expression is found in about 10-30% of cells may be considered to have weak CD38 positivity; and a population of cells in which CD38 expression is found in more than about 30% of cells can be considered as definitely 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. Expression density on cell surfaces can be determined using methods known in the art, such as, for example, flow cytometric measurement of the mean fluorescence intensity of cells that have been fluorescently labeled with antibodies specifically binding CD38.

[00186] The therapeutic anti-CD38 antibodies of the invention bind to CD38 positive cells, resulting in depletion of the cell population through a variety of mechanisms of action, including both the ADCC and CDC pathways.

[00187] It is known in the art that certain pathological conditions are associated with cells that express CD38, and that certain pathological conditions are associated with overexpression, high density expression, or overexpression of CD38 on cell surfaces. Whether or not a cell population expresses CD38 can be determined by methods known in the art, for example, flow cytometric determination of the percentage in a given population of cells labeled with an antibody that specifically binds CD38, or immunohistochemical assays, as generally described below for diagnostic applications. . For example, a population of cells in which CD38 expression is found in about 10-30% of cells may be considered to have weak CD38 positivity; and a population of cells in which CD38 expression is found in more than about 30% of cells can be considered as definitely 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. Expression density on cell surfaces can be determined using methods known in the art, such as, for example, flow cytometric measurement of the mean fluorescence intensity of cells that have been fluorescently labeled with antibodies specifically binding CD38.

[00188] In one aspect, this invention relates to methods of treating a pathological condition associated with the proliferation of cells expressing CD38, comprising administering to a patient a pharmaceutically effective amount of a disclosed antibody. In some embodiments, the pathological condition is a cancer, and in specific embodiments, the cancer is a hematological cancer. In some embodiments, the pathological condition is multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, as well as plasma cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-cell lymphoma, or Burkitt's lymphoma.

[00189] In some embodiments, the condition is multiple myeloma and the therapeutic anti-CD38 antibody does not bind or has reduced binding to human erythrocytes compared to daratumumab. In some embodiments, the condition is multiple myeloma and the therapeutic anti-CD38 antibody does not bind or has reduced binding to cynomolgus macaque erythrocytes compared to daratumumab. In some embodiments, the condition is multiple myeloma and the therapeutic anti-CD38 antibody does not bind or has reduced binding to human or cynomolgus macaque erythrocytes.

[00190] CLL is the most common adult leukemia in the Western world. CLL consists of a clonal expansion of lymphocytes that appear mature, involving lymph nodes and other lymphoid tissues with progressive bone marrow infiltration and presence in the peripheral blood. The majority of cases are represented by the B-cell form (B-CLL).

B-cell form of chronic lymphocytic leukemia (B-CLL)

[00191] B-CLL is an incurable disease characterized by a progressive increase in the number of anergic monoclonal cells of B-lymphocyte origin, which accumulate continuously in the bone marrow and peripheral blood for many years. CD38 expression has been considered as an independent negative predictor for B-CLL (Hamblin et al. (2002) Blood 99: 1023-9).

[00192] B-CLL is characterized by two subtypes - indolent and aggressive. These clinical phenotypes correlate with the presence or absence of somatic mutations in the immunoglobulin heavy chain variable region (IgVH) gene. As used herein, indolent B-CLL refers to a pathological disorder in a subject having a mutated IgVH gene and/or having one or more clinical phenotypes associated with indolent B-CLL. As used herein, "aggressive B-CLL" refers to a pathological disorder in a subject having a non-mutated IgVH gene (or) having one or more clinical phenotypes associated with aggressive B-CLL.

[00193] The current standard therapy for B-CLL is palliative and is mainly carried out using the cytotoxic drug chlorambucil or fludarabine. When relapses occur, combination therapy with fludarabine, cyclophosphamide plus rituximab (anti-CD20 monoclonal antibody) or alemtuzumab (anti-CD52 monoclonal antibody) is often attempted. In one study, 35 patients with relapsed or refractory aggressive B-cell NHL received high-dose chemotherapy (HDC) followed by rituximab 375 mg/m ² weekly for 4 doses starting on day 40 and repeated four doses starting on the 180th day. Rituximab infusions were well tolerated, with only one case of grade 3/4 infusion toxicity. An unexpected adverse event noted in this study was delayed neutropenia in more than half of the patients (19/35 patients with 46 episodes of grade 3 or 4 neutropenia; Kosmas et al. (2002) Leukemia 16: 2004-2015, which can be found on the Internet at: https://www.nature.com/articles/2402639). In another study, six patients received alemtuzumab by intravenous infusion every other day three times a week for 12 weeks. The dose was gradually increased daily (3, 10, then 30 mg) as long as the patient tolerated it. The main AEs were hematological adverse events - anemia, neutropenia (each in 6/6 patients) and thrombocytopenia (5/6 patients) (Ishizawa et al. (2017) Jpn. J. Clin. Oncol. 47(1): 54-60 ). Thus, there is a critical unmet medical need for methods of treating B-CLL with reduced hematological adverse events. In some embodiments, methods for treating B-CLL using the disclosed anti-CD38 antibodies are provided and, as demonstrated below, this can be accomplished using combination therapy, optionally and independently of any of the above drugs.

Multiple myeloma (MM)

[00194] Multiple myeloma is a malignant cell disease of B-lymphocytic origin, characterized by neoplastic proliferation of plasma cells in the bone marrow and/or extramedullary sites. Myeloma cell proliferation causes a variety of effects, including lytic bone lesions (holes), organ damage, anemia (decreased red blood cell count), abnormal protein production (with concomitant damage to the kidneys, nerves, and other organs), reduced immune system function, kidney failure, and increased levels of calcium in the blood (hypercalcemia). Current treatment options include chemotherapy, preferably associated with autologous stem cell transplantation (ASCT) when possible. These regimens have moderate levels of clinical response to therapy. However, only minor changes in overall survival are observed, and the median survival is approximately 3 years. Thus, there is an urgent unmet medical need for methods of treating multiple myeloma. In some embodiments, methods for treating multiple myeloma using the disclosed antibodies are provided.

[00195] Newly diagnosed MM (MMMM) is different from recurrent MM or relapsed and refractory MM (RPMM). Recurrent MM is considered as a recurrence of the disease after a prior clinical response to therapy and is defined based on objective laboratory and radiological criteria: an increase in serum or urine monoclonal protein (M-protein) by ≥25% or a difference of ≥25% between involved and uninvolved free light chains in serum relative to their minimum levels, respectively, or the development of new plasmacytomas or hypercalcemia. In patients with non-secretory disease, relapse is defined as an increase in bone marrow plasma cells. Typically, the indication for treatment of relapse is either the onset or reappearance of one or more of the MM symptoms described above, or a rapid and consistent biochemical relapse. Relapsed/refractory MM (RRMM) is defined as disease that becomes refractory or progressive on therapy or within 60 days of last treatment in patients who have achieved a minimal response (MR) or greater clinical response to prior therapy (Sonneveld and Broijl (2016 ) Haematologica 101 (4): 396-406).

Unexplained monoclonal gammopathy (MGNG) and smoldering multiple myeloma (TMM)

[00196] Unexplained monoclonal gammopathy (MGNG) and smoldering multiple myeloma (TMM) are asymptomatic premalignant disorders characterized by the proliferation of monoclonal plasma cells in the bone marrow and the absence of target organ damage.

[00197] Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell proliferative disorder with a high risk of progression to symptomatic or active multiple myeloma (Kyle et al. (2007) N. Engl. J. Med. 356(25): 2582-2590) . The internationally agreed criteria defining TMP were adopted in 2003 and imply a patient's M-protein level >30 g/L and/or bone marrow clonal plasma cell level >10% (Internat. Myeloma Working Group (2003) Br. J Haematol 121: 749-757). Patients should not have any organ or associated tissue dysfunction, such as bone lesions or symptoms. Recent studies have identified two subgroups of TMMs: i) patients with developing disease and ii) patients with non-developing disease (Internat. Myeloma Working Group (2003) Br. J. Haematol. 121: 749-757).

[00198] TMM resembles monoclonal gammopathy of unknown origin (MGUS) in that there is no target organ damage (Kyle et al. (2007) N. Engl. J. Med. 356(25): 2582-2590). However, clinically, TMM is much more likely to progress to active multiple myeloma or amyloidosis after 20 years (78% chance for TMM versus 21% for MGUS) (Kyle et al. (2007) N. Engl. J. Med. 356( 25): 2582-2590).

[00199] Internationally agreed criteria for defining MGNG include a patient's M-protein level <30 g/L, bone marrow plasma cell level <10%, and absence of organ or associated tissue dysfunction, including bone lesions or symptoms (Internat. Myeloma Working Group (2003) Br. J. Haematol 121: 749-757).

Pathological conditions associated with CD38

[00200] The antibodies, methods, and dosage units of this invention find use in a variety of applications, including the treatment or amelioration of diseases associated with CD38, such as diseases and conditions associated with inflammation and immunopathologies, in particular diseases associated with activated lymphocytes. . The anti-CD38 antibodies of the invention bind to CD38 positive cells, resulting in depletion of the population of these cells, such as activated lymphocytes, through a variety of mechanisms of action, including both the ADCC pathway and the CDC pathway.

[00201] Thus, any autoimmune disease that exhibits either increased expression of CD38 or an increased number of cells expressing CD38 as a component of the disease can be treated using the antibodies of this invention. These include, but are not limited to: rejection of allogeneic islet transplants, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune adrenal disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, neutropenia, immune myocarditis autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac dermatitis, chronic fatigue syndrome (immune dysfunction), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, limited scleroderma (CREST) syndrome), cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, primary mixed cryoglobulinemia, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia, fibromyosis itis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Goodpasture's syndrome, graft versus host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA-associated neuropathy, IgM-associated polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease, Wilson's lichen, systemic lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondria, polyendocrine syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, solid organ transplant rejection, muscle stiffness syndrome, systemic lupus erythematosus, arteritis ayasu, temporal arteritis/giant cell arteritis, purpura thrombotic thrombocytopenia, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis, vitiligo and Wegener's granulomatosis.

[00202] Of particular use in some embodiments is the use of the antibodies of this invention for use in the diagnosis and/or treatment of a number of diseases, including but not limited to autoimmune diseases, which include, but are not limited to: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis, and graft-versus-host disease.

[00203] Thus, for example, patients with high plasma cell counts can be treated, such as SLE patients who have high plasma cell levels, as well as RA patients who have been shown not to respond to CD20-based therapy.

Antibody compositions for administration in vivo

[00204] Antibody dosage forms used in accordance with this invention are prepared for storage by mixing an antibody having the required degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition (1980) Osol, A. Ed .) in the form of lyophilisates or aqueous solutions.

[00205] Dosage forms according to this document may also contain more than one active compound, which is necessary for a particular indication to be treated, preferably with complementary actions that do not adversely affect each other. For example, it may be useful to give antibodies other specificities. Alternatively, or in addition, the composition may contain a cytotoxic agent, a cytokine, a growth inhibitor, and/or a small molecule antagonist. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

Subcutaneous administration

[00206] The present invention is based on the surprising discovery that anti-CD38 antibodies such as AB79 described herein can be administered at sufficiently low doses that are therapeutically effective to allow small volumes of liquid dosage forms to be administered subcutaneously. Subcutaneous administration is a minimally invasive route of administration and is considered to be the most versatile and therefore more appropriate route of administration that can be used for both short and long term therapy. In some embodiments, subcutaneous administration may be by injection. In some embodiments, the injection site or devices may be alternated when multiple injections or devices are required.

[00207] Accordingly, subcutaneous dosage forms are much easier for a patient to self-administer, especially as it may be necessary to take the dosage form regularly throughout the life of the patient (eg, from the first year of a child's life). In addition, the ease and speed of subcutaneous administration improves patient compliance and provides faster access to drugs when needed. Thus, the subcutaneous formulations of anti-CD38 antibodies provided herein provide a significant advantage over the prior art and address certain previously unmet needs.

[00208] In some embodiments, the antibodies of this invention are administered to a subject according to known methods by the subcutaneous route. In some embodiments, the antibodies of this invention may be administered by subcutaneous injection. In specific embodiments, the dosage form is administered subcutaneously to the same site on the patient's body (eg, injected into the upper arm, anterior thigh, lower abdomen, or upper back) for repeated or continuous injections. In other embodiments, the subcutaneous dosage form is administered by subcutaneous injection at a different site on the patient's body, or the injection site alternates. Can be used single or multiple administration of dosage forms.

[00209] In some embodiments, the subcutaneous dosage forms described herein can be used to treat cancer. In some embodiments, the subcutaneous dosage unit forms described herein can be used to treat hematologic cancer. In some embodiments, the subcutaneous dosage forms described herein can be used to treat multiple myeloma.

[00210] In some embodiments, antibodies of the invention that transiently bind human erythrocytes have increased bioavailability. In some embodiments, the bioavailability of antibodies of this invention that temporarily bind red blood cells is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or more . In some embodiments, the bioavailability of antibodies of this invention that transiently bind human erythrocytes is 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250% or 300% or more.

[00211] In some embodiments, the increase in bioavailability provides subcutaneous administration. In some embodiments, the increase in bioavailability is due to the fact that the antibodies of this invention temporarily bind to red blood cells. In some embodiments, the increased bioavailability in humans is due to the fact that the antibodies of the invention bind differently to human erythrocytes.

[00212] In some embodiments, the antibodies of the invention result in depletion of a population of NK cells, B cells, and/or T cells. In some embodiments, the antibodies of this invention allow for increased depletion of a population of NK cells compared to depletion of populations of B cells or T cells. In some embodiments, the antibodies of the invention allow for increased depletion of a population of NK cells relative to B cells, as well as an increase in depletion of a population of NK cells relative to T cells. In some embodiments, the antibodies of the invention allow for increased depletion of a population of NK cells relative to B cells, as well as increased depletion of a population of B cells relative to T cells. In some embodiments, the antibodies of the invention allow for increased depletion of a population of NK cells relative to B cells and increased depletion of a population of B cells relative to T cells.

[00213] In certain embodiments, the bioavailability of the anti-CD38 antibodies described herein after subcutaneous administration is from at least 50% to at least 80% compared to intravenous administration normalized for the same dose. In certain embodiments, the bioavailability of the anti-CD38 antibodies described herein after subcutaneous administration is from at least 60% to at least 80% compared to intravenous administration normalized for the same dose. In certain embodiments, the bioavailability of the anti-CD38 antibodies described herein after subcutaneous administration is from at least 50% to 70% compared to intravenous administration normalized for the same dose. In certain embodiments, the bioavailability of the anti-CD38 antibodies described herein after subcutaneous administration is from at least 55% to 65% compared to intravenous administration normalized for the same dose. In certain embodiments, the bioavailability of the anti-CD38 antibodies described herein after subcutaneous administration is from at least 55% to 70% compared to intravenous administration normalized for the same dose.

[00214] In certain embodiments, the bioavailability of the anti-CD38 antibodies described herein after subcutaneous administration is at least 40%, at least 45%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, not less than 84%, or not less than 85% compared with intravenous administration normalized for the same dose.

[00215] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is 50-80% compared to intravenous administration normalized for the same dose.

[00216] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is at least 50% compared to intravenous administration normalized for the same dose.

[00217] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is at least 55% compared to intravenous administration normalized for the same dose.

[00218] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is at least 60% compared to intravenous administration normalized for the same dose.

[00219] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is at least 65% compared to intravenous administration normalized for the same dose.

[00220] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is at least 70% compared to intravenous administration normalized for the same dose.

[00221] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is at least 75% compared to intravenous administration normalized for the same dose.

[00222] In some embodiments, this disclosure relates to a method wherein the bioavailability of antibodies of the invention that temporarily bind human erythrocytes after subcutaneous administration is at least 80% compared to intravenous administration normalized for the same dose.

[00223] In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously as a single bolus injection. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously on a monthly basis. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously once every three weeks. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every two weeks. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously on a weekly basis. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously twice a week. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously daily. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every 12 hours. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every 8 hours. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every six hours. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every four hours. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every two hours.

[00224] In some embodiments, the subcutaneous dosage unit forms are administered at a dosage of from about 0.01 mg per kilogram of body weight to about 0.8 mg per kilogram of body weight. In some embodiments, subcutaneous dosage unit forms contain an amount sufficient to administer a dosage of from about 0.02 mg per kilogram of body weight to about 0.75 mg per kilogram of body weight. In some embodiments, subcutaneous dosage unit forms contain an amount sufficient to administer a dosage of from about 0.02 mg per kilogram of body weight to about 0.7 mg per kilogram of body weight. In some embodiments, subcutaneous dosage unit forms contain an amount sufficient to administer a dosage of from about 0.03 mg per kilogram of body weight to about 0.6 mg per kilogram of body weight. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.25 milliliters (ml) to about 3.5 milliliters (ml). In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 3 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 2.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 2 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 1 ml to about 2 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.25 ml to about 1.25 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 1.25 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.75 ml to about 1.25 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.75 ml to about 1 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.9 ml to about 1.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.9 ml to about 1.1 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 1 ml to about 1.0 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.95 ml to about 1.05 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 3.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 3 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 2.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 2 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 1.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 1 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 0.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 0.25 ml.

Standard dosage forms

[00225] In some embodiments, the therapeutic anti-CD38 antibodies are formulated as part of a unit dosage form. In some embodiments, the antibody comprises a heavy chain containing the following CDR amino acid sequences: GFTFDDYG (SEQ ID NO: 3; HCDR1 AB79), ISWNGGKT (SEQ ID NO: 4; HCDR2 AB79), and ARGSLFHDSSGFYFGH (SEQ ID NO: 5; HCDR3 AB79). In some embodiments, the antibody comprises a light chain containing the following CDR amino acid sequences: SSNIGDNY (SEQ ID NO: 6; LCDR1 AB79), RDS (SEQ ID NO: 7; LCDR2 AB79), and QSYDSSLSGS (SEQ ID NO: 8; LCDR3 AB79 ). In some embodiments, the antibody comprises a heavy chain containing the following CDR amino acid sequences: GFTFDDYG (SEQ ID NO: 3; HCDR1 AB79), ISWNGGKT (SEQ ID NO: 4; HCDR2 AB79), ARGSLFHDSSGFYFGH (SEQ ID NO: 5; HCDR3 AB79 ) and a light chain containing the following CDR amino acid sequences: SSNIGDNY (SEQ ID NO: 6; LCDR1 AB79), RDS (SEQ ID NO: 7; LCDR2 AB79) and QSYDSSLSGS (SEQ ID NO: 8; LCDR3 AB79). In some embodiments, the antibody comprises a heavy chain comprising the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 9.

EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLA (SEQ ID NO: 9).

[00226] In some embodiments, the antibody comprises a light chain comprising the light chain variable region (VL) amino acid sequence of SEQ ID NO: 10.

(SEQ ID NO: 10).

[00227] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of the VH region of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of the VL region of SEQ ID NO: 10.

[00228] As will be appreciated by those skilled in the art, variable heavy and light chains can be fused to human IgG constant domain sequences, typically IgG1, IgG2, or IgG4.

In some embodiments, the antibody comprises the heavy chain (HC) amino acid sequence of SEQ ID NO: 11.

EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

[00229] In some embodiments, the antibody comprises the light chain (LC) amino acid sequence of SEQ ID NO: 12.

QSVLTQPPSASGTPGQRVTISCSGSSSNIGDNYVSWYQQLPGTAPKLLIYRDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYTVSCAPHEGSQ2.

[00230] In some embodiments, the antibody comprises the HC amino acid sequence of SEQ ID NO: 11 and the LC amino acid sequence of SEQ ID NO: 12.

[00231] In some embodiments, the dosage form containing the anti-CD38 antibody is a unit dosage form. In some embodiments, the unit dosage form contains an amount sufficient to administer a dosage of from about 0.01 mg per kilogram of body weight to about 0.8 mg per kilogram of body weight. In some embodiments, the unit dosage form contains an amount sufficient to administer a dosage of from about 0.02 mg per kilogram of body weight to about 0.75 mg per kilogram of body weight. In some embodiments, the unit dosage form contains an amount sufficient to administer a dosage of from about 0.02 mg per kilogram of body weight to about 0.7 mg per kilogram of body weight. In some embodiments, the unit dosage form contains an amount sufficient to administer a dosage of from about 0.03 mg per kilogram of body weight to about 0.6 mg per kilogram of body weight. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.25 milliliters (ml) to about 3.5 milliliters (ml). In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 3 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 2.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 2 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 1 ml to about 2 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.25 ml to about 1.25 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.5 ml to about 1.25 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.75 ml to about 1.25 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.75 ml to about 1 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.9 ml to about 1.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.9 ml to about 1.1 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 1 ml to about 1.1 ml. In some embodiments, the amount is contained in a dosage form with a volume of from about 0.95 ml to about 1.05 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 3.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 3 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 2.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 2 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 1.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 1 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 0.5 ml. In some embodiments, the amount is contained in a dosage form with a volume of about 0.25 ml.

[00232] In some embodiments, the anti-CD38 antibody unit dosage forms provided herein may further include one or more pharmaceutically acceptable excipients, carriers, and/or diluents.

[00233] Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased according to the needs of the therapeutic situation. Compositions may be formulated in unit dosage form for ease of administration and dosage uniformity. As used herein, the term "unit dosage form" means physically separate units suitable as single doses for subjects to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect, in combination with the necessary pharmaceutical carrier.

[00234] The specification for a unit dosage form of this invention is driven 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 inherent in the art of formulating such an active compound for the treatment of an individual.

[00235] Effective doses and dosage regimens for anti-CD38 antibodies used in this invention depend on the type and severity of the disease or condition being treated and can be determined by those skilled in the art.

[00236] The dosage forms provided herein are based on subcutaneous administration, which is achieved, at least in part, based on a lower ability to bind or remain associated with red blood cells compared to daratumumab. Without wishing to be bound by any particular theory, this binding activity may be due to the transient nature of AB79 binding to erythrocytes. In some embodiments, the therapeutic antibodies bind human erythrocytes transiently. In some embodiments, the therapeutic anti-CD38 antibodies bind cynomolgus macaque erythrocytes transiently. In some embodiments, the therapeutic anti-CD38 antibodies bind human or cynomolgus macaque erythrocytes transiently. In some embodiments, the therapeutic anti-CD38 antibodies bind human and cynomolgus macaque erythrocytes transiently.

[00237] In one embodiment, the anti-CD38 antibody is administered by subcutaneous injection at a weekly dosage of about 0.01 to about 1 mg/kg, such as about 0.02 to about 0.8 mg/kg. Such administration may be repeated, for example, 1 to 14 times, such as 3 to 5 times. An exemplary non-limiting range for a therapeutically effective amount of an anti-CD38 antibody used in this invention is about 0.01-1 mg/kg, e.g., about 0.01-0.8 mg/kg, about 0.02-0.75 mg/kg. about 0.02-0.7 mg/kg; or about 0.03-0.6 mg/kg.

[00238] As non-limiting examples, treatment according to this invention can be provided as a daily dose of antibody in an amount of from about 0.01 to about 1 mg/kg, for example 0.009; 0.01; 0.03; 0.05; 0.07; 0.09; 0.11; 0.13; 0.15; 0.17; 0.19; 0.21; 0.23; 0.25; 0.27; 0.29; 0.31; 0.33; 0.35; 0.37; 0.39; 0.41; 0.43; 0.45; 0.47; 0.49; 0.51; 0.53; 0.55; 0.57; 0.59; 0.61; 0.63; 0.65; 0.67; 0.69; 0.71; 0.72; 0.73; 0.75; 0.77; 0.79; 0.81; 0.83; 0.85; 0.87; 0.89; 0.91; 0.93; 0.95; 0.97 or 0.99; 1.0; or 1.1 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, 18, 12, 8, 6, 4 or 2 hours, or any combination thereof.

[00239] In one embodiment, the anti-CD38 antibody is administered weekly at a dosage of about 0.01 to about 1 mg/kg, such as about 0.02 to about 0.8 mg/kg. Such administration may be repeated, for example, 1 to 14 times, such as 3 to 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours. This regimen can be repeated one or more times as needed, for example after 6 months or 12 months. Dosage can be determined or adjusted by measuring the amount of a compound of this invention in the blood immediately after administration, for example by taking a biological sample and using anti-idiotypic antibodies that target the antigen-binding region of an anti-CD38 antibody.

[00240] In a further embodiment, the anti-CD38 antibody is administered once a week for 2 to 12 weeks, eg for 3 to 10 weeks, eg for 4 to 8 weeks.

[00241] In one embodiment, the anti-CD38 antibody is administered as maintenance therapy, such as once a week for a period of 6 months or more.

[00242] In one embodiment, the anti-CD38 antibody is administered in a schedule comprising one infusion of the anti-CD38 antibody followed by an infusion of the radioisotope-conjugated anti-CD38 antibody. The regimen can be repeated, for example, after 7-9 days.

[00243] In some embodiments, an anti-CD38 antibody of this invention is used in combination with one or more additional therapeutic agents, such as a chemotherapeutic agent. Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (eg, irinotecan, topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (eg etoposide, teniposide and daunorubicin); alkylating agents (eg melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, methotrexate, mitomycin C and cyclophosphamide); DNA intercalators (eg cisplatin, oxaliplatin and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (eg, 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

[00244] Chemotherapeutic agents that disrupt cell replication include: paclitaxel, docetaxel, and related analogs; vincristine, vinblastine and related analogues; thalidomide, lenalidomide and related analogues (eg CC-5013 and CC-4047); protein tyrosine kinase inhibitors (eg, imatinib mesylate and gefitinib); proteasome inhibitors (eg, bortezomib); NF-κB inhibitors, including IκB kinase inhibitors; antibodies that bind to proteins that are overexpressed, mis-expressed, or activated in cancer and thereby suppress cell replication (eg, trastuzumab, rituximab, cetuximab, and bevacizumab); and other protein or enzyme inhibitors known to be upregulated, overexpressed, misexpressed, or upregulated in cancer, the inhibition of which suppresses cell replication.

[00245] In some embodiments, the antibodies of this invention may be used before, concurrently with, or after treatment with Velcade® (bortezomib).

Treatment Methods

[00246] In the methods of this invention, therapeutic agents are used to provide a positive therapeutic response to a disease or pathological condition. The term "positive therapeutic response" means the positive dynamics of the disease or pathological condition and (or) the weakening of the symptoms associated with the disease or pathological condition. For example, a positive therapeutic response may refer to one or more of the following types of positive disease dynamics: (1) a decrease in the number of neoplastic cells; (2) increased death of neoplastic cells; (3) inhibition of neoplastic cell survival; (5) inhibition (ie, slowing down to some extent, preferably stopping) tumor growth; (6) improved patient survival; and (7) some reduction in one or more of the symptoms associated with the disease or condition.

[00247] Positive therapeutic responses for any disease or condition can be determined using standardized response criteria specific to that disease or condition. Tumor response can be assessed for changes in tumor morphology (i.e., total tumor burden, tumor size, etc.) using screening techniques such as magnetic resonance imaging (MRI), radiography, computed tomography (CT), scanning skeletal bone, endoscopy and tumor biopsy, including bone marrow aspiration (BMA) and counting of tumor cells in the bloodstream.

[00248] In addition to these positive therapeutic responses, the subject being treated may experience a positive effect in terms of reducing the symptoms associated with the disease. In the case of B-cell tumors, the subject may experience a reduction in so-called B-symptoms - for example, nocturnal hot flashes, fever, weight loss and/or urticaria. In the case of pre-malignant pathological conditions, treatment with a therapeutic anti-CD38 antibody can block and/or prolong the time to development of an associated malignant condition, for example, the development of multiple myeloma in subjects suffering from unexplained monoclonal gammopathy (MGNG).

[00249] The positive dynamics of the disease can be characterized as a complete response. The term "complete response" means the absence of clinically detectable disease with normalization of any previously abnormal radiographic findings, bone marrow and cerebrospinal fluid (CSF) values, or abnormal monoclonal protein level in the case of myeloma.

[00250] Such a response may be maintained for at least 4-8 weeks, or sometimes 6-8 weeks, after treatment in accordance with the methods of this invention. In another embodiment, the positive dynamics of the disease can be classified as a partial response. The term "partial response" means at least about 50% reduction in the total measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured total value of tumor masses, or the amount of abnormal monoclonal protein) in the absence of new lesions, which may persist for 4-8 weeks or 6-8 weeks.

[00251] Treatment includes a "therapeutically effective amount" of drugs used. "Therapeutically effective amount" means that amount that is effective to achieve the desired therapeutic result in dosages and for the required periods of time.

[00252] The terms "therapeutically effective amount" and "therapeutically effective dosage" means an amount of a therapeutic agent that is sufficient to reduce or alleviate the severity and/or duration of a disorder or one or more of its symptoms; preventing the progression of the disorder; ensuring the reverse development of the violation; preventing the recurrence, development, manifestation or progression of one or more symptoms associated with the disorder; or enhancing or improving the prophylactic(s) or therapeutic(s) effect(s) of another therapy (eg, prophylactic or therapeutic agent), in dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on such factors as the course of the disease, the age, sex and body weight of the individual, and the ability of the drugs to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of an antibody or antibody portion are offset by therapeutically beneficial effects. A therapeutically effective amount of an antibody for tumor therapy can be measured by its ability to stabilize the progression of the disease. The ability of a compound to inhibit cancer can be assessed in an animal model system predictive of efficacy in human cancers.

[00253] In another embodiment, this property of the composition can be assessed by testing the ability of the compound to inhibit cell growth or induce apoptosis using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound may result in a reduction in tumor size or otherwise relieve symptoms in a subject. One of ordinary skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

Anti-CD38 antibody kits

[00254] In another aspect of the invention, kits are provided for the treatment of a disease or condition associated with hematologic cancer. In one embodiment, the kit contains a dose of an anti-CD38 antibody described herein, such as AB79. In some embodiments, the kits provided herein may contain one or more doses of a liquid or lyophilized dosage form, as provided herein. When the kits contain a lyophilized dosage form of an anti-CD38 antibody described herein, such as AB79, typically the kits also contain a suitable liquid for reconstitution of the liquid dosage form, such as sterile water or a pharmaceutically acceptable buffer. In some embodiments, kits may contain a dosage form of the anti-CD38 antibody described herein prepackaged in a syringe for hypodermic administration by a healthcare professional or for home use.

[00255] In certain embodiments, the kit will be for a single dose of an anti-CD38 antibody described herein, such as AB79. In other embodiments, the kit may contain multiple doses of an anti-CD38 antibody described herein, such as AB79, for subcutaneous administration. In one embodiment, the kit may contain a dosage form of the anti-CD38 antibody described herein, prepackaged in a syringe for hypodermic administration by a healthcare professional or for home use.

Products

[00256] In other embodiments, an article of manufacture is provided that contains materials useful in the treatment of the disorders described above. The product contains 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 in 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 stopper pierceable by a hypodermic needle). The active agent in the composition is an antibody. A label on or associated with the container indicates that the composition is being used to treat the selected pathological condition. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, or dextrose solution. In addition, it may include other commercially and user-friendly materials, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

EXAMPLES

Example 1: Model-Based Characterization of Anti-CD38 Antibody in Cynomolgus Macaque

[00257] The anti-CD38 antibody AB79 binds CD38 of the cynomolgus macaque (Javanese), which distinguishes it from daratumumab (Darzalex ^TM ), a cytolytic anti-CD38 monoclonal antibody recently approved for the treatment of multiple myeloma. This unique feature has justified the use of Javanese in preclinical studies to characterize the pharmacokinetics (PK), pharmacodynamics (PD) and safety of AB79. To this end, assays have been developed to measure drug concentration, immunogenicity, and quantify T, B, and NK cells in the blood of cynomolgus monkeys. We evaluated these parameters in 8 pharmacological and toxicological preclinical studies. Of the cell populations tested, CD38 is most highly expressed on NK cells; therefore, we assume that the effect of the drug on NK cells is closest to the effect on the target cells in question - plasmablasts, plasma cells and other activated lymphocytes.

[00258] Data were collected from 8 studies in healthy monkeys using a dose range of 0.03-100 mg/kg, and mathematical models were developed that describe pharmacokinetics and exposure-effect relationships for each of the cell types. Depletion of the NK cell population has been identified as the most sensitive pharmacodynamic effect of AB79. This depletion was described using a turnover model (EC50 = 34.8 μg/ml in depletion rate) and complete depletion was achieved at 0.3 mg/kg IV. There were also intermediate effects on T cell count using the direct response model (EC50=9.43 µg/mL) and on B cell count using the 4-chamber transit model (EC50=19.3 µg/mL for depletion rate) . These analyzes confirmed the observation that the AB79 antibody passed through each of the measured lymphocyte subpopulations at different rates and required different times to deplete the model blood chamber.

[00259] Mathematical models that describe PK and PD are useful tools for obtaining a mechanistic and quantitative understanding of the relationship between drug exposure and effect (Friberg et al. (2002) J. Clin. Oncol. 20: 4713-4721; Mager et al . (2003) Drug Metab Dispos 31 : 510-518 Han and Zhou (2011) Ther Deliv 2 : 359-368). Typical PK features of IgG antibodies, including distribution and elimination, physiological and genetic similarities between macaque and human, can be used to explain the pharmacology of AB79 (Glassman and Balthasar (2014) Cancer Biol. Med. 11: 20-33; Kamath (2016) Drug Discov. Today Technol. 21-22: 75-83). In addition, these models have been successfully applied to predict FA concentrations and PD effects in healthy subjects (Han and Zhou (2011) Ther. Deliv . 2: 359-368).

Materials and methods

[00260] The summarized results of studies on macaques are shown in Table 2 in chronological order. Single dose studies Nos. 2, 7, and 8 were primarily conducted to evaluate PK and PD for intravenous (IV) and subcutaneous (SC) administration of AB79 (FIG. 1). Repeated dose studies were conducted to evaluate safety, PK and PD, including two 4-week studies (studies #1 and 3) and three 13-week NLP studies (studies #4, 5 and 6). A dosing error occurred in 13-week Study #5. Animals in the lowest dose group received 0.01 mg/kg instead of the intended 0.1 mg/kg at a time (second dose) and then continued with 0.1 mg/kg. These data were added to the data set with the correct information about the actual doses administered. Study #6 replicated the low dose 0.1 mg/kg weekly treatment group of Study #5. All animal studies were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals adopted and published by the US National Institutes of Health.

Table 2. AB79 studies in macaques in chronological order Study No. Study Description Number of animals (females, males) Doses (mg/kg) Number of samples per animal (FC/PD) one Day 1 (1 mg/kg) + Day 28 (2 mg/kg), IV, FC, PD 6 (0, 6) plc; one; 2 19/10 2 Single dose, IV, FC, PD 9 (0.9) plc; 0.3; 3 14/9 3 Tox. 4 weeks, once a week, IV, FC, PD 12 (4, 8) plc; one; thirty; 100 15/8 four Tox. 13 weeks, every 2 weeks, IV, FC, PD 40 (20, 20) plc; 3; thirty; 80 47/29 5 Tox. 13 weeks, once a week, IV, FC, PD 52 (26, 26) plc; 0.1; 0.3; one 31/9 6 Tox. 13 weeks, once a week, IV, FC, PD 20 (20, 0) plc; 0.1 31/10 7 Single dose, i/v/s/c, FC, PD 12 (12, 0) 0.1 (0.3; 1) 16/16 eight Single dose, i/v/s/c, FC, PD 24 (24, 0) 0.03 (0.1; 0.3) 19/19 IV - intravenous 30-minute infusion (studies # 1-4) or bolus (studies # 5-8); s / c - subcutaneous injection (group 4 of study No. 7 and group 3 of study No. 8); FC - dense sample of FC; FD - a dense sample of whole blood samples for flow cytometry, giving data on the number of T-, B- and NK-cells; plc - placebo; "4 weeks" or "13 weeks" describe the length of the treatment period; tox. - toxicological study.

Bioanalytical technique

[00261] PK was analyzed using a validated method developed and implemented by Charles River Laboratories (Reno, Nevada). Briefly, AB79 concentration was measured in macaque serum using an indirect enzyme-linked immunosorbent assay (ELISA). A 96-well microtiter plate was coated with anti-idiotypic antibody against AB79. Blanks, standards and quality control (QC) samples containing AB79 at various concentrations were added to the plate and incubated for 55-65 minutes at room temperature. After washing the microtiter plate, peroxidase-conjugated mouse anti-human IgG (Jackson ImmunoResearch's Peroxidase AffiniPure Mouse Anti-Human IgG, Fcγ Fragment Specific) was added and incubated on the plate for an additional 55-65 minutes. The plate was washed again and tetramethylbenzidine (TMB) was added to the wells to form the chromophore and color development was stopped by adding stop solution (dvs sulfuric acid). Absorbance at 450 nm was measured using a SPECTRAmax® 190 microplate analyzer (Molecular Devices) and AB79 concentrations were calculated using a 4-parameter exponential weighted (1/y ² ) standard calibration curve. In Study #1 (Table 2), the lower limit of quantitation (LQLO) of AB79 antibody in serum was 0.061 μg/mL, while in all other studies it was 0.05 μg/mL.

Determination of the immunogenicity of anti-AB79 antibodies

[00262] Anti-drug antibody (AMDA) screening in macaque serum was performed using a qualitative electrochemiluminescent (ECL) method validated and performed by Charles River Laboratories (Reno, Nevada). Briefly, undiluted serum samples were incubated with 300 mM acetic acid. Acid dissociated samples were incubated in a mixture of biotinylated AB79, AB79 labeled with SULFO-TAG™ (Meso Scale Diagnostics, labeled from Charles River Laboratories), and 1.5M Trizma® base reagent to neutralize the acid and form the immune complex. This complex was then added to a streptavidin-coated MSD plate (Meso Scale Diagnostics) and allowed to bind. After washing, the complex was detected by adding the reagent "MSD read buffer T" (Meso Scale Diagnostics) to the plate and subsequent excitation of the SULFO-TAG ™ label through the electrochemical reaction of the [Ru(bpy)3] ²⁺ complex with the formation of luminescence (light), which was read using the MSD Sector 6000 analyzer (Meso Scale Diagnostics). Luminescence intensity correlated quantitatively with the level of macaque anti-AB79 antibodies present in the serum of individual samples.

Characterization of blood cells

[00263] To evaluate and compare the level of AB79 binding in humans and macaques, blood samples from each were collected in sodium heparin tubes. An aliquot of blood (100 μl) was mixed with the appropriate volume of characterizing antibody (Table 3) and incubated for 15-20 minutes at room temperature in the dark. After incubation, 1 ml BD FACS lyse reagent (single strength; BD Biosciences; San Jose, CA) was added to lyse erythrocytes and the cells were incubated for 10 minutes at room temperature in the dark, then centrifuged, decanted and resuspended in 1 ml staining bovine serum albumin buffer (BD Biosciences). Cells were centrifuged a second time, decanted and 250 µl of "Flow Fix" reagent (1% paraformaldehyde in Dulbecco's phosphate-buffered saline without calcium and magnesium (Life Technologies, Carlsbad, CA)) was added and fluorescence measured by flow cytometry using a FACSCanto flow cytometer. ™ II (BD Biosciences). Macaque NK cells (CD3-, CD159a+), B cells (CD3-, CD20+) and T cells (CD3+), and NK cells (CD3-, CD16/CD56+), B cells (CD3-, CD19+) and human T cells (CD3+). The average fluorescence intensity of AB79 staining for each cell population was converted to equivalent soluble fluorochrome (SERF) molecules using a standard curve generated using Rainbow Beads reagent (Spherotech; Lake Forest, Illinois).

Table 3. Antibodies used to characterize blood cells Antibody Volume of antibody per sample (µl/100 µl of sample) Source CD3-APCH7 (SK7)* 5 BD Biosciences CD3-APC Cy7 (SP34-2) 1.25 BD Biosciences CD3-PerCp Cy 5.5 (SP34-2) 5 BD Biosciences AB19-AF647 one Made by Takeda AB79-AF488 2 Made by Takeda AB19-AF488 0.42 Made by Takeda CD19-PerCp Cy 5.5 (HIB19)* 5 BD Biosciences CD20-PE (2H7) ten BD Biosciences CD20-APCH7 (2H7) 2.5 BD Biosciences CD16-PE (B73.1)* 5 BD Biosciences CD16-PerCP-Cy5.5 (3G8) twenty BD Biosciences CD56-PE (B159)* 5 BD Biosciences CD159a-PE (Z199) 5 Beckman Coulter (Brea, California) CD45-PerCP TruCount (DO58-1283) N/A BD Biosciences CD45-PeCy7 (HI30)* 2.5 BD Biosciences CD45-PeCy7 (DO58-1283) 2.5 BD Biosciences Mouse IgG1 kappa -AF488 or -AF647 (MOPC-21) 2.5 BioLegend (San Diego, California) *used to stain human cells only

[00264] In the studies shown in Table 2, cells were stained and analyzed using a validated method developed and implemented by Charles River Laboratories (Reno, Nevada). Blood samples from monkeys were collected in sodium heparin tubes before and several times after AB79 administration, and certain lymphocyte populations were analyzed by flow cytometry using a FACSCanto™ II flow cytometer (BD Biosciences). Commercial antibodies and anti-CD38 antibody (AB19; Table 3); US patent No. 8362111) were titrated to optimal concentrations for staining. Macaque CD38+/-, T cells (CD3+), B cells (CD3-/CD20+), and natural killer (NK) cells (CD3-/CD20-/CD16+) were identified and lymphocytes were quantified using CD45TruCount™ tubes (BD Biosciences). Aliquots of approximately 100 μl from each blood sample were placed in the appropriate well of a 96-well plate and antibodies were added in the indicated volume, mixed and incubated for a minimum of 30 minutes at room temperature in the dark. After incubation, the erythrocytes were lysed, the samples were mixed and incubated at room temperature for an additional 10 minutes in the dark. The plate was centrifuged and the supernatant was decanted. Then the cell pellet was resuspended in 1800 μl of staining buffer, the samples were mixed, centrifuged and the supernatant was decanted. The cell pellet was resuspended in 500 μl of fetal bovine serum staining buffer and approximately 300 μl of the cell suspension was transferred to a 96-well V-bottom plate for analysis. Percentages of NK cells, as well as total T cells and B cells, were converted from cell counts obtained using TruCount™ tubes (BD Biosciences; San Jose, CA) and used to determine the absolute cell count for each population . Studies #1-4 analyzed baseline levels of CD38+ NK-, B-, and T-cell subpopulations with labeled anti-CD38 antibodies, AB79 or AB19. Although AB19 binds to a different epitope, the results were very similar and therefore not presented separately. The processed samples were analyzed immediately.

Development of the FC model

[00265] In developing the FK model, the structures of the one-, two-, and three-chamber model were investigated. The two-chamber model clearly outperformed the one-chamber model as measured by the Model Fit Criteria (KAM) and Decreasing Objective Function (TFV) plots. Based on visual inspection of the diagnostic plots, the introduction of a third chamber was not necessary for a reliable description of the data. Bioavailability ( F ) was modeled using the logit transform F=exp(PAR)/(1+exp(PAR)) , where PAR denotes a model parameter to ensure that the range of estimates between 0 and 1 is limited. Non-linear PK at low concentrations was modeled using approximate quasi-stationary state (QSS) model of target-mediated drug distribution (MPD) (Gibiansky and Gibiansky (2009) Expert Opin. Drug Metab. Toxicol. 5: 803-812).

[00266] A schematic representation of the model is shown in FIG. 2C. The approximation of the KSS was based on the assumption that steady state concentrations of free drug C, target R and drug target complex RC are established very quickly compared to all other processes. This means that the binding process is balanced with the processes of dissociation and internalization, and that the following equation holds in appropriate units: K _ON *C*R = ( K _OFF +K _INT ) RC , where K _ON denotes the rate constant of binding, K _OFF is the constant dissociation rate, and K _INT is the internalization rate constant.

[00267] Intersubject variability (MSV) was investigated for all parameters and modeled using exponential models of the following type: PAR _i =TVPAR*e ^ETAPAR _i , where PAR _i is an estimate of the value of an individual parameter, TVPAR is an estimate of the value of a typical parameter, and ETAPAR _i - assessment of the deviation of the individual parameter i . The ETAPAR _i values were assumed to follow a normal distribution with a mean value of zero. The residuals were described using a combined additive and proportional error model (Beal and Sheiner (1992) NONMEM User Guides, in University of California CA).

[00268] The following parameters were investigated to identify potential covariate effects on AB79 FC: body weight, gender, dose, route of administration, and study.

Development of the FK-PD model

[00269] For each of the three cell types, PKPD model development was performed separately. Note that measurements close to the time of drug administration (<8 hours post-dose) were not used for model development, as they were affected by a non-specific, drug-independent effect, possibly due to taking several blood samples. The FK model and parameter estimates were fixed. Turnover, transit chamber, and direct response models of various forms have been tested (Friberg et al. (2002) J. Clin. Oncol. 20: 4713-4721; Mager et al. (2003) Drug Metab. Dispos. 31: 510-518 ). In turnover models, the effect of the drug was introduced into the cell elimination rate parameter in the form of a generic model Emax with or without Hill factors. In our notation, the Emax model is a function f of drug concentration c of the following form: f(c)=EMAX*c ^H /(c ^H +C50 ^H ) , where EMAX denotes the maximum effect, C50 is the concentration at which half of the maximum effect is achieved , and H is the Hill factor. In the transit chamber model (MTC), the drug effect was introduced and tested in different parameters: in the proliferation rate, in circulating cells and in the third transit chamber. Combinations of these effects were also tested and whether the data support a feedback mechanism from circulation to proliferation rate. In addition, direct response models of the Emax type with and without Hill factors were tested to describe the drug concentration-effect curve.

[00270] Random effect parameters were introduced to assess intersubject variability for the following: base cell number, cell production rate (KIN), mean transit time (MTT) in the transit chamber model, C50, and EMAX. Individual mean baseline cell levels were presented in the data set (column BU). These values were used in the model as typical values. A random effect parameter has been added to allow adjustments to be made to individual baseline scores based on all of the individual's scores. The PD residuals were described using a proportional error model.

[00271] For model validation during simulation (PK and PK-PD), FFTs, standard errors, QAM plots, and individual predictions versus data plots were used to evaluate the models and compare them to alternative models.

[00272] The following software packages were used: NONMEM (version 7.2), KIWI (version 1.6), Berkeley Madonna (version 8.3.14), PSN (version 4) and R (version 3.3.0).

Data set preparation

[00273] Datasets from 8 macaque studies were collected, reorganized in one format, and combined into three separate NONMEM-matched PK-PD datasets. Each of the three datasets contained individual macaque characteristics (study, identifier, group, body weight, sex), dosage information, PK data, and data for NK, B, or T cells. For control animals, only cell counts were included in the datasets, but no PK data were included, provided there were no apparent serum levels of AB79. Information taking into account the time parameter on the status of antidrug immunogenicity (ADLP), namely TITR, containing the result of a quantitative measurement and the variable 0/1-flag AALP-F (AALP-F=1 if APL affects the concentration of AB79, AMLP-F=0 if not) has been added for each observation result in separate columns. AALP titers were measured using different methodological specifications in different studies, and therefore values are not directly comparable quantitatively between studies. In order to consistently use AALP information across all studies, we applied the following procedure on an animal-by-animal basis: AALP titers that increased at time points later than 7 days from levels originally measured were considered AALP-positive and labeled in the data set (APPL- F=1). If a sample at one time point was labeled as AALP-positive, all samples taken after that time point were also labeled as AALP-positive in that animal, regardless of the titer measured. Positive ALP values were not used to estimate the parameters in the development of the model. Note that the PD measurements at times when AALP affected FA concentrations were also labeled as AALP-F=1.

[00274] For cell count data, individual baseline values for each cell type (NK, B, and T cells) were calculated as the average of all available measurements prior to administration of a single animal. In most studies, this was a single measurement. The baseline parameter value for each animal was then added as an observation at the time of the first dosing (TIME=0) and as a constant value in the BU column to each observation of the respective animal. Based on this baseline, a percentage of baseline was calculated for each measured number of cells that was added to the data set.

Scaling of PK parameters derived from macaque studies

[00275] The final PK and PK-PD models were used as a starting point for modeling PK and PK-PD profiles for the first human clinical trial. Although such an analysis is not conclusive, a comparative analysis of therapeutic monoclonal antibody data has demonstrated that PK parameters derived from macaque studies can be scaled to help predict human PK profiles with acceptable accuracy (Han and Zhou ( 2011) Ther. Deliv . 2: 359-368). The above publication indicated that using a fixed exponent of 0.85 can reliably predict the clearance rates of monoclonal antibodies in humans. Accordingly, this ratio was applied to calculate the clearance parameters for a person (CL, Q), while the volume parameters (V _C , V _P ) were calculated using a direct relationship between body weight (BW):

results

Pharmacokinetics of AB79

[00276] Data from all 8 studies in healthy monkeys (excluding placebo groups) were pooled into a PK data set (Table 2). In total, these data were obtained from 140 animals, 58 of which were males and 82 were females. The body weight of the studied animals ranged from 2.1 to 4.7 kg, and the doses ranged from 0.03 to 100 mg per kg of body weight (mg/kg). In one group from Study No. 7 and in three groups from Study No. 8, doses of 0.03; 0.1; 0.3 and 1 mg/kg were administered subcutaneously (15 animals in total). The pooled dataset contained 2199 measurable PK values above the LLQL (FIGS. 2A and 2B). Samples for PK analysis were most frequently taken after the first dose, and even in long-term toxicological studies, most animals were euthanized before day 98. Only study #4 included recovery groups and we could only collect PK data from 4 animals - 2 from the 80mg/kg group and one from each of the 30mg/kg and 3mg/kg groups (Fig. 2B) . In parallel with AB79 concentrations, ALLP was measured. AALP affected 229 FC observations (Figure 3).

[00277] Initially, for each of the macaque studies, PK was analyzed using standard non-chamber methods (NCM). Based on single dose studies (bolus IV injection or 30-minute IV infusion), the volume of distribution during the terminal phase (V _T ) was calculated, which varied from 64 to 116 ml / kg, clearance - from 6.04 to 14.7 ml / kg / day and the half-life in the terminal phase (T1 / 2) - from 4.75 to 11.2 days. It was found that the area under the curve (AUC) of concentration versus time and the maximum concentration (Cmax) increase in proportion to dose over a wide range. Only the PK profiles of the lowest dose groups (<1 mg/kg, Figs. 2D-2F) provide evidence of a non-linear increase in clearance at concentrations below 0.5 µg/mL, likely driven by target-mediated drug distribution (DDR) mechanisms (Kamath (2016 ) Drug Discov. Today Technol. 21-22: 75-83). Based on data from all macaque studies, excluding the two lowest dose groups (dose >0.3 mg/kg), a linear 2-chamber model was constructed. When modeling PK for the lowest dose groups and overlaying measured concentrations, it became apparent that the linear model predicted overestimated concentrations (FIGS. 2D-2F).

[00278] The available data on FC after a single sc injection demonstrated that Cmax was 70-80% lower in the sc groups compared to the i.v. groups at the same dose, and that their AUCs were comparable. No differences in FC parameters were observed between male and female macaques. The results of this preliminary analysis were used as a starting point for model development.

Development of the FC model

[00279] Model development began with data from a single intravenous dose, and then the initial model was gradually expanded using more complex data. Like other therapeutic antibodies, the resulting FA strictly follows a linear 2-chamber model (Kamath (2016) Drug Discov. Today Technol. 21-22: 75-83). The non-linear elimination component (NEC) describing accelerated clearance at low concentrations was modeled using an approximate quasi-stationary state (QSS) model (Gibiansky and Gibiansky (2009) Expert Opin. Drug Metab. Toxicol. 5: 803-812). The assumption that the association process between the drug and the target is much faster than the processes of dissociation, distribution and elimination of the drug, and also much faster than the elimination of the target and the drug-target complex, leads to a simplified model of ORPM (Fig. 2, Table four). The volume of data for low concentrations was relatively small, so not all parameters were calculated with a single download of the software. Therefore, the parameters of the MRPM model were first calculated from data from the low single dose studies Nos. 7 and 8. The resulting MRPM parameter values were then held fixed during the final calculations for the entire data set (Table 4).

Table 4. Results of PK population modeling, values parameters and standard errors in percent (%SOS) Final parameter values Intersubject variability/
residual variability Parameter typical value %SOS Value %SOS F 0.227 121 N/R - K _A (l/day) 0.399 20.5 CV: 42.1% 53.8 CL (l/day) 0.0187 5.17 CV: 42.9% 20.1 V _C (l) 0.141 3.23 CV: 19.8% 22.5 Q (l/day) 0.127 14.7 N/R - V _P (l) 0.127 6.45 CV: 39.4% 20.8 K _INT (1/day) 0.1 FIX. CV: 49.3% 36.7* _KSS (1/day) 5.68 38.7* N/R - K _SYN (u/l/day) ^$ 0.04 FIX. N/R - K _DEG (1/day) 0.00452 30.1* N/R - PATH on V _C -0.697 6.51 N/R - RV add 3.17E-04 21.2 CO: 0.0178 - RV prop 0.0677 1.58 CV: 26.0% - Minimum objective function value=5748.412 ^$ K _SYN - CD38 receptor synthesis rate. Because actual concentration measurements or information on the rate of CD38 synthesis or degradation in vivo were not available, "u" was used as the unit for some unknown amount of CD38.
* Parameter values and standard errors for MRPM parameters were obtained from a single program run that focused on data from low dose groups and then fixed for final evaluation of other PK parameters. N/R - not calculated; SOS - standard error of the mean; KV - coefficient of variation; FIX. - fixed value; SD - standard deviation.

[00280] The values of the absorption parameters - K _A and F - were obtained by adding data obtained from groups of s/c. All s.c. data were from the four lower single dose cohorts from studies 7 and 8. These lower doses (≤1 mg/kg) cover a clinically relevant range but may limit the ability to generalize parameter values to higher doses.

[00281] Intersubject variability (MSV) for PK parameters has been described using exponential models. The absorption coefficient (K _A ), clearance (CL) and peripheral volume of distribution (V _P ) have an MSV value of about 40%, and a central volume of distribution (V _C ) is about 20% (Table 4). Covariance analysis revealed the influence of the route of administration on V _C . The typical value for V _C was 0.141 L for IV administration, and 0.043 L (approximately 70% less) for SC administration. No other significant covariance effects were found. Due to data limitations at low concentrations, inter-subject variability and individual predictions of MRPM parameters were calculated only for the K _INT internalization rate (MSV: 49%). Model evaluation based on residual error, PCF, standard error, KAM plots, and individual curve functions confirmed that the final model reliably described PK for AB79 in healthy monkeys (Table 4, Figs. 4A-4J).

Pharmacodynamics

[00282] The level of binding of AB79 by NK-, T- and B-cells of human and macaque blood was compared using flow cytometry. As shown in FIG. 5, macaque lymphocytes had levels of CD38 expression, as measured by the MERF (equivalent soluble fluorochrome molecule) score for AB79, which were slightly lower compared to human lymphocytes, but with a similar relationship between cell types, for example, CD38 expression followed the following hierarchy: NK- cells > B cells > T cells. These data support the use of this non-human primate species as a relevant model to help model the potential activity of AB79 in humans.

[00283] For an in-depth quantitative analysis of the relationship between drug exposure (PK) and the extent and duration of cell depletion (PD), we compiled datasets on PK concentrations, NK cell, B cell, and T cell counts from all 8 macaque studies, including placebo groups where available (Table 2). Initial characterization of the data set demonstrated that, at baseline, T cells had a median value of 3732 cells per µL (interquartile range (ICI): 2,881–5,176), and were the most common lymphocyte subset compared to B cells, with a score of 1279 cells per µl (MCI: 860.8-1890) and NK cells with a rate of 685 cells per µl (MCI: 482.8-970.1). Baseline levels of CD38 expression in these cell subpopulations were assessed in studies #1-4 (Table 2, n=67). 86.7% (SD: 11.3) of NK cells expressed CD38 with less variability. In contrast, 58.7% (SD: 27.0) of B cells and 34.5% (SD: 24.5) of T cells expressed CD38 with greater variability.

[00284] Data from placebo-treated animals demonstrated that the mean number of cells of each type varied over time in each individual animal more than would be expected based on intrasubject variability (FIG. 6). For example, the mean coefficient of variation for B cell counts on individual placebo curves was 27%, but individual mean B cell counts ranged from 436.6 to 4389. In addition, there were also differences between mean baseline lymphocyte levels in males and females, and in animals from different studies, which increases the variability (Fig. 7). Based on these results, each post-drug cell count was calculated relative to its individual baseline as a percentage, rather than as absolute values at each time point. For example, a value of 33% means that the number of cells in the sample was 1/3 of the base number of cells. This approach provided standardized values that can be compared across the entire dataset.

[00285] The rapid onset of depletion of AB79-binding cells suggests that the decrease in lymphocyte count is related to the initial blood concentration (FIG. 8). At intravenous doses of AB79 at 0.3 mg/kg, the median maximum effect on NK cells was 93.9% (i.e., 6.1% of baseline cell count remained). At 0.1 mg/kg, the depletion peak was 71% (remaining 29% of baseline). At doses >0.3 mg/kg, NK cells were almost completely depleted in the simulated blood chamber (minimum and range were 1.06% of baseline and 0.17-6.23, respectively; Fig. 8A). After a single dose of 0.3 mg/kg, NK cells took approximately 7 days to recover to an average of 50% of baseline, although recovery kinetics had high inter-subject variability (FIGS. 8B and 8C). Consistent with these results, NK cell function was also tested in a subgroup of animals in Study #7 (n=3/group; Table 2). This experiment demonstrated a dose-dependent decrease with minimal changes in the activity of NK cells in the blood 48 hours after administration of the drug in animals treated with 0.1 mg/kg AB79 (% lysis ± SD at the ratio of effector : target value = 100 : 1 was 44.5 % ± 23.6% compared to 41.4% ± 25.8%) and almost complete loss of NK cell activity in animals treated with 1.0 mg/kg (% lysis ± SD at ratio effector : target = 100 : 1 was 37.4% ± 10.3% compared to 6.8% ± 12.5%). The function of NK cells was restored on the 57th day, and at the next measured time point, % lysis ± SD at the ratio effector : target value = 100 : 1 was 16.0% ± 11.9%.

[00286] B cell and T cell populations were depleted to a lesser extent compared to NK cells, consistent with their lower levels of CD38 expression (FIG. 5). For example, at an intravenous dose of AB79 of 0.3 mg/kg, the median maximum depletion rate for B cells was 45% of baseline, and T cells were depleted to 43% of baseline (FIGS. 8D and 8G). At this dose, a 50% reduction in B cells from baseline was not achieved in all animals. Only at the highest doses ≥30 mg/kg were B cells almost completely depleted (Fig. 8D). T cells were depleted to the same extent as B cells, but recovery was faster (FIGS. 8G-8I).

[00287] Two studies, Nos. 7 and 8, compared i.v. and s.c. dosing (FIGS. 8C, 8F, and 8I). There were no obvious differences in cell depletion with respect to routes of administration. At lower doses (Study #8), sustained (>24 hours) cell depletion 50% below baseline was observed only in the NK cell population, but not in T and B cells; although all cells showed specific depletion at early time points. The time of onset of NK cell depletion was similar between dose groups regardless of the route of administration, and the duration of depletion was dose dependent. Cell recovery in all tested groups was observed on the 57th day.

FK-FD models

[00288] Separate PKPD models have been developed to describe the effects of exposure to AB79 on NK, B, and T cells. In the PK-PD simulation, the PK parameters were kept fixed and equal to the values obtained in the final PK model, and various PD models were tried. The population of NK cells in the peripheral blood was reliably described using a turnover model, and the depleting effect of the drug was associated through the PK concentration and the type model Emax with the rate of depletion. In this model, EMAX denotes the maximum rate of additional NK cell depletion, and C50 is the concentration at which the rate of additional NK cell depletion is half the maximum. The structural model of PKPD for NK cells had the following form:

[00289] In this formula, NK is the actual number of NK cells, K _IN is the rate of production, and K _OUT is the rate of elimination in the absence of drug. Note that with a given baseline measurement of BL K _OUT defined by the equation K _OUT =K _IN/ BL, c denotes the concentration of AB79 in the central chamber of the model. With the simultaneous calculation of all parameters, the program did not give stable results. Individual parameters KIN, EMAX and C50 were highly correlated. In addition, due to the limited differentiation between the maximum effects of different doses (see previous section) and the high intersubject variability, it was not possible to expect an accurate assessment of all parameters. In a series of calculations, one or two of the three parameters KIN, EMAX, and C50 were equated to different values and the remaining parameters were calculated. With fixed parameters KIN=10000 and EMAX=322, stable operation of the program and an acceptable value of the model adequacy criteria were achieved. A typical calculated C50 value was 29.0 µg/mL (Table 5). In addition, the sensitivity of the selected KIN and EMAX values was tested by selecting different combinations of higher and lower values. Inter-subject variability was high and for NK cells was 113% for production rate (K _IN ) and 149% for C50, corresponding to large individual differences in baseline levels and between treated animals. The model was evaluated based on residual error, FCF, standard error, QAM plots, and individual curve functions (Table 5, Fig. 9).

Table 5. PD simulation results, parameter values and standard errors in percent (%SOS) Final parameter values Intersubject variability/residual variability Parameter typical value %SOS Value %SOS NK cells KIN (number/day) 10000 FIX. CV: 113% 19.2 C50 (mcg/ml) 29.0 18.8 CV: 149% 25.1 EMAX 322 FIX. N/R - Baseline (NK cells)* N/R - CV: 28.3% 20.8 Residual NK cells 0.291 2.42 CV: 53.9% - B cells SVP (day) 8.48 15.4 CV: 135% 17.4 C50 (mcg/ml) 19.5 7.58 N/R - EMAX 2.37 FIX. N/R - Baseline (B cells)* N/R - CV: 24.1% 10.7 Residual B cells 0.136 2.20 CV: 36.9% - T cells C50 (mcg/ml) 11.86 7.267 N/R - EMAX 0.4656 6.578 CV: 69.46% 29.50 Baseline (T cells)* N/R - CV: 29.08% 15.50 Residual T cells 0.1343 2.406 CV: 36.65% - * For each individual animal, a typical baseline value was calculated as the average of all pre-dose measurements. N/R - not calculated; SOS - standard error of the mean; KV - coefficient of variation; FIX. - fixed value.

[00290] The transit chamber model was superior to direct response or turnover models in describing depletion of the B cell population induced by the AB79 antibody. Four transit chambers were found to be suitable for the model, and the effect of the drug on the depletion rate was described using a generic Emax model. Similar to the NK cell depletion model, EMAX denotes the maximum rate and C50 the concentration at which the rate is half of the maximum. Thus, the structural model of PKPD for B cells is defined by the following five equations:

[00291] TR _i (i=1-4) denotes four transit chambers. K _TR , K _PROL and K _CIRC are defined by the following equations: K _TR =K _PROL =K _CIRC =4/MTR where MTR is the mean transit time (Friberg et al. (2002) J. Clin. Oncol. 20: 4713-4721 ). B denotes the number of B cells in the blood, and c is the concentration of AB79 in the central chamber.

[00292] At a fixed EMAX=2.37, the typical C50 was 19.5 μg/mL and the typical mean transit time (MTT) was 8.48 days (Table 5). The delay of the maximum effect relative to the maximum concentration of AB79 was clearly recorded. The model indicates that AB79 primarily affects circulating B cells. No additional effects and no feedback loops to progenitor cells were required to describe the available macaque B-cell data. The intersubject variability in SVP of 135% and baseline (BU) B cells of 24.1% indicates large individual differences between animals.

[00293] Drug-induced rapid recovery T cell depletion was reliably described using a direct response model: T(c) = BU _T * (1 - EMAX*c/(c+C50)), where T denotes the actual amount of T -cells, BU _T is the baseline of T cells, and c is the concentration of AB79 in the central chamber of the model. The typical C50 value was 11.86 μg/mL and the typical EMAX value was 0.47, indicating that in this case, AB79 could only deplete about half of the T cell population (Table 5). Note, however, that the intersubject variability for EMAX was nearly 70%. In this model, in contrast to the NK and B cell depletion models, C50 is the concentration at which T cell depletion was half the maximum.

[00294] As with NK cells, evaluation of the final PKPD models for B and T cells based on residual error, PCF, standard error, RAM plots, and individual curve functions confirmed that they reliably described the available data obtained from macaques (Table 5, Fig. 9).

Modeling of FC and depletion of human cell populations

[00295] The PK and PK-PD models derived from macaque studies were used as the starting point for model-based PK simulation and human cell count data to inform the design and dose selections for the first human clinical trial ( PKIL) - healthy volunteers. To this end, it has been suggested that model structures, including MRPM derived from macaque studies, also describe the main features of human FC and subsequent depletion of the lymphocyte population. To obtain predictions for human FC parameters, we scaled estimates of the following macaque FC parameters: central and peripheral volumes of distribution (V _C, V _P ), and clearance (CL) and intercameral clearance (Q) with a direct approach for monoclonal antibodies (Han and Zhou (2011) Ther. Deliv . 2: 359-368). AB79 is a fully human monoclonal antibody and therefore we would expect less immunogenicity in humans than in macaques. Therefore, for modeling and simulation purposes, we excluded AALP-positive samples from the data set.

[00296] Using a scaled model, the exposure and depletion profiles of NK-, B- and T-cells were modeled for single doses administered by infusion (IV) for 2 hours or by subcutaneous injection (SC) ranging from 0.0003 to 1.0 mg/kg as planned for the PIL study (Fig. 10). In accordance with the simulation results, after an IV dose of 0.0003 mg/kg, no observed drug effect on the number of lymphocytes and even measurable FA concentrations above the LLQL should be expected. Due to variability and due to the limited size of dosage groups, it was assumed that the minimum detectable effect of the drug on the number of NK cells would be a reduction of at least 10%. At doses of 0.01 mg/kg IV and 0.03 mg/kg sc, NK cells were predicted to be depleted to levels below the remaining 90% of baseline.

[00297] At an IV dose of 0.3 mg/kg, we predicted depletion of the NK cell population to the remaining 17% of baseline within 3 hours after the end of the infusion and recovery to more than 50% after 11 days (Fig. 10 ). At the same dose, the model predicts that the B cell population is maximally depleted to 67% of baseline after 2.5 days, and the T cell population is immediately depleted to 86% of baseline. For the same dose of 0.3 mg/kg subcutaneously, the model predicted a smaller and longer maximum depletion (minimum relative to baseline: NK cells - 37%, B cells - 74%, T cells - 94%).

[00298] These in vitro and in vivo preclinical studies demonstrate that the macaque is a suitable animal model for studying the pharmacology of AB79. Data on NK-, B-, and T-cell numbers based on multiple selected samples from eight studies in macaques at different doses and dosing regimens provide a rich source of data for a broad and quantitative understanding of the relationships between AB79 dose, exposure, and cell population depletion. The generated PK and PKPD population models reliably describe the data obtained and provide a powerful tool for predicting exposure and depletion of the lymphocyte population not only for future studies in macaques, but also for clinical trials in humans.

[00299] The first human trial (PTL) was conducted using a single dose escalation in healthy volunteers (www.clinicaltrials.gov: NCT02219256) (FIG. 11). The target pharmacological effect of AB79 is the depletion of the population of activated lymphocytes. Excessive and prolonged depletion of the lymphocyte population (enhanced pharmacology) can lead to immune system disorders that are unacceptable for patients or healthy study participants. Therefore, a safe initial IV dose of 0.0003 mg/kg was chosen for PKIL.

[00300] Evidence from macaque studies suggests that depletion of the NK cell population has been identified as the most sensitive biological effect. The results of modeling PK for NK cells helped to determine the minimum dose level - 0.01 mg/kg IV, at which the most sensitive pharmacological effect (depletion of the NK cell population) could be detected in humans. Emerging data from the PKIL study demonstrated that the overall pattern of dose-dependent and cell-type-specific debilitating effects of AB79 is in line with model-based predictions (manuscript in preparation). Apparently, AB79 is even more efficient than predicted. For example, at an IV dose of 0.03 mg/kg, the NK cell population in humans was depleted to less than 10% of baseline. The median minimum depletion rate in macaques at this dose was 20.0% (FIG. 8).

[00301] Three cytolytic anti-CD38 monoclonal antibodies (daratumumab, SAR650984, and MOR202) are in clinical trials for the treatment of multiple myeloma (van de Donk et al. (2016) Immunol. Rev. 270: 95-112). Daratumumab (Darzalex™ given as an intravenous infusion) has recently been approved for the treatment of multiple myeloma in the US and non-Hodgkin's lymphoma in Europe. Unlike AB79, daratumumab does not cross-react with macaque CD38. Thus, comparing our results for AB79 derived from studies in macaques with those for daratumumab is not possible. In addition, patients with multiple myeloma have a large number of CD38-positive malignant cells, which may require higher effective antibody concentrations for the treatment of this type of cancer (de Weers et al. (2011) J. Immunol. 186: 1840-1848).

[00302] Notably, however, daratumumab at a weekly IV dose of 16 mg/kg is approved for the treatment of multiple myeloma, although AB79 provides complete depletion of the peripheral NK cell population at a dose of about 1 mg/kg and B cells at dose of about 3 mg/kg (Fig. 8).

[00303] Despite a rich database based on 8 studies conducted on macaques, a number of limitations have been identified. AB79 effectively depleted the NK cell population even at the lowest dose studied of 0.03mg/kg. At these low doses, PK parameters rapidly fall below the limit of quantitation of the bioanalytical assay, preventing exposure-effect relationships from being determined at lower doses. In addition, during preclinical testing it was found that the maximum cell depletion occurs soon after the maximum drug concentration is reached, but the definition of the early phase of depletion is technically limited by the total number of samples and, potentially, non-specific cell depletion due to repeated blood sampling (blood sampling effect). ). The effect of blood sampling was observed as a transient pancytopenia characterized by depletion of those cell types that do not bind AB79 (eg, erythrocytes) and was not dose dependent, indicating that this effect is due to the loss of blood volume due to multiple blood draws, and not with any AB79-specific effects (Fig. 12). Therefore, the possibility of accurately estimating the model parameters, especially for the depletion of the NK cell population, was limited, and in order to achieve stable and reliable estimation results, the typical values of KIN and EMAX should be fixed.

[00304] The effect of AB79 on tissue plasma cells or plasma blasts cannot be measured. However, like plasma cells and plasma blasts, NK cells have high levels of CD38 on their surface, and the efficiency of cell depletion in a particular lymphocyte subset depended, at least in part, on CD38 expression levels. Therefore, the cytolytic effect of AB79 on plasmablasts and plasma cells may be comparable to the effect on NK cells. There is currently limited information on the long-term effects of AB79 treatment of macaques. Only a small subgroup of animals in the 13 week toxicology studies were tested in the convalescent group for a longer period of time, and most animals in all dose groups developed AALP (FIG. 3). In addition, baseline values and depletion profiles of different lymphocyte subpopulations had high intersubject variability. Therefore, the long-term effects of AB79 cannot be investigated in macaques and must be studied in humans.

[00305] With the availability of new data from human studies, it will be interesting to compare PK and PD data for humans and macaques in detail. Building a PK model based on data from human studies and comparing it to a model based on data from studies in macaques will improve the AB79 PBM model. Data obtained from studies in patients will provide insight into how AB79-mediated depletion of a population of B-lymphocyte-derived cells may be comparable between patients with RA and SLE, as well as between patients with multiple myeloma and healthy subjects. Investigation of subject or disease related factors that may influence cell depletion efficiency in addition to CD38 expression levels is also important and may lead to personalized treatment. In addition, a thorough head-to-head comparison of AB79 with daratumumab and/or other anti-CD38 antibodies in vitro and in vivo will provide valuable information on the pharmacology of anti-CD38 antibodies and their optimal use.

[00306] Rich pharmacological data and models of PK and PK-PD made it possible to characterize the exposure-effect relationship for the cynomolgus monkey. Model-based analysis of NK, B and T cells confirmed and quantified the observation that each of the blood lymphocyte subpopulations depletes antibody at a different rate and requires different time intervals to fill the model blood chamber. Models have proven to be an excellent tool for modeling PK and PD data in various dosing scenarios in preparation for clinical trials.

Example 2: Depletion of a population of CD38-positive cells with AB79 antibody

[00307] Table 6 demonstrates that AB79 mediates cell depletion by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Cell lines with increased CD38 expression were more susceptible to ADCC. ADCC was not observed in a human lymphoblast cell line that did not express CD38 (MV-4-11) or in a Chinese hamster ovary cell line transfected with CD157, a molecule closely related to CD38 (data not shown). Unlike other B cell selective therapies that target CD20 and do not directly deplete plasma blasts, which are low or negative for CD20 expression, CD38 is highly expressed on plasma blasts and plasma cells, making these cells a direct target. for AB79. In vitro studies with human blood cells and cell lines have demonstrated that binding of AB79 to CD38 does not result in PBMC cytokine activation, demonstrating that AB79 is not an agonist, as discussed below. Rather, AB79 mediated depletion of human B-lymphocyte-derived cells by ADCC and CDC, and cell lines overexpressing CD38 were, in most cases, more prone to cell lysis.

Table 6. AB79 mediates depletion of human cell lines of B-lymphocyte origin by ADCC and CDC cell line CD38
Number of antigenic sites of the receptor ADCC
EU _fifty ± SD (nM) KZTs
EU _fifty ± SD (nM) Molp8 623891 0.05±0.04
n=9 1.1 ± 1.0
n=7 Daudi 417874 0.03±0.04
n=4 1.6±0.4
n=3 NCI-H929 82341 0.14 ± 0.11
n=6 N/A RPMI-8226 98080 0.46 ± 0.53
n=8 N/A OPM2 54556 0.65 ± 0.64
n=4 N/A EU ₅₀ -50% effective concentration; N/A - not fulfilled; SD - standard deviation.

[00308] This is consistent with observational findings from studies in healthy cynomolgus monkeys where depletion efficiency correlated with CD38 expression level and AB79 dose level. The NK cell population, which express high levels of CD38, was depleted to a greater extent than CD20+ B cells and CD3+ T cells, which express less CD38 (FIG. 13). AB79 effectively suppressed human B cell secondary immunity responses to antigen in vivo in a mouse model of adoptive transfer (FIG. 14). Together, these data support the need for further study of AB79 in autoimmune diseases.

[00309] Human PBMCs were treated with AB79 antibody under several conditions and the release of inflammatory cytokines was measured. The cynomolgus macaque was used to demonstrate the relationship of cell type-specific depletion and dose of AB79 because AB79 cross-reacts with macaque CD38, whose CD38 protein is 91% identical to human CD38 protein. The second animal, adoptive transfer of human PMCs into mice, was used to determine if AB79 could target human antibody-producing cells.

AB79 binds CD38 and mediates ADCC and CDC

[00310] The number of receptor antigenic sites was determined using the "QIFIKIT®" reagent kit (DAKO, cat. no. K0078) using mouse anti-human CD38 antibody (clone HIT2) and calculated by converting the average fluorescence intensity (MFI) of stained samples into a quantitative result according to the calibration curve generated from the SIF of 5 populations of microbeads associated with a certain number of antibody molecules. The number of receptor antigenic sites was calculated by subtracting the CIF of the isotype control (mouse IgG1) from the CIF of the anti-CD38 antibody.

[00311] CSC was assessed by seeding cell lines at 10,000 cells/well and adding AB79, IgG control or medium. A 5-point dose-response curve (0.001-10 mg/mL) was plotted according to standard practice. Rabbit complement (2-15 µl; CedarLane Laboratories; Cat #: CL 3441) was added to each well except controls. The CytoTox-Glo reagent kit (Promega, G7571/G7573) was used to determine cytotoxicity by luminescence. Test groups: cells only; cells + complement; cells+IgG-control+complement; cells + AB79 + complement. %CSC calculation equation: %CSC=100 - ((REL (test) / REL (complement only)) X 100), where REL are relative units of luminescence.

[00312] ADCC was tested by seeding target cells ("C", cell lines) at 5000 cells/well with 50 ml of AB79, IgG control, Triton X-100 (1%; Sigma Chemical) or medium alone, and with 50 ml of effector ("E") human PBMC in the ratio of C:E cells from 1:25 to 1:50. A dose-response curve was plotted using 9 antibody concentration points (0.000001-100 nM) according to standard practice. Experimental lysis=PBMC+cell line+antibody. Spontaneous lysis=PBMC+cell line without antibody. Maximum lysis=cell line+Triton X-100. Cytotoxicity was assessed using the CytoTox-Glo™ Cytotoxicity Luminescence assay kit (Promega).

AB79 has no agonistic activity

[00313] The ability of AB79 to induce cytokine production in antibody-treated human PBMCs was compared to a negative IgG1 isotype control and a positive control, PHA, anti-CD3 (clone OKT3) or anti-CD52 (Campath) antibodies (FIGS. 15 and 16).

[00314] Soluble preparation AB79 did not increase IL-6 levels (mean ± SD) in PBMCs collected from 4 different subjects after 24 hours of incubation compared to IgG1 isotype control. PHA resulted in an increase in cytokine levels in all subjects, demonstrating that the cells have the ability to produce IL-6 (FIG. 15). Similar results were observed with PBMC stimulation for 48 hours and when analyzed for IL-2, IL-4, IL-10, GM-CSF, IFN-γ, and TNF-α (data not shown).

[00315] The method by which an antibody is presented to a cell can influence the antibody's effect of ligand capture and cellular response (Stebbings et al. (2007) J. Immunol. 179: 3325-3331). Stebbings et al. demonstrated that the maximum cellular response (release of cytokines) to an agonistic antibody occurs when the antibody is highly concentrated and bound to the surface of the well - for example, when the antibody is added to the well in solution and the liquid is allowed to evaporate (dry binding), compared to the antibody, which binds to the surface of the well in solution (wet binding) or to the antibody added directly to the PBMC suspension (solution binding) (FIG. 16A). The AB79 preparation did not stimulate cytokine production with any of these approaches (FIG. 16B).

[00316] AB79 (100 mg/mL) did not stimulate IL-2, -4, -6, -8, -10, GM-CSF, IFN-γ, or TNF-α in any of the experimental conditions after 24 hours . AB79 did not induce IL-10 or GM-CSF, but both cytokines were induced by an anti-CD3 antibody (not demonstrated; all values except anti-CD3 were below the LEL). IL-8 was produced constitutively in the PBMC and was not altered by any of the treatments (data not shown) (Table 7).

Table 7. AB79 and stimulation of cytokine production Presentation of antibodies Without stimulation Isotype control AB79 Anti-CD52 FGA Anti-CD3 IL-2 Binding in solution NPPO NPPO NPPO NPPO 14953 ± 3117 N/A wet bondage NPPO NPPO NPPO NPPO N/A 395.0±64.8 Dry bonding NPPO NPPO NPPO NPPO N/A 167.9 ± 90.1 IL-4 Binding in solution 7.3±1.4 4.1±0.8 6.4 ± 3.2 6.9±4.0 50.4 ± 8.9 N/A wet bondage 5.3±0.08 3.8±1.5 6.4±0.6 8.5 ± 3.7 N/A 17.8±2.4 Dry bonding 4.4 ± 1.8 7.8 ± 2.7 9.1 ± 2.6 10.5±2.1 N/A 16.2 ± 3.1 IL-6 Binding in solution 325.1 ± 65.3 236.0 ± 98.9 170.5 202.0±48.0 18880±0 N/A wet bondage 216.8 ± 95.1 191.2 ± 47.0 194.6 ± 66.0 207.2 ± 60.2 N/A 902.7 ± 114.1 Dry bonding 165.0±79.4 369 ± 143 465.8 ± 230 811.1 ± 473.7 N/A 500±17 IFN-γ Binding in solution 1190.2 ± 117.5 857.1 ± 311.7 770.1 ± 203.6 1116.9 ± 330.8 15857 ± 4614.8 N/A wet bondage 1052.8 ± 385.8 657.2 ± 222.6 993.4 ± 198.1 1138.9 ± 339.1 N/A 5674.6 ± 564.7 Dry bonding 768.1 ± 110.0 1583.1 ± 418.6 1927.6 ± 517.6 1827.0±281.5 N/A 3513.2 ± 708.3 TNF-α Binding in solution 28.71 ± 8.1 11.6 ± 7.1 59.6 ± 90.1 79.9 ± 18.2 9270.0±0 N/A wet bondage 16.5 ± 3.4 16.1 ± 3.9 14.8 ± 8.8 166.1 ± 21.7 N/A 2123.3 ± 239.7 Dry bonding 16.2±7.4 919.2 ± 77.4 745.1 ± 141 984.4 ± 317.0 N/A 2231.6 ± 687

To measure the concentrations of IL-2, -4, -6, -8, -10, GM-CSF, IFN-γ and TNF-α, a multiplex analysis of cytokines was carried out in accordance with the manufacturer's instructions (reagent kit "Bio-Plex ProTM Human Cytokine Standard 8-Plex"). Abbreviations: LLQL, lower limit of quantitation; N/A - not fulfilled; PHA - phytohemagglutinin; PBMCs are peripheral blood mononuclear cells.

AB79 depletes the population of CD38+ cells

[00317] AB79 binds CD38 with high affinity and mediates CDC and ADCC. AB79 is not an agonist and does not induce cytokine release from human PBMC. AB79 binds both human and cynomolgus monkey CD38. Lymphocytes of both species had similar cell-specific CD38 expression patterns with a NK cell > B cell > T cell hierarchy based on mean fluorescence intensity when stained for AB79. Treatment with AB79 resulted in depletion of the macaque lymphocyte population in a reversible, cell-specific and dose-dependent manner. AB79 effectively suppressed secondary immunity responses in a mouse model of adoptive transfer.

Example 3 Evaluation of AB79 Binding to Human and Cynomolgus Macaque Erythrocytes and Platelets

[00318] AB79, a fully human, high-affinity, non-agonistic anti-human CD38 isotype IgG1 monoclonal antibody, was tested to determine its binding to human or cynomolgus macaque erythrocytes or platelets. Thirty blood samples from healthy human volunteers and thirty whole blood samples from healthy Javanese were tested for binding to AB79 compared to palivizumab, a control monoclonal antibody of the same isotype that has different specificity from AB79 and does not bind to red blood cells or platelets.

Methods

[00319] Blood samples from 30 healthy human subjects (15 males and 15 females) for platelet testing, from 25 males and 5 females for erythrocyte testing, and 30 samples from Chinese cynomolgus monkeys (15 males and 15 females) were purchased from Bioreclamation Company (Long Island, New York). Whole blood was collected in sodium citrate tubes and delivered overnight at ambient temperature.

[00320] To evaluate AB79 binding to platelets, 50 µl of whole blood was stained with macaque anti-human CD61 monoclonal cross-reactive antibody FITC-fluorescein isothiocyanate (FITC) conjugated (BD Biosciences; catalog #: 555753) for identification platelets, in combination with Alexa Fluor 647 (AF647)-conjugated AB79 antibody or isotype control, and AF647-conjugated palivizumab (MedImmune; catalog #: 60574-4113-1), a humanized monoclonal antibody that is specific for epitope A in the antigenic region of the respiratory syncytial virus (RSV) F protein, i. an antigen that is not present on red blood cells or platelets. RBC lysis was performed after staining and samples were analyzed using a BD Canto flow cytometer. CD61+ platelets were isolated on cytometry graphs for analysis.

[00321] To assess the binding of AB79 to erythrocytes, 50 μl of whole blood was stained with AF647-conjugated AB79 antibody or isotype control, and AF647-conjugated palivizumab. In some experiments, a cross-reactive anti-human CD45 antibody conjugated to PerCP-peridinine-chlorophyll protein (PerCP) (BD Biosciences; catalog #: 552724) was used to stain lymphocytes to demonstrate the binding of AB79 to a subpopulation of lymphocytes, such as this has been observed in previous studies. Samples were analyzed using a BD Canto flow cytometer. Data were expressed as the mean fluorescence intensity (MFI) of the AB79 antibody or isotype control.

Results and discussion

[00322] The ability of AB79 to bind to erythrocytes from thirty healthy human volunteers and thirty healthy Javanese was assessed by flow cytometry. RBC staining for AB79 did not exceed the level of isotype control antibody staining in any of the human blood samples (FIG. 17 and Table 8) or Javanese blood samples (FIG. 18 and Table 8). No measurable binding was observed in either species, and no difference was observed in the ratio of FIF for AB79/isotype control between human and Javanese.

Table 8. Mean fluorescence intensity values of the AB79/isotype control ratio for erythrocytes Donor No. Human Javanese macaque one 1.047 1.025 2 1.018 0.904 3 1.006 0.912 four 0.867 0.878 5 0.800 0.805 6 0.937 0.980 7 0.900 0.893 eight 0.920 0.861 9 0.896 0.839 ten 0.958 0.868 eleven 0.896 0.863 12 0.950 0.866 13 1.016 1.027 fourteen 0.946 0.989 fifteen 0.985 0.969 16 0.829 0.990 eleven 0.861 0.985 eighteen 0.903 1.022 19 0.914 0.915 twenty 0.914 0.911 21 0.813 0.882 22 0.859 0.888 23 0.947 0.960 24 0.917 0.940 25 0.976 0.948 26 0.933 0.931 27 0.889 0.967 28 0.938 0.949 29 0.976 0.941 thirty 0.934 0.951 Average 0.928 0.929 Art. off 0.056 0.057

The ability of AB79 to bind to CD61+ platelets from thirty healthy human volunteers and thirty healthy Javanese (donors 1-15 were male; donors 16-30 were female) was assessed by flow cytometry. Platelet staining for AB79 did not exceed the level of isotype control antibody staining in any of the human blood samples (FIG. 19 and Table 9) or Javanese blood samples (FIG. 20 and Table 9). No measurable binding was observed in either species, and no difference was observed in the ratio of FIF for AB79/isotype control between human and Javanese.

Table 9 Mean Fluorescence Intensity AB79/Isotype Control Ratios for Platelets Donor No. Human Javanese macaque one 1.46 0.96 2 1.10 0.88 3 1.76 0.90 four 1.16 0.88 5 1.21 1.28 6 1.53 1.19 7 2.14 0.85 eight 1.14 0.78 9 1.14 0.97 ten 0.53 1.37 eleven 0.38 0.63 12 0.60 0.98 13 1.06 0.71 fourteen 1.33 0.93 fifteen 0.78 1.06 16 1.06 1.03 17 0.95 0.83 eighteen 1.25 0.80 19 1.09 1.55 twenty 0.89 1.28 21 0.95 1.61 22 1.24 1.24 23 0.36 0.99 24 1.34 1.40 25 0.53 1.04 26 0.91 1.29 27 0.85 1.14 28 0.68 1.05 29 1.03 1.46 thirty 1.13 2.28 Average 1.05 1.11 Art. off 0.39 0.33

[00323] As a positive control for AB79 staining, AB79 staining was measured in a portion of the blood samples. Due to the high prevalence of erythrocytes in the blood, 200,000 cytometric events were analyzed, and a small population of CD45+ lymphocytes was taken for further evaluation. For isotype controls, no binding was found; however, the AB79 antibody bound to a small population of CD45+ lymphocytes (FIG. 21). This observation confirmed that AB79 has the ability to stain blood cells under conditions where there is no measurable binding of AB79 to erythrocytes or platelets.

Example 4: Assessing AB79 Binding to Human and Cynomolgus Macaque RBCs and Platelets Using High Sensitivity Flow Cytometry

[00324] To further assess the binding of AB79 to human or cynomolgus macaque erythrocytes, a highly sensitive flow cytometry method was developed to detect low levels of CD38 expression on erythrocytes in case the previous assay may not have been sufficiently sensitive. Blood samples from 4 healthy human volunteers were incubated with fluorescently labeled AB79 or fluorescently labeled daratumumab, another anti-CD38 antibody known to bind to CD38 on erythrocytes. Each sample was preincubated with the appropriate unlabeled drug to block CD38 binding to fluorescently labeled drugs; these samples served as negative controls for the assay. Anti-CD45 and CD235a antibodies were added to the RBC identification samples and then analyzed on a flow cytometer. Fluorescently labeled daratumumab was used as a positive control. The results demonstrated that AB79 and daratumumab bind to human erythrocytes obtained from healthy donors.

Methods

[00325] Human blood samples from four healthy donors (Millennium Blood Donor Program) were collected according to company protocol. Briefly, peripheral blood samples for erythrocyte binding were collected in sodium heparin tubes and for lymphocyte staining, peripheral blood samples were collected in BD Vacutainer® CPT™ tubes (BD Biosciences, Franklin Lakes, NJ, USA). A separate tube was used to collect peripheral blood mononuclear cells (PBMCs) to confirm binding of fluorescently labeled AB79 and fluorescently labeled daratumumab to CD38+ lymphocytes. In both erythrocyte binding and lymphocyte staining experiments, peripheral blood samples were stored at room temperature and taken to the experiment within 2 hours of collection to maintain cell viability. For erythrocyte binding, peripheral blood samples were first diluted 1:10,000 in staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) to reduce the high concentration of erythrocytes present in the sample. Since the normal range of red blood cells in healthy human blood samples is approximately 5 million cells per microliter, the samples had to be diluted substantially to obtain an acceptable number of red blood cells for staining and analysis by flow cytometry. Samples were then transferred to a 96-well V-bottom plate and incubated overnight at 4°C on a soft shaker with unlabeled AB79 (500 µg/mL), unlabeled daratumumab (500 µg/mL), or BD buffer alone (no drug). ), all at 25 µl/well. A plate shaker was used to prevent erythrocyte sedimentation during the incubation period. Peripheral blood samples were preincubated with unlabeled drugs in order to block CD38 antigenic sites on the surface of erythrocytes; these samples served as negative controls for the assay. After this incubation period, clinically significant concentrations of biotin-streptavidin-BV421 daratumumab (0; 0.1; 1; 10 and 100 µg/mL) or biotin-streptavidin-BV421 AB79 (0; 0.1; 1; 10 and 100 µg/mL) ml) was added to the samples for 3 hours at room temperature on a soft shaker. The samples were then washed several times with BD buffer and stained with cell surface markers CD45 and CD235a to identify erythrocytes (erythrocytes: CD45-CD235a+) and streptavidin-BV421 complex to bind biotinylated antibodies. The use of biotin-streptavidin makes it possible to enhance a possible signal from a low level of CD38 expression on erythrocytes. The fluorescent dye BV421 was chosen because it is one of the brightest commercially available fluorophores and uses the violet laser of the flow cytometer, thereby minimizing the level of spectral overlap with other channels/markers from the staining panel. In general, this approach to signal amplification makes it possible to detect molecules on the cell surface that would otherwise be lost in the noise performance of the instrument. Samples were then washed and analyzed for BD FACSCanto™ II (BD Biosciences, Franklin Lakes, NJ, USA). The target cell collection level was set at 10,000 CD235a+ cytometric events. For lymphocyte binding, peripheral blood from the same healthy donors, collected in BD Vacutainer® CPT™ tubes, was centrifuged and isolated peripheral blood mononuclear cells (PBMCs) using standard techniques. Cells were washed, stained and processed as described for erythrocyte experiments.

[00326] To obtain biotinylated antibodies, AB79 (21.4 mg/ml, Takeda, CA, USA) and daratumumab (20 mg/ml, Janssen Biotech, Horsham, PA, USA) were purified simultaneously on the same day using a commercial kit " Protein A column kit” (Abcam, Cambridge, UK) to remove substances that could potentially interfere with the biotinylation procedure. The protein concentration in the purified products was determined by the A280/260 ratio and equal amounts of each antibody protein were conjugated to biotin using the Lightning Link Rapid Biotin Conjugation kit (Innova Biosciences, Cambridge, UK). At the end of the procedure, the protein concentration was again measured by the ratio A280/260. Both antibodies were conjugated to commercial polystyrene microspheres, which have the ability to bind antibodies of any isotype, or to inert microbeads (negative microbeads). After mixing microbeads with biotin-strepavidin-BV421-AB79 complex or biotin-strepavidin-BV421-daratumumab complex, each of the two resulting reagents provides a distinct positive control with a high signal for the respective negative population, which can be used to evaluate the mean fluorescence (MIF) of each analyte. antibodies by flow cytometry. Samples were analyzed on a BD Biosciences FACSCANTO™ II instrument using BD Biosciences FACSDiva™ software (BD Biosciences, Franklin Lakes, NJ, USA). Raw data files were transferred to a secure server and then analyzed offline using FlowJo® software version 10 (FlowJo, LLC; Ashland, Oregon, USA). To identify erythrocytes, CD235a-positive cells were first isolated on cytometry graphs. Next, the CIF geometry and the percentage of positive biotin-strepavidin-BV421-AB79 and biotin-strepavidin-BV421-daratumumab cytometric events for the isolated cells were determined and a corresponding histogram plotted (FIG. 22). These data were compared with isotype controls (samples preincubated with unlabeled AB79 or unlabeled daratumumab and then incubated with their respective labeled biotin-strepavidin-BV421 drugs). To identify lymphocytes, debris was first excluded based on forward and side scatter (FSC-A vs. SSC-A). Then, the CD45-positive population was isolated on the cytometry graph and the CIF and the percentage of positive biotin-strepavidin-BV421-AB79 and biotin-strepavidin-BV421-daratumumab events for the isolated cells were determined, and the corresponding histogram was plotted (Fig. 22). These data were compared with isotype controls (samples preincubated with unlabeled AB79 or unlabeled daratumumab and then incubated with their respective labeled biotin-strepavidin-BV421 drugs).

Table 10. Summary of results from RBC binding studies with AB79 and daratumumab (geometric mean fluorescence) AB79 biotin-streptavidin-BV421 Conc.
(µg/ml) Donor 1 Donor 2 Donor 3 Donor 4 Average SO 0 138 155 202 284 195 65.37 0.1 175 201 207 355 235 81.53 one 193 224 282 392 273 87.64 ten 196 205 557 521 370 196.02 100 143 139 533 445 315 204.11 Unlabeled AB79 and AB79 biotin-streptavidin-BV421 Conc.
(µg/ml) Donor 1 Donor 2 Donor 3 Donor 4 Average SO 0 139 143 220 BUT 167 45.65 0.1 133 137 198 BUT 156 36.43 one 132 142 223 BUT 166 49.90 ten 136 148 421 BUT 235 161.19 100 133 140 447 BUT 240 179.30 Daratumumab biotin-streptavidin-BV421 Conc.
(µg/ml) Donor 1 Donor 2 Donor 3 Donor 4 Average SO 0 150 158 182 274 191 56.98 0.1 227 297 215 411 288 89.92 one 304 394 387 527 403 92.22 ten 317 352 556 599 456 142.11 100 162 190 643 493 372 234.74 Unlabeled daratumumab and daratumumab biotin-streptavidin-BV421 Conc.
(µg/ml) Donor 1 Donor 2 Donor 3 Donor 4 Average SO 0 143 164 180 BUT 162 18.56 0.1 133 146 191 BUT 157 30.44 one 135 147 216 BUT 166 43.71 ten 155 170 431 BUT 252 155.20 100 133 142 BUT BUT 138 6.36

N/O=not determined; SD=standard deviation

Results and discussion

[00327] The amount of biotin on AB79 and daratumumab was determined using highly sensitive flow cytometry. As the results show in FIG. 23, biotin-strepavidin-BV421 daratumumab binds 1.6-2.0 times more antibody-bound microbeads than biotin-strepavidin-BV421 AB79. Therefore, biotin-strepavidin-BV421 daratumumab is a "brighter" antibody compared to biotin-strepavidin-BV421 AB79. Therefore, comparisons of SIF scores should be calibrated to account for this difference in staining intensity.

[00328] Confirmation of the specificity of biotin labeled drugs was an important step to ensure that the parameters observed were the result of specific binding to the target protein. Competitive assay is a common way to test the specificity of antibodies. Flow cytometry was used for this competitive analysis. Briefly, peripheral blood samples from healthy volunteers were pre-incubated with unlabeled AB79 or unlabeled daratumumab and then stained with biotinylated AB79 or biotinylated daratumumab, respectively. It was expected that samples preincubated with unlabeled drug would block the binding of biotinylated drugs. As shown in FIG. 24, binding of biotinylated AB79 and biotinylated daratumumab can be blocked in peripheral blood lymphocytes if unlabeled drugs are used.

[00329] The percentage binding of biotin-strepavidin-BV421 AB79 and biotin-strepavidin-BV421 daratumumab to erythrocytes was determined from four peripheral blood samples from healthy volunteers. As shown in FIG. 25 and 26, AB79 bound CD38-expressing erythrocytes in a dose-dependent manner in all four donors studied. Peak erythrocyte binding levels varied between healthy volunteers and were between 1 and 10 µg/mL for both drugs. At 10 and 100 µg/mL, both biotin-strepavidin-BV421 AB79 antibodies and biotin-streptavidin-BV421 daratumumab had lower erythrocyte levels in 3 of 4 donors studied. There are several factors that may explain the reduced level of drug binding to CD38, including increased erythrocyte hemolysis or flipping of the catalytic domain from the outside of the cell to the inside of the cell (Yoshiga et al. (2008) Int. J. Mol. Med. 22: 369-374 ). In addition, daratumumab reduced CD38 expression levels, at least in part, through trogocytosis (Cole et al. (2018) Arthritis Res. Ther. 20 (1): 85); CD38 complexes and the accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes (Kraan et al. (1999) Rheumatology (Oxford) 38 (11): 1074-1080). In one donor (donor 3), erythrocyte levels increased with increasing amount of drug, possibly as a result of greater surface expression of CD38 on the surface of erythrocytes. More experiments are needed to better understand the reduction in drug binding at the highest concentration tested.

[00330] RBC binding profiles were compared with AB79 and daratumumab. As shown in Table 10 and in FIG. 27 and 28, there appears to be a difference in the amount of erythrocyte binding (ie, in SIF) between drugs in 3 of the 4 donors studied; however, this difference may be due to differences in biotin levels on each of the antibodies, since daratumumab contains 1.6-2.0 times more biotin than AB79. An alternative assay that controls the potential difference in fluorescent labeling of antibodies is to compare the concentration with the binding profile of each antibody, and a useful indicator is the concentration at which maximum binding occurs (i.e. maximum specific antigen binding (Bmax)). The Bmax is the same for both antibodies in 3 out of 4 donors (for example, 1 μg/ml for donor 1). Taken together, these data indicate that both antibodies bind with the same affinity within the current assay resolution limit of 10. In conclusion, in this assay, both AB79 and daratumumab bound RBCs with affinities that were within from each other; There was no 10-fold or greater difference in the binding affinity of these antibodies to erythrocytes in this assay system.

Example 5: Assessment of hemolysis by AB79 antibody of human or macaque erythrocytes in vitro

[00331] Fresh whole blood from healthy human volunteers and cynomolgus monkeys, five (n=5) subjects of each species, was analyzed for in vitro hemolysis in response to in vitro treatment with AB79 antibody (27.3 mg/mL; Takeda, California, USA), human IgG1 isotype control (7.14 mg/mL; Bio X Cell) and daratumumab (20 mg/mL; Janssen Biotech, Horsham, PA, USA). The dose-dependent effect for experimental samples was studied at 0; 0.03; 0.08; 0.25; 0.74; 2.2; 6.6 and 20 µg/ml. The semi-log dilution of saponin was evaluated starting with the highest concentration (1%) saponin solution used as an engineering positive control for blood reactivity. To measure acute hemolysis, treatment was carried out for 1 hour at 37° C., 5% CO ₂ . The absorbance was measured at a wavelength of 540 nm using a spectrophotometer and the percentage of hemolysis was calculated as follows:

[00332] The hemolytic index was scored as follows (hemolytic index=degree of hemolysis): 0-2=no hemolysis; 2-5=weak hemolysis; >5=hemolysis.

Results: Erythrocytes from all individuals of each species, human and Javanese, responded with acute in vitro hemolysis in response to a dilution series of 1% saponin solution with a hemolytic index greater than 5. AB79, daratumumab and human IgG1 isotype control did not cause measurable hemolysis in any samples. erythrocytes from both species, and the hemolytic index was zero (FIGS. 29 and 30).

Example 6: AB79 score in a cynomolgus macaque model of collagen-induced arthritis

[00333] The expression of CD38 ectoenzyme is increased on lymphocytes in response to antigen challenge, and it is hypothesized that targeting these activated lymphocytes may attenuate pathological activity in autoimmune diseases. The cynomolgus macaque is a suitable model for evaluating the potential effects of CD38 targeting in humans as this species exhibits similar CD38 expression profiles and the human anti-CD38 antibody AB79 binds to macaque CD38 with an affinity (EC ₅₀ =4.5 nM) that allows pharmacological intervention. Therefore, the potential activity of AB79 was investigated in the cynomolgus macaque collagen-induced arthritis (CIA) model. Prophylactic administration of the AB79 antibody (3 mg/kg iv per week) was well tolerated and prevented the development of arthritis, in contrast to vehicle-treated control animals, which developed progressive disease with radiographic damage and clinical deterioration during the study. Therapeutic exposure to AB79 antibody (3 mg/kg iv per week) in arthritis monkeys was also well tolerated and reduced disease progression and symptoms. Clinical scores for arthritis and joint swelling were significantly lower than for vehicle controls and were accompanied by decreases in blood levels of CRP, ALP, NK-, B-, and T-cells. Histopathological, morphometric, and radiological studies revealed significantly less joint damage in animals treated prophylactically with AB79 antibody compared to animals treated with vehicle, and significantly (p < 0.05) less damage in animals treated with therapeutic treatment with AB79 antibody or dexamethasone ( daily at 0.1 mg/kg po), illustrating the potential effect on disease progression. In conclusion, these data indicate that depletion of the CD38-expressing cell population may be a therapeutic alternative in the treatment of autoimmune diseases without the negative effects of steroids.

Methods and materials

Antibodies

[00334] The AB79 formulation was available locally. Fluorochrome-conjugated anti-mouse IgG was purchased from Jackson Immunoresearch Laboratories (West Grove, PA). The pharmaceutical buffer was purchased from BD Biosciences (San Jose, CA). Fluorochrome-conjugated antibodies to macaque proteins were purchased from various sources: CD20 from BD Biosciences (San Jose, CA); mouse anti-CD3 antibody from eBioscience (San Diego, CA); mouse anti-CD16 antibody from Miltenyi Biotech (Auburn, CA); mouse anti-CD4 and CD8 antibodies from R&D Systems. Non-conjugated anti-AB79 antibodies and a humanized control antibody with different antigenic specificity from AB79 but with the same IgG1 Fc fragment were available locally, in addition to Alexa Fluor 647-conjugated AB79. The primary antibodies for macaque tissue cross-reactivity studies were rabbit anti-AB79 antibodies (generated in our laboratory) and a negative control for human IgG1 (Millipore Bioscience Research Reagents, Temecula, CA).

Immunohistochemical study of tissues

[00335] The CD38 antigen expression profile was compared in 15 different tissue types obtained from healthy human and cynomolgus monkey donors using immunohistochemical methods. The suitability of each tissue for CD38 detection was verified using a positive control antibody against the related CD31 transmembrane receptor (Dako North America, Inc.). Sections (5 µm) were cut from fresh frozen tissue samples embedded in OCT Compound (Sakura Finetek USA, Inc., Torrance, CA) and fixed in acetone for 10 minutes at room temperature. Immediately before staining, slides were fixed for 10 seconds in 10% neutral buffered formalin. Cryosections fixed with acetone or formalin were washed twice in phosphate buffered saline (PBS) and incubated for 20 minutes with a protein blocker (PBS; 0.5% casein; 5% human gamma globulins; 0.02% goat IgG; 1 mg/ml of heat-aggregated human IgG) designed to reduce non-specific binding. Unconjugated AB79 or a negative control to human IgG1 (Millipore Bioscience Research Reagents) were applied to sections at a concentration of 5 or 25 μg/ml and incubated at room temperature for 1 hour. Then the micropreparations were washed twice in PBS and these primary reagents were detected by an indirect immunoperoxidase method. Then a secondary antibody, rabbit anti-AB79, was applied at a concentration of 5 μg/ml for 30 minutes and washed twice in PBS. Endogenous peroxidase was blocked by incubating the micropreparations with the peroxidase solution provided in the Dako EnVision+ kit for 5 minutes and then washing twice in PBS. The slides were then treated for 30 minutes with peroxidase-labeled goat anti-rabbit IgG polymer supplied in the Dako EnVision+ kit, washed twice in PBS, and treated for 8 minutes with the substrate-chromogen solution (DAB+) supplied in the Dako EnVision+ kit. . All slides were washed with tap water, stained with hematoxylin, washed, stained blue with saturated lithium carbonate, washed, dehydrated by passing through alcohols, cleared in xylene, and covered with coverslips according to standard procedures. Staining intensity was assessed semi-quantitatively in a blinded analysis performed by an American College of Veterinary Pathologist (ACVP) certified pathologist.

AB79 binds to recombinant CD38

[00336] For cell surface binding studies of anti-CD38 antibodies, Chinese Hamster Ovary K1 (CHO-K1) cells stably expressing human, mouse, or macaque CD38 were generated. CHO-K1 cells (Lonza, USA) were transfected with full-length cDNA clones encoding human, mouse, or cynomolgus macaque CD38 (Origene Technologies, Rockville, MD). After selection, pools were sorted by flow cytometry and clones expressing the highest levels of human, mouse or cynomolgus macaque CD38 (clones with an average fluorescence intensity (MFI) of 85-100% of maximum) were used for binding studies. 200,000 cells per well were plated in a 96-well round bottom plate and stained with 66.7 nM Alexa Fluor® 488 direct conjugated antibody in 50 µl of cytometry buffer (1% ABS in PBS) on ice for 30 minutes or more. up to 1 hour. Cells were washed 3-4 times in a final volume of 200 to 250 µl of cytometric buffer. The final cell pellet was resuspended in 100 μl of cytometry buffer containing 1% paraformaldehyde. Samples were evaluated on FACS Canto II HTS (BD Biosciences) and analyzed using Flojo software (Tree Star, USA).

Flow cytometry of cell lines and whole blood

[00337] For cell line staining, cells were resuspended at 2 x 10 ⁶ /ml in cytometry buffer (5% fetal bovine serum and 0.05% sodium azide in D-PBS (Dulbecco's phosphate buffer in saline without calcium and magnesium)) (VWR, West Chester, PA) and 200 μl samples were stained with the respective monoclonal antibodies at 4° C. for 30 minutes. Samples were washed with cytometric buffer and analyzed by flow cytometry (FACSCalibur, BD). To stain macaque whole blood, 200 µl samples were stained with the appropriate monoclonal antibody at 4° C. for 30 minutes. The erythrocytes were lysed with the BD FACS lysing solution, and then the samples were washed with cytometric buffer and analyzed by flow cytometry. Saturating antibody concentrations were used in all cases, and in many cases up to four antibodies were used per sample.

AB79 activity in cynomolgus macaque whole blood

[00338] Whole blood of cynomolgus monkeys was purchased from Charles River Laboratories (Wilmington, Massachusetts, USA). For binding studies, peripheral whole blood of cynomolgus monkeys mixed with anticoagulant (total volume of each sample - 100 μl) was incubated with increasing concentrations of AB79 antibody (43-690 nM) for 30 minutes at room temperature. Binding of AB79 to cells was analyzed using phycoerythrin-labeled goat anti-human IgG Fc (Thermo Fisher Scientific). After antibody binding, erythrocytes were lysed by high productivity lysis without fixatives (Thermo Fisher Scientific). The cells were then washed twice with magnetic field activated cell sorting buffer (MACS buffer) containing 0.5% ABS (Miltenyi Biotec). Cell staining results were analyzed by flow cytometry (on an Attune NxT Acoustic Focusing Cytometer) using FlowJo software.

[00339] For cytolysis assay, 90 μl of whole blood was plated into each well of a 96-well U-bottomed plate. Immediately after seeding, whole blood was stimulated with AB79 at concentrations of 0.69; 2.06; 6.17; 18.52; 55.56; 166.67 and 500 nM, or PBS control for 6, 24 and 48 hours. At each time point, blood stimulated with a control or test molecule from a 96-well U-bottom plate was transferred to a deep well plate for erythrocyte lysis. After lysis, cells were resuspended in staining buffer with CD16-BV605, CD56-FE, and CD38-FITC antibodies to stain surface markers. Cells were protected from light and incubated at 4 ^° C. for 20 minutes. After incubation, cells were centrifuged at 350 x g at room temperature for 5 minutes and washed with 1x Annexin V binding buffer. Cells were resuspended in 1x Annexin V binding buffer containing Annexin V conjugated to Alexa Fluor 647 at room temperature in within 15 minutes. Stained cells were analyzed using a BD FACSCelesta flow cytometer and data was recorded using BD FACSDiva version 8.0.1 software. Graphs of NK cell viability parameters at each tested concentration and IC50 values of NK cell population depletion induced by AB79 antibody at each time point were plotted and analyzed using the GraphPad Prism 7.04 software.

Dose range studies in healthy monkeys

[00340] In a series of studies, healthy, purpose-bred, previously unexperimented cynomolgus monkeys received a vehicle control, 0.03; 0.1; 0.3 and 1 mg/kg compound AB79 weekly, or 3; 30 or 80 mg/kg every two weeks by 20-minute intravenous infusion (Charles River Laboratories). In all studies, animals were assessed for changes in clinical signs (animal cage observations twice daily, post-dose observations, weekly detailed examinations, food intake observations, and weekly weighing). Blood samples were collected before and after dosing in all studies to evaluate pharmacokinetics, pharmacodynamics, and primate antibodies against human antigens (APHA). Studies were conducted on cynomolgus monkeys in accordance with the Standard Operating Procedures (SOP) of the test facility (Charles River Laboratories), which adheres to the standards set forth in the USDA Animal Welfare Act (US Code of Federal Regulations). , Section 9, Parts 1, 2, and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press).

Method for bioanalytical determination of AB79 concentrations in blood serum

[00341] The concentration of AB79 in the blood serum of cynomolgus monkey was measured using the method of enzyme-linked immunosorbent assay (ELISA). This method is an indirect ELISA miniaturized for use with 96-well plates. The plate was coated with mouse anti-idiotype antibody against AB79. Blanks, standards, and quality control (QC) samples containing AB79 at various concentrations, as well as macaque blood serum samples, were added to a microtiter plate and incubated for 55-65 minutes at room temperature. After washing the microtiter plate, detection antibody (peroxidase-conjugated high affinity mouse anti-human IgG) was added and the plate was incubated for an additional 55-65 minutes. The plate was washed again and tetramethylbenzidine (TMB) was added to the wells to form the chromophore and color development was stopped by adding stop solution (dvs sulfuric acid). Absorbance at 450 nm was measured and AB79 concentrations were calculated using a 4-parameter exponential weighted (1/y ² ) standard calibration curve.

Method for the bioanalytical determination of anti-AB79 antibodies

[00342] Screening for anti-AB79 antibodies in cynomolgus macaque serum was performed using the enhanced electrochemiluminescence (ECL) method. The plan of the experiment using this qualitative ECL method was as follows. Undiluted cynomolgus monkey serum samples were incubated with 300 mM acetic acid. Acid dissociated samples were incubated in a mixture of biotinylated AB79, AB79 labeled with SULFO-TAG™, and 1.5 M Trizma® base reagent to neutralize the acid and form an immune complex. This complex was then added to a streptavidin-coated plate manufactured by MSD and left for the coupling reaction. After washing, the complex was detected by adding the “MSD read buffer T” reagent to the plate and subsequent excitation of the SULFO-TAG™ label through the electrochemical reaction [Ru(bpy)3] ²⁺ with the formation of luminescence, which was measured on an MSD Sector 6000 analyzer. Luminescence intensity correlates with the level of macaque anti-AB79 antibodies present in the serum of individual samples. The minimum required dilution (MTR) of cynomolgus monkey serum for this assay was set at 1/30. For graphical representation, the performance of all animals was plotted individually as a function of antibody titer and duration of the study in days (Fig. 31). Data for serum samples with values equal to or below the plate-specific limit of detection were plotted using the nominal titer value for ease of plotting.

Collagen-induced arthritis model in the cynomolgus macaque

[00343] The ethical use of non-human primates was ensured by modeling the pharmacodynamic responses of cynomolgus macaque to AB79 and then extrapolating the minimum number of animals per treatment group sufficient to obtain statistically significant differences in PD responses. Thirty-four female cynomolgus macaques, not previously used in experiments, aged 3 to 4 years, weighing 2.5 to 3.3 kg, were purchased from Biomedical Research (GZ) Ltd (SNBL, China). After receiving them, a medical examination of each animal was carried out by a contract research organization (PharmaLegacy Laboratories, Inc., China). Animals were kept one per cage and acclimated for a minimum of 14 days prior to the start of experimental procedures. The animal cage room was maintained at a temperature of 20-29°C, a relative humidity of 40-70%, and a 12-hour light-dark cycle. Animals were accustomed to receiving intravenous infusions or the introduction of an oral tube prior to the start of the study. Macaques had free access to vegetables, fruits, food (Shanghai Shilin Biologic Science & Technology Co. Ltd., China) and water according to a standard protocol. The cells were arranged in tiers within the racks to reduce the impact of any environmental influences on the study. This experimental design, all research protocols and experimental procedures have been reviewed and approved by the Host Ethics Committee (PharmaLegacy Laboratories Inc) in accordance with China's animal experimentation law.

[00344] Macaques for the study were selected in accordance with the pre-selection criteria. One animal not previously used in any experiments was not injected with collagen and was kept as a negative control for disease induction. The rest of the animals were injected with type II bovine collagen (Sichuan, China) dissolved in 0.01 N acetic acid (SPGC Sinopharm Chemical Reagent Co., Ltd; Shanghai, China) to a final concentration of 4 mg/ml (Mihara M., et al ., Clin Immunol. 2001; 98(3): 319-26; Uchiyama Y., et al. , Biol Pharm Bull. 2008; 31(6): 1159-63; Uchiyama Y, Koike N, Mihara M. Anemia in monkey collagen-induced arthritis is correlated with serum IL-6, but not TNFa Rheumatol Int 2008 28: 879-883; Kato A., et al. , Experimental and Molecular Pathology 2008 84:262-270), at 0-e and day 21 sc (Mihara et al. (2001) Clin. Immunol. 98 (3): 319-26; Uchiyama et al. (2008) Biol. Pharm. Bull. 31 (6): 1159-63; Uchiyama et al (2008) Rheumatol Int 28: 879-883 Kato et al (2008) Experimental Mol Path 84: 262-270). Collagen was emulsified with an equal volume of complete Freund's adjuvant (CFA) (Sigma-Aldrich; St. Louis, USA). Animals were pre-treated with ketamine (4 mg/kg IM) to provide a hypnotic effect, and, if necessary, additional anesthesia was applied in the form of 1.5-5% isoflurane (using an inhalation anesthesia machine Matrix vip3000 isoflurane) with an oxygen consumption of 0.8- 1.5 liters. Any ulcerative skin lesions developing at the immunization sites were treated with iodine each time the animal was sedated to prevent infection.

[00345] Seven animals treated with collagen were included in prophylactic group AB79 on day 0 (Fig. 31A). Animals in the AB79 prophylactic group received weekly infusions of AB79 antibody at 3 mg/kg starting on day 7, with the last dose on day 56, for a total of 8 doses (FIG. 31A). Animals of this group were sacrificed on the 63rd day. The remaining immunized animals were examined as one group and were treated weekly with vehicle (saline) by 30-minute intravenous infusion starting on day 7 until the animal reached or exceeded ≥15% of the maximum clinical arthritis score (for CIA). From this point on, the animal was included in the vehicle control group, AB79 treatment group, or dexamethasone treatment group, and enrollment continued on an ad-hoc basis due to differences in time of disease onset (FIG. 31A). Animals included in the vehicle control group received weekly vehicle infusions for 5 weeks (FIG. 31A). Animals of this group were sacrificed 7 days after the last dose. For animals included in the AB79 treatment group, weekly infusions were performed for 5 weeks (FIG. 31A). Animals of this group were sacrificed 7 days after the last dose. Animals included in the therapeutic group of dexamethasone were administered daily by oral gavage for 5 weeks (FIG. 31A) and sacrificed 1 day after the last dose. Animals were monitored daily for signs of malaise and general response to the administered compounds. All exceptions to normal healthy appearance and behavior were recorded and detailed on standard clinical observation forms.

Assessing Arthritis Activity

[00346] The body weight of the monkeys was measured once during the acclimation period (5 days before the start of the experiment), the day before each cycle of disease induction, and then once a week until the end of the study. The number of animals in the group with joint swelling was recorded on day 0 and day 21, and then daily until the end of the study. The number of proximal interphalangeal (PIP) joints (on each hand and foot, respectively) with joint swelling was recorded for each animal on day 0, day 21, and then once a week until the end of the study. The longitudinal and transverse axes of the HMF of the joints of the fore and hind limbs (without the thumb) were measured with calipers on day 0, day 21, and then once a week after the onset of the disease until the end of the study for all HMF of joints with arthritis. The mean oval area of 16 PMF joints was calculated and recorded as an individual parameter. The oval area of each PMF joint was calculated using the following formula: oval area=longitudinal axis × transverse axis × 3.14 × 1/4. Percent change in oval area and joint swelling was calculated using the following formula: % change in oval area = (mean oval area on day X/mean oval area on day 1 sensitization) × 100. Joint swelling = (mean oval area on day X - mean oval area on the day of the 1st sensitization).

Clinical Assessment of Arthritis

[00347] The severity of arthritis in each limb in monkeys was assessed on day 0 and day 21, and then once a week until the end of the study according to the following criteria: (0) normal; (1) mild arthritis, subtle but definite; (2) moderate swelling; (3) severe arthritis with significant swelling and/or marked deformity of the joint. The following joints were examined and evaluated in each paw: a total of 15 joints, including 5 metacarpophalangeal (MP) joints, 4 proximal interphalangeal (PMF) joints, 4 distal interphalangeal (DIP) joints, 1 first proximal interphalangeal joint; each wrist or ankle was evaluated as one complex joint. The knee/elbow of each limb was also assessed for disease severity. The arthritis score for each animal was the total score for each individual joint, with a maximum score of 192 (16 × 3 × 4), where 16 is the total number of joints plus knee/elbow for each limb; 3 - maximum score for each individual joint; 4 - the number of limbs of one macaque.

Collection and analysis of blood

[00348] Blood samples were used for complete blood count, biochemical analysis of blood and obtaining serum for the measurement of antibodies, FA and APPL, and were taken from animals at the time points indicated below. Vehicle control and AB79 prophylaxis: During weeks 1 and 5, blood was collected twice, once before dosing and once the next day. During weeks 2, 3, 4, 6, 7 and 8, blood samples were collected just prior to AB79 administration. During the 9th week, blood samples were collected immediately before the sacrifice of the animal. All other animals were bled weekly until the disease reached a threshold of 15% of the maximum arthritis score. After inclusion in the vehicle group, AB79 therapy group, or dexamethasone therapy group, blood samples were collected weekly immediately before dosing and before sacrificing the animal. In addition, samples were collected the day after the first dose of the drug and the day after the fifth dose. For the vehicle control, AB79 prophylaxis, and AB79 and dexamethasone treatment groups, blood samples for flow cytometry were collected before the first dose (on the day of infusion), the day after the first dose, before the second dose (on the day of dosing), before administration of the fifth dose (on the day of dosing) and on the day the animal is sacrificed. For therapeutic group AB79, blood samples for flow cytometry were collected before the first dose of the drug, the day after the first dose, before the 8th dose, before the 29th dose, and on the day of sacrifice of the animal.

X-ray examination

[00349] Radiography was performed for each joint (DMF, PMF, 1st MF and PF - sites that are often affected in humans with RA) on the forelimbs and hind limbs of anesthetized animals at the end of the study. Radiographic assessment was performed blinded based on a 0-4 scoring system: (0) normal; (1) slight deformity in the layers of articular cartilage and/or in areas of subchondral bone; (2) severe deformity in the layers of articular cartilage and areas of subchondral bone, a small number of osteophytes present on the periosteal surfaces and joint margins, which are indistinct but still visible; (3) changes of the same type as in grade 2, but progressing further and with more osteophytes present on the periosteal surfaces, and the joint cavity is indistinguishable or not visible; (4) changes of the same type as in grade 3, but progressing further, the joint cavity is not detected, the bones appear scleral or ankylosed, significant deformities occur.

Histopathology and tissue histomorphometry in macaques with arthritis

[00350] At the end of the study, animals were sacrificed by exsanguination under anesthesia. Paws, spleen, colon and lymph nodes (mesenteric and inguinal) were collected and fixed in neutral buffered formalin. PMF and DMF joints were decalcified with 15% EDTA, dehydrated and embedded in paraffin (8 blocks/paw×4 paws×22 animals=704 blocks). Frontal and coronal sections of the joints were obtained using a rotary microtome. Histopathology was assessed on toluidine blue stained sections (32 × 22 animals = 704 slides) by a blinded bone histopathologist. Histological sections were qualitatively assessed for the following histopathological features: cellular infiltration, pannus, cartilage involvement, and bone resorption. Quantitative histomorphometry was performed using Osteomeasure software (OsteoMetrics, Inc., Atlanta, GA) coupled to a Nikon Eclipse E400 light/fluorescence microscope and video subsystem. Histomorphometric measurements of the articular area and surface were performed by a bone histopathologist blinded using micropreparations stained with toluidine blue (704 micropreparations).

Statistical analysis

[00351] Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was carried out using the GraphPad Prism program for each parameter among experimental-naive, model and control (dexamethasone) and test groups. Differences were considered statistically significant at p <0.05.

results

CD38 expression profile in cynomolgus macaque

[00352] To determine whether the cynomolgus macaque could be a valid model for evaluating the potential effects of CD38 targeting in humans, the CD38 antigen expression profile was compared in 15 different tissue types collected from healthy human and cynomolgus monkey donors. Immunohistochemistry demonstrated that AB79 binds to mononuclear leukocytes in the colon, stomach, small intestine, bone marrow, and lymph node, as well as to some endothelial sites in the lamina propria of the colon, stomach, and small intestine in both species (Table 11).

Table 11. Comparison of CD38 expression profiles in human and macaque tissues. Human Toque Anti-CD38 mAb Human IgG1 control Anti-CD38 mAbs Human IgG1 control Textile 5 µg/ml 25 µg/ml 5 µg/ml 25 µg/ml Cells stained with AB79 5 µg/ml 25 µg/ml 5 µg/ml 25 µg/ml Cells stained with AB79 Bone marrow 3, C/M 2+, M Neg. Neg. Bone marrow cells, possibly white blood cells 1+, S/M 1+, M Neg. Neg. Few bone marrow cells, possibly white blood cells Heart Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Colon 3+, C/M 4+, C/M Neg. Neg. Leukocytes, including lymphocytes, in the lamina propria of the mucous membrane; some endothelial cells 2+, C/M Neg. Neg. Neg. Cells in the lamina propria, possibly lymphocytes; some endothelial cells Stomach Neg. Neg. Neg. Neg. Leukocytes, including lymphocytes, in the lamina propria of the mucous membrane; some endothelial cells 1+, S/M 1+, C Neg. Neg. Fusiform cells in the lamina propria; presumably endothelial cells Small intestine 4+, C/M 4+, C/M Neg. Neg. Leukocytes, including lymphocytes, in the lamina propria of the mucous membrane; some endothelial cells 2+, C/M 1+, S/M Neg. Neg. Cells in the lamina propria, possibly lymphocytes; some endothelial cells renal glomerulus Neg. Neg. Neg. Neg. In his own record; few endothelial cells Neg. Neg. Neg. Neg. renal tubules Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Liver Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Lymph node 3+, C/M 4+, C/M Neg. Neg. Cells in medullary ligaments and sinuses; possibly plasma cells; few cells in the cortex 4+, M 3+, C Neg. Neg. Cells in medullary ligaments and sinuses, less in cortex Lung 3+, C/M 3+, C/M Neg. Neg. Interstitial and peribronchiolar cells Neg. Neg. Neg. Neg. Pancreas Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Prostate 3+, C 3+, C Neg. Neg. acinar epithelium; some interstitial leukocytes Neg. Neg. Neg. Neg. Leather Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Cervix 2+, C/M 1+, S/M Neg. Neg. Submucosal leukocytes, probably lymphocytes Neg. Neg. Neg. Neg. Endometrium of the uterus Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Staining intensity: 1+ = minimal, 2+ = weak, 3+ = moderate, 4+ = strong, Neg. = negative, O=none. Frequency of staining for a specific cell type: very low (OH: <25% of cells); low (H: 25-50% cells); medium (C: >50-75% of cells); high (B: 76-100% of cells). The nature of staining includes the type of cells or tissue element - features of the corresponding cell type (for example, epithelium) or tissue element (for example, basement membrane) - subcellular or extracellular localization (membrane, cytoplasm, cytoplasmic filaments, basement membrane).

[00353] In contrast to macaques, in humans, AB79 also binds to mononuclear leukocytes in the liver, lungs, prostate, and cervix, as well as prostate acinar epithelium. Binding to AB79 was not observed in the heart, kidney, pancreas, skin and uterine endometrium of both species. In general, AB79 staining was moderate to significant (3+ to 4+) in human tissues and minimal to weak (1+ to 2+) in cynomolgus monkey tissues (Table 11), which may reflect differences in AB79 affinity for human and macaque CD38 and/or differences in CD38 expression. The staining pattern was mostly cytoplasmic, and cell membranes were stained in some cells (Table 11). Overall, it was concluded that the macaque is a valid model species for evaluating the potential pharmacological effect(s) of CD38 targeting in an autoimmune disease model.

AB79 binds to CD38 expressed by cynomolgus monkey

[00354] The amino acid sequence of the human CD38 protein has 91% amino acid identity with its cynomolgus macaque ortholog protein, 59% identity with that of the mouse and rat, and 54% with the rabbit. Comparison of the AB79 binding epitope in human CD38 with the corresponding sequence in macaque CD38 revealed one amino acid substitution, a lysine substitution with glutamate at position 274. To determine whether AB79 could be used to evaluate the potential effects of CD38 targeting in macaques, macaque CD38 was expressed in Chinese hamster ovary cells. (CHO) and bound macaque CD38 to AB79 at a half maximum effective concentration (EC ₅₀ ) of 4.5 nM (FIG. 32A). This value is approximately 10-fold lower than the binding affinity of AB79 for human CD38 expressed by CHO cells (KD=0.7 nM), indicating that AB79 binds macaque CD38 with less activity than human CD38. AB79 also bound to B-, NK-, and T-cells in macaque whole blood, and NK-cells had a higher average fluorescence intensity than B-cells and T-cells (Fig. 32B), indicating that NK- macaque cells have a higher density of CD38 expression per cell. After incubation with macaque whole blood in vitro , the AB79 antibody elicited cytolysis of NK cells in a dose-dependent manner (FIG. 32C), showing a mean EC ₅₀ of 29.6 nM, representing approximately 30 times less activity than NK cell cytolysis. from human peripheral blood (data not shown). Taken together, these data indicate that the cynomolgus macaque may be less sensitive to the pharmacological effects of AB79 than humans. However, this cross-reactivity profile, as well as the similarity of CD38 expression profiles, suggests that the cynomolgus macaque is a suitable model species to investigate the potential pharmacological effect(s) of the AB79 antibody in vivo .

Pharmacological effects of AB79 in healthy monkeys

[00355] To characterize the in vivo pharmacological effects of AB79, healthy cynomolgus monkeys were administered AB79 intravenously at a dose of 0.03; 0.1; 0.3 and 1 mg/kg weekly, and 3, 30 and 80 mg/kg every other week for 3 months, and a subgroup of animals was observed for 3 months after the last dose (Fig. 31A). Peak concentrations of AB79 usually occurred at the end of the infusion and were generally dose proportional (Table 12).

Table 12 Mean AB79 pharmacokinetics in female cynomolgus macaque after intravenous infusion Dose (mg/kg) Dose Schedule Sample collection time C _max (µg/ml) PPK ₃₆₆ (h*mcg/ml) T _1/2
(h) 0.1 Weekly 1st week 1.77 104 N/R 13th week 2.49 218 N/R 0.3 Weekly 1st week 4.32 327 N/R 13th week 7.78 665 N/R 1.0 Weekly 1st week 21.1 1630 N/R 13th week 28.6 2700 N/R 3 Once in two weeks 1st week 62.1 7600 N/R 13th week 90.1 18400 237 thirty Once in two weeks 1st week 680 92800 N/R 13th week 962 228000 N/R 80 Once in two weeks 1st week 1630 220000 N/R 13th week 3140 456000 415

[00356] Drug exposure generally increased in proportion to dose at steady state in animals that did not have anti-AB79 antibodies, and increased 1.7-2.4-fold by week 13 (Table 12), which is consistent with reduced clearance due to target-mediated distribution of AB79. The elimination half-life after the last dose in animals from the cohorts treated with 3 and 80 mg/kg of anti-AB79 antibodies was 237 and 415 hours, respectively (Table 12). Sex differences in FC characteristics were not observed.

[00357] The NK cell population expressed CD38 at a uniformly high density (Fig. 32B), which decreased in peripheral blood after the first infusion of AB79, with an ED ₅₀ of 0.3 mg/kg (Fig. 31B), corresponding to C _max in 7.63 μg/ml and a total mean exposure of 665 h*μg/ml at week 13 (Table 12). In contrast, the B cell population expressed CD38 heterogeneously, with lower mean densities than NK cells (Fig. 32B), and which decreased in peripheral blood after the first AB79 infusion, with an ED ₅₀ of 1.0 mg/kg (Fig. . 31C), C _max at 21.1 μg/ml and with a total mean exposure of 2700 h*μg/ml at week 13 (Table 12). The T cell population also expressed CD38 heterogeneously, with an even lower mean density than B cells (data not shown), and which decreased in peripheral blood after the first AB79 infusion, and had an ED ₅₀ of 30 mg/kg (Fig. 31D), C _max of 62.1 µg/mL, and cumulative mean exposure of 18400 h*µg/mL at week 13 (Table 12). Taken together, these data from the dose range study indicate that a weekly dose of 3 mg/kg will maintain total peripheral blood NK, B, and T cell populations at levels that are 80%, 60%, and 20% % above base levels, respectively.

Action profile of AB79 in collagen-induced arthritis

[00358] The potential efficacy of AB79 was investigated in a CIA model in the cynomolgus macaque (a species whose immune system, joint and skeletal anatomy is reasonably similar to that of humans) to allow for the use of macaque clinical indicators. Collagen type II was injected intradermally to macaques on days 0 and 21 to induce arthritis (FIG. 33), and the appearance of anti-collagen antibodies confirmed successful immunization in each animal (data not shown). One group of animals was injected with 3 mg/kg of AB79 antibody according to the prophylactic regimen, starting on the 7th day and continuing for 8 weeks. Macaques from three treatment groups that had overt disease were randomized and administered vehicle control, 3 mg/kg AB79 or 0.1 mg/kg dexamethasone. Therapeutic exposure continued for 5 weeks after the start of exposure (Fig. 33).

[00359] AB79 was well tolerated in both prophylactic and therapeutic regimens. The healthy control macaque gained weight over time, while the collagen immunized macaques from the other four groups began to lose weight from day 7 (FIG. 34A), indicating a systemic effect associated with the development of arthritis. Based on the percentage change in body weight relative to baseline body weight at baseline, animals in the AB79 prophylaxis group, AB79 treatment group, and dexamethasone treatment group were found to regain lost body weight from day 14 post-exposure, suggesting therapeutic benefit from both AB79, and from treatment with a positive control - dexamethasone. Weight gain approached normal levels in macaques treated with AB79 and dexamethasone compared to healthy intact controls; arthritic animals treated with vehicle lost weight (FIG. 34A). Arthritis severity was assessed using the Clinical Arthritis Score, which is a cumulative disease activity score that takes into account each measurable joint in an animal over the course of the study using a 192-point scoring system. Vehicle-treated animals experienced disease progression with increasing clinical scores over the course of the study (FIG. 34B). Prophylactic exposure with AB79 antibody prevented the development of arthritis with statistical significance ( p <0.01) compared to vehicle control. Similarly, treatment with AB79 antibody or dexamethasone inhibited the development of arthritis with statistical significance ( p < 0.05) compared to vehicle control, and reduced arthritis scores from pre-treatment baseline levels (FIG. 34B). Similar effects were observed for the PMF joint subgroup, both in terms of the number of inflamed PMF joints (Fig. 34C) and the mean joint swelling score for all PMF joints (Fig. 34D). To obtain a comprehensive assessment of arthritic joints and the ability of AB79 to exhibit "disease modifying" activity in human arthritis, x-rays were taken of each MF and PF joint. Prophylactic exposure with AB79 antibody prevented damage to PMF joints (data not shown), DMF (Fig. 34E) and PF joints (Fig. 34F) ( p <0.01) compared to vehicle control. Similarly, treatment with AB79 antibody or dexamethasone resulted in a significant ( p <0.05) reduction in the extent of damage in PMF joints (data not shown), DMF (Fig. 34E), and PF joints (Fig. 34F) compared to vehicle control. . To characterize the components of progressive arthritis, histological analysis of DMF and PMF joints was performed for cellular infiltration, pannus severity, cartilage damage, bone resorption, and osteophyte formation. Prophylactic treatment with AB79 resulted in composite scores that were significantly lower ( p <0.01) than the vehicle control and comparable in magnitude to non-collagen immunized animals (FIG. 35A). AB79 had a similar effect on every component; their values were significantly lower ( p < 0.01) than vehicle controls for pannus (Fig. 35B), leukocyte infiltration (Fig. 35C), cartilage damage (Fig. 35D), bone resorption (Fig. 35E), and osteophytes (Fig. 35F). Similar effects, albeit less strong, were also observed with AB79 antibody or dexamethasone treatment (FIGS. 35A-E), although not all differences reached statistical significance.

[00360] Quantitative histomorphometry was performed on all DMF and PMF joints in blind studies by a certified veterinary pathologist, and parameters were measured including articular cartilage area (Fig. 36A), thickness of articular cartilage lesion (Fig. 36B), percentage of articular cartilage surface lesion (Fig. 36C) and osteophyte area relative to the total periosteal surface (Fig. 36D). All these parameters illustrate that prophylactic exposure to AB79 antibody prevented the development of joint damage with a statistically significant probability. Therapeutic exposure to the AB79 antibody reduced the severity of the disease, although some histomorphometric parameters did not reach the level of statistical significance. Therapeutic effects of AB79 were similar to those of therapeutic administration of dexamethasone.

[00361] C-reactive protein (CRP) (FIG. 37A), alkaline phosphatase (AP) (FIG. 37B), and albumin (ALB) levels (data not shown) correlated with disease severity. Prophylactic treatment with AB79 prevented the inflammation-associated increase in CRP (FIG. 37A) and ALP (FIG. 37B) observed in the vehicle control animal. Therapeutic exposure to AB79 resulted in a rapid reduction in CRP and ALP, while exposure to dexamethasone required long-term treatment to reduce CRP and ALP levels (FIGS. 37A and 37B, respectively). Serum chemistry showed no evidence of liver injury (eg, increased ALT or AST), suggesting that the increase in ALP was associated with the development of bone disease in CIA animals, and that the decrease in ALP in animals treated with AB79 was associated with reduced bone loss. Serum biochemical parameters - creatinine, blood urea nitrogen, glucose and total protein - changed over time, but did not have a constant correlation with arthritis severity or treatment regimen (data not shown).

[00362] During the study, CBC, CBC, and leukocyte counts were performed, and key parameters were published. No change in RBC count was observed after exposure to AB79 or dexamethasone (FIG. 38A). In addition, hematocrit levels decreased after the development of arthritis and rose back to baseline naive control levels after exposure to both AB79 and dexamethasone (FIG. 38B). Reticulocytes were elevated after the development of arthritis, and exposure to both AB79 and dexamethasone reduced the reticulocyte count to the normal level found in naive controls (Fig. 38C). Arthritis platelet and neutrophil levels were elevated, and AB79 exposure decreased both platelet and neutrophil levels compared to naive controls, while dexamethasone had no effect (FIGS. 38D and 38E, respectively). In contrast, total lymphocyte counts decreased with disease compared to naive controls, and exposure to AB79 antibody further lowered lymphocyte levels, as expected, while dexamethasone treatment had no effect (FIG. 38F).

[00363] Among subpopulations of peripheral blood lymphocytes, NK cells decreased >95% below baseline levels (Fig. 39A) within 24 hours of exposure to AB79 antibody, while baseline levels of B cells (Fig. 39B), T cells (Fig. 39C) and monocytes (Fig. 39D) decreased by a maximum of 60%, 55% and 50%, respectively. The decrease in the number of NK and B cells was maintained throughout the entire dosing period, while the decrease in the number of T cells and monocytes was transient and was observed only after the first infusion. A similar pattern of decline was observed in each cell population after prophylactic exposure to AB79 antibody, except that the corresponding group of animals had lower monocyte levels prior to exposure and monocyte levels did not change in response to exposure. No differences were observed in B, NK, T cell counts or monocyte counts between vehicle treated animals and dexamethasone treated animals (FIGS. 39A, 29D and 29F, respectively).

Conducted biological analysis of blood serum to measure the concentration of AB79 and anti-AB79 antibodies. Each animal treated with a prophylactic (FIG. 40A) or therapeutic (FIG. 40B) dose of AB79 achieved a significant effect with a C _max ranging from 24 to 97 μg/mL. All animals exposed to AB79 had minimum concentrations that exceeded EC ₅₀ for CD38 saturation (FIG. 32A) and NK cell lysis (FIG. 32C) in vitro . Anti-AB79 antibodies were detected in 4 of 7 animals from the prophylactic group (Fig. 40C) 14-30 days after the first dose and until the end of the study. Two animals had high titers that corresponded to low AB79 concentrations in these animals by day 31 (FIG. 40A), indicating that these anti-AB79 antibodies may affect clearance. Anti-AB79 antibodies were also detected in 4 of 5 animals from the treatment group (Fig. 40D) from 21 days post-first dose until the end of the study, and two animals also had high titers that corresponded to low AB79 concentrations in these animals by the end of the study. research (Fig. 40B). However, all animals were included in the data analysis because most target cells remained reduced from baseline throughout the duration of the study (Figures 39E and 39F), with the exception of T cells after the second therapeutic dose ( Fig. 39G).

Discussion

[00364] CD38 deficiency in mice has been reported to result in attenuated forms of CIA, illustrating the irreplaceable function(s) of this molecule in an autoimmune disease model, but the role of CD38-expressing cells in primate models has not been studied. In addition, numerous studies have demonstrated that the overall level of CD38 expression on peripheral blood cells positively correlates with disease activity in human patients with RA and SLE (Cole S., et al. , Arthritis Res Ther. 2018 May 2; 20(1) : 85; Kraan MC, et al. , Rheumatology (Oxford) 1999 Nov; 38 (11): 1074-80; Vital EM, et al., Arthritis Rheum. 2011 Oct. 63 (10): 3038-47; or Banchereau R., et al. , Cell 2016 Apr 21;165(3):551-65). The anti-CD38 monoclonal antibody AB79 caused depletion of plasma cells in blood or bone marrow samples from patients with RA or SLE in vitro (Smithson et al. (2017) J. Immunol. 198 (1 Supplement) 224.20; Cole et al. (2018) Arthrit Res. Ther. 20 (1): 85; Wang et al. (2016) Arthrit. Rheumatol. 68 (suppl 10) 2016 ACR/ARHP Annual Meeting, 1085; Mihara M., et al., Clin Immunol. 2001 ; 98 (3): 319-26; and Uchiyama Y., et al., Biol Pharm Bull 2008; 31 (6): 1159-63). Unlike other anti-CD38 mAbs under development (eg, daratumumab, isatuximab, and MOR202), AB79 binds to CD38 expressed by cynomolgus monkeys and provides a unique opportunity to determine whether a decrease in CD38-expressing cells can prevent and/or ) reduce inflammation and tissue damage in a non-human primate model of autoimmune disease. AB79 is a high affinity monoclonal antibody that effectively mediates the depletion of the CD38+ cell population (Smithson, G., et al. , J Immunol May 1, 2017, 198 (1 Supplement) 224.20). Integrative analysis demonstrates that CD38 is a therapeutic target in pre-disease with high plasma cell counts, established rheumatoid arthritis, and systemic lupus erythematosus (Cole S., et al. , Arthritis Res Ther. 2018 May 2; 20 (1): 85).

[00365] The cynomolgus monkey has been found to be a suitable model for evaluating the potential effects of AB79 in autoimmune disease, as CIA in these primates is characterized by symmetrical small joint polyarthritis that resembles human RA (Mihara et al. (2001) Clin. Immunol. 98 ( 3): 319-26 Uchiyama et al (2008) Biol Pharm Bull 31 (6): 1159-63 Uchiyama et al (2008) Rheumatol Int 28: 879-883 Kato et al. (2008) Experimental Mol. Path. 84: 262-270), expression profiles for CD38 are similar between these species (Table 11) and AB79 binds to macaque CD38 with an affinity that is 10 times lower than human CD38 (Fig. 32). We concluded that a weekly dose of 3 mg/kg of AB79 would be sufficient to evaluate the potential role of CD38 in CIA because dose range studies in healthy monkeys demonstrated that administration of a weekly dose of 3 mg/kg of AB79 resulted in a decrease in overall NK populations. -, B - and T cells by more than 80%, 60% and 20% of baseline levels in peripheral blood, respectively (Fig. 31B, 31C and 31D). Similar reductions in NK, B and T cell levels were achieved in the prophylactic CIA model (FIGS. 37E, 37F and 37G) despite the appearance of anti-AB79 antibodies in some animals (FIGS. 39C and 39D). The decrease in AB79 over time in the 3 prophylactic treated animals (Fig. 39A) indicates that these antibodies may have increased clearance, but B-, NK-, and T-cells did not recover to baseline levels by the end of the study ( Fig. 37E, 37F and 37G), indicating that exposure to AB79 was sufficient to maintain the PD effects throughout the study. Therefore, these animals were included in all analyses. This pattern is also broadly consistent with the therapeutic effects of AB79. NK and B cells did not recover to baseline after completion of the study (FIGS. 37E, 37F and 37G), indicating that exposure to AB79 was sufficient to maintain the PD effects throughout the study. In contrast, T cell counts recovered to baseline by the end of the study (Fig. 37G), suggesting that anti-AB79 antibodies may partially confound the interpretation of therapeutic data (i.e., underestimate the contribution of T cells expressing CD38, in KIA macaques).

[00366] Prophylactic administration of AB79 prevented the development of arthritis, as consistently demonstrated in all measures, while therapeutic exposure to the AB79 antibody suppressed the development of arthritis and joint damage (FIGS. 34, 35 and 36). Histological analysis of the joints demonstrated that the AB79 antibody had a relatively broad effect, preventing pannus formation (Fig. 35B), infiltration (Fig. 35C), cartilage damage (Fig. 35D), bone erosion (Fig. 35E), and osteophyte formation (Fig. 35F). It should be noted that the therapeutic treatment with AB79 antibody and dexamethasone also suppressed these progressive histological changes, although to a lesser extent than prophylactic administration. Arthritis scores decreased with treatment over time (FIGS. 34B, 34C, and 34D), indicating potential reversibility of the damage. Taken together, these data demonstrated consistent disease-modifying effects with prophylactic and therapeutic exposure to the AB79 antibody.

[00367] Within the framework of the studied regimens, the effect of this therapeutic effect appears to be comparable for AB79 and dexamethasone. The therapeutic use of steroids such as dexamethasone is highly effective in the treatment of human autoimmune diseases, including RA and SLE, but negative side effects (eg, osteoporosis, hypertension, diabetes, weight gain, cataracts, glaucoma, thinning of the skin, and bruising) limit long-term use these medicinal products. The possibility of targeted depletion of CD38-expressing cells to provide comparable efficacy in human autoimmune diseases without the negative side effects of steroids requires further clinical studies.

[00368] The prophylactic and therapeutic effects of AB79 have been associated with sustained decreases in total blood lymphocytes (FIG. 38F), NK-, B-, and T-cells (FIGS. 37C-37E), transient decreases in monocytes (FIG. 37F), and the absence of changes in erythrocytes (Fig. 38A), platelets (Fig. 38D) and neutrophils (Fig. 38E). These pharmacodynamic data demonstrate that there are differences in the sensitivity of cells expressing CD38 to AB79-mediated depletion. The decrease in these indicators mainly correlated with the density of cellular expression of CD38; the NK cell population uniformly exhibited the highest median CD38 density among all cell types examined (FIG. 32B) and was the most sensitive to AB79 (FIGS. 31B and 37C). The highest density of CD38 expressed on B cells was approximately 3-fold lower than on NK cells (Fig. 32B), and the B cell population was less sensitive to AB79 than NK cells (Fig. 31C and 37C ). CD38 was expressed on T cells at generally lower mean densities than on B cells (Fig. 32B), and T cells were less sensitive to AB79-mediated depletion than B and NK cells (Fig. 31D and 37D). The AB79 preparation had no effect on cells that express low densities of CD38 (eg erythrocytes) or do not express CD38 (eg neutrophils) (FIGS. 37A, 37D and 37E). The exceptions are monocytes, which express CD38 uniformly at an intermediate density and were only transiently downregulated by the AB79 antibody (FIG. 37F). The kinetics of the transient decline in monocyte levels differs from the sustained decline seen in B-, T-, and NK cells and points to different mechanisms. For NK cells, direct cytolysis by AB79 antibody in vitro was observed (Fig. 32C), and after a single administration of AB79 in vivo , there was a steady decrease in the number of NK, B, and T cells (data not shown), indicating that this decrease mediated by the CDC and/or ADCC mechanism in vivo . In contrast, no cytolysis of monocytes was observed in vitro (data not shown), and there was no sustained decrease in their number in vivo (Fig. 37F), indicating an alternative mechanism (eg, leukocyte edge standing) mediating transient decreases in cell number in vivo . Resistance of human monocytes to cytolysis by AB79 has also been observed in healthy subjects (unpublished) and has been described for daratumumab in patients with multiple myeloma (Nijhof et al. (2016) Blood 128 (7): 959-70). Expression of CDC and ADCC inhibitors in human monocytes did not significantly correlate with resistance to cytolysis by daratumumab, and it remains unknown why monocytes are relatively insensitive to daratumumab and AB79.

[00369] Although the reduction in NK cells observed with AB79 exposure is qualitatively consistent with the reduction in NK cells with daratumumab in patients with refractory myeloma, there are quantitative differences. IV infusion dose (0.3 mg/kg), maximum concentration (C _max =4.32 μg/ml) and exposure (AUC ₃₆₆ =327 μg h/ml) of AB79 required to maintain peripheral blood NK cells at 50% below baseline in macaque (Fig. 31B) were approximately 80, 116, and 297 times lower than the corresponding IV infusion dose (24 mg/kg), maximum concentration (C _max ~ 573 µg/mL) and exposure (AUC _inf ~ 97175 μg h/mL) required for a comparable reduction in NK cells by daratumumab in patients with refractory myeloma (Casneuf et al. (2017) Blood Adv. 1 (23): 2105-2114; Clemens et al (2017) Clin Pharmacokinet 56 (8) : 915-924 Notably, AB79 binds to monkey lymphocytes with an affinity (KD=4 nM) that is similar to that of daratumumab to cells expressing human CD38 (KD=4 nM). ) (Center For Drug Evaluation And Research Application Number: 761036orig1s00 Pharmacology Review(s)), however, it is not known whether these differences are due to potential species differences, the course of the disease and (or) the activities of the corresponding antibodies. However, pharmacokinetic and pharmacodynamic data for AB79 in patients with refractory myeloma are needed for a more accurate comparison because daratumumab does not cross-react with mouse, rat, rabbit, porcine, cynomolgus and rhesus monkey CD38 (1 April 2016 EMA/278085/ 2016 Committee for Medicinal Products for Human Use (CHMP) Assessment report Darzalex International non-proprietary name: daratumumab Procedure No. EMEA/H/C/004077/0000).

[00370] In conclusion, the reduction of CD38-expressing cells by the cytolytic antibody AB79 prevented the development of CIA in macaques when administered prophylactically and reversed the disease when administered therapeutically. A previous study using blood and bone marrow samples from SLE patients demonstrated that AB79 depletes 80% of the short-lived and long-lived plasma cell population and reduces autoantibodies (eg, VH4-34 9G4+, anti-Ro and anti-dsDNA) in vitro (Wang et al (2016) Arthritis Rheumatol 68 (suppl 10) 2016 ACR/ARHP Annual Meeting, 1085). These pooled data suggest that this therapeutic strategy may be effective in the treatment of RA, human SLE, as well as other autoimmune diseases.

Example 7: Assessment of the AB79 profile in healthy human volunteers

[00371] This study characterized the safety, tolerability, pharmacokinetics, and pharmacodynamics of AB79 using a randomized, double-blind, placebo-controlled study of a single intravenous (IV) infusion or subcutaneous (SC) injection of AB79 at increasing doses in healthy subjects.

results

[00372] AB79 was well tolerated. All adverse events (AEs) were mild to moderate and there were no early withdrawals of participants due to AEs or development of infusion or injection site reactions with IV and SC administration of study doses up to 0.06 and 0.6 mg kg ^-1 , respectively. At higher doses, a transient increase in cytokine levels, mainly after intravenous administration, coincided with a decrease in the number of cells expressing CD38; clinical symptoms primarily included mild pyrexia, headache, and postural hypotension. No identified abnormalities in laboratory tests, electrocardiograms, vital signs, or physical examinations have been reported associated with AB79 antibody treatment. AB79 reduced levels of plasma blasts and natural killer (NK) cells at similar doses and with a 50% maximum effective dose of approximately 0.003 and 0.1 mg·kg ^-1 for intravenous and subcutaneous administration, respectively. The decrease in the level of immunoglobulin (Ig) M and IgA occurred without comparable changes in the level of IgG. The total number of leukocytes, granulocytes, lymphocytes, erythrocytes and platelets remained within the normal range for all dose levels.

Conclusion

[00373] AB79 reduced the levels of plasma blasts and NK cells in the peripheral blood of healthy subjects when administered intravenously or subcutaneously, and was generally safe and well tolerated. Subcutaneous dosing was better tolerated and caused more persistent depletion of target cell populations than intravenous dosing. This plasmacytolytic profile may be useful in the treatment of disorders caused by plasmacytes or NK cells, their malignant counterparts (eg, multiple myeloma cells and NK cell leukemia), and pathogenic immunoglobulins.

Plan and objectives of the study

[00374] This study is the first phase 1, randomized, double-blind, placebo-controlled, single-dose human drug (SIL) study of AB79 in healthy adult subjects. The primary aim of the study was to evaluate the safety and tolerability of single escalating doses of AB79 following intravenous infusion or subcutaneous injection. Secondary objectives were to assess PK and PD in blood cell populations and immunogenicity. A total of 74 people took part in this study. After two screening visits separated by at least five days during the 28 day pre-randomization period, subjects were admitted to the study (Day -2) for baseline pre-dose evaluation. AB79 was administered on Day 1 via a 2-hour IV infusion at successively increasing doses of 0.0003; 0.001; 0.003; 0.01; 0.03 or 0.06 mg·kg ^-1 in six cohorts, or by SC injection with doses of 0.03; 0.1; 0.3 or 0.6 mg·kg ^-1 in four other cohorts. Dose selection was based on a PK/PD model derived from a series of cynomolgus monkey studies (Roepcke et al. (2018) Pharmacol Res Perspect. 6 (3): c00402). At each dose level, six to eight subjects were randomly assigned to the AB79 group (n=4-6) or the corresponding placebo group (n=2).

[00375] Signal dosing was used for each cohort and the first two subjects received either AB79 or placebo (1:1). Prior to dosing, the remaining subjects in each group were analyzed for daily post-dose safety and tolerability data for these two subjects. Participants were admitted to the inpatient Clinical Research Center (ICC) until day 8, followed by once or twice weekly follow-up visits, with the last scheduled clinic visit on day 78 for an overall safety assessment and analysis of PK, PD, and immunogenicity. The last follow-up phone call took place on the 92nd day.

[00376] Dose escalation was based primarily on the severity of the AE, which was assessed using the Common Terminology Criteria for Adverse Events. Dose escalation should have been stopped if at least two subjects from the same cohort experienced cytokine release syndrome (CRS), resulting in moderate clinical syndromes or moderate to severe drug reactions. Given that AB79 is an antibody that destroys lymphocyte cells, no further dose escalation was allowed if there was a clinically significant decrease in total lymphocyte count or subset of lymphocytes (nominally: >50% decrease from the subject's lowest pre-dose value and less than the lower limit of the normal reference range). range (NRD, English "NRR")) were observed and persisted for ≥29 days. The investigator and sponsor reviewed all blinded safety data for all participants at each dose level before proceeding to the next higher dose.

[00377] The study was conducted in accordance with good clinical practice guidelines at Parexel International Phase 1 CPU, located at Northwick Park Hospital, Harrow, UK. The protocol was reviewed and approved (approval number: 16/LO/2067) by the local independent ethics committee, the London-Brent Research Ethics Committee (London, UK). All subjects signed an informed consent form prior to beginning any study procedures.

Study participants

[00378] Eligible participants were healthy males or females (not of childbearing potential) aged 18 to 55 years, weighing 50 to 100 kg, and with a body mass index (BMI) of 18.5 to 30 kg m ^-2 .

[00379] Flow cytometry-based counts of CD45+ lymphocytes, T cells, CD4+ T cells, and B cells were required to be above the lower limit of the NRT, and NK cell levels to be within the upper 50th percentile of the NRT, given that CD38 is highly expressed on NK cells. The NRDs were determined by Takeda and analyzed by flow cytometry.

[00380] Participants were excluded from the study if they met the exclusion criteria defined in the protocol, such as diagnosed immunodeficiency, an increased risk of infection, a history of malignancy, or other investigational drug prior to this study that could affect the activity of the experimental drug from this study. research.

[00381] To measure serum AB79 antibody concentrations, serial blood samples were collected pre-dose and up to 168 hours post-dose, and additional blood samples were collected at intermediate post-dose or early termination (ED) time points. Serum AB79 concentration was determined by enzyme-linked immunosorbent assay certified by ICON (Whitesboro, NY).

[00382] To evaluate the pharmacodynamic response to AB79, peripheral blood samples were collected during screening visits, on days -1, 1, 2 (s.c. only), 3 (s.c. only), 4 (s.c. only), 4 (s.c. only). /c), 5 (for s.c. only), 6, 8, 15, 22, 29, 50 and 78 post-dose or DP. The primary and secondary pharmacodynamic measures assessed were blood levels of plasma blasts and NK cells, respectively. Additional measures included total white blood cell count and leukogram count, total T cell count and cell count in their subpopulations of CD4 and CD8 T cells, B cell count, monocyte count, and granulocyte count. Lymphocyte subpopulations, monocytes and plasmablasts were analyzed by flow cytometry at Covance (Brussels, Belgium). Electrochemiluminescent assay has been validated at ICON for the detection of anti-AB79 antibodies in human serum.

[00383] Usual drug safety parameters, such as AEs, clinical laboratory parameters, physical examinations, electrocardiogram (ECG) and vital signs, were monitored at screening, before dosing, and throughout the stay at the CCT and on subsequent visits.

[00384] Infusion reactions have been reported in clinical studies of other anti-CD38 antibodies administered to patients with MM following an IV infusion (Voorhees et al. (2015) Blood 126: 1829). Although ex vivo experiments did not show evidence of AB79 agonist activity in human blood cells, and AB79 infusion did not produce measurable effects indicative of an infusion response in preclinical macaque studies, there was careful monitoring for signs of an infusion reaction or CRS (cephalic pain, fever, chills, hypotension, nausea, vomiting). Levels of inflammatory mediators, including serum C-reactive protein (CRP), tumor necrosis factor (TNF) α, and interleukins 1 (IL-1) and 6 (IL-6), were measured at various time points on days 1 and 2, and also 4 (only for cohorts with SC administration). Reducing the infusion rate and/or pre-treatment with oral prophylactic paracetamol (acetaminophen) and antihistamines (anti-H1 and anti-H2) minimized side effects when needed.

[00385] Sc cohorts were monitored for injection site reaction (ISR), such as pain, burning, redness, itching, swelling, or induration at the injection site.

[00386] Computation of summary statistics and data analysis were performed using SAS version 9.2 and R version 3.5.1. Pharmacokinetic parameters were calculated by non-chamber analysis. Pharmacodynamic variables were evaluated and compared between active dose groups and between each dose level of AB79 and placebo.

[00387]

results

[00388] Seventy-four subjects were taken into the study, who were administered a single dose of AB79 (n=54) or placebo (n=20). For six IV cohorts treated with AB79 at doses of 0.0003; 0.001; 0.003; 0.01; 0.03 or 0.06 mg·kg ^-1 or placebo, and four cohorts with subcutaneous administration, treated with AB79 at doses of 0.03; 0.1; 0.3 or 0.6 mg·kg ^-1 or corresponding placebo, observed for 92 days; all subjects participated in the study until completion. All participants were male except for one female in the s.c. placebo group. The studied population consisted of representatives of Caucasoid (n=51), Mongoloid (n=13), Negroid (n=4) or interracial (n=6) race. Mean age (34.4 years, range 19 to 55 years) and BMI (24 to 25 kg m ^-2 ) were similar between the IV and SC cohorts and between the AB79 and placebo groups .

[00389] All doses of AB79 in this study were well tolerated. The severity of AEs was mild to moderate, with most AEs being mild and well balanced between the placebo and AB79 groups (Table 13). There were no serious AEs (SAEs) or deaths, and none of the AEs resulted in termination of the study or visits. No notable lab results, ECG, vital signs, or physical examinations related to exposure to AB79 have been reported.

Table 13 General characteristics of AEs and AEs reported in two or more subjects in any exposure group IV infusion AB79 (mg kg ^-one )
Number of subjects (%) Placebo pool (n=12) 0.0003 (n=4) 0.001
(n=4) 0.003
(n=4) 0.01
(n=6) 0.03
(n=6) 0.06
(n=6) Subjects with any AELV 11 (91.7) 3 (75.0) 3 (75.0) 3 (75.0) 6 (100.0) 6 (100.0) 6 (100.0) pyrexia 0 0 0 0 1 (16.7) 1 (16.7) 3 (50.0) Chills 0 0 0 0 0 0 2 (33.3) Nasopharyngitis 2 (16.7) 0 1 (25.0) 0 0 0 2 (33.3) Headache 4 (33.3) 0 0 1 (25.0) 2 (33.3) 3 (50.0) 5 (83.3) Postural dizziness 0 0 0 0 0 1 (16.7) 5 (83.3) Drowsiness 0 0 0 0 0 1 (16.7) 2 (33.3)

S / c injection AB79 (mg kg ^-one )
Number of subjects (%) Placebo pool (n=8) 0.03
(n=6) 0.1
(n=6) 0.3
(n=6) 0.6
(n=6) Subjects with any AELV 6 (75.0) 6 (100.0) 6 (100.0) 5 (83.3) 5 (83.3) Erythema at the injection site 1 (12.5) 5 (83.3) 0 1 (16.7) 1 (16.7) Pain at the injection site 0 3 (50.0) 3 (50.0) 0 0 feeling hot 0 0 1 (16.7) 1 (16.7) 2 (33.3) Nasopharyngitis 1 (12.5) 2 (33.3) 3 (50.0) 1 (16.7) 3 (50.0) Headache 2 (25.0) 2 (33.3) 1 (16.7) 1 (16.7) 3 (50.0) Pain in the oropharynx 1 (12.5) 0 2 (33.3) 0 1 (16.7)

AEs were defined as AEs that occur or worsen after the first dose of study drug and within 94 days after the last dose of study drug. Subjects with one or more AEs in the intervention group and the term level according to the Medical Dictionary of Regulatory Activity (MedDRA) were counted only once at the respective level. Percentages are based on the number of subjects in the security group relative to one type of exposure. The MedDRA dictionary (version 18.0) was used to encode the AE. AE - adverse event; in / in - intravenously; MedDRA - Medical Dictionary of Regulatory Activities; s / c - subcutaneously; NJVL is an undesirable phenomenon that arose during treatment.

[00390] As shown in Table 13, the majority of AEs were sporadic, with no trend towards dose dependence, with the exception of headache, dizziness, and chills, which were more common in the higher AB79 dose groups, consistent with a higher incidence of CRS. These effects (Table 14) were mainly observed in subjects receiving high doses (one subject at an IV dose of 0.03 mg·kg ^-1 and six at an IV dose of 0.06 mg·kg ^-1 ; one subject at a s.c. dose of 0.3 mg·kg ^-1 and two at a s.c. dose of 0.6 mg·kg ^-1 ). In these subjects, a decrease in the number of plasma blasts and NK cells was observed, suggesting that these symptoms are the result of depletion of cell populations expressing CD38.

Table 14. Number and percentage of subjects with clinical CRS IV infusion AB79 (mg kg ^-one ) Number of subjects (%) Placebo pool (n=12) 0.0003
(n=4) 0.001
(n=4) 0.003
(n=4) 0.01
(n=6) 0.03
(n=6) 0.06
(n=6) SVC 0 0 0 0 0 1 (16.7) 6 (100) Severity Light All lungs S / c injection AB79 (mg kg ^-one )
Number of subjects (%) Placebo pool (n=8) 0.03
(n=6) 0.1
(n=6) 0.3
(n=6) 0.6
(n=6) SVC 0 0 0 1 (16.7) 2 (33.3) Severity Light All lungs

[00391] Only mild, transient CRS cases have been observed following s.c. injection, most of which resolved within 7 days. These reactions were inversely dose-response as reactions occurred in five of the six subjects receiving the lowest s.c. dose and only one subject in each of the two highest dose groups.

[00392] The concentrations of the AB79 antibody in all blood serum samples taken at control time points in the IV cohort from No. 1 (0.0003 mg kg ^-1 ) to No. 4 (0.01 mg kg ^-1 ) were less than the lower limit of quantitation (LOQL) for this detection method (i.e. 10 ng·ml ^-1 ), presumably due to low doses and the absence of anti-drug antibodies (data not shown). After an IV infusion of 0.03 and 0.06 mg·kg ^-1 AB79, the maximum observed serum concentration ( C _max ) was 21.4 and 100.4 ng·ml ^-1 , respectively, and occurred 5 minutes after the end of infusion (Table 15 and Fig. 41). Subsequently, serum concentrations declined rapidly to levels below the LEL within 1 or 4 hours after the end of the infusion, respectively, and exposures could not be accurately calculated. It turned out that C _max increases approximately five times with a doubling of the dose from 0.03 to 0.06 mg·kg ^-1 . Due to limited data on AB79 serum concentrations (samples were taken at one to three time points per subject) available for the 0.03 and 0.06 mg kg ^-1 IV dose cohorts, there were it is impossible to accurately calculate the PK parameters, except for the time to reach the maximum concentration in the blood serum ( t _max ) and C _max .

Table 15 Summary of AB79 pharmacokinetic parameters following a single 2-hour IV infusion of AB79 at doses of 0.03 and 0.06 mg kg ^-1 or a single s.c. injection of AB79 at a dose of 0.6 mg kg ^-1 in healthy controls subjects

Route of administration Dose n t _max (h) n=6 C _max (ng ml ^-one ) n=6 PPK _last (ng day ^-one ml ^-one ) n=6 I/V 0.03 mg kg ^-1 6 2.09 (2.07; 2.67) 21.4 (39) N/A 0.06 mg kg ^-1 6 2.09 (2.07; 2.13) 100.4 (52) N/A PC 0.6 mg kg ^-1 6 23.87 (7.98; 96.02) ^a 23.0 (67) 90.4 (92)

^and n = 5. Values are mean (%CV), except for t _max where the medians (minimum and maximum) are presented. AUC _last - area under the curve serum concentration-time from time 0 to the time of the last concentration measurement; C _max - maximum observed concentration in blood serum; KV - coefficient of variation; in / in - intravenously; N/A - not applicable; s / c - subcutaneously; t _max is the time to reach the maximum serum concentration.

[ ⁰⁰³⁹³ ] Serum concentrations of ^AB79 in all subjects in s.c. points in time. Following a single s.c. injection of AB79 at 0.6 mg.kg ⁻¹ , five subjects from this cohort experienced a median t _max at about 24 hours post-injection. The mean C _max of all six subjects (including one subject with serum concentrations below the LLQL) in this group was 23.0 ng ml ^-1 , which is approximately 23% of the C _max after a 2-hour IV infusion into dose of 0.06 mg·kg ^-1 (one tenth of the SC dose in this cohort). Serum concentrations of AB79 gradually decreased to below the LLOQ 3-14 days after injection (Fig. 41). One subject from the 0.6 mg·kg ⁻¹ dose cohort had an AB79 level below the detection threshold during the entire PK sampling period. Compared to PK parameters from IV cohorts, higher inter-subject variability was observed after s.c. injection of 0.6 mg·kg ^-1 . Individual t _max ranged from about 8 to 96 hours (0.33 to 4 days) after injection into five subjects at measurable concentrations in the respective cohort.

[00394] In cohorts administered with AB79 by intravenous infusion, a dose-dependent decrease in NK cell levels was observed at doses of ≥0.003 mg·kg ^-1 , with a ≥90% decrease in all subjects receiving infusions with a dosage of 0.06 mg·kg ^{- 1} (Fig. 42). The effective concentration at half-maximal response (EC ₅₀ ) was below the LRQ of the PK assay method (i.e. 10 ng ml ^-1 ), however, the 75% maximum effective concentration (EC ₇₅ ) for reducing NK cells at in/in the introduction of AB79 was 21.4 ng·ml ^-1 (table 15). While NK cell levels were consistently below baseline by the end of the infusion, the duration of recovery to baseline levels (<-20%) was variable and usually dose-related; to restore NK cell levels to baseline for doses of 0.003; 0.01; 0.03 and 0.06 mg·kg ^-1 required an average of 4, 4, 6 and 8 days, respectively (Fig. 43). No clinically significant reductions in total lymphocytes, B and T cells, T helper and cytotoxic T cells, granulocytes, erythrocytes, or platelets were observed with intravenous administration of AB79 (data not shown).

[00395] In cohorts administered AB79 by sc injection, a dose-dependent decrease in NK cells (Fig. 42) and plasma blasts (Fig. 43) was observed at doses ≥0.1 mg kg^-one with ≥90% reduction in plasma blasts in all subjects treated with 0.6 mg kg injections^-one. 75% reduction in NK cells occurred at 0.6 mg kg^-one (data not shown) withC _maxequal to 23.0 ng ml^-one (Table 15). Plasma blast and NK cell levels were down from baseline within 8 hours post-injection and hadt _maxequal to 48 hours. The duration of recovery to baseline levels was variable; to restore to baseline (i.e. within -20% of baseline levels) for doses of 0.1; 0.3 and 0.6 mg kg^-one it took an average of 4, 78 and 50 days, respectively (data not shown). Decrease was minimal or absent for measures of total lymphocytes, B and T cells, cytotoxic T cells, T helpers, monocytes (Fig. 43), and granulocytes, erythrocytes, or platelets (data not shown).

[00396] While total immunoglobulin levels remained within the NWP, total IgM levels were decreased in the AB79 0.03 and 0.06 mg kg ^-1 IV cohorts, and on days 15-64 were significantly (P < 0.05) lower than the corresponding timed placebo control group (FIG. 44, upper panels). A significant ( P <0.01) decrease in total IgM was also observed on days 15-64 for AB79 administered subcutaneously at doses of 0.3 and 0.6 mg·kg ^-1 (Fig. 44, lower panels). IgM levels showed a tendency to recover to the initial level on the 78th day. No significant effect of AB79 on IgA and IgG levels was observed either with IV or SC administration (data not shown).

[00397] Of the 54 subjects who received ^AB79 , one subject from the s.c. , high (namely 160, 1280 and 320) anti-AB79 antibody titer (data not shown). Serum AB79 concentrations in this subject were below the LLQV throughout the study period, as were AB79 levels in anti-drug antibody (AMD) negative subjects from the same cohort, so a potential effect of ANTI on AB79 pharmacokinetics could not be determined. . It should also be mentioned that APLP did not coincide with AEs.

[00398] One of the therapeutic strategies for the treatment of SLE and RA is to reduce the level of CD38+ lymphocytes, based on several publications that demonstrate the relationship between the level of CD38+ lymphocytes and disease activity (Cole et al. (2018) Arthritis Res. Ther. 20 (1) : 85; Kraan et al. (1999) Rheumatology (Oxford) 38 (11): 1074-1080; Vital et al. (2011) Arthritis Rheum. 63 (10): 3038-3047; Banchereau et al. (2016) Cell 165 (3): 551-565 and Grammer et al (2003) J. Clin Invest 112 (10): 1506-1520). This can be achieved with the AB79 antibody as it specifically binds to CD38 and depletes cells (Smithson et al. (2017) J. Immunol. 198 (1 Suppl): 224.20). The addition of AB9 to blood or bone marrow samples from patients with SLE or RA resulted in a reduction in the plasma cell population and decreased production of autoantigen-specific antibodies (Wang et al. (2016 ACR/ARHP Annual Meeting abstract 1085) Arthritis Rheum. 68 (suppl 10) The AB79 antibody also caused a reduction in the number of lymphocytes expressing high levels of CD38 in monkeys (Roepcke et al. (2018) Pharmacol Res Perspect. 6 (3): e00402), prevented the development of CIA when administered prophylactically, and suppressed the progression of arthritis when administered therapeutically (Smithson et al. (2017) J. Immunol. 198 (1 Suppl): 127.17) Therefore, the aim of this study was to characterize the safety, tolerability, PK and PD for a single intravenous infusion and subcutaneous injection of AB79 in healthy subjects. and to determine whether a clinical trial is warranted in patients.

[00399] This study represents the first characterization of tolerability, PK and PD of an anti-CD38 cytolytic antibody administered to healthy subjects. All doses of AB79 in this study were well tolerated. The intensity of adverse events was mild to moderate, but most were mild (Table 13). There were no serious adverse events or deaths, and none of the adverse events resulted in termination of the study or visits. No notable lab results, ECG, vital signs, or physical examinations related to exposure to AB79 have been reported. While no infusion-related reactions were observed in this study, CRS occurred in seven subjects in the AB79 IV infusion group and in three subjects in the AB79 s.c. group (Table 14). CRS cases were accompanied by a decrease in the number of plasma blasts and NK cells (Figs. 42 and 43), a moderate increase in the level of cytokines (eg, TNF-α, IL-1β and IL-6; data not shown) and C-reactive protein in the blood ( data not shown). These data are consistent with macaque studies illustrating that cell depletion coincided with an increase in serum levels of TNF-α (Roepcke et al. (2018) Pharmacol Res Perspect. 6 (3): e00402). Compared with the intravenous groups, the subcutaneous groups had a lower incidence of CRS and a minimal increase in cytokines at comparable peripheral blood concentrations of AB79 (e.g., 0.03 mg kg ^-1 iv vs. 0.6 mg kg ^{- 1} subcutaneously). These results are consistent with the lower rate of infusion reactions for subcutaneous daratumumab compared to intravenous administration in patients with refractory myeloma. For intravenous and subcutaneous administration of daratumumab, dosage forms of different composition were used, while for intravenous and subcutaneous administration of AB79 to healthy subjects, the dosage form of the same composition was used. The data for AB79 demonstrate that the lower incidence of CRS is primarily due to the subcutaneous route of administration rather than differences in formulation.

[00400] Preclinical evaluation of AB79 in macaques suggested that the serum area under the concentration-time curve increased more than proportionally to dose after single IV and SC administration of AB79 (Roepcke et al. (2018) Pharmacol Res Perspect. 6 (3): e00402). Due to the limited amount of data on serum concentration in healthy people, a formal assessment of dose proportionality or bioavailability with subcutaneous administration of AB79 could not be carried out. However, C _max after IV infusion seemed to increase more than proportionally to the dose (namely, C _max increased approximately five-fold with a two-fold increase in dose from 0.03 to 0.06 mg kg ^-1 ), which is comparable with a similar trend observed in macaques with AB79 administration, and with data published for other target-mediated elimination monoclonal antibodies. Normalized relative to the dose C _max after s / c injection was significantly lower than after a 2-hour / infusion. After reaching _Cmax , serum concentrations decreased much more slowly after s / c injection and were maintained above LLQL much longer compared to iv infusion.

[00401] The pharmacodynamic response of healthy subjects to intravenous administration of AB79 is the most compelling example of depletion of a population of NK cells by a monoclonal anti-CD38 antibody described to date. Antibody AB79 at a dose of 0.06 mg·kg ^-1 resulted in an average C _max of 100.4 ng·ml ^-1 and a decrease in peripheral blood NK cells in each healthy subject at least 90% below baseline levels (Fig. 42). This degree of NK cell reduction was not achieved in patients with relapsed/refractory MM treated with intravenous _daratumumab up to 24 mg kg ^-1 and whose mean Cmax was up to 573 µg ml ^-1 (Clemens et al (2017) Clin Pharmacokinet 56 (8) : 915-924 Xu et al (2017) Clin Pharmacol Ther 101 (6): 721-724). It is not known whether this difference in drug potency is the result of differences in study populations and/or properties of the respective antibodies. While normal subjects and myeloma patients have similar numbers of NK cells and these cells express a similar density of CD38 (Krejcik et al. (2016) Blood 128 (3): 384-394), myeloma patients have more high levels of other CD38+ target cells (such as myeloma cells), which may contribute to differences in NK cell depletion activity between these populations. A more precise analysis requires PK and PD data for AB79 in a similar patient population with relapsed/refractory myeloma.

[00402] An attribute of higher potency is that less amount and volume of therapeutic agent is required to obtain comparable pharmacodynamic effects. Patients treated with an IV infusion of anti-CD38 mAb may benefit from administration of this antibody by small volume SC injection, as it can be administered within a minute, as opposed to the 2-4 hours typically required to receive an IV infusion, and in chronic therapy, s.c. injection can potentially be safely administered by the patient at home with convenience and pharmacoeconomic advantages. This study represents the first demonstration of cell reduction following SC injection of a cytolytic anti-CD38 antibody in human subjects, demonstrating that SC injection is better tolerated than iv infusion. This study is also the first report that s.c. injection of anti-CD38 antibody with a volume of 1 ml and a dose of 0.1 to 0.6 mg kg ^-1 causes the maximum pharmacodynamic effect (for example, a decrease in the level of plasmablasts by >90% ) in the peripheral blood. Antibody AB79 led to a reduction in the number of plasma blasts and NK cells in a dose-dependent manner at intravenous doses ≥0.1 mg·kg ^-1 (Fig. 42 and 43). The corresponding EC ₉₀ concentration for plasmablast reduction was approximately 23.0 ng·ml ⁻¹ , while for NK cell reduction it was 100.4 ng·ml ⁻¹ (Table 15). This potential difference in sensitivity may be related to the level of CD38 expression that is characteristic of these cell populations; plasmablasts express about five times higher density of CD38 than NK cells (Krejcik et al. (2016) Blood 128 (3): 384-394). Whether such a difference exists in patients with SLE, RA, and/or MM is not known, as data describing the levels of plasma blasts and NK cells in these patient populations have not been reported to date. If patients have comparable activity, then the efficacy of a small volume SC injection could potentially provide therapy for patients without access to an infusion center and reduce overall healthcare costs.

[00403] Circulating monocytes, B and T cells express lower levels of CD38 in healthy subjects and these cell types have not been reduced by AB79 to the same extent as plasmablasts and NK cells. These data are consistent with those described for cynomolgus monkeys after an initial dose of AB79 as well as after 3 months of chronic exposure; NK cells uniformly expressed high levels of CD38 and were more sensitive to AB79 than B and T lymphocytes, which express CD38 heterogeneously and usually at lower levels than NK cells (Roepcke et al. (2018) Pharmacol Res Perspect. 6(3): e00402). These data are also consistent with those described for patients with relapsed/refractory myeloma who were treated chronically with daratumumab; no effect on monocytes was observed, and subpopulations of CD38+ B- and T-cells were reduced in peripheral blood (Krejcik et al. (2016) Blood 128 (3): 384-39).

[00404] Ig is mainly produced by plasma cells found in lymphoid tissues (eg, bone marrow, lymph nodes, spleen, etc.). A similar effect has been described in SLE patients exposed to multiple doses of the proteasome inhibitor bortezomib; there was a significant decrease in the level of total IgM (34%), IgA (34%) and IgG (15%) in the blood serum, which corresponded to a decrease in the level of blood plasma blasts (54%), bone marrow plasma cells (50%) and indicators of disease activity scores (Alexander et al. (2015) Ann. Rheum. Dis. 74 (7): 1474-1478). Taken together, these data indicate that AB79 may be effective in reducing Ig levels and potentially in selectively reducing both IgM and IgA levels compared to IgG. This activity profile may provide therapeutic benefit in IgM-, IgA- and IgG-associated nephropathies while minimizing suppression of other components of the immune system.

[00405] In conclusion, a single dose of AB79 administered intravenously or subcutaneously was well tolerated by healthy subjects. AB79 caused depletion of the plasmablast and NK cell population and decreased levels of serum IgM and IgA. This plasmacytolytic activity may be useful in the treatment of various hematological malignancies and/or immunological disorders associated with dysregulation of plasma cells, pathogenic antibodies, immunoglobulins and/or NK cells.

Inclusion by reference

[00406] The contents of all cited references (including references to literature, patents, patent applications, and websites) that may be cited in this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited in this document, to the same extent as if each individual reference were specifically and individually designated to be incorporated as a reference in its entirety for any purpose.

Equivalents

The disclosure of the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Thus, the foregoing embodiments are to be considered in all respects as illustrative, and not as limiting, of the disclosure of the present invention. Therefore, the scope of the disclosure of this invention is indicated by the appended claims and not by the foregoing description, and therefore, all changes that fall within the meaning and equivalence range of the claims are considered to be included in this document. Modifications to carry out the invention that are obvious to those skilled in the art are intended to be within the scope of the appended claims.

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SEQUENCE LIST

<110> TAKEDA PHARMACEUTICAL COMPANY LIMITED

Dahl, Martin

Fedik, Eric

Evans, Robert

Zhao, Lin

<120> SUBCUTANEOUS ANTI-CD38 ANTIBODIES

<130> 101588-5009-WO

<140> PCT/US19/13547

<141> 2019-01-14

<150> US 62/617,146

<151> 2018-01-12

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 300

<212> PROTEIN

<213> Artificial sequence

<220>

<223> human CD38

<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> Artificial sequence

<220>

<223> CD38 cynomolgus macaque

<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> HCDR1 AB79

<400> 3

Gly Phe Thr Phe Asp Asp Tyr Gly

fifteen

<210> 4

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HCDR2 AB79

<400> 4

Ile Ser Trp Asn Gly Gly Lys Thr

fifteen

<210> 5

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HCDR3 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> LCDR1 AB79

<400> 6

Ser Ser Asn Ile Gly Asp Asn Tyr

fifteen

<210> 7

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR2 AB79

<400> 7

Arg Asp Ser

one

<210> 8

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR3 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> Chain segment amino acid sequence (VH)

<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> Chain segment amino acid sequence (VL)

<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> 453

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Chain segment amino acid sequence (HC)

<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 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> 12

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Chain segment amino acid sequence (LC)

<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 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> 13

<211> 318

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human CD157

<400> 13

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

<---

Claims (30)

1. A method of treating a disease with increased expression of CD38 in an individual, wherein the method comprises subcutaneously administering to an individual a unit dosage form of an isolated human anti-CD38 antibody, where the anti-CD38 antibody contains heavy chain variable region (VH) containing CDR1 with the amino acid sequence of SEQ ID NO:3, CDR2 with the amino acid sequence of SEQ ID NO:4 and CDR3 with the amino acid sequence of SEQ ID NO:5; and light chain variable region (VL) containing CDR1 with the amino acid sequence SEQ ID NO:6, CDR2 with the amino acid sequence of SEQ ID NO:7 and CDR3 with amino acid sequence SEQ ID NO:8, where the standard dosage form is in a volume of 3 ml or less at a dose of 0.03 to 0.6 mg per 1 kg of body weight. 2. The method according to claim 1, where the VH region has the amino acid sequence of SEQ ID NO:9 and the VL region has the amino acid sequence of SEQ ID NO:10. 3. The method of claim 1, wherein the anti-CD38 antibody comprises a heavy chain with the amino acid sequence of SEQ ID NO:11 and light chain with the amino acid sequence of SEQ ID NO:12. 4. The method of any one of the preceding claims, wherein the unit dosage form is in a volume of 2 ml or less. 5. The method of any one of the preceding claims, wherein the unit dosage form is in a volume of 1 ml or less. 6. The method of any one of the preceding claims, wherein administration of the anti-CD38 antibody does not cause hemolytic anemia or thrombocytopenia. 7. The method according to any one of paragraphs. 1-5, where administration of an anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of treatment-related adverse events (AEs), 3 -th or 4th degree, selected from the group consisting of anemia, hemolytic anemia, thrombocytopenia, fatigue, infusion reactions (IR), leukopenia and lymphopenia. 8. The method of any one of the preceding claims, wherein administration of the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% reduction in the red blood cell population. 9. The method of any one of the preceding claims, wherein administration of the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% reduction in the platelet population. 10. The method of any one of the preceding claims, wherein the disease is an autoimmune disease or cancer. 11. The method according to any one of paragraphs. 1-9, wherein the disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), myasthenia gravis (MG), optic neuromyelitis (ONN) , immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), antiphospholipid syndrome (APS), pemphigus vulgaris (PO), pemphigus foliaceus (PL), anti-NMDA receptor encephalitis (ANRE), autoimmune hemolytic anemia (AHA), Graves' disease, membranous nephropathy, Sjögren's syndrome (SS), ANCA-associated vasculitis, acquired epidermolysis bullosa (PBE), bullous pemphigoid (BP), Hashimoto's thyroiditis, scleroderma, IgG4-associated disease, and graft versus host disease. 12. The method according to any one of paragraphs. 1-9, wherein the disease is selected from the group consisting of multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-cell lymphoma, and Burkitt's lymphoma. 13. The method of claim 12 wherein the disease is multiple myeloma. 14. The method of claim 13, wherein the disease is relapsed multiple myeloma (RMM), relapsed and refractory multiple myeloma (RRMM), or newly diagnosed multiple myeloma (BMM). 15. The method of any one of the preceding claims, wherein the human anti-CD38 antibody is administered in the form of a pharmaceutically acceptable composition.

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