Subcutaneous administration of anti-CD38 antibody

Abstract

A method for subcutaneously administering an isolated anti-CD38 antibody at low doses is disclosed. This method provides effective treatment for autoimmune diseases, including hematological disorders, and cancer. A unit dosage form of the anti-CD38 antibody is also disclosed. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 617,146, filed January 12, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials Incorporated by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing, filed concurrently herewith, created on December 4, 2018, identified as follows: a 20 kilobyte ASCII (text) file named "101588-5009-WO-Sequence-Listing.txt,"

[0003] A method of administering isolated anti-CD38 antibodies at low doses and volumes via subcutaneous (SC) administration is disclosed. [Background technology]

[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 Aplysia ADPR cyclase. This enzyme family has the unique ability to convert NAD to cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate. CD38 is a Ca 2+CD38 is involved in signal transduction through the recruitment and tyrosine phosphorylation of numerous signaling molecules, including phospholipase Cγ, ZAP-70, Syk, and c-cbl. Based on these observations, CD38 is a key signaling molecule in the maturation and activation of lymphoid cells during normal development. Among hematopoietic cells, CD38-mediated signaling has been ascribed various functional consequences, including regulation of lymphocyte proliferation, cytokine release, B cell and myeloid cell development and survival, and induction of dendritic cell (DC) maturation.

[0005] CD38 is expressed on immature hematopoietic cells, downregulated in mature hematopoietic cells, and re-expressed at high levels on activated lymphocytes and plasma cells. For example, high CD38 expression is observed on activated B cells, plasma cells, activated CD4+ T cells, activated CD8+ T cells, NK cells, NKT cells, mature DCs, and activated monocytes (U.S. Patent No. 8,362,211). CD38 deficiency in mice is associated with reduced levels of peripheral T regulatory and invariant NKT cells, defective humoral B cell responses and DC trafficking, and an attenuated form of collagen-induced arthritis (CIA) (Chiba et al. (2005) Arthritis. Rheum. 52:1941-48).

[0006] The presence of autoantibodies against CD38 is associated with many diseases, such as 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 reported in a variety of diseases, including autoimmune diseases and cancer, including 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), neuromyelitis optica (NMO) (Chihara et al. (2011) Proc. Natl. Acad. Sci. USA 108(9):3701-6), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP) (Behzad et al. (2018) APMIS 126(6):523-532), and antiphospholipid syndrome (APS) (Alvarez-Rodriguez et al. (2018) APMIS 126(6):523-532). et al. (2018) Int. J. Mol. Sci. 19(2):pii), pemphigus vulgaris (PV), pemphigus foliaceus (PF), anti-NMDAR encephalitis (NMDR), autoimmune hemolytic anemia (AIHA), Graves' disease, membranous nephropathy, Sjögren's syndrome (SS), ANCA vasculitis, epidermolysis bullosa pustulosis (EBA), bullous pemphigoid (BP), Hashimoto's thyroiditis, scleroderma, and IgG4-related disease. Patients with RA have increased plasma cells in joint tissue compared with controls. Patients with SLE have increased plasmablasts in the peripheral blood of patients with more active disease. However, current CD20-based B cell depletion therapies, such as rituximab, effectively deplete CD20+ B cells but cannot directly and effectively deplete plasma cells or plasmablasts because they do not express CD20. Therefore, patients with RA or SLE who have high levels of plasma cells or plasmablasts are unlikely to derive substantial clinical benefit from CD20-based therapy.

[0008] Therapies targeting CD38, which is highly expressed on plasma cells, plasmablasts, NK cells, activated B lymphocytes, plasmacytoid dendritic cells, and activated T cells, may provide effective treatment for RA and SLE, as well as other diseases characterized by CD38 expression. The level of CD38-expressing plasmablasts in the peripheral blood of adult SLE patients treated with rituximab and oral steroids was the best predictor of time to relapse. Furthermore, circulating immunoglobulin (Ig)-secreting cells expressing high levels of CD38 have been identified in the peripheral blood of SLE patients with active disease, and levels of this subset are associated with reduced treatment-induced anti-double-stranded DNA (anti-dsDNA) antibody levels, proteinuria, and disease activity (Grammer et al. (2003) J. Clin. Invest. 112:1506-1520). Furthermore, plasma cells are sensitive to proteasome inhibition and are reduced in the peripheral blood of highly refractory SLE patients exposed to bortezomib (Alexander et al. (2015) Ann. Rheum. Dis. 74(7):1474-1478). This reduction coincided with a decrease in anti-dsDNA antibodies and a corresponding improvement in disease activity in each patient. Unfortunately, the therapy was associated with treatment-emergent adverse events (TEAEs, e.g., neuropathy, diarrhea) that may be attributable to proteasome inhibition of non-lymphoid cells (e.g., neurons, epithelial cells). Collectively, these data indicate that specifically reducing CD38-expressing plasma cells may offer benefits that improve the risk profile of refractory SLE patients. Current therapies for both RA and SLE produce major clinical responses and sustained remission in only a minority of patients, necessitating the urgent exploration of additional therapeutic mechanisms.

[0009] Increased expression of CD38 has been reported in various diseases derived from hematopoietic system and cell lines derived from it, and has been reported as a negative prognostic marker for blood cancer.Such diseases include but are not limited to multiple myeloma (MM), chronic lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia (B-CLL), including B and T acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia, Waldenstrom's macroglobulinemia, mantle cell lymphoma, prolymphocytic / myeloid 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 (BL), T-cell lymphoma (TCL), hairy cell leukemia (HCL), and Hodgkin's lymphoma (HL). Furthermore, CD38 expression is a prognostic indicator for patients with conditions such as B-CLL (Durig et al. (2002) Leukemia 16:30-35; and Morabito et al. (2001) Leukemia Res. 25:927-932) and acute myeloid leukemia (Keyhani et al. (1999) Leukemia Res. 24:153-159). Thus, CD38 provides a useful target for the treatment of hematopoietic diseases.

[0010] Several anti-CD38 antibodies are currently undergoing clinical trials for the treatment of CD38-associated cancers. However, these prior art therapeutic anti-CD38 antibodies bind to red blood cells (RBCs) and platelets, which may explain why higher doses are required to overcome the sink caused by RBC binding. For example, treatment with daratumumab (anti-CD38 IgG1 mAb Darzalex®, FDA-approved and commercially available from Janssen Oncology) requires very high doses (≥ 16 mg / kg) and an intensive regimen (8 doses weekly, 8 doses 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 RBCs and platelets results in a positive antiglobulin test (indirect Coombs test), which can persist for 6 months after the last daratumumab infusion (Sullivan et al. (2017) Blood 129(22):3033-3037). This is an important property of daratumumab and other antibodies that bind to red blood cells. Although CD38 is expressed at approximately 1,000-fold lower levels on RBCs than on myeloma cells (deWeers et al. (2011) J. Immunol. 186(3):1840-1848), there are approximately 36,000 red blood cells for every myeloma cell in the blood of MM patients with active disease (Witzig et al. (1993) Cancer 72(1):108-113). Thus, there are 36 times more CD38 molecules expressed by RBC populations compared with tumor cell populations. It has therefore been hypothesized that RBCs bind to a significant amount of the anti-CD38 antibody administered, resulting in the need to administer large doses in order to have sufficient levels of anti-CD38 antibody to achieve a therapeutic effect on tumor cells.

[0011] Therefore, treatment using anti-CD38 antibodies currently focuses on intravenous (IV) administration due to the large amounts of antibody required to achieve therapeutic efficacy, which are not suitable for subcutaneous administration, and the limitations of concentrated Ab formulations. For example, a 1200 mg dose of daratumumab administered IV over two hours has been approved for the treatment of relapsed and refractory multiple myeloma (RRMM) and newly diagnosed multiple myeloma (NDMM). A subcutaneous (SC) formulation of daratumumab, consisting of 1800 mg in 15 mL, which must be co-formulated with Enhance™ (containing halozyme to speed absorption), is administered eight times weekly, eight times every other week, and then monthly, and is currently in phase 3 trials for RRMM.

[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, which corresponds to 700–1400 mg per 70 kg patient, consistent with a projected infusion volume of 3.5–14 mL.

[0013] The higher doses and volumes required for prior art anti-CD38 antibodies currently in the clinic can also cause serious side effects such as hemolytic anemia, a condition in which RBCs are destroyed faster than they are replaced. In an open-label, single-arm study, isatuximab was administered intravenously to a total of 97 patients at 3 mg / kg every 2 weeks (Q2W; n=23), 10 mg / kg Q2W for two cycles followed by Q4W (n=25), 10 mg / kg Q2W (n=24), and 20 mg / kg weekly for four doses (1 cycle) followed by Q2W (n=25). The most common severe (grade 3 / 4) adverse event was anemia, affecting 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 daratumumab trial, 45% of all patients experienced anemia (19% of which were grade 3) and 48% of patients experienced thrombocytopenia (10% of which were grade 3 and 8% of which were grade 4) (see, e.g., 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 challenges of administering large amounts of anti-CD38 mAb subcutaneously to patients illustrate the need in the art for more potent anti-CD38 antibodies, as more potent antibodies can achieve the desired pharmacological effect with smaller amounts / volumes, thereby making the administration more effective. More potent antibodies may result in smaller doses being administered more effectively SC than daratumumab (Darzalex) in patients for whom depletion of CD38-expressing cells is indicated, such as for the treatment of autoimmune diseases and hematological cancers. The advantages of administering smaller amounts of drug include administration requiring seconds, compared to minutes for SC-administered daratumumab and hours for IV-administered daratumumab, isatuximab, and MOR202, as well as fewer infusion reactions (as seen with SC administration of daratumumab). Reducing the time patients need to spend at an infusion center means enabling home treatment options, increased efficiency of administration and increased productivity at infusion centers (reducing healthcare costs per patient as a result of more efficient facilities), and wider availability when medications can be used by patients without accessing an infusion center.

[0015] AB79 is a fully human immunoglobulin IgG1 monoclonal antibody that specifically binds to CD38 with high affinity (Kd = 3.5 nM) (U.S. Patent No. 8,362,211, the entire contents of which are incorporated herein by reference). AB79 inhibits the growth of CD38-expressing tumor cells by cell depletion via antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). AB79 also reduces the levels of plasma cells and plasmablasts in blood isolated from healthy subjects and patients with systemic lupus erythematosus (SLE). In SLE, 80% of the plasma cell population, including short-lived and long-lived plasma cells, was reduced. Furthermore, the number of cells producing pathogenic autoantibodies, such as VH4-34 9G4+ antibodies (70% reduction), anti-Ro antibodies (70% reduction), and anti-dsDNA antibodies (80% reduction), was also reduced. The anti-human CD38 mAb daratumumab also dose-dependently depletes CD38-expressing plasmablasts and plasma cells in vitro in samples from SLE and RA patients. In contrast to daratumumab, AB79 cross-reacts with CD38 expressed by cynomolgus monkeys, providing a unique opportunity to determine whether reducing the level of CD38-expressing cells affects inflammation and tissue damage in non-human primate models of autoimmune disease. In healthy cynomolgus monkeys, the depletion efficiency of lymphocytes, B, T, and NK cells was positively correlated with CD38 expression levels and AB79 dose levels (PCT Application No. PCT / US2017 / 042128, U.S. Patent No. 8,362,211).

[0016] Because many CD38 antibodies in the clinic are not suitable for low-dose or low-volume subcutaneous administration and have dangerous side effects, there remains a need in the art for safer, more convenient, and more effective antibody formulations for treating diseases in which binding to CD38 is demonstrated, such as autoimmune diseases and forms of hematologic cancer. Summary of the Invention

[0017] Provided herein are methods for treating diseases exhibiting binding to CD38, such as, for example, autoimmune diseases and hematological cancers, comprising subcutaneous administration of isolated anti-CD38 antibodies at unexpectedly low and / or small doses.

[0018] In one aspect, the invention provides a method of treating a disease exhibited by binding to CD38 in a subject, the method comprising subcutaneously administering to a subject having a disease exhibited by binding to CD38 a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the disease, wherein the anti-CD38 antibody comprises a variable heavy (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 SEQ ID NO: 5, and a variable light (VL) chain region 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 SEQ ID NO: 8.

[0019] In another aspect, the invention provides a method of treating a disease exhibited by binding to CD38 in a subject, the method comprising subcutaneously administering to a subject having a disease exhibited by binding to CD38 a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the disease, wherein the anti-CD38 antibody comprises a VH chain region comprising 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 SEQ ID NO: 5, and a VL chain region comprising 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 SEQ ID NO: 8, wherein the anti-CD38 antibody is administered in a volume of 3 milliliters or less.

[0020] In another aspect, the present invention provides a method of treating a disease exhibited by binding to CD38 in a subject, the method comprising subcutaneously administering to a subject having a disease exhibited by binding to CD38 a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the disease, wherein the anti-CD38 antibody comprises a VH chain region comprising 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 SEQ ID NO: 5, and a VL chain region comprising 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 SEQ ID NO: 8, and the anti-CD38 antibody is administered at a dose of 0.03 to 0.6 milligrams per kilogram of body weight.

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

[0022] In one aspect, administration of the 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% incidence of Grade 3 or 4 treatment-emergent adverse events (TEAEs) selected from the group consisting of anemia, hemolytic anemia, thrombocytopenia, fatigue, infusion-related reactions (IRR), leukopenia, and lymphopenia.

[0023] In one embodiment, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, less than 50% depletion of RBCs.

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

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

[0026] In one embodiment, 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), neuromyelitis optica (NMO), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), antiphospholipid syndrome (APS), pemphigus vulgaris (PV), pemphigus foliaceus (PF), anti-NMDAR encephalitis (NMDR), autoimmune hemolytic anemia (AIHA), Graves' disease, membranous nephropathy, Sjogren's syndrome (SS), ANCA vasculitis, epidermolysis bullosa (EBA), pemphigoid (BP), Hashimoto's thyroiditis, scleroderma, IgG4-related 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 embodiment, the hematological 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 myelogenous leukemia, B cell lymphoma, and Burkitt's lymphoma.

[0028] In one aspect, the hematological cancer is multiple myeloma. In certain embodiments, the multiple myeloma is selected from the group consisting of RRMM and EDMM.

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

[0030] In one embodiment, 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 embodiment, the therapeutically effective amount is a dosage of 0.03 to 0.6 milligrams per kilogram of body weight.

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

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

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

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

[0036] In another aspect, the invention provides a method of treating a hematological cancer in a subject, the method comprising subcutaneously administering to a subject having a hematological cancer a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the hematological cancer, 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 SEQ ID NO: 5, and a variable VL chain region 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 SEQ ID NO: 8.

[0037] In another aspect, the invention provides a method of treating a hematological cancer in a subject, the method comprising subcutaneously administering to a subject having a hematological cancer a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the hematological cancer, 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 SEQ ID NO:5, and a VL chain region 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 SEQ ID NO:8, wherein the anti-CD38 antibody is administered in a volume of 3 milliliters or less, 2 mL or less, or 1 mL or less.

[0038] In another aspect, the invention provides a method of treating a hematological cancer in a subject, the method comprising subcutaneously administering to a subject having a hematological cancer a therapeutically effective amount of an isolated human anti-CD38 antibody sufficient to treat the hematological cancer, wherein the anti-CD38 antibody comprises a VH chain region comprising 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 SEQ ID NO: 5, and a VL chain region comprising 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 SEQ ID NO: 8, wherein the anti-CD38 antibody is administered at a dose of 0.03 to 0.6 milligrams per kilogram of body weight.

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

[0040] In one aspect, administration of the 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% incidence of Grade 3 or 4 of one or more TEAEs selected from the group consisting of anemia, including hemolytic anemia, thrombocytopenia, fatigue, infusion-related reactions (IRR), leukopenia, and lymphopenia.

[0041] In one embodiment, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% depletion of RBCs.

[0042] In one embodiment, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% depletion of platelets.

[0043] In one embodiment, the hematological cancer is selected from the group consisting of multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myelogenous leukemia, B-cell lymphoma, NK / T-cell lymphoma, and Burkitt's lymphoma.

[0044] In one aspect, the hematological cancer is multiple myeloma. In certain embodiments, the multiple myeloma is selected from the group consisting of RRMM and EDMM.

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

[0046] In one embodiment, 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 embodiment, the therapeutically effective amount is a dosage of 0.03 to 0.6 milligrams per kilogram of body weight.

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

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

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

[0051] In one embodiment, 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 comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 9 and a light chain variable region amino acid sequence of SEQ ID NO: 10, wherein the isolated antibody binds to CD38, and 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 embodiment, the heavy chain comprises the amino acid sequence of SEQ ID NO:11 and the light chain comprises the amino acid sequence of SEQ ID NO:12.

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

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

[0056] In one embodiment, 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 the antibody in the treatment of a hematological cancer selected from the group consisting of multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myelogenous leukemia, B-cell lymphoma, and Burkitt's lymphoma.

[0058] In one aspect, the hematological cancer is multiple myeloma. In certain embodiments, the multiple myeloma is selected from the group consisting of RRMM and EDMM.

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

[0060] In one embodiment, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% depletion of RBCs.

[0061] In one embodiment, the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% depletion of platelets.

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

[0063] The objects and features of the present invention may be better understood by referring to the drawings described below. [Figure 1] Cynomolgus monkey (cyno) PK data for the SC-administered groups are shown in Table 2. Anti-drug antibodies (ADAs) were detected with a validated qualitative electrochemiluminescence (ECL) assay. The incidence increased over time and was affected when PK reached a certain threshold titer of approximately 1000 (approximately log(7)). [Figure 2] Figure 1 shows the cyno PK data and PK model for AB79. Panels A and B show the raw IV PK data from an eight-monkey study; Panel A represents the first 7 days after the first dose, and Panel B represents the entire observation period. SC data were omitted (see Figure 1 for SC data). Panel C depicts the final PK model structure, including the target-mediated pharmacokinetics (TMDD) model marked with a blue box. VC indicates the volume of the central compartment where AB79 concentration is observed (marked Conc). VP indicates the volume of the peripheral compartment. Rtotal represents the compartments of bound antibody and unbound receptor CD38. KSYN and KDEG represent the receptor production and degradation rate constants, and KINT represents the internalization rate constant (complex elimination rate constant). KSS is the steady-state constant, defined as KSS = (KOFF + KINT) / KON, where KOFF is the dissociation rate constant and KON is the association rate constant. Panels D–F show the overlay of the linear two-compartment model predictions (median, 95% prediction interval) without the TMDD component and the observed data for the lowest three doses (Study 8). Note the different time scales between Panels D, E, and F. [Figure 3]ADA efficacy and PK in a cynomolgus monkey toxicity study at week 13 are shown. The evaluation refers to the final population PK model (Figure 2, Table 2). The following goodness-of-fit (GOF) plots stratified by dose and route of administration are presented (Keizer et al. (2013) CPT Pharmacometrics Syst. Pharmacol. 2:e50): (1) conditional weighted residuals (CWRES) versus time; (2) observed concentration versus population model prediction; (3) CWRES versus population model prediction; and (4) observed concentration versus individual model prediction. [Figure 4] GOF plots for the final population PK model stratified by dose and route of administration (IV - cross, SC - triangle) are shown. Figure 4A shows the overall, Figure 4B is for the 0.03 mg / kg dose, Figure 4C is for the 0.1 mg / kg dose, Figure 4D is for the 0.3 mg / kg dose, Figure 4E is for the 1.0 mg / kg dose, Figure 4F is for the 2.0 mg / kg dose, Figure 4G is for the 3.0 mg / kg dose, Figure 4H is for the 30 mg / kg dose, Figure 41 is for the 80 mg / kg dose, and Figure 4J is for the 100 mg / kg dose. [Figure 5]Comparison of CD38 expression on the surface of human and monkey NK, B, and T cells is shown. Flow cytometry measurements were normalized, and signals are reported in molecules of equivalent soluble fluorescence (MOEF). Human and monkey blood lymphocytes bind similar levels of AB79. Direct comparison of CD38 expression levels on monkey NK cells (CD3-, CD159a+), B cells (CD3-, CD20+), T cells (CD3+), and human NK cells (CD3-, CD16 / CD56+), B cells (CD3-, CD19+), and T cells (CD3+) was assessed by flow cytometry. The median fluorescence intensity (MFI) of AB79 staining for each cell population was converted to MOEF units using a standard curve generated using Rainbow Beads (Spherotech; Lake Forest, IL). Data shown are from triplicates of each species and represent MOEF ± SD for each cell type. There are differences in CD38 expression among blood lymphocytes, with higher levels of AB79 binding to NK cells > B cells > T cells. The pattern of AB79 binding is similar among monkey blood cells, but the levels of AB79 binding / CD38 expression are lower. [Figure 6] Figure 6 shows inter- and intra-individual variability in T cell, B cell, and NK cell counts in placebo-treated animals from the study shown in Figure 5. Figure 6A is NK cells, Figure 6B is B cells, and Figure 6C is T cells. [Figure 7] Pre-dose NK, B, and T cell counts (cells / μL) stratified by study (top row) or gender (bottom row) are shown. Figure 7A shows NK cells by study, Figure 7B shows B cells by study, Figure 7C shows T cells by non-study prefecture, Figure 7D shows NK cells by male / female, Figure 7E shows B cells by male / female, and Figure 7F shows T cells by male / female. [Figure 8]Figure 8 shows AB79-dependent depletion of NK cells, B cells, and T cells. Graphs focus on changes occurring within the first 7 days after treatment with the first dose of AB79. Data from single- and multiple-dose studies with weekly or biweekly dosing schedules could be pooled. Figure 8A shows nadir cell depletion of NK cells, Figure 8B shows 1 week after the first dose of NK cells, Figure 8C shows the average NK cell profile per dose group, Figure 8D shows nadir cell depletion of B cells, Figure 8E shows 1 week after the first dose of B cells, Figure 8F shows the average B cell profile per dose group, Figure 8G shows nadir cell depletion of T cells, Figure 8H shows 1 week after the first dose of T cells, and Figure 8I shows the average T cell profile per dose group. Graphs A-C show the individual minimum cell counts (i.e., maximum PD effect), individual cell counts 7 days after the first dose, and the average NK cell depletion profile per dose and the PK-PD model structure, respectively. Graphs D-F show the same information for B cells, and graphs G-I show the same information for T cells. [Figure 9] Study 7 (Table 2) shows the effect of AB79 treatment on RBCs on day 2 post-dose (FIG. 9A) and total lymphocyte counts on the first day post-dose (FIG. 9B). [Figure 10]Figure 1 shows simulated human PK and NK cell, B cell, and T cell depletion profiles for AB79. Based on the scaled monkey PK and PK-PD models, five single IV and SC dose PK and cell depletion profiles were simulated (0.0003–1 mg / kg). The plots on the left show data after IV administration, while the plots on the right show data after SC administration. The first column of plots displays the PK profiles. The lower limit of quantitation (LLOQ) of 0.05 μg / mL is indicated by a horizontal dashed line. The PK of the lowest dose was completely overlaid by noise, and only at the 0.03 mg / kg dose did PK reach levels above the LLOQ. Figure 9A is AB79 via IV, Figure 9B is AB79 via SC, Figure 9C is IV in NK cells, Figure 9D is SC in NK cells, Figure 9E is IV in B cells, Figure 9F is SC in B cells, Figure 9G is IV in T cells, and Figure 9H is SC in T cells. [Figure 11] The design for the AB79 single-dose escalation toxicity study in healthy volunteers is shown. A total of six IV and four SC cohorts of 74 subjects were randomized to receive a single dose of AB79. Extensive blinded safety, PK, and PD data were reviewed after each cohort before dose escalation. Discontinuation criteria included target cell depletion to avoid potential immunosuppression in healthy volunteers. Subjects were followed for 92 days after dosing. [Figure 12] GOF plots of the PK-PD model stratified by route of administration (IV-red, SC-blue) are shown: FIG. 12A is NK cells, FIG. 12B is B cells, and FIG. 12C is T cells. [Figure 13]These results demonstrate that AB79 mediates depletion of monkey lymphocytes. Following a single IV dose of AB79, female cynomolgus monkeys (n = 4 / treatment group) showed dose-dependent depletion of blood NK cells > B cells > T cells, as quantified by flow cytometry with Flow-Count™ Fluorospheres (Beckman-Coulter). Samples were collected at pretreatment (week 1), day 1 (pre-dose), 15, 30 minutes, 1, 4, 8, 24, 48, 96, and 168 hours post-dose on days 10, 15, 22, 29, 36, 43, 50, and 57. For clarity, only data from the 2-week period are shown. Mean cell count values ​​were calculated at each time point and used to calculate % of baseline counts. Figure 13A shows T cells, Figure 13B shows B cells, and Figure 13C shows NK cells. [Figure 14] We demonstrate that AB79 treatment reduces the human tetanus toxoid (TTd) recall response. CB17 / SCID mice were treated with anti-asialoGM1 to deplete NK cells and then administered 25 x 106 human peripheral blood lymphocytes. Seven to 10 days later, serum samples were collected to assess human Ig levels, and Ig levels served as the criterion for randomization. Mice were given TTd to elicit the recall response and then treated with the indicated antibodies twice weekly for 10 days. Three days after the last treatment, serum was collected and analyzed for anti-TTd antibodies. AB79 dose-dependently suppressed the TTd recall response to a similar extent to Rituxan (Rtx) (isotype (Iso), Rtx, and AB79, all at 10 mg / kg). [Figure 15]This shows that AB79 does not induce cytokine induction. AB79 did not increase IL-6 levels in PBMCs collected from four different subjects after 24 hours of incubation compared to the IgG1 isotype control. The positive controls, PHA and anti-CD3, increased cytokine levels in all subjects, confirming the cells' ability to produce IL-6. Similar results were seen in PBMCs stimulated for 48 hours when IL-2, IL-4, IL-10, GM-CSF, IFNγ, and TNFα were tested (data not shown). Bars represent the following for each subject: (i) no Ab, (ii) isotype control, (iii) AB79, (iv) anti-CD3, and (v) PHA. Each value is the mean ± SD of triplicate wells measured 24 hours after incubation. [Figure 16A] Figure 16B shows the experimental setup for dry binding, dry binding, wet binding, and solubility (modified from Stebbings et al. (2007) J. Immunol. 179:3325-3331). [Figure 16B] This indicates that AB79 has no agonist activity. AB79 was highly concentrated when added to the wells in solution and the liquid was allowed to evaporate (dry-bound), as opposed to when it was bound to the wells in solution (wet-bound) or added directly to PBMCs (soluble). AB79 did not stimulate IL-6 or IL-2, IL-4, IL-8, IL-10, GM-CSF, IFNγ, or TNFα under any of the conditions tested after 24 hours. IL-8 was constitutively produced by PBMCs and was not altered by any treatment (data not shown). Each measurement is the average of triplicate wells measured 24 hours after incubation from a single subject. [Figure 17] Figure 1 shows an evaluation of the binding of AB79 to human RBCs. In this study, no binding of AB79 (solid histogram) or the isotype control (shaded histogram) to RBCs was observed in the whole blood of 30 human volunteers. Representative data from 15 of the 30 human volunteers is shown. [Figure 18] Figure 1 shows an evaluation of AB79 binding to cynomolgus monkey RBCs. In this study, no binding of AB79 (solid histogram) or isotype control (shaded histogram) to RBCs in whole blood from 30 cynomolgus monkeys was observed. Representative data from 15 of the 30 cynomolgus monkeys are shown. [Figure 19] Figure 1 shows an evaluation of the binding of AB79 to human platelets. In this study, no binding of AB79 (filled histogram) or isotype control (shaded histogram) to CD61+ gated platelets in whole blood of 30 human volunteers was observed. Representative data from 15 of the 30 human volunteers is shown. [Figure 20] Figure 1 shows an evaluation of AB79 binding to cynomolgus monkey platelets. In this study, no binding of AB79 (filled histogram) or isotype control (shaded histogram) to CD61+ gated platelets in whole blood from 30 cynomolgus monkeys was observed. Representative data from 15 of 30 cynomolgus monkeys are shown. [Figure 21] Figure 1 shows an assessment of AB79 binding to cynomolgus monkey CD45+ lymphocytes. Binding of AB79 to CD45+ lymphocytes in unlysed cynomolgus monkey whole blood. CD45+ lymphocytes were gated and then assessed for AB79 binding (solid histogram) or isotype control binding (shaded histogram). AB79 binding was detected in a subset of lymphocytes, as indicated by the percentage of cells to the right of the vertical dashed line. Little or no binding of the isotype control to lymphocytes was observed. [Figure 22] (A) Gating hierarchy for RBC identification and (B) lymphocyte identification of human RBCs is shown. [Figure 23]Antibody binding capacity for biotin-streptavidin-BV421 AB79 and biotin-streptavidin-BV421 daratumumab is shown. Bars represent (i) negative beads, (ii) negative control (Sav-Bv421 only), (iii) AB79-biotin / Sav-BV421 (5 μg / ml), (iv) daratumumab-biotin / Sav-BV421 (5 μg / ml), (v) AB79-biotin / Sav-BV421 (10 μg / ml), and (vi) daratumumab-biotin / Sav-BV421 (10 μg / ml). MFI = median fluorescence; Sav = streptavidin. A photomultiplier tube (PMT) voltage of 275 was used. [Figure 24] 1 shows the binding of biotin-streptavidin-BV421 AB79 and biotin-streptavidin-BV421 daratumumab 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), and (vi) daratumumab-biotin-strep-BV421 (10 μg / ml). Vertical lines represent standard deviation, n = 4 donors (3 donors for unlabeled). MFI = median fluorescence. [Figure 25] Binding of AB79 and daratumumab to human RBCs is shown (% positive results for individual donors). Peripheral blood from four healthy volunteers was incubated for 3 hours at RT on a gentle shaker with biotin-streptavidin-BV421 AB79 (0, 0.1, 10, 100 μg / ml) or biotin-streptavidin-BV421 daratumumab (0, 0.1, 1, 10, 100 μg / ml) in the presence or absence of unlabeled AB79 (500 μg / ml) or unlabeled daratumumab (500 μg / ml). Key:

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[0064] The present invention relates to a method for treating CD38-associated diseases by subcutaneous administration of low doses (≦600 mg) and volumes (≦3 mL) of anti-CD38 antibodies.

[0065] AB79, daratumumab, isatuximab, and MOR202 are IgG1-antibody-mediated tumor killing mechanisms that primarily involve antibody-dependent cellular cytotoxicity (ADCC). This mechanism requires effector cells, such as NK cells, to bind to antibodies on target cells, form a lytic synapse, and secrete cytotoxic agents. The frequency of these effector cells in the blood is orders of magnitude lower than that of RBCs and platelets. For example, the ratio of RBCs to NK cells in the blood is 20,000:1. Furthermore, CD38 molecules are expressed approximately 36 times more frequently on RBCs than on myeloma cells in patients with active disease. It is hypothesized that the effector activity of daratumumab, isatuximab, and MOR202 is primarily diverted from the tumor by anti-CD38 antibodies bound to RBCs and platelets, preventing the formation of a lytic synapse with the tumor, resulting in low ADCC efficiency. In contrast, reduced or more transient binding of RBCs and platelets by AB79 compared with daratumumab may allow effector cells to concentrate at the tumor, resulting in more efficient ADCC, higher tumoricidal activity, and a lower effective dose.

[0066] Treatment of patients with anti-CD38 antibodies that bind to RBCs and platelets can also cause life-threatening side effects. For example, treatment of RRMM with MOR202 has resulted in several serious treatment-emergent adverse events (TEAEs) (see, e.g., Raab et al. (2015) Blood 126:3035). The most common TEAEs of any grade were anemia (15 patients, 34%), fatigue (14 patients, 32%), infusion reactions (IRR) and leukopenia (13 patients, 30% each), lymphopenia and nausea (11 patients, 25% each). Grade ≥3 TEAEs were reported in 28 patients (64%), the most common of which included lymphopenia (8 patients, 18%), leukopenia (5 patients, 11%), and hypertension (4 patients, 9%). IRRs occurred primarily during the first infusion and were all grade 1-2 except for one patient (grade 3). Infections were commonly reported (26 patients, 59%) but were not considered treatment-related in most cases. MOR202 is only used clinically via IV infusion. This contrasts with the present invention, which allows for subcutaneous administration of AB79 at low doses and volumes, as described herein.

[0067] Other Morphosys antibodies that target CD38 are known (see, for example, WO 2006 / 125640, which discloses four human antibodies: MOR03077, MOR03079, MOR03080, and MOR03100, and two murine antibodies: OKT10 and IB4). These prior art antibodies are inferior to AB79 for various reasons. MOR03080 binds to human CD38 and cynomolgus monkey CD38, but has low affinity for human CD38 (Biacore K). D = 27.5 nm). OKT10 binds to human CD38 and cynomolgus monkey CD38, but has low / moderate affinity for human CD38 (Biacore K D =8.28 nm). MOR03079 binds to human CD38 with high affinity (Biacore K D=2.4 nm) but do not bind to cynomolgus monkey CD38. MOR03100 and MOR03077 bind to human CD38 with moderate or low affinity (Biacore K D In comparison, AB79 binds with high affinity to human CD38 and cynomolgus monkey CD38 (Biacore K for human CD38). D =5.4 nm). Furthermore, prior art antibodies have poor ADCC as well as CDC activity.

[0068] The advantage of more efficient ADCC is that anti-CD38 therapeutics can be delivered as small injections. When AB79 is formulated at a concentration of 135 mg / mL, an effective dose for an 80 kg myeloma patient can be administered as a single SC injection of less than 2.5 mL. In contrast, the SC dose of daratumumab being tested in Phase 3 clinical trials is 1800 mg suspended in a 15 mL co-formulation of Enhance™ (Halozyme).

[0069] The safety, tolerability, pharmacokinetics, and pharmacodynamics of AB79 administered IV and SC were initially characterized in monkeys. AB79 was administered as a single dose to cynomolgus monkeys (4 females / group) at 0.03, 0.1, and 0.3 mg / kg in sterile saline (IV) or SC diluents by IV bolus or SC injection. With the IV route, Cmax was approximately proportional to the dose of AB79 from 0.03 to 0.3 mg / kg, and AUC(0-t) was greater than that of 0.03 to 0.1 mg / kg, but was approximately proportional to the dose of AB79 from approximately 0.1 to 0.3 mg / kg. With the SC route, Cmax and AUC(0-t) increased with dose, but AUC(0-t) increased proportionally to dose over the range of 0.03 to 0.3 mg / kg after SC administration. 1 / 2The mean bioavailability via the SC route was estimated at 120–144 hours. The mean bioavailability via the SC route was approximately 100% (120%, 73%, and 120% at 0.03, 0.1, and 0.3 mg / kg, respectively). Administration of AB79 at 0.03, 0.1, or 0.3 mg / kg via a single intravenous or subcutaneous injection to female cynomolgus monkeys was well tolerated. The expected pharmacological effects of mild to moderate depletion of lymphocytes (T and B lymphocytes) and a dose-dependent reduction in NK cell populations were observed at all dose levels and with both routes, with the maximum cell depletion effect following SC administration being similar to or slightly less than that observed following IV administration at the same dose level (Roepcke et al. (2018) Pharmacol. Res. Perspect. 6(3):e00402). Based on the results of this study, the no-observed-adverse-effect level (NOAEL) was considered to be 0.3 mg / kg by both the IV and SC routes. Changes in lymphocytes and NK cells resolved after a 56-day AB79-free period. The serum AUC and Cmax associated with the NOAEL were 574 h*μg / mL and 7.94 μg / mL by IV and 698 h*μg / mL and 2.15 μg / mL by SC, respectively.

[0070] 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 1 mL subcutaneous (SC) injection in escalating dose cohorts of healthy human subjects. AB79 was well tolerated, with all adverse events (AEs) mild or moderate, and no withdrawals due to AEs or infusion reactions (Fedyk et al. (2018) Blood 132:3249). In the high-dose cohort, transient, mild-to-moderate increases in cytokine levels coincided with a reduction in CD38-expressing cells, and clinical symptoms primarily included fever, headache, and orthostatic hypotension. No significant findings in laboratory tests, electrocardiograms, vital signs, or physical examinations were reported in association with AB79 treatment. AB79 reduced plasmablast and natural killer (NK) cell levels at similar doses, with a maximum effective dose (ED ). 50) at 0.003 mg / kg IV and 0.1 mg / kg SC. Reductions in total immunoglobulin (Ig) M and A occurred without comparable changes in IgG. Total white blood cell, granulocyte, lymphocyte, red blood cell, and platelet counts were within normal ranges at all dose levels. In summary, AB79 selectively reduced plasmablast and NK cell levels in the peripheral blood of healthy subjects when administered IV or SC and was overall safe and well-tolerated. This plasmacytolytic profile could be useful in treating disorders caused by plasma cells or NK cells, their malignant counterparts (e.g., multiple myeloma and NK cell leukemia), and pathogenic antibodies or Ig.

[0071] Unlike therapeutics that target B-cell precursors of plasma cells (e.g., anti-BAFF mAbs (e.g., belimumab), anti-CD20 mAbs (e.g., rituximab), and BTK inhibitors (e.g., baricitinib)), AB79 directly targets plasma cells, potentially inducing a relatively rapid therapeutic response (e.g., disease remission) in Ig- or antibody-mediated diseases. These latter strategies indirectly target plasma cells and inhibit the differentiation of the former, thereby essentially eliminating the de novo generation of plasma cells. The existing pool of plasma cells remains relatively unaffected and continues to produce pathogenic antibodies / Ig throughout their lifespan. Therefore, the longevity of existing plasma cells, some of which may survive for decades, potentially causing a decline in pathogenic antibodies / Ig, and therefore, the onset of activity and efficacy may be slower than with AB79 in indirect strategies. The only other therapeutic agents demonstrated to directly reduce plasma cells are proteasome inhibitors (e.g., bortezomib), a class of drugs that are relatively poorly tolerated and include dose-limiting adverse events (e.g., neuropathy, diarrhea) attributable to proteasome inhibition in non-plasma cells because proteasomes are ubiquitously expressed in these tissues. Therefore, the specificity of AB79 for CD38 combined with the restricted expression profile of CD38 creates a mechanism of action that directly targets plasma cells while minimizing the impact of non-plasma cells, potentially resulting in rapid efficacy in diseases caused by plasma cells, their transformed counterparts, and / or pathogenic antibodies / Ig.

[0072] The anti-CD38 methods and unit dosages of the present disclosure provide, for the first time, therapeutically effective subcutaneous administration of low doses and volumes of anti-CD38 antibodies, thereby providing an unexpected benefit and avoiding the side effects, inconvenience, and expense of administering high-dose, systemic anti-CD38 antibody therapies.

[0073] 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 to treat a disease exhibiting binding to CD38, including hematological cancers. In some embodiments, the antibody for subcutaneous administration comprises a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10. The anti-CD38 antibodies provided herein can be therapeutically effective when administered at unexpectedly low doses, and thus can be administered in surprisingly small amounts, facilitating subcutaneous administration.

[0074] Another advantage of the anti-CD38 antibodies of the present invention is that, unlike some other anti-CD38 antibodies in the clinic, the anti-CD38 antibodies of the present invention (e.g., AB79) can bind to cynomolgus monkey (cyno) CD38, providing a useful animal model for preclinical evaluation of tolerability, toxicity, and efficacy of administration.

[0075] Another advantage of the anti-CD38 antibodies of the present invention is that they can be used to screen other antibodies that compete for binding to CD38 at the same epitope and may be useful in the methods and unit dosages of the present invention.

[0076] Another advantage of the anti-CD38 antibodies of the invention is that they can be used to screen for other antibodies that have reduced or alternative (e.g., more transient) binding to RBCs and / or platelets compared to daratumumab, e.g., antibodies that compete with or bind to the same epitope as AB79, and may be useful in the methods and unit dosages of the invention.

[0077] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of terms shall be clear. However, in the event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or extrinsic definitions. Furthermore, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular. The term "or" shall include "and / or" unless otherwise specified. Furthermore, the use of the terms "including," "includes," or "comprising" is not limiting. Terms such as "element" and "component" encompass both elements and components that constitute a single unit and elements and components that constitute two or more subunits, unless otherwise specified.

[0078] The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and, unless otherwise specified, as described in the various general and more specific references cited and discussed throughout this specification. Laboratory procedures and techniques in cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization, analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and patient treatment. Commercially available enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein.

[0079] All heading and section designations are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will understand the utility of combining various aspects of the disclosure from different headings and sections as necessary in accordance with the spirit and scope of the invention described herein.

[0080] In order that the present invention may be more readily understood, selected terms are defined below.

[0081] The terms "human CD38" and "human CD38 antigen," as defined herein (Table 1), refer to the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, such as an epitope. Generally, CD38 has a short intracytoplasmic tail, a transmembrane domain, and an extracellular domain. The terms "cynomolgus CD38" and "cynomolgus 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-ribosyl cyclase; ADP-ribosyl cyclase 1; cADPr hydrolase 1; CD38-rs1; I-19; NIM-R5 antigen; 2'-phospho-cyclic-ADP-ribose transferase; 2'-phospho-ADP-ribosyl cyclase; 2'-phospho-cyclic-ADP-ribose transferase; 2'-phospho-ADP-ribosyl cyclase; and T10. [Table 1]

[0082] The terms "therapeutically effective amount" and "therapeutically effective dosage" refer to an amount of a therapeutic agent sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent the progression of a disorder, cause regression of a disorder, prevent the recurrence, occurrence, onset, or progression of one or more symptoms associated with a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent) at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount of an antibody is also an amount in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A therapeutically effective amount of an antibody for tumor therapy can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer may also be assessed in animal model systems predictive of efficacy in human cancer.

[0083] 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 treatment. In one embodiment, the patient is a mammal, e.g., a human.

[0084] The term "disease exhibiting binding to CD38" refers to a disease in which binding of a binding partner (e.g., an anti-CD38 antibody of the present disclosure) to CD38 provides a prophylactic or therapeutic benefit, including amelioration of one or more symptoms of the disease. Such binding may result in blockage 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 autoantibodies to CD38 that are blocked by the anti-CD38 antibodies of the present invention. Such binding may be exhibited as a result of expression of CD38 by cells or a subset of cells, e.g., MM cells, such that providing a CD38 binding partner to a subject results in removal, e.g., lysis, of those cells, e.g., via necrosis or apoptosis. Such CD38 expression may be the result of normal, overexpressed, inappropriately expressed, or activated CD38, e.g., compared to normal cells or compared to other cell types during either a non-disease or disease state.

[0085] The term "hematological cancer" refers to malignant neoplasms of blood-forming tissues and includes leukemia, lymphoma, and multiple myeloma. Non-limiting examples of conditions associated with aberrant CD38 expression include multiple myeloma (MM), including relapsed refractory MM (RRMM) or newly diagnosed MM (NDMM) (Jackson et al. (1988) Clin. Exp. Immunol. 72:351-356); B-cell chronic lymphocytic leukemia (B-CLL) (Durig 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 (Keyhani et al.(1999) Leukemia Res.24:153-159; and 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 myeloid leukemia or chronic myeloid leukemia (CML); acute myeloid leukemia or acute myeloid leukemia (AML); acute lymphocytic leukemia (ALL); hairy cell leukemia (HCL); NK / T cell lymphoma, myelodysplastic syndrome (MDS) (Nurulhuda et al.(2017) Blood 130:2814); and all subtypes and stages of these leukemias and other hematologic disorders (e.g., CML acute phase (BP), chronic phase (CP), or accelerated phase (AP)) as defined by morphological, histochemical, and immunological techniques well known to those skilled in the art.

[0086] The terms "neoplasm" and "neoplastic condition" refer to a pathological condition associated with the proliferation of cells 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.

[0087] An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to CD38 is substantially free of antibodies that specifically bind to antigens other than CD38. However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human CD38 or cynomolgus monkey CD38 may have cross-reactivity with other related antigens from other species, such as CD38 species homologs. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0088] The terms "red blood cells," "RBC," and "erythrocytes" refer to hemoglobin-containing blood cells derived from bone marrow that carry oxygen to cells and tissues and carbon dioxide back to the respiratory tract. RBCs are also called red blood cells, red blood corpuscles, hematides, erythroid cells, etc.

[0089] The terms "specific binding," "specifically binds," and "being specific" in reference to the interaction of a particular antibody, protein, or peptide with an antigen, epitope, or other chemical species refer to a measurably different interaction from a nonspecific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that lacks binding activity. For example, specific binding can be determined by competition with a control molecule that is identical to the target. The anti-CD38 antibodies of the present invention specifically bind to a CD38 ligand. The terms "specific binding," "specifically binds," and "being specific" also mean that the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to specific protein structures rather than proteins in general. If an antibody is specific for epitope "A," then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody. Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12Specific binding to a particular antigen or epitope can be demonstrated by an antibody having a KD of 20, 50, 100, 500, 1000, 5,000, 10,000, or more times greater than the antigen or epitope relative to a control molecule, where KD refers to the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD that is 20, 50, 100, 500, 1000, 5,000, 10,000, or more times greater than the antigen or epitope relative to a control molecule. Specific binding to a particular antigen or epitope can also be demonstrated by, for example, an antibody whose K or Ka for the antigen or epitope is at least 20, 50, 100, 500, 1000, 5,000, 10,000, or more times greater than the epitope relative to a control molecule, where K or Ka refers to the on-rate of a particular antibody-antigen interaction.

[0090] The term "over a period of time" refers to any period of time, such as minutes, hours, days, months, or years. For example, over a period of time can 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 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 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 1 month, at least 1 year, or any time interval in between. In other words, the antibody from the composition can be absorbed by the individual to whom the antibody is administered over a period of 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 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 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 1 month, at least 1 year, or any time interval therebetween.

[0091] A composition that "substantially" comprises an ingredient means that the composition contains greater than about 80% by weight, in some embodiments greater than about 90% by weight, in some embodiments greater than about 95% by weight, in some embodiments greater than about 97% by weight, in some embodiments greater than about 98% by weight, and in some embodiments greater than about 99% by weight of the ingredient.

[0092] The term "about" refers to an approximation of a number, degree, volume, time, etc., with slight variations in dimensions of up to 10%.

[0093] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle suitable for administering a compound of the present invention to a mammal. Carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that are involved in carrying or transporting the compound of interest from one organ or body part to another. A carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. In one embodiment, a pharmaceutically acceptable carrier is suitable for intravenous administration. In another embodiment, a pharmaceutically acceptable carrier is suitable for locoregional injection. In another embodiment, a pharmaceutically acceptable carrier is suitable for subcutaneous administration. In another embodiment, a pharmaceutically acceptable carrier is suitable for subcutaneous injection.

[0094] The term "pharmaceutical composition" refers to a preparation suitable for administration to a subject and treatment of a disease. When the anti-CD38 antibodies of the present invention are administered as pharmaceuticals to a mammal, e.g., a human, they can be administered "as is" or as a pharmaceutical composition containing the anti-CD38 antibody in combination with a pharmaceutically acceptable carrier and / or other excipients. The pharmaceutical composition can be in the form of a unit dosage form for administering a specific dose of the anti-CD38 antibody at a specific concentration, amount, or volume. Pharmaceutical compositions comprising the anti-CD38 antibody alone or in combination with a prophylactic agent, therapeutic agent, and / or pharmaceutically acceptable carrier are provided.

[0095] Conventional antibody structural units typically comprise a tetramer. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having one "light" chain (typically approximately 25 kDa in molecular weight) and one "heavy" chain (typically approximately 50-70 kDa in molecular weight). Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, and epsilon, defining antibody isotypes 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 immunoglobulin subclasses defined by the chemical and antigenic characteristics of their constant regions. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. Therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses.

[0096] Each variable heavy (VH) and variable light (VL) chain region (approximately 100-110 amino acids in length) is composed of three hypervariable regions called "complementarity-determining regions" (CDRs) and four framework regions (FRs) (approximately 15-30 amino acids in length), arranged from amino terminus to carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. "Variable" refers to the fact that the sequences of the CDRs vary widely among antibodies, thereby determining unique antigen-binding sites.

[0097] The hypervariable regions generally consist of approximately amino acid residues 24-34 (LCDR1; "L" refers to the light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and approximately amino acid residues 31-35B (HCDR1; "H" refers to the 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, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), and / or those residues that form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917).

[0098] When referring to residues within the variable domains (approximately residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), the Kabat numbering system is commonly used (see, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD), for the Fc region, the EU numbering system is used.

[0099] The term "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin having a defined tertiary structure. In addition to the variable domain, each heavy and light chain contains constant domains: a constant heavy (CH) domain, a constant light (CL) domain, and a hinge domain. In the context of IgG antibodies, IgG isotypes each contain three CH regions. The carboxy-terminal portions of each HC and LC define the constant region primarily responsible for effector function. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as in Kabat; "CH2" refers to positions 237-340 according to the EU index as in Kabat; and "CH3" refers to positions 341-447 according to the EU index as in Kabat.

[0100] Another type of Ig domain in the heavy chain is the hinge region. The term "hinge region" refers to a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at EU position 237. Thus, for IgG, the antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), with numbering according to the EU index as in Kabat. In some embodiments, for example, in relation to the Fc region, the lower hinge is included, and "lower hinge" generally refers to positions 226 or 230.

[0101] As used herein, "Fc region" refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain, and optionally a portion of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may also include the J chain. For IgG, the Fc domain includes immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). While the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined as including residues C226 or P230 at its carboxy terminus, and numbering herein follows the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region to, for example, alter binding to one or more FcγR or FcRn receptors, as described in more detail below.

[0102] CD38 antibody Thus, the present invention provides isolated anti-CD38 antibodies that specifically bind to human and primate CD38 protein and have reduced, or less than 10%, 20%, 30%, 40%, or 50% binding to human RBCs when compared to daratumumab, thereby finding use in subcutaneous administration methods and unit dosage forms. Particularly useful in the present invention are antibodies that bind to both human and primate CD38 protein, particularly primates used in clinical trials, such as cynomolgus monkeys (Macaca fascicularis, also referred to herein as "cyno").

[0103] In some embodiments, the anti-CD38 antibodies of the invention interact with CD38 at a number of amino acid residues in the AB79 epitope, including K121, F135, Q139, D141, M142, D202, V203, H205, Q236, E239, W241, S274, C275, K276, F284, C287, V288, K289, N290, P291, E292, and D293. Any antibody that interacts with these residues also finds use in the therapeutic methods and unit dosages of the invention.

[0104] 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. Note that these residues are identical in both humans and cynomolgus monkeys, except that S274 is actually F274 in cynomolgus monkeys. These residues may represent residues within the footprint of immunodominant epitopes and / or specific antigen-binding peptides.

[0105] In some embodiments, the anti-CD38 antibody comprises a heavy chain comprising 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 comprising 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 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 comprising 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 variable heavy (VH) chain amino acid sequence of SEQ ID NO: 9.

[0106] [ka]

[0107] In some embodiments, the antibody comprises a light chain comprising the variable light (VL) chain amino acid sequence of SEQ ID NO:10.

[0108] [ka]

[0109] In some embodiments, the antibody comprises a heavy chain comprising the VH chain amino acid sequence of SEQ ID NO:9 and a light chain comprising the VL chain amino acid sequence of SEQ ID NO:10.

[0110] As will be appreciated by those skilled in the art, the variable heavy and light chains may be linked to human IgG constant domain sequences, generally IgG1, IgG2 or IgG4.

[0111] In some embodiments, the antibody comprises the heavy (HC) chain amino acid sequence of SEQ ID NO:11.

[0112] [ka]

[0113] In some embodiments, the antibody comprises the light chain (LC) amino acid sequence of SEQ ID NO:12.

[0114] [ka]

[0115] 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.

[0116] The present invention encompasses antibodies that bind to both human and cyno CD38 and interact with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of these amino acid residues.

[0117] In some embodiments, the antibody is full-length. By "full antibody" herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions, and including one or more modifications as outlined herein.

[0118] Alternatively, antibodies may be of various structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each. Specific antibody fragments include, but are not limited to, (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a Fd fragment consisting of the VH and CH1 domains, (iii) a Fv fragment consisting of the VL and VH domains of a single antibody, (iv) a dAb fragment consisting of a single variable region (Ward et al. (1989) Nature 341:544-546), (v) an isolated CDR region, (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments, and (vii) a single-chain Fv molecule (scFv), in which the VH and VL domains 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 (WO03 / 11161), and (ix) "diabodies" or "triabodies," which are multivalent or multispecific 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).

[0119] antibody modification The present invention further provides mutant anti-CD38 antibodies, i.e., the antibodies of the present disclosure may have numerous modifications, including, but not limited to, amino acid modifications in the CDRs (affinity maturation), amino acid modifications in the Fc region, glycosylation variants, other types of covalent modifications, etc.

[0120] The term "variant" refers to a polypeptide that differs from the parent polypeptide. Amino acid variants can include amino acid substitutions, insertions, and deletions. Generally, as described herein, variants can include any number of modifications, so long as the protein's function remains intact. That is, for example, in the case of amino acid variants generated in any CDR of AB79, the antibody still specifically binds to both human and cynomolgus CD38 and does not bind to RBCs, or has reduced binding, or less than 10%, 20%, 30%, 40%, or 50% of that of daratumumab. A "variant Fc region" refers to an Fc sequence that differs from that of a wild-type or parent Fc sequence by at least one amino acid modification. An Fc variant can refer to the Fc polypeptide itself, a composition comprising an Fc variant polypeptide, or an amino acid sequence. For example, when amino acid variants are generated in the Fc region, the variant antibody must maintain the functionality required for the antibody's particular use or indication. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions can be utilized, e.g., 1-10, 1-5, 1-4, 1-3, and 1-2 substitutions. Appropriate modifications can be made at one or more positions, particularly for specific amino acid substitutions that increase binding to Fc receptors, as outlined, for example, in U.S. Patent Nos. 6,086,875, 6,737,056, 7,317,091, 7,670,600, 8,084,582, 8,188,231, 8,367,805, 8,937,158, and 9,040,041 (all of which are expressly incorporated by reference in their entireties).

[0121] For example, it may be desirable to have 1-5 modifications in the Fc region and 1-5 modifications in the Fv region of a wild-type or engineered protein. Preferably, the variant polypeptide sequence has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the parent sequence (e.g., the variable region, constant region, and / or heavy and light chain sequences of AB79).

[0122] The term "amino acid substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution S100A refers to a variant polypeptide in which the serine at position 100 is replaced with an alanine. The term "amino acid insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. The term "amino acid deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence.

[0123] The terms "parent antibody" and "precursor antibody" refer to an unmodified antibody that is subsequently modified to generate a variant. In one embodiment, the parent antibody herein is AB79. The parent antibody may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence encoding it. Thus, the term "parent Fc polypeptide" refers to an Fc polypeptide that has been modified to generate a variant.

[0124] As used herein, "wild-type" or "WT" and "native" refer to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0125] In some embodiments, one or more amino acid modifications are made in one or more of the CDRs of an anti-CD38 antibody. Generally, only 1, 2, or 3 amino acid substitutions are made in any single CDR, and usually no more than 4, 5, 6, 7, 8, 9, or 10 amino acid changes are made within a set of CDRs. However, it should be understood that any combination of no substitutions, 1, 2, or 3 substitutions in any CDR can be independently and optionally combined with any other substitution.

[0126] In some cases, amino acid modifications in the CDRs are referred to as "affinity maturation." An "affinity matured" antibody is one that has one or more alterations in one or more CDRs that result in improved affinity of the antibody for an antigen compared to a parent antibody that does not have those alterations. In some cases, it may be desirable to reduce the affinity of an antibody for its antigen.

[0127] Affinity maturation can be performed to increase the binding affinity of an antibody for an antigen by at least about 10% to 50%, 100%, 150% or more, or 1 to 5 fold compared to the "parent" antibody. Preferred affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by known procedures (see, 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).

[0128] Alternatively, "silent" amino acid modifications can be made in one or more of the CDRs of an antibody of the invention, for example, that do not significantly alter the affinity of the antibody for antigen. These can be made for several reasons, including optimization of expression (as can be made to nucleic acids encoding antibodies of the invention).

[0129] Thus, variant CDRs and antibodies are included within the definition of the CDRs and antibodies of the present invention, i.e., antibodies of the present invention may contain amino acid modifications in one or more of the AB79 CDRs. Furthermore, as outlined below, amino acid modifications may be made independently and optionally in any region outside the CDRs, including the framework and constant regions.

[0130] In some embodiments, a variant antibody of AB79 is described that is specific for human CD38 (SEQ ID NO: 1) and cynomolgus monkey CD38 (SEQ ID NO: 2). This antibody consists of six CDRs, each of which can differ 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 by zero, one, two, or more amino acid substitutions without significantly altering or inhibiting function.

[0131] In addition to the modifications outlined above, other modifications can be made. For example, the molecule can be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al. (1996) Nature Biotech. 14:1239-1245). Furthermore, there are a variety of covalent modifications of antibodies that can be made, as outlined below.

[0132] Covalent modifications of antibodies are included within the scope of the present invention and are usually, but not always, done post-translationally. For example, several types of covalent modifications of antibodies are introduced into the molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0133] In some embodiments, the anti-CD38 antibodies of the invention specifically bind to one or more residues or regions of CD38 but do not cross-react with other proteins homologous to CD38, such as BST-1 (bone marrow stromal cell antigen-1) and Mo5 (also known as CD157).

[0134] Typically, lack of cross-reactivity means that the relative competitive inhibition between the molecules is less than about 5% when assessed by ELISA and / or FACS analysis using sufficient amounts of the molecules under appropriate assay conditions.

[0135] Inhibition of CD38 activity and reduction of side effects The disclosed antibodies may find use in blocking ligand-receptor interactions or inhibiting receptor component interactions. Anti-CD38 antibodies of the present invention may be "blocking" or "neutralizing." A "neutralizing antibody" refers to an antibody that, upon binding to CD38, inhibits the biological activity of CD38, such as its ability to interact with ligands, its enzymatic activity, its signaling activity, and in particular its ability to generate activated lymphocytes. Inhibition of CD38 biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art.

[0136] The terms "inhibit binding" and "blocking binding" (e.g., when referring to inhibiting / blocking the binding of a CD38 antibody to CD38) encompass both partial and complete inhibition / blocking. Inhibiting / blocking the binding of a CD38 antibody to CD38 may reduce or alter the normal level or type of cell signaling that occurs when the CD38 antibody binds to CD38 without inhibition or blockage. Inhibition and blocking are also intended to include a measurable decrease in the binding affinity of a CD38 antibody to CD38 when contacted with an anti-CD38 antibody, compared to a ligand not contacted with the anti-CD38 antibody, such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% blocking of binding of a CD38 antibody to CD38.

[0137] The disclosed anti-CD38 antibodies can also inhibit cell proliferation. The term "inhibit proliferation" refers to a measurable decrease in cell proliferation when contacted with an anti-CD38 antibody compared to the proliferation of the same cells not contacted with the anti-CD38 antibody, such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% inhibition of cell culture proliferation.

[0138] In some embodiments, the disclosed anti-CD38 antibodies are capable of depleting activated lymphocytes and plasma cells. The term "depletion" in this context refers to a measurable reduction in serum levels of activated lymphocytes and / or plasma cells in a subject compared to an untreated subject. Generally, depletion of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% is observed. As shown in the Examples below, one particular advantage of the antibodies of the present invention is that these cells can be restored after administration; that is, as is known with some treatments (e.g., with anti-CD20 antibodies), prolonged cell depletion can result in undesirable side effects. As shown herein, the effects on activated lymphocytes and / or plasma cells are reversible.

[0139] The anti-CD38 antibodies of the present invention allow for reduced side effects compared to prior art anti-CD38 antibodies. In some embodiments, AB79 does not induce TEAEs. In some embodiments, AB79 allows for reduced TEAEs compared to other anti-CD38 antibodies, such as MOR202. TEAEs are typically designated grades 1, 2, 3, 4, and 5, with grade 1 being the least severe and grade 5 being the most severe TEAE. Based on FDA and other guidelines regarding the Common Terminology Criteria for Adverse Events (CTCAE) criteria for oncology drugs (e.g., https: / / evs.nci.nih.gov / ftp1 / CTCAE / CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf; and https: / / ctep.cancer.gov / protocoldevelopment / electronic_applications / ctc.htm; and Nilsson and Koke (2001) Drug Inform. J. 35:1289-1299), the general method for determining these grades is as follows: Grade 1 is mild, asymptomatic or with mild symptoms, requiring only clinical or diagnostic observation and no intervention; Grade 2 is moderate, requiring minimal, local, or noninvasive intervention and limiting age-appropriate activities of daily living (ADL); Grade 3 is severe or medically significant, but not immediately life-threatening, requiring hospitalization or prolonged hospitalization, being disabling, and limiting self-care ADL; Grade 4 is life-threatening and requiring urgent intervention; and Grade 5 is AE-related death.

[0140] In some embodiments, AB79 allows for a reduction in the grade of TEAEs compared to other anti-CD38 antibodies, such as MOR202. In some embodiments, AB79 allows for a reduction in the grade of TEAEs from grade 5 to grade 4 compared to other anti-CD38 antibodies. In some embodiments, AB79 allows for a reduction in the grade of TEAEs from grade 4 to grade 3 compared to other anti-CD38 antibodies. In some embodiments, AB79 allows for a reduction in the grade of TEAEs from grade 3 to grade 2 compared to other anti-CD38 antibodies. In some embodiments, AB79 allows for a reduction in the grade of TEAEs from grade 2 to grade 1 compared to other anti-CD38 antibodies.

[0141] In some embodiments, AB79 allows for a reduction in the grade of one or more TEAEs selected from the group consisting of anemia (including hemolytic anemia), thrombocytopenia, fatigue, infusion-related reactions (IRR), leukopenia, lymphopenia, and nausea. In some embodiments, AB79 allows for a reduction in the occurrence of one or more TEAEs selected from the group consisting of anemia (including hemolytic anemia), thrombocytopenia, fatigue, infusion-related reactions (IRR), leukopenia, lymphopenia, and nausea.

[0142] In some embodiments, diagnostic tests are used to determine the presence and / or grade of anemia, including hemolytic anemia. Diagnostic tests for anemia, including hemolytic anemia, involve measuring hemoglobin levels. Generally, hemoglobin levels are interpreted as follows: (i) very mild anemia / absence of 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, involve measuring haptoglobin levels. Generally, a haptoglobin level ≤ 25 mg / dL indicates the presence of anemia, including hemolytic anemia. Other diagnostic tests include the direct antiglobulin test (DAT) (also known as the direct Coombs test), which is used to determine whether RBCs are coated in vivo with immunoglobulins, complement, or both.

[0143] In some embodiments, diagnostic tests are used to determine the presence and / or grade of thrombocytopenia. Generally, diagnostic tests for thrombocytopenia involve measuring the number of platelets per microliter (μL) of blood. Normally, there are 150×10 platelets per μL of blood. 3 ~450×10 3 Generally, thrombocytopenia is defined as a platelet count of 150 x 10 per μL of blood. 3 Mild thrombocytopenia is diagnosed when there are fewer than 70–150 × 10 platelets per μL of blood. 3 Moderate thrombocytopenia is generally diagnosed when the level is between 20 and 70 × 10 per μL. 3 Severe thrombocytopenia is generally diagnosed when thrombocytopenia is <20 × 10 per μL of blood. 3 It is generally diagnosed if there are only a few.

[0144] Disease indications The antibodies, methods, and dosage units of the present invention find use in a variety of applications, including treating or ameliorating CD38-associated diseases.

[0145] CD38 is expressed on immature hematopoietic cells, downregulated on mature cells, and re-expressed at high levels on activated lymphocytes and plasma cells. For example, high CD38 expression is observed on 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 conditions are associated with cells expressing CD38, and certain conditions are associated with overexpression, high density expression, or upregulated expression of CD38 on the cell surface. Whether a cell population expresses CD38 can be determined by methods known in the art, such as flow cytometry measurement of the proportion of cells in a given population labeled with an antibody specifically binding to CD38 or immunohistochemical assays, as generally described below for diagnostic applications. For example, a population of cells in which CD38 expression is detected in approximately 10-30% of the cells can be considered weakly positive for CD38, and a population of cells in which CD38 expression is detected in approximately 30% or more of the cells can be considered clearly positive for CD38 (as in Jackson et al. (1988) Clin. Exp. Immunol. 72:351-356). However, other criteria can be used to determine whether a cell population expresses CD38. The density of expression on the surface of cells can be determined using methods known in the art, such as flow cytometry measurement of the mean fluorescence intensity of fluorescently labeled cells using an antibody that specifically binds to CD38.

[0146] The therapeutic anti-CD38 antibodies of the present invention bind to CD38-positive cells and result in the depletion of these cells through multiple mechanisms of action, including both CDC and ADCC pathways.

[0147] It is known in the art that certain conditions are associated with cells expressing CD38, and that certain conditions are associated with overexpression, high density expression, or upregulated expression of CD38 on the cell surface. Whether a cell population expresses CD38 can be determined by methods known in the art, such as flow cytometry measurement of the proportion of cells in a given population that are labeled with an antibody that specifically binds to CD38 or by immunohistochemical assays, as generally described below for diagnostic applications. For example, a population of cells in which CD38 expression is detected in approximately 10-30% of the cells can be considered weakly positive for CD38, and a cell population in which CD38 expression is detected in approximately 30% or more of the cells can be considered clearly positive for CD38 (Jackson et al. (1988) Clin. Exp. Immunol. 72:351-356), although other criteria can be used to determine whether a cell population expresses CD38. The density of expression on the surface of a cell can be determined using methods known in the art, such as, for example, flow cytometry measurement of the mean fluorescence intensity of cells fluorescently labeled using an antibody that specifically binds to CD38.

[0148] In one aspect, the invention provides a method for treating a condition associated with the proliferation of cells expressing CD38, comprising administering to a patient a pharmaceutically effective amount of the disclosed antibody. In some embodiments, the condition is cancer, and in particular embodiments, the cancer is a hematological cancer. In some embodiments, the condition is multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, and plasma cell leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, B-cell lymphoma, or Burkitt's lymphoma.

[0149] In some embodiments, the condition is multiple myeloma and the therapeutic anti-CD38 antibody does not bind to human RBCs or has reduced binding to human RBCs compared to daratumumab. In some embodiments, the condition is multiple myeloma and the therapeutic anti-CD38 antibody does not bind to cynomolgus monkey RBCs or has reduced binding to cynomolgus monkey RBCs compared to daratumumab. In some embodiments, the condition is multiple myeloma and the therapeutic anti-CD38 antibody does not bind to human or cynomolgus monkey RBCs or has reduced binding to human or cynomolgus monkey RBCs.

[0150] CLL is the most common leukemia in adults in Western countries. It is a clonal proliferation of mature-appearing lymphocytes that involve lymph nodes and other lymphoid tissues, with progressive infiltration of the bone marrow and presence in the peripheral blood. The B-cell subtype (B-CLL) represents the majority of cases.

[0151] B-cell type of chronic lymphocytic leukemia (B-CLL) B-CLL is an incurable disease characterized by the progressive increase in refractory monoclonal B-lineage cells that accumulate in the bone marrow and peripheral blood over many years. CD38 expression is considered an independent poor prognostic factor for B-CLL (Hamblin et al. (2002) Blood 99:1023-9).

[0152] B-CLL is characterized by two subtypes: indolent and progressive. 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 disorders in subjects who have a mutated IgVH gene and / or exhibit one or more clinical phenotypes associated with indolent B-CLL. As used herein, progressive B-CLL refers to disorders in subjects who have an unmutated IgVH gene and / or exhibit one or more clinical phenotypes associated with progressive B-CLL.

[0153] The current standard of care for B-CLL is palliative, primarily achieved with the cytostatic drugs chlorambucil or fludarabine. In the event of relapse, combination therapy is often initiated using fludarabine and cyclophosphamide in combination with rituximab (a monoclonal antibody against CD20) or alemtuzumab (a monoclonal antibody against CD52). In one study, 35 patients with relapsed or refractory aggressive B-cell NHL underwent high-dose chemotherapy (HCT) followed by rituximab 375 mg / m 2 This was repeated for four doses starting on day 40 and four more doses starting on day 180. Rituximab infusions were well tolerated with only one grade 3 / 4 infusion-related 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, available online 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) until tolerated by the patient. The major TEAEs were anemia, neutropenia (6 / 6 patients each), and thrombocytopenia (5 / 6 patients) in hematological adverse events (Ishizawa et al. (2017) Jpn. J. Clin. Oncol. 47(1):54-60). Thus, there is a significant unmet medical need for the treatment of B-CLL with reduced hematological adverse events. In some embodiments, methods of treating B-CLL using the disclosed anti-CD38 antibodies are provided, which may be performed using combination therapy, optionally and independently, including any of the above-mentioned drugs, as outlined below.

[0154] Multiple myeloma (MM) Multiple myeloma is a malignant disorder of the B-cell system characterized by the neoplastic proliferation of plasma cells in the bone marrow. The proliferation of myeloma cells leads to a variety of effects, including lytic lesions (holes) in the bones, a decrease in red blood cell count, abnormal protein production (with associated damage to the kidneys, nerves, and other organs), decreased immune system function, and elevated blood calcium levels (hypercalcemia). Currently, treatment options include chemotherapy, preferably in conjunction with autologous stem cell transplantation (ASCT) if possible. These treatment regimens have shown moderate response rates, although only modest changes in overall survival have been observed, with a median survival of approximately 3 years. Thus, there is a significant unmet medical need for treatment of multiple myeloma. In some embodiments, methods for treating multiple myeloma using the disclosed antibodies are provided.

[0155] Newly diagnosed MM (NDMM) is distinguished from relapsed MM or relapsed and refractory MM (RRMM). Relapsed MM is considered a recurrence of disease after a previous response and is defined based on objective laboratory and radiological criteria: a ≥25% increase in serum or urinary monoclonal protein (M protein), or a ≥25% difference between involved and uninvolved serum-free light chains from nadir, respectively, or the development of new plasmacytomas or hypercalcemia. In patients with nonsecretory disease, relapse is defined as an increase in bone marrow plasma cells. In general, indications for relapse treatment are defined as either the appearance or recurrence of one or more of the above MM symptoms or a rapid and consistent biochemical relapse. Relapsed / refractory MM (RRMM) is defined as disease that becomes non-responsive or progressive to therapy, or becomes non-responsive or progressive to therapy within 60 days of the last treatment in patients who achieved a minimal response (MR) or good response to previous therapy (Sonneveld and Broijl (2016) Haematologica 101(4):396-406).

[0156] Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are asymptomatic preneoplastic disorders characterized by monoclonal plasma cell proliferation in the bone marrow and the absence of end-organ damage.

[0157] Smoldering multiple myeloma (SMM) is an asymptomatic proliferative disorder of plasma cells with a high risk of progression to symptomatic or active multiple myeloma (Kyle et al. (2007) N. Engl. J. Med. 356(25):2582-2590). International consensus criteria for defining SMM were adopted in 2003 and require patients to have an M protein level of >30 g / L and / or >10% bone marrow clonal plasma cells (Internat. Myeloma Working Group (2003) Br. J. Haematol. 121:749-757). Patients must not have organ or associated tissue damage, including bone lesions or symptoms. Recent studies have identified two SMM subsets: i) patients with progressive disease and ii) patients with non-progressive disease (Internat. Myeloma Working Group (2003) Br. J. Haematol. 121:749-757).

[0158] SMM resembles monoclonal gammopathy of undetermined significance (MGUS) in the absence of end-organ damage (Kyle et al. (2007) N. Engl. J. Med. 356(25):2582-2590). However, clinically, SMM is much more likely to progress to active multiple myeloma or amyloidosis over a 20-year period (78% chance for SMM vs. 21% chance for MGUS) (Kyle et al. (2007) N. Engl. J. Med. 356(25):2582-2590).

[0159] International consensus criteria for defining MGUS require that patients have an M protein level of <30 g / L, a bone marrow plasma cell level of <10%, and no organ or associated tissue damage, including bone lesions or symptoms (Internat. Myeloma Working Group (2003) Br. J. Haematol. 121:749-757).

[0160] CD38-related pathologies The antibodies, methods, and dosage units of the invention find use in a variety of applications, including the treatment or amelioration of diseases and conditions associated with inflammation and immune disorders, particularly CD38-associated diseases, such as diseases associated with activated lymphocytes. The anti-CD38 antibodies of the invention bind to CD38-positive cells and result in the depletion of these cells, e.g., activated lymphocytes, through multiple mechanisms of action, including both CDC and ADCC pathways.

[0161] Thus, the antibodies of the present invention may be used to treat any autoimmune disease that exhibits increased CD38 expression or increased numbers of CD38-expressing cells as a disease component, including, but not limited to, allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic antibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behçet's disease, bullous pemphigoid, fungal myopathy, Castleman's syndrome, and sericulture. Axe Sprue dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hematopoietic stem cell transplantation (HTTR), hematopoietic stem cell transplantation (HST ... Myelopathy A, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary Agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, solid organ transplant rejection, stiff-man syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

[0162] Of particular use, in some embodiments, is the use of the antibodies of the present invention for use in the diagnosis and / or treatment of a number of diseases, including but not limited to autoimmune diseases, including but not limited to systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis, and graft-versus-host disease.

[0163] Thus, patients with high plasma cell content can be selected, for example, SLE patients who exhibit high plasma cell levels, as well as RA patients who have been shown to be unresponsive to CD20-based therapy.

[0164] Antibody Compositions for In Vivo Administration Formulations of antibodies used according to the present invention are prepared in lyophilized or aqueous form for storage by mixing antibodies of the desired purity with any pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition (1980) Osol, A. Ed.).

[0165] The formulations herein may also contain two or more active compounds as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to provide antibodies with other specificities. Alternatively, or in addition, the compositions may include cytotoxic agents, cytokines, growth inhibitors, and / or small molecule antagonists. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0166] Subcutaneous administration The present invention is based on the unexpected discovery that anti-CD38 antibodies described herein, such as AB79, can be administered at a sufficiently low dose to be therapeutically effective, allowing for subcutaneous administration of a low-volume liquid formulation. Subcutaneous administration is the least invasive method of administration and is considered the most versatile and therefore desirable method for short- and long-term treatment. In some embodiments, subcutaneous administration can be performed by injection. In some embodiments, the site of injection or device can be rotated when multiple injections or devices are required.

[0167] Therefore, subcutaneous formulations are much easier for patients to self-administer, especially since the formulation may need to be taken periodically throughout the patient's lifetime (e.g., as early as the first year of life in children). Furthermore, the ease and speed of subcutaneous delivery improves patient compliance and allows for more rapid access to the drug when needed. Thus, the subcutaneous formulations of anti-CD38 antibodies provided herein offer substantial advantages over the prior art and address specific unmet needs.

[0168] In some embodiments, the antibody of the present invention is administered to a subject via subcutaneous route according to known methods. In some embodiments, the antibody of the present invention can be administered by subcutaneous injection. In certain embodiments, the subcutaneous formulation is subcutaneously injected into the same site of the patient for repeated or continuous injections (e.g., into the upper arm, front of the thigh, lower abdomen, or upper back). In other embodiments, the subcutaneous formulation is subcutaneously injected into different or alternating sites of the patient. Single or multiple administrations of the formulation may be used.

[0169] In some embodiments, the subcutaneous unit dosage forms described herein can be used to treat cancer. In some embodiments, the subcutaneous unit dosage forms described herein can be used to treat hematological cancers. In some embodiments, the subcutaneous unit dosage forms described herein can be used to treat multiple myeloma.

[0170] In some embodiments, antibodies of the invention that bind to human RBCs have temporarily increased bioavailability. In some embodiments, the bioavailability of antibodies of the invention that transiently bind to RBCs is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. In some embodiments, the bioavailability of antibodies of the invention that transiently bind to RBCs is increased by 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, or 300% or more.

[0171] In some embodiments, the increased bioavailability allows for subcutaneous administration. In some embodiments, the increased bioavailability is due to the fact that the antibodies of the present invention bind transiently to RBCs. In some embodiments, the increased bioavailability in humans is due to the fact that the antibodies of the present invention bind differentially to RBCs.

[0172] In some embodiments, antibodies of the invention result in depletion of NK cells, B cells, and / or T cells. In some embodiments, antibodies of the invention allow for increased depletion of NK cells compared to depletion of B cells or T cells. In some embodiments, antibodies of the invention allow for increased depletion of NK cells compared to B cells, and increased depletion of NK cells compared to T cells. In some embodiments, antibodies of the invention allow for increased depletion of NK cells compared to B cells, and increased depletion of B cells compared to T cells. In some embodiments, antibodies of the invention allow for increased depletion of NK cells compared to B cells and increased depletion of B cells compared to T cells.

[0173] In certain embodiments, the bioavailability of an anti-CD38 antibody described herein after subcutaneous administration is at least 50% to at least 80% compared to normalized intravenous administration at the same dose. In certain embodiments, the bioavailability of an anti-CD38 antibody described herein after subcutaneous administration is at least 60% to at least 80% compared to normalized intravenous administration at the same dose. In certain embodiments, the bioavailability of an anti-CD38 antibody described herein after subcutaneous administration is at least 50% to at least 70% compared to normalized intravenous administration at the same dose. In certain embodiments, the bioavailability of an anti-CD38 antibody described herein after subcutaneous administration is at least 55% to at least 65% compared to normalized intravenous administration at the same dose. In certain embodiments, the bioavailability of an anti-CD38 antibody described herein after subcutaneous administration is at least 55% to at least 70% compared to normalized intravenous administration at the same dose.

[0174] In certain embodiments, the bioavailability of an anti-CD38 antibody described herein following 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%, at least 84%, or at least 85% compared to normalized intravenous administration at the same dose.

[0175] In some embodiments, the present disclosure provides methods in which the bioavailability of antibodies of the invention that transiently bind to human RBCs after subcutaneous administration is 50% to 80% compared to normalized intravenous administration at the same dose.

[0176] In some embodiments, the present disclosure provides methods in which the bioavailability of an antibody of the invention that transiently binds to human RBCs after subcutaneous administration is at least 50% compared to normalized intravenous administration at the same dose.

[0177] In some embodiments, the disclosure provides methods wherein the bioavailability of an antibody of the invention that transiently binds to human RBCs after subcutaneous administration is at least 55% compared to normalized intravenous administration at the same dose.

[0178] In some embodiments, the present disclosure provides methods in which the bioavailability of an antibody of the invention that transiently binds to human RBCs after subcutaneous administration is at least 60% compared to normalized intravenous administration at the same dose.

[0179] In some embodiments, the present disclosure provides methods wherein the bioavailability of an antibody of the invention that transiently binds to human RBCs after subcutaneous administration is at least 65% compared to normalized intravenous administration at the same dose.

[0180] In some embodiments, the present disclosure provides methods wherein the bioavailability of an antibody of the invention that transiently binds to human RBCs after subcutaneous administration is at least 70% compared to normalized intravenous administration at the same dose.

[0181] In some embodiments, the present disclosure provides methods in which the bioavailability of an antibody of the invention that transiently binds to human RBCs after subcutaneous administration is at least 75% compared to normalized intravenous administration at the same dose.

[0182] In some embodiments, the present disclosure provides methods in which the bioavailability of an antibody of the invention that transiently binds to human RBCs after subcutaneous administration is at least 80% compared to normalized intravenous administration at the same dose.

[0183] In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously in a single bolus injection. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously monthly. 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 weekly. 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 6 hours. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every 4 hours. In certain embodiments, the anti-CD38 antibodies described herein are administered subcutaneously every two hours.

[0184] In some embodiments, the subcutaneous unit dosage form is administered at a dose of about 0.01 mg / kg body weight to about 0.8 mg / kg body weight. In some embodiments, the subcutaneous unit dosage form contains an amount sufficient to administer at a dose of about 0.02 mg / kg body weight to about 0.75 mg / kg body weight. In some embodiments, the subcutaneous unit dosage form contains an amount sufficient to administer at a dose of about 0.02 mg / kg body weight to about 0.7 mg / kg body weight. In some embodiments, the subcutaneous unit dosage form contains an amount sufficient to administer at a dose of about 0.03 mg / kg body weight to about 0.6 mg / kg body weight. In some embodiments, the amount is formulated in a volume between about 0.25 milliliters (mL) and about 3.5 milliliters (mL). In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 3 mL. In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 2.5 mL. In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 2 mL. In some embodiments, the amount is formulated in a volume between about 1 mL and about 2 mL. In some embodiments, the amount is formulated in a volume between about 0.25 mL and about 1.25 mL. In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 1.25 mL. In some embodiments, the amount is formulated in a volume between about 0.75 mL and about 1.25 mL. In some embodiments, the amount is formulated in a volume between about 0.75 mL and about 1 mL. In some embodiments, the amount is formulated in a volume between about 0.9 mL and about 1.5 mL. In some embodiments, the amount is formulated in a volume between about 0.9 mL and about 1.1 mL. In some embodiments, the amount is formulated in a volume between about 1 mL and about 1.0 mL. In some embodiments, the amount is formulated in a volume between about 0.95 mL and about 1.05 mL. In some embodiments, the amount is formulated in a volume of about 3.5 mL. In some embodiments, the amount is formulated in a volume of about 3 mL. In some embodiments, the amount is formulated in a volume of about 2.5 mL. In some embodiments, the amount is formulated in a volume of about 2 mL. In some embodiments, the amount is formulated in a volume of about 1.5 mL. In some embodiments, the amount is formulated in a volume of about 1 mL.In some embodiments, the amount is formulated in a volume of about 0.5 mL. In some embodiments, the amount is formulated in a volume of about 0.25 mL.

[0185] Unit dosage form In some embodiments, the therapeutic anti-CD38 antibody is formulated as part of a unit dosage form. In some embodiments, the antibody comprises a heavy chain comprising 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 comprising 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 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 comprising 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 variable heavy (VH) chain amino acid sequence of SEQ ID NO: 9.

[0186] [ka]

[0187] In some embodiments, the antibody comprises a light chain comprising the variable light (VL) chain amino acid sequence of SEQ ID NO:10.

[0188] [ka]

[0189] In some embodiments, the antibody comprises a heavy chain comprising the VH chain amino acid sequence of SEQ ID NO:9 and a light chain comprising the VL chain amino acid sequence of SEQ ID NO:10.

[0190] As will be appreciated by those skilled in the art, the variable heavy and light chains may be linked to human IgG constant domain sequences, generally IgG1, IgG2 or IgG4. In some embodiments, the antibody comprises the heavy (HC) chain amino acid sequence of SEQ ID NO:11.

[0191] [ka]

[0192] In some embodiments, the antibody comprises the light chain (LC) amino acid sequence of SEQ ID NO:12.

[0193] [ka]

[0194] 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.

[0195] In some embodiments, the formulation comprising an anti-CD38 antibody is in unit dosage form. In some embodiments, the unit dosage form comprises an amount sufficient to administer at a dose of about 0.01 mg / kg body weight to about 0.8 mg / kg body weight. In some embodiments, the unit dosage form comprises an amount sufficient to administer at a dose of about 0.02 mg / kg body weight to about 0.75 mg / kg body weight. In some embodiments, the unit dosage form comprises an amount sufficient to administer at a dose of about 0.02 mg / kg body weight to about 0.7 mg / kg body weight. In some embodiments, the unit dosage form comprises an amount sufficient to administer at a dose of about 0.03 mg / kg body weight to about 0.6 mg / kg body weight. In some embodiments, the amount is formulated in a volume between about 0.25 milliliters (mL) and about 3.5 milliliters (mL). In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 3 mL. In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 2.5 mL. In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 2 mL. In some embodiments, the amount is formulated in a volume between about 1 mL and about 2 mL. In some embodiments, the amount is formulated in a volume between about 0.25 mL and about 1.25 mL. In some embodiments, the amount is formulated in a volume between about 0.5 mL and about 1.25 mL. In some embodiments, the amount is formulated in a volume between about 0.75 mL and about 1.25 mL. In some embodiments, the amount is formulated in a volume between about 0.75 mL and about 1 mL. In some embodiments, the amount is formulated in a volume between about 0.9 mL and about 1.5 mL. In some embodiments, the amount is formulated in a volume between about 0.9 mL and about 1.1 mL. In some embodiments, the amount is formulated in a volume between about 1 mL and about 1.1 mL. In some embodiments, the amount is formulated in a volume between about 0.95 mL and about 1.05 mL. In some embodiments, the amount is formulated in a volume of about 3.5 mL. In some embodiments, the amount is formulated in a volume of about 3 mL. In some embodiments, the amount is formulated in a volume of about 2.5 mL. In some embodiments, the amount is formulated in a volume of about 2 mL. In some embodiments, the amount is formulated in a volume of about 1.5 mL.In some embodiments, the amount is formulated in a volume of about 1 mL. In some embodiments, the amount is formulated in a volume of about 0.5 mL. In some embodiments, the amount is formulated in a volume of about 0.25 mL.

[0196] In some embodiments, unit dosage forms of the anti-CD38 antibodies provided herein can further comprise one or more pharmaceutically acceptable excipients, carriers, and / or diluents.

[0197] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. The composition may be formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers, in some embodiments, to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0198] The specifications for the unit dosage forms of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of individuals.

[0199] The effective dosage and administration regimen of the anti-CD38 antibodies used in the present invention will depend on the disease or condition being treated and can be determined by one skilled in the art.

[0200] The dosage forms provided herein are based on subcutaneous administration, which is achieved at least in part based on AB79's lower ability to bind to or remain bound to RBCs compared to daratumumab. Without being bound to a particular theory, such binding activity may be due to the primary nature of AB79's binding to red blood cells. In some embodiments, the therapeutic antibody transiently binds to human RBCs. In some embodiments, the therapeutic anti-CD38 antibody transiently binds to cynomolgus monkey RBCs. In some embodiments, the therapeutic anti-CD38 antibody transiently binds to human or cynomolgus monkey RBCs. In some embodiments, the therapeutic anti-CD38 antibody transiently binds to human and cynomolgus monkey RBCs.

[0201] In one embodiment, the anti-CD38 antibody is administered subcutaneously at a weekly dose of about 0.01 to about 1 mg / kg, e.g., about 0.02 to about 0.8 mg / kg. Such administration may be repeated, for example, 1 to 14 times, e.g., 3 to 5 times. An exemplary, non-limiting range for a therapeutically effective amount of an anti-CD38 antibody used in the present invention is about 0.01 to 1 mg / kg, e.g., about 0.01 to 0.8 mg / kg, about 0.02 to 0.75 mg / kg, about 0.02 to 0.7 mg / kg, or about 0.03 to 0.6 mg / kg.

[0202] As a non-limiting example, treatment according to the present invention can be administered using single or divided doses every 24, 18, 12, 8, 6, 4, or 2 hours, or any combination, at least once on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, or 40th day after initiation of treatment, or at least once on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th week, or any combination thereof, at a dose of about 0.01 to about 1 mg / kg per day. g, e.g., 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. Amounts of antibody of 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 may be provided as a daily dose.

[0203] In one embodiment, the anti-CD38 antibody is administered at a weekly dose of about 0.01 to about 1 mg / kg, e.g., about 0.02 to about 0.8 mg / kg. Such administration may be repeated, for example, 1 to 14 times, e.g., 3 to 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours. Such a regimen may be repeated one or more times as needed, e.g., after 6 or 12 months. Dosage can be determined or adjusted by measuring the amount of the compound of the invention in the blood at the time of administration, e.g., by collecting a biological sample and using an anti-idiotypic antibody targeting the antigen-binding region of the anti-CD38 antibody.

[0204] In a further embodiment, the anti-CD38 antibody is administered once weekly for 2 to 12 weeks, such as 3 to 10 weeks, for example 4 to 8 weeks.

[0205] In one embodiment, the anti-CD38 antibody is administered by maintenance therapy, eg, once weekly for a period of six months or more.

[0206] In one embodiment, the anti-CD38 antibody is administered in a regimen comprising one infusion of the anti-CD38 antibody followed by an infusion of the anti-CD38 antibody conjugated to a radioisotope, which may be repeated, for example, 7-9 days later.

[0207] In some embodiments, the anti-CD38 antibodies of the invention are used in combination with one or more additional therapeutic agents, e.g., chemotherapeutic agents. Non-limiting examples of DNA-damaging chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camtothecin and its analogs or metabolites, doxorubicin, etc.); topoisomerase II inhibitors (e.g., etoposide, teniposide, daunorubicin, etc.); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, methotrexate, methicillin-resistant Staphylococcus aureus ... DNA intercalators (e.g., cisplatin, oxaliplatin, carboplatin); DNA intercalators and free radical generators (e.g., bleomycin); and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, hydroxyurea).

[0208] Chemotherapeutic agents that interfere with cell replication include: paclitaxel, docetaxel and related analogs; vincristine, vinblastine and related analogs; thalidomide, lenalidomide and related analogs (e.g., CC-5013, CC-4047, etc.); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κB inhibitors (including inhibitors of IκB kinase); antibodies that bind to proteins overexpressed in cancer, thereby downregulating cell replication (e.g., trastuzumab, rituximab, cetuximab, bevacizumab, etc.); and other inhibitors of proteins or enzymes known to be upregulated, overexpressed or activated in cancer, whose inhibition downregulates cell replication.

[0209] In some embodiments, the antibodies of the invention can be used before, concurrently with, or after treatment with Velcade® (bortezomib).

[0210] treatment method In the method of the present invention, treatment is used to provide a positive therapeutic response for a disease or condition. The term "positive therapeutic response" refers to an improvement in a disease or condition and / or an improvement in symptoms associated with a disease or condition. For example, a positive therapeutic response can refer to one or more of the following improvements in a disease: (1) a decrease in the number of tumor cells; (2) an increase in tumor cell death; (3) an inhibition of tumor cell survival; (5) an inhibition (i.e., a slowdown to some extent, preferably a halt) of tumor growth; (6) an increase in patient survival rate; and (7) some relief from one or more symptoms associated with a disease or condition.

[0211] Positive therapeutic response in any given disease or condition can be determined by standardized response criteria specific to that disease or condition.Tumor response can be evaluated for changes in tumor morphology (i.e., tumor burden, tumor size, etc.) using screening techniques such as magnetic resonance imaging (MRI) scan, X-ray, computed tomography (CT) scan, bone scan, endoscopy, and tumor biopsy sampling, including bone marrow aspiration (BMA) and counting circulating tumor cells.

[0212] In addition to these positive therapeutic responses, the subject undergoing treatment may experience the beneficial effect of improving the symptoms associated with the disease.In the case of B cell tumors, the subject may experience the alleviation of so-called B symptoms, such as night sweats, fever, weight loss, and / or hives.In the case of pre-malignant conditions, treatment with anti-CD38 therapeutic antibody may block or prolong the time to the onset of related malignant conditions, for example, the onset of multiple myeloma in subjects suffering from monoclonal gammopathy of undetermined significance (MGUS).

[0213] Improvement in disease may be characterized as a complete response, which in the case of myeloma refers to the absence of clinically detectable disease accompanied by normalization of any previously abnormal radiological studies, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal proteins.

[0214] Such a response may last for at least 4-8 weeks, or at least 6-8 weeks, following treatment with the methods of the invention. Alternatively, the improvement in disease may be classified as a partial response. The term "partial response" refers to at least about a 50% reduction in all measurable tumor burden (i.e., the number of malignant cells present in a subject, or the measured bulk or amount of abnormal monoclonal protein in the tumor mass) in the absence of new lesions, and may last for 4-8 weeks, or 6-8 weeks.

[0215] Treatment according to the present invention includes a "therapeutically effective amount" of the pharmaceutical agent used. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.

[0216] The terms "therapeutically effective amount" and "therapeutically effective dosage" refer to an amount of a therapeutic agent sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent the progression of a disorder, cause regression of a disorder, prevent the recurrence, occurrence, onset, or progression of one or more symptoms associated with a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent) at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A therapeutically effective amount of an antibody for tumor therapy can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer may also be assessed in animal model systems predictive of efficacy in human tumors.

[0217] Alternatively, this property of the composition can be evaluated by examining the ability of the compound to inhibit cell proliferation or induce apoptosis by in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Those skilled in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0218] Anti-CD38 antibody kit In another aspect of the present invention, a kit for treating a disease or condition associated with hematological cancer is provided. In one embodiment, the kit includes a dose of an anti-CD38 antibody described herein, such as AB79. In some embodiments, the kit provided herein may include one or more doses of a liquid or lyophilized formulation provided herein. When the kit includes a lyophilized formulation of an anti-CD38 antibody described herein, such as AB79, the kit generally also contains a liquid suitable for reconstituting the liquid formulation, such as sterile water or a pharmaceutically acceptable buffer. In some embodiments, the kit may include an anti-CD38 antibody formulation described herein pre-packaged in a syringe for subcutaneous administration by a medical professional or for home use.

[0219] In certain embodiments, the kit is for a single administration or dose of an anti-CD38 antibody described herein, such as AB79. In other embodiments, the kit may include multiple doses of an anti-CD38 antibody described herein, such as AB79, for subcutaneous administration. In one embodiment, the kit may include an anti-CD38 antibody formulation described herein pre-packaged in a syringe for subcutaneous administration by a medical professional or for home use.

[0220] manufactured goods In other embodiments, an article of manufacture containing materials useful for treating the above-mentioned disorders is provided. The article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. The container can hold a composition effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is an antibody. A label on or associated with the container indicates that the composition is used to treat a selected condition. The article of manufacture may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. [Example]

[0221] Example 1: Model-based characterization of anti-CD38 antibodies in cynomolgus monkeys The anti-CD38 antibody AB79 binds to cynomolgus monkey (cyno) CD38, linking it to daratumumab (Darazalex), a cytolytic CD38 monoclonal antibody recently approved for the treatment of multiple myeloma. 商標 This unique feature supported the use of cynomolgus monkeys in preclinical studies to characterize the pharmacokinetics (PK), pharmacodynamics (PD), and safety of AB79. To this end, assays were developed to measure drug concentrations, immunogenicity, and quantify T, B, and NK lymphocytes in the blood of cynomolgus monkeys (cynomolgus monkeys). We evaluated these parameters in eight pharmacological and toxicological preclinical studies. Of the cell populations tested, CD38 is most highly expressed on NK cells; therefore, we assume that the drug's effects on NK cells will most closely resemble the effects on the target cells considered: plasmablasts, plasma cells, and other activated lymphocytes.

[0222] Data from eight studies in healthy monkeys using a dose range of 0.03–100 mg / kg were pooled to create mathematical models describing the pharmacokinetics and exposure-effect relationships for each cell type. NK cell depletion was identified as the most sensitive pharmacodynamic effect of AB79. This depletion was described by a metabolic turnover model (EC50 = 34.8 μg / mL depletion rate), with complete depletion achieved using an IV dose of 0.3 mg / kg. Intermediate effects on T cell numbers using a direct response model (EC50 = 9.43 μg / mL) and B cell numbers using a four-transit compartment model depletion rate (EC50 = 19.3 μg / mL) were also observed. These analyses substantiated the observation that each of the measured lymphocyte subsets was cleared at different rates by AB79 and required different time intervals to deplete the blood compartment.

[0223] Mathematical models that explain PK and PD data are useful tools for gaining mechanistic and quantitative insight into 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 characteristics of IgG antibodies, including distribution and elimination, physiological and genetic similarities between monkeys and humans, can be leveraged 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 PK concentrations and PD effects in healthy human subjects (Han and Zhou (2011) Ther. Deliv. 2:359-368).

[0224] material and method A summary of the monkey studies, in chronological order, is shown in Table 2. Single-dose studies 2, 7, and 8 were primarily conducted to evaluate the PK and PD of intravenously (IV) and subcutaneously (SC) administered AB79 (Figure 1). Repeat-dose studies, including two 4-week studies (Studies 1 and 3) and three 13-week studies under GLP conditions (Studies 4, 5, and 6), were conducted to evaluate safety, PK, and PD. A dosing error occurred in the 13-week study 5. Animals in the lowest dose group received a single dose of 0.01 mg / kg (second dose) instead of the intended 0.1 mg / kg, and then continued at 0.1 mg / kg. These data were added to the dataset along with the correct information on the actual administered dose. Study 6 repeated the lower dose of the 0.1 mg / kg QW group from Study 5. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals adopted and promulgated by the US National Institutes of Health. [Table 2]

[0225] biological analysis PK was analyzed using a validated method developed and implemented by Charles River Laboratories (Reno, NV). Briefly, AB79 concentrations were measured in monkey serum using an indirect enzyme-linked immunosorbent assay (ELISA). A 96-well microtiter plate was coated with an anti-idiotypic antibody against AB79. Blanks, standards, and quality control (QC) samples containing various concentrations of AB79 were added to the plate and incubated for 55–65 minutes at room temperature (RT). After washing the microtiter plate, peroxidase-conjugated affinity-purified mouse anti-human IgG (Peroxidase AffiniPure Mouse Anti-Human IgG, Fcγ fragment specific; Jackson ImmunoResearch) 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 generate the chromophore, followed by the addition of a stop solution (2N sulfuric acid) to stop the color development. Absorbance was measured at 450 nm using a SPECTRAmax® 190 microplate reader (Molecular Devices) and calculated using a four-parameter logistic weighting (1 / y 2 ) AB79 concentrations were calculated using a standard calibration curve. In Study 1 (Table 2), the lower limit of quantitation (LLOQ) of AB79 in serum was 0.061 μg / mL, and in all other studies, it was 0.05 μg / mL.

[0226] Determination of anti-AB79 antibodies (immunogenicity) Anti-drug antibody (ADA) screening of monkey sera was analyzed using a qualitative electrochemiluminescence (ECL) method, validated and performed by Charles River Laboratories (Reno, NV). Briefly, undiluted serum samples were incubated with 300 mM acetic acid. The acid-dissociated samples were incubated in a mixture of biotinylated AB79, AB79 labeled with SULFO-TAG (Meso Scale Diagnostics, Charles River Laboratories), and 1.5 M Trizma base to neutralize the acid and allow immune complex formation. The complexes were then added to streptavidin-coated MSD plates (Meso Scale Diagnostics) and allowed to bind. After washing, MSD Read Buffer T (Meso Scale Diagnostics) was added to the plate, and the complexes were detected by exciting the SULFO-TAG™ via the electrochemical reaction of Ru(bpy)3 to produce luminescence (light), which was read using an MSD Sector 6000 (Meso Scale Diagnostics). The amount of luminescence correlated with the level of monkey anti-AB79 antibodies present in the serum of each sample.

[0227] Blood cell characterization To assess and compare the level of AB79 binding between humans and monkeys, blood samples from each were collected in sodium heparin tubes. An aliquot (100 μL) of blood was mixed with the appropriate amount of characterized antibody (Table 3) and incubated for 15–20 minutes at room temperature in the dark. After incubation, 1 mL of BD FACS Lysing (1X; BD Biosciences; San Jose, CA) was added to lyse red blood cells. The cells were incubated for 10 minutes at room temperature in the dark, then centrifuged, decanted, and resuspended in 1 mL of staining buffer containing bovine serum albumin (BD Biosciences). Cells were centrifuged twice and decanted with 250 μL of Flow Fix (1% paraformaldehyde in calcium- and magnesium-free Dulbecco's PBS (Life Technologies, Carlsbad, CA)), and fluorescence was measured by flow cytometry analysis using a FACSCanto™ II flow cytometer (BD Biosciences). Monkey NK cells (CD3-, CD159a+), B cells (CD3-, CD20+), and T cells (CD3+), as well as human NK cells (CD3-, CD16 / CD56+), B cells (CD3-, CD19+), and T cells (CD3+), were measured. The mean fluorescence intensity of AB79 staining for each cell population was converted to units of molecular weight equivalents (MOEF) using a standard curve generated using Rainbow Beads (Spherotech; Lake Forest, IL). [Table 3]

[0228] For the studies outlined in Table 2, cells were stained and analyzed using validated methods developed and implemented by Charles River Laboratories (Reno, NV). Monkey blood samples were collected multiple times before and after AB79 treatment in sodium heparin tubes, and specific lymphocyte populations were measured by flow cytometry analysis using a FACSCanto™ II flow cytometer (BD Biosciences). Commercially available antibodies and a CD38 antibody (AB19; Table 3; U.S. Patent No. 8,362,211) were titrated to optimal concentrations for staining. Monkey CD38+ / -, T cell (CD3+), B cell (CD3- / CD20+), and natural killer (NK) cell (CD3- / CD20- / CD16+) populations were identified, and lymphocytes were quantified using CD45 TruCount™ tubes (BD Biosciences). Approximately 100 μL aliquots of each blood sample were placed into the appropriate wells of a 96-well plate, and the indicated amount of antibody was added. The samples were mixed and incubated at room temperature in the dark for a minimum of 30 minutes. After incubation, red blood cells were lysed, and the samples were mixed and incubated at room temperature in the dark for an additional 10 minutes. The plates were centrifuged and the supernatant decanted. The cell pellets were then resuspended in 1,800 μL of staining buffer, the samples were mixed, centrifuged, and the supernatant decanted. The cell pellets were resuspended in 500 μL of staining buffer containing fetal bovine serum, and approximately 300 μL of the cell suspension was transferred to a 96-well V-bottom plate for analysis. The percentage of NK cells and the total T and B cells were applied to the cell count values ​​obtained using TruCount™ tubes (BD Biosciences; San Jose, CA) and used to determine the absolute cell numbers of each cell population. In studies 1-4, CD38+ NK, B, and T cell subsets were assessed at baseline using labeled anti-CD38 antibodies AB79 or AB19. Although AB19 binds to a different epitope, the results were very similar and therefore are not presented separately. Processed samples were analyzed immediately.

[0229] Development of PK model During PK model development, one-, two-, and three-compartment model structures were investigated. As judged by goodness-of-fit (GOF) plots and reduced objective function values ​​(OFV), two-compartment models were clearly superior to one-compartment models. Based on visual inspection of diagnostic plots, it was not necessary to introduce a third compartment to adequately describe the data. Bioavailability (F) was modeled using the logit transformation F = exp(PAR) / (1 + exp(PAR)), where PAR represents the model parameter, ensuring estimates fell within the 0-1 range. Nonlinear PK at low concentrations was modeled with a quasi-steady-state (QSS) approximation model of the target-mediated pharmacokinetic (TMDD) process (Gibiansky and Gibiansky (2009) Expert Opin. Drug Metab. Toxicol. 5:803-812).

[0230] A schematic of the model is provided in Figure 2C. The QSS approximation assumes that the steady-state concentrations of free drug C, target R, and drug-target complex RC are established very rapidly compared to all other processes. This means that the binding process is balanced with the dissociation and internalization processes, and that the following equation holds in the appropriate units: K ON *C*R=(K OFF +K INT )*RC, where K ON denotes the binding rate constant, and K OFF denotes the dissociation rate constant, and K INT indicates the internalization rate constant.

[0231] Intersubject variability (BSV) was investigated for all parameters and modeled with the following type of exponential model: PAR i =TVPAR*e ETAPAR i , where PAR i is the individual, TVPAR is the typical parameter estimate, and ETAPAR i is the estimate of the deviation of individual i. iValues ​​were assumed to follow a normal distribution with mean zero. Residuals were described by a combined additive and proportional error model (Beal and Sheiner (1992) NONMEM User Guides, in University of California, CA).

[0232] To identify potential covariate effects on the PK of AB79, the following parameters were investigated: body weight, sex, dose, route of administration, and study.

[0233] PK-PD model development PK-PD model development was performed separately for each of the three cell types. Note that model development measurements close to drug administration (less than 8 hours post-dose) were not used because multiple blood samples were drawn over a short period of time, potentially subject to nonspecific drug-independent effects. PK model and parameter estimates were modified. Turnover, transit compartment, and various forms of direct response models were tested (Friberg et al. (2002) J. Clin. Oncol. 20:4713-4721; Mager et al. (2003) Drug Metab. Dispos. 31:510-518). In the turnover model, the drug effect was introduced into the cell elimination rate in the form of an Emax-type model with or without the Hill coefficient. In this 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 ), EMAX indicates the maximum effect, C50 indicates the concentration at which half of the maximum effect is achieved, and H indicates the Hill coefficient. In the transit compartment model (TCM), the drug effect was introduced and tested at different locations: proliferation rate, circulation, and a third transit compartment. The combination of these effects and whether the data support the existence of a feedback mechanism from circulation to proliferation rate were also tested. Furthermore, an EMAX-type direct response model with or without the Hill coefficient was tested to describe the drug concentration-effect curve.

[0234] Random effect parameters were introduced to estimate inter-subject variability for baseline cell count, cell production rate (KIN), transit time of the transit compartment model (MTT), C50, and EMAX. Individual mean baseline cell levels were provided in the dataset (dataset BL). This was used as a representative value in the model. Random effect parameters were added to allow adjustment of individual baseline estimates based on all measurements for an individual. PD residuals were described with a proportional error model.

[0235] Model validation during modeling (PK and PK-PD) OFV used standard errors, GOF plots, and individual prediction versus data plots to evaluate models and compare them with alternative models.

[0236] The following software packages were utilized: NONMEM (version 7.2), KIWI (version 1.6), Berkeley Madonna (version 8.3.14), PSN (version 4), and R (version 3.3.0).

[0237] Preparing the dataset Datasets from eight monkey studies were collected, reorganized, and merged into three separate NONMEM-readable PK-PD datasets. Each of the three datasets included individual monkey characteristics (study, ID, group, weight, and sex), dosing information, PK, and either NK, B, or T cell data. For control animals, only cell counts were added to the dataset, assuming no serum AB79 levels were present. Time-resolved information regarding antidrug immunogenicity status (ADA), i.e., TITER, containing the quantitative measurement result and a 0 / 1 flag variable ADAF (ADAF = 1 if ADA affects AB79 concentrations, and ADAF = 0 if it does not), was added to each observation in a separate column. Because ADA titers were measured with different method specifications in different studies, values ​​cannot be directly compared across studies. To utilize ADA information in a consistent manner across all studies, the following procedure was applied individually to each animal: ADA titers that increased beyond the initially measured level at time points later than 7 days were considered ADA-positive and flagged in the dataset (ADAF = 1). If a sample at a time point was flagged as ADA positive, all samples taken after that time point were also flagged as ADA positive in that animal, regardless of the titer measured. ADA-positive observations were not used for parameter estimation during model development. Note that PD measurements from sampling time points of ADA-affected PK concentrations were also flagged with ADAF=1.

[0238] For cell count data, individual baseline values ​​for each cell type (NK, B, and T cells) were calculated as the average of all available pre-dose measurements for a given animal. In most studies, this was a single measurement. The baseline value for each animal was then added to each observation for each animal as the observation at the first dosing event (TIME=0) and as a constant value in column BL. Based on this baseline value, the percent baseline for each observed cell count was calculated and added to the dataset.

[0239] Scaling of monkey PK parameters The final PK and PK-PD models were used as starting points to simulate the first PK and PK-PD profiles of human clinical trials. While by no means conclusive, a comparative analysis of data from a therapeutic monoclonal antibody indicates that PK parameters derived from monkey studies can be scaled to aid in predicting human PK profiles with acceptable accuracy (Han and Zhou (2011) Ther. Deliv. 2:359-368). This publication demonstrated that a fixed exponent of 0.85 can be used to reliably predict human clearance of a monoclonal antibody. Consequently, this relationship was applied to scale human clearance parameters (CL, Q), while volumetric parameters (VC, VP) were scaled using a direct relationship between body weight (BW).

[0240]

number

[0241] result Pharmacokinetics of AB79 The PK dataset was pooled from all eight studies in healthy monkeys, excluding the placebo group (Table 2). In total, the dataset included data from 140 animals, 58 of which were male and 82 were female. The weights of the animals tested 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 in Study 7 and three groups in Study 8, SC doses of 0.03, 0.1, 0.3, and 1 mg / kg were administered (total of 15 animals). The pooled dataset contained 2,199 measurable PK observations greater than the LLOQ (Figures 2A and 2B). PK was most densely sampled after the first dose, and even in the long-term toxicity studies, most animals were terminated before day 98. Only Study 4 included a recovery group, allowing collection of PK data from only four animals: two from the 80 mg / kg group and one each from the 30 mg / kg and 3 mg / kg groups (Figure 2B). ADA was assessed in parallel with AB79 concentration. 229 PK observations were affected by ADA (Figure 3).

[0242] First, PK analysis was performed using standard noncompartmental techniques (NCA) for each of the monkey studies. Based on single-dose studies (IV bolus injection or 30-minute IV infusion), we calculated a terminal volume of distribution (Vz) ranging from 64 to 116 mL / kg, clearance from 6.04 to 14.7 mL / kg / day, and terminal elimination half-life (T1 / 2) ranging from 4.75 to 11.2 days. Area under the concentration-time curve (AUC) and maximum concentration (Cmax) values ​​were found to increase dose-proportionally over a wide range. Only the PK profile for the lowest dose group (<1 mg / kg, Figures 2D-2F) provided evidence of nonlinearly increasing clearance at concentrations below 0.5 μg / mL (TMDD) (Kamath (2016) Drug Discov. Today Technol. 21-22:75-83). A linear two-compartment model was constructed based on data from all monkey studies except for the two lowest dose groups (doses >0.3 mg / kg). When the PK of the lowest dose groups was simulated and overlaid with the measured concentrations, it was clear that the linear model predicted the concentrations (Figures 2D-2F).

[0243] Available PK data after a single SC administration revealed that Cmax was 70-80% lower in the SC group compared to the IV group at the same dose, and AUC was comparable. No differences in PK parameters were observed between male and female monkeys. The results of these initial analyses were used as the starting point for model development.

[0244] PK model development Model development began with IV administration data from the competition, and the initial model was gradually expanded using more complex data. Similar to other therapeutic antibodies, PK follows an overall linear two-compartment model (Kamath (2016) Drug Discov. Today Technol. 21-22:75-83). The nonlinear elimination component (TMDD), describing accelerated clearance at low concentrations, was modeled using a quasi-steady-state (QSS) approximation (Gibiansky and Gibiansky (2009) Expert Opin. Drug Metab. Toxicol. 5:803-812). The assumption that the drug-target association process is much faster than the drug dissociation, distribution, and elimination processes, as well as the complex target-drug elimination process, resulted in a simplified TMDD model (Figure 2, Table 4). The amount of data at low concentrations was relatively small, and therefore not all parameters were estimated in a single run of the software program. Therefore, the parameters of the TMDD model were first estimated by focusing on the data from low-dose single-dose studies 7 and 8. The resulting TMDD parameter estimates were then left unfixed during the final estimation for the entire data set (Table 4).

[0245] [Table 4]

[0246] Absorption parameter K A Estimates for and F were obtained when data from the SC groups were added. All SC data were obtained from the four low-dose single-dose groups in Studies 7 and 8. These lower doses (≤1 mg / kg) covered the clinically relevant range but may limit the generalizability of parameter estimates for higher doses.

[0247] Inter-subject variability (BSV) for PK parameters was described by an exponential model. A ), clearance (CL), and peripheral volume of distribution (V P) has an estimated BSV of approximately 40% and a central volume of distribution (V C ) had an estimated BSV of approximately 20% (Table 4). Covariate analysis revealed that V C The effect of the administration route on V C Typical values ​​for TMDD were 0.141 L for IV administration and 0.043 L for SC administration (approximately 70% smaller). No other significant covariate effects were identified. Due to the limited amount of data at low concentrations, the correlation between subject variability and individual predictions for TMDD parameters was limited by the internalization rate K INT The BSV was estimated only for AB79 (BSV: 49%). Model evaluation based on residuals, OFV, standard error, GOF plots, and individual curve fits confirmed that the final model adequately described the PK of AB79 in healthy monkeys (Table 4, Figures 4A-4J).

[0248] Pharmacodynamics The levels of AB79 binding to human and monkey blood NK cells, T cells, and B cells were compared by flow cytometry analysis. As shown in Figure 5, monkey lymphocytes had CD38 expression levels based on AB79 molecular equivalents of fluorescence (MOEF), which were slightly lower compared to their human counterparts, but with a similar relationship between cell types: CD38 expression on NK cells > CD38 expression on B cells > CD38 expression on T cells. These data support the use of this non-human primate species as a relevant model to help predict the potential for AB79 PD activity in humans.

[0249] For a thorough quantitative analysis of the relationship between drug exposure (PK) and the extent and duration of cell depletion (PD), we compiled datasets from PK concentrations, NK cell, B cell, and T cell counts across all eight monkey studies, including placebo-treated animals when available (Table 2). Initial characterization of the datasets showed that at baseline, T cells were the most abundant lymphocyte subtype, with a median of 3,732 cells per µL (interquartile range (IQR): 2,881–5,176), compared to B cells with 1,279 cells per µL (IQR: 860.8–1,890) and NK cells with 685 cells per µL (IQR: 482.8–970.1). Baseline CD38 expression in these cell populations was assessed in studies 1–4 (Table 2, n = 67). 86.7% (SD 11.3) of NK cells expressed CD38, with smaller 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.

[0250] Data from placebo-treated animals showed that the mean counts of each cell type varied over time between individual animals beyond what would be expected from variation within a single individual (Figure 6). For example, the mean coefficient of variation for B cell counts across individual placebo curves was 27%, while individual mean B cell levels ranged from 436.6 to 4,389. Furthermore, there were differences between the mean baseline lymphocyte counts from male and female animals and between animals from different studies, adding to the variability (Figure 7). Based on these results, cell counts after each treatment were calculated as a percentage relative to their individual baseline value, rather than absolute cell counts at each time point. For example, a value of 33% means that the cell count in a sample was 1 / 3 of the baseline cell count. This provided standardized values ​​that could be compared across datasets.

[0251] The rapid onset of depletion of AB79-bound cells suggests that initial blood concentrations accelerate the reduction in lymphocyte counts (Figure 8). At an IV dose of 0.3 mg / kg of AB79, the median maximal effect on NK cells was 93.9% depletion (i.e., 6.1% of baseline cell counts remaining). At 0.1 mg / kg, peak depletion was 71% (29% of baseline remaining). At doses >0.3 mg / kg, NK cells were almost completely depleted in the blood compartment (Nadir (range): 1.06% of baseline (0.17, 6.23); Figure 8A). After a single dose of 0.3 mg / kg, it took approximately 7 days for NK cells to recover to an average of 50% of baseline, although the kinetics of recovery were highly variable between individuals (Figures 8B and 8C). Consistent with these results, NK function was also tested in a subset of animals from Study 7 (n = 3 / group; Table 2). This experiment demonstrated a dose-dependent decrease with minimal change in blood NK activity at 48 hours post-treatment in animals treated with 0.1 mg / kg AB79 (lysis rate at 100:1 effector:target ratio ± SD; 44.5% ± 23.6% vs. 41.4% ± 25.8%) and a near complete loss of NK activity in animals treated with 1.0 mg / kg (lysis rate at 100:1 effector:target ratio ± SD; 37.4% ± 10.3% vs. 6.8% ± 12.5%). NK cell function showed recovery over the 57 days measured (lysis rate at 100:1 effector:target ratio ± SD, 16.0% ± 11.9%).

[0252] B cells and T cells were depleted to a relatively lesser extent than NK cells, consistent with their lower CD38 expression levels (Figure 5). For example, at 0.3 mg / kg IV AB79, B cells had a median maximum depletion of 45% of baseline, and T cells were depleted to 43% of baseline (Figures 8D and 8G). At this dose level, a 50% reduction from baseline in B cell numbers was not achieved in all animals. Only at the highest dose of ≥30 mg / kg were B cells nearly completely depleted (Figure 8D). T cells were depleted to a similar extent as B cells, but recovery was more rapid (Figures 8G-8I).

[0253] Two studies, 7 and 8, compared IV and SC administration (Figures 8C, 8F, and 8I). There were no apparent differences in cell depletion between the routes of administration. At the low dose (study 8), all cells showed some cell depletion at early time points. Sustained cell depletion (>24 hours) below baseline values ​​was only seen in the NK cell population, not in T and B cells. The timing of onset of NK cell depletion appeared similar between dose groups, regardless of route of administration, and the duration of depletion was dose-dependent. Cell recovery in all test groups was seen by day 57.

[0254] PK-PD model Separate PK-PD models were developed to describe the effects of AB79 exposure on NK, B, and T cells. During PK-PD modeling, PK parameters were fixed at the final PK model estimates, and various PD models were tested. The NK cell population in peripheral blood was adequately described by a turnover model, and the depletion drug effect was related to the depletion rate via PK concentration using an Emax-type model. In this model, Emax represents the maximum rate of additional NK cell depletion, and C50 represents the concentration at which the rate of additional NK cell depletion is half-maximal. The structural PK-PD model for NK cells was of the following form:

[0255]

number

[0256] In the formula, NK represents the actual number of NK cells, and K IN represents the production rate, and K OUT represents the excretion rate in the absence of drug. Using a given baseline measurement, BLK OUT is the equation K OUT =K INNote that the K value is defined as K / BL. c represents the AB79 concentration in the central compartment. When all parameters were estimated at once, the software program did not produce stable results. Individual estimates of K, E, and C were highly correlated. Furthermore, due to limited differences between the maximum effects of different doses (see the previous section) and large interindividual variability, accurate estimation of all parameters was not expected. In a series of estimations, one or two of the three parameters K, E, and C were fixed to different values, and the other parameters were estimated. K was fixed at 10,000 and E at 322, achieving stable performance and a reasonable goodness of fit. A typical C estimate was 29.0 μg / mL (Table 5). In addition, the sensitivity of the selected K and E values ​​was tested by selecting different combinations of higher and lower values. Intersubject variability was also investigated by the NK production rate K. IN The C50 was 113% and C50 was 149%, which was due to the large individual variability at baseline and between treated animals. The model was evaluated based on residuals, OFV, standard error, GOF plots, and individual curve fits (Table 5, Figure 9).

[0257] [Table 5]

[0258] The transit compartment model was superior to the direct response or turnover models for describing AB79-induced B cell depletion. Four transit compartments were found to be appropriate, and the drug effect was described by an Emax-type model for depletion rate. Similar to the NK cell depletion model, Emax represents the maximum rate, and C50 represents the concentration at which the rate is half-maximal. Thus, the structural PK-PD model for B cells is given by the following five equations:

[0259]

number

[0260] TR i (i=1~4) represents the four transit compartments. K TR , K. PROL and K. CIRC is expressed by the following equation, K TR =K PROL =K CIRC = 4 / MTT, where MTT is the mean transit time (Friberg et al. (2002) J. Clin. Oncol. 20:4713-4721). B represents the number of B cells in the blood, and c represents the concentration of AB79 in the central compartment.

[0261] With an EMAX fixed at 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 in maximum effect relative to the maximum AB79 concentration was adequately captured. The model indicates that AB79 primarily affects circulating B cells. Additional effects on progenitor cells and feedback loops were not necessary to describe the available monkey B cell data. The inter-subject variability of 135% MTT and 24.1% baseline B cell levels (BASE) indicates large individual differences between animals.

[0262] Drug-induced T cell depletion with rapid recovery is aptly described by the direct response model: T(c)BL T *(1-EMAX*c / (+C50)), where T represents the actual T cell count and BL T where σ represents the T cell count at baseline, and c represents the AB79 concentration in the central compartment. A typical C was estimated to be 11.86 μg / mL, and a typical E was 0.47, indicating that in this case, only about half of the T cells could be depleted by AB79 (Table 5). Note, however, that the inter-subject variability in E was approximately 70%. In this model, unlike the NK and B cell depletion models, C represents the concentration at which T cell depletion was half-maximal.

[0263] For NK cells, model evaluation of the final PK-PD models for B and T cells based on residuals, OFV, standard errors, GOF plots, and individual curve fits supported an adequate description of the available monkey data (Table 5, Figure 9).

[0264] Simulation of human PK and cell depletion The monkey PK and PK-PD models were used as starting points for model-based simulations of human PK and cell count data to support the design and justify the selected doses for first-in-human (FIH) clinical trials in healthy volunteers. To this end, the model structure, including the TMDD derived from the monkey data, was also assumed to describe key features of human PK and subsequent lymphocyte depletion. To obtain predictions of human PK parameters, the following monkey PK parameter estimates were scaled: central and peripheral volume of distribution (V). C , V P ), and clearance (CL) and intercompartmental clearance (Q) using a head-on approach to monoclonal antibodies (Han and Zhou (2011) Ther. Deliv. 2:359-368). Because AB79 is a fully human monoclonal antibody, it is expected to be less immunogenic in humans than observed in monkeys. Therefore, for modeling and simulation, ADA-positive samples were excluded from the dataset.

[0265] Using a scale model, we simulated the exposure and depletion profiles of NK, B, and T cells for a single dose of 0.0003–1.0 mg / kg administered via a 2-h infusion (IV) or via subcutaneous injection (SC), as planned in the FIH trial (Figure 10). Simulations predict that after an IV dose of 0.0003 mg / kg, no observable drug-induced effect on lymphocyte counts, and even measurable PK concentrations above the LLOQ, are expected. Due to variability and the limited size of the dose groups, the minimal detectable drug effect on NK cell counts was assumed to be at least a 10% decline. At doses of 0.01 mg / kg IV and 0.03 mg / kg SC, NK cells were predicted to deplete to less than 90% of baseline remaining.

[0266] At the 0.3 mg / kg IV dose, we predicted NK cell depletion to a remaining 17% of baseline within 3 hours after the end of the infusion, with recovery to over 50% after 11 days (Figure 10). At the same dose, the model predicts maximal depletion of B cells to 67% of baseline after 2.5 days, and immediate depletion of T cells to 86% of baseline. For the same dose of 0.3 mg / kg subcutaneously, the model predicted it would result in less, and then maximal, depletion (nadir relative to baseline: NK cells 37%, B cells 74%, T cells 94%).

[0267] These in vitro and in vivo preclinical studies demonstrate that monkeys are an appropriate animal model for testing the pharmacology of AB79. The meticulous sampling of NK, B, and T lymphocyte PK and cell count data from eight monkey studies using diverse doses and administration schedules provides a rich data source for a comprehensive and quantitative understanding of the relationship between AB79 dose, exposure, and cell depletion. The generated population PK and PK-PD models adequately explain the observed data and provide powerful tools for predicting exposure and lymphocyte depletion in future studies in monkeys as well as in human clinical trials.

[0268] A first-in-human (FIH) single ascending dose study in healthy volunteers was conducted (www.clinicaltrials.gov:NCT02219256) (Figure 11). The intended pharmacological effect of AB79 is the depletion of activated lymphocytes. However, profound and sustained lymphocyte depletion (enhanced pharmacology) may lead to immune system impairment, which is unacceptable for patients or healthy study participants. Therefore, a safe IV starting dose of 0.0003 mg / kg was selected for the FIH study.

[0269] The monkey data suggest that NK cell depletion was determined to be the most sensitive biological effect. PK-NK simulation results helped determine the minimum dose level of 0.01 mg / kg IV at which the most sensitive pharmacological effect (NK cell depletion) detectable in humans was predicted. New data from the FIH study revealed that the overall pattern of AB79's dose-dependent, cell type-specific depletion effect followed model-based predictions (manuscript in preparation). AB79 appeared even more efficient than predicted. For example, at an IV dose of 0.03 mg / kg, NK cells in human subjects were depleted to less than 10% of baseline. The median nadir (lowest depletion point) in monkeys at this dose was 20.0% (Figure 8).

[0270] Three cytolytic anti-CD38 monoclonal antibodies (daratumumab, SAR650984, and MOR202) are in clinical development for multiple myeloma (van de Donk et al. (2016) Immunol. Rev. 270:95-112). Daratumumab (Darzalex™, administered as an intravenous infusion) was recently approved for multiple myeloma in the United States and for non-Hodgkin's lymphoma in Europe. Unlike AB79, daratumumab does not cross-react with monkey CD38. Therefore, comparison of our results with those of AB79 in cynomolgus monkeys using daratumumab was not possible. Furthermore, multiple myeloma patients have high levels of CD38-positive malignant cells, which may require higher effective antibody concentrations for this cancer indication (de Weers et al. (2011) J. Immunol. 186:1840-1848).

[0271] However, even though AB79 achieved complete peripheral NK cell depletion at approximately 1 mg / kg and complete B cell depletion at approximately 3 mg / kg ( Figure 8 ), it is noteworthy that daratumumab is approved in multiple myeloma at a weekly IV dose of 16 mg / kg.

[0272] Despite the extensive database from eight monkey studies, several limitations were recognized. AB79 effectively depletes NK cells even at the lowest dose tested, 0.03 mg / kg. At such low doses, PK quickly fell below the limit of quantification of the bioanalytical assay, preventing elucidation of the relationship between exposure and effect at low doses. Furthermore, during preclinical development, it was recognized that maximum cell depletion occurred shortly after maximum drug concentration, but elucidation of the early stages of depletion was technically limited by the overall sample size and potentially by nonspecific cell depletion due to repeated blood draws (blood draw effects). The blood draw effects were observed as transient pancytopenia characterized by the depletion of cell types that do not bind to AB79 (e.g., RBCs) and were not dose-dependent, suggesting that this was due to blood loss resulting from multiple blood draws rather than any specific effect of AB79 (Figure 12). As a result, our ability to accurately estimate model parameters, especially for NK cell depletion, was limited, and typical values ​​of KIN and EMAX required modification to achieve stable and appropriate estimation results.

[0273] The effects of AB79 on tissue plasma cells or plasmablasts could not be measured. However, like plasma cells and plasmablasts, NK cells have high levels of CD38 on their surface, and the efficiency of cell depletion of specific lymphocyte subsets depended, at least in part, on the expression level of CD38. Therefore, the cytolytic effect of AB79 on plasmablasts and plasma cells may be comparable to its effect on NK cells. Currently, information on the long-term effects of AB79 treatment in monkeys is limited. Only a small proportion of animals in the 13-week toxicology study were investigated long-term in the recovery group, and most animals in all dose groups developed ADA (Figure 3). Furthermore, the baseline values ​​and depletion profiles of different lymphocyte subsets were highly variable between individuals. Therefore, the long-term effects of AB79 cannot be investigated in monkeys and need to be tested in humans.

[0274] As human data emerge, detailed comparisons of human and monkey PK and PD data will be interesting. Building a PK model based on human data and comparing it with a monkey model will enable refinement of the TMDD model for AB79. Data generated in patient trials will provide insight into how AB79-mediated depletion of B-lineage cells compares between RA and SLE patients, and between multiple myeloma and healthy subjects. Investigating subject- or disease-related factors that may affect cell depletion efficiency in addition to CD38 expression levels will also be important, potentially leading to personalized treatment. Furthermore, thorough in vitro and in vivo comparisons of AB79 with daratumumab and / or other CD38 antibodies will reveal valuable information about the pharmacology of anti-CD38 antibodies and their optimal applications.

[0275] Extensive pharmacological data and PK and PK-PD models enabled characterization of exposure-effect relationships in cynomolgus monkeys. Model-based analysis of NK, B, and T cells supported and quantified the discovery that each blood lymphocyte subset is depleted by antibody at different rates and requires different periods to replenish the blood compartment. The models proved to be an excellent tool for simulating PK and PD data under various dosing scenarios in preparation for clinical trials.

[0276] Example 2: CD38+ cell depletion with AB79 Table 6 shows 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 lymphoblastoid cell line (MV-4-11) that does not express CD38, 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 but do not directly deplete plasmablasts, CD20 低 / 陰性CD38 is expressed at high levels on plasmablasts and plasma cells, making these cells direct targets of AB79. In vitro studies with human blood cells and cell lines demonstrated that AB79 binding to CD38 did not result in PBMC cytokine activation, demonstrating that AB79 is not an agonist, as discussed below. Rather, AB79 mediated cell depletion of human B-lineage cell lines by ADCC and CDC, and in most cases, cell lines with increased CD38 expression were susceptible to cytolysis.

[0277] [Table 6]

[0278] This is consistent with findings in healthy cynomolgus monkeys, where the efficiency of depletion correlated with CD38 expression levels and AB79 dose levels. NK cells expressing high levels of CD38 were depleted to a greater extent than CD20+ B cells and CD3+ T cells, which expressed less CD38 (Figure 13). In vivo, AB79 potently suppressed the recall response of human B cells to antigen in a mouse adoptive transfer model (Figure 14). Together, these data support further investigation of AB79 in autoimmune diseases.

[0279] Human PBMCs were treated with AB79 under multiple conditions and the release of proinflammatory cytokines was measured. Because AB79 cross-reacts with monkey CD38, which shares 91% protein identity with the human protein, cynomolgus monkeys were used to demonstrate cell-type-specific intrinsic depletion and the relationship with AB79 dose. A second animal model, adoptively transferred human PBMCs into mice, was used to determine whether AB79 targets human antibody-producing cells.

[0280] AB79 binds to CD38 and mediates ADCC and CDC. Receptor counts were determined with FIKIT (DAKO, cat #K0078) using a mouse anti-human CD38 antibody (clone HIT2) and calculated by converting the mean fluorescence intensity (MFI) of the stained samples to a standard curve generated from the MFI of five populations of beads with defined numbers of antibody molecules coupled. Absolute receptor counts were calculated by subtracting the isotype control (mouse IgG1) MFI from the anti-CD38 antibody MFI.

[0281] CDC was assessed by seeding cell lines at 10,000 cells / well and adding AB79, control IgG, or medium. A five-point dose-response curve (0.001–10 mg / ml) was typically performed. Rabbit complement (2–15 μl; #CL 3441 CedarLane Laboratories) was added to each well except the control well. CytoTox-Glo reagent (Promega, G7571 / G7573) was used to detect cytotoxicity by luminescence. Groups tested: cells only; cells + complement; cells + IgG control + complement; cells + AB79 + complement. %CDC equation: %CDC = 100 – ((RLU(test) / RLU(complement only)) × 100).

[0282] ADCC was tested by seeding 5,000 target cells / well (T, cell line) with 50 ml of AB79, control IgG, Triton X-100 (1%; Sigma Chemical), or medium alone, and 50 ml of human effector (E) PBMCs at a T:E cell ratio of 1:25 to 1:50. A nine-point antibody dose-response curve (0.000001 to 100 nM) was typically performed. Experimental lysis = PBMCs + cell line + antibody. Spontaneous lysis = PBMCs + cell line without antibody. Maximum lysis = cell line + Triton X-100. Cytotoxicity was assessed using the CytoTox-Glo™ luminescent cytotoxicity assay (Promega).

[0283] AB79 has no agonist activity. The ability of AB79 treatment to induce cytokine production in human PBMC was compared to a negative IgG1 isotype control and positive controls, PHA, anti-CD3 (clone OKT3) or anti-CD52 (Campath) antibodies (Figures 15 and 16).

[0284] Soluble AB79 did not increase IL-6 levels (mean ± SD) in PBMCs collected from four different subjects after 24 hours of incubation compared to the IgG1 isotype control. PHA increased cytokine levels in all subjects, indicating that the cells had the capacity to produce IL-6 (Figure 15). Similar results were seen in PBMCs stimulated for 48 hours when IL-2, IL-4, IL-10, GM-CSF, IFNγ, and TNFα were tested (data not shown).

[0285] The method by which antibodies are presented to cells can contribute to the antibody outcome: ligand binding and cellular response (Stebbings et al. (2007) J. Immunol. 179:3325-3331). Stebbings et al. showed that the greatest cellular response (cytokine release) to an agonist antibody occurred when the antibody was added to the well in solution and attached to the well surface at a high concentration, such as when the liquid was evaporated (dry binding), compared to antibody bound to the well in solution (wet binding) or antibody added directly to PBMCs (soluble) (Figure 16A). AB79 did not stimulate cytokine production using either of these approaches (Figure 16B).

[0286] AB79 (100 mg / ml) did not stimulate IL-2, -4, -6, -8, -10, GM-CSF, IFNγ, or TNFα under any of the conditions tested after 24 hours. AB79 did not induce IL-10 or GM-CSF, both of which were induced by anti-CD3 (not shown, but all values ​​except for anti-CD3 were below the LLOQ). IL-8 was constitutively produced by PBMC and was not altered by any treatment (data not shown) (Table 7).

[0287] [Table 7-1]

[0288] [Table 7-2]

[0289] AB79 depletes CD38+ cells AB79 binds CD38 with high affinity and mediates CDC and ADCC. AB79 is not an agonist and did not induce cytokine release from human PBMCs. AB79 bound to CD38 from both humans and cynomolgus monkeys. Lymphocytes from both species had a similar cell-specific pattern of CD38 expression: NK cells > B cells > T cells, based on the median fluorescence intensity of AB79 staining. Treatment with AB79 depleted monkey lymphocytes in a reversible, cell-specific, and dose-dependent manner. AB79 effectively blocked human antibody recall responses in a mouse adoptive transfer model.

[0290] Example 3: Evaluation of AB79 binding to human and cynomolgus monkey erythrocytes and platelets AB79, a fully human, high-affinity, non-agonistic IgG1 monoclonal Ab directed against human CD38, was evaluated to determine its binding to human or cynomolgus monkey (cyno) red blood cells (RBCs) or platelets. Thirty blood samples from healthy human volunteers and 30 whole blood samples from healthy cynos were evaluated for AB79 binding compared to palivizumab, an isotype-matched control monoclonal antibody of irrelevant specificity that does not bind to RBCs or platelets.

[0291] method Blood samples from 30 normal human subjects (15 males and 15 females for platelets, 25 males and 5 females for RBCs) and 30 rhinos (15 males and 15 females) of Chinese origin were purchased from Bioreclamation (Long Island, NY). Whole blood was collected in sodium citrate tubes and shipped overnight at ambient temperature.

[0292] To assess AB79 binding to platelets, 50 μL of whole blood was stained with a cytochrome-cross-reactive anti-human CD61 FITC mAb (BD Biosciences Cat# 555753) in combination with Alexa Fluor 647 (AF647)-conjugated AB79 or an isotype-matched control, AF647-conjugated palivizumab (MedImmune Cat# 60574-4113-1), a humanized monoclonal antibody specific for an epitope in the A antigenic site of the F protein of respiratory syncytial virus (RSV), an antigen not present in RBCs or platelets. After staining, red blood cell lysis was performed, and samples were analyzed using a BD Canto flow cytometer. CD61+ platelets were gated for analysis.

[0293] To assess AB79 binding to RBCs, 50 μl of whole blood was stained with AF647-conjugated AB79 or the isotype-matched control, AF647-conjugated palivizumab. In some experiments, lymphocytes were stained with a cytochrome-cross-reactive anti-human CD45 PERCP (BD Biosciences Cat# 552724) to provide evidence of AB79 binding to lymphocyte subsets, as observed in previous studies. Samples were analyzed using a BD Canto flow cytometer. Data were expressed as mean fluorescence intensity (MFI) of AB79 or the isotype control.

[0294] Results and Discussion The ability of AB79 to bind to RBCs from 30 healthy human volunteers and 30 healthy cynos was assessed using flow cytometry. RBC staining by AB79 did not exceed the level of staining seen with the isotype control antibody in either human ( FIG. 17 and Table 8 ) or cyno blood samples ( FIG. 18 and Table 8 ). No detectable binding was observed in either species, and no difference in the ratio of MFI for AB79 / isotype control was observed between humans and cynos.

[0295] [Table 8]

[0296] The ability of AB79 to bind to CD61+ platelets from 30 healthy human volunteers and 30 healthy cynos (donors 1-15 were male, and donors 16-30 were female) was assessed using flow cytometry. AB79 staining of platelets did not exceed the staining level observed with the isotype control in either human blood samples (Figure 19 and Table 9) or cyno blood samples (Figure 20 and Table 9). No detectable binding was observed in either species, and no difference in the ratio of MFI for AB79 / isotype control was observed between humans and cynos.

[0297] [Table 9]

[0298] As a positive control for AB79 staining, AB79 staining was measured in a portion of the blood sample. Due to the high predominance of red blood cells in the blood, 200,000 events were generated, and a minor population of CD45+ lymphocytes was gated for further evaluation. While no binding was observed with the isotype control, AB79 bound to a minor population of CD45+ lymphocytes (Figure 21). This confirmed AB79 was able to stain blood cells under conditions where no detectable binding of AB79 to RBCs or platelets was observed.

[0299] Example 4: Evaluation of AB79 binding to human and cynomolgus monkey erythrocytes and platelets using more sensitive flow cytometry To further evaluate whether AB79 binds to human or cynomolgus monkey (cynomolgus monkey) RBCs, we developed a highly sensitive flow cytometry assay in case previous assays were not sensitive enough to detect low levels of CD38 expression on RBCs. Blood samples from four healthy human volunteers were incubated with fluorophore-labeled AB79 or fluorescently labeled daratumumab, another anti-CD38 antibody known to bind to CD38 on RBCs. Each sample was preincubated with the respective unlabeled drug to block CD38 binding by the fluorescently labeled drug and serve as a negative control for the assay. Antibodies against CD45 and CD235a were added to the samples to identify RBC-positive cells, which were then analyzed by flow cytometry. Fluorescently labeled daratumumab was used as a positive control. Results demonstrated that AB79 and daratumumab bind to RBCs from healthy human donors.

[0300] method Blood samples from four healthy human donors (Millennium Blood Donor Program) were collected according to the company's protocol. Briefly, peripheral blood samples for RBC binding determination were collected in sodium heparin tubes. For lymphocyte staining, peripheral blood samples were collected in BDVacutainer® CPT™ tubes (BD Biosciences, Franklin Lakes, NJ, USA). Separate tubes were used to collect peripheral blood mononuclear cells (PBMCs) and confirm the binding of fluorescently labeled AB79 and fluorescently labeled daratumumab to CD38+ lymphocytes. For both RBC binding and lymphocyte staining experiments, peripheral blood samples were stored at room temperature and processed within 2 hours of collection to maintain cell viability. For RBC binding, peripheral blood samples were first diluted 1:10,000 in staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) to dilute the large number of RBCs present in the sample. Because the normal range for RBCs in healthy human blood samples is approximately 5 million cells per microliter, samples must be substantially diluted to obtain an acceptable number of RBCs for staining in flow cytometry analysis. Samples were then transferred to a V-bottom 96-well plate and incubated overnight at 4°C on a gentle shaker with 25 μL / well of unlabeled AB79 (500 μg / mL), unlabeled daratumumab (500 μg / mL), or BD buffer alone (no drug). The plate shaker was used to prevent RBC sedimentation during the incubation period. Peripheral blood samples were preincubated with unlabeled pharmaceuticals to block CD38 antigen sites on the surface of RBCs. This served as the negative control for the assay. After this incubation period, clinically relevant 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) were added to the samples for 3 h at room temperature on a gentle shaker.The samples were then washed several times with BD buffer and stained with the cell surface markers CD45 and CD235a (RBCs are CD45-CD235a+) for RBC identification, using streptavidin-BV421 conjugated to a biotinylated antibody. The use of biotin-streptavidin allows for amplification of low levels of CD38 expression on RBCs. The BV421 fluorophore is one of the brightest commercially available fluorophores and was chosen because the flow cytometer uses a violet laser, thereby minimizing the amount of spectral overlap with other channels / markers in the panel. Together, this signal amplification approach provides an opportunity to detect cell surface molecules that would otherwise fall within the instrument's noise range. The samples were then washed and acquired on a BD FACSCanto™ II (BD Biosciences, Franklin Lakes, NJ, USA). Target cell acquisition was set to 10,000 CD235a+ events. For lymphocyte binding, peripheral blood from the same healthy donor collected in CPT tubes was centrifuged and peripheral blood mononuclear cells (PBMCs) were isolated using standard techniques. Cells were washed, stained, and processed as described for RBC experiments.

[0301] To prepare biotinylated antibodies, AB79 (21.4 mg / mL, Takeda, California, USA) and daratumumab (20 mg / mL, Janssen Biotech, Horsham, PA, USA) were purified simultaneously on the same day using a commercially available Protein A column kit (Abcam, Cambridge, UK) to remove substances that might interfere with the biotinylation procedure. The protein concentration of the purified product was determined by A280 / 260, and an equal amount of protein from each antibody was conjugated to biotin using a Lightning Link Rapid Biotin Conjugation Kit (Innova Biosciences, Cambridge, UK). At the end of the procedure, the protein concentration was measured again using A280 / 260. Both antibodies were conjugated to commercially available polystyrene microspheres with antibody-binding capacity for any antibody isotype or inert beads (negative beads). After mixing the beads with biotin-streptavidin-BV421 AB79 or biotin-strepavidin-BV421 daratumumab, the two components mixed together provide a clear, high-signal positive control with an appropriate negative population that can be used to estimate the median fluorescence (MFI) of each test antibody by flow cytometry. Samples were acquired using a BD Biosciences FACSDiva™ (BD Biosciences, Franklin Lakes, NJ, USA) on a BD Biosciences FACSCANTO™ II instrument. Raw data files were transferred to a secure server and then analyzed offline using FlowJo® version 10 (FlowJo, LLC; Ashland, OR, USA). For RBC identification, CD235a+ cells were first gated. The geometric MFI and percent positive biotin-strepavidin-BV421 AB79 and biotin-strepavidin-BV421 daratumumab events were then determined for the gated cells and plotted as a histogram (FIG. 22).This was compared with an isotype control (samples were preincubated with unlabeled AB79 or unlabeled daratumumab, then incubated with their respective biotin-strepavidin-BV421 formulations). Lymphocyte identification was first performed by excluding debris using forward and side scatter (FSC-A and SSC-A). The CD45-positive population was then gated, and the MFI and percent positive biotin-strepavidin-BV421 AB79 and biotin-strepavidin-BV421 daratumumab were determined for the gated cells and plotted as a histogram (Figure 22). This was compared with an isotype control (samples were preincubated with unlabeled AB79 or unlabeled daratumumab, then incubated with their respective biotin-strepavidin-BV421 formulations).

[0302] [Table 10]

[0303] Results and Discussion The amount of biotin on AB79 and daratumumab was determined using a highly sensitive flow cytometry assay. As the results shown in Figure 23, biotin-strepavidin-BV421 daratumumab binds 1.6-2.0 times more antibody-conjugated beads than biotin-strepavidin-BV421 AB79. Therefore, biotin-strepavidin-BV421 daratumumab is a "brighter" antibody compared to biotin-strepavidin-BV421 AB79. Consequently, comparisons of MFI intensities need to be adjusted for this difference in labeling.

[0304] Confirming the specificity of biotin-labeled preparations was an important step in ensuring that the observed results were the result of specific binding to the target protein. Competitive assays are a common method for testing antibody specificity. Flow cytometry assays were used for this competitive evaluation. Briefly, peripheral blood samples from healthy volunteers were preincubated with unlabeled AB79 or unlabeled daratumumab, and then stained with biotinylated AB79 or biotinylated daratumumab, respectively. Samples preincubated with unlabeled preparations were expected to block the binding of biotinylated preparations. As illustrated in Figure 24, biotinylated AB79 and biotinylated daratumumab were able to block peripheral blood lymphocytes using unlabeled preparations.

[0305] The percentage of binding of biotin-strepavidin-BV421 AB79 and biotin-strepavidin-BV421 daratumumab to RBCs was determined using a total of four peripheral blood samples from healthy volunteers. As shown in Figures 25 and 26, AB79 bound to CD38-expressing RBCs in a dose-dependent manner in all four donors tested. Peak RBC binding levels varied among healthy volunteers and were observed between 1 and 10 μg / mL for both formulations. At 10 and 100 μg / mL of either biotin-streptavidin-BV421 AB79 or biotin-streptavidin-BV421 daratumumab, RBC binding levels were low in three of the four donors tested. Several factors may explain the reduced levels of drug binding to CD38, including increased RBC hemolysis and reversal of the catalytic domain from extracellular to intracellular (Yoshiga et al. (2008) Int. J. Mol. Med. 22:369-374). Additionally, daratumumab reduced CD38 expression levels, at least in part, through phagocytosis (Cole et al. (2018) Arthritis Res. Ther. 20(1):85); CD38 complexes and associated cell membranes actively translocated from multiple myeloma cells to monocytes and granulocytes (Kraan et al. (1999) Rheumatology (Oxford) 38(11):1074-1080). One donor (Donor 3) had increased RBC levels with increasing drug dose, likely as a result of having more surface RBC CD38 expression. Additional experiments are needed to better understand the reduced drug binding at the highest concentration tested.

[0306] We compared the RBC binding profiles of AB79 and daratumumab. As depicted in Table 10 and Figures 27 and 28, there appeared to be differences in the magnitude of RBC binding (i.e., MFI) between the formulations for three of the four donors tested; however, this difference may be due to differences in the biotin concentration of each antibody, with daratumumab having 1.6-2.0 times more biotin than AB79. An alternative analysis to control for potential differences in antibody fluorescent labeling is to compare the concentration versus binding profile of each antibody; a useful metric is the concentration at which maximal binding occurs (i.e., maximum specific binding of antigen (Bmax)). Bmax is identical for both antibodies in three of the four donors (e.g., 1 μg / mL for Donor 1). Collectively, these data indicate that both antibodies bind with similar affinities, within the resolution limit of the current assay, which is a factor of 10. In conclusion, both AB79 and daratumumab bound to RBCs with affinities within 10-fold of each other in this assay, and no greater than 10-fold differences in the binding affinities of these antibodies to red blood cells existed within this assay system.

[0307] Example 5: Evaluation of AB79 hemolysis of human or monkey RBCs in vitro Fresh whole blood from normal, healthy human volunteers and cynomolgus monkeys (cynomolgus monkeys), five individuals per species (n = 5), was evaluated for in vitro hemolysis in response to in vitro treatment with AB79 (27.3 mg / mL; Takeda, California, USA), a human IgG1 isotype control (7.14 mg / mL Bio X Cell), and daratumumab (20 mg / mL; Janssen Biotech, Horsham, PA, USA). Dose responses for the desired test articles were examined at 0, 0.03, 0.08, 0.25, 0.74, 2.2, 6.6, and 20 μg / mL. Half-log dilutions of saponin were evaluated, starting with a 1% saponin solution at the top, which served as a technical positive control for blood reactivity. For acute hemolysis measurements, treatment was performed for 1 hour at 37°C and 5% CO2. The absorbance was measured at a wavelength of 540 nm using a spectrophotometer, and the hemolysis rate was calculated as follows:

[0308]

number

[0309] The hemolytic index is evaluated as follows (hemolytic index = hemolysis grade): 0–2 = non-hemolytic; 2–5 = slightly hemolytic; >5 = hemolytic. Results: RBCs from all individuals of each species, human and cyno, exhibited acute hemolysis in vitro in response to a 1% saponin solution titration with a hemolytic index greater than 5. AB79, daratumumab, and the human IgG1 isotype control did not induce detectable hemolysis in any red blood cell samples across species, which were assessed as having a nonhemolytic index of zero (Figures 29 and 30).

[0310] Example 6: Evaluation of AB79 in a collagen-induced arthritis model in cynomolgus monkeys Expression of the extracellular enzyme CD38 is increased on lymphocytes in response to antigen challenge, and it has been hypothesized that targeting these activated lymphocytes could ameliorate the pathological activity of autoimmune diseases. Cynomolgus monkeys are an appropriate model to evaluate the potential impact of targeting human CD38 because this species displays a similar CD38 expression profile and the human anti-CD38 antibody AB79 has affinity (EC) for monkey CD38. 50=4.5 nM), allowing for pharmacological intervention. Therefore, the potential activity of AB79 was investigated in the monkey collagen-induced arthritis (CIA) model of autoimmune disease. Prophylactic administration of AB79 (3 mg / kg weekly intravenous injection) was well tolerated and prevented the development of arthritis, in contrast to vehicle-treated control animals, which showed progressive disease with radiographic lesions and worsening clinical scores over the course of the study. Therapeutic treatment of arthritic monkeys with AB79 (3 mg / kg weekly iv) was also well tolerated and attenuated disease progression and symptoms. Arthritis scores and joint swelling were significantly lower than vehicle controls, accompanied by decreases in blood levels of CRP, ALP, NK, B, and T cells. Histopathology, morphometry, and radiology revealed significantly less joint damage in animals prophylactically exposed to AB79 treatment compared with vehicle-treated animals, and significantly less damage in animals treated with AB79 or dexamethasone (0.1 mg / kg po daily) (p<0.05), indicating potential disease-modifying activity. In conclusion, these data indicate that depletion of CD38-expressing cells may be a therapeutic option for treating autoimmune diseases without the deleterious effects of steroids.

[0311] Methods and Materials antibody AB79 was available in-house. Fluorochrome-conjugated anti-mouse IgG was purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Pharmlyse buffer was obtained from BD Biosciences (San Jose, CA). Fluorochrome-conjugated antibodies against monkey proteins were purchased from various sources: CD20 from BD Biosciences (San Jose, CA), mouse antibody against CD3 from eBioscience (San Diego, CA), rabbit mouse antibody against CD16 from Miltenyi Biotech (Auburn, CA), and mouse antibodies against CD4 and CD8 from R&D Systems. In addition to Alexa Fluor 647-conjugated AB79, a non-conjugated antibody against AB79 and a humanized control antibody with different antigen specificity but the same Fc IgG1 were available in-house. Primary antibodies for monkey tissue cross-reactivity studies were rabbit anti-AB79 (generated in-house) and negative control human IgG1 (Millipore Bioscience Research Reagents, Temecula, CA).

[0312] Tissue immunohistochemistry The CD38 antigen expression profiles were compared across 15 different tissue types collected from healthy human and cynomolgus monkey donors using immunohistochemistry. The suitability of each tissue for CD38 detection was verified using a positive control antibody against the related transmembrane receptor, CD31 (Dako North America, Inc.). Sections (5 μm) were cut from freshly 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 in 10% neutral-buffered formalin for 10 seconds. Acetone / formalin-fixed frozen sections were rinsed twice in phosphate-buffered saline (PBS) and incubated for 20 minutes with a protein block designed to reduce nonspecific binding (PBS; 0.5% casein; 5% human gamma globulin; 0.02% goat IgG; 1 mg / ml heat-aggregated human IgG). Unconjugated AB79 or negative control human IgG1 (Millipore Bioscience Research Reagents) was applied to the sections at 5 or 25 μg / ml and incubated for 1 hour at room temperature. Slides were then rinsed twice with PBS, and an indirect immunoperoxidase procedure was performed to detect these primary reagents. A secondary antibody, rabbit anti-AB79, was then applied at 5 μg / ml for 30 minutes and rinsed twice with PBS. Endogenous peroxidase was blocked by incubating the slides in the peroxidase solution provided in the Dako EnVision+ kit for 5 minutes, followed by rinsing twice with PBS. Slides were then treated with the peroxidase-labeled goat anti-rabbit IgG polymer provided in the Dako EnVision+ kit for 30 minutes, rinsed twice with PBS, and treated with the substrate chromogen (DAB+) solution provided in the Dako EnVision+ kit for 8 minutes. All slides were rinsed in tap water, counterstained with hematoxylin, washed, "blued" with saturated lithium carbonate, cleared, dehydrated through alcohol, cleared in xylene, and coverslipped according to standard methods. Staining intensity was graded semiquantitatively by a blinded American College of Veterinary Pathologists (ACVP)-certified anatomic pathologist.

[0313] AB79 binds to recombinant CD38 Chinese hamster ovary K1 (CHO-K1) cells stably expressing either human, mouse, or monkey CD38 were generated to test the cell surface binding of anti-CD38 antibodies. CHO-K1 cells (Lonza, USA) were transfected with full-length cDNA clones of human, mouse, or cynomolgus monkey CD38 (Origene Technologies, Rockville, MD). After selection, pools were sorted by flow cytometry, and the highest human, mouse, or cynomolgus monkey CD38-expressing clones (top 15% mean fluorescence intensity [MFI]) were used for binding studies. 200,000 cells per well were seeded into 96-well round-bottom plates and stained with 66.7 nM AlexaFluor® 488 directly conjugated to Abs in 50 μl of FACS buffer (1% BSA in PBS) on ice for 30 min to 1 h. Cells were washed 3-4 times with a final volume of 200-250 μl of FACS buffer. The final cell pellet was resuspended in 100 μl of FACS buffer containing 1% paraformaldehyde. Samples were evaluated on a FACS Canto II HTS (BD Biosciences) and analyzed using Flojo software (Tree Star, USA).

[0314] Flow cytometry of cell lines and whole blood For staining of cell lines, 2 x 10 cells 6The cells were resuspended at 1 / ml in FACS buffer (D-PBS (Dulbecco's phosphate-buffered saline without calcium and magnesium) containing 5% fetal bovine serum and 0.05% sodium azide (VWR, West Chester, PA)), and 200 μl samples were stained with the appropriate monoclonal antibodies for 30 minutes at 4°C. Samples were washed with FACS buffer and analyzed by flow cytometry (FACSCalibur, BD). For whole blood staining, 200 μl samples from monkeys were stained with the appropriate monoclonal antibodies for 30 minutes at 4°C. Red blood cells were lysed with BD FACS lysing solution, and samples were washed with FACS buffer and analyzed by flow cytometry. In all cases, antibodies were used at saturating concentrations; in many cases, up to four antibodies were used per sample.

[0315] AB79 activity in whole blood from cynomolgus monkeys Cynomolgus monkey whole blood was obtained from Charles River Laboratories (Wilmington, MA, USA). For binding assays, anticoagulated cynomolgus monkey peripheral whole blood (100 μl) was incubated with increasing concentrations of AB79 antibody (43-690 nM) for 30 minutes at room temperature. AB79 binding to cells was assayed using a PE-labeled goat anti-human IgG Fc antibody (Thermo Fisher Scientific). After antibody binding, red blood cells (RBCs) were lysed using fixative-free high-yield lysis solution (Thermo Fisher Scientific). Cells were then washed twice with magnetic-activated cell sorting (MACS) buffer containing 0.5% BSA (Miltenyi Biotec). Cell staining data were collected by flow cytometry (Attune NxT Acoustic Focusing Cytometer) and analyzed using FlowJo software.

[0316] For the cell lysis assay, 90 μL of whole blood was seeded into each well of a 96-well U-bottom plate. Immediately after seeding, the whole blood was treated with 0.69, 2.06, 6.17, 18.52, 55.56, 166.67, or 500 nM AB79 or PBS control for 6, 24, and 48 hours. At each time point, the control and test molecule-treated whole blood in the 96-well U-bottom plate was transferred to a deep-well plate for RBC lysis. After lysis, cells were resuspended in staining buffer with CD16-BV605, CD56-PE, and CD38-FITC antibodies for surface marker staining. Protected from light, the cells were incubated at 4°C for 20 minutes. After incubation, the cells were centrifuged at 350 x g for 5 minutes at room temperature and washed with 1X Annexin V binding buffer. Cells were resuspended in 1X Annexin V binding buffer containing Annexin V Alexa Fluor 647 for 15 minutes at room temperature. Stained cells were analyzed using a BD FACSCelesta flow cytometer, and data were recorded using BD FACSDiva software, version 8.0.1. NK cell viability at each test concentration and the IC50 value for NK cell depletion of AB79 at each time point were graphed and fitted using GraphPad Prism 7.04.

[0317] Dose-ranging study in healthy monkeys In a series of studies, healthy, purpose-bred, experimentally naive cynomolgus monkeys received vehicle control or AB79 at 0.03, 0.1, 0.3, and 1 mg / kg weekly or biweekly at 3, 30, or 80 mg / kg via a 20-minute intravenous infusion (Charles River Laboratories). In all studies, animals were evaluated for changes in clinical signs (twice-daily cageside observations, post-dose observations, weekly detailed examinations, food consumption, and weekly body weights). In all studies, blood samples were collected pre- and post-dose for evaluation of pharmacokinetics, pharmacodynamics, and primate anti-human antibodies (PAHA). Studies were conducted in cynomolgus monkeys in accordance with the test facility SOPs (Charles River Laboratories), which adhered to regulations outlined in the USDA Animal Welfare Act (9 CFR 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).

[0318] Bioanalytical procedure for determination of AB79 serum concentrations The concentration of AB79 in cynomolgus monkey serum was measured using an enzyme-linked immunosorbent assay (ELISA). This method is an indirect ELISA using a 96-well microtiter plate. Plates were coated with a mouse anti-idiotypic antibody against AB79. Blanks, standards, and quality control (QC) samples containing various concentrations of AB79, along with monkey serum samples, were added to the coated microtiter plate and incubated at room temperature for 55–65 minutes. After washing the microtiter plate, a detection antibody (peroxidase-conjugated Affinipure 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 generate the chromophore, followed by the addition of a stop solution (2N sulfuric acid) to stop the color development. The absorbance at 450 nm was measured, and the AB79 concentration was calculated using a four-parameter logistic-weighted (1 / y²) standard calibration curve.

[0319] Bioanalytical procedure for the determination of anti-AB79 antibodies Anti-AB79 antibody screening of cynomolgus monkey serum was measured using enhanced chemiluminescence (ECL) assay. This qualitative ECL assay was designed by incubating undiluted cynomolgus monkey serum samples with 300 mM acetic acid. The acid-dissociated samples were incubated in a mixture of biotinylated AB79, SULFO-TAG-labeled AB79, and 1.5 M Trizma base to neutralize the acid and allow immune complex formation. The complexes were then added to a streptavidin-coated MSD plate and allowed to bind. After washing, MSD read buffer T was added to the plate, and the complexes were detected by exciting SULFO-TAG™ via the electrochemical reaction of Ru(bpy)3 to generate luminescence, which was then read using an MSD Sector 6000. The amount of luminescence correlated with the level of cynomolgus monkey anti-AB79 antibodies present in each sample's serum. The minimum required dilution (MRD) of cynomolgus serum for this assay was set at 1 / 30. For graphical representation, all animals were plotted individually as a function of antibody titer and enrollment days (Figure 31). Serum samples with values ​​below the plate-specific cutpoint were plotted using the nominal titer value for ease of plotting.

[0320] Monkey collagen-induced arthritis model Ethically responsible use of nonhuman primates was ensured by modeling the pharmacodynamic response to AB79 in cynomolgus monkeys and extrapolating the minimum number of animals required per treatment group to produce a statistically significant difference in PD response. Thirty-four untreated female cynomolgus monkeys, 3-4 years old and weighing 2.5-3.3 kg, were obtained from Biomedical Research (GZ) Ltd. (SNBL China). Upon receipt, health examinations for each animal were performed by a contract research organization (PharmaLegacy Laboratories, Inc., China). Animals were housed one per cage and allowed to acclimate for a minimum of 14 days before the start of experimental procedures. The animal room was maintained at a temperature of 20-29°C, a relative humidity of 40-70%, and a 12-h light / dark cycle. Prior to the start of the study, animals were trained to receive intravenous infusions or oral gavage. According to the conventional protocol, the monkeys had free access to vegetables, fruits, solid food (Shanghai Shilin Biologic Science & Technology Co. Ltd., China), and water. Cages were stratified within racks to reduce the impact of any environmental influences on the study. This experimental plan, all study protocols, and experimental procedures were reviewed and approved by the sponsor's ethical committee (PharmaLegacy Laboratories Inc.) in accordance with Chinese law on animal experimentation.

[0321] Monkeys were selected for the study based on pre-screening criteria. One naive animal received no collagen and served as a negative control for disease induction. The remaining animals received subcutaneous injections of bovine type II collagen (Sichuan, China) dissolved in 0.01N acetic acid (SPGC Sinopharm Chemical Reagent Co., Ltd.; Shanghai, China) at 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 TNFα. Rheumatol Int. 2008 28:879-883; Kato A., et al., Experimental and Molecular Pathology 2008 84:262-270) on days 0 and 21 (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 presedated with ketamine (4 mg / kg intramuscularly). If necessary, additional anesthesia was administered with 1.5–5% isoflurane (Matrix vip3000 isoflurane inhalation anesthesia machine) at a flow rate of 0.8–1.5 liters of oxygen. Ulcerative skin lesions that developed at the vaccination site were treated with iodine each time the animals were sedated to prevent infection.

[0322] Seven animals receiving collagen were assigned to the prophylactic AB79 group on day 0 (Figure 31A). Prophylactic AB79 animals received weekly injections of 3 mg / kg AB79 starting on day 7, with the final dose administered on day 56, for a total of eight doses (Figure 31A). Animals in this group were sacrificed on day 63. The remaining immunized animals were treated as a single group and received weekly 30-minute intravenous infusions of vehicle (saline) starting on day 7 until the animals reached or exceeded a maximum clinical arthritis score (CIA) of ≥15%. At this point, animals were enrolled in the vehicle control group, therapeutic AB79 group, or therapeutic dexamethasone group, and continued enrollment due to the staggered onset of disease (Figure 31A). Animals enrolled in the vehicle control group received weekly injections of vehicle for 5 weeks (Figure 31A). Animals in this group were sacrificed 7 days after the last injection. For animals enrolled in the therapeutic AB79 group, weekly injections were administered for 5 weeks (Figure 31A). Animals in this group were sacrificed 7 days after the last injection. Animals enrolled in the therapeutic dexamethasone group were administered daily by oral gavage for 5 weeks (Figure 31A) and sacrificed 1 day after the last injection. Animals were observed daily for signs of health and general response to treatment. Any exceptions to normal healthy appearance and behavior were noted and detailed on standard clinical observation forms.

[0323] Assessment of arthritis activity The monkeys' body weights were measured once during the acclimation period (5 days before the start of the experiment), the day before each cycle of disease induction, and then weekly until the end of the study. The number of animals in each group with joint swelling was recorded on days 0 and 21, and then daily until the end of the study. The number of proximal interphalangeal (PIP) joints (hands and feet, respectively) with joint swelling was recorded on days 0 and 21, and then weekly until the end of the study. The vertical and horizontal axes of the PIP joints of the forelimbs and hindlimbs (without the thumb) were measured with calipers on days 0 and 21, and then weekly from the onset of disease until the end of the study for all PIPs with arthritis. The average elliptical area of ​​the 16 PIP joints was calculated and used as individual data. The elliptical area of ​​each PIP was calculated using the following formula: elliptical area = vertical axis × horizontal axis × 3.14 × 1 / 4. The percent change in oval area and joint swelling were calculated using the following formulas: percent 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 day 1 sensitization).

[0324] Clinical Arthritis Score The severity of arthritis in each limb of each monkey was recorded on days 0 and 21, and then weekly until the end of the study, according to the following criteria: (0) normal; (1) mild arthritis, mild but distinct; (2) moderate swelling; (3) severe arthritis with significant swelling and / or marked joint deformity. The following joints in each paw were examined and scored: five metacarpophalangeal (MCP) joints, four proximal interphalangeal (PIP) joints, for a total of 15 joints; four distal interphalangeal (DIP) joints, and one interphalangeal joint of the first finger. Each wrist or ankle was scored as a single composite joint. The knees / elbows of each limb were also evaluated for disease severity. The arthritis score for each animal was the sum of the scores of the individual joints, with a maximum score of 192 (16 × 3 × 4) (16 = total number of joints and knees / elbows in each limb, 3 = maximum score for each individual joint, 4 = number of limbs per monkey).

[0325] Blood collection and analysis Blood samples were collected from animals at the following time points for CBC, blood chemistry, antibody serum preparation, PK, and ADA measurements. Animals in the vehicle control and prophylactic AB79 groups were bled twice during weeks 1 and 5: once before dosing and once the day after. Blood samples were collected immediately before AB79 administration during weeks 2, 3, 4, 6, 7, and 8. During week 9, blood samples were collected when the animals were terminated. All other animals were bled weekly until disease reached a threshold of 15% of the maximum arthritis score. After assignment to the vehicle, therapeutic AB79, or therapeutic dexamethasone group, blood samples were collected weekly immediately before dosing and at the end of treatment. Additionally, samples were collected the day after the first and fifth drug administrations. In the vehicle control, prophylactic AB79, therapeutic AB79, and dexamethasone groups, blood samples for flow cytometry were collected before the first dose (day of infusion), the day after the first dose, before the second dose (day of administration), before the fifth dose (day of administration), and on the termination day. In the therapeutic AB79 group, blood samples for flow cytometry were collected before the first dose of drug, the day after the first dose, before the eighth dose, before the 29th dose, and on the termination day.

[0326] X-ray examination At the end of the study, each joint of the hands and feet (DIP, PIP, and 1st joint) of the anesthetized live animals was examined. 1 Examinations were performed on the interphalangeal joint (IP) and medial chondral cortex (MCP)—sites typically involved in human RA. Radiographic grading was performed blindly based on a 0-4 grading system: (0) normal; (1) mild deformation of the articular cartilage and / or subchondral bone; (2) severe deformation of the articular cartilage and subchondral bone, with a small number of osteophytes present on the periosteal surface and joint margins, although obscured; (3) the same type of changes observed in grade 2, but more advanced, with a large number of osteophytes present on the periosteal surface and the joint space being indistinguishable or not visible; and (4) the same type of changes as grade 3, but more advanced, with the joint space becoming undetectable, the bone becoming sclerotic or avascular, and significant deformation.

[0327] Histopathology and histomorphometry of tissues from arthritic monkeys At the end of the study, animals were euthanized by exsanguination under anesthesia. Paws, spleens, colons, and lymph nodes (mesenteric and inguinal) were harvested and fixed in neutral-buffered formalin. PIP and DIP joints were decalcified in 15% EDTA, dehydrated, and embedded in paraffin (8 blocks / paw × 4 paws × 22 animals = 704 blocks). Frontal coronal sections of the joints were obtained using a rotary microtome. Histopathological scoring was performed in a blinded manner by a bone histopathologist using toluidine blue-stained sections (32 × 22 animals = 704 slides). Histological sections were qualitatively evaluated for the following histopathological features: tissue infiltration, pannus, cartilage lesions, and bone resorption. Quantitative histomorphometry was performed using a Nikon Eclipse E400 light / fluorescence microscope and Osteomeasure software (OsteoMetrics, Inc., Atlanta, GA) interfaced with a video subsystem. Histomorphometric measurements of the joint area and surface were performed in a blinded manner by a bone histopathologist using toluidine blue stained slides (704 slides).

[0328] statistical analysis Data are presented as mean ± standard error (SEM). Statistical analysis was performed for each parameter between untreated, model, and relevant agonist (dexamethasone) and test article groups using GraphPad Prism. p<0.05 was considered to indicate a significant difference.

[0329] result CD38 expression profile in cynomolgus monkeys To determine whether cynomolgus monkeys are an appropriate model for evaluating the potential impact of targeting CD38 in humans, the expression profiles of the CD38 antigen were compared in 15 different tissue types collected from healthy humans and cynomolgus donors. Immunohistochemistry revealed that AB79 bound to mononuclear leukocytes in the colon, stomach, small intestine, bone marrow, and lymph nodes, as well as to the lamina propria endothelium of the colon, stomach, and small intestine of both species (Table 11).

[0330] [Table 11]

[0331] Conversely, monkey, but not human, AB79 also bound to mononuclear leukocytes in the liver, lung, prostate, and uterus (cervix), as well as to prostate acinar epithelium. AB79 binding was not observed in the heart, kidney, pancreas, skin, or endometrium of either species. AB79 staining was generally moderate to marked (3+-4+) in intensity in human tissues and minimal to mild (1+-2+) in intensity in cynomolgus monkey tissues (Table 11). This may reflect differences in the affinity of AB79 for human versus monkey CD38 and / or differences in the magnitude of CD38 expression. The staining pattern was primarily cytoplasmic, with some cells exhibiting plasma membrane staining (Table 11). Overall, we conclude that monkeys are an appropriate model species for evaluating the potential pharmacological effect(s) of targeting CD38 in models of autoimmune disease.

[0332] AB79 binds to CD38 expressed by cynomolgus monkeys The amino acid sequence of human CD38 protein shows 91% amino acid identity with the cynomolgus monkey orthologue, 59% with those of mouse and rat, and 54% with that of rabbit. Comparison of the binding epitope of AB79 on human CD38 with the corresponding sequence of monkey CD38 revealed a single amino acid substitution of lysine with glutamic acid at position 274. To determine whether AB79 could be used to evaluate the potential impact of targeting CD38 in monkeys, monkey CD38 was expressed in Chinese hamster ovary (CHO) cells, and AB79 was administered at a half-maximal effective concentration (EC) of 4.5 nM. 50) bound to monkey CD38 (Figure 32A). This value was approximately 10-fold less than the binding affinity of AB79 for human CD38 expressed by CHO cells (KD = 0.7 nM), indicating that AB79 binds less strongly to CD38 in cynomolgus monkeys than to human CD38. AB79 also bound to monkey B, NK, and T cells in whole blood, with NK cells exhibiting higher mean fluorescence intensity than B and T cells (Figure 32B), indicating that monkey NK cells have a higher density of CD38 per cell. After incubation with monkey whole blood in vitro, AB79 cytolyzed NK cells in a dose-dependent manner (Figure 32C), with a mean EC 50 The cytolysis of NK cells from human peripheral blood was 29.6 nM, approximately 30-fold less potent than AB79-induced cytolysis of NK cells from human peripheral blood (data not shown). Collectively, these data indicate that cynomolgus monkeys may be less sensitive than humans to the pharmacological effects of AB79. Nevertheless, this cross-reactivity profile, as well as the similarity in the CD38 expression profile, suggests that cynomolgus monkeys are a suitable model species for investigating the potential pharmacological effect(s) of AB79 in vivo.

[0333] Pharmacological effects of AB79 in healthy monkeys To characterize the pharmacology of AB79 in vivo, healthy cynomolgus monkeys were infused IV with AB79 at 0.03, 0.1, 0.3, or 1 mg / kg weekly and 3, 30, or 80 mg / kg every other week for 3 months, and a subset of animals was monitored for 3 months after the last dose (Figure 31A). Peak AB79 concentrations generally occurred toward the end of the infusion and were generally dose-proportional (Table 12).

[0334] [Table 12]

[0335] Exposure generally increased dose-proportionally at steady state in animals that did not develop anti-AB79 antibodies, accumulating 1.7- to 2.4-fold by week 13 (Table 12), consistent with reduced clearance due to target-mediated pharmacokinetics of AB79. Half-lives after the last dose in animals in the 3 and 80 mg / kg cohorts that did not develop anti-AB79 antibodies were 237 and 415 hours, respectively (Table 12). No gender differences in PK characteristics were observed.

[0336] The NK cell population uniformly and densely expressed CD38 (Figure 32B), and at 0.3 mg / kg ED 50 was decreased in peripheral blood after the first infusion of AB79 with β-glucan (Figure 31B), and the corresponding C max The ED at 1.0 mg / kg was 7.63 μg / mL, for an overall mean exposure of 665 h*μg / mL at week 13 (Table 12). In contrast, B cell populations heterogeneously expressed CD38 with a lower median NK density (Figure 32B), and the ED at 1.0 mg / kg was 7.63 μg / mL. 50 was reduced in peripheral blood after the first injection of AB79 (Figure 31C), and C max The ED at 30 mg / kg was 21.1 μg / mL, for an overall mean exposure of 2700 μg / mL at week 13 (Table 12). T cell populations also heterogeneously expressed CD38 at an even lower median density than B cells (not shown), and the ED at 30 mg / kg was 2700 μg / mL. 50 was decreased in peripheral blood after the first infusion of AB79 with C (Fig. 31D). max The mean exposure at week 13 was 62.1 μg / mL, with an overall mean exposure of 18,400 h*μg / mL (Table 12). Overall, these dose-ranging findings demonstrate that the 3 mg / kg weekly dose maintains total NK, B, and T cell counts in peripheral blood at greater than 80, 60, and 20%, respectively, from baseline levels.

[0337] Profile of AB79 in collagen-induced arthritis The potential efficacy of AB79 was investigated in a CIA model in cynomolgus monkeys, a species whose immune system, joints, and skeletal anatomy are sufficiently similar to their human counterparts to allow the use of clinical endpoints. To induce arthritis, monkeys were treated intradermally with type II collagen on days 0 and 21 (Figure 33). Successful immunization of each animal was confirmed by the appearance of anti-collagen antibodies (data not shown). One group of animals was treated on day 7 with a prophylactic AB79 treatment regimen of 3 mg / kg, continued for 8 weeks. In three therapeutic groups, monkeys with overt disease were randomized to receive either 3 mg / kg TAK 079 as a vehicle control, or 0.1 mg / kg dexamethasone. Therapeutic treatment continued for 5 weeks after treatment initiation (Figure 33).

[0338] AB79 was well tolerated in both prophylactic and therapeutic regimens. While healthy control monkeys gained weight over time, the collagen-immunized monkeys in the other four groups began losing weight starting on day 7, indicating systemic effects associated with the development of arthritis (Figure 34A). Based on the percentage change in weight relative to baseline weight at enrollment, animals in the prophylactic AB79, therapeutic AB79, and therapeutic dexamethasone groups regained weight 14 days after treatment initiation, suggesting a therapeutic effect from both the AB79 and dexamethasone positive control treatments. Weight gain approached normal levels in monkeys treated with AB79 and dexamethasone compared with healthy untreated controls; vehicle-treated arthritic animals lost weight (Figure 34A). Arthritis severity was assessed using a clinical arthritis score, a global assessment of disease activity that considers all measurable joints in the animal over the course of the study using a 192-point scoring system. Vehicle-treated animals showed progressive disease, with increasing clinical scores over the course of the study (Figure 34B). Prophylactic exposure to AB79 significantly prevented the development of arthritis compared to vehicle controls (p<0.01). Similarly, treatment with AB79 or dexamethasone significantly inhibited the development of arthritis compared to vehicle controls (p<0.05) and reduced arthritis scores from pretreatment baseline (Figure 34B). Similar effects were observed in a subset of PIP joints, both in terms of the number of inflamed PIP joints (Figure 34C) and the overall mean swelling of all PIP joints (Figure 34D). To obtain a comprehensive assessment of arthritic joints and the potential for AB79 to have "disease-modifying" activity in human arthritis, radiographic examination of all IP and MCP joints was performed. Prophylactic exposure to AB79 significantly prevented damage to PIP (data not shown), DIP (Figure 34E), and MCP (Figure 34F) joints compared with vehicle controls (p<0.01). Similarly, therapeutic exposure to AB79 or dexamethasone resulted in significantly (p<0.05) less damage to PIP (data not shown), DIP (Figure 34E), and MCP (Figure 34F) joints than vehicle control animals.To characterize the components of progressive arthritis, DIP and PIP joints were analyzed histologically for cellular infiltration, pannus severity, cartilage damage, bone resorption, and osteophyte formation. Prophylactic exposure to AB79 resulted in composite scores that were significantly lower (p<0.01) than vehicle control values ​​and comparable in magnitude to animals not immunized with collagen (Figure 35A). AB79 had a similar effect on each component; scores were significantly lower (p<0.01) than vehicle control values ​​for pannus (Figure 35B), infiltrating leukocytes (Figure 35C), cartilage lesions (Figure 35D), bone resorption (Figure 35E), and osteophyte formation (Figure 35F). Similar, smaller differences were also observed following treatment with AB79 or dexamethasone (Figures 35A-E), although not all differences reached statistical significance.

[0339] Quantitative histomorphometric measurements, including articular cartilage area (Figure 36A), damaged articular cartilage thickness (Figure 36B), percentage of damaged articular surface (Figure 36C), and osteophyte area versus total periosteal surface (Figure 36D), were performed on all DIP and PIP joints in a blinded manner by a board-certified veterinary pathologist. All parameters indicated that prophylactic exposure to AB79 significantly prevented the development of joint damage. Treatment with AB79 reduced disease severity, although some histomorphometric measurements did not achieve statistical significance. The therapeutic effect of AB79 was similar to that of therapeutically administered dexamethasone.

[0340] Levels of C-reactive protein (CRP) (Figure 37A), alkaline phosphatase (ALP) (Figure 37B), and albumin (ALB) (data not shown) correlated with disease severity. Prophylactic treatment with AB79 prevented the inflammation-associated increases in CRP (Figure 37A) and ALP (Figure 37B) observed in vehicle-control animals. Treatment with AB79 caused a rapid decrease in CRP and ALP, whereas chronic treatment with dexamethasone was required to lower CRP and ALP levels (Figures 37A and 37B, respectively). No evidence of liver damage was found in serum chemistry parameters (e.g., increases in ALT or AST), suggesting that the increase in ALP is due to the development of bone disease in animals with CIA and the reduction in ALP is due to reduced bone damage in animals exposed to AB79. Serum chemistry values ​​for creatinine, blood urea nitrogen, glucose, and total protein fluctuated over time but did not show a consistent correlation with arthritis severity or treatment regimen (data not shown).

[0341] Hematology, complete blood count, and differential analysis were performed throughout the study, and key parameters were reported. No changes in RBC levels were observed after exposure to AB79 or dexamethasone (Figure 38A). Furthermore, hematocrit counts decreased after the onset of arthritis and increased toward untreated control levels after treatment with AB79 or dexamethasone (Figure 38B). Reticulocyte counts increased after the onset of arthritis, and treatment with both AB79 and dexamethasone reduced reticulocyte counts toward normal levels found in untreated controls (Figure 38C). Both platelet and neutrophil levels were elevated in arthritis, and AB79 treatment reduced both platelet and neutrophil levels toward untreated controls, whereas dexamethasone had no effect (Figures 38D and 38E, respectively). In contrast, total lymphocyte levels were further reduced by disease compared to untreated controls and treatment with AB79, as expected, whereas treatment with dexamethasone had no effect (FIG. 38F).

[0342] Among peripheral blood lymphocyte subsets, NK cells were reduced by more than 95% from baseline levels (Figure 39A) within 24 hours of exposure to AB79, whereas baseline levels of B cells (Figure 39B), T cells (Figure 39C), and monocytes (Figure 39D) were reduced by up to 60%, 55%, and 50%, respectively. While the reduction in NK and B cells persisted throughout treatment, the reduction in T cells and monocytes was transient and observed only after the first infusion. Similar patterns of reduction were observed in each cell population after AB79 prophylactic treatment, although this group of animals had low monocyte levels before treatment and monocyte levels did not change in response to treatment. No differences were observed in the numbers of B, NK, T cells, or monocytes between vehicle- and dexamethasone-treated animals (Figures 39A, 29D, and 29F, respectively). Serum bioassays were performed to measure the concentrations of AB79 and anti-AB79 antibodies. C max Substantial exposure was achieved in the range of 24-97 μg / mL. All animals exposed to AB79 achieved EC 300 for CD38 saturation (Figure 32A) and NK cell lysis (Figure 32C) in vitro. 50 Anti-AB79 antibodies were detected in 4 of 7 animals in the prophylactic group, 14 to 30 days after the first dose and until the end of the study (Figure 40C). Two animals showed high titers by day 31, corresponding to the low concentrations of AB79 in these animals (Figure 40A), indicating that these anti-AB79 antibodies may be affecting clearance. Anti-AB79 antibodies were also detected in 4 of 5 animals in the treatment group (Figure 40D), from 21 days after the first dose until the end of the study. Two animals also showed high titers, corresponding to the low concentrations of AB79 in these animals, until the end of the study (Figure 40B). Nevertheless, with the exception of T cells after the second treatment dose (Figure 39G), all animals were included in the data analysis because most target cells remained reduced from baseline levels over the study period (Figures 39E and 39F).

[0343] Consideration Although CD38 deficiency in mice has been reported to result in attenuated CIA, indicating a nonredundant function of this molecule in autoimmune disease models, the role of CD38 expression in primate models has not been investigated. Additionally, numerous studies have shown that the overall level of CD38 expression on peripheral blood cells positively correlates with disease activity in human RA and SLE patients (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 mAb AB79 depleted 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). In contrast to other anti-CD38 mAbs in development (e.g., daratumumab, isatuximab, and MOR202), AB79 binds by CD38 expressed by cynomolgus monkeys, providing a unique opportunity to determine whether reducing the levels of cells expressing CD38 would prevent and / or ameliorate inflammation and tissue damage in a non-human primate autoimmune model.AB79 is a high-affinity monoclonal antibody that effectively mediates depletion of CD38+ cells (Smithson, G., et al., J Immunol May 1, 2017, 198(1 Supplement) 224.20). Integrated analyses have revealed that CD38 is a therapeutic target for plasma cell-rich pre-disease and established rheumatoid arthritis and systemic lupus erythematosus (Cole S., et al., Arthritis Res Ther. 2018 May 2;20(1):85).

[0344] Cynomolgus monkeys were determined to be a suitable model for evaluating the potential efficacy of AB79 in autoimmune disease because CIA in these monkeys is characterized by symmetric small-joint polyarthritis similar to 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), the expression profiles for CD38 are similar between these species (Table 11), and AB79 bound to monkey CD38 with one-tenth the affinity of human CD38 (Figure 32). Because a dose-ranging study in healthy monkeys showed that AB79 administered at a weekly dose of 3 mg / kg reduced total NK, B, and T cell populations by >80%, >60%, and >20% from baseline levels in peripheral blood, respectively (Figures 31B, 31C, and 31D), we concluded that a weekly dose of AB79 of 3 mg / kg was sufficient to evaluate the potential role of CD38 in CIA. Similar reductions in NK, B, and T cells were achieved in the prophylactic CIA model (Figures 37E, 37F, and 37G), despite the appearance of anti-AB79 antibodies in some animals (Figures 39C and 39D). Although the decrease in AB79 concentrations over time in the three prophylactically exposed animals (Figure 39A) indicates that these antibodies may have increased clearance, B, NK, and T cell counts did not recover to baseline levels by the end of the study (Figures 37E, 37F, and 37G), indicating that exposure to AB79 was sufficient to maintain PD effects throughout the study. Therefore, these animals were included in all analyses. This was also generally true for therapeutic treatment with AB79.NK and B cells had not recovered to baseline levels by the end of the study (Figures 37E, 37F, and 37G), indicating that exposure to AB79 was sufficient to maintain the PD effect throughout the study. In contrast, T cell numbers recovered to baseline levels by the end of the study (Figure 37G), suggesting that anti-AB79 antibodies may partially confound the interpretation of treatment data (i.e., underestimate the contribution of CD38-expressing T cells in monkey CIA).

[0345] Prophylactic administration of AB79 consistently inhibited the development of arthritis across all assessments, whereas therapeutic treatment with AB79 inhibited the development of arthritis and joint damage (Figures 34, 35, and 36). Histological evaluation of the joints demonstrated that AB79 exerted a relatively broad effect, preventing pannus formation (Figure 35B), infiltration (Figure 35C), cartilage lesions (Figure 35D), bone erosion (Figure 35E), and osteophyte formation (Figure 35F). It is noteworthy that therapeutic treatment with AB79 and dexamethasone also inhibited these progressive histological changes, albeit to a lesser extent than prophylactic administration. Arthritis scores decreased over time with therapeutic treatment (Figures 34B, 34C, and 34D), indicating potential reversal of damage. Collectively, these data demonstrate consistent disease-modifying effects of prophylactic and therapeutic exposure to AB79.

[0346] The efficacy of these therapeutic AB79 and dexamethasone treatments appears comparable to each other in the regimes studied. While the therapeutic use of steroids such as dexamethasone is highly effective in treating human autoimmune diseases such as RA and SLE, adverse side effects (e.g., osteoporosis, hypertension, diabetes, weight gain, cataracts, glaucoma, skin thinning, and purpura) limit the chronic use of these therapeutics. The potential for targeted CD38 cell depletion to provide comparable efficacy in human autoimmune diseases without the adverse side effects of steroids warrants future clinical investigation.

[0347] The prophylactic and therapeutic effects of AB79 were associated with sustained reductions in blood levels of total lymphocytes (Figure 38F), NK, B, and T cells (Figures 37C-37E), transient reductions in monocytes (Figure 37F), and no changes in red blood cells (Figure 38A), platelets (Figure 38D), and neutrophils (Figure 38E). These pharmacodynamic data indicate that CD38-expressing cells differ in their sensitivity to AB79 reduction. Reduction generally correlated with the density of CD38 expression by the cells, with the NK cell population uniformly expressing the highest median density of CD38 among all cell types examined (Figure 32B) and being the most sensitive to AB79 (Figures 31B and 37C). The highest density of CD38 expressed on B cells was approximately three-fold lower than that on NK cells (Figure 32B), indicating that the B cell population was less sensitive to AB79 than the NK cells (Figures 31C and 37C). CD38 was generally expressed at a lower median density on T cells than on B cells (Figure 32B), and T cells were less sensitive to depletion by AB79 than B cells and NK cells (Figures 31D and 37D). Cells expressing low densities of CD38 (e.g., erythrocytes) or cells not expressing CD38 (e.g., neutrophils) were unaffected by AB79 (Figures 37A, 37D, and 37E). The exception was monocytes, which uniformly expressed CD38 at intermediate densities and were only transiently reduced by AB79 (Figure 37F). The kinetics of the transient reduction in monocytes differed from the sustained reduction observed in B, T, and NK cells, suggesting a different mechanism. Direct cytolysis of NK cells by AB79 was observed in vitro (Figure 32C), and sustained reduction of NK, B, and T cells occurred after a single dose of AB79 in vivo (data not shown), indicating that CDC and / or ADCC mediate the reduction of these B, T, and NK cells in vivo. In contrast, monocytes were not lysed in vitro (data not shown) and did not exhibit sustained reduction in vivo (Figure 37F), indicating that alternative mechanisms (e.g., margination) mediate their transient reduction in vivo.Resistance of human monocytes to AB79-induced cytolysis has also been observed in healthy subjects (unpublished work) and described for daratumumab in patients with multiple myeloma (Nijhof et al. (2016) Blood 128(7):959-70). Expression of inhibitors of CDC and ADCC by human monocytes did not significantly correlate with resistance to daratumumab-induced cytolysis, and the reasons for the relative insensitivity of monocytes to daratumumab and AB79 remain unclear.

[0348] While the NK cell reduction observed with AB79 is qualitatively consistent with that observed with daratumumab in patients with refractory myeloma, quantitative differences exist. The IV infusion dose (0.3 mg / kg) and maximum concentration (C) of AB79 required to maintain peripheral blood NK cells at less than 50% of baseline levels in monkeys were significantly different. max = 4.32 μg / mL) and exposure (AUC366 = 327 μg h / mL) (Figure 31B) are comparable to the corresponding IV infusion dose (24 mg / kg), maximum concentration (C max Approximately 573ug / mL) and exposure (AUC infThe AB79 binding affinity was approximately 80-fold, 116-fold, and 297-fold lower than that of daratumumab (approximately 97,175 μg h / mL) (Casneuf et al. (2017) Blood Adv. 1(23):2105-2114; Clemens et al. (2017) Clin. Pharmacokinet. 56(8):915-924). It is noteworthy that AB79 binds to monkey lymphocytes with similar affinity (KD = 4 nM) to that of daratumumab (KD = 4 nM) to cells expressing human CD38 (Center for Drug Evaluation and Research Application Number: 761036orig1s00 Pharmacology Review(s)). However, it is unclear whether these differences are due to potential differences in species, disease state, and / or potency of each antibody. Nevertheless, a more definitive comparison requires pharmacokinetic and kinetic data for AB79 in patients with refractory myeloma, as daratumumab does not cross-react with CD38 from mice, rats, rabbits, pigs, cynomolgus monkeys, and rhesus monkeys (April 1, 2016 EMA / 278085 / 2016 Committee for Medicinal Products for Human Use (CHMP) Evaluation Report Darzalex International non-proprietary name: daratumumab Procedure No. EMEA / H / C / 004077 / 0000).

[0349] In conclusion, reduction of CD38-expressing cells with the cytolytic antibody AB79 prevented the development of CIA in monkeys when administered prophylactically and reversed disease progression when administered therapeutically. Previous studies utilizing blood and bone marrow samples from SLE patients showed that AB79 depleted 80% of short- and long-lived plasma cells and reduced autoantibodies (e.g., 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 collective data suggest that this therapeutic strategy may be effective in treating RA, SLE, and other autoimmune diseases in humans.

[0350] Example 7: Evaluation of AB79 in healthy human volunteers This study characterized the safety, tolerability, pharmacokinetics, and pharmacodynamics of AB79 through a randomized, double-blind, placebo-controlled trial of a single intravenous (IV) infusion or subcutaneous (SC) injection of AB79 at escalating doses in healthy subjects.

[0351] result AB79 was well tolerated. All adverse events (AEs) were mild or moderate, with up to 0.06 and 0.6 mg kg-1 doses, respectively. -1 There were no withdrawals due to AEs or infusion or injection site reactions at the IV and SC doses tested. At the higher doses, a transient increase in cytokine levels, primarily after IV administration, coincided with a reduction in CD38-expressing cells, and clinical symptoms included primarily mild fever, headache, and orthostatic hypotension. No notable findings in laboratory tests, electrocardiograms, vital signs, or physical examinations were reported in association with AB79 treatment. AB79 reduced phenotype and natural killer (NK) cell levels at similar doses, approximately 0.003 and 0.1 mg kg−1 for IV and SC administration, respectively. -1 The reduction in immunoglobulin (Ig)M and IgA occurred without comparable changes in IgG. Total counts of leukocytes, granulocytes, lymphocytes, erythrocytes, and platelets were within normal ranges at all dose levels.

[0352] conclusion AB79, when administered intravenously or subcutaneously, reduced plasmablast and NK cell levels in the peripheral blood of healthy subjects and was generally safe and well-tolerated. SC administration was better tolerated than IV administration, resulting in durable target cell depletion. This plasmacytolytic profile may be useful in the treatment of disorders caused by plasma or NK cells, their malignant counterparts (e.g., multiple myeloma and NK cell leukemia), and pathogenic immunoglobulins.

[0353] Study design and objectives This was a first-in-human (FIH), phase 1, randomized, double-blind, placebo-controlled, single-dose study of AB79 in healthy adult subjects. The primary objective of the study was to evaluate the safety and tolerability of single ascending doses of AB79 following IV infusion or SC injection. Secondary objectives were to evaluate the PK and PD of blood cell populations and immunogenicity. A total of 74 subjects were enrolled in the study. After two screening examinations, separated by a minimum of 5 days within a 28-day window prior to randomization, subjects were admitted on day 2 prior to dosing for baseline assessments. AB79 was administered in six cohorts at doses of 0.0003, 0.001, 0.003, 0.01, 0.03, or 0.06 mg kg. -1 or in another four cohorts, 0.03, 0.1, 0.3, or 0.6 mg kg -1 AB79 was administered by SC injection at a dose of 0.001 mg / kg on day 1. Dose selection was based on a PK / PD model derived from a series of studies in cynomolgus monkeys (Roepcke et al. (2018) Pharmacol Res Perspect. 6(3):e00402). At each dose level, 6–8 subjects were randomly assigned to receive AB79 (n = 4–6) or placebo (n = 2).

[0354] Sentinel dosing was used for each cohort, with the first two subjects receiving either AB79 or placebo (1:1). 24-hour postdose safety and tolerability data from these two subjects were reviewed before dosing the remaining subjects in each cohort. Participants remained in the inpatient clinical pharmacology unit (CPU) until Day 8, followed by weekly or biweekly follow-up, with a final scheduled clinic visit on Day 78 for general safety assessments and PK, PD, and immunogenicity analyses. A final follow-up phone call was conducted on Day 92.

[0355] Dose escalation was primarily based on the severity of AEs, graded using Common Terminology Criteria for Adverse Events grading criteria. Dose escalation was halted if at least two subjects in a cohort experienced cytokine release syndrome (CRS), resulting in a moderate clinical syndrome or a moderate-to-severe infusion reaction. Given that AB79 is a lymphocyte-depleting antibody, if a clinically relevant reduction in total or subtype lymphocyte counts was observed (nominal, typically a >50% decrease from the subject's predose nadir and below the lower limit of the normal reference range (NRR)), further dose escalation was not permitted and was maintained for ≥29 days. The investigators and sponsor reviewed all blinded safety data for all participants at each dose level before proceeding to the next higher dose.

[0356] This study was conducted in accordance with Good Clinical Practice guidelines at Parexel International's Phase 1 Clinical Trials Center, Northwick Park Hospital, Harrow, UK. The study 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 informed consent forms before the start of study procedures.

[0357] Study participants Eligible participants were between 18 and 55 years old, weighed between 50 and 100 kg, and had a mean body mass of 18.5 to 30 kg m -2 Participants were healthy men or women (not of childbearing potential) with a body mass index (BMI) of 0.05 or lower.

[0358] Flow cytometry-based counts of CD45+ lymphocytes, T cells, CD4+ T cells, and B cells were required to exceed the lower limit of NRR and NK cell count within the upper 50th percentile of NRR, given that CD38 is highly expressed on NK cells. NRR was defined by Takeda, who performed the flow cytometry analysis.

[0359] Participants were excluded if they met the exclusion criteria defined in the study protocol, such as known immunodeficiency, increased risk of infection, history of malignancy, or taking another investigational drug prior to the study that may affect the efficacy of the investigational drug.

[0360] Serial blood samples were collected pre-dose and up to 168 hours post-dose, with additional blood samples collected at intermediate time points post-dose or on the early discontinuation date (ET) to measure serum AB79 concentrations. Serum AB79 concentrations were determined using a validated enzyme-linked immunosorbent assay at ICON (Whitesboro, NY).

[0361] To assess PD responses to AB79, peripheral blood samples were collected at screening, post-dose, and on days -1, 1, and 2 (SC only), 3 (SC only), 4 (IV only), 5 (SC only), 6, 8, 15, 22, 29, 50, and 78, or ET. Primary and secondary PD endpoints were plasmablast and NK cell counts measured in the blood, respectively. Additional assessments included total white blood cell count and differential, total T cell, CD4 and CD8 T cell subsets, B cell, monocyte, and granulocyte counts. Lymphocyte subsets, monocytes, and plasmablasts were measured by flow cytometry at Covance (Brussels, Belgium). An electrochemiluminescence assay was validated at ICON for the detection of anti-AB79 antibodies in human serum.

[0362] Routine safety parameters, including AEs, clinical laboratory parameters, physical examination, electrocardiogram (ECG), and vital signs, were monitored at screening, pre-dose, and during the containment period and follow-up visits.

[0363] Infusion reactions have been reported in clinical trials of other anti-CD38 antibodies administered to MM patients after IV infusion (Voorhees et al. (2015) Blood 126:1829). Ex vivo experiments showed no evidence of agonist activity by AB79 in human blood cells, and AB79 infusion did not induce observable findings suggestive of infusion reactions in preclinical studies in monkeys. However, patients were closely monitored for signs of infusion reactions or CRS (headache, fever, chills, hypotension, nausea, and vomiting). Serum C-reactive protein (CRP) and tumor necrosis factor (TNF)-α, as well as inflammatory mediators including interleukin-1 (IL-1) and IL-6 (IL-6) levels, were assessed at various time points on days 1, 2, and 4 (SC cohort only). Side effects could be minimized by reducing the rate of injectable and / or oral prophylactic premedication of paracetamol (acetaminophen) and antihistamines (anti-H1 and anti-H2), if necessary.

[0364] The SC cohort was monitored for signs of injection site reactions (ISR), including pain, burning, redness, itching, swelling, and induration at the injection site.

[0365] Summary statistics and data analysis were performed using SAS version 9.2 and R version 3.5.1. PK parameters were calculated using noncompartmental analysis. PD variables were assessed and compared between active treatment groups and between each AB79 dose level and placebo.

[0366] result

[0367] Seventy-four subjects were enrolled and received a single dose of AB79 (n = 54) or placebo (n = 20) at doses of 0.0003, 0.001, 0.003, 0.01, 0.03, or 0.06 mg kg -1Six IV cohorts receiving AB79 at doses of 0.03, 0.1, 0.3, or 0.6 mg kg-1 or a matching placebo, and four SC cohorts receiving AB79 at doses of 0.03, 0.1, 0.3, or 0.6 mg kg-1 or a matching placebo, were monitored for 92 days and all completed the study. Participants were all male, except for one female in the SC placebo group. The study population consisted of Caucasian (n=51), Asian (n=13), African (n=4), and multiracial (n=6) ethnicities. Mean age (34.4 years, range 19-55 years) and BMI (24-25 kg m-1) were 92 days. -2 ) were similar between the IV and SC cohorts and between the AB79 and placebo treatment groups.

[0368] All doses of AB79 were well tolerated in this study. AEs were mild to moderate in intensity, with most AEs being mild and balanced between the placebo and AB79 treatment groups (Table 13). There were no serious adverse events (SAEs) or deaths, and no study or visit discontinuations due to adverse events. No notable findings on laboratory tests, ECGs, vital signs, or physical examinations were reported related to AB79 treatment.

[0369] [Table 13]

[0370] As shown in Table 13, most AEs were sporadic with no dose-related trend, except for headache, dizziness, and chills, which were seen more frequently with higher IV AB79 doses, consistent with a higher incidence of CRS. These effects (Table 14) were more pronounced in subjects receiving the higher doses (1 subject and 6 subjects at 0.03 and 0.06 mg kg−1, respectively). -1 IV, 0.3 and 0.6 mg kg in one and two subjects, respectively. -1 These subjects exhibited a reduction in plasmablasts and NK cells, suggesting that these symptoms result from a depletion of cells expressing CD38.

[0371] [Table 14]

[0372] Only mild, transient ISRs were observed after SC injection, most of which resolved within 7 days. These reactions demonstrated an inverse dose-effect relationship, with 5 of 6 subjects treated with the lowest SC dose responding and only 1 subject in each of the two highest dose cohorts responding.

[0373] IV Cohort 1 (0.0003 mg kg -1 ) to cohort 4 (0.01 mg kg -1 ) serum concentrations of AB79 at all PK sampling time points up to the lower limit of quantitation (LLOQ) of the detection assay (i.e., 10 ng mL -1 ), likely due to the low dose and the absence of anti-drug antibodies (data not shown). -1 The maximum observed serum concentration (C max ) are 21.4 and 100.4 ng mL -1 , which occurred 5 minutes after the end of the infusion (Table 15 and Figure 41). Serum concentrations then rapidly declined to below the LLOQ within 1 hour or 4 hours, respectively, after the end of the infusion, preventing accurate calculation of exposure. max is 0.03 to 0.06 mg kg -1 There appeared to be an approximately five-fold increase beyond a two-fold dose increase to 0.03 and 0.06 mg kg IV. -1 Due to the limited availability of AB79 serum concentrations from the cohort (1–3 time points per subject), the time to maximum serum concentration (t max ) and C max Other PK parameters could not be reliably estimated.

[0374] [Table 15]

[0375] SC cohort 1 (0.03 mg kg -1 ) to SC cohort 3 (0.3 mg kg -1 ) serum concentrations of AB79 in all subjects were below the LLOQ of the detection assay at all time points. -1 After SC injection of AB79, five subjects in this cohort experienced t max The mean C of all 6 subjects in this cohort (including 1 subject with serum concentrations below the LLOQ) max is 23.0ng mL -1 , which is 0.06 mg kg -1 C after 2 hours of IV infusion at max The serum concentration of AB79 gradually decreased between days 3 and 14 post-injection, falling below the LLOQ (Figure 41). -1 One subject in the SC cohort did not exhibit detectable levels of AB79 throughout the PK sampling period. Compared with PK parameters from the IV cohort, -1 There was high inter-subject variability after SC injection at 100 μg / mL. max ranged from approximately 8 to 96 hours (0.33 to 4 days) post-injection for the five subjects with measurable concentrations in this cohort.

[0376] In the cohort receiving AB79 by IV infusion, there was a dose-dependent reduction in NK cells > 0.003 mg kg -1 ≥90% reduction was observed at doses of 0.06 mg kg -1 The 50% effective concentration (EC 50 ) is the LLOQ (i.e., 10 ng mL) of the PK assay. -1 Although the IV administration of AB79 occurred at less than 75% of the maximum effective concentration (EC 75 ) is 21.4ng ML -1NK cell levels consistently declined from baseline by the end of the infusion, but the duration of recovery to baseline levels (<-20%) was variable and generally dose-related, with doses of 0.003, 0.01, 0.03, and 0.06 mg kg -1 Recovery to baseline levels for the 100 mg / kg / day dose required an average of 4, 4, 6, and 8 days, respectively (Figure 43). No clinically meaningful reductions were observed in total lymphocytes, B and T cells, helper and cytotoxic T cells, granulocytes, red blood cells, or platelets with IV administration of AB79 (data not shown).

[0377] In the cohort treated with AB79 by SC injection, there was a dose-dependent reduction in NK cells (Figure 42) and plasmablasts (Figure 43) at doses > 0.1 mg kg -1 ≥90% reduction was observed at doses of 0.6 mg kg -1 A 75% reduction in NK cells occurred in all subjects receiving 0.6 mg kg -1 (data not shown) and 23.0 ng mL -1 C max Plasmablast and NK cell levels declined from baseline within 8 hours after injection and were associated with a 48-hour t max The duration of recovery to baseline levels was variable, with 0.1, 0.3, and 0.6 mg kg -1 Recovery to baseline (i.e., within -20% of baseline levels) for each dose required an average of 4, 78, and 50 days, respectively (data not shown). Minimal or no reductions were observed in total lymphocytes, B and T cells, cytotoxic T cells, helper T cells, monocytes (Figure 43), and granulocytes, red blood cells, and platelets (data not shown).

[0378] Total Ig levels remained within the NRR, but were between 0.03 and 0.06 mg kg -1Total IgM levels were reduced in the AB79 IV cohort and were significantly lower (P<0.05) than the time-matched placebo control cohort on days 15-64 (Figure 44, top panel). -1 A significant (P<0.01) reduction in total IgM levels was also observed for AB79 administered SC from days 15 to 64 (Figure 44, bottom panel). IgM levels tended to return to baseline by day 78. No significant effect on IgA and IgG levels was observed for AB79 administered IV or SC (data not shown).

[0379] Of the 54 subjects who received AB79, 0.03 mg kg -1 One subject in the SC cohort developed persistent (i.e., days 15, 29, and 78) and higher titers (i.e., 160, 1280, and 320) of anti-AB79 (data not shown). This subject's serum AB79 concentration, similar to the AB79 levels of anti-drug antibody (ADA)-negative subjects in the same cohort, remained below the LLOQ throughout the study period, and therefore the potential impact of ADA on the PK of AB79 could not be determined. It should also be noted that ADA was not a concordant AE.

[0380] One therapeutic strategy for treating SLE and RA is to reduce the level of CD38+ lymphocytes, based on several reports showing an association between CD38+ lymphocyte levels 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 AB79, as it specifically binds to CD38 and depletes cells (Smithson et al. (2017) J. Immunol. 198(1 Suppl):224.20). Addition of AB79 to blood or bone marrow samples from patients with SLE or RA reduced PC populations and decreased the production of autoantigen-specific antibodies (Wang et al. (2016 ACR / ARHP Annual Meeting abstract 1085) Arthritis Rheum. 68(suppl 10)). AB79 also reduced 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 inhibited the progression of arthritis when administered therapeutically (Smithson et al. (2017) J. Immunol. 198(1 Suppl):127.17). Therefore, the objective of this study was to characterize the safety, tolerability, PK, and PD of a single IV infusion and SC injection of AB79 in healthy subjects and to determine whether clinical trials in patients are warranted.

[0381] This is the first characterization of the tolerability, PK, and PD of a cytolytic CD38 antibody administered to healthy subjects. All doses of AB79 were well tolerated in this study. AEs were mild to moderate in intensity, with most being mild (Table 13). There were no SAEs or deaths, and no study or visit discontinuations due to adverse events. No notable findings on laboratory tests, ECGs, vital signs, or physical examinations were reported in relation to AB79 treatment. No infusion-related reactions were observed in this study, and CRS was observed in seven subjects in the AB79 IV infusion group and three subjects in the AB79 SC group (Table 14). Cases of CRS were consistent with reductions in plasmablasts and NK cells (Figures 42 and 43), moderate increases in cytokines (e.g., TNF-α, IL-1β, IL-6; data not shown), and circulating C-reactive protein (data not shown). These data are consistent with studies in monkeys, showing that cell depletion coincided with elevated serum levels of TNF-α (Roepcke et al. (2018) Pharmacol Res Perspect. 6(3):e00402). Compared with the IV treatment group, the SC treatment cohort had comparable concentrations of AB79 in the peripheral blood (e.g., 0.03 mg kg). -1 IV vs. 0.6 mg kg -1 SC administration of daratumumab resulted in a low incidence of CRS and minimal increases in cytokine levels. These results are consistent with the lower incidence of infusion reactions with SC administration of daratumumab compared with IV administration in patients with refractory myeloma. While a different formulation is used for IV administration of daratumumab compared with SC administration, the same formulation was used for IV administration of AB79 compared with SC administration in healthy subjects. The AB79 data indicate that the lower rate of CRS is primarily attributable to the SC administration route, as opposed to differences in formulation.

[0382] Nonclinical evaluation of AB79 in monkeys suggested that the area under the serum concentration-time curve increased proportionally across doses after single IV and SC administration of AB79 (Roepcke et al. (2018) Pharmacol Res Perspect. 6(3):e00402). Due to the limited amount of serum concentration data in healthy subjects, a formal assessment of dose proportionality or SC bioavailability of AB79 could not be performed. However, C after IV infusion max appears to increase proportionally across doses (i.e., doses of 0.03 to 0.06 mg kg -1 The dose-normalized C after SC injection was greater than two-fold, resulting in a roughly five-fold increase in C (Fig. 1B). This is consistent with similar trends observed in monkeys for AB79 and those published for other monoclonal antibodies with target-mediated elimination. max was substantially lower than that after 2 hours of IV infusion. max After reaching LLOQ, serum concentrations declined much more slowly after SC injection and remained much longer above LLOQ compared with IV infusion.

[0383] The PD response of healthy subjects to AB79 IV is the most potent example of NK cell depletion by an anti-CD38 monoclonal antibody described to date. -1 AB79 at 100.4ng mL -1 Average C max , resulting in a reduction of peripheral blood NK cells in each healthy subject by at least 90% below baseline levels (Figure 42). This range of NK cell reduction was observed up to 24 mg kg -1 of daratumumab administered IV infusion up to 573 μg mL -1 Average C maxIn patients with relapsed / refractory MM, efficacy was not achieved using AB79 (Clemens et al. (2017) Clin. Pharmacokinet. 56(8):915-924; Xu et al. (2017) Clin. Pharmacol. Ther. 101(6):721-724). It is unclear whether this difference in efficacy is due to differences in the study populations and / or the characteristics of each antibody. Although healthy subjects and myeloma patients have similar numbers of NK cells, and these cells express similar densities of CD38 (Krejcik et al. (2016) Blood 128(3):384-394), myeloma patients have higher levels of other CD38+ target cells (i.e., myeloma cells), which may contribute to the difference in efficacy of NK cell depletion between these populations. PK and PD data for AB79 in a similar population of relapsed / refractory myeloma patients are needed for a more definitive analysis.

[0384] An attribute of higher potency is that a smaller amount and volume of therapeutic agent is required to elicit comparable PD effects. The efficacy of treating patients with IV infusion of anti-CD38 mAb can be improved by administering it as a small SC injection, because SC injections can be administered within 1 minute, as opposed to the 2–4 hours typically required for IV infusions. For chronic treatment, patients can safely self-administer it at home, offering convenience and pharmacological advantages. This is the first demonstration of cytoreductive therapy following SC injection of a cytolytic anti-CD38 antibody in human subjects, and it has been shown that SC injections are better tolerated than IV infusions. Furthermore, SC injections of 0.1–0.6 mg kg−1 were administered at a dose of 0.1–0.6 mg kg−1. -1 It has also been reported for the first time that a 1 mL SC injection of an anti-CD38 antibody AB79 induces maximal PD activity (e.g., >90% reduction in plasmablasts) in peripheral blood. -1 IV doses of 100 mg / kg / day reduced plasmablasts and NK cells in a dose-dependent manner (Figures 42 and 43). 90 is approximately 23.0 ng mL -1 However, the reduction in NK cells was 100.4ng mL -1(Table 15). This potential difference in sensitivity may be related to the level of CD38 expression expressed by these populations, with plasmablasts expressing CD38 at approximately five-fold higher densities than NK cells (Krejcik et al. (2016) Blood 128(3):384-394). Whether this difference exists in SLE, RA, and / or MM patients is unclear, as no data describing the levels of plasmablasts and NK cells in these patient populations have been reported to date. If comparable activity is observed in patients, then the efficiency of small-volume SC injections could potentially provide treatment to patients without access to infusion facilities, lowering overall healthcare costs.

[0385] Circulating monocytes, B cells, and T cells express lower levels of CD38 in healthy subjects, and these cell types are not reduced to the same extent by AB79 as plasmablasts and NK cells. These data are consistent with those described in cynomolgus monkeys after the first dose of AB79 and after 3 months of chronic exposure, where NK cells uniformly express high levels of CD38 and are more sensitive to AB79 than B and T lymphocytes, which express CD38 heterogeneously and generally at lower levels than NK cells (Roepcke et al. (2018) Pharmacol Res Perspect. 6(3):e00402). These data are also consistent with those described in relapsed / refractory myeloma patients chronically exposed to daratumumab, where monocytes are unaffected and CD38+ B and T cell subpopulations are reduced in peripheral blood (Krejcik et al. (2016) Blood 128(3):384-39).

[0386] Ig is primarily produced by PCs present in lymphoid tissues (e.g., bone marrow, lymph nodes, spleen, etc.). Similar effects have been reported in SLE patients exposed to multiple doses of the proteasome inhibitor bortezomib, resulting in significant reductions in serum levels of total IgM (34%), IgA (34%), and IgG (15%), corresponding to reductions in blood plasmablasts (54%), bone marrow PCs (50%), and disease activity scores (Alexander et al. (2015) Ann. Rheum. Dis. 74(7):1474-1478). Collectively, these data suggest that AB79 may be effective in lowering Ig levels, selectively reducing IgM and IgA levels relative to IgG. This profile may offer therapeutic benefit for IgM, IgA, and IgG nephropathy while minimizing suppression of other components of the immune system.

[0387] In conclusion, a single dose of AB79 administered intravenously or subcutaneously was well tolerated by healthy subjects. AB79 depleted plasmablasts and NK cells and reduced serum IgM and IgA levels. This plasmacytolytic activity may be beneficial for the treatment of various hematological malignancies and / or immunological disorders involving dysregulated PC, pathogenic antibodies, IgA, and / or NK cells.

[0388] Incorporation by Reference The contents of all references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are expressly incorporated by reference in their entirety for all purposes, as well as the references cited therein, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0389] equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments should be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein. Modifications of the above-described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the appended claims.

Claims

1. 1. A formulation for treating a disease exhibiting binding to CD38 in a subject, the formulation comprising an isolated human anti-CD38 antibody, the anti-CD38 antibody comprising a variable heavy (VH) chain region comprising CDR1 having the amino acid sequence of SEQ ID NO: 3, CDR2 having the amino acid sequence of SEQ ID NO: 4, and CDR3 having the amino acid sequence of SEQ ID NO: 5, and a variable light (VL) chain region comprising 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 disease being an autoimmune disease or a blood cancer, and the formulation being administered subcutaneously to the subject in a volume of 1 mL or less at a dose of 0.1 to 0.6 milligrams of antibody per kilogram of body weight.

2. 2. The formulation of claim 1, wherein the VH chain region has the amino acid sequence of SEQ ID NO: 9 and the VL chain region has the amino acid sequence of SEQ ID NO:

10.

3. 3. The formulation of claim 1 or 2, wherein the anti-CD38 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 11 and a light chain amino acid sequence of SEQ ID NO:

12.

4. The disease may be autoimmune thrombocytopenia, immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), myasthenia gravis (MG), neuromyelitis optica (NMO), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), antiphospholipid syndrome (APS), or rheumatoid arthritis (HPA). The formulation of any one of claims 1 to 3, wherein the treatment is selected from the group consisting of pemphigus vulgaris (PV), pemphigus foliaceus (PF), anti-NMDAR encephalitis (NMDR), autoimmune hemolytic anemia (AIHA), Graves' disease, membranous nephropathy, Sjogren's syndrome (SS), ANCA vasculitis, epidermolysis bullosa (EBA), pemphigoid (BP), Hashimoto's thyroiditis, scleroderma, IgG4-related disease, and graft-versus-host disease.

5. 4. The formulation of any one of claims 1 to 3, wherein the disease is selected from the group consisting of myasthenia gravis, autoimmune thrombocytopenia, immune-mediated thrombocytopenia, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myelogenous leukemia, B-cell lymphoma, and Burkitt's lymphoma.

6. The formulation according to any one of claims 1 to 3, wherein the disease is multiple myeloma.

7. 7. The formulation of claim 6, wherein the disease is selected from the group consisting of relapsed multiple myeloma (RMM), relapsed and refractory multiple myeloma (RRMM), and newly diagnosed multiple myeloma (NDMM).

8. The formulation of any one of claims 1 to 7, wherein the human anti-CD38 antibody is administered in the form of a pharmaceutically acceptable composition.

9. 1. A formulation comprising an isolated antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10, wherein the isolated antibody binds to CD38, the formulation being formulated for subcutaneous administration of the antibody at a dose of 0.1 to 0.6 milligrams per kilogram of body weight, and the formulation having a volume of 1 mL or less.

10. 10. The formulation of claim 9, wherein the isolated antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:

12.

11. 11. The formulation of claim 9 or 10, wherein the formulation is formulated for subcutaneous administration of the antibody in the treatment of a hematological cancer selected from the group consisting of multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myelogenous leukemia, B-cell lymphoma, and Burkitt's lymphoma.

12. 12. The formulation of claim 11, wherein the hematological cancer is multiple myeloma.

13. 13. The formulation of claim 12, wherein the multiple myeloma is selected from the group consisting of relapsed multiple myeloma (RMM), relapsed and refractory multiple myeloma (RRMM), and newly diagnosed multiple myeloma (NDMM).

14. The formulation of any one of claims 1 to 13, wherein the anti-CD38 antibody does not cause hemolytic anemia or thrombocytopenia.

15. 14. The formulation of any one of claims 1-13, wherein the anti-CD38 antibody results in less than a 10%, less than 20%, less than 30%, less than 40%, or less than 50% incidence of Grade 3 or 4 treatment-emergent adverse events (TEAEs) selected from the group consisting of anemia, hemolytic anemia, thrombocytopenia, fatigue, infusion-related reactions (IRR), leukopenia, and lymphopenia.

16. 14. The formulation of any one of claims 1-13, wherein the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% depletion of RBCs.

17. 14. The formulation of any one of claims 1-13, wherein the anti-CD38 antibody results in less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% depletion of platelets.

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