Anti-CD38 antibodies

Abstract

The present invention discloses isolated antibodies that bind to human CD38 and macaque CD38. Also disclosed are pharmaceutical compositions comprising the disclosed antibodies, as well as therapeutic and diagnostic methods of using the disclosed antibodies.

Description

[0001] This divisional application is a divisional application based on the original Chinese patent application with application number 201180063232.6, application date December 30, 2011, applicant "Takeda Pharmaceutical Company Limited", and invention name "Anti-CD38 Antibody" (original international application number PCT / US2011 / 068235).

[0002] background

[0003] This application claims the benefit of USSN 61 / 428,699, filed December 30, 2010; USSN 61 / 470,382, filed March 31, 2011; USSN 61 / 470,406, filed March 31, 2011; and USSN 61 / 485,104, filed May 11, 2011, all of which are incorporated herein by reference.

[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 include CD157 and the Aplysia ADPR cyclase. Enzymes in this family have the unique ability to convert NAD into cyclic ADP ribose or nicotinic acid-adenine dinucleotide phosphate.

[0005] In addition, CD38 has been reported to be involved in Ca 2+ CD38 is involved in signal transduction via tyrosine phosphorylation of many signaling molecules, including phospholipase Cγ, ZAP-70, syk, and c-cbl. Based on these observations, CD38 is proposed to be an important signaling molecule in the maturation and activation of lymphoid cells during normal development.

[0006] In hematopoietic cells, a range of functional effects have been attributed to CD38-mediated signaling, including lymphocyte proliferation, cytokine release, regulation of B cell and myeloid cell development and survival, and induction of dendritic cell maturation.

[0007] However, the precise role of CD38 in signal transduction and hematopoiesis remains unclear, as most signal transduction studies have used cell lines ectopically overexpressing CD38 and anti-CD38 monoclonal antibodies, which are nonphysiological ligands.

[0008] The putative natural ligand of CD38 is CD31 (PECAM-1; Platelet endothelial cell adhesion molecule-1). CD31 is a 130 kD member of the immunoglobulin superfamily that is expressed on the surface of circulating platelets, neutrophils, monocytes, and naive B-lymphocytes. Functionally, CD31 is thought to act as an adhesion molecule. It has been suggested that the interaction of CD38 with CD31 may play a role in promoting the survival of leukemic cells.

[0009] In many cases, animal models lacking a single molecule have become an essential tool for understanding the biological role of that molecule in the animal. The implicit assumption is that if a protein performs a non-redundant function, then its complete absence will result in a complete loss of that function.

[0010] Mice with a knockout of CD38 have been generated. These animals exhibit an almost complete loss of tissue-associated NADase activity. However, these animals are viable, leading to the conclusion that CD38 and its activity are not essential for life. Nevertheless, these mice show defects in their innate immunity and a reduction in T-cell-dependent humoral responses.

[0011] In contrast to the results in mice, there is strong indirect evidence that the absence of CD38 in humans is incompatible with life. Analysis of more than 5,000 blood samples from newborns failed to identify a single CD38 - individuals; suggesting that, unlike in mice, CD38 is required for survival. Therefore, it is unclear whether the observations made in mice regarding CD38 function can be extrapolated to humans.

[0012] CD38 is upregulated in many hematopoietic malignancies and cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL) and chronic myeloid leukemia (CML). On the other hand, the most primitive pluripotent stem cell of the hematopoietic system is CD38. - ( Figure 1 ).

[0013] Despite recent advances in the discovery and development of anticancer agents, many cancer forms involving CD38-expressing tumors still have a poor prognosis. Therefore, there is a need for improved methods for treating such cancer forms. SUMMARY OF THE INVENTION

[0015] Provided herein are reagents and methods for binding CD38, and methods for treating CD38-related diseases and detecting CD38 using CD38-specific binding agents including antibodies specific for CD38.

[0016] Thus, in some embodiments, isolated antibodies specific for human CD38 (SEQ ID NO: 1) and macaque CD38 (SEQ ID NO: 2) are described. This antibody consists of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of three complementary determining regions (CDRs), namely HCDR1, HCDR2 and HCDR3, and wherein the light chain variable region also consists of three CDRs, namely LCDR1, LCDR2 and LCDR3. The sequences of the CDRs are represented by: HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4), HCDR3 (SEQ ID NO: 5), LCDR1 (SEQ ID NO: 6), LCDR2 (SEQ ID NO: 7) and LCDR3 (SEQ ID NO: 8).

[0017] In other embodiments, the isolated antibody consists of a heavy chain variable region, wherein the sequence of the heavy chain variable region is encompassed by SEQ ID NO: 9. In other embodiments, the isolated antibody consists of a light chain variable region, wherein the sequence of the light chain variable region is encompassed by SEQ ID NO: 10.

[0018] In some embodiments, the isolated antibody consists of a heavy chain variable region, wherein the sequence of the heavy chain variable region is encompassed by SEQ ID NO: 9. In other embodiments, the isolated antibody consists of a light chain variable region, wherein the sequence of the light chain variable region is encompassed by SEQ ID NO: 10. This combination of heavy chain variable region and light chain variable region is referred to as Ab79.

[0019] In some embodiments, the isolated antibody consists of a heavy chain and a light chain, wherein the heavy chain sequence is encompassed by SEQ ID NO:11 and the light chain is encompassed by SEQ ID NO:12.

[0020] In some embodiments, the isolated antibody comprises an Fc domain. In other embodiments, the Fc domain is a human Fc domain. In other embodiments, the Fc domain is a variant Fc domain.

[0021] In some embodiments, an isolated nucleic acid encoding the heavy chain of SEQ ID NO: 11 is provided. In other embodiments, an isolated nucleic acid encoding the light chain of SEQ ID NO: 12 is provided.

[0022] In some embodiments, a host cell is provided, the host cell containing an isolated nucleic acid encoding the heavy chain of SEQ ID NO: 11 and an isolated nucleic acid encoding the light chain of SEQ ID NO: 12.

[0023] In some embodiments, a method of producing an antibody of the present invention is provided. The method encompasses culturing a host cell containing an isolated nucleic acid encoding the heavy chain of SEQ ID NO: 11 and an isolated nucleic acid encoding the light chain of SEQ ID NO: 12 under conditions that express the isolated nucleic acid and produce the antibody.

[0024] In some embodiments, isolated antibodies specific for human CD38 (SEQ ID NO: 1) and macaque CD38 (SEQ ID NO: 2) are described. This antibody consists of six CDRs, wherein each CDR of the antibody may differ from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 by 0, 1, or 2 amino acid substitutions.

[0025] In other embodiments, isolated antibodies specific for human CD38 (SEQ ID NO: 1) and macaque CD38 (SEQ ID NO: 2) are described. This antibody consists of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of three complementary determining regions (CDRs), namely HCDR1, HCDR2 and HCDR3, and wherein the light chain variable region also consists of three CDRs, namely LCDR1, LCDR2 and LCDR3. The sequences of the CDRs are represented by: HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14), HCDR3 (SEQ ID NO: 15), LCDR1 (SEQ ID NO: 16), LCDR2 (SEQ ID NO: 17) and LCDR3 (SEQ ID NO: 18).

[0026] In other embodiments, the isolated antibody consists of a heavy chain variable region, wherein the sequence of the heavy chain variable region is encompassed by SEQ ID NO: 19. In other embodiments, the isolated antibody consists of a light chain variable region, wherein the sequence of the light chain variable region is encompassed by SEQ ID NO:20.

[0027] In some embodiments, the isolated antibody consists of a heavy chain variable region, wherein the sequence of the heavy chain variable region is encompassed by SEQ ID NO: 19. In other embodiments, the isolated antibody consists of a light chain variable region, wherein the sequence of the light chain variable region is encompassed by SEQ ID NO: 20. This combination of heavy chain variable region and light chain variable region is referred to as Ab19.

[0028] In some embodiments, the isolated antibody consists of a heavy chain and a light chain, wherein the heavy chain sequence is encompassed by SEQ ID NO:21 and the light chain is encompassed by SEQ ID NO:22.

[0029] In some embodiments, an isolated nucleic acid encoding the heavy chain of SEQ ID NO: 21 is provided. In other embodiments, an isolated nucleic acid encoding the light chain of SEQ ID NO: 22 is provided.

[0030] In some embodiments, a host cell is provided, the host cell containing an isolated nucleic acid encoding the heavy chain of SEQ ID NO: 21 and an isolated nucleic acid encoding the light chain of SEQ ID NO: 22.

[0031] In some embodiments, a method of producing an antibody of the present invention is provided. The method encompasses culturing a host cell containing an isolated nucleic acid encoding the heavy chain of SEQ ID NO: 21 and an isolated nucleic acid encoding the light chain of SEQ ID NO: 22 under conditions that express the isolated nucleic acid and produce the antibody.

[0032] In other embodiments, isolated antibodies specific for human CD38 (SEQ ID NO: 1) and macaque CD38 (SEQ ID NO: 2) are described. This antibody consists of six CDRs, wherein each CDR of the antibody may differ from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 by 0, 1, or 2 amino acid substitutions.

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

[0034] In some embodiments, antibodies are provided that compete with Ab79 and / or Abl9 for binding to human CD38 and / or cynomolgus CD38.

[0035] These and other embodiments, features and potential advantages will become apparent with reference to the following description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 CD38 expression profile of lymphoid lineage cells is depicted, with asterisks indicating high CD38 expression. + CD19 + CD20), activated B cells (CD19 + CD20 + ), plasma cells (CD138 + CD19 - CD20 - ), activated CD4 + and CD8 + T cells, NKT cells (CD3 + CD56 + ) and NK cells (CD56 + CD16 + ) were identified on lymphocyte progenitor cells (CD34 + CD45RA + CD10 + CD19 - CD38 expression was found on mature DCs and activated monocytes.

[0038] FIG2 shows the heavy chain sequence and light chain sequence of Ab79 and Ab19.

[0039] Figure 3 The sequences of human CD38 and macaque CD38 are depicted.

[0040] Figure 4 The antigenic epitope of human CD38 is displayed, which binds each of the antibodies: Benchmark 1 and Benchmark 2, Ab19 and Ab79.

[0041] Figure 5 Depicts the increased expression of CD38 in PMBCs from SLE patients using a commercially available human CD38 antibody.

[0042] Figure 6 Depicted are the percent changes in cell number in cyno monkeys 24 hours after dosing.

[0043] Figure 7 Restoration of cell depletion after a single dose of Ab79 is demonstrated.

[0044] Figure 8 Results from a single HuSCID mouse showing a significant reduction of all Ig isotypes following a single dose of Ab79.

[0045] Fig. 9 Similar to Figure 8 , as described in the Examples, with respect to Ab79 depletion activity in HuSCID mice.

[0046] Fig.10 Depicts a significant reduction in anti-tetanus response in the HuSCID model following Ab79 treatment.

[0047] Fig.11 The significant increase in survival following Ab79 treatment was again demonstrated in the HuSCID model, which is essentially a graft-versus-host model.

[0048] Fig.12 Depicts the difference in CD38 antigen expression in human and mouse PBMCs using the respective commercial antibodies.

[0049] Fig.13 Demonstrating therapeutic efficacy in an inflammatory setting, surrogate mouse anti-CD38 antibodies were shown to deplete immune cells from peripheral blood. DETAILED DESCRIPTION OF THE INVENTION

[0051] Overview

[0052] It has been confirmed that the extracellular domain of CD38 has bifunctional enzymatic activity, namely ADP-ribosyl cyclase activity and ADP-ribosyl hydrolase activity. + Converted to cADPR (cyclase), which can further hydrolyze it to ADP-ribose (hydrolase). cADPR is involved in the mobilization of calcium from cellular memory stores, which is a second messenger activity important for cell proliferation, differentiation, and apoptosis.

[0053] Increased expression of CD38 has been documented in various diseases of hematopoietic origin and has been described as a negative prognostic marker for chronic lymphoblastic leukemia. Such diseases include, but are not limited to, multiple myeloma (Jackson et al. (1988)), chronic lymphoblastic leukemia (Moribito et al. (2001), Jelinek et al. (2001), Chevalier et al. (2002), Dürig et al. (2002)), B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia (Keyhani et al. (2000)), including B-cell acute lymphocytic leukemia, Waldenstrom macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic leukemia / promyelocytic leukemia, acute myeloid leukemia (Keyhani et al. (1993)), chronic myeloid leukemia (Marinov et al. (1993)), follicular lymphoma, NK cell leukemia, and plasma cell leukemia. Therefore, CD38 provides an effective target for the treatment of hematopoietic diseases.

[0054] Several anti-CD38 antibodies are in clinical trials for the treatment of CD38-related cancers. Therefore, antibodies to CD38 with therapeutic effects and / or diagnostic applications are useful. The present invention provides two different sets of anti-CD38 CDRs that bind to different antigenic epitopes of CD38 and bind to human and macaque forms of CD38, and the present invention provides antibodies containing these CDRs.

[0055] In addition, the present invention demonstrates that anti-CD38 antibodies are useful for diagnosing and / or treating inflammatory and / or immune disorders associated with activated lymphocytes, including, inter alia, autoimmune diseases. As shown herein, CD38 is expressed in immature hematopoietic cells, downregulated in mature cells, and re-expressed at high levels in activated lymphocytes and plasma cells. For example, high CD38 expression is found in activated B cells, plasma cells, activated CD4+T cells, activated CD8+T cells, NK cells, NKT cells, mature dendritic cells (DCs), and activated monocytes.

[0056] The findings herein are surprising since the presence of autoantibodies to CD38 has been associated with diabetes, chronic autoimmune thyroiditis, and Graves' disease (see Antonelli et al., Clin. Exp. Immunol. 2001 126:426-431; Mallone et al., Diabetes 50:752 (2001) and Antonelli et al., J. Endocrinol. Invest. 27:695-707 (2004), all incorporated by reference.

[0057] Thus, the antibodies of the invention are useful for the diagnosis and / or treatment of a number of diseases including, but not limited to, autoimmune diseases as described below, including but not limited to systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and ulcerative colitis.

[0058] Thus, for example, patients with high plasma cell content, such as SLE patients who display high plasma cells, and RA patients who do not display a response to CD20-based therapy, can be selected.

[0059] The therapeutic anti-CD38 antibodies of the present invention bind to CD38-positive cells via multiple mechanisms of action, thereby leading to the depletion of these cells (such as activated lymphocytes) to achieve the treatment and / or improvement of autoimmune diseases, and the mechanisms of action include but are not limited to CDC, ADCC and apoptosis pathways as outlined herein.

[0060] An advantage not seen in some anti-CD38 antibodies in oncology clinical testing is the ability to bind to macaque CD38, since these primates are suitable for preclinical testing, thus enabling early evaluation of dosing, toxicity, efficacy, etc.

[0061] CD38 protein

[0062] Thus, the present invention provides isolated anti-CD38 antibodies that specifically bind to human CD38 protein (and, as described below, additionally and preferably specifically bind to primate CD38 protein). As is known in the art, CD38 protein is found in many species. Antibodies that are particularly useful in the present invention bind to human CD38 protein and primate CD38 protein, particularly primates used in clinical testing, such as macaques (Macaca fascicularis, cynomolgus monkeys, sometimes referred to herein as "cyno"). As defined herein, "human CD38" or "human CD38 antigen" refers to protein SEQ ID NO: 1 or a functional fragment, such as an antigenic epitope. Generally, CD38 possesses a short intracytoplasmic tail, a transmembrane domain, and an extracellular domain. In a specific embodiment, the antibodies of the present invention bind to the extracellular portion of the CD38 protein. "Macaque CD38" herein means SEQ ID NO: 2, which is 92% identical to human CD38.

[0063] Synonyms for CD38 include ADP ribosyl cyclase 1, cADPr hydrolase 1, Cd38-rs1, cyclic ADP ribosyl hydrolase 1, 1-19, and NIM-R5 antigen.

[0064] In some embodiments, the anti-CD38 Ab79 antibodies of the invention interact with CD38 at a number of amino acid residues, including K121, F135, Q139, D141, M142, D202, V203, H205, Q236, E239, W241, S274, C275, K276, F284, C287, V288, K289, N290, P291, E292, D293. As outlined herein, other antibodies that interact with the residues are also suitable for therapeutic and diagnostic applications.

[0065] In some embodiments, the anti-CD38 antibodies of the invention optionally (and preferably in some cases) do not bind to other members of the CD38 family, such as CD 157. For example, preferred embodiments herein do not bind to human CD 157 (GenBank Accession No. NP_004325) of SEQ ID NO: 23.

[0066] Antibody

[0067] The present invention provides anti-CD38 antibodies, generally providing therapeutic and / or diagnostic antibodies as described herein. Antibodies suitable for use in the present invention can take many formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics as described below. In essence, the present invention provides antibody structures containing a set of 6 CDRs as defined herein (including minor amino acid changes as described below).

[0068] Traditional antibody structural units usually include tetramers. Each tetramer is usually composed of two pairs of identical polypeptide chains, each pair having a "light" chain (usually having a molecular weight of about 25kDa) and a "heavy" chain (usually having a molecular weight of about 50-70kDa). Human light chains are divided into kappa light chains and lambda light chains. Heavy chains are divided into μ, δ, γ, α or ε, and the isotypes of the antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. The subclasses of IgM include but are not limited to IgM1 and IgM2. Therefore, "isotype" used herein means any subclass of immunoglobulin defined by the chemical characteristics and antigenic characteristics of its constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE. It will be appreciated that therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses.

[0069] The amino terminus of each chain includes a variable region with about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. In the variable region, each of the V domains with heavy and light chains gathers three loops to form an antigen binding site. Each of the loops is called a complementary determining region (hereinafter referred to as "CDR"), in which the change in amino acid sequence is the most significant. "Variable" refers to that the sequence of certain segments of the variable region is widely different between antibodies. The variability within the variable region is not evenly distributed. In fact, the V region is composed of a relatively constant elongated region called a framework region (FR) with 15-30 amino acids, which is separated by a highly variable shorter region called a "hypervariable region" that is 9-15 amino acids long or longer.

[0070] Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0071] The hypervariable region generally encompasses amino acid residues 24-34 (LCDR1; "L" for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) from the light chain variable region and approximately 31-35B (HCDR1; "H" for heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) from the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Public Health Service, 5th ed., National Institutes of Health, Bethesda, MD Md. (1991) and / or those residues forming the hypervariable loop in the light chain variable region (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3)) and those residues forming the hypervariable loop in the heavy chain variable region, 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3); Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the invention are described below.

[0072] Throughout this specification, the Kabat numbering system (e.g., Kabat et al., supra (1991)) is generally used when referring to residues in the variable domains (roughly, residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), while the EU numbering system is used for the Fc region.

[0073] CDR promotes the formation of the antigen binding site of the antibody, or more particularly the antigen epitope binding site. "Epitope" refers to the determinant that interacts with the specific antigen binding site called the paratope in the variable region of the antibody molecule. Epitopes are molecules such as amino acids or sugar side chains that are grouped, and generally have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. For example, as shown herein, two different antibodies referred to herein as "Ab19" and "Ab79" bind to different epitopes on the CD38 molecule.

[0074] An antigenic epitope may contain amino acid residues that are directly involved in binding (also called the immunodominant component of the antigenic epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by the specific antigen binding peptide; in other words, the amino acid residues are within the footprint of the specific antigen binding peptide.

[0075] Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes may differ in that binding to the former is lost in the presence of denaturing solvents while binding to the latter is not lost.

[0076] Antigenic epitopes typically include at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same antigenic epitope can be validated in a simple immunoassay that demonstrates the ability of one antibody to block the binding of another antibody to the target antigen, for example, "binning" as outlined in the Examples. X-ray crystallography studies as shown in the Examples have identified amino acid residues that bind antibodies of the present invention (including Ab19 and Ab79) and antibodies of the prior art (Benchmark 1 and Benchmark 2), such as Figure 4 shown.

[0077] In the present invention, as outlined in the Examples, Ab79 interacts with a number of amino acid residues of CD38, including K121, F135, Q139, D141, M142, E239, W241, S274, C275, K276, F284, V288, K289, N290, P291, E292, and D293. It should be noted that the residues are identical in humans and cyan monkeys, with the exception that S274 is actually F274 in cyan. The residues may represent immunodominant epitopes and / or residues within the region occupied by the specific antigen binding peptide.

[0078] In the present invention, Ab19 binds to different epitopes, including G91, E103, E104, D105, Q107, M110, K111, T114, Q115, T148, V192, R194, R195, F196, A199, H228, N229, Q231, E233 and K234. It should be noted that the residues are the same in humans and cyan monkeys, with the exception that M110 is V110 in cyan and A199 is T199 in cyan.

[0079] Thus, in some embodiments, antibodies that compete with AB79 and Abl9 by binding to any of the epitopes described herein can be used to treat autoimmune diseases. It should be noted that Ab79 and BM1 have some overlap; therefore, antibodies that compete with Ab79 and are not BM1 are suitable for use in the present invention.

[0080] Thus, the invention provides antibodies that bind human CD38 and cyan CD38 and interact with at least 80%, 90%, 95% or 98% of said residues. In other words, the surface area of ​​the interaction region does not exceed the area of ​​said residues.

[0081] The carboxyl terminus of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected many primary sequences of the variable regions of the heavy and light chains. Based on sequence conservation, Kabat et al. divided individual primary sequences into CDRs and frameworks and tabulated them (see SEQUENCES OF IMMUNOLOGICAL INTEREST, Fifth Edition, NIH publication, No. 91-3242, EA Kabat et al., incorporated by reference in its entirety).

[0082] In the IgG subclass of immunoglobulin, there are several immunoglobulin domains in the heavy chain. "Immunoglobulin (Ig) domain" herein means the immunoglobulin region with significantly different tertiary structures. What the present invention is concerned with is the heavy chain domain, including the constant heavy chain (CH) domain and the hinge domain. In the case of IgG antibodies, each IgG isotype has three CH regions. Therefore, the "CH" domain in the case of IgG is as follows: "CH1" refers to the position 118-220 according to the EU index as in Kabat. "CH2" refers to the position 237-340 according to the EU index as in Kabat, and "CH3" refers to the position 341-447 according to the EU index as in Kabat.

[0083] The Ig domain of another type of heavy chain is the hinge region. "Hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" herein means a flexible polypeptide comprising the amino acids between the first constant domain and the second constant domain of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Therefore, for IgG, antibody hinges are defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein numbering is according to the EU index as in Kabat. In some embodiments, for example, in the case of the Fc region, a low-level hinge is included, wherein the "low-level hinge" generally refers to position 226 or 230.

[0084] The present invention is particularly concerned with the Fc region. "Fc" or "Fc region" or "Fc domain" as used herein means a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain, and in some cases, excluding part 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-termini of these domains. For IgA and IgM, Fc may include a J chain. For IgG, the Fc domain includes immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and a low-order hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region generally defined includes residues C226 or P230 at its carboxyl terminus, wherein numbering is according to the EU index as in Kabat. In some embodiments, as described more fully below, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcγR receptors or FcRn receptors.

[0085] In some embodiments, the antibody is a full-length antibody. "Full-length antibody" herein means 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.

[0086] Alternatively, the antibody may be a variety of structures, including but not limited to antibody fragments, monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof. Still depending on the structure

[0087] In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL and CH1 domains, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of VL and VH domains of a single antibody, (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546, incorporated by reference in its entirety), (v) isolated CDR regions, (vi) F(ab')2 fragments, which are bivalent fragments comprising two linked Fab fragments, (vii) single-chain Fv molecules (scFv), in which the VH domain and the VL domain are connected by a peptide linker, which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, incorporated by reference in its entirety), (viii) bispecific single-chain Fv (WO 03 / 11161, incorporated herein by reference), and (ix) "diabodies" or "tribodies", which are multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO 94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all incorporated by reference).

[0088] Chimeric and humanized antibodies

[0089] In some embodiments, the antibody may be a mixture of different species from, for example, chimeric and / or humanized antibodies. That is, in the present invention, CDR sets may be used with frameworks and constant regions other than those specifically described by the sequences herein.

[0090] In general, "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine multiple regions from more than one species. For example, a "chimeric antibody" generally comprises a variable region from a mouse (or in some cases from a rat) and a constant region from a human. A "humanized antibody" generally refers to a non-human antibody in which the variable domain framework region has been exchanged for a sequence present in an adult antibody. In general, in a humanized antibody, the entire antibody, except for the CDR, is encoded by a polynucleotide derived from a human or is identical to this antibody except for its CDR. The CDRs encoded in part or in whole by nucleic acids derived from non-human organisms are transplanted into the beta-folded framework of the variable region of a human antibody to produce an antibody whose specificity is determined by the transplanted CDRs. The generation of such antibodies is described in, for example, WO 92 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all of which are incorporated by reference in their entirety. "Backmutation" of selected acceptor framework residues to corresponding donor residues is often required to regain affinity lost in the initially transplanted construct (US 5530101, US 5585089, US 5693761, US 5693762, US 6180370, US 5859205, US5821337, US 6054297, US 6407213, all of which are incorporated by reference in their entirety). Humanized antibodies will also optimally comprise at least a portion of an immunoglobulin constant region, typically at least a portion of a human immunoglobulin, and thus will typically comprise a human Fc region. Humanized antibodies can also be produced using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20: 639-654, the entire contents of which are incorporated by reference. Various techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein, all of which are incorporated by reference in their entirety).Humanization methods include, but are not limited to, those described in Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160:1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all of which are incorporated by reference in their entirety. Humanization methods or other methods to reduce the immunogenicity of non-human antibody variable regions may include surface reshaping (resurfacing) methods, for example, as described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, the entire contents of which are incorporated by reference. In one embodiment, the parent antibody has been affinity matured as known in the art. Humanization and affinity maturation can be performed using structure-based methods, for example, as described in USSN 11 / 004,590. Humanization and / or affinity maturation of antibody variable regions can be performed using selection-based methods, including but not limited to those described in Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272(16): 10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10): 753-759, all of which are incorporated by reference in their entirety. Other humanization methods may involve grafting only portions of the CDRs, including but not limited to the methods described in USSN 09 / 810,510; Tan et al., 2002, J. Immunol. 169: 1119-1125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084, all of which are incorporated by reference in their entirety.

[0091] In one embodiment, the antibody of the present invention may be a multispecific antibody, in particular a bispecific antibody, sometimes also referred to as a "diabodies". The antibody is an antibody that binds to two (or more) different antigens or different antigenic epitopes on the same antigen. Diabodies can be made in various ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4: 446-449, the entire contents of which are incorporated by reference), for example, by chemical preparation or from hybridized hybridomas.

[0092] In one embodiment, the antibody is a minibody. Minibodies are minimized antibody-like proteins that contain a scFv connected to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, the entire contents of which are incorporated by reference. In some cases, the scFv may be connected to the Fc region and may include part or all of the hinge region.

[0093] The antibodies of the present invention are generally isolated or recombinant antibodies. When "isolated" is used to describe the various polypeptides disclosed herein, it means that the polypeptide has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Generally, the isolated polypeptide can be prepared by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to CD38 is substantially free of antibodies that specifically bind to an antigen different from CD38.

[0094] However, an isolated antibody that specifically binds to an antigenic epitope, isoform or variant of human CD38 or macaque CD38 may have cross-reactivity to other related antigens, such as related antigens from other species (such as CD38 species homologs). In addition, the isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0095] Isolated monoclonal antibodies with different specificities may be combined in well-defined compositions. Thus, for example, Ab79 and Ab19 may be combined in a single preparation if desired.

[0096] The anti-CD38 antibodies of the present invention specifically bind to CD38 ligands (e.g., human and macaque CD38 proteins SEQ ID NO: 1 and SEQ ID NO: 2. "Specific binding" or "specifically binds" or "specific for a particular antigen or antigenic epitope" means binding that is significantly different from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule, which is generally a structurally similar molecule that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0097] Specific binding to a particular antigen or antigenic epitope can be determined, for example, by a KD for the antigen or antigenic epitope of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 - 10 M, at least about 10 -11 M, at least about 10 -12 Antibodies with KD of M or higher are displayed, where KD refers to the dissociation rate of a specific antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a KD for a control molecule that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or higher times that for the antigen or antigenic epitope.

[0098] Furthermore, specific binding for a particular antigen or antigenic epitope can be exhibited, for example, by an antibody having a KA or Ka for the antigen or antigenic epitope that is at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times greater than that of a control, where KA or Ka refers to the on-rate of the specific antibody-antigen interaction.

[0099] Antibody modification

[0100] The present invention further provides variant antibodies. That is, many modifications can be made to the antibodies of the present invention, including but not limited to amino acid modifications in the CDR (affinity maturation), amino acid modifications in the Fc region, glycosylation variations, other types of covalent modifications, etc.

[0101] "Variant" herein means a polypeptide sequence that differs from the sequence of a parent polypeptide by at least one amino acid modification. Amino acid modifications may include substitutions, insertions and deletions, wherein the former is preferred in many cases.

[0102] In general, variants may include many modifications as long as the functionality of the protein remains, as described herein. That is, where, for example, the CDRs of Ab79 or Ab19 are used to generate amino acid variants, the antibodies should still specifically bind to human and macaque CD38. Similarly, if, for example, the Fc region is used to generate amino acid variants, the variant antibodies should retain the receptor binding function required for the specific application or indication of the antibody.

[0103] However, in general, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions are usually utilized, as the goal is often to utilize the minimum number of modifications to change function. In some cases, there are 1 to 5 modifications, and in many embodiments, 1 to 2, 1 to 3 and 1 to 4 modifications may also be used.

[0104] It should be noted that the number of amino acid modifications can be within a functional domain: for example, it may be desirable to have 1 to 5 modifications in the Fc region of a wild-type or engineered protein, and 1 to 5 modifications in the Fv region. The variant polypeptide sequence will preferably have at least about 80%, 85%, 90%, 95%, or up to 98% or 99% identity to the parent sequence (e.g., for the variable region, constant region, and / or heavy and light chain sequences of Ab79 and / or Ab19). It should be noted that the percentage identity will depend on the number of amino acids, depending on the size of the sequence.

[0105] As used herein, "amino acid substitution" or "substitution" means that an amino acid at a specific position in a parent polypeptide sequence is replaced by another amino acid. For example, the substitution S100A refers to a variant polypeptide in which the serine at position 100 is replaced by alanine. As used herein, "amino acid insertion" or "insertion" means adding an amino acid at a specific position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" means removing an amino acid at a specific position in a parent polypeptide sequence.

[0106] As used herein, "parent polypeptide", "parent protein", "precursor polypeptide", or "precursor protein" means an unmodified polypeptide that is subsequently modified to produce a variant. Generally speaking, the parent polypeptides herein are Ab79 and Ab19. The parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or an amino acid sequence encoding it. Therefore, as used herein, "parent Fc polypeptide" means an Fc polypeptide that is modified to produce a variant, and "parent antibody" as used herein means an antibody that is modified to produce a variant antibody.

[0107] "Wild type" or "WT" or "original" herein means the amino acid sequence or nucleotide sequence found in nature, including allelic gene variations. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, etc. have amino acid sequences or nucleotide sequences that have not been intentionally modified.

[0108] By "variant Fc region" herein is meant an Fc sequence that differs from the wild-type Fc sequence by at least one amino acid modification. Fc variants may refer to the Fc polypeptide itself, a composition comprising an Fc variant polypeptide, or an amino acid sequence.

[0109] In some embodiments, one or more amino acid modifications may be made in one or more of the CDRs of the antibody (Ab79 or Ab19). Generally, only 1 or 2 or 3 amino acids are substituted in any single CDR, and generally no more than 4, 5, 6, 7, 8, 9 or 10 changes are made within a group of CDRs. However, it should be understood that any combination of no substitution, 1, 2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitutions.

[0110] In some cases, amino acid modifications in CDRs are referred to as "affinity maturation." An "affinity matured" antibody is one that has one or more changes in one or more CDRs that result in an improvement in the antibody's affinity for the antigen compared to a parent antibody that does not have those changes. In some cases, although rare, it may be desirable to reduce the affinity of an antibody for its antigen, but this is generally not preferred.

[0111] Affinity maturation can be performed to increase the binding affinity of the antibody for the antigen by at least about 10% to 50-100-150% or more, or 1 to 5 times, compared to the "parent" antibody. Preferred affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by known procedures. For example, see Marks et al., 1992, Biotechnology 10:779-783, which describes affinity maturation by variable heavy chain (VH) and variable light chain (VL) domain shuffling. Random mutagenesis of CDR and / or framework residues is described in, e.g., 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.

[0112] Alternatively, "silent" amino acid modifications, such as modifications that do not significantly change the affinity of the antibody for the antigen, may be made in one or more of the CDRs of the antibodies of the invention. These modifications may be made for many reasons, including optimizing expression (such as modifications that may be made to nucleic acids encoding antibodies of the invention).

[0113] Therefore, variant CDRs and antibodies are included within the definition of the CDRs and antibodies of the present invention; that is, the antibodies of the present invention may include amino acid modifications in one or more of the CDRs of Ab79 and Ab19. In addition, as outlined below, amino acid modifications may also be independently and optionally performed in any region outside of the CDRs, including the framework and constant regions.

[0114] In some embodiments, variant antibodies of Ab79 and Ab19 specific for human CD38 (SEQ ID NO: 1) and macaque CD38 (SEQ ID NO: 2) are described. The antibody consists of six CDRs, wherein each CDR of the antibody differs from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 by 0, 1, or 2 amino acid substitutions. In other embodiments, the variant anti-CD38 antibody consists of six CDRs, wherein each CDR of the antibody differs from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 by 0, 1, or 2 amino acid substitutions.

[0115] In some embodiments, the anti-CD38 antibodies of the invention consist of variant Fc domains. As is known in the art, the Fc region of an antibody interacts with many Fc receptors and ligands, thereby conferring a series of important functional capabilities, known as effector functions. Such Fc receptors include, but are not limited to (in humans) FcγRI (CD64), including isoforms FcγRIa, FcγRib and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158, which are involved in antibody-dependent cellular cytotoxicity (ADCC)) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), FcRn (neonatal receptor), C1q (complement protein involved in complement dependent cytotoxicity (CDC)) and FcRn (neonatal receptor involved in serum half-life). Suitable modifications can be made, for example, at one or more positions as described in US patent application Ser. No. 11 / 841,654 and references cited therein, US 2004 / 013210, US2005 / 0054832, US2006 / 0024298, US2006 / 0121032, US2006 / 0235208, US2007 / 0148170, USSN 12 / 341,769, US Patent No. 6,737,056, US Patent No. 7,670,600, US Patent No. 6,086,875, all of which are expressly incorporated by reference in their entirety, particularly with respect to specific amino acid substitutions that increase binding to Fc receptors.

[0116] In addition to the modifications outlined above, other modifications may be made. For example, the molecule may be stabilized by incorporating disulfide bridges connecting the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14: 1239-1245, the entire contents of which are incorporated by reference). In addition, there are various antibody covalent modifications that may be made as outlined below.

[0117] Covalent modifications of antibodies are included within the scope of the present invention and are usually, but not always, performed post-translationally. For example, several types of antibody covalent modifications are introduced into the molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents that react with selected side chains or N-terminal or C-terminal residues.

[0118] Cysteinyl residues are most commonly reacted with α-haloacetates (and the corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0119] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0, as the reagent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0120] Lysyl and amino terminal residues are reacted with succinic acid or other carboxylic acid anhydrides. Derivatization with such reagents has the effect of reversing the charge of the lysyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0121] Arginyl residues are modified by reaction with one or several conventional reagents, among which are phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues requires the reaction to be carried out under alkaline conditions because of the high pKa of the guanidine functional group. In addition, the reagents react with the groups of lysine as well as with the arginine epsilon-amino group.

[0122] The specific modification of tyrosyl residues can be carried out by reacting with aromatic diazo compounds or tetranitromethane, with particular attention to introducing spectral labels into tyrosyl residues. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl substances and 3-nitro derivatives respectively. Use 125I or 131I iodinated tyrosyl residues to prepare labeled proteins for use in radioimmunoassays, and the chloramine T method described above is suitable.

[0123] The carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N--R'), wherein R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0124] Derivatization with bifunctional agents is useful for crosslinking antibodies to water-insoluble carrier matrices or surfaces for use in a variety of methods in addition to those described below. Common crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane; glutaraldehyde; N-hydroxysuccinimide esters, such as the ester with 4-azidosalicylic acid; homobifunctional imidoesters, including disuccinimidyl esters, such as 3,3'-dithiobis(succinimidylpropionate); and bifunctional maleimides, such as bis-N-maleimido-1,8-octane. Derivatization agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate generate photoactivatable intermediates capable of forming crosslinks in the presence of light. Alternatively, for immobilization of proteins, reactive water-insoluble matrices such as cynomolgusogen bromide-activated carbohydrates and reactive matrices are employed as described in U.S. Pat. Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440, all of which are incorporated by reference in their entirety.

[0125] Glutaminyl and asparaginyl residues are often subjected to deamidation to obtain the corresponding glutamyl and aspartyl residues, respectively. Alternatively, the residues can be deamidated under slightly acidic conditions. Any form of the residues falls within the scope of the present invention.

[0126] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86

[1983] , the entire contents of which are incorporated by reference), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0127] Additionally, as will be appreciated by those of skill in the art, labels including fluorescent, enzymatic, magnetic, radioactive labels, etc. may be added to the antibodies (as well as other compositions of the invention).

[0128] Glycation

[0129] Another type of covalent modification is a change in glycosylation. In another embodiment, the antibodies disclosed herein may be modified to include one or more engineered glycoforms. "Engineered glycoforms" as used herein means a carbohydrate composition covalently attached to an antibody, wherein the carbohydrate composition is chemically different from the carbohydrate composition of a parent antibody. Engineered glycoforms can be useful for various purposes, including but not limited to increasing or decreasing effector function. A preferred form of engineered glycoforms is afucosylation, which has been shown to be associated with an increase in ADCC function, which may be achieved via a tighter binding to the FcγRIIIa receptor. In this case, "afucosylation" means that most antibodies produced in host cells are substantially free of afucosylation, e.g., 90-95-98% of the antibodies produced do not have a significant amount of afucosylation as a component of the carbohydrate portion of the antibody (generally attached to N297 in the Fc region). Functionally defined afucosylated antibodies generally exhibit at least 50% or higher affinity to the FcγRIIIa receptor.

[0130] Modified glycoforms can be produced by various methods known in the art ( et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; US6,602,684; USSN 10 / 277,370; USSN 10 / 113,929; PCT WO 00 / 61739A1; PCT WO 01 / 29246A1; PCT WO 02 / 31140A1; PCT WO 02 / 30954A1, all of which are incorporated by reference in their entirety; ( Technology [Biowa, Inc., Princeton, NJ]; Glycoengineering technologies [Glycart Biotechnology AG, Zürich, Switzerland]). Many of these technologies are based on controlling the amount of fucosylated and / or bisecting oligosaccharides covalently linked to the Fc region, for example by expressing the IgG in various engineered or other organisms or cell lines (e.g., Lec-13 CHO cells or rat hybridoma YB2 / 0 cells), by regulating enzymes involved in the glycosylation pathway (e.g., FUT8 [α1,6-fucosyltransferase] and / or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifying carbohydrates after the IgG is expressed. For example, Seattle Genetics' "sugar engineered antibody" or "SEA technology" works by adding modified sugars that inhibit fucosylation during production; see, e.g., 20090317869, the entire contents of which are incorporated herein by reference. Engineered glycoforms generally refer to different carbohydrates or oligosaccharides; thus antibodies may include engineered glycoforms.

[0131] Alternatively, engineered glycoforms may refer to IgG variants comprising different carbohydrates or oligosaccharides. As known in the art, the glycosylation pattern may depend on the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues discussed below) or the host cell or organism that produces the protein. Specific expression systems are discussed below.

[0132] Glycation of polypeptides is usually N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are discrimination sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of the tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0133] The addition of glycosylation sites to the antibody is preferably achieved by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may also be performed by adding one or more serine or threonine residues to the starting sequence or by replacing one or more serine or threonine residues (for O-linked glycosylation sites). For convenience, the antibody amino acid sequence is preferably altered via changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at a preselected base so that codons that will be translated into the desired amino acids are generated.

[0134] Another means of increasing the number of carbohydrate moieties on the antibody is chemical or enzymatic coupling of glycosides to proteins. These procedures are advantageous in that they do not require the production of proteins with saccharification capacity in the host cell for N-linked or O-linked saccharification. Depending on the coupling method used, the sugar may be linked to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) the acylamino group of glutamic acid. Such methods are described in WO 87 / 05330 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both of which are incorporated by reference in their entirety.

[0135] Removal of carbohydrate moieties present on the starting antibody (e.g., post-translation) can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. The treatment results in the cleavage of most or all sugars other than the linked sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch.Biochem.Biophys.259:52 and Edge et al., 1981, Anal.Biochem.118:131, the entire contents of which are incorporated by reference. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using various endoglycosidases and exoglycosidases as described in Thotakura et al., 1987, Meth.Enzymol.138:350, the entire contents of which are incorporated by reference. Glycation at potential glycosylation sites can be prevented by using the compound tunicamycin as described in Duskin et al., 1982, J. Biol. Chem. 257: 3105. Tunicamycin blocks the formation of protein-N-glycosidic linkages.

[0136] Another type of covalent modification of antibodies comprises linking the antibody to various non-protein polymers, including but not limited to various polyols, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in a manner such as that mentioned in the 2005-2006 PEG Catalog of Nektar Therapeutics (available from the Nektar website), U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337, all of which are incorporated by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made at various positions within the antibody to facilitate the addition of polymers such as PEG. See, for example, U.S. Publication No. 2005 / 0114037A1, the entire contents of which are incorporated by reference.

[0137] Specific CDR and variable region embodiments

[0138] The present invention provides a number of antibodies each having a specific set of CDRs (including some amino acid substitutions as outlined above). As outlined above, antibodies can be defined by a set of 6 CDRs, by a variable region, or by a full-length heavy chain and light chain (including a constant region). In addition, as outlined above, amino acid substitutions can also be performed. In general, in the case of changes within the CDR, since the CDR has a relatively short length, the amino acid modification is usually described in terms of the number of amino acid modifications that can be performed. Although this is not applicable to discussing the number of amino acid modifications that can be introduced into the variable sequence, constant sequence, or full-length sequence, it is also appropriate to define the changes in terms of "identity %" in addition to the number of changes. Therefore, as described herein, the antibodies included in the present invention have 80%, 85%, 90%, 95%, %, 98% or 99% identity with the sequence identification numbers listed herein.

[0139] In the case of Ab79 antibody, the sets of CDRs are as follows: the three CDRs of the heavy chain encompass HCDR1 SEQ ID NO: 3 (HCDR1), SEQ ID NO: 4 (HCDR2) and SEQ ID NO: 5 (HCDR3), and the three CDRs of the light chain encompass SEQ ID NO: 6 (LCDR1), SEQ ID NO: 7 (LCDR2) and SEQ ID NO: 8 (LCDR3).

[0140] In the case of Abl9, the set of CDRs is as follows: HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14), and HCDR3 (SEQ ID NO: 15), and LCDR1 (SEQ ID NO: 16), LCDR2 (SEQ ID NO: 17), and LCDR3 (SEQ ID NO: 18).

[0141] Specifically excluded from the present invention are antibodies of SEQ ID NO: 24 and SEQ ID NO: 25 (heavy and light chains of Benchmark 1) and SEQ ID NO: 26 and SEQ ID NO: 27 (heavy and light chains of Benchmark 2). It should be noted that the antibodies may not cross-react with macaque CD38 discussed below.

[0142] The antibodies of the present invention may cross-react with human and macaque CD38 and are therefore species cross-reactive antibodies. A "species cross-reactive antibody" is an antibody that has a binding affinity to an antigen from a first mammalian species and a binding affinity to a homologue of the antigen from a second mammalian species that is almost the same. Species cross-reactivity can be expressed, for example, as the ratio of the KD of an antibody to an antigen of a first mammalian species to the KD of the same antibody to a homologue of the antigen from a second mammalian species, wherein the ratio is 1.1, 1.2, 1.3, 1.4, 1.5, 2, 5, 10, 15, up to 20. Alternatively or additionally, when an antibody is administered to a second species and exhibits therapeutic or diagnostic efficacy, it has "species cross-reactivity". Therefore, in the present case, the antibodies of the present invention may cross-react with macaque CD38, exhibit preclinical efficacy when administered to macaque primates, and therefore may be considered to have cross-reactivity.

[0143] In some embodiments, antibodies that compete with antibodies of the invention (e.g., Ab79 and / or Ab19) for binding to human CD38 and / or macaque CD38 are provided, but do not include BM1 or BM2. Competition between two or more anti-CD38 antibodies for binding to CD38 or a portion of CD38 can be determined by any suitable technique as known in the art.

[0144] Competition in the context of the present invention refers to any detectable significant reduction in the propensity of an antibody of the invention (e.g., Ab79 or Ab19) to bind to its specific binding partner (e.g., CD38) in the presence of a test compound. Typically, competition means ... At least about 10-100% reduction in binding of an antibody of the invention to CD38 in the presence of a competitor as measured by standard techniques for the assay. Thus, for example, it is possible to set criteria for competitiveness where an antibody is deemed sufficiently competitive after at least about 10% relative inhibition is detected, at least about 15% relative inhibition is detected, or at least about 20% relative inhibition is detected. In the case where the antigens belonging to the competing antibodies are located in close positions in the antigen, competition can be indicated as a relative inhibition of CD38 binding greater than about 40% (e.g., at least about 45% inhibition, such as at least about 50% inhibition, such as at least about 55% inhibition, such as at least about 60% inhibition, such as at least about 65% inhibition, such as at least about 70% inhibition, such as at least about 75% inhibition, such as at least about 80% inhibition, such as at least about 85% inhibition, such as at least about 90% inhibition, such as at least about 95% inhibition, or a higher level of relative inhibition).

[0145] In some cases, as discussed below in the context of diagnostic applications, one or more of the components of the competition binding assay are labeled.

[0146] Situations may also arise where there is competition between anti-CD38 antibodies directed against more than one CD38 epitope and / or portion of CD38, for example where the antibody binding properties to a specific region of CD38 are retained in its fragment, such as where a well-defined linear epitope is present in the various tested fragments or where a conformational epitope is presented in a sufficiently large CD38 fragment as well as in CD38.

[0147] Evaluation of competition generally involves evaluating relative inhibitory binding using an antibody of the invention, CD38 (human or macaque CD38 or both) and a test molecule. The test molecule may include any molecule, including other antibodies, small molecules, peptides, etc. The compounds are mixed in amounts sufficient to form a comparison that gives information about the selectivity and / or specificity of the molecule in question relative to other molecules present.

[0148] The amounts of test compounds, CD38, and antibodies of the invention may vary. For example, for ELISA evaluation, about 5-50 μg (e.g., about 10-50 μg, about 20-50 μg, about 5-20 μg, about 10-20 μg, etc.) of anti-CD38 antibody and / or CD38 target are needed to assess whether there is competition. The conditions should also be suitable for binding. Typically, physiological conditions or near-physiological conditions (e.g., a temperature of about 20-40° C., a pH of about 7-8, etc.) are suitable for anti-CD38 antibody:CD38 binding.

[0149] Competition is often indicated as a relative inhibition significantly greater than about 5% as determined by ELISA and / or FACS analysis. It may be desirable to set a higher relative inhibition cutoff as a criterion / determinant of the appropriate level of competition in a particular situation (e.g., when using competition analysis to select or screen for new antibodies designed with the intended function of blocking the binding of another peptide or molecule that binds to CD38 (e.g., a natural binding partner of CD38, such as CD31, also known as CD31 antigen, EndoCAM, GPIIA, PECAM-1, platelet / endothelial cell adhesion molecule, or a naturally occurring anti-CD38 antibody).

[0150] In some embodiments, the anti-CD38 antibodies of the invention specifically bind to one or more residues or regions in CD38 and also 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.

[0151] Generally, the absence of cross-reactivity means that there is less than about 5% relative competitive inhibition between the molecules when assessed by ELISA and / or FACS using sufficient amounts of the molecules under appropriate assay conditions.

[0152] Inhibition of CD38 activity

[0153] The disclosed antibodies can block ligand-receptor interactions or inhibit receptor component interactions. The anti-CD38 antibodies of the present invention can be "blocking" or "neutralizing" antibodies. "Neutralizing antibody" means an antibody that binds to CD38 resulting in inhibition of the biological activity of CD38, such as its ability to interact with ligands, enzymatic activity, signaling ability, and in particular its ability to cause activated lymphocytes. Inhibition of the biological activity of CD38 can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see Examples below).

[0154] "Inhibiting binding" or "blocking binding" (e.g., when referring to inhibition / blocking of binding of a CD38 binding partner to CD38) encompasses both partial and complete inhibition / blocking. Inhibition / blocking of binding of a CD38 binding partner to CD38 can reduce or alter the normal level or type of cellular signaling that occurs when the CD38 binding partner binds to CD38 in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of a CD38 binding partner to CD38 when contacted with an anti-CD38 antibody compared to when the ligand is not contacted with an anti-CD38 antibody, such as binding of a CD38 binding partner to CD38 is blocked by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.

[0155] The disclosed anti-CD38 antibodies can also inhibit cell growth. "Inhibition of growth" includes any measurable decrease in cell growth when contacted with an anti-CD38 antibody compared to the growth of the same cells when not contacted with an anti-CD38 antibody, such as an inhibition of growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0156] In some embodiments, the disclosed anti-CD38 antibodies are capable of depleting activated lymphocytes and plasma cells. "Depletion" in this context means a measurable decrease in serum levels of activated lymphocytes and / or plasma cells (e.g., tested in macaques) compared to untreated animals. In general, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% depletion can be seen. In addition, as shown in the following examples, a particular advantage exhibited by the antibodies of the present invention is the recoverability of the cells after administration; that is, as known for some treatments (e.g., treatments using anti-CD20 antibodies), cell depletion can last for a long period of time, causing unwanted side effects. As shown herein, the effect on activated lymphocytes and / or plasma cells is recoverable.

[0157] Method for preparing the antibody of the present invention

[0158] The present invention further provides methods for preparing the disclosed anti-CD38 antibodies. The methods encompass culturing host cells containing isolated nucleic acids encoding antibodies of the present invention. As will be appreciated by those skilled in the art, the culturing can be performed in various ways depending on the nature of the antibodies. In some embodiments, where the antibodies of the present invention are, for example, full-length conventional antibodies, the heavy chain variable regions and light chain variable regions are under conditions where the antibodies can be produced and the antibodies can be separated.

[0159] In general, nucleic acids encoding antibodies of the invention are provided. Such polynucleotides encode the variable and constant regions of each of the heavy and light chains, although other combinations are also contemplated by the invention according to the compositions described herein. The invention also contemplates oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to the polynucleotides.

[0160] The polynucleotides may be in the form of RNA or DNA. Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogs and synthetic DNA are within the scope of the present invention. DNA may be double-stranded or single-stranded, and if single-stranded, may be coding strands (sense strands) or non-coding strands (antisense strands). The coding sequence of the encoded polypeptide may be identical to the coding sequence provided herein or may be different coding sequences, and the sequence encodes the same polypeptide as the DNA provided herein due to the redundancy or degeneracy of the genetic code.

[0161] In some embodiments, the nucleic acid encoding the antibody of the present invention is incorporated into an expression vector, which may be an extrachromosomal vector or designed to be integrated into the genome of the host cell into which it is introduced. The expression vector may contain many appropriate regulatory sequences (including but not limited to transcription and translation control sequences, promoters, ribose binding sites, enhancers, replication origins, etc.) or other components (selection genes, etc.), which are all operably connected as well known in the art. In some cases, two nucleic acids are used and each is placed in a different expression vector (e.g., the heavy chain is in the first expression vector and the light chain is in the second expression vector), or it may be placed in the same expression vector. Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of host cells, the desired protein expression level, and the like.

[0162] In general, nucleic acid and / or expression can be introduced into a suitable host cell to create a recombinant host cell, and the introduction uses any method suitable for the selected host cell (e.g., transformation, transfection, electroporation, infection) so that the nucleic acid molecule is operably linked to one or more expression control elements (e.g., in a vector, in a construct created by each process in a cell, or integrated into the host cell genome). The resulting recombinant host cell can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide. In some cases, the heavy chain is produced in one cell and the light chain is produced in another cell.

[0163] Mammalian cell lines available as expression hosts are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) (Manassas, VA), including but not limited to Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and many other cell lines. Non-mammalian cells can also be used to express recombinant antibodies, including but not limited to bacteria, yeast, insect, and plant cells. In some embodiments, antibodies can be produced in transgenic animals such as cattle or chickens.

[0164] General methods for antibody molecular biology, expression, purification and screening are described in, e.g., Antibody Engineering, Kontermann & Dubel, eds., Springer, Heidelberg, 2001 and 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

[0165] Applications and Indications

[0166] The antibodies of the present invention can be used in various applications after being prepared, including the diagnosis of CD38-related diseases and the treatment thereof.

[0167] CD38-related conditions

[0168] In one aspect, the present invention provides methods for diagnosing and treating conditions associated with inflammatory and immune diseases, particularly diseases associated with activated lymphocytes. As shown herein, CD38 is expressed in immature hematopoietic cells, downregulated in mature cells, and re-expressed at high levels in activated lymphocytes and plasma cells. For example, high CD38 expression is seen in activated B cells, plasma cells, activated CD4+T cells, activated CD8+T cells, NK cells, NKT cells, mature dendritic cells (DCs), and activated monocytes.

[0169] The therapeutic anti-CD38 antibodies of the invention bind to CD38-positive cells, thereby leading to the depletion of such cells, such as activated lymphocytes, via a variety of mechanisms of action including CDC and ADCC pathways.

[0170] Thus, any autoimmune disease that exhibits increased expression of CD38 or an increased number of cells expressing CD38 can be treated using the antibodies of the invention. Such diseases include, but are not limited to, allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies (ANCA), adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac dermatitis, spruce-dermatitis, chronic fatigue immune disfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease, Guillain-Barre syndrome, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditisthyroiditis, hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki's disease, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobinulinemia, primary biliary cirrhosis, psoriasis, psoriasis arthritis, Reynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjorgen's syndrome, solid organ transplant rejection, stiff-man syndrome, systemic lupus erythematosus, takayasuarteritis, temporal arteristis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegner's granulomatosis.

[0171] Of particular use in some embodiments is the use of the antibodies of the invention for 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.

[0172] Thus, for example, patients with high plasma cell content can be selected, such as SLE patients who exhibit high plasma cells, and RA patients who have been shown to be unresponsive to CD20-based therapy.

[0173] In one aspect, the invention provides a method of treating a condition associated with proliferation of cells expressing CD38, comprising administering to a patient a pharmaceutically effective amount of a disclosed antibody. In certain embodiments, the condition is cancer, and in specific embodiments, the cancer is a hematological cancer. In other specific embodiments. The condition is multiple myeloma, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-cell leukemia, or Burkitt's lymphoma.

[0174] It is known in the art that 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 of the percentage of cells in a given population labeled by an antibody that specifically binds to CD38, or immunohistochemistry, as generally described below for diagnostic applications. For example, a cell population in which CD38 expression is detected in about 10-30% of cells can be considered weakly positive for CD38; and a cell population in which CD38 expression is detected in more than about 30% of cells can be considered clearly positive for CD38 (as in 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 expression density on the cell surface can be determined using methods known in the art, such as, for example, flow cytometric measurement of the mean fluorescence intensity of cells that have been fluorescently labeled using an antibody that specifically binds CD38.

[0175] In some embodiments, the compositions and methods of the invention are applied to cancers, such as "hematological cancers," a term referring to malignant neoplasms of blood-forming tissues and encompassing leukemias, lymphomas, and multiple myeloma. Non-limiting examples of conditions associated with CD38 expression include, but are not limited to, multiple myeloma (Jackson et al. (1988), Clin. Exp. Immunol. 72:351-356), B chronic lymphocytic leukemia (B-CLL) Dürig et al. (2002), Leukemia 16:30-5; Morabito et al. (2001), Leukemia Research 25:927-32; Marinov et al. (1993), Neoplasma 40(6):355-8; and Jelinek et al. (2001), Br. J. Haematol. 115:854-61), acute lymphoblastic leukemia (Keyhani et al. (1999), Leukemia Research 24:153-9; and Marinov et al. (1993), Neoplasma 40(6):355-8), chronic myeloid leukemia (Marinov et al. (1993), Neoplasma 40(6):355-8), acute myeloid leukemia (Keyhani et al. (1999), Leukemia Research 24:153-9), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia or chronic myelogenous leukemia (CML), acute myeloid leukemia or acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), hairy cell leukemia (HCL), myeloid dysplasia syndrome (MDS) or chronic myeloid leukemia in blast phase (CML-BP), and all subtypes of the said leukemias defined by morphological, histochemical and immunological techniques well known to those skilled in the art.

[0176] "Neoplasm" or "neoplastic condition" refers to a condition associated with cellular proliferation characterized by loss of normal control resulting in one or more symptoms including unregulated growth, lack of differentiation, local tissue invasion, and metastasis.

[0177] In some embodiments of the invention, the hematological cancer is selected from the group of chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).

[0178] Furthermore, it is known in the art that CD38 expression is a prognostic indicator for patients with conditions such as, for example, B chronic lymphocytic leukemia (Dürig et al. (2002), Leukemia 16:30-5; and Morabito et al. (2001), Leukemia Research 25:927-32) and acute myeloid leukemia (Keyhani et al. (1999), Leukemia Research 24:153-9).

[0179] CLL is the most common leukemia in adults in the Western world. CLL involves clonal expansion of mature lymphocytes involving lymph nodes and other lymphoid tissues, with progressive infiltration of the bone marrow and presence in peripheral blood. The B-cell form (B-CLL) represents almost all cases.

[0180] B-CLL

[0181] B-CLL is an incurable disease characterized by a progressive increase in anergic monoclonal B-lineage cells that accumulate in the bone marrow and peripheral blood for many years in a persistent manner. The expression of CD38 is considered an independent poor prognostic factor for B-CLL. Hamblin et al., Blood 99: 1023-9 (2002).

[0182] Today's standard therapy for B-CLL is palliative and is primarily performed with the cytostatic chlorambucil or fludarabine. When relapse occurs, a combination therapy using fludarabine, cyclophosphamide in combination with rituximab (a monoclonal antibody to CD20) or campath (a monoclonal antibody to CD52) is often initiated. Therefore, there is a critical unmet medical need for the treatment of B-CLL. In some embodiments, methods of treating B-CLL using the disclosed anti-CD38 antibodies are provided (as outlined below, the treatment may be performed using a combination therapy that optionally and independently includes any of the above-mentioned drugs).

[0183] B-CLL is characterized by two subtypes, i.e., indolent and aggressive. The clinical phenotype is related to the presence or absence of somatic mutations in immunoglobulin heavy chain variable region (IgVH) genes. As used herein, indolent B-CLL refers to a disease in a subject with a mutated IgVH gene and / or presenting one or more clinical phenotypes associated with indolent B-CLL. As used herein, the phrase aggressive B-CLL refers to a disease in a subject with unmutated IgVH and / or presenting one or more clinical phenotypes associated with aggressive B-CLL.

[0184] Multiple Myeloma

[0185] Multiple myeloma is a malignant disease of the B cell spectrum characterized by the neoplastic proliferation of plasma cells in the bone marrow. Current treatment regimens exhibit moderate response rates. However, only marginal changes are observed throughout the survival period and the median survival period is approximately 3 years. Therefore, there is a critical unmet medical need for the treatment of multiple myeloma. In some embodiments, methods of treating multiple myeloma using the disclosed antibodies are provided.

[0186] CD38 is highly expressed on plasma cells, which are terminally differentiated into B cells.

[0187] The proliferation of myeloma cells causes a variety of effects, including lytic lesions (holes) in bones, a decrease in the number of red blood cells, the production of abnormal proteins (with damage to the kidneys, nerves, and other organs), a weakened immune system, and elevated blood calcium levels (hypercalcemia).

[0188] Current treatment options include chemotherapy, preferably combined with autologous stem cell transplantation (ASCT) when possible.

[0189] Monoclonal gammaglobulinopathy of undetermined significance and smoldering multiple myeloma

[0190] In some embodiments, methods of using the disclosed antibodies to treat monoclonal gamma-immunoglobulinopathy are provided. In other embodiments, methods of using the disclosed antibodies to treat smoldering multiple myeloma are provided.

[0191] Monoclonal gammaglobulinopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are asymptomatic, pre-malignant disorders characterized by the proliferation of monoclonal plasma cells in the bone marrow and the absence of end-organ damage.

[0192] Smoldering multiple myeloma (SMM) is an asymptomatic proliferative disorder of plasma cells with a high risk of progression to symptomatic or active multiple myeloma (N. Engl. J. Med. 356(25):2582-2590 (2007)).

[0193] In 2003, international consensus criteria for defining SMM were adopted, which require patients to have an M-protein content of >30 g / L and / or >10% of bone marrow clonal plasma cells (Br. J. Haematol. 121:749-57 (2003)). Patients must have no organ or related tissue damage, including bone lesions or symptoms (Br. J. Haematol. 121:749-57 (2003)).

[0194] Recent studies have identified two subgroups of SMM; i) patients with progressive disease and ii) patients with non-progressive disease (Br. J. Haematol. 121: 631-636 (2003)). International consensus criteria defining MGUS require patients to have an M-protein content of <30 g / L, <10% bone marrow plasma cells, and the absence of organ or related tissue damage, including bone lesions or symptoms (Br. J. Haematol. 121: 749-57 (2003)).

[0195] SMM is similar to monoclonal gammaglobulinopathy of undetermined significance (MGUS) in the absence of end-organ damage (N. Engl. J. Med. 356 (25): 2582-2590 (2007)). However, clinically, SMM is likely to progress to symptomatic multiple myeloma or amyloidosis within 20 years (78% probability for SMM vs. 21% for MGUS) (N. Engl. J. Med. 356 (25): 2582-2590 (2007)).

[0196] Antibody compositions for in vivo administration

[0197] The preparation of the antibody used according to the present invention is prepared by mixing the antibody having the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed.

[1980] ) into a lyophilized preparation or an aqueous solution for storage. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, chloride), phenol, butyl or benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium ions; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0198] The formulations herein may also contain more than one active compound required for the specific indication being treated, preferably compounds having complementary activities and not adversely affecting each other. For example, it may be necessary to provide antibodies with other specificities. Alternatively or in addition, the composition may contain cytotoxic agents, cytokines, growth inhibitors and / or small molecule antagonists. Such molecules are appropriately present in combination in an amount effective for the intended purpose.

[0199] The active ingredient may also be enclosed in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0200] Preparations to be administered in vivo should be sterile or nearly sterile. This can be easily achieved by filtration through sterile filtration membranes.

[0201] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-co-glycolic acid and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-co-glycolic acid can release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time.

[0202] When encapsulated antibodies remain in the body for a long time, they may denature or aggregate due to exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be designed to achieve stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be the formation of intramolecular S--S bonds via sulfo-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0203] Application Mode

[0204] The antibodies and chemotherapeutic agents of the invention are administered to a subject according to known methods, such as intravenous administration as a bolus or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred.

[0205] Treatment modality

[0206] In the methods of the present invention, therapy is used to provide a positive therapeutic response to a disease or condition. A "positive therapeutic response" means an improvement in a disease or condition, and / or an improvement in symptoms associated with the disease or condition. For example, a positive therapeutic response would refer to one or more of the following improvements in a disease: (1) a decrease in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (5) inhibition of tumor growth (that is, slowing to some extent, preferably stopping); (6) an increase in patient survival; and (7) some relief from one or more symptoms associated with the disease or condition.

[0207] A positive treatment response for any given disease or condition can be determined by standardized response criteria specific for that disease or condition. Screening techniques such as magnetic resonance imaging (MRI) scans, x-ray imaging, computed tomography (CT) scans, bone scan imaging, endoscopy, and tumor biopsy sampling, including bone marrow aspiration (BMA) and enumeration of circulating tumor cells, can be used.

[0208] In addition to such positive therapeutic responses, subjects receiving therapy may experience the beneficial effect of amelioration of symptoms associated with the disease.

[0209] Thus, for B cell tumors, subjects may experience a reduction in so-called B symptoms, that is, night sweats, fever, weight loss, and / or urticaria. For pre-malignant conditions, therapy with anti-CD38 therapeutics may block the development of and / or prolong the time prior to the development of the relevant malignant condition, such as the development of multiple myeloma in subjects suffering from monoclonal gammaglobulinopathy of undetermined significance (MGUS).

[0210] Improvement of disease can be characterized as a complete response. "Complete response" means the absence of clinically detectable disease and normalization of any previously abnormal radiographic studies, bone marrow and cerebrospinal fluid (CSF) or abnormal monoclonal proteins in the case of myeloma.

[0211] The response may last for at least 4 to 8 weeks or sometimes 6 to 8 weeks after treatment according to the methods of the invention. Alternatively, the improvement of the disease may be classified as a partial response. A "partial response" means that all measurable tumor burden (that is, the number of malignant tumor cells present in the subject, or the volume of the tumor mass or the amount of abnormal monoclonal protein measured) is reduced by at least about 50% in the absence of new lesions, which may last for 4 to 8 weeks or 6 to 8 weeks.

[0212] Treatments according to the invention include a "therapeutically effective amount" of the agent used. A "therapeutically effective amount" refers to an amount effective, at dosages and durations necessary, to achieve the desired therapeutic result.

[0213] A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and 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.

[0214] A "therapeutically effective amount" for tumor treatment can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated in an animal model system that predicts efficacy in human tumors.

[0215] Alternatively, the properties of the composition can be evaluated using in vitro assays known to skilled practitioners to detect the ability of a compound to inhibit cell growth or induce apoptosis. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise improve symptoms in a subject. One skilled in the art will be able to determine such amounts based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0216] The dosage regimen is adjusted to provide the optimal desired response (e.g., a 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 severity of the therapeutic situation. For ease of administration and uniformity of dosage, parenteral compositions may be formulated in dosage unit form. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

[0217] The specification for the dosage unit forms of the 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 an active compound for the treatment of sensitivity in individuals.

[0218] The effective dosage and dosage regimen of the anti-CD38 antibodies used in the present invention depends on the disease or condition to be treated and can be determined by one skilled in the art.

[0219] An exemplary, non-limiting range of a therapeutically effective amount of an anti-CD38 antibody used in the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, for example about 0.5, about 0.3, about 1 or about 3 mg / kg. In another embodiment, the antibody is administered at a dose of 1 mg / kg or more, such as a dose of 1 to 20 mg / kg, for example a dose of 5 to 20 mg / kg, for example a dose of 8 mg / kg.

[0220] A medical professional of skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can initiate the dosage of the agent employed in the pharmaceutical composition at a level below the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0221] In one embodiment, the anti-CD38 antibody is administered by infusion at a weekly dose of 10 to 500 mg / kg, such as 200 to 400 mg / kg. This administration can be repeated, for example, 1 to 8 times, such as 3 to 5 times. The administration can be performed by continuous infusion for 2 to 24 hours, such as 2 to 12 hours.

[0222] In one embodiment, the anti-CD38 antibody is administered as needed by slow continuous infusion over a prolonged period of time, such as greater than 24 hours, to reduce side effects including toxicity.

[0223] In one embodiment, the anti-CD38 antibody is administered up to 8 times, such as 4 to 6 times, at a weekly dose of 250 mg to 2000 mg, such as (for example) 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg. The administration can be performed by continuous infusion for 2 to 24 hours, such as 2 to 12 hours. The regimen can be repeated one or more times as needed, for example after 6 months or 12 months. The dosage can be determined or adjusted by measuring the amount of the compound of the invention in the blood after administration, for example, by taking out a biological sample and using an anti-idiotypic antibody targeting the antigen binding region of the anti-CD38 antibody.

[0224] In yet another embodiment, the anti-CD38 antibody is administered once a week for 2 to 12 weeks, such as 3 to 10 weeks, such as 4 to 8 weeks.

[0225] In one embodiment, the anti-CD38 antibody is administered by maintenance therapy, such as, for example, once a week for 6 months or more.

[0226] In one embodiment, the anti-CD38 antibody is administered by a regimen comprising one infusion of the anti-CD38 antibody followed by an infusion of the anti-CD38 antibody conjugated to a radioisotope. The regimen may be repeated, for example, 7 to 9 days later.

[0227] As a non-limiting example, treatment according to the present invention can be performed in an amount of about 0.1-100 mg / kg per day, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day as a daily dose of the antibody, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0228] In some embodiments, the anti-CD38 antibody molecules thereof are used in combination with one or more other therapeutic agents, such as chemotherapeutic agents. Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and its analogs or metabolites, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine); The invention relates to drugs that can be used to treat type 2 diabetes, for example, metronidazole, metronidazole, metronidazole, metronidazole, metronidazole, and metronidazole); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators, such as bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

[0229] 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 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate); mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κB inhibitors, including IκB kinase inhibitors; antibodies that bind to proteins overexpressed in cancer and thereby downregulate cell replication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab); and other inhibitors of proteins or enzymes known to be upregulated, overexpressed, or activated in cancer that inhibit downregulation of cell replication.

[0230] In some embodiments, the use of the antibodies of the invention can be in before, concurrently with, or after (bortezomib).

[0231] Diagnostic Uses

[0232] The anti-CD38 antibodies provided can also be used for in vitro or in vivo imaging of tumors or autoimmune disease states associated with CD38. In some embodiments, the antibodies described herein are used for both diagnosis and treatment, or are used alone for diagnosis. When anti-CD38 antibodies are used for both diagnosis and treatment, some embodiments rely on two different anti-CD38 antibodies for two different antigenic epitopes so that the diagnostic antibody does not compete with the therapeutic antibody for binding, but in some cases the same antibody can be used for both. For example, in some cases, the Ab19 antibody is used for diagnosis (generally labeled as discussed below) and Ab79 is used for treatment, or conversely the Ab19 antibody is used for treatment and Ab79 is used for diagnosis. Therefore, the present invention includes a composition comprising a diagnostic antibody and a therapeutic antibody, and in some embodiments, the diagnostic antibody is labeled as described herein. In addition, the composition of the therapeutic and diagnostic antibodies can also be co-administered with other drugs as outlined herein.

[0233] In many embodiments, the diagnostic antibody is labeled. "Label" herein means that one or more elements, isotopes or chemical compounds are attached to the antibody disclosed herein to achieve detection in a screening or diagnostic procedure. In general, labels are divided into several categories: a) immunolabeling, which can be an antigenic epitope incorporated as a fusion partner recognized by the antibody, b) isotopic labeling, which can be a radioactive or heavy isotope, c) small molecule labeling, which can include fluorescent and colorimetric dyes, or molecules such as biotin that can perform other labeling methods, and d) labels such as particles (including bubbles for ultrasound labeling) or paramagnetic labels that allow body imaging. As known in the art, labels can be incorporated into any position in the antibody and can be incorporated in vitro or in vivo during protein expression.

[0234] Diagnosis can be performed in vivo by administering diagnostic antibodies that allow whole body imaging as described below, or in vitro on a sample removed from a patient. A "sample" in this context includes many things, including but not limited to body fluids (including but not limited to blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen) and tissue samples such as biopsies obtained from relevant tissues.

[0235] In some embodiments, in vivo imaging is performed, including but not limited to ultrasound, CT scans, X-rays, MRI, and PET scans, as well as optical techniques such as the use of optical markers of tumors near the surface of the body.

[0236] In vivo imaging of diseases associated with CD38 can be performed by any suitable technique. For example, 99The anti-CD38 antibody may be labeled with a Tc label or with another beta-emitting isotope. Variations on the technique may include the use of magnetic resonance imaging (MRI) to improve imaging relative to gamma camera techniques. Similar immunoscintigraphy methods and principles are described, for example, in Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum Press 1988), Chase, "Medical Applications of Radioisotopes," in Remington's Pharmaceutical Sciences, 18th ed., Gennaro et al., (eds.), pp. 624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies," in Biotechnology And Pharmacy 227-49, Pezzuto et al., (eds.) (Chapman & Hall 1993).

[0237] In one embodiment, the invention provides an in vivo imaging method in which an anti-CD38 antibody is conjugated to a detection facilitating agent, the conjugated antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled antibody in the host is determined. Via the described techniques and any other diagnostic methods provided herein, the invention provides methods for screening for the presence of disease-associated cells in a biological sample in or taken from a human patient.

[0238] For diagnostic imaging, the radioisotope can be directly or indirectly conjugated to the anti-CD38 antibody by using an intermediate functional group. Useful intermediate functional groups include chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, e.g., U.S. Pat. No. 5,057,313). In such diagnostic assays involving anti-CD38 antibodies conjugated to radioisotopes, the dose of the conjugated anti-CD38 antibody delivered to the patient is usually maintained at as low a level as possible via the choice of isotope to achieve the best combination of minimum half-life, minimum residence time in vivo, and minimum isotope amount that will allow detection and accurate measurement.

[0239] In addition to radioisotopes and radio-opaque agents, diagnostic methods can also be performed using anti-CD38 antibodies conjugated to dyes (e.g., biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., U.S. Pat. No. 6,331,175, which describes MRI techniques and methods for preparing antibodies conjugated to MRI enhancing agents). Such diagnostic / detection agents can be selected from agents used in magnetic resonance imaging and fluorescent compounds.

[0240] In order to load the anti-CD38 antibody with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a long tail to which are attached a plurality of chelating groups for binding the ions. This tail may be a polymer such as polylysine, a polysaccharide, or other derivatizable or derivatizable chain having side groups to which chelating groups may be attached, such as, for example, porphyrins, polyamines, crown ethers, bithiosemicarbazones, polyoximes, and similar groups known to be useful for the purpose.

[0241] Chelating agents can be coupled to the anti-CD38 antibody using standard chemistry. Chelating agents are typically linked to the anti-CD38 antibody via a group that is capable of forming a bond with the molecule with minimal loss of immunoreactivity and with minimal aggregation and / or internal cross-linking.

[0242] Examples of potentially useful metal-chelator combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which for radioimaging are used with diagnostic isotopes in the overall energy range of 60 to 4,000 keV, such as 125 I. 123 I. 124 I. 62 Cu, 64 Cu, 18 F. 111 In, 67 Ga, 99 Tc, 94 Tc, 11 C. 13 N. 5 O and 76 Br.

[0243] Labels include radionuclides, radiocontrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photoactive agents. Such diagnostic agents are well known and any such known diagnostic agents may be used. Non-limiting examples of diagnostic agents may include radionuclides, such as 110 In, 111 In,177 Lu, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 90 Y. 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I. 123 I. 124 I. 125 I. 131 I. 154- 158 Gd, 32 P. 11 C. 13 N. 15 O. 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co. 72 As, 75 Br, 76 Br, 82 mRb 83 Sr, or other gamma-, beta- or positron emitters.

[0244] Useful paramagnetic ions may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), or erbium (III). Metal contrast agents may include lanthanum (III), gold (III), lead (II), or bismuth (III).

[0245] The ultrasound contrast agent may comprise liposomes, such as gas-filled liposomes. The radiopaque diagnostic agent may be selected from a compound, a barium compound, a gallium compound, and a thallium compound.

[0246] These and similar chelators, when complexed with non-radioactive metals such as manganese, iron, and gadolinium, are useful in MRI diagnostics in conjunction with anti-CD38 antibodies. Macrocyclic chelators such as NOTA, DOTA, and TETA can be used with a variety of metals and radioactive metals, most notably with the radionuclides gallium, yttrium, and copper, respectively. Such metal-chelator complexes can be made very stable by tailoring the ring size to suit the metal of interest. Other ring-type chelators such as macrocyclic polyethers are known for their ability to stabilize binding to radionuclides such as cyclohexane. 223 Ra has attracted attention as a radionuclide and can also be used in diagnostic methods.

[0247] Thus, the present invention provides diagnostic anti-CD38 antibody conjugates, wherein the anti-CD38 antibody conjugate is conjugated to a contrast agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound contrast enhancement agent) or a radionuclide which may be, for example, an isotope emitting gamma, beta, alpha, Auger electrons, or positrons.

[0248] Anti-CD38 antibodies may also be useful, for example, in detecting the expression of an antigen of interest in a particular cell, tissue, or serum. For diagnostic applications, the antibody is typically labeled with a detectable moiety for in vitro assays. As will be appreciated by those skilled in the art, there are a wide variety of suitable labels for use in in vitro testing. Dyes suitable for use in this aspect of the invention include, but are not limited to, fluorescent lanthanide complexes, including fluorescent complexes of europium and terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, quantum dots (also known as "nanocrystals"; see U.S. Pat. No. 09 / 315,584, which is incorporated herein by reference), pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., Texas Red, Cy dyes (Cy3, Cy5, etc.), alexa dyes (including Alexa, phycoerythrin, bodipy, and other dyes described in Molecular Probes Handbook, 6th edition, edited by Richard P. Haugland, which is specifically incorporated herein by reference.

[0249] The stained tissue can then be assessed for radioactivity counts as an indication of the amount of CD38-related peptide in the tumor. Images obtained using such techniques can be used to assess the biodistribution of CD38 in a patient, mammal, or tissue, for example in the case of using CD38 as a biomarker for the presence of invasive cancer cells.

[0250] Products

[0251] In other embodiments, a product containing materials suitable for treating the above-mentioned diseases is provided. The product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed of various materials such as glass or plastic. The container stores a composition effective for treating the condition and can have a sterile access port (for example, the container can be an intravenous bag or vial with a stopper that can be punctured by a hypodermic injection needle). The active agent in the composition is an antibody. The label on or associated with the container indicates that the composition is used to treat the selected condition. The product may further include a second container, which contains a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, screening procedures, needles, syringes and a drug package insert with instructions for use. Example

[0252] The following examples are provided to illustrate but not to limit the present invention.

[0253] Example 1: Construction of expression vectors containing polynucleotides encoding human, macaque and mouse CD38

[0254] To construct a vector expressing human CD38 (huCD38), the The polynucleotide encoding huCD38 was isolated from human cDNA. The isolated huCD38 was cloned into a strain containing neomycin resistance (neo R ) gene in a stable expression vector (XOMA, Inc.), which allows selection of G418 (Geneticin) resistant transfectants. The huCD38 gene present in the selected transfectants was sequenced to identify any sequence errors. Errors in the sequence derived from GenBank Accession No. NM_001775 were corrected by PCR site-directed mutagenesis. The final vector DNA was confirmed by 5' sequencing.

[0255] To construct a vector expressing macaque CD38 (cyCD38), a polynucleotide encoding cyCD38 was isolated from DNA obtained from normal spleen tissue of cDNA-monkey (Macaca mulatta) of Biochain Institute's. The isolated cyCD38 was cloned into a culture medium containing neo R The cyCD38 gene was expressed in a stable expression vector (XOMA, Inc.) that allows selection of G418 (Geneticin) resistant transfectants. The cyCD38 gene present in the selected transfectants was sequenced to identify any sequence errors. Errors in the sequence derived from GeneBank Accession No. AY555148 were corrected by PCR site-directed mutagenesis. The final vector DNA was confirmed by sequencing.

[0256] To construct a vector expressing mouse CD38 (moCD38), the polynucleotide encoding moCD38 was isolated from DNA obtained from Origene's TrueORF collection. The isolated moCD38 was cloned into a culture medium containing neo R The moCD38 gene was expressed in a stable expression vector (XOMA, Inc.) that allows selection of G418 (Geneticin) resistant transfectants. The moCD38 gene present in the selected transfectants was sequenced to identify any sequence errors. Errors in the sequence derived from GenBank Accession No. NM_007646 were corrected by PCR site-directed mutagenesis. The final vector DNA was confirmed by sequencing.

[0257] Example 2: Development of Chinese Hamster Ovary (CHO) cells expressing CD38

[0258] To develop CHO cells expressing huCD38, muCD38 and cyCD38, CHO cells were transfected with linearized DNA. After one week of selection, cells were sorted by flow cytometry, and cells expressing the highest huCD38, muCD38 or cyCD38 (top 15%) were seeded in 96-well plates to produce single colonies. The remaining cells were also seeded under selection to produce spare colonies. After about 12-14 days of inoculation, single colonies were identified and transferred to 96 deep-well plates. Clones were screened by FACS analysis after the second passage. The top-producing clones were passaged and expanded to shake flasks. Freeze and / or culture the top 2 clones for mycoplasma AVA testing and scale-up.

[0259] To construct a luciferase reporter gene for the disseminated xenograft model, a commercial vector containing a CMV promoter luciferase gene / neomycin selectable marker (Promega, Madison, WI) was used to generate stable transfections in Daudi Burkitt's lymphoma cells.

[0260] Example 3: Phage display library and screening of agents that bind CD38

[0261] Target-specific antibodies were selected from phage display libraries according to the method described by Marks et al. (2004, Methods Mol. Biol. 248: 161-76). Briefly, the phage display library was incubated with 100 pmol of biotinylated CD38 at room temperature for 1 hour, followed by 100 μL of streptavidin bead suspension ( The formed complex was captured by ELISA (M-280 Streptavidin, Invitrogen). The beads were washed with washing buffer (5% milk in PBS) to remove non-specific phage. The bound phage was eluted with 0.5 ml of 100 nM triethylamine (TEA) and immediately neutralized by adding an equal volume of 1 M TRIS-CI (pH 7.4). The eluted phage pool was used to infect TG1 E. coli cells grown in the logarithmic growth phase, and the phagemid was rescued as described in Marks et al. above. A total of three rounds of selection were repeated.

[0262] Alternatively, phage display libraries were panned against fixed CD38 (R&D systems) to identify groups of antibody fragments capable of binding to CD38. Panning was performed using standard protocols (see, e.g., Methods in Molecular Biology, Vol. 178: Antibody Phage Display: Methods and Protocols Edited by: PMO'Brien and R.Aitken, Humana Press; "Panning of Antibody Phage-Display Libraries," Coomber, DWJ, pp. 133-145, and "election of Antibodies Against Biotinylated Antigens," Chames et al., pp. 147-157). Briefly, phage display libraries were panned against fixed CD38 (R&D systems) to identify groups of antibody fragments capable of binding to CD38. Panning was performed using standard protocols (see, e.g., Methods in Molecular Biology, Vol. 178: Antibody Phage Display: Methods and Protocols Edited by: PMO'Brien and R.Aitken, Humana Press; "Panning of Antibody Phage-Display Libraries," Coomber, DWJ, pp. 133-145, and "election of Antibodies Against Biotinylated Antigens," Chames et al., pp. 147-157). Three wells of the MAXISORP plate were coated with 50 μL recombinant CD38 (R&D Systems) at a concentration of 10 μg / ml in PBS. After overnight incubation at 4°C, free binding sites were blocked with 5% milk in PBS for one hour at room temperature. Then about 200 μL of phage library in 5% milk / PBS was added to the blocked wells and incubated at room temperature for about one to two hours. The wells were washed and the bound phages were dissolved using standard methods (see, for example, Sam brook and Russell, Molecule Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001). The dissolved phages were amplified by infecting Escherichia coli TG 1 host cells in the logarithmic growth phase. The infected TG 1 cells were recovered by centrifugation at 2,500 RPM for five minutes, inoculated on 15 cm2 YT-ampicillin-2% glucose agar plates, and incubated overnight at 30°C. The panning process was then repeated using the amplified phages. The cycle of panning, elution, and amplification was repeated three times.

[0263] After panning, a single colony from the seeded TG1 cells was used to immunize the culture medium in a 96-well plate. The microculture was grown to an OD600 of 0.6, at which time 1 mM IPTG was added and incubated overnight at 30°C in a shaker to induce the expression of soluble scFv. The bacteria were pelleted by centrifugation, and the binding of the scFv to immobilized CD38 was tested using a standard ELISA assay and a FACS binding assay using periplasmic extracts.

[0264] For FACS binding screening, scFvs in periplasmic extracts (PPE) were screened for their ability to bind to naive, membrane-bound CD38 using CHO cells stably expressing CD38. Parental and CHO transfected strains (cell lines expressing human CD38 or macaque CD38 or mouse CD38) were each incubated at 2 × 10 6 Cells / ml were resuspended in PBS (Life Technologies), 0.5% BSA (Sigma-Aldrich) and 0,1% NaN3 (Sigma-Aldrich) (FACS buffer). Parental CHO cells that do not express CD38 were used as negative controls. 25 μL of cell aliquots were seeded in V-bottom 96-well plates (Costar Cat#3897) and 25 μL of periplasmic extract containing myc-tagged scFv antibody fragments were added to the cells, followed by incubation of the mixture at 4°C for 30 minutes. The cells were then washed twice, after which the pellet was resuspended in 25 μL of mouse anti-c-myc antibody (1 / 1000 in FACS buffer) (Roche) and incubated again at 4°C for 30 minutes. The cells were then washed twice and resuspended in 25 μL of 1 / 200 diluted anti-mouse IgG-PE antibody (Jackson labs) in FACS buffer and incubated again at 4°C for 30 minutes. The cells were then washed twice to remove excess unbound antibody, resuspended in 70 μL FACS buffer and cultured on BD Biosciences. The acquired data were evaluated using FlowJo software (TreeStar, Inc.). The median fluorescence intensity of CD38-transfected CHO cells was compared with that of the parental CHO cell line (CD38 - ) to identify positive samples.

[0265] Antibody clones that bind human CD38 were sequenced to identify unique clones. The off-rates determined by the analysis were used to rank unique scFV clones. 200 RU to 500 RU of human recombinant CD38 (R&D Systems' cat# 2404-AC or equivalent) were coupled to the scFV clones using standard amine coupling chemistry. Immobilize to CM5 or equivalent chip. Reference spots are also prepared, which can be activated and then blocked when the protein is not immobilized. This is done by diluting the antigen to 1-3 μg / ml in acetate buffer (pH 5.0) and injecting onto the activated surface until the desired content is fixed (3-5) minutes. The surface is then blocked with ethanolamine. The periplasmic extract is diluted one to one with the assay electrophoresis buffer 10mM HEPES, 150mM NaCl, 3mM EDTA (ethylenediaminetetraacetic acid) and 0.05% polysorbate 20 (pH 7.4) containing 2mg / mL BSA (bovine serum albumin). The diluted periplasmic extract is injected onto the surface plasmon resonance (SPR) surface at 30 minutes for 300 seconds, with an additional 900 seconds of dissociation time monitored. Regeneration is performed using a single 8 second injection of 100mM HCl. The data obtained from the reference spot is subtracted from the data obtained from the active surface and then a 1:1 dissociation model is used at The dissociation curves were fitted in T100 software.

[0266] The top-level scFV clones were transformed to IgG1 antibodies. The IgG1 reformatted clones were screened by repeated FACS binding to ensure that binding properties were retained and species cross-reactivity was evaluated using parental CHO cells and CHO cells expressing human, mouse and macaque CD38. The FACS characterization of IgG reformatted clones was performed as described above, but the steps consisting of adding anti-c-myc antibody and anti-mouse IgG-PE antibody were replaced by a single step of detecting the binding of full-length human IgG by adding anti-human IgG antibody (JacksonLabs) conjugated to phycoerythrin.

[0267] Example 4: In vitro cell-based assay of IgG engineered clones

[0268] Approximately 150 clones were transformed into human IgG1 antibodies and five antibodies (Ab19, Ab43, Ab72, Ab79, and Ab110) were fully evaluated using a series of assays as described below. The IgG transformations were compared in in vitro and in vivo assays with the expression of two antibodies, BMTK4-1 (also referred to as benchmark-1, BM-1, or BMTK-1) (SEQ ID NO: 24 and SEQ ID NO: 25; heavy and light chain variable regions) and BMTK4-2 (also referred to as benchmark-2, BM-2, or BMTK-2) (SEQ ID NO: 26 and 27; heavy and light chain variable regions), the amino acid sequences of which are derived from the sequences of the known anti-CD38 antibodies daratumumab (also referred to as HuMax-CD38, disclosed in International Publication No. WO 06 / 099875) and SAR650984 (disclosed in International Publication No. WO 08 / 047242), respectively. Palivizumab ) (MedImmune), a clinically approved antibody recognizing respiratory syncytial virus, served as a negative control for CD38 binding.

[0269] Example 5: Detection of Ab79 binding by immunofluorescence

[0270] Alexa Dye-labeled Ab79 was applied to frozen sections of normal human colorectal tissue, prostate, and lymph nodes. Dye-labeled palivizumab Served as a negative staining control. The staining pattern observed for Ab79 was consistent with that seen on normal human colorectal tissue, prostate, and lymph nodes using multiple commercially available anti-CD38 antibodies (data not shown).

[0271] Via Alexa Dye-labeled Ab79 was also applied to normal and multiple myeloma bone marrow samples (data not shown).Ab79 bound to approximately 10% of cells in normal bone marrow, while >90% of multiple myeloma bone marrow cells showed Ab79 binding in 4 of 4 samples tested.

[0272] Ab79 was also tested for its ability to bind to a variety of cell lines (MOLP-8, DAUDI, RPMI and MCF7). MOLP-8 (human multiple myeloma), DAUDI (lymphoblasts derived from patients with Burkitt's lymphoma) and RPMI (cell lines established from patients with chronic myelogenesis leukemia) cells all showed binding by Ab79. Breast cancer cell line MCF7 expressed a high degree of negativity for Ab79 binding (data not shown).

[0273] Conjugated Alexa Antibodies were stained on 8 μm cryostat frozen sections, which were fixed in an ethanol / acetone mixture for 5 minutes and then incubated with antibodies for 1 hour at room temperature in a humidity-controlled chamber. The sections were then washed, mounting medium containing DAPI (Vector Laboratories, cat#H1500) was added, and coverslips were applied.

[0274] Example 6: Evaluation of Ab79 expression in multiple myeloma (MM) and chronic lymphocytic leukemia (CLL)

[0275] Binding of Ab79 to bone marrow samples from multiple myeloma patients against CD138 + After cell enrichment, flow cytometry or CD138 + CD45 - / lo The gating of cells was analyzed. Ab79 was found to be expressed on >95% of cells from four of six multiple myeloma samples. The binding pattern of Ab79 showed high similarity to that of anti-CD38 antibodies used in clinical trials. In addition, Ab79 bound to cells from patients with chronic lymphocytic leukemia.

[0276] To measure the binding of Ab79 to MM and CLL by FACS, patient samples were processed within 24 hours. TM Peripheral blood mononuclear cells were isolated from 1% paraformaldehyde (Ab79) (GE Healthcare) according to the manufacturer's instructions. Expression analysis was performed using the following antibody panels and clones in parentheses. MM panel: Ab79-Alexa CD45-PerCP (2D1), CD138-APC (MI15). CLL group: Ab79-Alexa CD5-PE (UCHT2), CD45-PerCP (2D1), CD19-APC (SJ25C1). 5 μL of PE, PerCP, or APC-labeled antibodies or 10 μL of Alexa Fluor Labeled antibodies or isotype controls were added to each well or tube containing 100 μL of 0.2x106 PBMC or CD138-enriched cells from bone marrow aspirates. Samples were incubated for 30 minutes at room temperature, after which red blood cells were lysed using BD Pharmlyse according to the manufacturer's instructions. All samples were washed three times in FACS buffer. Samples were fixed in 1% paraformaldehyde and analyzed on a BD FACSCanto TM II or BD FACSCaliber TM On analysis.

[0277] Example 7: Anti-CD38 antibody-induced CDC assay

[0278] The ability of macaque cross-reactive clones to induce complement-dependent cytotoxicity (CDC) was tested. MOLP-8 cells were seeded in 50 μL complete medium (supplemented with 10% foetal calf serum RPMI) in black 96-well flat-bottom tissue culture plates at a density of 10,000 cells per well. 50 μL 2x anti-CD38 antibodies, control IgG antibodies or separate culture media were added to each well, and incubated at room temperature for 10 minutes. Depending on cell line, purified rabbit complement (cat#CL 3441Cedarlane Laboratories, Canada) of varying amounts (2-15 μL) was added to each well, except for control wells. After incubation at 37°C for one hour, the plate was allowed to reach room temperature, and 100 μL cell titration CytoTox Glo was added to each well. TM Reagent (Promega G7571 / G7573), shake the plate for 5 to 7 minutes at Luminescence was read on a (Perkin Elmer) luminescence plate reader. Conditions tested: cells alone; cells + complement; cells + IgG control + complement; cells + antibody + complement. %CDC was calculated using the following equation:

[0279] 100-(RLU T / RLU C )×100),

[0280] Where RLU T RLU is the relative luminescence unit of the test sample. C is the relative luminescence unit of the complement-only sample. Statistical analysis was performed using PRISM software. EC was determined from the curve of %CDC versus antibody concentration. 50 Values, as shown in Table 1.

[0281] Example 8: Anti-CD38 antibody induced ADCC assay

[0282] Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated using Daudi, MOLP-8, and RPMI-8226 cell lines as target cells. TM Separate and separate PBMC as effector cells. Dilute the sample 1:3 with 2% FBS in PBS. 15 mL Ficoll-Plaque TM (GE Healthcare) was gently layered under the 35 mL diluted sample and centrifuged at 1800 rpm (brake) for 25 minutes. The turbid interphase containing PBMCs was collected, washed 3 times in 2% FBS in PBS, and aliquots of 50×106 cells / mL per aliquot were frozen in 10% DMSO / FBS. When needed, frozen aliquots of PBMCs were thawed and cultured overnight at 2×106 / mL in 10% FBS / RPMI+5ng / mL recombinant human IL2 (R&D systems #202-IL).

[0283] For ADCC assays, all steps were performed in complete medium. 5000 target cells were seeded per well of a 96-well plate and 50 μL of 3x anti-CD38 antibody, control IgG, or medium alone was added, followed by 50 μL of human effector PBMCs at a ratio of between 1:25 and 1:50 target:effector (T:E) cells. The plate was briefly centrifuged at 800 rpm for approximately 30 seconds to bring all cells into close proximity. After 4 hours at 37°C, the plate was centrifuged at 1100 rpm for 5 minutes and 100 μL of supernatant was transferred to a white plate. 100 μL of CytoTox Glo was added to the supernatant. TM Reagent (Promega cat# G9292) and shake the plate at room temperature (RT) for 20-30 minutes. Luminescence was read on a (Perkin Elmer) luminescence plate reader and the specific lysis percentage was calculated using the following equation:

[0284] (RLU T / RLU E / T ) / (RLU L / RLU E / T )x 100

[0285] Where RLU T The relative luminescence unit (RLU) of the test sample E / T is the relative luminescence unit of a sample containing individual target cells and effector cells, and RLU Lis the relative luminescence unit of cells lysed with Triton X-100. Statistical analysis was performed using PRISM software. EC was determined from the curve of % specific lysis versus antibody concentration. 50 Values, as shown in Table 1.

[0286] Table 1 CDC, ADCC and agonist activities of IgG modified antibodies

[0287]

[0288]

[0289] Example 9: FACS determination of affinity

[0290] The MOLP-8 cells expressing CD38 are suspended in 1% FBS buffer with a viable cell concentration of about 2 million cells / mL. The monoclonal antibody (mAb) to be tested is serially diluted (2 times) between the wells on two 96-well plates in 1x PBS. The last well of each titration contains only buffer. Other PBS and cell suspensions are added to each well so that the final volume is 300~L / well and each well contains about 100,000 cells. mAb is listed below, as well as the corresponding mAb binding site final concentration (2X molecular concentration) range for titration:

[0291] Benchmark 1, [mAb] binding site = 50.8nM-49.7pM

[0292] Benchmark 2, [mAb] binding site = 49.5 nM-48.3 pM

[0293] Ab 43, [mAb] binding site = 49.3 nM-48.2 pM

[0294] Ab 110, [mAb] binding site = 204 nM - 49.9 pM

[0295] Ab 79, [mAb] binding sites = 103 nM - 25.3 pM

[0296] Ab 72, [mAb] binding sites = 103 nM - 25.2 pM

[0297] Ab 19, [mAb] binding sites = 100 nM - 12.2 pM.

[0298] The plate was placed on a plate shaker at 4°C for 5 hours, after which the plate was washed 3 times with 1X PBS at 4°C. 200 μl of 99 nM Cy5 goat anti-human IgG Fc specific polyclonal antibody (Jackson ImmunoResearchLaboratories, #109-175-008) was then added to each well and the plate was shaken for 30 minutes at 4°C. The plate was washed again with 1X PBS at 4°C twice, and then the FACSCanto was used. TM The mean fluorescence intensity (MFI) of 5000 events was recorded for each well containing a unique mAb binding site concentration using a II HTS flow cytometer. The KD was estimated by nonlinearly fitting the mean fluorescence intensity as a function of antibody binding site concentration using Scientist 3.0 software using the following equation:

[0299] F=p[(KD+LT+n(M))-{(KD+LT+n(M))2–4n(M)(LT)}1 / 2] / 2+B

[0300] Where F (mean fluorescence intensity), LT (total mAb binding site concentration), p (proportionality constant relating any fluorescence unit to bound mAb), M (cell concentration in molarity; 0.553 fM is based on 100,000 cells in 300 μl), n (number of receptors per well), B (background signal), and KD = equilibrium dissociation constant.

[0301] For each antibody titration curve, KD estimates were obtained when P, n, B, and KD were allowed to float freely in the nonlinear analysis. For a detailed derivation of the above equation, see Drake and Klakamp (2007), "A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants," J. Immunol. Methods 318: 157-62, which is incorporated herein by reference. Table 2 lists the KD obtained for all antibodies in order of decreasing affinity, and the 95% confidence interval for each fit in parentheses. Nonlinear curve fitting was performed using antibody binding site concentration (2X molecular concentration).

[0302] Example 10: Affinity Determination

[0303] Surface plasmon resonance (SPR) analysis was performed on Biacore TMThe affinity of IgG antibodies to the soluble CD38 extracellular domain (ectodomain; ECD) was determined on A100 at 22°C. Goat anti-human IgG polyclonal antibodies (Caltag H10500) were immobilized to the CM5 biosensor chip using standard amine coupling to spots 1, 2, 4 and 5 within all four flow cells of the chip. The immobilization content on each spot ranged from 5865RU to 6899RU. Human CD38 (Cat#2404-AC, Lot#PEH020812A) was obtained from R&D Systems. The stock concentration of CD38 was determined using the methods detailed in Pace et al. (1995) "How to measure and predict molar absorption coefficient of a protein" Protein Science 4(11):2411-23 and Pace and Grimsley (2004) "Spectrophotometric determination of protein concentration," in Current Protocols in Protein Science, Chapter 3: Section 3.1, the teachings of each reference being incorporated herein by reference.

[0304] The electrophoresis buffer was prepared by degassing HEPES buffered saline, 0.005% polysorbate 20 and adding filtered BSA to a final concentration of 100 μg / mL. All eight purified mAbs were diluted to approximately 2 μg / mL using electrophoresis buffer. Preliminary experiments estimated the amount of each mAb to be captured in order to maintain the surface capacity (R max ) is not more than about 100RU.For each mAb capture / antigen injection cycle, mAb is captured on spots 1 and 5 in each flow cell, wherein juxtaposed spots 2 and 4 serve as respective reference surfaces.Each diluted mAb is captured at a flow rate of 10 μL / min for 1 minute, followed by running electrophoresis buffer for three minutes to achieve surface stability.HuCD38 is injected for 120 seconds at a concentration range of 193.7nM-3.0nM (2x serial dilutions) on all four flow cells, followed by a 15-minute dissociation period.Samples are prepared in electrophoresis buffer and randomly injected in triplicate, wherein for dual reference, seven buffer injections are interspersed.Surface regeneration is achieved using two 20-second 10mM glycine (pH 1.7) pulses.

[0305] All sensorgram data were processed using Scrubber 2.0c software and were fitted to a 1:1 interaction model in Scrubber 2.0c. The resulting binding constants are shown in Table 2.

[0306] Table 2

[0307]

[0308] Example 11: Immunofluorescence internalization assay

[0309] Immunofluorescence was used to evaluate the internalization of anti-CD38 antibodies in MOLP-8 cells. MOLP-8 cells were harvested and incubated in RPMI-1640 at 4°C with 1 μg of directly conjugated Alexa Fluor 5 × 10 cells were stained with each anti-CD38 antibody at 488 6 The cells were washed in PBS containing 1% BSA and incubated at 4°C or 37°C for 1×10 6 cells for 3 or 6 hours at 4°C using 2 μg rabbit anti-Alexa Antibodies (Invitrogen) were used to quench surface staining for 30 min. Cells were washed, fixed in PBS containing 1% PFA, transferred to Microtest 96-well plates (BD Biosciences), and analyzed using FACSCanto TM II (BD Biosciences) flow cytometer or use Image Micro (Molecular Devices) was imaged at 20x magnification.

[0310] Example 12: By Reclassification of antigenic epitopes (Binning)

[0311] use The A100 instrument re-ranked two benchmark antibodies as well as Ab19 and Ab 79. Antibodies were first immobilized at high and low density on a CM5 wafer using NHS / EDC coupling chemistry. For each cycle of the epitope re-ranking experiment, CD38 was first injected on the surface. Similar to a sandwich assay in an ELISA format, a unique antibody (taken from the set of immobilized antibodies) was then injected on the surface containing the CD38 / antibody complex. A pulse of phosphoric acid was used to regenerate the surface at the end of each cycle. Data were collected at 22°C using HBS-P (10mM 10HEPES pH 7.4, 150mM NaCI, 0.005% P-20) supplemented with BSA. The resulting sensorgrams are shown in Figure 2. The A100Evaluation software package and the experimental version of Scrubber for the A100 dataset were processed using the "Epitope Mapping" module. Binary 4×4 matrices were generated for the above 4 mAbs using replicate data from two independent experiments, as shown in Table 3.

[0312] Table 3

[0313] Ab79 BM1 Ab19 BM2 Ab79 0 0 1 1 BM1 0 0 1 0 Ab19 1 1 0 0 BM2 1 0 0 0

[0314] Example 13: In vivo oncology analysis

[0315] The in vivo efficacy of Ab19 and Ab79 was tested in the disseminated Daudi-luciferase model of human lymphoma. Female CB.17 SCID mice from Taconic Laboratories, 6 to 8 weeks old, were injected intravenously with 1×10 6 Daudi-Luc tumor cells. On study day 7, mice were treated intraperitoneally with the following: palivizumab, Ab 79, Ab19, Benchmark 1, and Benchmark 2. Bioluminescent imaging was performed weekly starting on day 21 using the IVIS Xenogen system (Caliper Life Sciences) to monitor tumor burden. For imaging, animals were injected intraperitoneally with luciferase substrate (150 mg / kg) 10 minutes before imaging, then anesthetized under isoflurane and imaged.

[0316] Example 14: Inflammatory / immune disease correlation

[0317] Show that CD38 is sharply upregulated in the PBMC cells after activation. In the static PMBC from normal donors, less than 20% of static cells express CD38, and wherein the number of receptors is calculated to be about 10,000-20,000 per cell. Activated PMBC from normal donors again show that 60-75% of cells express CD38, and wherein the number of receptors is shown to be 110,000 to 160,000 per cell.

[0318] In addition, if Figure 5 As shown in , CD38 showed increased expression in PMBCs from SLE patients.

[0319] Overexpression of CD38 in samples was tested by immunohistological methods. 19 samples from SLE patients were analyzed and observations of increased CD38 expression on B and T memory cells and pDC were obtained. Analysis of 3 RA patients showed infiltration of CD38 plasma cells in the synovial tissue of all three patients, and increased staining of synovial tissue with Ab79. Analysis of 7 Crohn's disease patients and 6 ulcerative colitis patients showed infiltration of plasma cells expressing CD38 in the smooth muscle of the colon (two primary cell samples from patients were tested. It should be noted that the same number of normal patients were tested and the results were not shown.

[0320] Example 15: Depletion using anti-CD38 antibodies

[0321] Administration of Ab79 to macaques showed a significant reduction in lymphocytes, B and T cells, and NK cells. The antibody was administered via 30-minute intravenous infusion at a dose of Figure 6 , where data were collected at 24 hours. Similar data are shown for days 4, 7, 11 and 18.

[0322] However, the pK / pD data show the recovery of animals after dosing. Figure 7 As shown in , lymphocyte counts returned similar to control counts on days after a single intravenous 30-minute infusion dose, even after the highest dose, indicating that there was no problem with lymphocyte progenitor cells.

[0323] Example 16: Autoimmune disease model

[0324] Three different models were used.

[0325] The HuScid mouse model serves as a pseudo-gravt-v-host model and a model for anti-tetanus reaction, IgG depletion, and total survival. CB17 / HuSCID mice were injected with ASGM1 to deplete NK cells, followed by injection of human PBMCs one day later. Implantation was continued for 7-10 days, and serum was collected for human Ig randomization. One day later, mice were injected with 10 mg / kg of Ab79 followed by injection of TTd, the second dose after 4 days, the third dose after 3 days, serum was collected afterwards for human Ig counting, and anti-tetanus detection was performed after 3 days (10 days apart from the first dose). Figure 8 Results from a single mouse donor are shown, showing a significant decrease in all Ig isotypes following treatment. Fig. 9 Results are shown for the mean Ig content of each group of recipients, wherein each data point represents the mean value of each group (n=5 to n=10). Fig.10 showed a significant reduction in anti-tetanus response after AB79 treatment. Fig.11 Overall significant survival was shown with Ab79 treatment.

[0326] Surrogate mouse experiment

[0327] Since Ab79 does not cross-react with rodent CD38, a surrogate antibody was developed. As a preliminary study, a commercial antibody against human CD38 and a commercial antibody against mouse CD38 were used, and CD38 expression levels showed significant differences between human and mouse cell types (see Fig.12 ), indicating that the biology of the disease is different. Therefore, using antibodies that cross-react with monkey CD38 in the monkey model gave significantly better results.

[0328] Surrogate antibodies were developed and expressed from spleen, blood and bone marrow (data not shown) as well as from peripheral blood (see Fig.13 ) were harvested from CD38+ cells. Fig.13 The kinetics of depletion are shown in Figure 1, where spleen, blood, and bone marrow were collected 1, 2, or 3 days after a single dose of 10 mg / kg mouse surrogate. Rapid depletion of B cells in the blood was shown within 24 hours, while slower depletion was shown in the spleen and bone marrow.

[0329] Mouse SLE Model

[0330] Surrogate mouse anti-CD38 antibodies were tested in the MRL / lpr model using the following interim readouts (e.g., every two weeks) from blood: anti-dsDNA autoantibodies, CBC, FACS for T / B cell depletion, albumin in urine, and skin inflammation. Endpoint readouts included, but were not limited to, from blood: anti-dsDNA autoantibodies, CBC, FACS for T / B cell depletion, from spleen, lymph nodes, and bone marrow: organ weights, immune cell numbers, FACS for T / B cell depletion, for monkeys: organ weights, histopathology (H&E and PAS), IC treatment, inflammatory cells, and skin inflammation (histopathology).

[0331] Collagen-induced arthritis (CIA) model

[0332] The mouse CIA model was utilized with 7 groups of mice using mouse surrogate antibodies to evaluate pro-prophylatic, preventive and therapeutic efficacy. All mice were immunized with CII / CFA on day 0, and CII / CFA was added on day 21, generally making the development of the disease reach day 42 (study termination) from day 21. Group 1 (10 mice) was injected intraperitoneally with 10 mg / kg surrogate antibody twice a week, starting on day 0 (pro-prophylactic). Group 2 (n=10) was similarly administered, but started on day 21. Group 3 (n=10) was similarly injected at the onset of the disease (days 25-28). Group 4 (n=10) was administered at the same level, but hIgG1 (e.g., isotype) was given on day 0. Group 5 (n=10) was administered at the same level of hIgG1, but started on day 21. Group 6 (n=10) was administered with 0.5 mg / l dexamethasone aqueous solution on day 21, and Group 7 (n=5) was not treated.

[0333] A macaque CIA study was conducted with n=3 in each group, Group 1 was untreated animals, vehicle only. Group 2 was a single dose of Ab79 at 3 mg / kg or 10 mg / kg, q1w (prophylactic treatment started on day 7 after collagen immunization). Group 3 was Ab79 at 3 mg / kg or 10 mg / kg, q1w for therapeutic treatment, starting at the onset of disease, i.e., day 21 or day 28. Group 4 was also treated with 0.1 mg / kg of dexamethasone, q1d, at the onset of disease.

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340] Having described the present invention in detail and with reference to specific embodiments of the present invention, it is apparent that modifications and variations are possible without departing from the scope of the present invention as defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as being particularly advantageous, it should be understood that the present invention is not necessarily limited to the described specific aspects of the present invention.

Claims

1. An isolated antibody that specifically binds to human CD38 (SEQ ID NO: 1) and macaque CD38 (SEQ ID NO: 2), comprising: a) a heavy chain variable region comprising: i) a first CDR comprising SEQ ID NO: 3; ii) a second CDR comprising SEQ ID NO: 4; iii) a third CDR comprising SEQ ID NO: 5; and b) a light chain variable region comprising: i) a first CDR comprising SEQ ID NO: 6; ii) a second CDR comprising SEQ ID NO: 7; iii) a third CDR comprising SEQ ID NO:

8.

2. The isolated antibody of claim 1, wherein the heavy chain variable region comprises SEQ ID NO:

9.

3. The isolated antibody of claim 1, wherein the light chain variable region comprises SEQ ID NO:

10.

4. The isolated antibody of claim 1, wherein the heavy chain variable region comprises SEQ ID NO: 9 and the light chain variable region comprises SEQ ID NO:

10.

5. The isolated antibody of claim 1, wherein the heavy chain comprises SEQ ID NO: 21 and the light chain comprises SEQ ID NO:

22.

6. An isolated antibody that specifically binds to human CD38 (SEQ ID NO: 1) and macaque CD38 (SEQ ID NO: 2), comprising: a) a heavy chain variable region comprising: i) a first CDR comprising SEQ ID NO: 13; ii) a second CDR comprising SEQ ID NO: 14; iii) a third CDR comprising SEQ ID NO: 15; and b) a light chain variable region comprising: i) a first CDR comprising SEQ ID NO: 16; ii) a second CDR comprising SEQ ID NO: 17; iii) a third CDR comprising SEQ ID NO:

18.

7. The isolated antibody of claim 6, wherein the heavy chain variable region comprises SEQ ID NO:

11.

8. The isolated antibody of claim 6, wherein the light chain variable region comprises SEQ ID NO:

12.

9. The isolated antibody of claim 6, wherein the heavy chain variable region comprises SEQ ID NO: 19 and the light chain variable region comprises SEQ ID NO:

20.

10. The isolated antibody of claim 6, wherein the heavy chain comprises SEQ ID NO: 34 and the light chain comprises SEQ ID NO: 35.

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