Anti-CD38 antibody and use thereof

Abstract

To provide a compositions and a method concerning an antibody or an antibody fragment that specifically binds to CD38.SOLUTION: A composition comprising an antibody or an antibody fragment, the antibody or antibody fragment comprising, as a constituent thereof, one or more complementarity determining regions or framework regions of a light chain variable region or a heavy chain variable region comprising an amino acid sequence selected from the group consisting of predetermined amino acid sequences.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] (cross reference) This application claims priority to U.S. Provisional Application No. US63042773, filed in the United States Patent and Trademark Office on June 23, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] CD38, also known as cyclic ADP-ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 is a member of a group of related membrane-bound or soluble enzymes that includes CD157 and Aplysia ADPR cyclase. This enzyme family has the unique ability to convert NAD to cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate.

[0003] Furthermore, CD38 inhibits Ca 2+ It has been reported that CD38 is involved in the signal transduction of many signaling molecules (including phospholipase Cγ, ZAP-70, syk, and c-cbl) through the recruitment and tyrosine phosphorylation of CD38. Based on these observations, CD38 is considered to be an important signaling molecule for the maturation and activation of lymphoid cells during normal development.

[0004] In hematopoietic cells, various functional roles have been attributed to CD38-mediated signaling, including regulation of lymphocyte proliferation, cytokine release, development and survival of B cells and bone marrow cells, and induction of dendritic cell maturation.

[0005] However, because most signaling studies use cell lines that ectopically overexpress CD38 and anti-CD38 monoclonal antibodies (non-physiological ligands), the exact role of CD38 in signaling and hematopoiesis remains unclear.

[0006] CD31 (PECAM-1, platelet endothelial cell adhesion molecule-1) is thought to be the natural ligand for CD38. CD31 is a 130 kD member of the immunoglobulin superfamily and is expressed on the surface of circulating platelets, neutrophils, monocytes, and naive B lymphocytes. It is thought to function as an adhesion molecule. It has been shown that the interaction between CD38 and CD31 can function to promote the survival of leukemia cells.

[0007] In many cases, animal models lacking a single molecule have become essential tools for understanding the biological role of that molecule in animals, the basic assumption being that if a protein exerts a non-redundant function, then its complete deletion will result in a complete loss of that function.

[0008] A CD38 knockout mouse model was constructed. These animals showed a near-complete loss of tissue-associated NAD enzyme activity but were viable, leading to the conclusion that CD38 and its activity are not necessary for life. However, these mice did have defects in innate immunity and reduced T cell-dependent humoral responses.

[0009] However, strong indirect evidence suggests that, contrary to the findings from mice, CD38 is essential for life in humans. Analysis of blood samples from over 5,000 newborns revealed that a single CD38 - Because it was not possible to identify individuals, it was suggested that CD38 is necessary for human survival, unlike in mice. Therefore, it is unclear whether the function of CD38 is essential for human survival. It is unclear whether the observations made apply to humans.

[0010] CD38 is upregulated in many 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), as well as in cell lines derived from various hematopoietic malignancies. On the other hand, most primitive pluripotent stem cells of the hematopoietic system express CD38. - (Figure 1).

[0011] Despite recent advances in the discovery and development of anti-cancer drugs, many forms of cancer associated with CD38 expression still have poor prognoses, and improved methods for treating these forms of cancer are needed. Summary of the Invention

[0012] Provided herein are reagents and methods for binding to CD38, methods for treating CD38-associated diseases, and methods for detecting CD38 using CD38-specific binding agents (including CD38-specific antibodies or antibody fragments).

[0013] In some embodiments, an isolated antibody or antibody fragment specific for human CD38 (SEQ ID NO: 1) is described. The antibody or antibody fragment consists of a heavy chain variable region and a light chain variable region, where the heavy chain variable region consists of three complementary determining regions (CDRs): HCDR1, HCDR2, and HCDR3, and the light chain variable region also consists of three CDRs: LCDR1, LCDR2, and LCDR3. The sequences of the CDRs are represented as HCDR1 (SEQ ID NO: 9), HCDR2 (SEQ ID NO: 13), HCDR3 (SEQ ID NO: 17), LCDR1 (SEQ ID NO: 25), LCDR2 (SEQ ID NO: 29), and LCDR3 (SEQ ID NO: 33).

[0014] In some embodiments, the isolated antibody or antibody fragment consists of a heavy chain variable region, wherein the sequence of the heavy chain variable region is included in SEQ ID NO:5.

[0015] In some embodiments, the isolated antibody or antibody fragment consists of a light chain variable region, wherein the sequence of the light chain variable region is included in SEQ ID NO:21.

[0016] In some embodiments, the isolated antibody or antibody fragment consists of a heavy chain variable region, and the sequence of the heavy chain variable region is included in SEQ ID NO: 5. In other embodiments, the isolated antibody consists of a light chain variable region, and the sequence of the light chain variable region is included in SEQ ID NO: 21. Such a combination of a heavy chain variable region and a light chain variable region is referred to as scFv418.

[0017] 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.

[0018] In some embodiments, an isolated nucleic acid is provided that encodes the heavy chain set forth in SEQ ID NO: 37. In other embodiments, an isolated nucleic acid is provided that encodes the light chain set forth in SEQ ID NO: 41.

[0019] In some embodiments, a host cell is provided comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO:5 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO:21.

[0020] In some embodiments, a host cell is provided comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO:37 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO:41.

[0021] In some embodiments, methods of producing an antibody of the invention are provided, the methods comprising culturing a host cell comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO:5 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO:21 under conditions that express the isolated nucleic acids to produce the antibody.

[0022] In some embodiments, methods of producing an antibody of the invention are provided, the methods comprising culturing a host cell comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO: 37 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO: 41 under conditions such that the isolated nucleic acids are expressed to produce the antibody.

[0023] In some embodiments, an isolated antibody specific for human CD38 (SEQ ID NO: 1) is described. The antibody consists of six CDRs, and each CDR of the antibody can differ by 0, 1, or 2 amino acid substitutions: SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33.

[0024] In other embodiments, the isolated antibody consists of a heavy chain variable region, wherein the sequence of the heavy chain variable region is included in SEQ ID NO:5.

[0025] In some alternative embodiments, the isolated antibody consists of a light chain variable region, and the sequence of the light chain variable region is included in SEQ ID NO: 21. Such a combination of a heavy chain variable region and a light chain variable region is referred to as scFv418.

[0026] In some embodiments, an isolated nucleic acid is provided that encodes the heavy chain set forth in SEQ ID NO: 37. In other embodiments, an isolated nucleic acid is provided that encodes the light chain set forth in SEQ ID NO: 22. This combination of heavy and light chains is referred to as an IgG418 intact antibody.

[0027] In some embodiments, a host cell is provided comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO:5 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO:21.

[0028] In some embodiments, a host cell is provided comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO:37 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO:41.

[0029] In some embodiments, methods of producing an antibody of the invention are provided, the methods comprising culturing a host cell comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO:5 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO:21 under conditions that express the isolated nucleic acids to produce the antibody.

[0030] In some embodiments, methods of generating antibodies according to the invention are provided. comprises culturing a host cell comprising an isolated nucleic acid encoding the heavy chain set forth in SEQ ID NO: 37 and an isolated nucleic acid encoding the light chain set forth in SEQ ID NO: 41 under conditions to express the isolated nucleic acids and produce the antibody.

[0031] In some alternative embodiments, an isolated antibody specific for human CD38 (SEQ ID NO: 1) is described, which consists of six CDRs, each of which can differ from SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33 by 0, 1, or 2 amino acid substitutions.

[0032] In some embodiments, an isolated anti-CD38 antibody that specifically binds to human CD38 (SEQ ID NO: 1) is provided, wherein the antibody binds to human CD38 at a concentration of about 10 -6 , 10 -7 , 10 -8 , 10 -9 or bind with a higher KD.

[0033] In some embodiments, antibodies that bind to human CD38 in competition with IgG418 are provided.

[0034] In some embodiments, compositions comprising the CD38 antibody or antibody fragment are provided.

[0035] In some embodiments, methods are provided for treating a subject suffering from a disease associated with CD38 expression, the methods comprising administering to the subject an effective amount of a CD38 antibody or antibody fragment according to the present invention, or a composition comprising the CD38 antibody or antibody fragment.

[0036] In some embodiments, there is provided the use of a CD38 antibody or antibody fragment or composition thereof according to the invention in the manufacture of a medicament for treating a disease associated with CD38 expression.

[0037] In some embodiments, a pharmaceutical composition comprising a CD38 antibody or antibody fragment according to the invention is provided for treating a disease associated with CD38 expression.

[0038] These and other embodiments, features and potential advantages will become apparent with reference to the following description and drawings. [Brief explanation of the drawings]

[0039] The following detailed description of preferred embodiments of the invention will be better understood when taken in conjunction with the drawings. For the purpose of illustrating the invention, there is shown in the drawings preferred embodiments. It is to be understood, however, that the invention is not limited to the specific manner and instrumentality of the embodiments shown in the drawings.

[0040] [Figure 1] FIG. 1 shows the results of detecting the affinity of IgG418 to CD38 recombinant protein using a capture ELISA, with Darzalex as a control. [Figure 2] Figure 2 shows the results of detecting the affinity of IgG418 for CD38 expressed on Daudi cells using flow cytometry, with Darzalex as a control. [Figure 3] Figure 3 shows the results of competitive ELISA, which demonstrate that the epitope of CD38 that binds to IgG418 is different from the epitope that binds to Darazalex. [Figure 4-1] Figure 4-1 shows a statistical analysis of the CDC activity of various concentrations of IgG418 against Daudi cells, with Darzalex as a control. [Figure 4-2] Figure 4-2 shows a statistical analysis of the CDC activity of various concentrations of IgG418 against Daudi cells, with Darzalex as a control. [Figure 4-3] Figure 4-3 shows a statistical analysis of the CDC activity of various concentrations of IgG418 against Daudi cells, with Darzalex as a control. [Figure 5] Figure 5 shows the results of the statistical analysis of the CDC data. [Figure 6] FIG. 6 shows the ADCC activity of wild-type and defucosylated IgG418 against Daudi cells, with Darzalex as a control. [Figure 7] FIG. 7 shows the inhibitory effect of anti-human CD38 monoclonal antibody on the growth of human B-cell lymphoma Daudi xenograft tumors in SCID mice at a dose of 1 mg / kg. [Figure 8] FIG. 8 shows the inhibitory effect of anti-human CD38 monoclonal antibody on the growth of human B-cell lymphoma Daudi xenograft tumors in SCID mice at a dose of 10 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0041] (overview) The extracellular domain of CD38 is known to possess bifunctional enzymatic activity, namely, ADP-ribosyl cyclase and ADP-ribosyl hydrolase activities. Thus, CD38 can catalyze the conversion of NAD to cADPR (cyclase) and further hydrolyze it to ADP-ribose (hydrolase). cADPR is involved in mobilizing calcium from intracellular stores, which has second messenger activity important for cell proliferation, differentiation, and apoptosis.

[0042] Increased CD38 expression has been demonstrated in various diseases of hematopoietic origin and is considered a negative prognostic marker in chronic lymphocytic leukemia (CLL). These diseases include multiple myeloma (Jackson et al. (1988)), chronic lymphocytic leukemia (Moribito et al. (2001), Jelinek et al. (2001), Chevalier et al. (2002), Durig et al. (2002)), B-cell chronic lymphocytic leukemia, acute lymphocyte These include, but are not limited to, leukemia (Keyhani et al. (2000)), B-cell acute lymphocytic leukemia, Waldenstrom's macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic / granulocytic leukemia, acute myeloid leukemia (Keyhani et al. (1993)), chronic myeloid leukemia (Marinov et al., (1993)), follicular lymphoma, NK cell leukemia, and plasma cell leukemia. Thus, CD38 provides a useful target for the treatment of hematopoietic disorders.

[0043] Several anti-CD38 antibodies are in clinical trials for treating CD38-associated cancers. Therefore, antibodies against CD38 with therapeutic and / or diagnostic uses would be useful. The present invention provides various anti-CD38 CDRs that bind to different epitopes of CD38, and antibodies comprising these CDRs.

[0044] Since 2015, CD38 antibody therapy has been approved for bone marrow myeloma. Furthermore, several other recent studies have shown that CD38 antibody therapy may overcome tumor cell resistance to PD1 / PDL1 therapy. Therefore, the anti-CD38 antibody of the present invention can be used not only for bone marrow myeloma but also for the diagnosis and treatment of all other types of cancer.

[0045] The present invention also demonstrates that anti-CD38 antibodies are useful for the diagnosis and / or treatment of inflammatory and / or immunological disorders associated with activated lymphocytes, including, in particular, autoimmune diseases. As shown herein, CD38 is expressed on immature hematopoietic cells, downregulated on mature cells, and re-expressed at high levels on activated lymphocytes and plasma cells. For example, high CD3 8 Expression is seen on activated B cells, plasma cells, and activated CD4 + cells, activated CD8 + It is found in cells, NK cells, NKT cells, mature dendritic cells (DCs), and activated monocytic cells.

[0046] Surprisingly, the presence of autoantibodies against 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 of which are incorporated herein by reference.

[0047] Therefore, the antibodies of the present invention can be used in the diagnosis and / or treatment of various diseases, including, but not limited to, autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), multiple sclerosis (MS), inflammatory bowel disease (IBD), diabetes, and ulcerative colitis.

[0048] Thus, patients with high plasma cell content, such as SLE patients who exhibit high plasma cell counts or RA patients who have not responded to CD20-based therapy, can be selected for inclusion.

[0049] Therapeutic anti-CD38 antibodies of the present invention bind to CD38-positive cells and result in the depletion of these cells (e.g., activated lymphocytes) through a variety of mechanisms of action, including, but not limited to, CDC, ADCC, ADCP, and apoptosis pathways as described herein, resulting in the treatment and / or amelioration of autoimmune diseases.

[0050] (antibody) The present invention provides anti-CD38 antibodies, generally referred to as therapeutic and / or diagnostic antibodies, as described herein. The antibodies used in the present invention can take a variety of forms as described herein, including conventional antibodies as described below, and antibody variants, derivatives, fragments, and analogs that retain their antigen-binding ability. Essentially, the present invention provides antibody structures comprising six CDR sets as defined herein (with minor amino acid changes as described below).

[0051] Conventional antibody structural units typically comprise a tetramer. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair comprising a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, and IgE. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Therefore, as used herein, "isotype" refers to any subclass of immunoglobulin defined by the chemical and antigenic properties of their constant regions. Known human immunoglobulin isotypes include IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE, although it is understood that therapeutic antibodies can also comprise hybrids of isotypes and / or subclasses.

[0052] The amino-terminal portion of each chain contains approximately 100-110 or more amino acids that are primarily responsible for antigen recognition. The variable regions of antibodies contain three loops in each V domain of the heavy and light chains that aggregate to form the antigen-binding site. Each loop is called a complementarity-determining region (CDR), and is where the most significant amino acid sequence variation occurs. "Variability" refers to the fact that the sequences of specific segments in the variable regions vary significantly among antibodies. The variability within the variable regions is not uniformly distributed. Instead, the V regions consist of relatively invariant segments of 15-30 amino acids (called framework regions (FRs)), separated by short regions of extreme variability (called "hypervariable regions") that are 9-15 amino acids or longer.

[0053] Each VH and VL is composed of three hypervariable regions ("complementarity-determining regions", "CDRs") and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0054] The hypervariable region of the light chain variable region generally comprises amino acid residues located at about positions 24-34 (LCDR1, "L" represents the light chain), 50-56 (LCDR2), and 89-97 (LCDR3). The hypervariable region of the heavy chain variable region generally comprises amino acid residues located at about positions 31-35 (HCDR1, "H" represents the heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, of Health, Bethesda, Md. (1991)), and / or those residues forming the hypervariable loops, e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region, and residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). Specific CDRs of the present invention are as follows:

[0055] Throughout this specification, when referring to residues in the variable domains (residues from about 1 to 107 for light chain variable regions and about 1 to 113 for heavy chain variable regions), the Kabat numbering system (e.g., Kabat et al., supra (1991)) is generally used, with the EU numbering system being used for the Fc region.

[0056] CDRs contribute to the formation of antigen binding of an antibody, more specifically, the formation of the epitope-binding site. An "epitope" refers to a determinant that interacts with a specific antigen-binding site (called a paratope) in the variable region of an antibody molecule. An epitope is a group of molecules, such as amino acids or sugar side chains, that generally have specific structural and charge characteristics. An antigen can have one or more epitopes. For example, as shown herein, two different antibodies, referred to herein as "IgG418" and Darzalex, bind to different epitopes on the CD38 molecule.

[0057] An epitope can include amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by peptides that are involved in specific binding of the antigen; in other words, these amino acid residues are within the space occupied by peptides that are involved in specific binding of the antigen.

[0058] Epitopes can be conformational or linear. Conformational epitopes arise from spatially juxtaposed amino acids in different segments of a linear polypeptide chain. Linear epitopes arise from adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes lose their binding in the presence of denaturing solvents. The difference is that the binding to the latter is not lost.

[0059] An epitope generally includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be identified by a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

[0060] In the present invention, the binding epitopes of IgG418 and Darzalex are different because they do not compete for the same epitope in a competitive ELISA assay.

[0061] Thus, in some embodiments, antibodies that competitively bind to epitopes of either scFv418 or IgG418 are useful in the treatment of cancer and autoimmune diseases. Note that antibodies that compete with scFv418 or IgG418 find use in the present invention.

[0062] The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector functions. Kabat et al. collected several primary sequences of the variable regions of the heavy and light chains. Based on the degree of sequence conservation, they divided each primary sequence into CDRs and frameworks and compiled a list (SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 th See, for example, E.A. Kabat et al., NIH Publication No. 91-3242, E.A. Kabat et al., incorporated herein by reference.

[0063] The IgG subclass of immunoglobulins has several immunoglobulin domains in the heavy chain. As used herein, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin with a different tertiary structure. The focus of the present invention is on the heavy chain domain, which includes a constant heavy chain (CH) domain and a hinge domain. In IgG antibodies, each IgG isotype has three CH regions. Therefore, the "CH" domains of IgG are as follows: "CH1" refers to positions 118 to 220 according to the EU index of Kabat et al., "CH2" refers to positions 237 to 340 according to the EU index of Kabat et al., and "CH3" refers to positions 341 to 447 according to the EU index of Kabat et al.

[0064] Another Ig domain of the heavy chain is the hinge region. The terms "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" herein refer to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain stops at EU position 220, and the IgG CH2 domain starts at EU residue 237. Thus, for IgG, the antibody hinge herein is defined as comprising positions 221 (221 for IgG1) to 236 (G236 for IgG1), where the numbering is according to the EU index as in Kabat. In some embodiments, for example in the case of the Fc region, the lower hinge is included. However, "lower hinge" generally refers to positions 226 or 230.

[0065] Of particular interest in the present invention is the Fc region. As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody, but not including the first constant region immunoglobulin domain, and optionally including a portion of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. In IgA and IgM, Fc can include the J chain. In IgG, the Fc domain includes the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region might vary, the human IgG heavy chain Fc region is generally defined to include residues C226 or P230 at its carboxy terminus, where numbering is according to the EU index as in Kabat. In some embodiments, amino acid modifications are made in the Fc region to alter binding, for example, to one or more FcγR receptors or FeRn receptors, as described below.

[0066] In some embodiments, the antibody is full-length. By "full-length antibody" herein is meant the structure that constitutes the native biological form of an antibody, including variable and constant regions, including one or more modifications described herein.

[0067] Alternatively, antibodies may be of various structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody analogs"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each.

[0068] In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to, (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341:544-546, fully incorporated by reference); (v) an isolated CDR region; (vi) a bivalent F(ab')2 fragment comprising two linked Fab fragments; and (vii) a single-chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to bind to form an antigen-binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 2002). al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, incorporated herein by reference in its entirety), (viii) bispecific single-chain Fvs (WO 03 / 11161, incorporated herein by reference), and (ix) "diabodies" or "tribodies," 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, incorporated herein by reference in its entirety).

[0069] (Chimeric and humanized antibodies) In some embodiments, antibodies may be a mixture from different species, including, for example, chimeric and / or humanized antibodies, i.e., CDR sets may be used in the present invention with framework and constant regions other than those specifically represented in the sequences herein.

[0070] In general, both "chimeric antibody" and "humanized antibody" refer to antibodies that combine regions derived from more than one species. For example, a "chimeric antibody" typically contains variable regions derived from a mouse (or occasionally a rat) and constant regions derived from a human. A "humanized antibody" generally refers to an antibody in which the variable domain framework regions of a non-human antibody have been replaced with sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, excluding the CDRs, is encoded by a polynucleotide of human origin, or the portion of the antibody excluding the CDRs is identical to an antibody of human origin. An antibody is generated by grafting some or all of the CDRs encoded by nucleic acid derived from a non-human organism onto the beta-sheet framework of a human antibody variable region, and the structure of this antibody is The specificity is determined by the grafted CDRs. The production of such antibodies is described, for example, in WO92 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536 (all of which are incorporated herein by reference). Generally, it is necessary to "backmutate" selected acceptor framework residues to the corresponding donor residues to restore affinity lost in the originally grafted construct (U.S. Patent No. 5,530,101; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No. 6,180,370; U.S. Patent No. 5,859,205; U.S. Patent No. 5,821,337; U.S. Patent No. 6,054,297; U.S. Patent No. 6,407,213, all incorporated by reference in their entireties). Ideally, a humanized antibody will comprise at least a portion of an immunoglobulin constant region, typically a human Fc region, which is generally a portion of a human immunoglobulin. Humanized antibodies can also be produced using mice with genetically engineered immune systems (Roque et al., 2002). (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA) and references cited therein, which are incorporated by reference in their entireties.) Techniques and methods for humanizing and remodeling 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, which are incorporated by reference in their entireties).Examples of humanization methods include, but are not limited to, those described by 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. al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8 (incorporated by reference in their entirety). Other methods for reducing the immunogenicity of humanized or non-human antibody variable regions include, for example, the resurfacing method described in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973 (incorporated by reference in their entirety). In one embodiment, the parent antibody is an affinity-matured antibody, as known in the art. Structure-based methods, such as those described in U.S. Serial No. 11 / 004,590, can be used for humanization and affinity maturation.Selection-based methods, such as 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 (incorporated by reference in their entirety), can be used to humanize and / or affinity mature antibody variable regions. Other humanization methods include, but are not limited to, those described in U.S. Series No. 09 / 810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084 (incorporated by reference in their entireties). One method is to transplant only a part of the DR.

[0071] In one embodiment, the antibodies of the present invention may be multispecific antibodies, particularly bispecific antibodies called "diabodies." These antibodies bind to two (or more) different antigens or different epitopes on the same antigen. Diabodies can be produced by a variety of methods known in the art, for example, chemical or hybridoma methods (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, incorporated by reference in its entirety).

[0072] In one embodiment, the antibody is a miniantibody. Miniantibodies are minimal antibody-like proteins comprising an scFv linked to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, is incorporated by reference in its entirety. The scFv may be linked to an Fc region, and may include part or all of the hinge region.

[0073] Antibodies of the present invention are generally isolated or recombinant. When any polypeptide disclosed herein is described as "isolated," it refers to a polypeptide that has been identified, isolated, or recovered from a cell or cell culture that expresses it. Generally, an isolated polypeptide is produced through at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to CD38 is substantially free of antibodies that specifically bind to antigens other than CD38.

[0074] An isolated antibody that specifically binds to an epitope, isotype, or variant of human CD38 or cynomolgus CD38 may, however, have cross-reactivity to other related antigens from other species, such as species homologs of CD38. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0075] Isolated monoclonal antibodies with different specificities can be combined into defined compositions, thus IgG418 can be combined into a single formulation if desired.

[0076] The anti-CD38 antibodies of the present invention specifically bind to its ligand, CD38 (e.g., the human CD38 protein of SEQ ID NO: 1). "Specific binding" or "specific" for a particular antigen or epitope refers to binding that is significantly different from nonspecific interactions. Specific binding can be measured, for example, by comparing the binding of an antibody to a corresponding molecule with the binding of the antibody to a control molecule, where the control molecule is a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0077] Specific binding to a particular antigen or epitope can be reflected by a KD value. For example, the KD of an antibody for an antigen or epitope is at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M or greater. KD refers to the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a KD with the antigen or epitope that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or greater than that of a control molecule.

[0078] Furthermore, specific binding to a particular antigen or epitope is reflected by K A or K B . For example, an antibody may have a K A or K B for an antigen or epitope that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more times greater than for a control epitope, where K A or K B refers to the on-rate of a particular antibody-antigen interaction.

[0079] (antibody modification) The present invention also provides antibody mutants. Specifically, various modifications can be made to the antibodies of the present invention. These modifications include, but are not limited to, amino acid modifications in the CDRs (affinity maturation), amino acid modifications in the Fc region, glycosylation variants, and other types of covalent modifications.

[0080] By "variant" herein is meant a polypeptide sequence that differs from a parent polypeptide by virtue of at least one amino acid modification(s), such as a substitution, insertion, or deletion, although the former is often preferred.

[0081] Typically, as described herein, variants can include any number of modifications as long as the protein remains functional. That is, for example, when generating amino acid variants using the CDRs of IgG418, the antibody should specifically bind to human CD38. Similarly, when generating amino acid variants using the Fc region, the antibody variants should maintain the receptor-binding function required for the particular use or indication of the antibody.

[0082] Typically, substitutions with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are common, aiming to alter function with minimal modifications. 1 to 5 modifications may be present. Also, 1 to 2, 1 to 3, and 1 to 4 modifications are often used in embodiments.

[0083] The number of amino acid modifications is preferably within a functional domain, for example, 1 to 5 modifications in the Fc region or 1 to 5 modifications in the FV region of a wild-type or engineered protein. The sequence of the variant polypeptide preferably has at least about 80%, 85%, 90%, 95%, or up to 98% or 99% identity with the parent sequence (e.g., the variable region, constant region, and / or heavy and light chain sequences of IgG418). It should be noted that, depending on the size of the sequence, the percent identity will depend on the number of amino acids.

[0084] As used herein, "amino acid substitution" or "substitution" refers to the substitution of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, an S100A substitution refers to a variant polypeptide in which the serine at position 100 is substituted with an alanine. As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence.

[0085] As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to generate a variant. The parent polypeptides described herein are generally scFv418 and IgG418. The parent polypeptide often refers to the polypeptide itself and includes the composition of the parent polypeptide or the amino acid sequence encoding it. Thus, as used herein, "parent Fc polypeptide" refers to an Fc polypeptide used for modification to generate a variant. Additionally, as used herein, "parent antibody" refers to an antibody used for modification to generate a variant antibody.

[0086] As used herein, "wild-type" or "WT" or "native" refers to an amino acid or nucleotide sequence found in nature, including allelic variations. For example, a WT protein, polypeptide, antibody, immunoglobulin, IgG, has an amino acid or nucleotide sequence that has not been intentionally modified.

[0087] As used herein, "Fc region variant" refers to an Fc sequence that differs from the wild-type Fc sequence by virtue of at least one amino acid modification. Fc variant may refer to an Fc variant polypeptide or an Fc polypeptide composition comprising the amino acid sequence.

[0088] In some embodiments, one or more amino acids in one or more CDRs of antibody IgG418 are modified. Generally, only one, two, or three amino acids are substituted in any CDR, and no more than four, five, six, seven, eight, nine, or ten amino acids are changed in a set of CDRs. However, it will be understood that any combination of none, one, two, or three substitutions in any CDR can be independently and optionally combined with any other substitution.

[0089] Amino acid modifications in the CDRs are sometimes referred to as "affinity maturation." An "affinity matured" antibody is one with one or more changes in one or more CDRs that improve the affinity of the affinity matured antibody for its antigen compared to a parent antibody that does not have those changes. Although there may be cases where the affinity of the antibody for its antigen is reduced, this is usually undesirable.

[0090] Affinity maturation can improve the binding affinity of an antibody for an antigen by at least about 10% to 50-100-150% or more, or 1- to 5-fold, compared to the "parent" antibody. Preferred affinity-matured antibodies have nanomolar or picomolar affinity for the target antigen. Affinity-matured antibodies can be generated by known procedures, such as affinity maturation by mixing variable heavy (VH) and variable light (VL) domains, as described by Marks et al., 1992, Biotechnology 10:779-783. Random mutagenesis of CDR and / or framework residues is described, for example, in Barbas, et al. 1994, Proc. Nat. Acad. Sci. USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226:889-896.

[0091] Alternatively, "silent" amino acid modifications can be made in one or more CDRs of an antibody of the invention, e.g., which do not significantly alter the affinity of the antibody for antigen. Such modifications can be made for a variety of reasons, including optimizing expression (which can be made by a nucleic acid encoding an antibody of the invention).

[0092] Thus, CDR variants and antibody variants are included in the definition of the CDRs and antibodies of the present invention. That is, antibodies of the present invention may contain amino acid modifications in one or more CDRs of IgG418. Furthermore, as described below, amino acid modifications may optionally be made independently in any region other than the CDRs, including framework and constant regions.

[0093] In some embodiments, the anti-CD38 antibodies of the present invention are comprised of Fc domain variants. As is known in the art, the Fc region of an antibody can be divided into several Fc receptors. Interactions with receptors and ligands confer a range of important functional capabilities called effector functions. Such Fc receptors include, but are not limited to, (human) FcγRI (CD64) including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isotypes (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD1.6) including isotypes FcγRIIIa (including allotypes V158 and F158, associated with antibody-dependent cellular cytotoxicity (ADCC)) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), FcRn (a neoplastic receptor), C1q (a complement protein involved in complement-dependent cytotoxicity (CDC)), and FcRn (a neoplastic receptor involved in serum half-life). Suitable modifications can be made at one or more positions, and examples include those described in U.S. Patent Application No. 11 / 841,654 and the references cited therein, including U.S. Patent Application No. 2004 / 013210, U.S. Patent Application No. 2005 / 0054832, U.S. Patent Application No. 2006 / 0024298, U.S. Patent Application No. 2006 / 0121032, U.S. Patent Application No. 2006 / 0235208, U.S. Patent Application No. 2007 / 0148170, U.S. Patent Application No. 12 / 341,769, U.S. Patent No. 6,737,056, U.S. Patent No. 7,670,600, and U.S. Patent No. 6,086,875, the entire contents of which are incorporated herein by reference. The modifications described in these patents can specifically enhance substitution of specific amino acids that bind to Fc receptors.

[0094] In addition to the modifications mentioned above, other modifications may be made, for example, disulfide bonds linking the VH and VL domains can be added to stabilize the molecule (Reiter (Et al., 1996, Nature Biotech. 14:1239-1245, incorporated herein by reference in its entirety.) Various covalent modifications to antibodies, as described below, may also be made.

[0095] Covalent modifications of antibodies are included within the scope of the present invention and are typically, but not necessarily, performed post-translationally. For example, several types of covalent modifications may be introduced into an antibody molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0096] Cysteine ​​residues are well known to react with α-haloacetates (and corresponding amines), and may be reacted, for example, with chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine ​​residues may also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, 2-pyridyl disulfide methyl, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0097] Histidine residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this reagent is relatively specific for the histidine side chain. p-Bromobenzoyl bromide may also be used. Preferably, the reaction is performed in 0.1 M sodium cacodylate at pH 6.0.

[0098] Lysinyl and amino terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable agents for derivatizing α-amino-containing residues include, for example, iminoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, and trinitrobenzenesulfonyl esters. sulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed glyoxylate reactions.

[0099] Arginine residues are modified by reaction with one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, the derivatization reaction of arginine residues must be performed under basic conditions. Note that these reagents may react with lysine groups and the ε-amino group of arginine.

[0100] Tyrosyl residues can be specifically modified. In particular, spectral labels are introduced into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl analogs and 3-nitro derivatives, respectively, and 125I- or 131I-iodinated tyrosyl residues are used to prepare labeled proteins for radioimmunoassay; the Chloramine T method is preferred.

[0101] Pendant carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-NCN-R', where R and R' are optionally different alkyl groups), such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Alternatively, aspartyl and glutamyl residues are converted to asparagine and glutamyl residues by reaction with ammonium ions.

[0102] Bifunctionalizing agent derivatization can be used to crosslink antibodies to water-insoluble support matrices or surfaces, and is used in a number of ways, including those described below. Commonly used crosslinking agents include homobifunctional imidoesters, including N-hydroxysuccinimide esters such as 1,1-bis(diazoniumacetyl)-2-phenylethane, glutaraldehyde, and 4-azidobenzoic acid, disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that can form crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices, such as those produced from cynomolgus monkeys by cynomolgus bromide activation of carbohydrates and those described in U.S. Patent 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 herein by reference), can be used for protein immobilization.

[0103] Glutamine and asparagine residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively, or these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the invention.

[0104] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxy group of serine or threonine residues, methylation of the α-amino group of the side chain of lysine, arginine, and histidine (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

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

[0105] Those of skill in the art will also appreciate that labels (eg, fluorescent, enzymatic, magnetic, radioactive, etc.) may be added to the antibodies and other compositions of the present invention.

[0106] (glycosylation) Another type of covalent modification is altered glycosylation. In another embodiment, the antibodies disclosed herein may be modified to contain one or more engineered glycoforms. As used herein, "engineered glycoform" refers to a carbohydrate composition covalently attached to an antibody, where the carbohydrate composition is chemically distinct from that of the parent antibody. Engineered glycoforms can be used for various purposes, including, but not limited to, enhancing or reducing effector function. A preferred form of engineered glycoform is defucosylated, which has been shown to be associated with increased ADCC function and is presumed to be achieved by tighter binding to the FcγRIIIa receptor. Thus, "defucosylated" means that the majority of antibodies produced in host cells are substantially free of fucose; for example, 90-95-98% of the produced antibodies do not contain any apparent fucose as a component of the carbohydrate moiety of the antibody (typically linked to N297 in the Fc region). Functionally, defucosylated antibodies typically exhibit at least 50% or greater affinity for the FcγRIIIa receptor.

[0107] Engineered glycoforms can be produced by a variety of methods known in the art (Umana et al., 1999, Nat Biotechnol 17). :176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Series No. 10 / 277,370; U.S. Series No. 10 / 113,929; PCT WO00 / 61739A1; PCT WO01 / 29246A1; PCT WO02 / 31140A1; PCT WO02 / 30954A1; the entire contents of which are incorporated herein by reference; (Potelligent® technology [Biowa, Inc., Princeton, NJ]; GlycoMAb® glycosylation engineering technology [Glycart Biotechnology AG, Zurich, Switzerland]). Many technologies are based on controlling the level of fucosylated and / or bisected oligosaccharides covalently attached to the Fc region. For example, this can be achieved by discovering engineered or otherwise modified IgG in various organisms or cell lines (e.g., Lec-13 CHO cells or rat hybridoma YB2 / 0 cells), by modulating enzymes involved in the glycosylation pathway (e.g., FUT8 [α1,6-fucosyltransferase] and / or β1-4 N-acetylglucosaminyltransferase III [GnTITT]), or by modifying the glycosylation of IgG after it has been expressed. For example, Seattle Genetics' "glycoengineered antibodies" or "SEA technology" works by adding modified sugars that inhibit fucosylation during manufacturing (see, e.g., 20090317869, incorporated herein by reference in its entirety). Engineered glycoforms typically refer to different glycans or oligosaccharides, and antibodies can contain engineered glycoforms.

[0108] Alternatively, engineered glycoforms may refer to IgG variants containing different glycans or oligosaccharides. As is known in the art, glycosylation patterns can be determined by the sequence of a protein (e.g., the presence or absence of specific glycosylated amino acid residues, as described below), or the structure of a protein. Depending on the host cell or organism in which it is produced, specific expression systems are described below.

[0109] Glycosylation of polypeptides can be either 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 represents any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these 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. Although 5-hydroxyproline or 5-hydroxylysine can also be used, serine or threonine are most common.

[0110] Addition of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may also be carried out by adding or substituting one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For convenience, alterations to the antibody amino acid sequence are preferably made at the DNA level, particularly by mutating selected bases in the DNA encoding the target polypeptide to obtain codons that can be translated into the desired amino acids.

[0111] Another method for increasing the number of carbohydrate moieties on an antibody is to attach glycosides to the protein by chemical or enzymatic coupling. These methods have the advantage that the protein does not need to be produced in a host cell capable of N- and O-glycosylation. Depending on the coupling method used, sugars can be linked to (a) arginine and histidine, (b) free carboxy groups, (c) free thiol groups, such as the thiol group of cysteine, (d) free hydroxy groups, such as the hydroxy groups of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan, or (I) the amide group of glutamine. These methods are described in WO 87 / 05330 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both of which are incorporated herein by reference.

[0112] Carbohydrate moieties present on the starting antibody (e.g., the antibody after translation) can be removed by chemical or enzymatic methods. Chemical deglycosylation requires exposure of the protein to trifluoromethanesulfonic acid or an equivalent compound. This treatment cleaves most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131, describe chemical deglycosylation, both of which are incorporated herein by reference. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved with a variety of endo- and exoglycosidases, as described in Thotakura et al., 1987, Meth. Enzymol. 138:350, incorporated herein by reference in their entireties. Glycosylation of potential glycosylation sites was confirmed by Duskin This can be avoided by using the compound tunicamycin, as described by W. et al., 1982, J. Biol. Chem. 257:3105 (incorporated herein by reference in its entirety), and protein-N-glycosidic bond formation is blocked by tunicamycin.

[0113] Other covalent modifications of antibodies include, for example, PEG, PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-31, PEG-32, PEG-33, PEG-34, PEG-35, PEG-36, PEG-37, PEG-48, PEG-49, PEG-41, PEG-42, PEG-43, PEG-4 Antibodies can also be linked to nonprotein polymers, including, but not limited to, polyols such as polyethylene glycol, polypropylene glycol, and polyoxyalkylenes, as described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337 (incorporated herein by reference in their entireties). Additionally, amino acid substitutions can be made at various positions within the antibody to facilitate the addition of PEG polymers, as is known in the art. See, e.g., U.S. Publication No. 2005 / 0114037A1 (incorporated herein by reference in its entirety).

[0114] Specific Embodiments of CDRs and Variable Regions The present invention provides various antibodies having specific sets of CDRs (including CDRs with some amino acid substitutions, as described above). As described above, antibodies are defined by a set of six CDRs, a variable region, or the full length of the heavy and light chains (including the constant region). Also, as described above, amino acid substitutions can be made. Because the CDRs are typically short, changes within the CDRs are typically described based on the number of amino acid modifications. This is also appropriate for discussing the number of amino acid modifications introduced into a variable sequence, a constant sequence, or a full-length sequence. In addition to the number of amino acid changes, it is also appropriate to define these changes in terms of "% identity." Thus, as described herein, the present invention also includes antibodies with 80%, 85%, 90%, 95%, 98%, or 99% identity to the SEQ ID NOs described herein. Note that when defining the percentage of amino acid sequence similarity, the term "homology" as used herein has the same meaning as "identity."

[0115] The IgG418 antibody has a CDR set consisting of three heavy chain CDRs comprising SEQ ID NO: 9 (HCDR1), SEQ ID NO: 13 (HCDR2), and SEQ ID NO: 17 (HCDR3), and three light chain CDRs comprising SEQ ID NO: 25 (LCDR1), SEQ ID NO: 29 (LCDR2), and SEQ ID NO: 33 (LCDR3).

[0116] In some embodiments, an antibody (e.g., scFv418 or IgG418) is provided that binds to human CD38 in competition with an antibody of the invention. As known in the art, competitive binding of two or more anti-CD38 antibodies to CD38 or a portion of CD38 can be determined by any suitable technique.

[0117] In the context of the present invention, competition refers to a detectable and significant decrease in the tendency of an antibody of the present invention (e.g., IgG418) to bind to its specific binding partner (e.g., CD38) in the presence of a test compound. Generally, competition refers to at least about a 10% to 100% decrease in binding of an antibody of the present invention to CD38 in the presence of a competitor, as measured, for example, by standard ELISA or Biacore® techniques. Competition criteria can be established such that at least about 10% relative inhibition, at least about 15% relative inhibition, or at least about 20% relative inhibition must be detected before an antibody is considered sufficiently competitive. When the epitope of the competing antibody is close to the antigen, competition can be determined by a relative inhibition of CD38 binding of greater than about 40%. The relative inhibition may be, for example, at least about 45% inhibition, at least about 50% inhibition, at least about 55% inhibition, at least about 60% inhibition, at least about 65% inhibition, at least about 70% inhibition, at least about 75% inhibition, at least about 80% inhibition, at least about 85% inhibition, at least about 90% inhibition, at least about 95% inhibition, or greater.

[0118] As discussed below in the diagnostic applications, one or more outcomes in competitive binding assays may be Minutes may also be labeled.

[0119] Anti-CD38 antibodies may have competition between multiple CD38 epitopes and / or portions of CD38, for example, if the particular segment of CD38 that binds to the antibody is located on or appears as a fragment, if well-presented linear antigens are located on different fragments tested, or if they are located on larger CD38 fragments and conformational epitopes of the CD38 molecule.

[0120] Evaluation of competition typically involves assessing relative binding inhibition using an antibody of the present invention, CD38, and a test molecule. The test molecule can be any molecule, including other antibodies, small molecules, or peptides. The compounds are combined in amounts sufficient for comparison to provide information about the selectivity and / or specificity of the molecule under discussion relative to other molecules present.

[0121] The amounts of the test compound, CD38, and antibody of the present invention can be varied. For example, ELISA assays require approximately 5 to 50 μg (e.g., approximately 10 to 50 μg, approximately 20 to 50 μg, approximately 5 to 20 μg, approximately 10 to 20 μg, etc.) of anti-CD38 antibody and / or CD38 target to assess the presence or absence of competition. Conditions must be suitable for binding, and typically, physiological or near-physiological conditions (e.g., a temperature of approximately 20 to 40°C, a pH of approximately 7 to 8, etc.) are suitable for binding of anti-CD38 to CD38.

[0122] Competition is generally determined as a relative inhibition significantly greater than about 5% in ELISA and / or FACS analysis. A relatively high threshold of relative inhibition can be set as a criterion / determinant of an appropriate level of competition under certain circumstances (e.g., when competition analysis is used to select or screen novel antibodies designed to block the binding of CD38 to another peptide or molecule (e.g., a natural binding partner of CD38, such as CD31, also known as the CD31 antigen, EndoCAM, GPIIA, PECAM-1, platelet / endothelial cell adhesion molecule, or a naturally occurring anti-CD38 antibody)).

[0123] In some embodiments, the anti-CD38 antibodies of the invention specifically bind to one or more residues or regions in CD38 but do not cross-react with other proteins that share homology with CD38, such as BST-1 (bone marrow stromal cell antigen-1) and Mo5 (also known as CD157).

[0124] Lack of cross-reactivity typically means that the relative competitive inhibition between the molecules is less than about 5% when assessed by ELISA and / or FACS analysis using sufficient amounts of the molecules under appropriate measurement conditions.

[0125] (Inhibition of CD38 activity) The antibodies disclosed herein can be used to block ligand-receptor interactions or inhibit receptor component interactions. Anti-CD38 antibodies of the present invention can be "blocking" or "neutralizing." A "neutralizing antibody" refers to an antibody that binds to CD38 and inhibits a biological activity of CD38, such as its ability to interact with ligands, enzymatic activity, signal transduction, and, in particular, its ability to activate lymphocytes. Inhibition of CD38 biological activity can be assessed by several standard in vitro or in vivo assays known in the art.

[0126] "Inhibition of binding" or "blocking of binding" (e.g., when referring to inhibiting / blocking binding of a CD38 binding partner to CD38) includes both partial and complete inhibition / blocking. Inhibiting / blocking binding of a CD38 binding partner to CD38 can reduce or alter the normal level or type of cell signaling that occurs when a CD38 binding partner binds to CD38 (with or without inhibition). Inhibition and blocking can occur when an anti-CD38 It is also intended to encompass any measurable decrease in binding affinity of a CD38 binding partner to CD38 when contacted with an anti-CD38 antibody compared to the ligand not contacted with the antibody, e.g., binding of the CD38 binding partner to CD38 is blocked by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0127] The anti-CD38 antibodies disclosed herein can also inhibit cell proliferation. "Inhibition of proliferation" encompasses a measurable decrease in proliferation of cells when contacted with an anti-CD38 antibody compared to proliferation of the same cells not contacted with the anti-CD38 antibody. For example, proliferation of a cell culture can be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0128] In some embodiments, the anti-CD38 antibodies disclosed herein can deplete activated lymphocytes and plasma cells. As used herein, "depletion" refers to a measurable reduction in serum levels of activated lymphocytes and / or plasma cells (e.g., when tested in cynomolgus monkeys) compared to untreated animals. The depletion observed is typically at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Furthermore, as shown in the Examples below, the antibodies of the present invention exhibit the particular advantage of the recoverability of these cells after administration. That is, as is known from some treatments (e.g., anti-CD20 antibodies), cell depletion can persist for long periods of time, causing undesirable side effects. As shown herein, the effects of the antibodies disclosed herein on activated lymphocytes and / or plasma cells are reversible.

[0129] (Method for producing an antibody according to the present invention) The present invention also provides methods for producing the disclosed anti-CD38 antibodies. These methods involve culturing host cells containing isolated nucleic acid encoding an antibody of the invention. Those skilled in the art will appreciate that this can be accomplished in a variety of ways, depending on the nature of the antibody. In some embodiments, the antibodies of the invention are full-length conventional antibodies, e.g., the heavy and light chain variable regions are in conditions that allow the produced antibody to be isolated.

[0130] The present disclosure provides nucleic acids encoding the antibodies of the present invention. Such polynucleotides encode the variable and constant regions of each heavy and light chain. The present invention also encompasses other combinations, based on the compositions described herein. Furthermore, the present invention includes oligonucleotide fragments derived from the polynucleotides disclosed above and nucleic acid sequences complementary to these polynucleotides.

[0131] 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. The DNA may be double-stranded or single-stranded. If single-stranded, it may be the coding (sense) strand or non-coding (antisense) strand. The coding sequence encoding a polypeptide may be the same as the coding sequence provided herein or may be a different coding sequence. Due to the redundancy and degeneracy of the genetic code, this sequence will encode the same polypeptide as the DNA provided herein.

[0132] In some embodiments, nucleic acids encoding the antibodies of the present invention are inserted into an expression vector. The expression vector may be extrachromosomal or may be designed to integrate into the genome of the host cell. The expression vector may include any number of appropriate regulatory sequences (including, but not limited to, transcriptional and translational regulatory sequences, promoters, ribosomal binding sites, enhancers, origins of replication, etc.) or other components (such as selection genes). As is known in the art, all of these components are operably linked. Furthermore, the two nucleic acids may be used and placed in different expression vectors (e.g., the heavy chain in one expression vector and the light chain in a second expression vector), or alternatively, 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 choice of host cell and the desired level of protein expression.

[0133] The nucleic acid and / or expression vector can be introduced into a suitable host cell, typically using any method appropriate for the selected host cell (e.g., transformation, transfection, electroporation, infection), to produce a recombinant host cell, whereby the nucleic acid molecule is operably linked to one or more expression control elements (e.g., in a vector, in a construct generated by intracellular processes, integrated into the genome of the host cell). The resulting recombinant host cell can be maintained under conditions appropriate for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a suitable medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide. Also, the heavy chain may be produced in one cell and the light chain in another.

[0134] Mammalian cell lines useful as expression hosts are known in the art and include, but are not limited to, Chinese hamster ovary (CHA). Examples of immortalized cell lines include those available from the American Type Culture Collection (ATCC) (Manassas, Va.), including human ovary (CHO) cells, HEK293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2), and many other cell lines. Non-mammalian cells (including, but not limited to, bacteria, yeast, insects, and plants) may also be used to express recombinant antibodies. In some embodiments, antibodies may be produced in transgenic animals (e.g., cows or chickens).

[0135] For general methods of antibody molecular biology, expression, purification and screening, see, for example, Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001 and 2010; Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

[0136] (Use and indications) The antibodies of the present invention can be used in a variety of applications, including the diagnosis and treatment of diseases associated with CD38.

[0137] (CD38-related disorder) In one aspect, the present invention provides methods for diagnosing and treating disorders associated with inflammatory and immune diseases, particularly diseases associated with activated lymphocytes. As shown herein, CD38 is expressed on immature hematopoietic cells, downregulated on mature cells, and re-expressed at high levels on activated lymphocytes and plasma cells. For example, high expression of CD38 is associated with activated B cells, plasma cells, activated CD4 + T cells, activated CD8 + It is found in T cells, NK cells, NKT cells, mature dendritic cells (DCs) and activated monocytes.

[0138] Therapeutic anti-CD38 antibodies of the present invention bind to CD38-positive cells (e.g., activated lymphocytes) and mediate their proliferation through several mechanisms, including CDC, ADCC, and ADCP pathways. This causes the depletion of these cells.

[0139] Thus, the antibodies of the present invention can be used to treat any autoimmune disease characterized by elevated expression of CD38 or elevated numbers of cells expressing CD38.These diseases include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), multiple sclerosis (MS), inflammatory bowel disease (IBD), ulcerative colitis, allogeneic islet transplant rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies, and the like. autoantibody (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 syndrome, abdominal sprue diarrhea-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Caucas syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, idiopathic mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Goodpasture syndrome, graft-versus-host disease Disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki disease, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, genotype These conditions include atopic dermatitis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, solid organ transplant rejection, stiff-man syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, dermatitis herpetiformis, vasculitis such as vasculitis, vitiligo, and Wegener's granulomatosis.

[0140] In some embodiments, the antibodies of the present invention are used in the diagnosis and / or treatment of several diseases, including, but not limited to, autoimmune diseases, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), multiple sclerosis (MS), inflammatory bowel disease (IBD), diabetes, graft-host disease, and ulcerative colitis.

[0141] Patients can be selected that have a high plasma cell content, such as SLE patients who exhibit high plasma cell counts, and RA patients who do not respond to CD20-based therapy.

[0142] In one aspect, the invention provides a method for treating a disorder associated with the proliferation of cells expressing CD38. The method comprises administering to a patient a pharmaceutically effective amount of the antibody. In certain embodiments, the disorder is cancer. In certain specific embodiments, the cancer is a hematological cancer. In certain other specific embodiments, the disorder is multiple myeloma, chronic lymphocytic leukemia, chronic lymphocytic leukemia, plasma cell leukemia, acute myeloid leukemia, chronic myelogenous leukemia, B-cell lymphoma, or Burkitt's lymphoma.

[0143] Certain disorders associated with cells expressing CD38, and with overexpression, high density expression, or upregulated expression of CD38 on the cell surface, are known in the art. As described below in diagnostic applications, whether a cell population expresses CD38 can be determined, for example, by CD38 expression can be determined by methods well known in the art, such as measuring the percentage of cells in a given population labeled with an antibody that specifically binds to CD38 by flow cytometry or immunohistochemical assays. For example, a cell population in which CD38 expression is detected in approximately 10% to 30% of the cells can be considered weakly positive for CD38. A cell population in which CD38 expression is detected in more than approximately 30% of the cells can be considered clearly positive for CD38 (e.g., Jackson et al. (1988), Clin. Exp. Immunol. 72:351-356). Other criteria can also be used to determine whether a cell population expresses CD38. The density of cell surface expression can also be determined by methods well known in the art, such as measuring the mean fluorescence intensity of cells fluorescently labeled with an antibody that specifically binds to CD38 by flow cytometry.

[0144] In some embodiments, the compositions and methods of the present invention are applied to cancers, such as hematological cancers, which refer to malignant tumors of blood-forming tissues and include leukemia, lymphoma, and multiple myeloma. Disorders associated with CD38 expression include, but are not limited to, multiple myeloma (Jackson et al. (1988), Clin. Exp. Immunol. 72:351-356), B-cell chronic lymphocytic leukemia (B-CLL) (Durig 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 lymphocytic leukemia (Keyhani et al. (1999), Leukemia Research 24:153-9; and Marinov et al. (1993), Neoplasma 40(6):355-8), chronic granulocytic 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 myeloid leukemia or chronic myeloid leukemia (CML), acute myeloid leukemia or acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), hairy cell leukemia (HCL), myelodysplastic syndrome (MDS) or blastic chronic myeloid leukemia, and subtypes of all these leukemias defined by morphological, histochemical, and immunological techniques well known to those skilled in the art.

[0145] "Tumor" or "neoplastic disorder" refers to a disease associated with cell proliferation characterized by loss of normal cell control resulting in one or more symptoms including uncontrolled proliferation, lack of differentiation, local tissue invasion, and metastasis.

[0146] In some embodiments of the invention, the hematological cancer is selected from chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).

[0147] Furthermore, CD38 expression is known in the art to be a prognostic indicator in patients with, for example, B-cell chronic lymphocytic leukemia (Durig 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).

[0148] CLL is the most common leukemia found in Western adults. It involves the clonal proliferation of mature lymphocytes in lymph nodes and other lymphoid tissues, with progressive infiltration of bone marrow and peripheral blood. Type B CLL (B-CLL) represents almost all cases.

[0149] (B-CLL) B-CLL is an incurable disease characterized by a persistent, long-term increase in unresponsive monoclonal B-lineage cells that accumulate in the bone marrow and peripheral blood. CD38 expression has been implicated as an independent factor in poor prognosis in B-CLL (Hamblin et al., Blood 99:1023-9 (2002)).

[0150] Currently, standard treatment for B-CLL is palliative, primarily through the cytostatic drugs chlorambucil or fludarabine. When relapse occurs, combination therapy, such as fludarabine, cyclophosphamide, and rituximab (a monoclonal antibody against CD20) or campath (a monoclonal antibody against CD52), is typically used. Thus, there remains a significant unmet medical need for the treatment of B-CLL. In some embodiments, the present disclosure provides methods for treating B-CLL using anti-CD38 antibodies (which, as described below, can be achieved using combination therapy, optionally and independently, including any of the above drugs).

[0151] B-CLL has two subtypes: indolent and aggressive. These clinical phenotypes are related to the presence or absence of somatic mutations in the immunoglobulin heavy chain variable region (IgVH) gene. As used herein, indolent B-CLL refers to disease in subjects who have a mutated IgVH gene and / or exhibit one or more clinical phenotypes associated with indolent B-CLL. As used herein, aggressive B-CLL refers to disease in subjects who have an unmutated IgVH gene and / or exhibit one or more clinical phenotypes associated with aggressive B-CLL.

[0152] (Multiple myeloma) Multiple myeloma is a malignant disease of the B-cell lineage characterized by neoplastic proliferation of plasma cells in the bone marrow. Current treatment regimens have shown moderate response rates. However, only modest changes in overall survival have been observed, with a median survival of approximately 3 years. Therefore, there is a significant unmet medical need for the treatment of multiple myeloma. In some embodiments, methods for treating multiple myeloma using the antibodies disclosed herein are provided.

[0153] CD38 is highly expressed on plasma cells, which are terminally differentiated B cells.

[0154] The growth of bone marrow cells can cause several adverse effects, including lytic lesions (holes) in the bone, a decrease in red blood cell count, abnormal protein production (with associated damage to the kidneys, nerves, and other organs), decreased immune system function, and elevated calcium levels in the blood (hypercalcemia).

[0155] Current treatment options include chemotherapy, preferably in combination with autologous stem cell transplantation (ASCT).

[0156] (Monoclonal Gammopathy of Undetermined Significance and Smoldering Multiple Myeloma) In some embodiments, methods are provided for treating monoclonal gammopathy using the antibodies of the present disclosure, hi other embodiments, methods are provided for treating smoldering multiple myeloma using the antibodies of the present disclosure.

[0157] Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are asymptomatic precancerous lesions characterized by monoclonal plasma cell proliferation in the bone marrow without end-organ damage.

[0158] Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell proliferative disorder with a high risk of progressing to symptomatic or active multiple myeloma (N. Engl. J. Med. 356(25):2582-2590(2007)).

[0159] The international consensus diagnostic criteria for SMM were adopted in 2003 and require patients to have M protein levels >30 g / L and / or bone marrow clonal plasma cells >10% (Br. J. Haematol. 121:749-57(2003)). Patients must also be free of organ or associated tissue damage, including bone lesions or symptoms (Br. J. Haematol. 121:749-57(2003)).

[0160] 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)). MGUS, as defined by the international consensus diagnostic criteria, requires that patients have M protein levels <30 g / L, bone marrow plasma cells <10%, and no organ or associated tissue damage, including bone lesions or symptoms (Br. J. Haematol. 121:749-57 (2003)).

[0161] In the absence of end-organ damage, SMM resembles monoclonal gammopathy of undetermined significance (MGUS) (N. Engl. J. Med. 356(25):2582-2590(2007)). However, clinically, SMM has a high probability of progressing to active multiple myeloma or amyloidosis within 20 years (78% probability for SMM, 21% probability for MGUS) (N. Engl. J. Med. 356(25):2582-2590(2007)).

[0162] Additionally, several other recent studies have shown that CD38 antibody therapy may overcome tumor cell resistance to PD1 / PDL1 therapy. Therefore, the anti-CD38 antibodies of the present invention, in combination with PD1 / PDL1 or other immune checkpoint-targeted therapies, have diagnostic and therapeutic applications not only in myeloma but also in all other types of cancer.

[0163] (Antibody Compositions for In Vivo Administration) The antibody used in the present invention can be prepared into a lyophilized formulation or an aqueous solution formulation for storage by mixing the antibody of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (see Remington's Pharmaceuticals, Inc. Sciences 16th edition, Osol, A. Ed.

[1980] . Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, for example, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkylparabens such as methylparaben and propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin, and immunoglobulins; and polyvinylpyrrolide. amino acids, such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other sugars, such as glucose, mannose, and dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions, such as sodium; metal complexes, such as zinc-protein complexes, and / or nonionic surfactants, such as TWEEN™, PLURONICS™, and polyethylene glycol (PEG).

[0164] The formulations herein may contain multiple active compounds as needed for the particular indication being treated, preferably with compounds having complementary activities that do not adversely affect each other. For example, antibodies with different specificities may be provided. Alternatively or additionally, the compositions may contain cytotoxic agents, cytokines, growth inhibitory agents, and / or small molecule antagonists. Such molecules are present in the combination in amounts effective for the intended purpose.

[0165] The active ingredient can be embedded in microcapsules prepared, for example, by aggregation techniques or interfacial polymerization, and can be embedded in, for example, hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or microemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0166] Formulations for in vivo administration must be sterile or nearly sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0167] The antibody can be prepared as a sustained-release formulation. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody. The matrices are 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) and poly(vinyl alcohol)), polylactide (U.S. Patent 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™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While certain polymers, such as ethylene-vinyl acetate and lactic acid-glycolic acid, can continuously release molecules for, for example, 100 days or more, certain hydrogels release proteins over a relatively short period of time.

[0168] If encapsulated antibodies remain in the body for a long time, they may denature or aggregate due to exposure to the humid environment at 37°C, resulting in loss of biological activity or altered immunogenicity. Rational strategies for stability can be designed based on the mechanism involved. For example, if the aggregation mechanism is thought to be the exchange of thio-disulfide bonds to form intermolecular S—S bonds, stabilization can be achieved through modification of sulfhydryl residues, lyophilization from acidic solution, control of water content, use of appropriate additives, and development of special polymer matrix compositions.

[0169] Administration Methods The antibodies or chemotherapeutic agents of the present invention are administered to a subject according to known methods, for example, intravenously by bolus injection or continuously infused over a period of time via intramuscular, intraperitoneal, intracerebral (intrabrospinal), subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Preferably, it is administered intravenously or subcutaneously.

[0170] (Treatment method) In the methods of the present invention, treating refers to providing a positive therapeutic response to a disease or disorder. A "positive therapeutic response" refers to an improvement in the disease or disorder and / or an improvement in symptoms associated with the disease or disorder. For example, a positive therapeutic response refers to one or more improvements in the disease, such as (1) a decrease in tumor cell count, (2) an increase in tumor cell death, (3) an inhibition of tumor cell survival, (5) an inhibition (i.e., a slowing to some extent, and preferably a halt) of tumor growth, (6) an improvement in patient survival, or (7) an alleviation of one or more symptoms associated with the disease or disorder.

[0171] Standardized response criteria for a particular disease or disorder can be used to determine a positive therapeutic response in any given disease or disorder. Tumor response can be assessed by changes in tumor morphology (i.e., overall tumor burden, tumor size, etc.) using, for example, magnetic resonance imaging (MRI), radiography, computed tomography (CT), bone imaging, endoscopy, and tumor biopsy sampling (including bone marrow aspirate (BMA) and circulating tumor cell counts).

[0172] In addition to these positive therapeutic responses, subjects undergoing treatment may benefit from an improvement in symptoms associated with the disease.

[0173] Thus, in the case of B-cell neoplasms, subjects may experience a reduction in so-called B symptoms (i.e., night sweats, fever, weight loss, and / or hives). In the case of pre-malignant symptoms, treatment with anti-CD38 therapeutic agents may block and / or prolong the time to progression of associated malignant symptoms, such as progression of multiple myeloma in subjects with monoclonal gammopathy of undetermined significance (MGUS).

[0174] Disease improvement can be characterized as a complete response, which means the absence of clinically detectable disease and, in the case of myeloma, normalization of previously abnormal radiographic studies, bone marrow and cerebrospinal fluid (CSF), or abnormal monoclonal proteins.

[0175] Following treatment with the methods of the present invention, such a response may persist for at least 4-8 weeks, and may persist for 6-8 weeks. Alternatively, disease improvement may be classified as a partial response. By "partial response," we mean a reduction in the total measurable tumor burden (i.e., the number of malignant cells present in a subject, or the volume of measured tumor masses or the number of abnormal monoclonal proteins) of at least about 50%, sustained for 4-8 or 6-8 weeks, in the absence of new lesions.

[0176] Treatment according to the present invention involves the use of a drug in a "therapeutically effective amount." A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.

[0177] A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. A therapeutically effective amount may be one in which any toxic or detrimental effects of the antibody or antibody portion are offset by the therapeutically beneficial effects.

[0178] A "therapeutically effective amount" for tumor therapy may be measured by its ability to stabilize the progression of the disease. Its efficacy against human tumors can be predicted by assessing the compound's ability to inhibit cancer in animal model systems.

[0179] Alternatively, the properties of such compositions may be evaluated by examining the ability of the compound to inhibit cell proliferation or induce apoptosis by in vitro assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject. A therapeutically effective amount can be determined by one of skill in the art 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.

[0180] The optimum desired response (e.g., therapeutic response) can be provided by adjusting the dosage regimen. For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suited to unitary dosages for the subjects 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.

[0181] The specifications for the dosage unit forms of the present invention are dictated by or 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 technology of formulating such compounds used to treat individual susceptibilities.

[0182] The effective dose and dosage regimen of the anti-CD38 antibodies used in the present invention will depend on the disease or disorder being treated and can be determined by one skilled in the art.

[0183] Exemplary, non-limiting ranges of therapeutically effective amounts of anti-CD38 antibodies used in the present invention include, for example, about 0.1 to 100 mg / kg, about 0, 1 to 50 mg / kg, about 0.1 to 20 mg / kg, about 0.1 to 10 mg / kg, about 0.5, about 0.3 mg / kg, about 1 mg / kg, or about 3 mg / kg. In another embodiment, the antibody is administered at a dose of 1 mg / kg or more, a dose of 1 to 20 mg / kg, a dose of 5 to 20 mg / kg, or a dose of 8 mg / kg, for example.

[0184] The effective amount of the pharmaceutical composition required can be readily determined and prescribed by a physician of ordinary skill in the art. For example, a physician or veterinarian can gradually increase the dosage of the drug to achieve the desired therapeutic effect, starting from a level lower than the level of the pharmaceutical composition required, up to a dose that achieves the desired effect.

[0185] In one embodiment, the anti-CD38 antibody is administered by infusion at a weekly dose of 10 to 500 mg / kg (e.g., 200 to 400 mg / kg). Such administration may be repeated 1 to 8 times (e.g., 3 to 5 times). Administration may be by continuous infusion over 2 to 24 hours (e.g., 2 to 12 hours).

[0186] In one embodiment, the anti-CD38 antibody is administered by slow continuous infusion over an extended period of time (eg, more than 24 hours) as needed to reduce side effects, including toxicity.

[0187] In one embodiment, the anti-CD38 antibody is administered in a weekly dose of 250 mg to 2000 mg (e.g., 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg, or 2000 mg) up to 8 times (e.g., 4 to 6 times). Administration can be by continuous infusion over 2 to 24 hours (e.g., 2 to 12 hours). Such a regimen may be repeated one or more times as needed, e.g., after 6 or 12 months. Dosage can be determined or adjusted by measuring the amount of a compound of the invention in the blood after administration, e.g., by removing a biological sample and using an anti-idiotypic antibody targeted to the antigen-binding region of the anti-CD38 antibody.

[0188] In another embodiment, the anti-CD38 antibody is administered once a week for, for example, 2 to 12 weeks, 3 to 10 weeks, or 4 to 8 weeks.

[0189] In one embodiment, the anti-CD38 antibody is administered as a maintenance treatment, eg, once weekly, for six months or more.

[0190] In one embodiment, the anti-CD38 antibody is administered in a regimen comprising a single infusion of the anti-CD38 antibody, followed by an infusion of the anti-CD38 antibody conjugated to a radioisotope, which can be repeated, for example, 7-9 days later.

[0191] Treatment according to the present invention may involve, by way of non-limiting example, a daily antibody dose in an amount of about 0.1 to 100 mg / kg, specifically, for example, 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, provided every 24, 12, 8, 6, 4, or 2 hours, and any combination thereof, using single or divided doses. and at least once at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after initiation of treatment, or at least once at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 16, 17, 18, 19, or 20 weeks after initiation of treatment, or any combination thereof.

[0192] In some embodiments, the anti-CD38 antibody molecule is used in combination with one or more other therapeutic agents (e.g., 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, semustine, streptozotocin, dacarbazine, methicillin-resistant Staphylococcus aureus ... These include cyclophosphamide, trehalose, mitomycin C, and cyclophosphamide), DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin), DNA intercalators and free radical generators such as bleomycin, and nucleoside analogs (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

[0193] Chemotherapeutic agents that disrupt 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 methanesulfonate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κB inhibitors, including IκB kinase inhibitors; antibodies that bind to proteins overexpressed in cancer and downregulate cell replication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab); and inhibitors of proteins or enzymes that are upregulated, overexpressed, or activated in cancer, whose inhibitory effects downregulate cell replication.

[0194] In some embodiments, antibodies of the present invention may be used before, concurrently with, or after Velcade® (bortezomib) treatment.

[0195] (diagnostic use) The anti-CD38 antibodies provided in this disclosure 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 diagnostic and therapeutic purposes, or for diagnostic purposes only.

[0196] In many embodiments, diagnostic antibodies are labeled. As used herein, "labeled" refers to an antibody disclosed herein having one or more elements, isotopes, or chemical compounds attached thereto to enable detection in screening or diagnostic procedures. Labels generally fall into the following categories: a) immunolabels, which can be incorporated into an epitope of a fusion partner recognized by the antibody; b) isotopic labels, which can be radioactive or heavy isotopes; c) small molecule labels, such as biotin molecules, which can include fluorescent or colorimetric dyes or allow other labeling methods; and d) paramagnetic labels, such as particle labels (including gas bubbles for ultrasound labeling) or paramagnetic labels, which allow imaging of the human body. Labels can be incorporated into antibodies at any position, as known in the art, and can be incorporated in vitro or in vivo during protein expression.

[0197] Diagnosis can be performed in vivo by administering diagnostic antibodies that allow for whole-body imaging, as described below, or in vitro on a sample removed from the patient. As used herein, the term "sample" encompasses various forms of matter, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen) and tissue samples from biopsies from relevant tissues.

[0198] 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 those that use optical markers for tumors near the surface of the body.

[0199] In vivo imaging of CD38-associated diseases can be performed by any suitable technique, for example, 99Anti-CD38 antibodies are labeled with a Tc label or another beta-emitting isotope label. Some variations of this technique include the use of magnetic resonance imaging (MRI) to improve imaging with gamma camera technology. Similar immunoscintigraphic 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" is described in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro et al. (eds), pp. 624-652 (Mack Publishing Co., 1990). Brown's "Clinical Uses of Monoclonal Antibodies" is described in Biotechnology and Pharmacy 227-49, Pezzuto et al. (eds) (Chapman & Hall 1993).

[0200] In one embodiment, the invention provides an in vivo imaging method in which an anti-CD38 antibody is conjugated to a detection-enhancing agent, the conjugated antibody is administered to a host, for example, by injection into the bloodstream, and the presence and location of the labeled antibody in the host is determined. Through this technique and other diagnostic methods provided herein, the invention provides a method for screening for the presence of disease-associated cells in a human patient or a biological sample obtained from a human patient.

[0201] For diagnostic imaging, radioisotopes are used either directly or indirectly via an intermediate functional group to deliver anti-CD38 antibodies. The CD38 antibody can be attached to a target substance. Useful intermediary functional groups include chelating agents such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, e.g., U.S. Patent No. 5,057,313). In diagnostic assays involving anti-CD38 antibodies conjugated to radioisotopes, the dose of conjugated anti-CD38 antibody delivered to the patient is typically kept as low as possible, which can be accomplished by selecting an isotope with the optimal combination of shortest half-life, shortest in vivo retention, and smallest isotope amount that can be detected and accurately measured.

[0202] In addition to radioisotopes and radio-opaque agents, anti-CD38 antibodies conjugated with dyes (e.g., biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancing agents (e.g., paramagnetic ions) may be used for magnetic resonance imaging (MRI) diagnostics (see, e.g., U.S. Patent No. 6,331,175, which describes MRI techniques and the preparation of antibodies conjugated with MRI enhancing agents). Such diagnostic / detection agents can be selected from reagents and fluorescent compounds used in magnetic resonance imaging.

[0203] To load an anti-CD38 antibody with a radiometal or paramagnetic ion, it may be necessary to react it with a reagent having a long chain bearing multiple chelating groups for binding the ions. Such chains may be polymers (e.g., polylysine, polysaccharides) or other derivative or derivatizable chains having side groups capable of binding to chelating groups. Chelating groups include porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and similar groups known for this purpose.

[0204] Chelates can be conjugated to anti-CD38 antibodies using standard chemical methods. Typically, the chelate is linked to the anti-CD38 antibody via a moiety that allows for the formation of a bond with the molecule and that has minimal loss of immunoreactivity, minimal aggregation and / or internal cross-linking.

[0205] Examples of potentially useful metal chelating compositions include 2-benzyl DTPA and its monomethyl and cyclohexyl analogs for radiation imaging. 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 It is used with diagnostic isotopes in the general energy range of 60 keV to 4,000 keV, such as Br.

[0206] Labels include radionuclides, radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photosensitizers. Such diagnostic agents are well known, and any such known diagnostic agent can be used. Non-limiting examples of diagnostic agents include, for example: 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 Radionuclides such as Sr or other gamma-, beta- or positron emitters are included.

[0207] Paramagnetic ions used include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(III), copper(III), neodymium(III), samarium(III), yterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III). Metallic contrast agents include lanthanum(III), gold(III), lead(II), or bismuth(III).

[0208] Ultrasound contrast agents include liposomes, such as gas-filled liposomes.Radiopaque diagnostic agents can be selected from compounds such as barium compounds, gallium compounds and thallium compounds.

[0209] These and similar chelates, when formulated with non-radioactive metals (e.g., manganese, iron, and gadolinium), can be used in MRI diagnostic procedures involving anti-CD38 antibodies. Macrocyclic chelates (e.g., NOTA, DOTA, and TETA) can be used with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium, and copper, respectively. Such metal chelate complexes can be made highly stable by adapting the ring size to the metal of interest. The nuclides (e.g., 223 Other cyclic chelates (eg, macrocyclic polyethers) that stably bind to Ra may also be applicable to diagnostic methods.

[0210] Thus, the present invention provides diagnostic anti-CD38 antibody conjugates that are linked to an imaging agent (e.g., a contrast agent used in magnetic resonance imaging, computed tomography, or ultrasound imaging) or a radionuclide (e.g., a gamma-, beta-, alpha-, Auger-, or positron-emitting isotope).

[0211] Anti-CD38 antibodies can also be used, for example, to detect the expression of an antigen of interest in specific cells, tissues, or serum. For diagnostic applications, the antibody is usually labeled with a detectable moiety and used for in vitro assays. Those skilled in the art will recognize that a variety of suitable labels are available for in vitro assays. Dyes suitable for use in this aspect of the invention include, but are not limited to, fluorescent lanthanide complexes (including those containing europium and terbium), fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, quantum dots (also called nanocrystals, see U.S. Serial No. 09 / 315,584, incorporated herein by reference), pyrene, malachite green, stilbene, firefly yellow, Cascade Blue™, Texas Red, Cy dyes (Cy3, Cy5, etc.), alexa dyes (Alexa, phycoerythrin, bodipy), and other dyes described in "Molecular Probes Handbook," 6th Edition, edited by Richard P. Haugland, incorporated herein by reference.

[0212] The radioactivity of the stained tissue can be assessed as an indicator of the CD38-related peptide content in the tumor. Images obtained using such techniques can be used to assess the biodistribution of CD38 in patients, mammals, or tissues, for example, using CD38 as a biomarker for the presence of invasive cancer cells.

[0213] (product) In another embodiment, an article of manufacture containing materials useful for treating the above-described diseases is provided, the article comprising a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed from a variety of materials, such as glass or plastic. The container contains a composition effective for treating the disease and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The active agent in the composition is an antibody. A label on or associated with the container indicates that the composition is used to treat the selected disease. The article of manufacture can further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. From a business and user perspective, the article of manufacture may also include other necessary materials, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. [Example]

[0214] The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but should be construed to encompass any and all variations that become apparent from the teachings provided herein.

[0215] It is believed, however, that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, practice the claimed methods of making and using the present invention. Accordingly, the following examples specifically set forth certain preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0216] [Example 1]: Screening of yeast-displayed human scFv library 1×10 11An initial human scFv library was constructed using yeast display, and the extracellular domain of human CD38 was purchased from ACRO biosystems. The library screening method was described previously (Zhao et al., J Immunol Methods. 2011;363(2):221-32). Briefly, recombinant biotinylated CD38-avi protein was incubated with the induced yeast display scFv library. Yeast cells that bound to the isolated CD38 were separated using streptavidin (SA)-conjugated microbeads followed by flow cytometer-activated cell sorting (FACS). The identified scFvs were engineered into intact antibodies and expressed in 293F cells.

[0217] ( Yeast display scFv library screening using magnetic beads The yeast display scFv library was thawed from -80°C and centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded, and the yeast cells were resuspended in 12 L of SD-CAA medium (1 liter of SD-CAA medium contains 5 g of casein amino acids, 1.7 g of yeast nitrogen base without ammonium sulfate and amino acids, 5.3 g of ammonium sulfide, 10.2 g of NaHPO·7H2O, 8.6 g of NaHPO·H2O, and 20 g of dextrose). The cells were grown overnight at 30°C with shaking at 200 rpm. The next day, the yeast cells were harvested by centrifugation at 3000 rpm for 5 minutes. An appropriate amount of yeast cells was resuspended in 12 L of S-CAA-GRD induction medium (1 L of S-CAA-GRD medium contains 5 g of casein amino acids, 1.7 g of ammonium sulfate and yeast nitrogen base without amino acids, 5.3 g of ammonium sulfate, 10.2 g of NaHPO 7H O, 8.6 g of NaHPO H O, 1 g of dextrose, 20 g of galactose, and 20 g of raffinose) to a final culture density of 0.5, and induced overnight at 20°C. The induced yeast cells were harvested by centrifugation at 3000 rpm for 5 minutes, washed twice with 2 L of PBE buffer (PBE buffer is a PBS buffer containing 2 mM EDTA and 0.5% BSA), and finally resuspended in 200 ml of PBE. Cells were incubated with 40 μg of biotinylated CD38 protein at room temperature (RT) for 1.5 hours, followed by 0.5 hours at 4°C. The following steps were performed at 4°C or on ice. Cells were collected by centrifugation at 3000 rpm for 5 minutes, washed twice with 2 L of PBE, and resuspended in 200 ml of PBE. Next, 2 ml of streptavidin microbeads (Miltenyi Biotec) were added to the cells and incubated with gentle shaking for 1 hour. One liter of PBE was added to the cells, shaken to disperse the cells into single cells, and filtered through a 70 μm filter. CD38-bound yeast cells were isolated using an AUTOMACS instrument. The collected cells were plated on SD-CAA plates and cultured at 30°C for 2 days. A total of 2.5 × 10 7Clones were obtained. Cells were scraped and induced for a second round of magnetic bead sorting. A portion of the cells was diluted in 10% glycerol The mixture was placed in SD-CAA containing HCl and stored at -80°C.

[0218] ( Yeast display scFv library screening using flow sorting Yeast cells obtained from magnetic bead sorting were further separated by flow sorting. Unless otherwise specified, all centrifugation was performed at 3000 rpm for 5 minutes, and all steps were carried out at 4°C or on ice. 2 × 10 cells separated from magnetic bead sorting were collected. 9 1 × 10 cells were induced in 100 ml of S-CAA-GRD medium overnight at 20 °C, and then 1 × 10 cells were cultured. 8 Cells were harvested and used for flow sorting. Cells were pelleted and washed twice with 15 ml of PBE, then resuspended in 1 ml of PBE and incubated with 0.2 mg of biotinylated CD38 protein at room temperature for 1.5 hours, followed by incubation at 4°C for 30 minutes. Cells were washed three times with PBE and then incubated with 50 μl of avidin-PE (Invitrogen) in 1 ml of PBE at 4°C in the dark for 1 hour. After staining, cells were washed three times with 15 ml of PBE and resuspended in 1 ml of PBE. Yeast cells that bound to CD38 were sorted by flow cytometry. The sorted cells were grown on SD-CAA plates at 30°C for 2 days.

[0219] Single clones were picked, expanded in 96-deep-well plates, and induced to express scFv. Single clones that specifically bound to CD38 were identified by flow cytometry.

[0220] [Example 2]: Modification of scFv418 to IgG418 Based on the V-base and IMGT databases, the germline sequences of the heavy and light chains of scFv418 were identified, and a signal peptide and constant region were added to the variable region to construct genes encoding the full-length heavy and light chain peptides. The heavy and light chain genes were cloned into a self-assembled intact antibody expression vector, Lh1, and expressed in 293F cells. The antibodies were purified using Protein A affinity chromatography.

[0221] [Example 3]: Measurement of IgG418 affinity by capture ELISA The affinity of IgG418 and Darzalex for CD38 recombinant protein was measured by capture ELISA. Briefly, anti-human Fc antibody was coated onto ELISA plates at a concentration of 10 μg / ml overnight at 4°C. The plates were washed twice with PBST, blocked for 2 hours at room temperature with PBS™, and incubated with 3 parts IgG418 diluted 4-fold from 50 nM to 0.012 nM. After washing six times with PBST, the plates were incubated with 0.5 μg / ml biotinylated CD38-avi recombinant protein in PBS™ for 1 hour at room temperature. After six washes, the plates were incubated with 1:1000 diluted streptavidin-HRP (BD Bioscience) in PBS™ for 30 minutes at room temperature. The plates were washed an additional six times and incubated for 20 minutes at room temperature with TMB. The colorimetric reaction was stopped with stop buffer, and the absorbance was read at OD450. The affinities were calculated using GraphPad Prism software, with Kd = 1.64 nM for Darzalex and Kd = 0.082 nM for IgG418 (Figure 1). Table 1 shows the ELISA measurements.

[0222] [Table 1]

[0223] [Example 4]: Measurement of IgG418 affinity by flow cytometry Using Darzalex as a control, the affinity of IgG418 for CD38 in its native conformation on the Daudi cell surface was measured by flow cytometry. Daudi cells were washed twice with PBS and incubated on ice for 1 hour with serially diluted antibodies in FACS buffer (PBS containing 2% FBS) as shown in Table 2. The cells were washed three times with PBS and incubated with anti-human IgG-Alexa647 diluted 1:200 in FACS buffer at 4°C for 30 minutes, protected from light. After washing three times with PBS, the cells were analyzed using a flow cytometer. Table 2 shows the antibody concentration and mean fluorescence readings for each sample. Affinities were calculated using GraphPad Prism software: Kd = 3.256 nM for Darzalex and Kd = 3.1 nM for IgG418 (Figure 2).

[0224] [Table 2]

[0225] Example 5: Using competitive ELISA to identify whether IgG418 shares the same binding epitope as Darzalex ELISA plates were coated with 2 μg / ml Darzalex at 50 μl / well (in PBS) overnight at 4°C. The plates were then blocked with PBS™ at room temperature for 2 hours. The ELISA wells were then incubated with either CD38 or antibody-CD38 complexes, which were obtained by pre-incubating the antibody (15 μg / ml) with CD38 (0.2 μg / ml) for 1 hour at room temperature. After 30 minutes of incubation at 4°C, the plates were washed with PBST and incubated with streptavidin-HRP for 30 minutes at room temperature. The plates were washed six times again and incubated with TMB for 20 minutes at room temperature. The colorimetric reaction was stopped with stop buffer, and the absorbance was read at OD450 (Figure 3). Competitive ELISA showed that IgG418 did not compete with Darzalex for the binding site on CD38, indicating that the epitopes are different from each other.

[0226] Example 6: CDC Activity of IgG418 Complement-dependent cytotoxicity (CDC) is one of the major mechanisms by which antibodies kill antigen-positive cells (e.g., tumor cells or pathogenic plasma cells). For CDC activity analysis of IgG418 and Darzalex, antibodies were serially diluted two-fold in complete medium in a 96-well plate from 20 nM to approximately 0.078 nM, 100 μl / well. To prevent evaporation, the surrounding wells were filled with 250 μl / well of water. To reduce aggregation after adding human serum, the plate was preheated in an incubator. Human serum was thawed and centrifuged at 6000 rpm for 5 minutes to remove aggregates. One volume of human serum was diluted with nine volumes of complete medium (containing 10% serum, equivalent to 2x), and 4 × 10 Daudi cells were cultured with the 1:9 diluted human serum. 4 The cells were resuspended at a density of 1 / 100 μl. Next, 100 μl of cells were added to each antibody-containing well (the total volume was 200 μl, since 100 μl of medium was already added) and incubated at 37°C for 2 hours. 7AAD was diluted 10-fold in complete medium, and 50 μl of the diluted 7AAD was added to each well and incubated for 5–10 minutes in the dark. The cells were transferred to 1.5 ml test tubes, and the samples were measured using an Accuri C6 flow cytometer. Figures 4-1, 4-2, and 4-3 show CDC data detected by flow cytometry. Here, only P1 were live cells (7AAD negative), P2 were dead cells that had completely released all cytoplasm and were therefore 7AAD negative (7AAD bound to DNA), and P3 were recently dead cells that had released some of their cytoplasm and were therefore 7AAD positive. Therefore, the percentage of P1 was analyzed to assess CDC activity. Figure 5 shows the statistical analysis of the CDC data. IC of IgG418, Darzalex, and IgG207 50 were 9.8 nM, 29.8 nM and 30459 nM, respectively. IgG207 was the negative control antibody.

[0227] [Table 3]

[0228] Example 7: Production of Defucosylated IgG418 Antibody-dependent cellular cytotoxicity (ADCC) is another important mechanism by which antibodies kill antigen-positive cells. It is recognized in the art that glycosylated antibodies at 297N may affect the efficacy of ADCC. Defucosylated antibodies generally have 10- to 100-fold enhanced ADCC activity. To further enhance ADCC activity, we produced defucosylated IgG418 using the FUT8 knockout CHO-K1 cell line from Antagen Pharmaceuticals Inc., and named it IgG418 AF.

[0229] Example 8: ADCC activity of IgG418 The ADCC activity of Darazalex and wild-type (WT) and defucosylated (AF) IgG418 was measured using a Jurkat cell line developed by Antagen Pharmaceuticals, which was engineered to use luciferase signal as an indicator of ADCC activity, as effector cells. ADCC measurements were performed on Daudi cells. Table 4 shows the antibody dilution concentrations and the corresponding luciferase measurements. As a result, wild-type IgG418 (WT) had a maximum ADCC activity 1.4-fold higher than Darazalex, and defucosylated IgG418 (AF) had a maximum ADCC activity 1.6-fold higher than Darazalex, with IC 50 was reduced by approximately 10-fold (Table 4 and Figure 6).

[0230] [Table 4]

[0231] Example 9: Inhibitory effect of IgG418 on tumor growth in SCID mice xenografted with human B-cell lymphoma Daudi CB-17 SCID mice, SPF grade, 16.6-21.5 g, male, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Daudi cells (Nanjing Kebai, Cat. No.: CBP60262) were cultured in 1640 complete medium (Hyclone, Cat. No.: SH30809.01), 10% FBS (Hyclone, Cat. No.: SH30087.03), 100 U / mL penicillin, and 100 μg / mL streptomycin (Hyclone, Cat. No.: SV30010) and maintained in a 5% CO -saturated humidity incubator at 37°C. Daudi cells in the logarithmic growth phase were collected, resuspended in 1640 complete medium, and Matrigel was added at a 1:1 ratio to a cell concentration of 2 × 10 7 Under sterile conditions, 0.1 ml of the cell suspension was diluted to 2 × 10 6 The tumor was subcutaneously inoculated into the right dorsal region of SCID mice at an inoculation concentration of 0.1 mL / mouse. 14 days after inoculation, tumor volumes were 100-200 mm 3 When tumor volume reached this level, the animals were randomly divided into four groups, each with nine animals, with the difference in tumor volume between groups being less than 10% of the mean. The day of grouping was designated Day 0. Group 1: Isotype control antibody (10 mg / kg) Group 2: Darazalex (10mg / kg) (JBS2Y20 Xi'an Yangsen Pharmaceutical Co., Ltd.) Group 3: IgG418-WT (10mg / kg) Group 4: IgG418-AF (10 mg / kg)

[0232] The experimental period was 31 days. Animal weights and tumor volumes were measured twice a week and recorded. Clinical symptoms were observed and recorded once a day. After the administration was completed, the mice were sacrificed and tumors were harvested.

[0233] Tumor volume (TV) was calculated using the formula: 1 / 2 × a × b², where a and b were the measured length and width of the tumor, respectively. Relative tumor volume (RTV) was calculated using the formula: Vt / V0, where V0 was the tumor volume at the time of grouping, and Vt was the tumor volume at each measurement. Tumor inhibition rate (TGI%) was calculated using the formula: (TWC-TWT) / TWC × 100%, where TWC was the mean tumor weight in the negative control group, and TWT was the mean tumor weight in the treatment group. Graphical analysis (mean ± SEM) was performed using Prism GraphPad graphing software. P values ​​between groups were statistically analyzed using the t-test. A difference of p<0.05 was considered significant between groups. A difference of p<0.01 was considered highly significant between groups. The results are shown in Table 5 and Figure 7.

[0234] [Table 5]

[0235] In vivo drug efficacy experiments in mice demonstrated that the humanized antibodies IgG418-AF and IgG418-WT of the present disclosure had significant inhibitory effects on a SCID mouse tumor model xenografted with the human lymphoma Daudi compared with the negative control isotype. Compared with the negative control isotype (10 mg / kg) group, the IgG418-AF (10 mg / kg) group and the IgG418-WT (10 mg / kg) group achieved tumor inhibition rates of 96% and 88%, respectively, at dose D21. These tumor inhibition rates were significantly higher than those of the positive control Darzalex (10 mg / kg) group (TGI: 79%), demonstrating superior tumor inhibition effects compared with Darzalex. The body weights of animals in each group were not significantly affected during the experimental administration period, suggesting that the antibodies of the present disclosure did not have any significant toxicity or side effects.

[0236] [Example 10]: Dose-dependent inhibitory effect of IgG418 on tumor growth in SCID mice xenografted with human B-cell lymphoma Daudi The experimental procedures and tumor measurements were the same as in Example 9. Group 1: Isotype control antibody (1 mg / kg) Group 2: Darazalex (1 mg / kg) (JBS2Y20 Xi'an Yangsen Pharmaceutical Co., Ltd.), Group 3: IgG418-WT (1mg / kg) Group 4: IgG418-AF (0.1 mg / kg) Group 5: IgG418-AF (0.3 mg / kg) Group 5: IgG418-AF (1 mg / kg) The results are shown in Table 6 and FIG.

[0237] [Table 6]

[0238] In vivo drug efficacy experiments in mice demonstrated that the humanized antibodies IgG418-AF and IgG418-WT of the present disclosure had significant inhibitory effects on tumor models in SCID mice xenografted with the human lymphoma Daudi compared with the negative control isotype. Compared with the negative control isotype (1 mg / kg) group, the IgG418-AF (1 mg / kg) group and the IgG418-WT (1 mg / kg) group achieved tumor inhibition rates of 76% and 84%, respectively, at dose D21. These tumor inhibition rates were significantly higher than those of the positive control Darazalex (1 mg / kg) group (TGI: 61%), demonstrating superior tumor inhibition compared with Darazalex. Furthermore, the tumor inhibition effect of each IgG418-AF treatment group was clearly dose-dependent, and the body weight of the animals in each group was not significantly affected during the experimental treatment period. These results suggest that the antibodies of the present disclosure have no significant toxicity or side effects.

[0239] The disclosures of each patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to certain specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the present invention may be designed without departing from the true spirit and scope of the invention. It is intended that the claims be construed to include all such embodiments and all equivalents. The sequences listed in this application are as follows:

Claims

1. An anti-CD38 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a complementarity-determining region 1 (CDR1), a complementarity-determining region 2 (CDR2), and a complementarity-determining region 3 (CDR3) of the heavy chain variable region; CDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9; CDR2 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13; and CDR3 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17; the light chain variable region comprises a complementarity-determining region 1 (CDR1), a complementarity-determining region 2 (CDR2), and a complementarity-determining region 3 (CDR3) of the light chain variable region; CDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; CDR2 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29; and CDR3 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:

33. An anti-CD38 antibody or an antigen-binding fragment thereof.

2. An anti-CD38 antibody or an antigen-binding fragment thereof described in claim 1, wherein the anti-CD38 antibody or an antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

5.

3. An anti-CD38 antibody or an antigen-binding fragment thereof described in claim 1, wherein the anti-CD38 antibody or an antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:

3.

4. The anti-CD38 antibody or its antigen-binding fragment described in claim 2, wherein the anti-CD38 antibody is an antibody that specifically binds to CD38 selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

5. An anti-CD38 antibody or antigen-binding fragment thereof described in claim 2, which is an antibody or antigen-binding fragment thereof that comprises part or all of the sequence of a λ or κ light chain constant region or a mutant thereof.

6. An anti-CD38 antibody or an antigen-binding antibody fragment thereof described in claim 1, wherein the anti-CD38 antibody or an antigen-binding fragment thereof is a human antibody, a humanized antibody, or a chimeric antibody or an antigen-binding fragment thereof.

7. A medicinal composition for treating a disease associated with CD38 expression, comprising an anti-CD38 antibody or its antigen-binding antibody fragment according to any one of claims 1 to 6, wherein the disease is a proliferative disease or an autoimmune disease.

8. A medicinal composition as described in claim 7, wherein the disease is cancer.

9. The medicinal composition according to claim 7, wherein the disease is an autoimmune disease.

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