Variant antibodies that bind to CD38

Abstract

The present disclosure provides anti-CD38 IgG class antibodies with improved ability to be produced in high yields compared to parent antibodies with the original wild-type sequence. The present disclosure provides mutated antibody light chains that reduce cleavage heterogeneity for improved production of a homogeneous population of antibody light chains. The present disclosure provides antigen-binding proteins that bind to CD38, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 1 and the light chain comprises the sequence of SEQ ID NO: 3. Such antigen-binding proteins may be, for example, single-chain antibodies, which may be fully human.

Description

[Technical field]

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Patent Application No. 62 / 687,695, filed June 20, 2018, and entitled "Improved Variant Antibodies That Bind to CD38," the entire contents of which are incorporated herein by reference.

[0002] Various publications, patents, and / or patent applications are referenced throughout this application. The disclosures of the publications, patents, and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains. In the event that any matter incorporated by reference conflicts with the express content of this application, the express content shall control.

[0003] The present disclosure provides anti-CD38 IgG class antibodies having variant light chains compared to the wild-type sequence. The disclosed antibodies may have improved ability to be produced in high yields compared to wild-type antibodies, and / or reduced cleavage heterogeneity, which may improve the homogeneity of the antibody light chain. [Background technology]

[0004] CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein binds NADPH to cyclic ADP-ribose (cADPR). + During ontogeny, CD38 is a bifunctional ectoenzyme that can catalyze the conversion of cADPR to ADP-ribose and also hydrolyze cADPR to ADP-ribose. + It appears on committed stem cells and lineage-committed progenitors of lymphoid, erythroid and myeloid cells. CD38 expression persists largely in the lymphoid lineage, with differential levels of expression during different stages of T and B cell development.

[0005] CD38 is upregulated in many hematopoietic malignancies and cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), chronic B-lymphocytic leukemia (B-CLL), acute B- and T-lymphocytic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). On the other hand, most primitive pluripotent stem cells of the hematopoietic system express CD38. - CD38 expression in hematopoietic malignancies and its correlation with disease progression makes CD38 an attractive target for anti-CD38 antibody therapy.

[0006] CD38 is Ca 2+mobilization (Morra et al., 1998, FASEB J., 12:581-592; Zilber et al., 2000, Proc. Natl. Acad. Sci. USA, 97:2840-2845), and signaling through tyrosine phosphorylation of a number of signaling molecules, including phospholipase C-gamma, ZAP-70, syk, and c-cbl, in lymphoid and myeloid cells or cell lines (Funaro et al., 1993, Eur. J. Immunol., 23:2407-2411; Morra et al., 1998, FASEB J., 12:581-592; Funaro et al., 1990, J Immunol, 145:2390-2396; Zubiaur et al., 1997, J Immunol., 145:2390-2396; Zubiaur et al., 1997, J Immunol., 145:2390-2396). Immunol, 159:193-205;Deaglio et al., 2003, Blood 102:2146-2155;Todisco et al., 2000, Blood, 95:535-542;Konopleva et al., 1998, J. Immunol., 161:4702-4708;Zilber et al. al., 2000, Proc. Natl. Acad. Sci. USA, 97:2840-2845;Kitanaka et al., 1997, J. Immunol., 159:184-192;Kitanaka et al., 1999, J. Immunol., 162:1952-1958;Mallone et al., 2001, Int. Immunol. 13:397-409). CD38 has been proposed to be a key signaling molecule in the maturation and activation of lymphoid and myeloid cells during normal development.

[0007] Evidence for the function of CD38 has been shown to be associated with defects in innate immunity, with defective dendritic cell migration resulting in reduced T cell-dependent humoral responses. - / -It is derived from knockout mice (Partida-Sanchez et al., 2004, Immunity, 20:279-291; Partida-Sanchez et al., 2001, Nat. Med., 7:1209-1216). However, the CD38 expression patterns during hematopoiesis are significantly different between humans and mice: a) unlike immature progenitor stem cells in humans, the similar progenitor stem cells in mice express high levels of CD38 (Randall et al., 1996, Blood, 87:4057-4067;Dagher et al., 1998, Biol. Blood Marrow Transplant, 4:69-74); b) during human B cell development, high levels of CD38 expression are found on germinal center B cells and plasma cells (Uckun, 1990, Blood, 76:1908-1923;Kumagai et al., 1995,J. Exp. Med., 181:1101-1110), whereas in mice the corresponding cells express lower levels of CD38 (Oliver et al., 1997, J. Immunol., 158:108-1115; Ridderstad and Tarlinton 1998, J. Immunol., 160:4688-4695), so it is unclear whether CD38 function in mice is identical to that in humans.

[0008] Several anti-human CD38 antibodies with different growth characteristics in various tumor cells and cell lines have been described in the literature. For example, chimeric OKT10 antibody with mouse Fab and human IgG1 Fc mediates antibody-dependent cell-mediated cytotoxicity (ADCC) against lymphoma cells very efficiently in the presence of peripheral blood mononuclear effector cells from MM patients or normal individuals (Stevenson et al., 1991, Blood, 77:1071-1079). CDR-grafted humanized version of anti-CD38 antibody AT13 / 5 has been shown to have strong ADCC activity against CD38-positive cell lines. Human monoclonal anti-CD38 antibodies have been shown to mediate in vitro killing of CD38-positive cell lines by ADCC and / or complement-dependent cytotoxicity (CDC) and to slow tumor growth in SCID mice carrying MM cell line RPMI-8226 (WO2005 / 103083A2). On the other hand, several anti-CD38 antibodies, except IB6, AT1, or AT2, IB4, SUN-4B7, and OKT10, induced proliferation of peripheral blood mononuclear cells (PBMCs) from normal individuals (Ausiello et al. 2000, Tissue Antigens, 56:539-547). Some of the prior art antibodies inhibit CD38 in a stromal cell-dependent or stromal-derived cytokine-dependent manner. +It has been reported that CD38 can trigger apoptosis in B cells. An agonistic anti-CD38 antibody (IB4) has been reported to prevent apoptosis in human germinal center (GC) B cells (Zupo et al. 1994, Eur. J. Immunol., 24:1218-1222) and induce proliferation of KG-1 and HL-60 AML cells (Konopleva et al. 1998, J. Immunol., 161:4702-4708), but induce apoptosis in Jurkat T lymphoblast cells (Morra et al. 1998, FASEB J., 12:581-592). Another anti-CD38 antibody, T16, induced apoptosis in immature lymphoid cells and leukemic lymphoblast cells from ALL patients (Kumagai et al. 1995, J. Exp. Med., 181:1101-1110) and leukemic myeloblast cells from AML patients (Todisco et al. 2000, Blood, 95:535-542), but only in the presence of stromal cells or stromal-derived cytokines (IL-7, IL-3, stem cell factor). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Zupo et al., Eur. J. Immunol. (1994) 24:1218-1222 [Non-Patent Document 2] Konopleva et al., J. Immunol. (1998) 161:4702-4708 [Non-Patent Document 3] Morra et al., FASEB J. (1998) 12:581-592 Summary of the Invention [Means for solving the problem]

[0010] Therefore, antibody drug conjugates (ADCs) targeted with anti-CD38 antibodies offer the promise and possibility of exerting strong antitumor activity with the advantage of reduced side effects.Therefore, there remains a need in the art for effective treatments based on CD38, especially anti-CD38 antibodies.The present disclosure provides variant antibody sequences that differ from the fully human wild-type sequence.The disclosed variant antibodies can be produced in much higher yields and / or provide improved light chain homogeneity.

[0011] The present disclosure provides an antigen-binding protein that binds to CD38, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO:1 and the light chain comprising the sequence of SEQ ID NO:3. Such an antigen-binding protein may be, for example, a single chain antibody, which may be fully human. The present disclosure provides a fully human antibody or antibody Fab fragment of the IgG class, comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 and a light chain comprising a light chain variable region having the amino acid sequence of SEQ ID NO:3. The present disclosure provides a nucleic acid encoding an antibody light chain comprising a light chain variable region having the amino acid sequence of SEQ ID NO:3, which may be combined with or further comprise a nucleic acid encoding an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:1.

[0012] The present disclosure also provides a population of variant antibodies of the IgG class, and a method for producing a population of variant antibodies. The population of variant antibodies includes two or more variant antibodies (e.g., a plurality of variant antibodies). Each individual variant antibody in the population includes an immunoglobulin having two antibody heavy chains and two antibody light chains having a variant sequence. The light chain variant sequence reduces cleavage heterogeneity during antibody preparation. For example, the population of variant antibodies can be prepared by host cell expression of recombinant antibody heavy and light chains. The light chain variant sequence can reduce the level of polypeptide length heterogeneity and the level of amino acid sequence heterogeneity by about 40-55% during antibody preparation, thereby reducing heterogeneity in the preparation of variant antibodies.

[0013] The present disclosure provides a population of variant antibodies of the IgG class that exhibit a reduced level of polypeptide length heterogeneity and a reduced level of amino acid sequence heterogeneity compared to a population of parent wild-type antibodies of the IgG class that exhibit a higher level of polypeptide length heterogeneity and a higher level of amino acid sequence heterogeneity. Each antibody in the population of variant antibodies comprises a heavy chain having a wild-type amino acid sequence and a light chain having a variant amino acid sequence. The variant amino acid sequence of the light chain can reduce the level of polypeptide length heterogeneity, reducing the level of amino acid sequence heterogeneity in the population of variant antibodies by about 40-55%.

[0014] In one embodiment, the population of variant antibodies comprises individual variant sequence antibodies having heavy chains comprising a heavy chain variable region having the wild-type amino acid sequence of SEQ ID NO:1, and individual variant antibodies having light chains comprising a light chain variable region having the variant amino acid sequence of SEQ ID NO:3.

[0015] In one embodiment, the population of variant antibodies binds to a CD38 epitope.

[0016] In one embodiment, the population of variant antibodies comprises fully human antibodies.

[0017] In one embodiment, each parent wild-type antibody in a population of parent wild-type antibodies comprises a light chain having a wild-type amino acid sequence that exhibits approximately 40-55% polypeptide length heterogeneity and amino acid sequence heterogeneity.

[0018] In one embodiment, each variant antibody in the population of variant antibodies comprises a light chain having a variant amino acid sequence that exhibits about 85-99%, 85-100%, 90-99%, or 90-100% polypeptide length homogeneity and amino acid sequence homogeneity. Homogeneity may be quantified as the percentage of members of the population (e.g., antibody light chains) that have a length and / or sequence that is more than or equal to the majority of the species (e.g., a light chain comprising a variable region having the sequence of SEQ ID NO:3).

[0019] In one embodiment, a population of variant antibodies of the IgG class exhibits a reduced level of polypeptide length heterogeneity and a reduced level of sequence heterogeneity compared to a population of parent wild-type antibodies of the IgG class that bind to a CD38 epitope and exhibit a higher level of polypeptide length heterogeneity and a higher level of sequence heterogeneity, wherein each antibody in the population of variant sequences comprises a heavy chain having a wild-type amino acid sequence and a light chain having a variant amino acid sequence, wherein the heavy chain of each variant antibody comprises a heavy chain variable region having the wild-type amino acid sequence of SEQ ID NO:1 and the light chain of each variant antibody comprises a light chain variable region having the variant amino acid sequence of SEQ ID NO:3, wherein the variant amino acid sequence of the light chain reduces the level of polypeptide length heterogeneity and the level of sequence heterogeneity by about 40-55%.

[0020] The present disclosure provides a population of fully human IgG class antibodies comprising a heavy chain having a wild-type heavy chain variable region amino acid sequence of SEQ ID NO:1 and a light chain having a light chain variable region variant amino acid sequence of SEQ ID NO:3.

[0021] The present disclosure provides a population of fully human Fab fragments comprising a heavy chain having a heavy chain variable region wild-type amino acid sequence of SEQ ID NO:1 and a light chain having a light chain variable region variant amino acid sequence of SEQ ID NO:3.

[0022] The present disclosure provides a population of antigen binding proteins (e.g., single chain antibodies, which may be fully human) comprising a heavy chain variable region wild-type amino acid sequence of SEQ ID NO: 1 and a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the single chain antibody comprises a heavy chain variable region and a light chain variable region linked together by a peptide linker.

[0023] The present disclosure provides a population of antigen binding proteins (e.g., fully human IgG class antibodies) comprising a heavy chain having a heavy chain variable region wild-type amino acid sequence of SEQ ID NO: 1, and a light chain comprising the amino acid sequence of SEQ ID NO: 2 with two point mutations near the N-terminus, specifically, alanine at position 2 is replaced with serine and glycine at position 3 is replaced with alanine. In one embodiment, the N-terminus of the mutant light chain contains the amino acid sequence QSALT (SEQ ID NO: 6).

[0024] The present disclosure provides a population of variant antibody light chains that exhibit a reduced level of polypeptide length heterogeneity and a reduced level of amino acid sequence heterogeneity compared to a population of parent wild-type antibody light chains that exhibit a higher level of polypeptide length and a higher level of amino acid sequence heterogeneity. Each antibody light chain in the population of variant antibody light chains comprises a light chain variable region having a variant amino acid sequence of SEQ ID NO:3.

[0025] In one embodiment, each variant sequence light chain in the population comprises a fully human antibody light chain.

[0026] In one embodiment, each parent wild-type sequence antibody light chain in a population of parent wild-type antibodies comprises a light chain variable region having a parent wild-type amino acid sequence that exhibits approximately 40-55% polypeptide length heterogeneity and amino acid sequence heterogeneity.

[0027] In one embodiment, each variant antibody in the population of variant antibodies comprises a light chain having a variant amino acid sequence that exhibits about 85-99%, 85-100%, 90-99%, or 90-100% polypeptide length homogeneity and amino acid sequence homogeneity.

[0028] In one embodiment, the variant antibody light chains of the IgG class exhibit reduced levels of polypeptide length heterogeneity and reduced levels of amino acid sequence heterogeneity compared to a population of parent wild-type antibodies having higher levels of polypeptide length heterogeneity and higher levels of amino acid sequence heterogeneity. Each antibody light chain in the population of variant antibody light chains comprises a light chain variable region having a variant amino acid sequence of SEQ ID NO: 3, and the variant amino acid sequence of the light chain reduces the level of polypeptide length heterogeneity and the level of amino acid sequence heterogeneity by about 40-55%.

[0029] In one embodiment, the fully human Fab fragment comprises a light chain having the light chain variable region variant amino acid sequence of SEQ ID NO:3.

[0030] In one embodiment, the single chain fully human antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO:3.

[0031] The present disclosure provides a fully human variant antibody of the IgG class that binds to a CD38 epitope, comprising a heavy chain having a heavy chain variable region having the wild-type amino acid sequence of SEQ ID NO:1 and a light chain having a light chain variable region having the variant amino acid sequence of SEQ ID NO:3.

[0032] The present disclosure provides a fully human antibody Fab fragment that binds to a CD38 epitope, comprising a heavy chain having a heavy chain variable region having the wild-type amino acid sequence of SEQ ID NO:1 and a light chain having a light chain variable region having the variant amino acid sequence of SEQ ID NO:3.

[0033] The present disclosure provides an antigen binding protein (e.g., a single chain antibody, which may be fully human) that binds to a CD38 epitope comprising a heavy chain having a heavy chain variable region having the wild-type amino acid sequence of SEQ ID NO: 1 and a light chain having a light chain variable region having the variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the single chain antibody comprises the heavy chain variable region and the light chain variable region linked together by a peptide linker.

[0034] The present disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a fully human antibody of the IgG class comprising a heavy chain having a wild-type heavy chain variable region amino acid sequence of SEQ ID NO:1 and a light chain having a mutant light chain variable region amino acid sequence of SEQ ID NO:3.

[0035] The present disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a fully human Fab fragment comprising a heavy chain having the wild-type heavy chain variable region amino acid sequence of SEQ ID NO:1 and a light chain having the light chain variable region variant amino acid sequence of SEQ ID NO:3.

[0036] The present disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and an antigen binding protein (e.g., a single chain antibody, which may be fully human) comprising a heavy chain having the heavy chain variable region wild-type amino acid sequence of SEQ ID NO:1 and a light chain having the light chain variable region variant amino acid sequence of SEQ ID NO:3.

[0037] The present disclosure provides a nucleic acid encoding a light chain disclosed herein. The present disclosure provides a nucleic acid encoding an antibody light chain comprising the light chain variable region amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid further comprises a nucleic acid encoding an antibody heavy chain comprising the heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1. In one embodiment, the nucleic acid encodes a fully human IgG class antibody. In one embodiment, the nucleic acid encodes an antigen binding protein (e.g., a single chain antibody, which may be fully human).

[0038] The present disclosure provides a nucleic acid encoding a Fab fragment having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid encodes a Fab fragment that also comprises an antibody heavy chain. In one embodiment, the nucleic acid further comprises a nucleic acid encoding an antibody heavy chain comprising the heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1.

[0039] The present disclosure provides a nucleic acid encoding a single chain antibody having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid encodes a single chain antibody that also comprises an antibody heavy chain. In one embodiment, the nucleic acid further comprises a nucleic acid encoding an antibody heavy chain comprising the heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1.

[0040] The disclosure provides an expression vector operably linked to a nucleic acid disclosed herein, for example, encoding an antibody light chain having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the vector is operably linked to a nucleic acid encoding an antibody heavy chain comprising the heavy chain variable region wild-type amino acid sequence of SEQ ID NO: 1. In one embodiment, the expression vector is further operably linked to one or more nucleic acids encoding a fully human IgG class antibody.

[0041] The present disclosure provides an expression vector operably linked to a nucleic acid encoding a Fab fragment having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the vector encodes a Fab fragment that also comprises an antibody heavy chain. In one embodiment, the vector is further operably linked to a nucleic acid encoding an antibody heavy chain comprising the heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1.

[0042] The present disclosure provides an expression vector operably linked to a nucleic acid encoding an antigen binding protein (e.g., a single chain antibody, which may be fully human) having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the vector encodes a single chain antibody that also comprises an antibody heavy chain. In one embodiment, the vector is further operably linked to a nucleic acid encoding an antibody heavy chain comprising the heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1.

[0043] The present disclosure provides a host cell comprising a nucleic acid disclosed herein encoding an antibody light chain. The present disclosure provides a host cell comprising an expression vector operably linked to a nucleic acid encoding an antibody light chain having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the vector contained in the host cell is further operably linked to a nucleic acid encoding an antibody heavy chain comprising a heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1. In one embodiment, the host cell comprises a second expression vector operably linked to a nucleic acid encoding an antibody heavy chain comprising a heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1. In one embodiment, the host cell expressed the antibody light chain and heavy chain. In one embodiment, the host cell expresses a fully human IgG class antibody having a light chain and a heavy chain.

[0044] The present disclosure provides a host cell comprising an expression vector operably linked to a nucleic acid encoding a Fab fragment having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the vector contained in the host cell is further operably linked to a nucleic acid encoding a Fab fragment comprising a heavy chain variable region wild-type amino acid sequence of SEQ ID NO: 1. In one embodiment, the host cell comprises a second expression vector operably linked to a nucleic acid encoding a Fab fragment having an antibody heavy chain comprising a heavy chain variable region wild-type amino acid sequence of SEQ ID NO: 1. In one embodiment, the host cell expresses a fully human Fab fragment having a light chain and a heavy chain.

[0045] The present disclosure provides a host cell comprising an expression vector operably linked to a nucleic acid encoding an antigen binding protein (e.g., a single chain antibody, which may be fully human) having a light chain variable region variant amino acid sequence of SEQ ID NO: 3. In one embodiment, the vector contained in the host cell is further operably linked to a nucleic acid encoding a single chain antibody comprising the heavy chain variable region wild type amino acid sequence of SEQ ID NO: 1. In one embodiment, the host cell expresses an antigen binding protein (e.g., a single chain antibody, which may be fully human) having a light chain variable region and a heavy chain variable region.

[0046] The present disclosure provides a method for preparing a fully human antibody light chain, comprising culturing a population of host cells under conditions suitable for expressing the antibody light chain, wherein each host cell in the population comprises an expression vector operably linked to a nucleic acid encoding an antibody light chain having a light chain variable region variant amino acid sequence of SEQ ID NO:3. In one embodiment, each host cell in the population comprises an expression vector also operably linked to a nucleic acid encoding an antibody heavy chain comprising a heavy chain variable region wild-type amino acid sequence of SEQ ID NO:1. In one embodiment, each host cell in the population comprises a second expression vector operably linked to a nucleic acid encoding an antibody heavy chain comprising a heavy chain variable region wild-type amino acid sequence of SEQ ID NO:1. In one embodiment, the population of host cells expresses the antibody light chain and heavy chain. In one embodiment, the population of host cells expresses a fully human IgG class antibody having a light chain and a heavy chain. In one embodiment, the method further comprises recovering the expressed antibody light chain from the population of host cells. In one embodiment, the method further comprises recovering the expressed antibody light chain and heavy chain from the population of host cells. In one embodiment, 90-100% of the expressed and recovered antibody light chains have the correct length, the correct N-terminal amino acid, and 100% sequence identity to the light chain variable domain of SEQ ID NO:3.

[0047] The present disclosure provides a method for preparing a fully human Fab fragment, comprising culturing a population of host cells under conditions suitable for expressing the Fab fragment, wherein each host cell in the population comprises an expression vector operably linked to a nucleic acid encoding an antibody light chain having a light chain variable region variant amino acid sequence of SEQ ID NO:3. In one embodiment, each host cell in the population comprises an expression vector also operably linked to a nucleic acid encoding a Fab fragment comprising a heavy chain variable region wild type amino acid sequence of SEQ ID NO:1. In one embodiment, each host cell in the population comprises a second expression vector operably linked to a nucleic acid encoding a Fab fragment heavy chain comprising a heavy chain variable region wild type amino acid sequence of SEQ ID NO:1. In one embodiment, the population of host cells expresses an antibody light chain and a heavy chain. In one embodiment, the population of host cells expresses a fully human Fab fragment having a light chain and a heavy chain. In one embodiment, the method further comprises recovering the expressed light chain from the population of host cells. In one embodiment, the method further comprises recovering the expressed light chain and the heavy chain from the population of host cells. In one embodiment, 90-100% of the expressed and recovered light chains have the correct length, the correct N-terminal amino acid, and 100% sequence identity to the light chain variable domain of SEQ ID NO:3.

[0048] The present disclosure provides a method for preparing an antigen binding protein (e.g., a single chain antibody, which may be fully human), comprising culturing a population of host cells under conditions suitable for expressing a single chain antibody, wherein each host cell in the population comprises an expression vector operably linked to a nucleic acid encoding an antibody light chain having a light chain variable region variant amino acid sequence of SEQ ID NO:3. In one embodiment, each host cell in the population comprises an expression vector also operably linked to a nucleic acid encoding a single chain antibody having a heavy chain variable region wild type amino acid sequence of SEQ ID NO:1. In one embodiment, the population of host cells expresses a fully human single chain antibody having a light chain variable region and a heavy chain variable region. In one embodiment, the method further comprises recovering the expressed single chain antibody from the population of host cells. In one embodiment, 90-100% of the expressed and recovered single chain antibodies have the correct length, the correct N-terminal amino acid, and 100% sequence identity to the light chain variable domain of SEQ ID NO:3. [Brief description of the drawings]

[0049] [Figure 1] 1A shows liquid chromatography-mass spectrometry (LC-MS) results of the parent wild-type CD38 A2 antibody, specifically the reduced and deglycosylated antibody heavy chain comprising the wild-type sequence of SEQ ID NO: 1. Peaks are labeled with molecular weight in amu.

[0050] Figure 1B shows liquid chromatography-mass spectrometry (LC-MS) results of a CD38 A2 SV variant antibody, specifically a reduced, deglycosylated antibody heavy chain comprising the wild-type sequence of SEQ ID NO: 1. Peaks are labeled with molecular weight in amu.

[0051] [Diagram 2] 2A shows liquid chromatography-mass spectrometry (LC-MS) results of the parent wild-type CD38 A2 antibody, specifically the antibody light chain comprising the wild-type sequence of SEQ ID NO: 2. Peaks are labeled with molecular weight in amu.

[0052] Figure 2B shows liquid chromatography-mass spectrometry (LC-MS) results for a variant CD38 A2 SV variant antibody, specifically an antibody light chain comprising the variant sequence of SEQ ID NO: 3. Peaks are labeled with molecular weight in amu.

[0053] [Diagram 3] FIG. 3A shows an SPR sensorgram of the binding kinetics of the parent antibody CD38 A2.

[0054] FIG. 3B shows an SPR sensorgram of the binding kinetics of variant antibody CD38 A2 SV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Unless otherwise defined, technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art unless otherwise defined.Generally, the terms related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization techniques described herein are well known and commonly used in the art.The methods and techniques provided herein are generally carried out according to conventional procedures well known in the art and described in various general and more specific references cited and discussed herein, unless otherwise indicated.See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992).Borrebaeck (ed) Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995;McCafferty et al. Antibody Engineering, A Practical Approach, IRL at Oxford Press, Oxford, England, 1996;and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ, 1995;Paul (ed.),Fundamental Immunology,Raven Press, NY, 1993;Coligan (1991) Current Protocols in Immunology Wiley / Greene, NY;Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif. and references cited therein;Coding Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New Several basic texts, including The Journal of Pharmacology, 1986, New York, NY, and Kohler and Milstein Nature 256:495-497, 1975, describe standard antibody production methods. All references cited herein are incorporated by reference in their entirety. Enzymatic reactions and concentration / purification methods are also well known and commonly accomplished in the art or performed according to manufacturer's specifications as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art.Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0056] The headings provided herein are not intended to be limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0057] As used herein, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as the singular use of any word, include plural referents unless expressly and unambiguously limited to one referent.

[0058] It is understood that the use of the alternative (eg, "or") herein is to be construed as meaning either one or both of the alternatives or any combination thereof.

[0059] The term "and / or" as used herein should be interpreted as meaning the specific disclosure of each of the specified features or components, whether or not the other is present. For example, when the term "and / or" is used in a phrase such as "A and / or B" herein, it is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, when the term "and / or" is used in a phrase such as "A, B, and / or C", it is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0060] As used herein, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be open-ended, such that one or more items in a list do not exclude other items that may be substituted for or added to the listed items. Whenever embodiments are described herein with the word "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0061] As used herein, the term "about" refers to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" may mean within one standard deviation or more than one standard deviation as is customary in the art. Alternatively, "about" or "approximately" may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is given in this disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range of that particular value or composition.

[0062] As used herein, the terms "peptide", "polypeptide" and "protein" and other related terms are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may include natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not yet been subjected to cleavage, such as cleavage by a secretory signal peptide or non-enzymatic cleavage at a particular amino acid residue. Polypeptides include mature molecules that have undergone cleavage. These terms encompass natural and artificial proteins, protein fragments and polypeptide analogs of protein sequences, such as muteins, variants, chimeric and fusion proteins, as well as post-translationally modified proteins or proteins that are otherwise covalently or non-covalently modified. Described herein are polypeptides that include the amino acid sequence of a binding protein that binds to CD38 (e.g., an anti-CD38 antibody or an antigen-binding portion thereof) prepared using recombinant procedures.

[0063] As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" and other related terms are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the present disclosure comprise contiguous open reading frames encoding an antibody, or a fragment or scFv, derivative, mutein, or variant thereof. In one embodiment, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides. Nucleic acids encoding antibody light chains, antibody heavy chains, anti-CD38 antibodies or antigen-binding portions thereof are described herein.

[0064] The terms "recover" or "recovery" or "recovering", and other related terms, refer to obtaining a protein (e.g., an antibody or antigen-binding portion thereof) from a host cell culture medium or from a host cell lysate or from a host cell membrane. In one embodiment, the protein is expressed by the host cell as a recombinant protein fused to a secretory signal peptide sequence that mediates secretion of the expressed protein. The secreted protein may be recovered from the host cell medium. In one embodiment, the protein is expressed by the host cell as a recombinant protein lacking a secretory signal peptide sequence and may be recovered from a host cell lysate. In one embodiment, the protein is expressed by the host cell as a membrane-bound protein and may be recovered using a detergent to release the expressed protein from the host cell membrane. In one embodiment, regardless of the method used to recover the protein, the protein may be subjected to a procedure to remove cellular debris from the recovered protein. For example, the recovered protein may be subjected to chromatography, gel electrophoresis and / or dialysis. In one embodiment, the chromatography comprises any one or any combination or two or more procedures including affinity chromatography, hydroxyapatite chromatography, ion exchange chromatography, reverse phase chromatography and / or silica chromatography. In one embodiment, the affinity chromatography comprises Protein A or G (cell wall components from Staphylococcus aureus).

[0065] The term "isolated" refers to a protein (e.g., an antibody or antigen-binding portion thereof) or polynucleotide that is substantially free of other cellular material. A protein can be rendered substantially free of components that are naturally associated with it (or components associated with the cellular expression system or chemical synthesis method used to produce the antibody) by isolation using protein purification methods well known in the art. The term isolated also refers in some embodiments to a protein or polynucleotide that is substantially free of other molecules of the same species, e.g., other proteins or polynucleotides that have different amino acid or nucleotide sequences, respectively. The purity of homogeneity of the desired molecule can be assayed using techniques well known in the art, including low-resolution methods such as gel electrophoresis and high-resolution methods such as HPLC or mass spectrometry. In one embodiment, either the antibody light chain, the antibody heavy chain, the anti-CD38 antibody, or an antigen-binding protein thereof, can be isolated.

[0066] As used herein, "antigen-binding protein" and related terms refer to a protein that includes a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the antigen-binding portion to assume a conformation that promotes binding of the antigen-binding protein to an antigen. Examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding portions of antibodies), antibody derivatives, and antibody analogs. Antigen-binding proteins may include alternative protein scaffolds or artificial scaffolds, including, for example, grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds that include mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic scaffolds, including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1: 121-129;Roque et al., 2004, Biotechnol. Prog. 20:639-654. Additionally, peptide antibody mimetics ("PAMs") may be used, as well as antibody mimetics based scaffolds that utilize fibronectin components as scaffolds. Described herein are antigen binding proteins that bind to CD38.

[0067] An antigen-binding protein may have the structure of, for example, an immunoglobulin. In one embodiment, an "immunoglobulin" refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 or more amino acids. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen-binding sites. In one embodiment, the antigen-binding protein may be a synthetic molecule that is distinct from a tetrameric immunoglobulin molecule but still has a structure that binds one target antigen or binds two or more target antigens. For example, the synthetic antigen-binding protein may comprise an antibody fragment, one to six or more polypeptide chains, an asymmetric assembly of polypeptides, or other synthetic molecules. Described herein are antigen-binding proteins with immunoglobulin-like properties that specifically bind to CD38.

[0068] The variable regions of immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) linked by three hypervariable regions, also called complementarity determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0069] One or more CDRs may be incorporated into a molecule covalently or non-covalently to make the molecule an antigen-binding protein.An antigen-binding protein may incorporate CDRs as part of a larger polypeptide chain, may covalently bind CDRs to another polypeptide chain, or may incorporate CDRs non-covalently.CDRs allow antigen-binding protein to specifically bind to a particular antigen of interest.

[0070] The amino acid assignments for each domain are given in Sequences of Proteins of Immunological Interest, 5 th According to the definition by Kabat et al., Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242,1991. Other numbering systems for the amino acids of immunoglobulin chains include IMGT.RTM. (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001); Chothia (Al-Lazikani et al., 1997 Journal of Molecular Biology 273:927-948; Contact (Maccallum et al., 1996 Journal of Molecular Biology 262:732-745, and Aho (Honegger and Pluckthun 2001 Journal of Molecular Biology 309:657-670).

[0071] As used herein, "antibody" and "antibodies" and related terms refer to an intact immunoglobulin or antigen-binding portion thereof that specifically binds to an antigen. Antigen-binding portions may be produced by recombinant DNA methods or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0072] Antibodies include recombinantly produced antibodies and antigen-binding portions. Antibodies include non-human, chimeric, humanized and fully human antibodies. Antibodies include monospecific antibodies, multispecific antibodies (e.g., bispecific, trispecific and higher specificity). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers. Antibodies include F(ab')2 fragments, Fab' fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragments (scFv), camelized antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), minibodies. Antibodies include monoclonal and polyclonal populations. Described herein are anti-CD38 antibodies, including antibody light and heavy chains.

[0073] As used herein, "antigen-binding domain", "antigen-binding region", or "antigen-binding site" and other related terms refer to a portion of an antigen-binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the specificity and affinity of the antigen-binding protein for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains. Antigen-binding domains derived from anti-CD38 antibodies are described herein.

[0074] The terms "specific binding," "specifically binds," or "specifically binding," and other related terms, as used herein in the context of an antibody or antigen-binding protein or antibody fragment, refer to preferential non-covalent or covalent binding to an antigen compared to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen compared to other available antigens). In one embodiment, an antibody is -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or a smaller dissociation constant K D If the antibody binds to the antigen at , the antibody specifically binds to the target antigen. Described herein are anti-Cd38 antibodies that specifically bind CD38.

[0075] In one embodiment, the dissociation constant (K D ) can be measured using a BIACORE surface plasmon resonance (SPR) assay. Surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example using a BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0076] As used herein, "epitope" and related terms refer to a portion of an antigen to which an antigen-binding protein (e.g., an antibody or antigen-binding portion thereof) binds. An epitope may include portions of two or more antigens to which an antigen-binding protein binds. An epitope may include one antigen or non-contiguous portions of two or more antigens (e.g., amino acid residues that are not contiguous in the primary sequence of the antigen, but are close enough to each other in the context of the antigen's tertiary and quaternary structure for an antigen-binding protein to bind). Generally, the variable region of an antibody, particularly the CDRs, interacts with the epitope. Described herein are anti-CD38 antibodies that bind to epitopes of a CD38 polypeptide (antigen), and antigen-binding proteins thereof.

[0077] As used herein, "antibody fragment", "antibody portion", "antigen-binding fragment of an antibody" or "antigen-binding portion of an antibody" and other related terms refer to molecules other than intact antibodies, including a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAbs; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide. Antigen-binding portions of antibodies may be produced by recombinant DNA methods or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer antigen-binding properties to the antibody fragment. Antigen-binding fragments of anti-CD38 antibodies are described herein.

[0078] The terms "Fab", "Fab fragment" and other related terms refer to the variable light chain region (V L ), the constant light chain region (C L ), variable heavy chain region (V H), and the first constant region (C H1 ) is a monovalent fragment that contains two Fab' fragments linked by a disulfide bridge at the hinge region. F(ab')2 has antigen-binding ability. Fd fragments are monovalent fragments that contain two Fab' fragments linked by a disulfide bridge at the hinge region. F(ab')2 has antigen-binding ability. H and C H1 The Fv fragment contains the V L and V H The Fv is capable of binding to an antigen. The dAb fragment comprises a V H Domain, V L Domain, or V H or an antigen-binding fragment of the VL domain (U.S. Patent Nos. 6,846,634 and 6,696,245; U.S. Patent Application Publication Nos. 2002 / 02512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958; and Ward et al., Nature 341:544-546,1989). Fab fragments containing an antigen-binding portion derived from an anti-CD38 antibody are described herein.

[0079] Single chain antibodies (scFv) are L and V H The antibody is a region that is linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain.Preferably, the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).A single-chain antibody comprising an antigen-binding portion derived from an anti-CD38 antibody is described herein.

[0080] Diabodies are bivalent antibodies that contain two polypeptide chains, each of which is too short to allow pairing between the two domains of the same chain and is therefore connected by a linker that allows each domain to pair with a complementary domain of another polypeptide chain. H and V L domains (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a diabody are identical, then the resulting diabody will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to create diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be the same or different. Diabody, tribody and tetrabody constructs can be prepared using antigen-binding portions from any of the anti-CD38 antibodies described herein.

[0081] The term "human antibody" refers to an antibody having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody). These antibodies can be prepared in a variety of ways, examples of which are described below, including recombinant methods or immunization of mice genetically modified to express antibodies derived from human heavy and / or light chain encoding genes with the antigen of interest. Fully human anti-CD38 antibodies and antigen-binding proteins thereof are described herein.

[0082] A "humanized" antibody refers to an antibody having a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that the humanized antibody is less likely to induce an immune response and / or induces a less severe immune response when administered to a human subject compared to the non-human species antibody. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when administered to a human subject. The amino acid sequence alterations made are conservative alterations, such that none of the altered amino acid residues are important for the immunospecific binding of the antibody to its antigen or the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies can be found in US Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

[0083] The term "chimeric antibody" and related terms as used herein refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are from a human antibody. In another embodiment, all of the CDRs are from a human antibody. In another embodiment, the CDRs from more than one human antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody may contain CDR1 from a light chain of a first human antibody, CDR2 and CDR3 from a light chain of a second human antibody, and CDRs from a heavy chain from a third antibody. In another example, the CDRs are from different species, such as human and mouse, or human and rabbit, or human and goat. Those skilled in the art will understand that other combinations are possible.

[0084] Furthermore, the framework regions may be derived from one of the same antibodies, one or more different antibodies, such as a human antibody, or a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical, homologous, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical, homologous, or derived from an antibody from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to a target antigen). Chimeric antibodies can be prepared from portions of the anti-CD38 antibodies described herein.

[0085] As used herein, the term "variant" polypeptide and "variant" of a polypeptide refers to a polypeptide comprising an amino acid sequence in which one or more amino acid residues are inserted, deleted and / or substituted into the amino acid sequence compared to a reference polypeptide sequence. Polypeptide variants include fusion proteins. Similarly, variant polynucleotides include nucleotide sequences in which one or more nucleotides are inserted, deleted and / or substituted into the nucleotide sequence compared to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

[0086] As used herein, the term "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, through conjugation to another chemical moiety, such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes antibodies comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0087] The term "Fc" or "Fc region" as used herein refers to the portion of an antibody heavy chain constant region beginning in or after the hinge region and ending at the C-terminus of the heavy chain. The Fc region includes at least a portion of the CH and CH3 regions, and may or may not include a portion of the hinge region. Two polypeptide chains, each with half an Fc region, can dimerize to form a complete Fc domain. The Fc domain can bind to Fc cell surface receptors and some proteins of the immune complement system. The Fc domain exhibits effector functions including any one or any combination of two or more activities including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization and / or cell binding. The Fc domain can bind to Fc receptors including FcγRI (e.g., CD64), FcγRII (e.g., CD32) and / or FcγRIII (e.g., CD16a).

[0088] The term "labeled antibody" or related terms, as used herein, refers to an antibody and its antigen-binding portion that is unlabeled or linked to a detectable label or moiety for detection, where the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, detectable beads (such as magnetic or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels can be used, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). Any of the anti-CD38 antibodies described herein may be unlabeled or linked to a detectable label or moiety.

[0089] "Percent identity" or "percent homology" as used herein and related terms refer to a quantitative measurement of similarity between two polypeptide sequences or two polynucleotide sequences.The percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions shared between two polypeptide sequences, taking into account the number of gaps that may need to be introduced to optimize the alignment of two polypeptide sequences, and the length of each gap.In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions shared between two polynucleotide sequences, taking into account the number of gaps that may need to be introduced to optimize the alignment of two polynucleotide sequences, and the length of each gap.Comparing sequences between two polypeptide sequences or two polynucleotide sequences and determining percent identity can be achieved using mathematical algorithms. For example, the "percent identity" or "percent homology" of two polypeptide sequences or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using default parameters. A phrase such as "comprises a sequence having at least X% identity to Y" with respect to a test sequence means that the test sequence, when aligned to sequence Y as described above, contains residues that are identical to at least X% of the residues of Y.

[0090] In one embodiment, the amino acid sequence of the test antibody may be similar, but not identical, to any of the anti-CD38 antibodies described herein, or any of the light and / or heavy chain polypeptides that make up the antigen binding protein. The similarity between the test antibody and the polypeptide may be at least 95%, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the anti-CD38 antibodies described herein, or any of the light and / or heavy chain polypeptides that make up the antigen binding protein. In one embodiment, the similar polypeptide may contain amino acid substitutions within the heavy and / or light chain. In one embodiment, the amino acid substitutions include one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference in its entirety. Examples of groups of amino acids with side chains that have similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) basic side chains: lysine, arginine and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains are cysteine ​​and methionine.

[0091] Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins with various antigen specificities. If such antibodies are subjected to affinity purification, the antibodies can be enriched for a particular antigen specificity. Such enriched preparations of antibodies are usually made with less than about 10% of the antibodies having specific binding activity for a particular antigen. These preparations can be subjected to several rounds of affinity purification to increase the percentage of antibodies having specific binding activity for the antigen. Antibodies prepared in this manner are often referred to as being "monospecific." Monospecific antibody preparations can be composed of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% of antibodies having specific binding activity for a particular antigen. Antibodies may be produced using recombinant nucleic acid techniques as described below.

[0092] As used herein, "vector" and related terms refer to a nucleic acid molecule (e.g., DNA or RNA) that can be operably linked to foreign genetic material (e.g., a nucleic acid transgene). A vector can be used as a vehicle to introduce foreign genetic material into a cell (e.g., a host cell). A vector may contain at least one restriction endonuclease recognition sequence for inserting a transgene into the vector. A vector may contain at least one gene sequence that confers antibiotic resistance or a selectable characteristic that aids in the selection of host cells that contain the vector-transgene construct. A vector may be a single-stranded or double-stranded nucleic acid molecule. A vector may be a linear or circular nucleic acid molecule. One type of vector is a "plasmid." A plasmid refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be linked to a transgene, replicate in a host cell, and transcribe and / or translate the transgene. Viral vectors typically contain viral RNA or DNA backbone sequences that can be linked to a transgene. Viral backbone sequences can be modified to abolish infection but retain insertion of the viral backbone and co-linked transgene into the host cell genome. Examples of viral vectors include retrovirus, lentivirus, adenovirus, adeno-associated, baculovirus, papovavirus, vaccinia virus, herpes simplex virus and Epstein-Barr virus vectors.Certain vectors can autonomously replicate in the host cell they are introduced into (e.g., bacterial vectors containing bacterial replication origins and episomal mammalian vectors).Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of host cell when introduced into host cell, and thus replicate together with the host genome.

[0093] An "expression vector" is a type of vector that can contain one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. An expression vector may contain a ribosome binding site and / or a polyadenylation site. The regulatory sequence directs the transcription, or the transcription and translation, of a transgene linked to the expression vector that is transduced into a host cell. The regulatory sequence can control the level, timing and / or location of expression of the transgene. The regulatory sequence may, for example, directly exert its effect on the transgene or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). The regulatory sequence may be part of a vector. Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606. The expression vector can include a nucleic acid encoding at least a portion of any of the light chains, heavy chains, or anti-CD38 antibodies described herein.

[0094] A transgene is "operably linked" to a vector if the linkage between the transgene and the vector is to allow for the function or expression of the transgene sequence contained in the vector. In one embodiment, a transgene is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.

[0095] The terms "transfected" or "transformed" or "transduced" or other related terms as used herein refer to a process in which an exogenous nucleic acid (e.g., a transgene) is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" host cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid (transgene). A host cell includes a primary subject cell and its progeny. An exogenous nucleic acid encoding at least a portion of any of the light chains, heavy chains or anti-CD38 antibodies described herein may be introduced into a host cell. An expression vector comprising at least a portion of any of the light chains, heavy chains or anti-CD38 antibodies described herein may be introduced into a host cell, and the host cell may express a polypeptide comprising at least a portion of the light chains, heavy chains or anti-CD38 antibodies.

[0096] The term "host cell" or "population of host cells" or related terms, as used herein, refers to a cell (or population thereof) into which a foreign (exogenous or transgene) nucleic acid has been introduced. The foreign nucleic acid may comprise an expression vector operably linked to a transgene, and the host cell may be used to express the nucleic acid and / or the polypeptide encoded by the foreign nucleic acid (transgene). The host cell (or population thereof) may be a cultured cell or may be extracted from a subject. The host cell (or population thereof) includes the primary subject cell and its progeny regardless of the number of passages. The progeny cell may or may not contain identical genetic material compared to the parent cell. The host cell encompasses the progeny cell. In one embodiment, a host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or engineered in any manner to express an antibody as disclosed herein. In one example, the host cell (or population thereof) may be introduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, or antigen-binding portion thereof, as described herein. The host cells and populations thereof may contain an expression vector that is stably integrated into the genome of the host, or may contain an extrachromosomal expression vector. In one embodiment, the host cells and populations thereof may contain an extrachromosomal vector that is present after a few cell divisions, or that is transiently present and disappears after a few cell divisions.

[0097] The host cell may be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. In one embodiment, the host cell may be introduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, thereby producing a transfected / transformed host cell that is cultured under conditions suitable for expression of the antibody by the transfected / transformed host cell, and the antibody may be recovered from the transfected / transformed host cell (e.g., from a host cell lysate) or from the culture medium, if desired. In one embodiment, the host cell includes non-human cells, including CHO, BHK, NS0, SP2 / 0, and YB2 / 0. In one embodiment, the host cell includes human cells, including HEK293, HT-1080, Huh-7, and PER.C6.Exemplary host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or the DHFR-deficient CHO line DX-B11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1 / EBNA cell line (ATCC CCL 70) derived from the African green monkey kidney cell line CV1 (McMahan et al., 1999, Cell 23:175), and the like. al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293EBNA or MSR293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, the host cell includes lymphoid cells such as Y0, NS0 or Sp20. In one embodiment, the host cell is a mammalian host cell, but is not a human host cell. Typically, the host cell is a cultured cell in which a polypeptide-encoding nucleic acid can be transformed or transfected and then expressed in the host cell. The phrases "transgenic host cell" or "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express a desired level of the nucleic acid unless a regulatory sequence is introduced into the host cell such that it is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell.Because certain modifications may occur in successive generations due to, for example, mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0098] Polypeptides (e.g., antibodies and antigen-binding proteins) of the disclosure can be produced using any method known in the art. In one example, the polypeptides are produced by recombinant nucleic acid methods by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector that is introduced into a host cell and expressed by the host cell under conditions that promote expression.

[0099] General techniques for recombinant nucleic acid manipulation are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and periodically updated versions, which are incorporated herein by reference in their entirety. The nucleic acid (e.g., DNA) encoding the polypeptide is operably linked to an expression vector having one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control the termination of transcription and translation. The expression vector may contain an origin of replication that confers the ability to replicate in a host cell. The expression vector may also contain a gene that confers selection to facilitate recognition of transgenic host cells (e.g., transformants).

[0100] Recombinant DNA can also code for any type of protein tag sequence that can be useful for protein purification.Examples of protein tags include, but are not limited to, histidine tag, FLAG tag, myc tag, HA tag, or GST tag.Appropriate cloning and expression vectors for use with bacteria, fungi, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, NY, 1985).

[0101] Expression vector construct can be introduced into host cell by using the method appropriate for the host cell.Various methods are known in the art for introducing nucleic acid into host cell, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; non-viral transfection; particle bombardment; lipofection; and infection (e.g., when vector is an infectious agent).Suitable host cell includes prokaryotic, yeast, mammalian cell, or bacterial cell.

[0102] Suitable bacteria include gram-negative or gram-positive organisms, such as E. coli or Bacillus spp. Yeast, preferably from the Saccharomyces species, such as S. cerevisiae, may also be used to produce polypeptides. Various mammalian or insect cell culture systems may also be used to express recombinant proteins. Baculovirus systems for the production of heterologous proteins in insect cells are reviewed by Luckow and Summers, (Bio / Technology, 6:47, 1988). Examples of suitable mammalian host cell systems include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines. Purified polypeptides are prepared by culturing a suitable host / vector system to express recombinant proteins. For many applications, the small size of many of the polypeptides disclosed herein will make expression in E. coli the preferred method of expression. The protein is then purified from the culture medium or cell extracts. Any of the light chain, heavy chain or anti-CD38 antibody, or antigen binding proteins thereof, can be expressed by a transgenic host cell.

[0103] The antibodies and antigen-binding proteins disclosed herein can also be produced using a cell translation system.For such purposes, the nucleic acid encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA, and to allow cell-free translation of the mRNA in the particular cell-free system used (eukaryotic cell-free translation system, such as mammalian or yeast cell-free translation system, or prokaryotic cell-free translation system, such as bacterial cell-free translation system).

[0104] Nucleic acids encoding any of the various polypeptides disclosed herein may be chemically synthesized. Codon usage may be selected to improve expression in cells. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See, for example, Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002(1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

[0105] The antibodies and antigen binding proteins described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the proteins can also be effected by chemical synthesis.

[0106] The antibodies and antigen-binding proteins described herein can be purified by protein isolation / purification methods generally known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination thereof. After purification, the polypeptide can be exchanged into a different buffer and / or concentrated by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis.

[0107] The purified antibodies and antigen-binding proteins described herein are preferably at least 65% pure, at least 75% pure, at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact numerical value of purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any of the light chain, heavy chain or anti-CD38 antibodies or antigen-binding proteins thereof described herein can be expressed by transgenic host cells and then purified to about 65-98% purity or a high level of purity using any method known in the art.

[0108] In certain embodiments, the antibodies and antigen-binding proteins herein may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. As a result, modified polypeptides can contain non-amino acid elements such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which conjugates one or more sialic acid moieties to a polypeptide. Sialic acid moieties improve solubility and serum half-life, but also reduce the immunogenic potential of the protein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

[0109] In one embodiment, the antibodies and antigen binding proteins described herein may be modified to become soluble polypeptides, which involves linking the antibodies and antigen binding proteins to a nonproteinaceous polymer. In one embodiment, the nonproteinaceous polymer comprises polyethylene glycol ("PEG"), polypropylene glycol, or polyoxyalkylenes, as described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337.

[0110] PEG is a water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or modification of the PEG termini, and has the formula: XO(CH2CHO). n-CH2CH2OH(1) (wherein n is 20 to 2300 and X is H or a terminal modification, e.g., C 1~4 In one embodiment, PEG is terminated at one end with hydroxy or methoxy (i.e., X is H or CH3 ("methoxy PEG")). PEG may further contain chemical groups necessary for conjugation reactions; this may result from chemical synthesis of the molecule; or it may be a spacer for optimal distance of parts of the molecule. Furthermore, such PEG may consist of one or more PEG side chains linked together. PEGs with more than one PEG chain are called multiarmed or branched PEGs. Branched PEGs can be prepared, for example, by addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-arm branched PEG can be prepared from pentaerythritol and ethylene oxide. Branched PEGs are described, for example, in EP-A 0473084 and U.S. Pat. No. 5,932,462. One form of PEG contains two PEG side chains (PEG2) linked through the primary amino groups of lysine (Monfardini et al., Bioconjugate Chem. 6(1995) 62-69).

[0111] The serum clearance rate of the PEG-modified polypeptides may be modulated (e.g., increased or decreased) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90% compared to the clearance rate of unmodified antibodies and antigen binding protein binding polypeptides. PEG-modified antibodies and antigen binding proteins have an increased half-life (t 1 / 2). The half-life of PEG-modified polypeptides may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even 1000%, compared to the half-life of unmodified antibodies and antigen-binding proteins. In some embodiments, the protein half-life is determined in vitro, such as in a buffered saline solution or serum. In other embodiments, the protein half-life is an in vivo half-life, such as the half-life of the protein in serum or other bodily fluids of an animal.

[0112] The present disclosure provides therapeutic compositions comprising any of the light chain, heavy chain or anti-CD38 antibodies or antigen-binding proteins thereof described herein in admixture with a pharma- ceutically acceptable excipient. Excipients include carriers, stabilizers and excipients. Pharmaceutically acceptable excipient excipients include, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Additional examples include buffers, stabilizers, preservatives, non-ionic surfactants, antioxidants, and isotonicity agents.

[0113] Therapeutic compositions and methods for preparing the same are well known in the art and can be found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. AR Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). Therapeutic compositions may be formulated for parenteral administration and can contain, for example, excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the antibodies (or their antigen-binding proteins) described herein. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of the antibodies (or their antigen-binding proteins). Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the antibody (or antigen-binding protein thereof) in the formulation will vary depending on several factors, including the dose of drug administered and the route of administration.

[0114] Any anti-CD38 antibody (or antigen-binding portion thereof) may be administered as a pharma- ceutically acceptable salt, such as a non-toxic acid addition salt or metal complex commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids such as tannic acid, carboxymethylcellulose; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like. Metal complexes include zinc, iron, and the like. In one example, the antibody (or antigen-binding portion thereof) is formulated in the presence of sodium acetate, which enhances thermal stability.

[0115] Any of the anti-CD38 antibodies (or antigen-binding portions thereof) may be formulated for oral use, including tablets containing the active ingredient in a mixture with non-toxic pharma- ceutically acceptable excipients. Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium.

[0116] The term "subject" as used herein refers to human and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject may be a human, a non-human primate, a monkey, an ape, a murine (e.g., a mouse and a rat), a cow, a pig, a horse, a dog, a cat, a goat, a wolf, a frog or a fish.

[0117] The terms "administering", "administered" and grammatical variations refer to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a route other than a parenteral route, for example orally. Other routes other than a parenteral route include topical, epithelial or mucosal routes of administration, for example, intranasal, intravaginal, intrarectal, sublingual or topical routes of administration. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time. Any of the anti-CD38 antibodies (or antigen binding proteins thereof) described herein can be administered to a subject using methods and delivery routes known in the art.

[0118] The terms "effective amount," "therapeutically effective amount," or "effective dose," or related terms, may be used interchangeably, and refer to an amount of an antibody or antigen binding protein (e.g., any of the anti-CD38 antibodies or antigen binding proteins thereof described herein) sufficient to result in a measurable improvement or prevention of a disease or disorder associated with tumor or cancer antigen expression when administered to a subject. Therapeutically effective amounts of the antibodies provided herein will vary depending on the relative activities (e.g., in inhibiting cell proliferation) of the antibodies and combinations when used alone or in combination, as well as on the subject and disease condition being treated, the subject's weight and age and sex, the severity of the disease condition in the subject, the mode of administration, etc., and can be readily determined by one of skill in the art.

[0119] In one embodiment, the therapeutically effective amount depends on the subject being treated and on certain aspects of the disorder being treated, and can be ascertained by one of skill in the art using known techniques. In general, the polypeptide is administered at about 0.01 g / kg to about 50 mg / kg per day, preferably 0.01 mg / kg to about 30 mg / kg per day, and most preferably 0.1 mg / kg to about 20 mg / kg per day. The polypeptide can be administered daily (e.g., 1, 2, 3, or 4 times daily), or preferably less frequently (e.g., weekly, biweekly, triweekly, monthly, or quarterly). Additionally, adjustments may be required for age and weight, general health, sex, diet, time of administration, drug interactions, and severity of disease, as known in the art.

[0120] The present disclosure provides a method for treating a subject with a disease associated with the expression of CD38. The disease includes cancer or tumor cells expressing tumor-associated antigens. In one embodiment, the cancer or tumor includes prostate cancer, breast cancer, ovarian cancer, head and neck cancer, bladder cancer, skin cancer, colorectal cancer, anal cancer, rectal cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), leiomyoma, brain cancer, glioma, glioblastoma, esophageal cancer, liver cancer, kidney cancer, stomach cancer, colon cancer, cervical cancer, uterine cancer, endometrial cancer, vulvar cancer, laryngeal cancer, vaginal cancer, bone cancer, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, oral cancer, oropharynx cancer, laryngeal cancer, hypopharynx cancer, salivary gland cancer, ureter cancer, urethral cancer, penile cancer and testicular cancer.

[0121] In one embodiment, the cancer comprises hematological cancers including leukemia, lymphoma, myeloma and B-cell lymphoma. Hematological cancers include multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) including Burkitt's lymphoma (BL), chronic B-lymphocytic leukemia (B-CLL), systemic lupus erythematosus (SLE), acute B- and T-lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's macroglobulinemia, mantle cell lymphoma, Hodgkin's lymphoma (HL), plasma cell myeloma, precursor B-cell lymphoblastic leukemia / lymphoma, plasmacytoma, giant cell myeloma. , plasma cell myeloma, heavy chain myeloma, light chain or Bence Jones myeloma, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunoregulatory disorders, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, and monoclonal gammopathy of undetermined significance.

[0122] Anti-CD38 antibodies are disclosed in U.S. Patent Application No. 15 / 094,384, filed April 8, 2016, the disclosure of which is incorporated herein by reference in its entirety. This antibody, referred to herein as the "parent" and / or "wild-type" antibody, comprises a parent wild-type heavy chain having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a parent wild-type light chain having a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0123] The present disclosure provides variant antibodies comprising a parent wild-type heavy chain having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain having a light chain variable region comprising the variant amino acid sequence of SEQ ID NO: 3. In some embodiments, the present disclosure provides methods of preparing variant sequence light chains or antigen binding proteins comprising variant sequence light chains, in which the variant amino acid sequence of the light chain reduces or eliminates the generation of a heterogeneous population of light chains containing a mixture of desirable and undesirable light chains. In contrast, the generation of a preparation of the parent wild-type light chain results in a population of antibody light chains with a high level of heterogeneity.

[0124] Recombinant antibody heavy and light chains are often designed to contain secretory signal peptides at the N-terminus to allow expression of precursor antibody chains in the host cell cytoplasm and translocation across the host cell membrane. The translocating precursor antibody chains undergo site-specific cleavage in the secretory signal peptide by a signal peptide peptidase enzyme expressed by the host cell, resulting in mature antibody chains. It is desirable to generate a homogeneous population of mature antibody chains from precursor antibody chains that have been cleaved at specific sites in the secretory signal peptide to obtain a population of mature antibody chains with the same length and sequence and containing the same N-terminus and C-terminus. However, the production of recombinant antibody chains often generates heterogeneous populations with different chain lengths and sequences, and different N-terminus and / or C-terminus. One source of heterogeneity results from cleavage of the secretory signal peptide or the N-terminal region of the antibody chain at variable sites, causing shortening and elongation of the chains. Cleavage heterogeneity is also known to result from non-enzymatic degradation of peptide bonds at certain amino acids, such as aspartic acid, glycine, serine, threonine, cysteine, methionine, or asparagine, in and near secretory signal peptides. Regardless of the mechanism that results in cleavage heterogeneity, imprecise cleavage causes antibody chain population heterogeneity. Cleavage heterogeneity can reduce the binding of the antibody to its target antigen. In some cases, cleavage heterogeneity can change the charge of the antibody chain, resulting in undesirable changes in the antibody pI (isoelectric point) value, thereby resulting in charge heterogeneity in the population of expressed antibody chains. In general, cleavage heterogeneity can reduce the efficacy of the antibody, reduce its shelf life, make it difficult to consistently produce a uniform product, and make the antibody chain less desirable or suitable for bioproduction.

[0125] The present disclosure provides variant antibody light chains having mutated amino acid sequences designed to reduce cleavage heterogeneity occurring during host cell expression, thereby reducing light chain population heterogeneity. The antibody light chain comprises a mutated amino acid sequence near its N-terminus adjacent to a secretory signal peptide to reduce cleavage heterogeneity. Without wishing to be bound by theory, it is hypothesized that the amino acid mutations of the antibody light chain described herein reduce cleavage heterogeneity due to enzymatic and / or non-enzymatic cleavage events.

[0126] A heterogeneous population of antibody light chains includes a mixture of desirable and undesirable light chains. Desired light chains have the correct length, the correct N-terminal and C-terminal amino acid residues, and include a light chain variable region with an amino acid sequence that is 100% identical to SEQ ID NO:3. In some embodiments, the desirable chains are the majority or majority species in the chain population. Undesirable light chains include light chain variable regions that have the incorrect length (e.g., longer or shorter), the incorrect N-terminal and / or C-terminal amino acid residues, and / or the amino acid sequence that is not 100% identical to SEQ ID NO:3.

[0127] In the discussion that follows, increases, decreases, and reductions are expressed relative to SEQ ID NO: 2. In some embodiments, light chain sequence mutations reduce the production of a heterogeneous population of light chains by increasing the percentage of desirable light chains and decreasing or even eliminating the percentage of undesirable light chains.

[0128] In some embodiments, the light chain sequence mutations reduce the percentage of undesirable light chains in a preparation of light chains (e.g., in a population of light chains) by about 10-30%, or about 30-50%, or about 50-70%, or about 70-90%, or about 90-95%, or about 95-99% or greater.

[0129] In some embodiments, the light chain sequence mutations increase the percentage of desirable light chains in a preparation of light chains (e.g., in a population of light chains) by about 10-30%, or about 30-50%, or about 50-70%, or about 70-90%, or about 90-95%, or about 95-99% or higher.

[0130] In one embodiment, the light chain sequence mutations include two point mutations near the N-terminus, specifically replacing an alanine at position 2 with a serine and a glycine at position 3 with a valine (see positions 2 and 3 of SEQ ID NO:3). The light chain sequence mutations increase the percentage of desired light chains such that a preparation of mutant light chains contains approximately 98-100% desired light chains with the correct length, correct N-terminal amino acid, and has 100% sequence (Figures 2A and B).

[0131] In one embodiment, the light chain sequence mutation comprises the amino acid sequence of SEQ ID NO:2 with two point mutations near the N-terminus, specifically replacing the alanine at position 2 with serine and replacing the glycine at position 3 with alanine. In one embodiment, the N-terminus of the mutant light chain contains the amino acid sequence QSALT (SEQ ID NO:6). The light chain sequence mutation increases the percentage of desired light chains such that a preparation of mutant light chains contains approximately 98-100% desired light chains with the correct length, correct N-terminal amino acid, and has 100% sequence (Table 2).

[0132] The light chain variant sequences described herein can provide reduced cleavage heterogeneity, improving the production and yield of the antibody light chain. The type and / or sequence of the secretory signal peptide operably linked to the antibody light chain is not critical.

[0133] In contrast, the parent wild-type light chain (SEQ ID NO:2) gives rise to a heterogeneous mixture of undesired and desired light chains. Preparations of the parent wild-type light chains can produce a high percentage of truncated light chains, with as much as 35-65% of the light chains being undesired truncated light chains (see Table 2 and Figures 2A and B). For example, preparations of the parent wild-type light chains can contain about 10-30%, or about 30-50%, or about 50-70%, or about 70-90%, or about 90-95%, or about 95-99% or higher levels of undesired truncated light chain forms.

[0134] The present disclosure provides a host cell expressing a precursor polypeptide comprising an amino acid sequence of a secretory signal peptide operably linked to a variant amino acid sequence of an antibody light chain having a light chain variable region comprising the amino acid sequence of SEQ ID NO:3. In one embodiment, the secretory signal peptide directs the translocation of the precursor polypeptide to the endoplasmic reticulum, secretory vesicles and / or the extracellular space. In one embodiment, the secretory signal peptide mediates the secretion of the precursor polypeptide from the host cell. In one embodiment, the secretory signal peptide serves as a substrate for enzymatic cleavage at a specific site (e.g., a desired cleavage site) of the secretory signal peptide resulting in a mature polypeptide that is shorter than the precursor polypeptide. In one embodiment, the cleavage event results in an antibody light chain having the correct length and with a new N-terminus, comprising an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3. The variant antibody light chain can assemble with a heavy chain to generate an immunoglobulin that binds to its target antigen CD38.

[0135] The present disclosure provides mutant light chains that assemble with heavy chains to form antigen binding proteins that specifically bind to the CD38 antigen, as well as nucleic acids encoding the antigen binding proteins, vectors containing the nucleic acids, host cells containing the vectors, and methods of use thereof.

[0136] Host cell expression of antibody heavy and light chains can be used to generate preparations of antibody populations that include variant antibodies and the parent wild-type antibody.

[0137] The host cell can express a parent wild-type heavy chain having a wild-type sequence. A preparation of the parent wild-type heavy chain can contain about 85-99% or more of the desired heavy chain sequence (e.g., low levels of heavy chain heterogeneity). The host cell can express a parent wild-type sequence light chain having a wild-type sequence. A preparation of the parent wild-type light chain can contain about 40-55% or more of the undesired light chain sequence (e.g., high levels of light chain heterogeneity).

[0138] The host cells can express a parent wild-type heavy chain having a wild-type sequence. A preparation of the parent wild-type heavy chain can contain about 85-99% or more of the desired heavy chain sequence (e.g., low levels of heavy chain heterogeneity). The host cells can express a variant sequence light chain having a mutant sequence. A preparation of the variant sequence light chain can contain about 85-99% or more of the desired light chain sequence, with reduced levels of light chain heterogeneity compared to a preparation of the parent wild-type light chain.

[0139] The present disclosure provides a fully human antibody of IgG class that binds to CD38 polypeptide. In one embodiment, the anti-CD38 antibody comprises a heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1; and the anti-CD38 antibody comprises a light chain variable region having 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the anti-CD38 antibody comprises an IgG1, IgG2, IgG3 or IgG4 class antibody. In one embodiment, the anti-CD38 antibody comprises an IgG1 or IgG4 class antibody.

[0140] In one embodiment, the anti-CD38 antibody, or fragment thereof, is -6 M or less, 10 -7 M or less, 10 -8M or less, 10 -9 M or less, or 10 -10 M or less binding affinity (K D ) (see Table 3). In one embodiment, the CD38 antigen comprises a cell surface CD38 antigen or a soluble CD38 antigen. In one embodiment, the CD38 antigen comprises an extracellular portion of a cell surface CD38 antigen. In one embodiment, the CD38 antigen comprises a human or non-human CD38 antigen. In one embodiment, the CD38 antigen is expressed by a human or non-human cell. In one embodiment, the anti-CD38 antibody binds to human CD38 expressed by human B cells or expressed by human multiple myeloma cells. In one embodiment, binding between anti-CD38 antibodies, or fragments thereof, can be detected and measured using surface plasmon resonance, flow cytometry and / or ELISA.

[0141] The present disclosure provides anti-CD38 antibodies that bind to an epitope of CD38 from a human or that can bind to an epitope (e.g., cross-reactive) of CD38 from any one or any combination of non-human animals, such as mouse, rat, goat, rabbit, hamster, and / or monkey (e.g., cynomolgus monkey). In one embodiment, the anti-CD38 antibody is -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less binding affinity K D In one embodiment, the anti-CD38 antibody binds to mouse CD38 at 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10-9 M or less, or 10 -10 M or less binding affinity K D It binds to cynomolgus monkey CD38.

[0142] The present disclosure provides fully human antibodies that bind to CD38, wherein the antibody comprises both a heavy and a light chain, and the heavy / light chain variable region amino acid sequences have at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence set SEQ ID NOs:1 and 3.

[0143] The present disclosure provides a Fab fully human antibody fragment comprising a heavy chain variable region from a heavy chain and a variable region from a light chain. The sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1. The sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0144] The present disclosure provides a Fab fully human antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy / light chain variable region amino acid sequences are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following sets of amino acid sequences: SEQ ID NOs: 1 and 3.

[0145] The disclosure provides a single chain fully human antibody comprising a polypeptide chain having a variable region derived from a fully human heavy chain and a variable region derived from a fully human light chain, and optionally a linker joining the variable heavy and variable light chain regions, wherein the variable heavy chain region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. The variable light chain region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0146] The present disclosure provides single-chain fully human antibodies comprising a polypeptide chain having a heavy chain variable region and a light chain variable region, wherein the heavy / light chain variable region amino acid sequence set is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOs: 1 and 3.

[0147] The present disclosure provides a therapeutic composition comprising any of the anti-CD38 antibodies described herein, or antigen-binding proteins thereof, in admixture with a pharma- ceutically acceptable excipient. Excipients include carriers and stabilizers. In one embodiment, the therapeutic composition comprises an anti-CD38 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy / light chain variable region amino acid sequences are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following sets of amino acid sequences: SEQ ID NOs: 1 and 3.

[0148] The present disclosure provides a method for treating a subject having a disease associated with CD38 expression, comprising administering to the subject an effective amount of a therapeutic composition comprising an anti-CD38 antibody or antigen-binding fragment thereof selected from the group consisting of any of the fully human anti-CD38 antibodies described herein, any of the Fab fully human anti-CD38 antibodies described herein, and any of the single chain human anti-CD38 antibodies described herein. In one embodiment, the disease associated with CD38 expression is a hematological cancer, including leukemia, lymphoma, myeloma, or B cell lymphoma. In one embodiment, hematological cancers include multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) including Burkitt's lymphoma (BL), chronic B-lymphocytic leukemia (B-CLL), systemic lupus erythematosus (SLE), acute B and T lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's macroglobulinemia, mantle cell lymphoma, Hodgkin's lymphoma (HL), plasma cell myeloma, precursor B cell lymphoblastic leukemia / lymphoma, plasmacytoma, giant cell lymphoma, and leukemia. These include follicular myeloma, plasma cell myeloma, heavy chain myeloma, light chain or Bence Jones myeloma, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunoregulatory disorders, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, and monoclonal gammopathy of undetermined significance.

[0149] Sequence Listing:

[0150] Table 1: [Table 1-1] [Table 1-2] EXAMPLES

[0151] The following examples are intended to be illustrative and may be used to further understand embodiments of the present disclosure, and should not be construed as limiting the scope of the present teachings in any way.

[0152] Example 1: Synthesis of mutant light chains of CD38-A2 antibody

[0153] A synthetic codon-optimized DNA fragment was obtained encoding the light chain of the CD38-A2 SV mutant antibody. The fragment contained two point mutations near the encoded N-terminus, specifically replacing alanine at position 2 with serine and glycine at position 3 with valine. Using standard restriction enzyme digestion and ligation, the fragment encoding the light chain was inserted in frame into the Lonza pXC-17.4 vector and a different fragment encoding the wild-type CD38-A2 heavy chain was inserted in frame into the Lonza pXC-18.4 vector (Lonza GS System from Lonza, Basel, Switzerland). Using standard enzyme digestion and ligation, the pXC-17.4 and pXC18.4 vectors were fused into a single vector and the resulting fusion vector was introduced into E. coli host cells.

[0154] Example 2: Liquid chromatography-mass spectrophotometric analysis of mutant light chains from anti-CD38 antibodies

[0155] A reduced LC-MS analysis was performed to compare CD38-A2 wild type (WT) and CD38-A2 SV variant antibodies. Briefly, 16 μL of antibody sample (20 μg) was mixed with 4 μL 5× Rapid PNGase Buffer (Cat: P0710S; NEB) and incubated at 80° C. for 5 min to reduce disulfide bonds and denature proteins. 0.5 μL of Rapid PNGase F (Cat: P0710S; NEB) was then added to release N-glycans at 50° C. for 15 min. Five micrograms of sample was injected into a Waters LC-ESI-MS system.

[0156] An ACQUITY UPLC® System equipped with a 2.1×150 mm BEH 300 Å 1.7 μm C4 column was used for separation of the reduced antibody chains, and the column temperature was 80° C. The antibody was eluted from the column with a 12 min gradient (25-40% acetonitrile / 0.1% formic acid, flow rate 0.4 mL / min).

[0157] A Waters Xevo G2 TOF MS System was run in sensitivity mode with detection of positive ions, 400-4000 m / z range. Source parameters were as follows: capillary voltage, 3.10 kV; sampling cone voltage, 40.0 V; extraction cone voltage, 3.0 V; source temperature, 125° C.; desolvation temperature, 350° C.; cone gas flow: 10 L / hr; desolvation gas flow, 500 L / hr. Protein peaks were deconvoluted by MassLynx MaxEnt1 function according to the following parameters: output resolution, 1.0 Da / channel; output mass range, 20,000-70,000 Da for reduced antibodies; uniform Gaussian width at half height, 0.75 Da; minimum intensity ratio, 33% for left and right; maximum number of iterations, 12.

[0158] The molecular masses of CD38A2 WT and SV variants were measured by LC-ESI-MS to confirm the protein ID. Both WT and SV heavy chain molecular masses were in good agreement with the theoretical mass. Meanwhile, a truncation of two amino acids from the N-terminal light chain was found in up to about 50% of all wild-type light chains. LC of the SV mutants did not show any detectable truncation.

[0159] Reduced CE-SDS was performed on the test samples using an Agilent Bioanalyzer 2100 according to the manufacture's instructions. Two light chain populations with a ratio of approximately 1:1 were detected by reduced CE-SDS for CD38-A2 wild type. The CD38-A2 SV mutant showed only one type of light chain by reduced CE-SDS. The results are consistent with the findings from LC-MS analysis that the wild type undergoes light chain truncation.

[0160] Mass spectrometry and reduced CE-SDS results indicate that approximately 50% of the population of CD38-A2 wild-type light chains are truncated molecules.

[0161] In conclusion, N-terminal light chain truncations occurred in CD38-A2 wild type but were not detectable for the CD38-A2 SV variant by LC-MS and CE-SDS analysis. The results are summarized in Table 2 and Figures 1A and B and 2A and B. Results are also shown for the CD38 A2-SA variant, in which the second and third amino acids of the light chain were serine and alanine, respectively.

[0162] Table 2: [Table 2-1] [Table 2-2]

[0163] Example 3: Comparative binding study of parent antibody CD38 A2 and variant antibody CD38 A2 SV

[0164] The binding kinetics of parental antibody CD38 A2 and variant antibody CD38 A2 SV were measured using surface plasmon resonance (SPR). The dynamic interaction between parental or variant antibodies and his-tagged CD38 protein was measured at 25°C using a Biacore T200 surface plasmon resonance (GE Healthcare). Anti-human fragment crystallizable region (Fc region) antibodies were immobilized to approximately 8000 resonance units (RU) on a CM5 sensor chip using standard N-hydroxysuccinimide / N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (NHS / EDC) coupling method. CD38 A2 parental or CD38 A2 SV variant antibodies (2 μg / mL) were captured for 60 seconds at a flow rate of 10 μL / min. Recombinant human his-tagged CD38 protein was serially diluted in a running buffer of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v Surfactant P20 (HBS EP+). All measurements were performed in HBS-EP+ buffer with a flow rate of 30 μL / min. Affinities were obtained using a steady-state affinity model. The sensorgrams of the CD38 A2 parental and CD38 A2 SV variant antibodies are shown in Figures 3A and B, respectively, and their corresponding binding kinetics are listed in Table 3 below.

[0165] Table 3: [Table 3]

Claims

1. 1. An antigen binding protein that binds to CD38, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:1 and the light chain variable region comprises the sequence of SEQ ID NO:3; An antigen-binding protein, wherein the N-terminus of the light chain variable region begins with the amino acid sequence QSVLT.

2. A fully human antibody or antibody Fab fragment of the IgG class, comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 and a light chain comprising a light chain variable region having the amino acid sequence of SEQ ID NO:3, A fully human antibody or antibody Fab fragment, wherein the N-terminus of the light chain variable region begins with the amino acid sequence QSVLT.

3. The fully human antibody or antibody Fab fragment of claim 2, which is a fully human antibody.

4. 3. The fully human antibody or antibody Fab fragment of claim 2, which is a Fab fragment.

5. The antigen-binding protein of claim 1, which is a fully human single-chain antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 and a light chain comprising a light chain variable region having the amino acid sequence of SEQ ID NO:

3.

6. A pharmaceutical composition comprising a pharma- ceutically acceptable excipient and an antigen-binding protein, fully human antibody or Fab fragment according to any one of claims 1 to 5.

7. A nucleic acid encoding an antibody heavy chain and an antibody light chain, the antibody heavy chain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1; the antibody light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3; A nucleic acid wherein the N-terminus of the light chain variable region begins with the amino acid sequence QSVLT.

8. A nucleic acid encoding an antigen-binding protein according to claim 1 or 5, or a fully human antibody or antibody Fab fragment according to any one of claims 2 to 4.

9. 9. An expression vector comprising a promoter operably linked to the nucleic acid of claim 7 or claim 8.

10. A host cell comprising the expression vector of claim 9.

11. 13. A method for preparing an antibody light chain, comprising culturing a population of host cells of claim 10 under conditions suitable for expressing the antibody light chain.

12. 12. The method of claim 11, further comprising the step of recovering the expressed antibody light chain from the host cell.

13. 13. The method of claim 12, wherein 90-100% of the expressed and recovered antibody light chains have 100% sequence identity to the light chain variable region of SEQ ID NO:

3.

14. The method of claim 11, further comprising recovering the expressed antibody light and heavy chains from the host cell.

15. 15. The method of claim 14, wherein 90-100% of the expressed and recovered antibody light chains have 100% sequence identity to the light chain variable region of SEQ ID NO:3.

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