JP2018087218A - Anti-cd38 antibodies and fusions with attenuated interferon alpha-2b - Google Patents

Abstract

PROBLEM TO BE SOLVED: To use anti-CD38 antibody-attenuated interferon alpha-2b fusion constructs to inhibit proliferation in cancerous cells that express both CD38 and the receptor for IFN-alpha2b, and to induce apoptosis in such cells, where the inhibition of proliferation and induction of apoptosis in cancerous cells may serve as the basis for the treatment of the underlying cancer.SOLUTION: The invention provides antibodies that specifically bind to CD38, and constructs comprising the antibodies fused with attenuated interferon alpha-2B proteins.SELECTED DRAWING: Figure 1

Description

Reference to Sequence Listing This application contains a sequence listing electronically filed as a text file named Anti-CD38_Antibodies_ST25, created on April 29, 2013, having a size of 462,000 bytes. The sequence listing is incorporated herein by reference.

  The present disclosure relates generally to the field of antibody engineering. More particularly, the present disclosure relates to antibodies that specifically bind CD38, as well as constructs comprising such antibodies and an attenuated interferon-alpha ligand, and methods of treatment using these constructs. In these constructs, the antibody directs the ligand to cells that express both CD38 and the receptor for the ligand, and attenuated interferon-alpha reduces interferon signaling in cells that do not express CD38. .

  Various publications are cited throughout this specification, including patents, published applications, technical papers, journal articles, and gene or protein accession numbers. Each of these materials is incorporated herein by reference in its entirety for all purposes.

  CD38 is a 46 kDa type II transmembrane glycoprotein. It has a short N-terminal cytoplasmic tail of 20 amino acids, a single transmembrane helix and a long extracellular domain of 256 amino acids. It is expressed on the surface of many immune cells such as CD4 and CD8 positive T cells, B cells, NK cells, monocytes, plasma cells, as well as on a significant proportion of normal bone marrow progenitor cells. In some examples, the expression of CD38 in lymphocytes may depend on the differentiation and activation status of the cells, for example, resting T and B cells may be negative for CD38 expression, Most immature and activated lymphocytes may be positive. CD38 mRNA expression has been detected in non-hematopoietic organs such as pancreas, brain, spleen and liver (Koguma, T. (1994) Biochim. Biophys. Acta 1223: 160).

CD38 is a multifunctional exoenzyme involved in transmembrane signaling and cell adhesion. It is also known as cyclic ADP ribose hydrolase because it can convert NAD ⁺ and NADP ⁺ to cADPR, ADPR and NAADP depending on the extracellular pH. These products can induce Ca ²⁺ mobilization inside the cell, leading to tyrosine phosphorylation and cell activation. CD38 is also a receptor capable of interacting with the ligand CD31. Receptor activation through CD31 results in intracellular events such as Ca ²⁺ migration, cell activation, proliferation, differentiation and migration.

  CD38 is high in multiple myeloma cells, often T and B cell acute lymphoblastic leukemia, some acute myelocytic leukemias, follicular central cell lymphomas, and T lymphoblastic lymphomas Expressed. CD38 is also expressed on B lineage chronic lymphoblastic leukemia (B-CLL) cells. In some cases, B-CLL patients presenting CD38 + clones are characterized by an unfavorable clinical course showing a more advanced disease stage, weak responsiveness to chemotherapy and shorter survival. The use of antibodies against CD38 has been proposed for the treatment of cancers and hematological malignancies that express CD38. Accordingly, it may be advantageous to provide alternative antibodies to CD38 that have desirable manufacturing, stability and immunogenic properties.

  Interact with cell surface receptors, thereby stimulating, inhibiting or otherwise modulating a biological response (usually involving signaling pathways internal to the cell carrying the receptor) Many peptide and polypeptide ligands have been reported that function by doing so. Examples of such ligands include peptide and polypeptide hormones, cytokines, chemokines, growth factors, and apoptosis inducing factors.

  Because of the biological activity of such ligands, many are potentially useful as therapeutic agents. Several peptide or polypeptide ligands are regulated as therapeutic products, such as human growth hormone, insulin, interferon (IFN) -alpha 2b, IFN-alpha 2a, IFN beta, erythropoietin, G-CSF and GM-CSF Authorized by the authorities.

  Although the potential of these and other ligands in therapeutic applications has been shown, they can also exhibit toxicity when administered to human patients. One reason for toxicity is that many of these ligands induce receptors on a variety of cells, including cells other than those that mediate the desired therapeutic effect. As a result of such “off target” activity of ligands, many ligands are not currently suitable for use as therapeutic agents. This is because the ligand cannot be administered at a sufficiently high dose to produce the maximum or optimal therapeutic effect on the target cells that mediate the therapeutic effect.

  For example, it has been known since the mid-1980s that interferons, particularly IFN-alpha, can increase apoptosis of certain cancer cells and reduce their proliferation. IFN-alpha has been approved by the FDA for the treatment of several cancers such as melanoma, renal cell carcinoma, B cell lymphoma, multiple myeloma, chronic myelogenous leukemia (CML) and hairy cell leukemia. The direct effect of IFN-alpha on tumor cells is mediated by IFN-alpha, which binds directly to type I IFN receptors on these cells and stimulates apoptosis, terminal differentiation or decreased proliferation. A further indirect effect of IFN-alpha on non-cancer cells is to stimulate the immune system, which can lead to further anticancer effects by causing the immune system to reject the tumor.

  These biological activities are mediated by type I interferon receptors on the surface of cancer cells and, when stimulated, initiate various signaling pathways that reduce proliferation and / or induce terminal differentiation or apoptosis. Bring. However, type I interferon receptors are also present on many non-cancerous cells. Activation of this receptor on non-cancerous cells by IFN-alpha causes the expression of several proinflammatory cytokines and chemokines, resulting in toxic and adverse effects. Such toxicity can cause severe cold-like symptoms and inhibit the administration of IFN-alpha to subjects at levels that exert maximal anti-proliferative and pro-apoptotic activity against cancer cells.

  When IFN-alpha 2b is used to treat multiple sclerosis, its utility is at least in part in its binding to type I interferon receptors on myeloma cells and then apoptosis And / or induce a reduction in proliferation and thus limit disease progression. Unfortunately, however, this IFN also binds to healthy cells in the body and triggers a variety of other cellular responses, some of which are harmful.

  A publication by Ozzello (Breast Cancer Research and Treatment 25: 265-76, 1993) describes tumors as a means of reducing tumor growth rate by chemically conjugating human IFN-alpha to tumor-targeted antibodies. Described the localization of the direct inhibitory activity of IFN-alpha and demonstrated that such conjugates have anti-tumor activity in a human cancer xenograft model. Since the human IFN-alpha used in the experiment did not appreciably interact with the mouse type I IFN receptor, which can provide an indirect anticancer effect, the mechanism of anticancer activity observed was , Due to the direct effect of IFN-alpha on cancer cells. Due to this lack of human IFN-alpha binding to mouse cells, the toxicity of the antibody-IFN-alpha conjugate compared to free IFN-alpha was not evaluated.

  The antibody and IFN-alpha can also be connected together in the form of a fusion protein. For example, WO01 / 97844 describes the direct fusion of human IFN-alpha to the C-terminus of the heavy chain of IgG specific for the tumor antigen CD20.

  In general, IFN can be targeted to cancer cells. While this approach can lead to an increase in the activity of IFN against cancer cells, it cannot fully address the problem of IFN's undesirable activity against healthy cells. Fusion of IFN-alpha to the C-terminus of the IgG heavy chain can extend the half-life of IFN alpha and lead to undesirable adverse events. Therefore, it is necessary to reduce the off-target activity of ligand-based drugs while retaining the “on-target” therapeutic effect of the ligand.

WO01 / 97844 U.S. Patent No. 7,732,578 U.S. Patent No. 7,083,784 U.S. Patent No. 7,217,797 WO00 / 42072 US Patent Application Publication No. 2009/0142340 US Patent Application Publication No. 2009/0068175 US Patent Application Publication No. 2009/0092599 U.S. Patent 6,602,684 U.S. Patent No. 7,326,681 U.S. Patent No. 7,388,081 PCT publication number WO08 / 006554 PCT application number PCT / AU2012 / 001323 US Patent Application Publication No. 2003/0039649

Koguma, T. (1994) Biochim. Biophys. Acta 1223: 160 Ozzello (Breast Cancer Research and Treatment 25: 265-76, 1993) Ku & Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995 Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 http://www.bioinfo.org.uk/mdex.html Edelman, GM et al. (1969) Proc. Natl. Acad. USA, 63, 78-85 Chothia et al. (1987) J. Mol. Biol. 196: 901-17 Chothia et al. (1989) Nature 342: 877-83 Al-Lazikani et al. (1997) J. Mol. Biol. 273: 927-48 Honnegher et al. (2001) J. Mol. Biol., 309: 657-70 Giudicelli et al. (1997) Nucleic Acids Res. 25: 206-11 Padlan et al. (1995) FASEB J. 9: 133-139 Shinkawa T. et al. (2003) J. Biol. Chem. 278: 3466-73 Labrin et al. (2009) Nature Biotechnology 27: 8; 767-773 Altschul SF (1997) Nucleic Acids Res. 25: 3389-3402 Hardin J. et al. (1994) Blood. 84: 3063-70 Gazdar, Blood 67: 1542-1549, 1986

  The present disclosure features new anti-CD38 antibodies and constructs comprising anti-CD38 antibodies and attenuated IFN-alpha. Antibodies that contain one or more mutations in the heavy and / or light chain variable regions retain the ability to specifically bind to CD38, including CD38 expressed on the surface of cells. The antibody can be fused, for example, to an attenuated form of interferon alpha to form an anti-CD38 antibody-attenuated interferon fusion construct.

  In some embodiments, an isolated antibody that specifically binds CD38 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 559 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 664. In some embodiments, the isolated antibody that specifically binds CD38 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 665 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 666. In some embodiments, the isolated antibody that specifically binds CD38 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 739 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 664. The heavy chain variable region amino acid sequence of SEQ ID NO: 559 does not include the amino acid sequence of SEQ ID NO: 13. The light chain variable region amino acid sequence of SEQ ID NO: 664 does not include the amino acid sequence of SEQ ID NO: 14. In some embodiments, the isolated antibody that specifically binds to CD38 is a heavy chain CDR1, SEQ ID NO: 202, SEQ ID NO: 516, SEQ ID NO: 200 comprising the amino acid sequence of SEQ ID NO: 200, SEQ ID NO: 514 or SEQ ID NO: 697: 544, heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 698 or SEQ ID NO: 737, and SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 518, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 699 or SEQ ID NO: 738 A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 233, SEQ ID NO: 319, SEQ ID NO: 583, SEQ ID NO: 590 or SEQ ID NO: 696, SEQ ID NO: 235, SEQ ID NO: 307, SEQ ID NO: 311 A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 585, SEQ ID NO: 591 or SEQ ID NO: 605, a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 237, SEQ ID NO: 321, SEQ ID NO: 324, SEQ ID NO: 587 or SEQ ID NO: 594 May be included.

  In a preferred embodiment, the heavy chain variable region comprises SEQ ID NO: 34, SEQ ID NO: 18, SEQ ID NO: 665, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 156, SEQ ID NO: 197, SEQ ID NO: 152, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO: 742, SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 731 including. In a preferred embodiment, the light chain variable region is SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, Sequence number 161, sequence number 184, sequence number 185, sequence number 188, sequence number 198 or sequence number 700, sequence number 701, sequence number 704, sequence number 705, sequence number 706, sequence number 707, sequence number 708, sequence number It includes the amino acid sequences of 709, SEQ ID NO: 710, SEQ ID NO: 711.

The antibody is preferably capable of binding to CD38 positive cells. The antibody can bind to CD38 positive cells with an EC50 value of less than about 100 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 75 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 50 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 30 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 25 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 20 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 15 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 13 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 10 nM.

  The antibody may be a monoclonal antibody, preferably a fully human antibody. The antibody may comprise a FAb. The antibody may comprise a human IgG1 constant region or a human IgG4 constant region. The IgG1 or IgG4 constant region may comprise tyrosine at position 252 according to the EU numbering system, threonine at position 254, and glutamic acid at position 256. The IgG4 constant region may contain a proline at position 228 according to the EU numbering system, and the proline at position 228 may be threonine at position 254 and glutamic acid at position 256 in addition to tyrosine at position 252.

  In some embodiments, the antibody is fused to attenuated interferon alpha-2b. Interferon alpha-2b may comprise a substitution of alanine at position 145 to glycine or aspartic acid, such as interferon alpha-2b having the amino acid sequence of SEQ ID NO: 649 or SEQ ID NO: 651. Attenuated interferon alpha-2b may be indirectly fused to the C-terminus of IgG1 or IgG4 constant region, and the antibody is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, sequence The amino acid sequence of SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, or SEQ ID NO: 694 may be included. An antibody, such as an antibody fused to attenuated interferon alpha-2b, may be included in a composition comprising a pharmaceutically acceptable carrier.

  Isolated polynucleotides encoding antibodies and antibodies fused to attenuated interferon alpha-2b are provided. The polynucleotide is SEQ ID NO: 667, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 675, SEQ ID NO: 676, or SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690 , SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 695, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: NO.718, SEQ ID NO: 719, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745 The nucleic acid sequence of SEQ ID NO: 746 may be included. The polynucleotide may comprise a vector. Vectors can be used, for example, to transform cells. Also provided are transformed cells containing such polynucleotides. The transformed cells may include mammalian cells, yeast cells, or insect cells.

  Stable cells expressing the antibody are also provided. The cell expressing the antibody may be a mammalian cell. Preferred cells are Chinese hamster ovary (CHO) cells.

  A kit comprising an antibody fused to attenuated interferon alpha-2b is provided. The kit uses an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct and the construct in a method for inhibiting growth of tumor cells that express CD38 and interferon alpha-2b receptors on the surface. Instructions, instructions for using said construct in a method for inducing apoptosis in tumor cells that express receptors of CD38 and interferon alpha-2b on the surface, CD38 and interferon alpha- in a subject in need thereof And instructions for using the construct in a method for treating a tumor comprising cells expressing the receptor of 2b on the surface, and optionally a pharmaceutically acceptable carrier. Kits comprising an anti-CD38 antibody are provided, such kits comprising an anti-CD38 antibody and instructions for using said antibody in a method for detecting CD38 positive tumor cells in a tissue sample isolated from a subject Wherein the antibody may optionally be fused to an attenuated interferon alpha-2b protein.

  The anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be used as a therapy in the treatment of tumors comprising cells that express CD38 and interferon alpha-2b receptors on their surface. In general, the method of treatment comprises administering to a subject having a tumor an amount of an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct effective to treat the tumor. The construct may include any construct described or exemplified herein. The subject is preferably a mammal, more preferably a non-human primate, most preferably a human. The tumor may include B cell lymphoma, multiple myeloma, non-Hodgkin lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia or acute myeloid leukemia.

  Optionally, an anti-CD38 antibody fused to an attenuated interferon alpha-2b protein can be used in a method for detecting CD38 or CD38 positive tumor cells in a tissue sample isolated from a subject. In general, the method comprises contacting an antibody that specifically binds to CD38 with a tissue sample isolated from a subject, and detecting a complex of the antibody and CD38 or CD38 positive cells in the tissue sample. including. The tissue sample may be known or suspected of having CD38 positive tumor cells. The tissue may include blood or bone marrow. The CD38 positive tumor cell may be a CD38 positive B cell lymphoma, multiple myeloma cell, non-Hodgkin lymphoma cell, chronic myeloid leukemia cell, chronic lymphocytic leukemia cell, or acute myeloid leukemia cell. The subject is preferably a mammal, more preferably a non-human primate, most preferably a human. The method may include the step of isolating a tissue sample from the subject. The method may further comprise contacting the antibody with, for example, a tissue sample that does not contain CD38 positive cells that serve as a negative control.

FIG. 2 shows an example of an anti-CD38 antibody-attenuated interferon fusion construct. FIG. 7 shows the heavy chain variable region sequences of X02.1, related constructs, and the most homologous germline antibody sequences. CDRs defined by Kabat numbering system are underlined. FIG. 7 shows the heavy chain variable region sequences of X02.1, related constructs, and the most homologous germline antibody sequences. CDRs defined by Kabat numbering system are underlined. FIG. 3 shows the sequence of the light chain variable region of X02.1, related constructs, and the most homologous germline antibody sequence. CDRs defined by Kabat numbering system are underlined. FIG. 3 shows the sequence of the light chain variable region of X02.1, related constructs, and the most homologous germline antibody sequence. CDRs defined by Kabat numbering system are underlined. FIG. 3 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 3 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 3 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 3 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 2 shows the consensus variable heavy chain sequence of A02.1 and related constructs. The boxed area contains the CDRs (as shown) defined by Kabat numbering system and enhanced Chothia numbering system. CDRs defined by Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 3 shows a consensus variable light chain sequence for A02.1 and related constructs. The boxed area contains the CDRs (as shown) defined by Kabat numbering system and enhanced Chothia numbering system. CDRs defined by Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. It is a figure which shows the arrangement | sequence of the heavy chain variable region of a humanized heavy chain variable region. CDRs defined by Kabat numbering system are underlined. It is a figure which shows the arrangement | sequence of the heavy chain variable region of a humanized heavy chain variable region. CDRs defined by Kabat numbering system are underlined. It is a figure which shows the arrangement | sequence of the heavy chain variable region of a humanized heavy chain variable region. CDRs defined by Kabat numbering system are underlined. It is a figure which shows the arrangement | sequence of the heavy chain variable region of a humanized light chain variable region. CDRs defined by Kabat numbering system are underlined. It is a figure which shows the arrangement | sequence of the heavy chain variable region of a humanized light chain variable region. CDRs defined by Kabat numbering system are underlined. It is a figure which shows the arrangement | sequence of the heavy chain variable region of a humanized light chain variable region. CDRs defined by Kabat numbering system are underlined. FIG. 6 shows the variable heavy chain of A10.0 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 6 shows the variable heavy chain of A10.0 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 6 shows the variable light chain of A10.0 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 6 shows the variable light chain of A10.0 and related constructs. CDRs defined by Kabat numbering system are underlined. FIG. 5 shows the variable heavy chain consensus sequence of A10.0 and related constructs. The boxed area contains the CDRs (as shown) defined by Kabat numbering system and enhanced Chothia numbering system. CDRs defined by Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 5 shows the variable heavy chain consensus sequence of A10.0 and related constructs. The boxed area contains the CDRs (as shown) defined by Kabat numbering system and enhanced Chothia numbering system. CDRs defined by Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 5 shows the variable light chain consensus sequence of A10.0 and related constructs. The boxed area contains the CDRs (as shown) defined by Kabat numbering system and enhanced Chothia numbering system. CDRs defined by Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. It is a figure which shows the binding activity of A02.1 variant to the multiple myeloma cell line ARP-1 which expresses CD38 measured by flow cytometry. Details of the assay are described in the Examples herein. It is a figure which shows the binding activity of A02.1 variant to multiple myeloma cell line NCI-H929 which expresses CD38 measured by flow cytometry. Details of the assay are described in the Examples herein. FIG. 5 shows the antiproliferative activity of A02.1 variant on multiple myeloma cell line ARP-1. The A-isotype is an irrelevant specificity antibody fused to attenuated interferon as a control. Details of the assay are described in the examples (cell proliferation assay). FIG. 5 shows the antiproliferative activity of A02.1 variant on multiple myeloma cell line ARP-1. The A-isotype is an irrelevant specificity antibody fused to attenuated interferon as a control. Details of the assay are described in the examples (cell proliferation assay). Anti-proliferative activity of IFN-alpha 2b (Intron A) compared to A02.1 and A10.0 and its corresponding non-fusion antibodies X02.1 and X10.0 on multiple myeloma cell line ARP-1 FIG. The A-isotype is an irrelevant specificity antibody fused to attenuated interferon as a control. Details of the assay are described in the examples (cell proliferation assay). In multiple myeloma cell line NCI-H929 expressing CD38 when treated with A02.1 and A10.0 and its corresponding non-fusion antibodies X02.1 and X10.0 for 24 hours compared to untreated controls It is a figure which shows the relative fold change of the annexin V production | generation in. The A-isotype is an irrelevant specificity antibody fused to attenuated interferon as a control. Details of the assay are described in the examples (Annexin V assay). Figure 2 shows the relative fold change in caspase activation in multiple myeloma cell line H929 expressing CD38 of IFN-alpha 2b (Intron A) vs. A02.1 and related constructs compared to untreated cells. is there. Isotype 145D is an irrelevant specificity antibody fused to attenuated interferon as a control. Details of the assay are described in the examples (caspase assay). FIG. 6 shows the off-target activity of A02.6 [A02.6 (wt.IFN)] fused to IFN-alpha 2b (Intron A) versus A02.6 and wild-type IFN-alpha 2b against CD38 negative cells. Details of the assay are described in the examples [HEK-BLUE ™]. Figure 5 shows the relative fold change in Annexin V production in the multiple myeloma cell line H929 expressing CD38, between the IgG1 and IgG4 subtypes of the anti-CD38 antibody-attenuated IFN-alpha fusion protein construct. is there. The A-isotype is a non-specific IgG4 antibody fused with attenuated interferon as a control. Antibodies A02.12 and A10.0 contain IgG4 constant regions fused to attenuated IFN-alpha, while A02.112 and A10.59 contain IgG1 constant regions fused to attenuated IFN-alpha. Details of the assay are described in the examples (Annexin V / 7AAD assay). It is a figure which shows the binding activity of A10.0 variant to multiple myeloma cell line NCI-H929 which expresses CD38 measured by flow cytometry. Details of the assay are described in the Examples herein. FIG. 6 shows caspase activation in multiple myeloma cell line H929 expressing A10.0 and A10.38 CD38 compared to untreated cells. The A-isotype is an irrelevant specificity antibody fused to attenuated IFN as a control. Details of the assay are described in the examples (caspase assay). FIG. 6 shows the relative fold change in caspase activation in multiple myeloma cell line H929 expressing CD38 with A10.0 variant compared to untreated cells. Details of the assay are described in the examples (caspase assay). FIG. 5 shows the relative fold change in the production of annexin V in multiple myeloma cell line H929 expressing CD38 with A10.0 variant. Details of the assay are described in the examples (Annexin V / 7AAD assay). FIG. 5 shows the antiproliferative activity of IFN-alpha 2b (Intron A) compared to A02.6, A10.0, A10.38 and parental A10A2.0 chimeric antibody constructs on Burkitt lymphoma cell line Daudi. The A-isotype is an irrelevant specificity antibody fused to attenuated IFN as a control. Details of the assay are described in the examples (cell proliferation assay). FIG. 5 shows the effect of humanized A10.0 vs. parental A10A2.0 chimeric antibody-attenuated interferon construct on the growth of subcutaneous H929 myeloma tumors in SCID mice. The bar labeled “treatment phase” indicates the duration of treatment with the compound. FIG. 5 shows non-antibody antigen targeted IFN activity of A10.0 variant fused to the same attenuated IFN-alpha 2b protein. Details of the assay are described in the Examples (“Off-Target Assay” —iLite Gene Reporter Assay). Shows “off-target” activity of IFN-alpha 2b (intron A) compared to parental A10A2.0 chimeric antibody [A10A2.0 chimera (wt.IFN)] fused to A10.0 variant and wild type IFN-alpha 2b FIG. Details of the assay are described in the Examples [“Off-Target Assay” —HEK-BLUE ™]. FIG. 9 shows a variable heavy chain consensus sequence of X910 / 12-HC-L0-interferon-alpha (A145D) and related sequences. The boxed area contains the CDRs (as shown) defined by Kabat numbering system and enhanced Chothia numbering system. CDRs defined by Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined.

  Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms should be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

  The terms subject and patient are used interchangeably and include any animal. Mammals, including companion and domestic mammals, and rodents such as mice, rabbits, and rats, and other rodents are preferred. Non-human primates such as cynomolgus monkeys are more preferred and humans are highly preferred.

  A molecule, such as an antibody, is “isolated” if it has been changed and / or removed from its natural environment by the human hand.

  As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise.

  The anti-CD38 antibody-attenuated interferon alpha-2b fusion construct includes, but is not limited to, interferon alpha-2b of SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, or SEQ ID NO: 651: Any antibody described or exemplified herein that specifically binds to CD38 fused to an attenuated interferon alpha-2b protein is included. In some embodiments, fusion of a non-mutated interferon alpha-2b protein, such as SEQ ID NO: 7, to an anti-CD38 antibody attenuates the biological activity of the interferon molecule. In the present disclosure, attenuated interferon, attenuated interferon alpha-2b, IFN-alpha 2b A145D, and IFN-alpha 2b A145G are used interchangeably.

  Specificity need not be in an absolute sense, but may be a relative term that indicates the degree of selectivity of an antibody-IFN-alpha fusion protein construct for antigen-positive cells compared to antigen-negative cells. The specificity of the antibody-IFN-alpha fusion protein construct for antigen positive cells is mediated by the variable region of the antibody, usually by the complementarity determining region (CDR) of the antibody. The construct may have a 100-fold specificity for antigen positive cells compared to antigen negative cells.

  Human CD38 includes the amino acid sequence of SEQ ID NO: 1, and cynomolgus CD38 includes the amino acid sequence of SEQ ID NO: 2.

  It is further possible that interferon-alpha 2b can be attenuated with respect to its biological activity mediated through interferon binding to cell surface interferon receptors by introducing certain amino acid changes in its protein sequence. Observed. Attenuated interferon molecule specifically binds to CD38 so that the antibody can serve as a delivery vehicle for attenuated interferon to CD38 positive cells resulting in reduced off-target interferon activity caused by the attenuated interferon molecule Can be fused to the antibody. It was further observed that the fusion of attenuated interferon to the CD38 antibody did not significantly affect the ability of the antibody to specifically bind CD38 on cells expressing CD38, such as cells in the animal's body. Manipulating CD38 antibody variants such that the antibody has reduced immunogenicity and enhanced stability and half-life without significantly losing the specificity or affinity of the antibody for the CD38 antigen; and Can be expressed. These variant antibodies can be fused to attenuated interferon.

  Thus, it features an antibody that specifically binds to CD38. It has also been observed that such anti-CD38 antibodies can be used as delivery vehicles for attenuated ligands such as interferon alpha. While not intending to be limited to any particular theory or mechanism of action, antibodies direct interferon alpha to bind them to CD38 positive cells, and interferon interacts with its receptor It is considered possible. Antibodies as delivery vehicles are believed to offset the reduced ability of interferon molecules to bind to their receptors. In this sense, attenuated interferon has a reduced ability to interact with its receptor on healthy cells, particularly cells that do not express CD38. By bringing attenuated interferon close to its receptor on CD38 positive cells, the antibody shows its associated receptor while showing a reduced ability to induce undesirable effects on healthy cells that do not express CD38. It is believed that the ability of attenuated interferon to bind to the body can be enhanced and induce therapeutic effects.

  The antibody may be polyclonal, but in some embodiments is not polyclonal. The antibody is preferably monoclonal. The antibody is preferably a full length antibody. Full-length antibodies generally comprise a variable region heavy chain and a variable region light chain. An antibody may comprise a derivative or fragment or portion of an antibody that retains antigen binding specificity and preferably retains much or all of the affinity of the parent antibody molecule (eg, for CD38). For example, the derivative may comprise at least one variable region (either heavy chain or light chain variable region). Other examples of suitable antibody derivatives and fragments include, but are not limited to, antibodies with multi-epitope specificity, bispecific antibodies, multispecific antibodies, diabodies, single chain molecules, and Fab, F (Ab ') 2, Fd, Fabc, and Fv molecules, single chain (Sc) antibodies, single chain Fv antibodies (scFv), individual antibody light chains, individual antibody heavy chains, antibody chains and other molecules Fusions, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy chain and one light chain, and other multimers. Single chain Fv antibodies may be multivalent. Any antibody isotype can be used to generate antibody derivatives, fragments, and portions. Antibody derivatives, fragments, and / or portions can be produced and expressed recombinantly in any prokaryotic or eukaryotic cell type.

  In some embodiments, an isolated antibody may refer to a monoclonal antibody in which IFN-alpha or attenuated IFN-alpha is fused to the C-terminus of a heavy chain IgG constant region. If the monoclonal antibody has binding specificity for CD38 and the IFN-alpha is attenuated IFN-alpha 2b, the isolated antibody herein is an anti-CD38 antibody-attenuated IFN-alpha fusion protein, or Also called anti-CD38 antibody-attenuated IFN-alpha fusion construct.

  In full-length antibodies, each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains, CH1, CH2 and CH3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FWR or FR). Each VH and VL includes three CDRs and four FWRs arranged in the following order from the amino terminus to the carboxy terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. Typically, the antigen binding properties of an antibody are less likely to be disturbed by changes to the FWR sequence than by changes to the CDR sequence. The immunoglobulin molecule may be of any type (eg, IgG, IgE, IgM, IgD, IgA and Igγ), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

  The antibody may be derived from any species. For example, the antibody may be a mouse, rat, goat, horse, pig, cow, camel, chicken, rabbit, donkey, llama, dromedary, shark, or human antibody, as well as an antibody from any other animal species. Good. For use in human treatment, non-human antibodies can be structurally altered such that they are less antigenic when administered to a human patient, such as by chimerization or humanization or superhumanization.

  In some embodiments, the antibody is a humanized antibody. Humanized antibodies are amino acids directly involved in antigen binding, e.g., heavy and / or light chain complementarity determining regions (CDRs), in some cases framework regions (FWRs), or portions thereof of human origin. Although not, the remaining amino acids in the antibody are human, or are otherwise of human origin, eg, a human antibody scaffold. A humanized antibody may also be a non-human residue in which one or more residues of a human protein are modified by one or more amino acid substitutions and / or one or more FWR residues of a human protein correspond Also included are antibodies that have been replaced by Humanized antibodies may also comprise residues that are found neither in human antibodies nor in non-human antibodies. The humanized antibody may be a superhumanized antibody as described, for example, in US Pat. No. 7,732,578. The antibody may be a humanized chimeric antibody.

  In a highly preferred embodiment, the antibody is a fully human antibody. A fully human antibody comprises the same amino acid sequence as a whole antibody, whether the entire molecule is human or otherwise of human origin. Completely human antibodies include, for example, those obtained from a human V gene library in which a human gene encoding the variable region of the antibody is recombinantly expressed. Fully human antibodies can be expressed in cells from other organisms (eg, mouse and xenomous technology) or from other organisms that have been transformed with genes encoding human antibodies. Nevertheless, fully human antibodies may contain amino acid residues that are not encoded by human sequences, eg, mutations introduced by random or site-specific mutations.

  The antibody may be a full-length antibody of any class, eg, IgG1, IgG2, or IgG4. The constant domain of such an antibody is preferably human. The variable region of such an antibody may be of non-human origin or preferably is of human origin or humanized. Antibody fragments can also be used in place of full length antibodies.

  The antibody may be a minibody. Minibodies contain small whole antibodies that encode the essential elements of all antibodies in a single chain. For example, a minibody may comprise the VH and VL domains of a natural antibody fused to the hinge region and CH3 domain of an immunoglobulin molecule.

  In some embodiments, the antibody may comprise a non-immunoglobulin derived protein framework. For example, a four-helix bundle protein cytochrome b562 with two loops selected for antigen binding and randomized to create a CDR is described (Ku & Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995).

  Natural sequence variations may be present in the heavy and light chains and the genes that encode them, and thus those skilled in the art will encode the amino acid sequences of the antibodies described and exemplified herein, or encode them One can expect to find some level of mutation in the gene. These variants preferably maintain the unique binding properties (eg, specificity and affinity) of the parent antibody. Such predictions are due, in part, to the known evolutionary success of conservative amino acid sequence variations due to the degeneracy of the genetic code as well as not appreciably altering the properties of the encoded protein. Accordingly, such variants and homologues are considered substantially the same as each other and are included within the scope of this disclosure. Thus, an antibody includes variants having one or more amino acid substitutions, deletions, additions or substitutions that retain the biological properties (eg, binding specificity and binding affinity) of the parent antibody. Variants are preferably conservative, but may be non-conservative.

  The amino acid positions assigned to CDRs and FWRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as Kabat numbering system). . Furthermore, the amino acid positions assigned to CDRs and FWRs can be defined according to the enhanced Chothia numbering scheme (http://www.bioinfo.org.uk/mdex.html). The heavy chain constant region of an antibody can be defined by the EU numbering system (Edelman, GM et al. (1969) Proc. Natl. Acad. USA 63, 78-85).

  According to Kabat's numbering system, VH FWRs and CDRs are expressed as follows: residues 1-30 (FWR1), 31-35 (CDR1), 36-49 (FWR2), 50-65 (CDR2), 66-94 (FWR3 ), 95-102 (CDR3) and 103-113 (FWR4), where VL FWR and CDR are: residues 1-23 (FWR1), 24-34 (CDR1), 35-49 ( FWR2), 50-56 (CDR2), 57-88 (FWR3), 89-97 (CDR3) and 98-107 (FWR4). In some examples, the variable region may increase in length, and according to Kabat's numbering system, several amino acids can be designated by letter after the number. This specification is not limited to FWRs and CDRs as defined by the Kabat numbering system, but includes Chothia et al. (1987) J. Mol. Biol. 196: 901-17; Chothia et al. (1989) Nature 342: 877- 83; and / or the standard numbering system of Al-Lazikani et al. (1997) J. Mol. Biol. 273: 927-48; Honnegher et al. (2001) J. Mol. Biol., 309: 657-70. Including any numbering system; such as the IMGT system discussed in Giudicelli et al. (1997) Nucleic Acids Res. 25: 206-11. In some embodiments, the CDRs are defined according to the Kabat numbering system.

  In some specific embodiments, for any of the heavy chain CDR2 subdomains described herein according to the Kabat numbering system, the 5 C-terminal amino acids may not be directly involved in antigen binding; Accordingly, it is understood that any one or more of these five C-terminal amino acids can be replaced with another natural amino acid without substantially adversely affecting antigen binding. In some embodiments, for any of the light chain CDR1 subdomains described herein, according to Kabat numbering system, the four N-terminal amino acids may not be directly involved in antigen binding, and thus It is understood that any one or more of these four amino acids can be replaced with another natural amino acid without substantially adversely affecting antigen binding. For example, as described by Padlan et al. (1995) FASEB J. 9: 133-139, the five C-terminal amino acids of heavy chain CDR2 and / or the four N-terminal amino acids of light chain CDR1 are involved in antigen binding. You don't have to. In some embodiments, both heavy chain CDR2 and light chain CDR1 are not directly involved in antigen binding.

  In some embodiments, chemical analogs of amino acids can be used in the antibodies described and / or exemplified herein. The use of chemical analogs of amino acids is useful for stabilizing the molecule, for example when it needs to be administered to a subject. Amino acid analogs contemplated herein include, but are not limited to, side chain modifications, incorporation of unnatural amino acids and / or derivatives thereof during peptide, polypeptide or protein synthesis and crosslinkers and proteins. Use of other methods of imposing conformational constraints on sex molecules or analogs thereof.

  An antibody may contain post-translational modifications or moieties that can affect the activity or stability of the antibody. These modifications or moieties include, but are not limited to, methylated, acetylated, glycosylated, sulfated, phosphorylated, carboxylated, and amidated moieties and other moieties well known in the art. It is done. A moiety includes any chemical group or combination of groups commonly found on immunoglobulin molecules, natural or otherwise added to antibodies by recombinant expression systems such as prokaryotic and eukaryotic expression systems. .

Examples of side chain modifications contemplated by this disclosure include reaction with an aldehyde followed by reductive alkylation by reduction with NaBH ₄ ; amidation with methylacetimidate; acylation with acetic anhydride; Carbamoylation; Trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); Acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and of lysine with pyridoxal-5-phosphate Amino group modifications such as pyridoxylation followed by reduction with NaBH ₄ may be mentioned.

  The guanidine group of arginine residues can be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

  The carboxyl group can be modified by O-acylisourea formation followed by carbodiimide activation, for example by subsequent derivatization to the corresponding amide.

  Carboxymethylation of sulfhydryl groups with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides; Formation of mercury derivatives with benzoic acid, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercury agents; modification by methods such as carbamoylation with cyanate at alkaline pH Can do.

  Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or halogenated sulfenyl. On the other hand, tyrosine residues can be changed by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

  Modification of the imidazole ring of a histidine residue can be achieved by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Crosslinkers, for example, homobifunctional crosslinkers such as bifunctional imide esters, glutaraldehyde, N-hydroxysuccinimide esters having (CH ₂ ) n spacer groups with n = 1 to n = 6 and usually N-hydroxy Using heterobifunctional reagents containing an amino reactive moiety such as succinimide and a reactive moiety specific for another group such as maleimide or dithio moiety (SH) or carbodiimide (COOH), 3D antibodies and constructs Conformation can be stabilized.

  The antibody may be affinity matured or may contain amino acid changes that reduce immunogenicity, eg, by removing a predicted MHC class II binding motif. The therapeutic utility of the antibodies described herein includes antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life, biodistribution and binding to Fc receptors. Further enhancement can be achieved by adjusting its functional characteristics, such as any combination of. This regulation can be achieved by protein engineering, glycoengineering or chemical methods. Depending on the therapeutic application required, it may be advantageous to increase or decrease any of these activities. As an example of glycoengineering, the Potelligent® method described in Shinkawa T. et al. (2003) J. Biol. Chem. 278: 3466-73 was used.

  The antibody protects IgG from catabolism and its serum, including modifications that modulate the interaction of the antibody with the neonatal Fc receptor (FcRn), a receptor that has an important role in maintaining high serum antibody concentrations Modifications that modulate half-life and biodistribution may be included. Modifications that modulate serum half-life are described in M252Y / S254T / T256E [EU numbering system (Edelman, GM et al. (1969) Proc. Natl. Acad. USA 63, 78-85, as described in US Pat. No. 7,083,784. May be present in the Fc region of IgG1 or IgG4, such as triple substitution (eg, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 694). Other substitutions may be present at positions 250 and 428 (see, e.g., U.S. Patent No. 7,217,797), and positions 307, 380, and 434 (see e.g., WO00 / 42072). . Examples of constant domain amino acid substitutions that regulate subsequent functions mediated by these receptors, such as binding to Fc receptors and FcRn binding and serum half-life are described in US Patent Application Publication Nos. 2009/0142340, 2009. / 0068175, and 2009/0092599. Naked antibodies may have the heavy chain C-terminal lysine omitted or removed to reduce heterogeneity. Substitution of S228P (EU numbering) in human IgG4 can stabilize antibody Fab arm exchange in vivo (Labrin et al. (2009) Nature Biotechnology 27: 8; 767-773).

  Glycans linked to antibody molecules are known to affect the interaction of antibodies with Fc receptors and glycan receptors, thereby affecting antibody activity such as serum half-life. Thus, certain glycoforms that modulate the desired antibody activity can provide therapeutic benefits. Methods for making engineered glycoforms include, but are not limited to, those described in US Pat. Nos. 6,602,684, 7,326,681, and 7,388,081 and PCT Publication No. WO08 / 006554. . Alternatively, antibody sequences can be modified to remove related glycoform binding sites.

  The antibody can be labeled or conjugated to any chemical or biological molecular moiety. Labeled antibodies can be useful in therapeutic, diagnostic, or basic research applications. Such labels / conjugates such as fluorescent dyes, radioactive labels, enzymes, fluorescent proteins, and biotin may be detectable. The label / conjugate may be a chemotherapeutic agent, a toxin, an isotope, and other agents used to treat conditions such as cancer cell killing. The chemotherapeutic agent may be any that is suitable for the purpose for which the antibody is used.

  Antibodies can be derivatized with known protecting / blocking groups to prevent protein cleavage or to enhance activity or stability.

The antibody preferably has binding affinity for an epitope on CD38 comprising a dissociation constant (Kd) of less than about 1 × 10 ⁻² M. In some embodiments, the Kd is less than about 1 × 10 ⁻³ M. In other embodiments, the Kd is less than about 1 × 10 ⁻⁴ M. In some embodiments, the Kd is less than about 1 × 10 ⁻⁵ M. In still other embodiments, the Kd is less than about 1 × 10 ⁻⁶ M. In other embodiments, the Kd is less than about 1 × 10 ⁻⁷ M. In other embodiments, the Kd is less than about 1 × 10 ⁻⁸ M. In other embodiments, the Kd is less than about 1 × 10 ⁻⁹ M. In other embodiments, the Kd is less than about 1 × 10 ⁻¹⁰ M. In still other embodiments, the Kd is less than about 1 × 10 ⁻¹¹ M. In some embodiments, the Kd is less than about 1 × 10 ⁻¹² M. In other embodiments, the Kd is less than about 1 × 10 ⁻¹³ M. In other embodiments, the Kd is less than about 1 × 10 ⁻¹⁴ M. In still other embodiments, the Kd is less than about 1 × 10 ⁻¹⁵ M. Affinity values are those obtained by standard methods including surface plasmon resonance, such as BiacoreTM analysis or analysis using Octet® Red 96 (Forte Bio) Dip-and-Read system. Point to.

  The antibody may comprise a single chain Fv molecule (scFv), Fab, or full length IgG. Any such antibody comprises the amino acid sequence of SEQ ID NO: 659 or SEQ ID NO: 665 or SEQ ID NO: 736, provided that the heavy chain comprising the amino acid sequence of SEQ ID NO: 659 or a variant thereof does not include the amino acid sequence of SEQ ID NO: 13. An amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity It may contain heavy chains. It is understood that antibodies that contain amino acid changes in the heavy chain retain the ability to specifically bind to CD38. The retained CD38 specific binding activity (including affinity) is preferably about the same as that of an antibody that does not contain amino acid changes in the heavy chain (including affinity), but its binding activity (affinity). May be lower or higher than antibodies that do not contain amino acid changes in the heavy chain. The antibody is at least about 85%, at least about 90% from the amino acid sequence of SEQ ID NO: 664 or SEQ ID NO: 666, provided that the light chain comprising the amino acid sequence of SEQ ID NO: 664 or a variant thereof does not include the amino acid sequence of SEQ ID NO: 14. %, At least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% light chains having amino acid sequences with sequence identity. It is understood that antibodies that contain amino acid changes in the light chain retain the ability to specifically bind to CD38. The retained CD38 specific binding activity (including affinity) is preferably about the same as that of an antibody that does not contain amino acid changes in the light chain (including affinity), but its binding activity (affinity). May be lower or higher than antibodies that do not contain amino acid changes in the light chain.

  In some embodiments, the heavy chain FWR1 comprises SEQ ID NO: 199, SEQ ID NO: 206, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 389, SEQ ID NO: 396, SEQ ID NO: 400, SEQ ID NO: 404. , SEQ ID NO: 408, SEQ ID NO: 412, SEQ ID NO: 416, SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 428, SEQ ID NO: 432, SEQ ID NO: 466, SEQ ID NO: 470, SEQ ID NO: 472, SEQ ID NO: 474, SEQ ID NO: 476, Sequence SEQ ID NO: 478, SEQ ID NO: 480, SEQ ID NO: 482, SEQ ID NO: 486, SEQ ID NO: 488, SEQ ID NO: 513, SEQ ID NO: 537, SEQ ID NO: 542, SEQ ID NO: 547, SEQ ID NO: 552, SEQ ID NO: 557, SEQ ID NO: 562, SEQ ID NO: 567 In some embodiments, the heavy chain FWR1 comprises SEQ ID NO: 199, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 572, SEQ ID NO: 572, SEQ ID NO: 577, or SEQ ID NO: 748. No. 389, SEQ ID No. 396, SEQ ID No. 400, SEQ ID No. 404, SEQ ID No. 408 SEQ ID NO: 412, SEQ ID NO: 416, SEQ ID NO: 420, SEQ ID NO: 424, SEQ ID NO: 428, SEQ ID NO: 432, SEQ ID NO: 466, SEQ ID NO: 472, SEQ ID NO: 474, SEQ ID NO: 476, SEQ ID NO: 478, SEQ ID NO: 480, SEQ ID NO: 482, SEQ ID NO: 486, SEQ ID NO: 488, SEQ ID NO: 513, SEQ ID NO: 537, SEQ ID NO: 542, SEQ ID NO: 547, SEQ ID NO: 552, SEQ ID NO: 557, SEQ ID NO: 562, SEQ ID NO: 567, SEQ ID NO: 572, At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 with the amino acid sequence of SEQ ID NO: 577 or SEQ ID NO: 748. %, At least about 98%, or at least about 99% amino acid sequences having sequence identity. In some embodiments, heavy chain FWR2 comprises SEQ ID NO: 201, SEQ ID NO: 211, SEQ ID NO: 229, SEQ ID NO: 391, SEQ ID NO: 397, SEQ ID NO: 401, SEQ ID NO: 405, SEQ ID NO: 409, SEQ ID NO: 413, SEQ ID NO: 417. , SEQ ID NO: 421, SEQ ID NO: 425, SEQ ID NO: 429, SEQ ID NO: 433, SEQ ID NO: 515, SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 538, SEQ ID NO: 538 Comprising the amino acid sequence of SEQ ID NO: 543, SEQ ID NO: 548, SEQ ID NO: 553, SEQ ID NO: 558, SEQ ID NO: 563, SEQ ID NO: 568, SEQ ID NO: 573, SEQ ID NO: 578, SEQ ID NO: 749 or SEQ ID NO: 750, in some embodiments, Heavy chain FWR2 is SEQ ID NO: 201, SEQ ID NO: 211, SEQ ID NO: 229, SEQ ID NO: 391, SEQ ID NO: 397, SEQ ID NO: 401, SEQ ID NO: 405, SEQ ID NO: 409, SEQ ID NO: 413, SEQ ID NO: 417, SEQ ID NO: 421, sequence SEQ ID NO: 425, SEQ ID NO: 429, SEQ ID NO: 433, SEQ ID NO: 515, SEQ ID NO: 520 SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 538, SEQ ID NO: 543, SEQ ID NO: 548, SEQ ID NO: 553, SEQ ID NO: 558, SEQ ID NO: 563, SEQ ID NO: 568, SEQ ID NO: 568 573, SEQ ID NO: 578, SEQ ID NO: 749 or SEQ ID NO: 750 with at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about at least about Amino acid sequences having 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, the heavy chain FWR3 comprises SEQ ID NO: 203, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 221, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 230. , SEQ ID NO: 393, SEQ ID NO: 399, SEQ ID NO: 403, SEQ ID NO: 407, SEQ ID NO: 411, SEQ ID NO: 415, SEQ ID NO: 419, SEQ ID NO: 423, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 435, SEQ ID NO: 468, Sequence SEQ ID NO: 517, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 540, SEQ ID NO: 545, SEQ ID NO: 550, SEQ ID NO: 555, SEQ ID NO: 560, SEQ ID NO: 565, SEQ ID NO: 570, SEQ ID NO: 575 In some embodiments, the heavy chain FWR3 comprises SEQ ID NO: 203, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 216, sequence SEQ ID NO: 580, SEQ ID NO: 751, or SEQ ID NO: 752. SEQ ID NO: 218, SEQ ID NO: 221, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 230 Sequence number 393, sequence number 399, sequence number 403, sequence number 407, sequence number 411, sequence number 415, sequence number 419, sequence number 423, sequence number 427, sequence number 431, sequence number 435, sequence number 468, sequence number 517, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 540, SEQ ID NO: 545, SEQ ID NO: 550, SEQ ID NO: 555, SEQ ID NO: 560, SEQ ID NO: 565, SEQ ID NO: 570, SEQ ID NO: 575, At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% with the amino acid sequence of SEQ ID NO: 580, SEQ ID NO: 751, or SEQ ID NO: 752. An amino acid sequence having at least about 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, heavy chain FWR4 comprises SEQ ID NO: 205, SEQ ID NO: 395, SEQ ID NO: 519, SEQ ID NO: 541, SEQ ID NO: 546, SEQ ID NO: 551, SEQ ID NO: 556, SEQ ID NO: 561, SEQ ID NO: 566, SEQ ID NO: 571. In some embodiments, the heavy chain FWR4 comprises SEQ ID NO: 205, SEQ ID NO: 395, SEQ ID NO: 519, SEQ ID NO: 541, SEQ ID NO: 546, SEQ ID NO: 576, SEQ ID NO: 576, SEQ ID NO: 581, or SEQ ID NO: 753. SEQ ID NO: 551, SEQ ID NO: 556, SEQ ID NO: 561, SEQ ID NO: 566, SEQ ID NO: 571, SEQ ID NO: 576, SEQ ID NO: 581 or SEQ ID NO: 753 and at least about 85%, at least about 90%, at least about 92%, Amino acid sequences having at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. It is understood that antibodies that contain amino acid changes in the heavy chain framework region (FWR1, FWR2, FWR3, and / or FWR4) retain the ability to specifically bind CD38. The retained CD38 specific binding activity (including affinity) is preferably about the same as the binding activity (including affinity) of antibodies that do not contain amino acid changes in any heavy chain framework region, Its binding activity (including affinity) may be lower or higher than antibodies that do not contain amino acid changes in any heavy chain framework region.

  In some embodiments, the heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 200, SEQ ID NO: 224, SEQ ID NO: 390, SEQ ID NO: 514, SEQ ID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, or SEQ ID NO: 697. In some embodiments, the heavy chain CDR1 comprises SEQ ID NO: 200, SEQ ID NO: 224, SEQ ID NO: 390, SEQ ID NO: 514, SEQ ID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, or SEQ ID NO: 697. At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, Or an amino acid sequence having at least about 99% sequence identity. In some embodiments, the heavy chain CDR2 comprises SEQ ID NO: 202, SEQ ID NO: 392, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 406, SEQ ID NO: 410, SEQ ID NO: 414, SEQ ID NO: 418, SEQ ID NO: 422, SEQ ID NO: 426. , SEQ ID NO: 430, SEQ ID NO: 434, SEQ ID NO: 467, SEQ ID NO: 471, SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 477, SEQ ID NO: 481, SEQ ID NO: 483, SEQ ID NO: 485, SEQ ID NO: 487, Sequence SEQ ID NO: 489, SEQ ID NO: 516, SEQ ID NO: 539, SEQ ID NO: 544, SEQ ID NO: 549, SEQ ID NO: 554, SEQ ID NO: 559, SEQ ID NO: 564, SEQ ID NO: 569, SEQ ID NO: 574, SEQ ID NO: 579, SEQ ID NO: 698 or SEQ ID NO: 737 In some embodiments, the heavy chain CDR2 comprises SEQ ID NO: 202, SEQ ID NO: 392, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 406, SEQ ID NO: 410, SEQ ID NO: 414, SEQ ID NO: 418, sequence No.422, SEQ ID NO: 426, SEQ ID NO: 430, SEQ ID NO: 434, SEQ ID NO: 467 Sequence number 471, sequence number 473, sequence number 475, sequence number 477, sequence number 479, sequence number 481, sequence number 483, sequence number 485, sequence number 487, sequence number 489, sequence number 516, sequence number 539, sequence number 544, SEQ ID NO: 549, SEQ ID NO: 554, SEQ ID NO: 559, SEQ ID NO: 564, SEQ ID NO: 569, SEQ ID NO: 574, SEQ ID NO: 579, SEQ ID NO: 698 or at least about 85% with the amino acid sequence of SEQ ID NO: 737, at least about 90 Amino acid sequences having%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity including. In some embodiments, the heavy chain CDR3 comprises SEQ ID NO: 204, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 228, SEQ ID NO: 231, SEQ ID NO: 394, SEQ ID NO: 469, SEQ ID NO: 518, SEQ ID NO: 534. In some embodiments, the heavy chain CDR3 comprises SEQ ID NO: 204, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 223, sequence SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 699, or SEQ ID NO: 738. SEQ ID NO: 228, SEQ ID NO: 231, SEQ ID NO: 394, SEQ ID NO: 469, SEQ ID NO: 518, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 699 or at least about 85% with the amino acid sequence of SEQ ID NO: 738, at least about Amino acids having 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity Contains an array. It is understood that antibodies that contain amino acid changes in the heavy chain complementarity determining regions (CDR1, CDR2, and / or CDR3) retain the ability to specifically bind to CD38. The retained CD38 specific binding activity (including affinity) is preferably about the same as the binding activity (including affinity) of an antibody that does not contain amino acid changes in any heavy chain complementarity determining region. The binding activity (including affinity) may be lower or higher than antibodies that do not contain amino acid changes in any heavy chain complementarity determining region.

  In some embodiments, the light chain FWR1 comprises SEQ ID NO: 232, SEQ ID NO: 247, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 436, SEQ ID NO: 443, SEQ ID NO: 447, SEQ ID NO: 451, SEQ ID NO: 455. , SEQ ID NO: 459, SEQ ID NO: 463, SEQ ID NO: 490, SEQ ID NO: 497, SEQ ID NO: 501, SEQ ID NO: 509, SEQ ID NO: 582, SEQ ID NO: 607, SEQ ID NO: 618, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 626, Sequence In some embodiments, the light chain FWR1 comprises SEQ ID NO: 232, SEQ ID NO: 247, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 436, comprising the amino acid sequence of SEQ ID NO: 630, SEQ ID NO: 634, or SEQ ID NO: 638. , SEQ ID NO: 443, SEQ ID NO: 447, SEQ ID NO: 451, SEQ ID NO: 455, SEQ ID NO: 459, SEQ ID NO: 463, SEQ ID NO: 490, SEQ ID NO: 497, SEQ ID NO: 501, SEQ ID NO: 509, SEQ ID NO: 582, SEQ ID NO: 607, SEQ ID NO: No. 614, SEQ ID No. 618, SEQ ID No. 622, SEQ ID No. 626, SEQ ID No. 630 At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97 with the amino acid sequence of SEQ ID NO: 634 or SEQ ID NO: 638. %, At least about 98%, or at least about 99% amino acid sequences having sequence identity. In some embodiments, the light chain FWR2 is SEQ ID NO: 234, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293. , SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 438, SEQ ID NO: 444, SEQ ID NO: 448, SEQ ID NO: 452, SEQ ID NO: 456, SEQ ID NO: 460, SEQ ID NO: 464, SEQ ID NO: 492, SEQ ID NO: 498, SEQ ID NO: 502, SEQ ID NO: 502 SEQ ID NO: 506, SEQ ID NO: 510, SEQ ID NO: 584, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 609, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 623, SEQ ID NO: 627, SEQ ID NO: 631, SEQ ID NO: 635 or SEQ ID NO: 639 In some embodiments, the light chain FWR2 comprises SEQ ID NO: 234, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, sequence SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 438 Sequence number 444, sequence number 448, sequence number 452, sequence number 456, sequence number 460, sequence number 464, sequence number 492, sequence number 498, sequence number 502, sequence number 506, sequence number 510, sequence number 584, sequence number 592, SEQ ID NO: 593, SEQ ID NO: 609, SEQ ID NO: 615, SEQ ID NO: 619, SEQ ID NO: 623, SEQ ID NO: 627, SEQ ID NO: 631, SEQ ID NO: 635 or at least about 90% with the amino acid sequence of SEQ ID NO: 639, at least about 90 Amino acid sequences having%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity including. In some embodiments, the light chain FWR3 comprises SEQ ID NO: 236, SEQ ID NO: 245, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 277. , SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 298 SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 323, SEQ ID NO: 327 , SEQ ID NO: 331, SEQ ID NO: 335, SEQ ID NO: 339, SEQ ID NO: 343, SEQ ID NO: 347, SEQ ID NO: 351, SEQ ID NO: 355, SEQ ID NO: 359, SEQ ID NO: 363, SEQ ID NO: 367, SEQ ID NO: 371, SEQ ID NO: 375, SEQ ID NO: 375 SEQ ID NO: 379, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 440, SEQ ID NO: 445, SEQ ID NO: 449, SEQ ID NO: 453, SEQ ID NO: 457, SEQ ID NO: 461, SEQ ID NO: 465, SEQ ID NO: 494, SEQ ID NO: 499, SEQ ID NO: 503, SEQ ID NO: 507, SEQ ID NO: 511, SEQ ID NO: 586, SEQ ID NO: 611, SEQ ID NO: 616, Comprising the amino acid sequence of SEQ ID NO: 620, SEQ ID NO: 624, SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 636 or SEQ ID NO: 640, and in some embodiments, the light chain FWR3 comprises SEQ ID NO: 236, SEQ ID NO: 245, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: No. 320, SEQ ID NO: 323, SEQ ID NO: 327, SEQ ID NO: 331, SEQ ID NO: 335, SEQ ID NO: 339, SEQ ID NO: 343, SEQ ID NO: 347, SEQ ID NO: 355, SEQ ID NO: 359, SEQ ID NO: 363, SEQ ID NO: 367 , SEQ ID NO: 371, SEQ ID NO: 375, SEQ ID NO: 379, SEQ ID NO: 383, SEQ ID NO: 387, SEQ ID NO: 440, SEQ ID NO: 445, SEQ ID NO: 453, SEQ ID NO: 457, SEQ ID NO: 461, SEQ ID NO: 465, SEQ ID NO: 465 SEQ ID NO: 494, SEQ ID NO: 499, SEQ ID NO: 503, SEQ ID NO: 507, SEQ ID NO: 511, SEQ ID NO: 586, SEQ ID NO: 611, SEQ ID NO: 616, SEQ ID NO: 620, SEQ ID NO: 624, SEQ ID NO: 628, SEQ ID NO: 632, SEQ ID NO: 636 Or at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least with the amino acid sequence of SEQ ID NO: 640 Amino acids having about 98%, or at least about 99% sequence identity Including acid sequence. In some embodiments, the light chain FWR4 comprises SEQ ID NO: 238, SEQ ID NO: 442, SEQ ID NO: 446, SEQ ID NO: 450, SEQ ID NO: 454, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 496, SEQ ID NO: 500, SEQ ID NO: 504. SEQ ID NO: 508, SEQ ID NO: 512, SEQ ID NO: 588, SEQ ID NO: 613, SEQ ID NO: 617, SEQ ID NO: 621, SEQ ID NO: 625, SEQ ID NO: 629, SEQ ID NO: 633, SEQ ID NO: 637 or SEQ ID NO: 641 In some embodiments, the light chain FWR4 comprises SEQ ID NO: 238, SEQ ID NO: 442, SEQ ID NO: 446, SEQ ID NO: 450, SEQ ID NO: 454, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 496, SEQ ID NO: 500, SEQ ID NO: 504. SEQ ID NO: 508, SEQ ID NO: 512, SEQ ID NO: 588, SEQ ID NO: 613, SEQ ID NO: 617, SEQ ID NO: 621, SEQ ID NO: 625, SEQ ID NO: 629, SEQ ID NO: 633, SEQ ID NO: 637 or SEQ ID NO: 641 About 85%, at least about 90%, at least about 92%, low At most about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or an amino acid sequence having at least about 99% sequence identity. It is understood that antibodies that contain amino acid changes in the light chain framework region (FWR1, FWR2, FWR3, and / or FWR4) retain the ability to specifically bind CD38. The retained CD38 specific binding activity (including affinity) is preferably about the same as the binding activity (including affinity) of an antibody that does not contain amino acid changes in any light chain framework region, Its binding activity (including affinity) may be lower or higher than antibodies that do not contain amino acid changes in any light chain framework region.

  In some embodiments, the light chain CDR1 is SEQ ID NO: 233, SEQ ID NO: 250, SEQ ID NO: 525, SEQ ID NO: 255, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 319, SEQ ID NO: 322, SEQ ID NO: 325, SEQ ID NO: 329. , SEQ ID NO: 333, SEQ ID NO: 337, SEQ ID NO: 341, SEQ ID NO: 345, SEQ ID NO: 349, SEQ ID NO: 353, SEQ ID NO: 357, SEQ ID NO: 365, SEQ ID NO: 369, SEQ ID NO: 373, SEQ ID NO: 377, SEQ ID NO: 377 Comprising the amino acid sequence of SEQ ID NO: 381, SEQ ID NO: 385, SEQ ID NO: 437, SEQ ID NO: 491, SEQ ID NO: 583, SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 608, or SEQ ID NO: 696, and in some embodiments, the light chain CDR1 SEQ ID NO: 233, SEQ ID NO: 250, SEQ ID NO: 525, SEQ ID NO: 255, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 319, SEQ ID NO: 322, SEQ ID NO: 325, SEQ ID NO: 329, SEQ ID NO: 333, SEQ ID NO: 337, SEQ ID NO: 341, SEQ ID NO: 345, SEQ ID NO: 349, SEQ ID NO: 353, SEQ ID NO: 35 7, SEQ ID NO: 361, SEQ ID NO: 365, SEQ ID NO: 369, SEQ ID NO: 373, SEQ ID NO: 377, SEQ ID NO: 381, SEQ ID NO: 385, SEQ ID NO: 437, SEQ ID NO: 491, SEQ ID NO: 583, SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 608, or at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about at least about the amino acid sequence of SEQ ID NO: 696 It comprises amino acid sequences having 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, the light chain CDR2 is SEQ ID NO: 235, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 264, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309. , SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 326, SEQ ID NO: 330, SEQ ID NO: 334, SEQ ID NO: 338, SEQ ID NO: 342, SEQ ID NO: 346, SEQ ID NO: 350, SEQ ID NO: 354, Sequence NO.358, SEQ ID NO: 362, SEQ ID NO: 366, SEQ ID NO: 370, SEQ ID NO: 374, SEQ ID NO: 378, SEQ ID NO: 382, SEQ ID NO: 386, SEQ ID NO: 439, SEQ ID NO: 493, SEQ ID NO: 585, SEQ ID NO: 591, SEQ ID NO: 605 In some embodiments, the light chain CDR2 comprises SEQ ID NO: 235, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 264, SEQ ID NO: 299, SEQ ID NO: 301, No. 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311 Sequence number 313, sequence number 315, sequence number 317, sequence number 326, sequence number 330, sequence number 334, sequence number 338, sequence number 342, sequence number 346, sequence number 350, sequence number 354, sequence number 358, sequence number 362, SEQ ID NO: 366, SEQ ID NO: 370, SEQ ID NO: 374, SEQ ID NO: 378, SEQ ID NO: 382, SEQ ID NO: 386, SEQ ID NO: 439, SEQ ID NO: 493, SEQ ID NO: 585, SEQ ID NO: 591, SEQ ID NO: 605, SEQ ID NO: 610 or At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about at least about the amino acid sequence of SEQ ID NO: 747 Contains amino acid sequences with 98%, or at least about 99% sequence identity. In some embodiments, the light chain CDR3 comprises SEQ ID NO: 237, SEQ ID NO: 244, SEQ ID NO: 251, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270. , SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 321, SEQ ID NO: 324, SEQ ID NO: 328, SEQ ID NO: 332, SEQ ID NO: 336, SEQ ID NO: 344, SEQ ID NO: 348, SEQ ID NO: 352, SEQ ID NO: 356, SEQ ID NO: 356 No. 360, SEQ ID NO: 364, SEQ ID NO: 368, SEQ ID NO: 372, SEQ ID NO: 376, SEQ ID NO: 380, SEQ ID NO: 384, SEQ ID NO: 388, SEQ ID NO: 441, SEQ ID NO: 495, SEQ ID NO: 587, SEQ ID NO: 594, SEQ ID NO: 595 SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 606 or SEQ ID NO: 612, In some embodiments, the light chain CDR3 comprises SEQ ID NO: 237, SEQ ID NO: 244. SEQ ID NO: 251, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 321, SEQ ID NO: 324, SEQ ID NO: 328, SEQ ID NO: 332, SEQ ID NO: 336, SEQ ID NO: 340, SEQ ID NO: 344, SEQ ID NO: 348, SEQ ID NO: 352, SEQ ID NO: 356, SEQ ID NO: 360, SEQ ID NO: 364, SEQ ID NO: 368, SEQ ID NO: 372, SEQ ID NO: 376, SEQ ID NO: 380, SEQ ID NO: 384, SEQ ID NO: 388, SEQ ID NO: 441, SEQ ID NO: 495, SEQ ID NO: 587, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 606 or SEQ ID NO: 612, at least about 85%, at least about 90%, at least about 92%, at least about 93%, At least about 94%, at least about 95%, at least about 96%, at least Also comprise amino acid sequences having about 97%, at least about 98%, or at least about 99% sequence identity. It is understood that antibodies that contain amino acid changes in the light chain complementarity determining regions (CDR1, CDR2, and / or CDR3) retain the ability to specifically bind to CD38. The retained CD38 specific binding activity (including affinity) is preferably about the same as the binding activity (including affinity) of an antibody that does not contain amino acid changes in any light chain complementarity determining region. The binding activity (including affinity) may be lower or higher than antibodies that do not contain amino acid changes in any light chain complementarity determining region.

  In some embodiments, the antibody comprises a particular heavy and light chain pair. Pair the heavy chain having the amino acid sequence of SEQ ID NO: 659 with any light chain having the amino acid sequence of SEQ ID NO: 664 or the heavy chain having the amino acid sequence of SEQ ID NO: 665 to the amino acid sequence of SEQ ID NO: 666 Or a heavy chain having the amino acid sequence of SEQ ID NO: 736 can be paired with any light chain having the amino acid sequence of SEQ ID NO: 664.

  The variable heavy chain and variable light chain pair may comprise pairs from the following table.

  An antibody-attenuated ligand construct that fuses an antibody to an attenuated ligand, e.g., exhibits an increased antigen-specificity index with respect to activation of signaling pathways due to the action of the attenuated ligand on cell surface receptors. Can be formed. These constructs are methods in which, in the context of antibody-ligand constructs, ligand activity on antigen-negative cells is dramatically attenuated, whereas ligand activity in antigen-positive cells, if any, is only moderately attenuated. This is based on the observation that the ligand moiety can be mutated. Such constructs are 1, 2, 3, 4 or 5 orders of magnitude more potent against antigen positive cells than antigen-negative cells compared to free ligand. In some embodiments, the antibody-attenuated ligand construct has at least 1%, at least 10%, at least 20%, at least 30% against antigen positive cells as a non-attenuated free (i.e. not bound to antibody) ligand, Retain at least 40% or at least 50% efficacy. In some embodiments, the antibody-attenuated ligand construct retains at least 30%, at least 50%, at least 75% or at least 90% of the maximum activity of the non-attenuated free (i.e., not bound to the antibody) ligand. To do. Maximum activity includes the amount of signaling activity (or its downstream effect) at the high plateau portion of the dose response curve where further increases in drug do not increase the response amount further.

In some embodiments, the antibody fusion to the interferon ligand and the inclusion of the attenuating mutation in the interferon ligand is more than 10 times, preferably more than 50 times compared to the antibody without the fusion. Preferably increasing the antigen specificity index (ASI) by more than 100-fold, preferably more than 1000-fold, or preferably more than 10,000-fold. ASI is an antibody-IFN compared to free non-mutated polypeptide ligand on target antigen positive cells, with a fold reduction efficacy of signaling activity compared to free non-mutated polypeptide ligand on target antigen negative cells Includes the fold increasing potency of the signaling activity of the ligand construct. By EC ₅₀ value, which is the numerical midpoint of the dose response curve, where dose refers to the concentration of ligand or antibody-ligand construct in the assay and response refers to the cellular quantitative response to the signaling activity of a particular dose of ligand. Efficacy can be expressed quantitatively. Thus, typically when measured by the same method, for example, the first compound has an EC ₅₀ (e.g. expressed in molar units) that is 10 times lower than the EC ₅₀ of the second compound on the same cell. The first compound is said to be 10 times more potent. Conversely, typically when measured by the same method, the first compound is shown to have an EC ₅₀ on the same cell that is 10 times higher than the EC ₅₀ of the second compound. The compound is said to have 10 times lower potency.

The antibody is preferably capable of binding to CD38 positive cells. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 100 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 75 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 50 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 30 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 25 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 20 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 18 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 15 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 13 nM. The antibody can bind to CD38 positive cells with an EC ₅₀ value of less than about 10 nM.

  The interferon linked to the antibody is preferably a point mutation and / or deletion that reduces the activity of the interferon in stimulating its respective receptor on the cell that lacks cell surface expression of the CD38 antigen to which the antibody binds. Including changes in its amino acid sequence. A highly preferred variant of interferon alpha comprises an amino acid change at position 168 of the interferon alpha 2b molecule of SEQ ID NO: 7. For example, the amino acid at position 168, which is an alanine in the parent IFN-alpha 2b molecule, is preferably changed to glycine (Gly / G) (SEQ ID NO: 650) or aspartic acid (Asp / D) (SEQ ID NO: 647). The In some embodiments, IFN-alpha 2b is truncated at its N-terminus when IFN-alpha 2b is fused to an IgG heavy chain constant domain, such as a human IgG1 or human IgG4 heavy chain constant domain. Truncated IFN-alpha 2b does not have the 23 N-terminal amino acids of SEQ ID NO: 7 (Met1 to Gly23 are deleted), and truncated IFN-alpha 2b contains the amino acid sequence of SEQ ID NO: 648 . Truncated IFN-alpha 2b may also contain an amino acid change that was previously at position 168 but became position 145 in the truncated protein (eg, alanine 168 becomes alanine 145). In the truncated IFN-alpha 2b, the alanine is preferably changed to glycine (Gly / G) (SEQ ID NO: 651) or aspartic acid (Asp / D) (SEQ ID NO: 649). Interferons with an A145D change (SEQ ID NO: 647 or SEQ ID NO: 649) are particularly preferred as attenuated ligands fused to the antibodies of the present disclosure. Any of these point-mutated, attenuated versions of IFN-alpha can be linked to any of the antibodies described herein, eg, as an antibody-attenuated interferon construct.

  The linkage between the antibody and interferon preferably comprises a peptide bond between the fusion, eg, the N or C terminus of the interferon and the N or C terminus of the antibody heavy or light chain. In a highly preferred embodiment, there is no linker between the antibody and interferon, thus the antibody and interferon are fused directly. A direct fusion that does not include an intervening linker peptide is believed to provide at least a measurable attenuation of the interferon protein, and this attenuation is also an interferon protein such as those described or exemplified herein. It is also thought to be additive with the attenuation of the interferon protein resulting from the mutation introduced therein.

  Polynucleotide sequences encoding antibodies and their subdomains (eg, FWR and CDR) are characterized in this disclosure. Polynucleotides include, but are not limited to, RNA, DNA, cDNA, RNA-DNA hybrids, and single-stranded, double-stranded, or triple-stranded RNA, DNA, or hybrids thereof.

  In some embodiments, the polynucleotide encodes an antibody heavy chain that specifically binds to an epitope on CD38. Polynucleotides are SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 695. , SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745 or SEQ ID NO: 746 A heavy chain containing the amino acid sequence may be encoded. The polynucleotide is SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, A light chain comprising the amino acid sequence of either SEQ ID NO: 718 or SEQ ID NO: 719 may be encoded. Polynucleotides are SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 695. , SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 746 No.669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690 , SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718 or SEQ ID NO: The nucleic acid sequence of any of number 719 may be included. Polynucleotides are SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 695. , SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 746 No.669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690 , SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718 or SEQ ID NO: At least about 80%, at least about 85%, small with any of the numbers 719 At least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% In some embodiments, such variants may preferably comprise a sequence of nucleic acids having at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 695, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, sequence 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 688, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 702 , SEQ ID NO: 703, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718 or the same amino acid encoded by the polynucleotide sequence of SEQ ID NO: 719. Preferably, the antibody encoded by the polynucleotide variant specifically binds CD38 with an affinity approximately equal to that of the antibody encoded by the parent (non-variant) polynucleotide sequence. For example, affinity can be measured according to any technique described or exemplified herein, such as those described in the Examples. The complements of polynucleotide sequences and variant polynucleotide sequences are also within the scope of this disclosure.

  Also encompassed within the scope of this disclosure are vectors comprising the polynucleotides of this disclosure. The vector may be an expression vector. Thus, a recombinant expression vector containing a sequence encoding a polypeptide of interest is provided. An expression vector may contain one or more additional sequences such as, but not limited to, regulatory sequences, selectable markers, purification tags, or polyadenylation signals. Such regulatory elements may include transcriptional promoters, enhancers, mRNA ribosome binding sites, or sequences that control the termination of transcription and translation.

  Expression vectors, particularly mammalian expression vectors, are origins of replication, suitable promoters and enhancers linked to the gene to be expressed, other 5 'or 3' flanking non-transcribed sequences, 5 'or 3' untranslated One or more non-transcribed elements such as sequences (such as essential ribosome binding sites), polyadenylation sites, splice donor and acceptor sites, or transcription termination sequences may be included. An origin of replication that confers the ability to replicate in a particular host can also be incorporated.

  The vector can be used to transform any variety of host cells well known to those of skill in the art, preferably any host cell capable of expressing the antibody. Vectors include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), and baculoviruses, as well as other bacteria, eukaryotic, yeast And viral vectors. Suitable host cells include, but are not limited to, CHO cells, HEK293 cells, or any known or generated stable eukaryotic cell line, including bacteria, yeast, and insect cells. .

  Antibodies can also be produced by hybridoma cells; methods for producing hybridomas are well known and established in the art.

  An anti-interferon alpha ligand having one or more mutations that substantially reduces the affinity of the ligand for the interferon receptor targets the mutant interferon alpha ligand to a target cell that exhibits the corresponding antigen of the antibody. It has been observed according to the present disclosure that when linked to a CD38 antibody, the activity of the ligand against target antigen positive cells is maintained while the activity of the ligand against non-target antigen negative cells is substantially reduced. The net result is a much higher potency in activating its receptor on antigen-positive target cells compared to antigen-negative non-target cells, providing a means to reduce the toxicity resulting from off-target ligand activity A ligand signaling molecule having

  In some embodiments, the polypeptide construct comprises an IFN-alpha variant linked to an anti-CD38 antibody or antigen binding portion thereof. Such polypeptides can exhibit IFN anti-proliferative activity against CD38 positive tumor cells with high potency, while exhibiting much lower potency against CD38 negative non-tumor cells in the body.

  The present disclosure also provides a composition comprising an antibody of the present disclosure and an antibody-attenuated interferon construct. These compositions include, but are not limited to, one or more diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or other suitable carriers and / or It may further comprise at least one any suitable adjuvant, such as an excipient. Pharmaceutically acceptable adjuvants are preferred. The composition may comprise any antibody described and / or exemplified herein, an antibody-attenuated interferon construct, and an acceptable carrier, such as a pharmaceutically acceptable carrier. Suitable carriers include any vehicle that does not interfere with the biological activity of the antibody and / or interferon, and is preferably non-toxic to the host to which it is administered. The carrier can be water, saline, or alcohol, or an aqueous solution such as a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. The carrier may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

  Pharmaceutical excipients and additives useful in the composition include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides and Sugars such as oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and other known sugars; and polysaccharides or sugar polymers), alone or in combination, in any suitable mass or It may be present alone or in combination, including volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and other known proteins. Representative amino acids that can also function in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. One preferred amino acid is histidine. The second preferred amino acid is arginine.

  Suitable carbohydrate excipients for use in the composition include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; Polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol. Preferred carbohydrate excipients for use in the present disclosure are mannitol, trehalose, and raffinose.

  The antibody composition may also include a buffer or pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carboxylic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts; Tris, tromethamine hydrochloride, or phosphate buffer . Preferred buffers for use in the compositions of the present invention are organic acid salts such as citrate.

  Furthermore, the composition of the present disclosure includes polyvinylpyrrolidone, ficoll (polymer sugar), dextrate (for example, cyclodextrin such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, antimicrobial agent, antioxidant, charging Inhibitors, surfactants (e.g. polysorbates such as `` TWEEN® 20 '' and `` TWEEN® 80 ''), lipids (e.g. phospholipids, fatty acids), steroids (e.g. cholesterol), and chelates Polymeric excipients / additives such as an agent (eg, EDTA) may be included.

  The composition can also be formulated in sustained release media or depot preparations. For example, the composition is formulated with a suitable polymeric or hydrophobic material (e.g., as an acceptable emulsion in oil) or ion exchange resin, or as a slightly less soluble derivative, e.g., as a slightly less soluble salt. can do. Liposomes and emulsions are well known examples of delivery vehicles suitable for use as carriers for hydrophobic drugs.

  The composition can be formulated for administration to a subject in any suitable dosage form. The composition can be formulated for oral, buccal, nasal, transdermal, parenteral, injection, intravenous, subcutaneous, intramuscular, rectal or vaginal administration. The composition can be formulated in a suitable controlled release medium with an adjuvant or as a depot preparation.

  Preparations for parenteral administration include sterile solutions ready for injection, sterile dry-dissolved products ready for use in combination with solvents, such as hypodermic tablets, and sterile ready for injection. Suspensions, sterile dry insoluble products ready to be combined with media just before use, as well as sterile emulsions.

  An anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be used, for example, to inhibit, reduce, reduce, block or prevent proliferation of cells expressing CD38 on the surface. In some embodiments, a method for inhibiting or reducing the growth of cells that express CD38 on a surface is generally effective to inhibit or reduce the proliferation of cells that express CD38 and cells. Contacting with an amount of anti-CD38 antibody-attenuated interferon alpha-2b fusion construct. The antibody that specifically binds to CD38 may be any antibody described or exemplified herein. The attenuated interferon alpha 2b may comprise IFN-alpha 2b A145D or IFN-alpha 2b A145G. The cells may be lymphocytes, autoimmune lymphocytes, or tumor cells such as leukemia cells, multiple myeloma cells, or lymphoma cells. An anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be, for example, a pharmaceutically acceptable carrier and optionally any such carrier, adjuvant, or excipient described or exemplified herein. Etc., may be included in the composition together with one or more adjuvants or excipients. The method can be performed in vitro, ex vivo, in vivo, or in situ.

  Anti-CD38 antibody-attenuated interferon alpha-2b fusion constructs can also be used, for example, to induce, facilitate, or enhance apoptosis of cells that express CD38 on their surface. In some embodiments, a method for inducing apoptosis in a cell that expresses CD38 generally comprises a cell that expresses CD38 and an amount of an anti-CD38 antibody-attenuated effective to induce apoptosis in the cell. Contacting with an interferon alpha-2b fusion construct. The antibody that specifically binds to CD38 may be any antibody described or exemplified herein. The attenuated interferon alpha 2b may comprise IFN-alpha 2b A145D or IFN-alpha 2b A145G. The cells may be lymphocytes, autoimmune lymphocytes, or tumor cells such as leukemia cells, multiple myeloma cells, or lymphoma cells. An anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be, for example, a pharmaceutically acceptable carrier and optionally any such carrier, adjuvant, or excipient described or exemplified herein. Etc., may be included in the composition together with one or more adjuvants or excipients. The method can be performed in vitro, ex vivo, in vivo, or in situ.

  An anti-CD38 antibody-attenuated interferon alpha-2b fusion construct may also be used to treat a subject comprising and / or at least partially mediated by a cell that expresses CD38 on its surface. it can. In some embodiments, a method for treating a tumor comprising cells that express CD38 on its surface generally provides a subject in need thereof with an amount of an anti-CD38 antibody effective to treat the tumor in the subject. Administering an attenuated interferon alpha-2b fusion construct. An effective treatment may include, for example, inhibiting or reducing the proliferation of CD38 positive cells in the tumor and / or inducing apoptosis of CD38 positive cells in the tumor. The antibody that specifically binds to CD38 may be any antibody described or exemplified herein. The attenuated interferon alpha 2b may comprise IFN-alpha 2b A145D or IFN-alpha 2b A145G. An anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be, for example, a pharmaceutically acceptable carrier and optionally any such carrier, adjuvant, or excipient described or exemplified herein. Etc., may be included in the composition together with one or more adjuvants or excipients.

  An anti-CD38 antibody-attenuated interferon alpha-2b fusion construct or a composition comprising such a construct can be administered to a tumor by administering the construct of the composition to blood. An anti-CD38 antibody-attenuated interferon alpha-2b fusion construct or a composition comprising such a construct can be administered such that the construct diffuses through the bloodstream and / or into tumor cells. The construct may be internalized by tumor cells.

Use of an anti-CD38 antibody or anti-CD38 antibody-attenuated interferon alpha-2b fusion construct in the treatment of a tumor is provided. Methods are provided for treating tumors using anti-CD38 antibodies or anti-CD38 antibody-attenuated interferon alpha-2b fusion constructs. Any anti-CD38 antibody or anti-CD38 antibody-attenuated interferon alpha-2b fusion construct described or exemplified herein can be used. Tumors that can be treated include but are not limited to AIDS-related cancers, acoustic neuromas, acute lymphoblastic leukemia, acute myeloid leukemia, adenocarcinoma, adrenocortical carcinoma, idiopathic myelogenous metaplasia, Alopecia, alveolar soft tissue sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, telangiectasia ataxia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestine Cancer, brain stem glioma, brain tumor and CNS tumor, breast cancer, CNS tumor, carcinoid tumor, cervical cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, Chronic myelogenous leukemia, colorectal cancer, cutaneous T-cell lymphoma, elevated dermal fibrosarcoma, fibrogenic small round cell tumor, ductal carcinoma, endocrine cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, eye: me Normal, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer, germ cell tumor, gestational choriocarcinoma Disease, glioma, gynecological cancer, hematological malignancy, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia , Hypopharyngeal cancer, intraocular melanoma, islet cell cancer, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Lee Fraumeni syndrome, lip cancer, liposarcoma , Liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, male breast cancer, renal malignant rhabdoid tumor, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelial , Metastatic cancer, oral cancer, multiple endocrine adenoma, mycosis fungoides, myelodysplastic syndrome, multiple myeloma, myeloproliferative disorder, nasal cavity cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma Cell tumor, neurofibromatosis, Nimiehen chromosomal instability syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCL)
C), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cancer, parathyroid cancer, parotid gland Cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, rare cancer and related disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rotomund Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), stomach cancer , Synovial sarcoma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), trophoblast cancer, urethral cancer, urological cancer, uroplakin (uroplakin), uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia Wilms' tumor, and the like to. In certain embodiments, the tumor is selected from the group of multiple myeloma or non-Hodgkin lymphoma.

  In a preferred embodiment, the method is used for the treatment of multiple myeloma, leukemia, or lymphoma in a subject in need thereof. Such methods may further comprise treating the subject with a retinoid, such as all-trans retinoic acid. In some preferred embodiments where the antigen bound to the cell surface is CD38, the tumor or cancer is selected from multiple myeloma, non-Hodgkin lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia or acute myeloid leukemia be able to.

  The anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be used in combination with other drugs and / or in addition to other cancer treatment regimens or modalities, such as radiation therapy or surgery. When an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct is used in combination with a known therapeutic agent, the combination is either sequentially (sequentially or divided by a period of no treatment), or simultaneously, or It can be administered as a mixture. In the case of cancer, there are several known anticancer agents that can be used in this context. Combination treatment may also include treatment with anti-CD38 antibody-attenuated interferon alpha-2b fusion construct, followed by treatment with a known treatment or agent, followed by anti-CD38 antibody-attenuated interferon alpha- It is also contemplated to include treatment with the 2b fusion construct. For example, in the treatment of cancer, an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct may be combined with an alkylating agent (such as mechloretamine, cyclophosphamide, chlorambucil, ifosfamide cisplatin, or cisplatin, carboplatin and oxaliplatin Platinum-containing alkylating agents), antimetabolites (such as purine or pyrimidine analogs or antifolates such as azathioprine and mercaptopurine), anthracyclines (daunorubicin, doxorubicin, epirubicin idarubicin, valrubicin, mitoxantrone, or anthra Cyclin analogs, etc.), plant alkaloids (vinca alkaloids or taxanes such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel or docetaxel), Can be administered in combination with topoisomerase inhibitors (such as type I or type II topoisomerase inhibitors), podophyllotoxins (such as etoposide or teniposide), or tyrosine kinase inhibitors (such as imatinib mesylate, nilotinib, or dasatinib) Is contemplated.

  For the treatment of multiple myeloma, an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct may be combined with a steroid such as dexamethasone, a proteasome inhibitor (such as bortezomib or carfilzomib), an immunomodulator (such as thalidomide, lenalidomide or pomalidomide), Or other suitable, such as administration of induction chemotherapeutic agents, followed by treatment of subjects by autologous hematopoietic stem cell transplantation with or without other chemotherapeutic agents such as melphalan hydrochloride or the chemotherapeutic agents listed above Can be administered in combination with other therapies.

  For the treatment of Hodgkin's lymphoma, anti-CD38 antibody-attenuated interferon alpha-2b fusion constructs are either ABVD (adriamycin (doxorubicin), bleomycin, vinblastine, and decarbazine), or Stanford V (doxorubicin, bleomycin, vinblastine, vincristine, mechlorethamine, Etoposide, prednisone), or BEACOPP (doxorubicin, bleomycin, vincristine, cyclophosphamide, procarbazine, etoposide, prednisone) and the like can be administered in combination.

  In the case of non-Hodgkin lymphoma or other lymphomas, the anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be administered in combination with current therapeutic approaches. Examples of drugs approved for non-Hodgkin lymphoma include Abitrexate (Methotrexate), Adriamycin PFS (Doxorubicin hydrochloride), Adriamycin RDF (Doxorubicin hydrochloride), Ambochlorin (chlorambucil), Ambochlorin (chlorambucil), Arranon (nerarabine) , Bendamustine hydrochloride, Bexxar (tositumomab and iodine I131 tositumomab), Blenoxane (bleomycin), bleomycin, bortezomib, chlorambucil, Clafen (cyclophosphamide), cyclophosphamide, Cytoxan (cyclophosphamide), DenileukinDiftitox, DepoCyt Liposomal cytarabine), doxorubicin hydrochloride, DTIC-Dome (dacarbazine), Folex (methotrexate), Folex PFS (methotrexate), Folotyn (pralatrexate), ibritumomab thixetane, Istodax (romidepsin), Leukeran (chlorambucil) (Chlorambu ), Liposomal cytarabine, Maturane (procarbazine hydrochloride), methotrexate, Methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), Mozobil (prerixafor), nelarabine, Neosar (cyclophosphamide), Ontak (Denileukin diftitox), Prerixafor, Pralatrexate, Rituxan (Rituximab), Rituximab, Romidepsin, Tositumomab and iodine I131 Tositumomab, Treanda (Bendamustine hydrochloride), Velban (Vinblastine sulfate), Velcade (Voltezo) And Velsar (vinblastine sulfate), vinblastine sulfate, Vincasar PFS (vincristin sulfate), vincristine sulfate, vorinostat, Zevalin (ibritumomabtiuxetane), and Zolinza (vorinostat). Examples of combination drugs used to treat non-Hodgkin lymphoma include CHOP (C = cyclophosphamide, H = doxorubicin hydrochloride (hydroxydaunomycin), O = vincristine sulfate (Oncovin), P = prednisone) COPP (C = cyclophosphamide, O = vincristine sulfate (Oncovin), P = procarbazine hydrochloride, P = prednisone); CVP (C = cyclophosphamide, V = vincristine sulfate, P = prednisone); EPOCH [E = Etoposide, P = Prednisone, O = Vincristine sulfate (Oncovin), C = Cyclophosphamide, H = Doxorubicin hydrochloride (hydroxydaunomycin)]; ICE (I = Ifosfamide, C = Carboplatin, E = Etoposide) ) And R-CHOP (R = rituximab, C = cyclophosphamide, H = doxorubicin hydrochloride (hydroxydaunomycin), O = vincristine sulfate (Oncovin), P = prednisone) That.

  An anti-CD38 antibody, or an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct can be used to detect CD38 positive cells, such as CD38 positive tumor cells. In some embodiments, contacting these constructs with an anti-CD38 antibody, or anti-CD38 antibody-attenuated interferon alpha-2b fusion construct, and a tissue sample isolated from a subject, and antibodies in the tissue sample Alternatively, it can be used in a method for detecting CD38 positive tumor cells in a tissue sample isolated from a subject, which may generally comprise detecting a complex of the construct and CD38 positive cells. The tissue sample is preferably blood. The cells may be CD38 positive B cell lymphoma cells, multiple myeloma cells, non-Hodgkin lymphoma cells, chronic myeloid leukemia cells, chronic lymphocytic leukemia cells, or acute myeloid leukemia cells. The method may further comprise isolating a tissue sample from the subject.

  The present disclosure also features a kit comprising any of the antibodies described and exemplified herein and an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct. The kit can be used to supply antibodies and other agents, particularly for use in diagnostic, basic research, or therapeutic methods.

  In some embodiments, the kit comprises an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct and a tumor that expresses CD38 on a surface, the composition optionally comprising a pharmaceutically acceptable carrier. One or more methods for inhibiting or reducing the proliferation of cells, methods for inducing apoptosis in tumor cells expressing CD38 on the surface, and / or cells expressing CD38 on the surface, And / or instructions for using the kit in a method for treating tumors mediated thereby. Such a method may be any method described or exemplified herein. The kit may include a pharmaceutically acceptable carrier. The kit may include pharmaceutically acceptable adjuvants and / or one or more pharmaceutically acceptable excipients. In the kit, the anti-CD38 antibody can be any antibody described or exemplified herein, and the attenuated interferon alpha-2b is any attenuated interferon alpha-2b described or exemplified herein. May be included. The construct may be contained in a sterile solution ready for injection or intravenous administration, or may comprise a sterile lyophilized form ready to be combined with a carrier just prior to use.

  In some embodiments, the kit comprises an anti-CD38 antibody and instructions for using the kit in a method for detecting CD38 positive cells in a sample, such as a tissue sample isolated from a subject. The anti-CD38 antibody may be any antibody described or exemplified herein. The antibody can optionally be fused to an attenuated interferon alpha-2b protein.

  The following examples are provided to describe the present disclosure in more detail. They are intended to illustrate the present disclosure and are not intended to be limiting.

Optimization of X355 / 02-HC-L0-IFN-alpha (A145D) IgG4 Other anti-CD38 antibody-attenuated IFN fusion proteins are described in PCT application number PCT / AU2012 / 001323. These include the antibody construct designated X355 / 02-HC-L0-IFN-alpha (A145D) IgG4 in this PCT application. Herein, X355 / 02-HC-L0-IFN-alpha (A145D) IgG4 has been renamed A02.1. The heavy chain sequence of the antibody comprises the amino acid sequence of SEQ ID NO: 11, and the light chain sequence comprises the amino acid sequence of SEQ ID NO: 12. The variable light chain of A02.1 (SEQ ID NO: 14) is formatted on its human IgG4 constant region (SEQ ID NO: 3) containing the S228P substitution (EU numbering) (SEQ ID NO: 13). ). This antibody is referred to herein as X02.1 and A02.1 contains a fusion with IFN-alpha 2b, but both antibodies share the same heavy and light chain sequences. .1 does not include it.

A BLAST search (Altschul SF (1997) Nucleic Acids Res. 25: 3389-3402) against a database of human germline immunoglobulin genes was performed using the amino acid sequence of the variable heavy chain of X02.1. The closest human germline variable heavy chain gene was IGHV4-61 ^* 01 (SEQ ID NO: 16). Figure 2 shows the alignment of X02.1 VH and IGHV4-61 ^* 01. The X02.1 variable heavy chain region differs from its nearest germline amino acid sequence by 8 amino acids. To reduce the immunogenicity of the X02.1 heavy chain variable region, generate germline amino acid residue substitutions at residues that differ from the germline sequence, and test the resulting antibody variants for anti-CD38 antibody binding activity. Can do.

Several heavy chain variants of the X02.1 parent sequence are detailed in FIG. These heavy chain variable regions were formalized on the IgG4 S228P constant region and co-expressed with the A02.1 light chain. Table 1a and Table 1b detail the sequences of variants tested along with their ability to bind human CD38 when assessed using flow cytometry and surface plasmon resonance (SPR). It is. Briefly, antibody chains were transiently co-expressed in CHO cells and purified by protein A chromatography as described in Example 5. Variants were evaluated using the flow binding assay described in Example 5. The EC _{50 of the} dose response curve obtained for each antibody is also listed in Table 1a and Table 1b.

The SPR binding of the variants detailed in Table 1a was evaluated separately from that of Table 1b. The K _D (M) of parent antibody X02.1 ranged from 2.7 × 10 ^{−8 to} 3.78 × 10 ⁻¹⁰ in SPR binding experiments. Flow cytometry binding experiments showed that antibodies X02.8, X02.11, X02.69 and X02.71 bind strongly to the CD38 positive cell line ARP-1.

Subsequently, antibodies with the above amino acid substitutions were searched in the context of a fusion protein by conjugation to attenuated IFN-alpha 2b (referred to as A02 and when linked to IFN represents the same variant with the X02 designation With digits after the decimal point). These heavy chain variable regions are formatted on an IgG4 constant region containing the S228P substitution fused to A145D attenuated IFN-alpha 2b and co-expressed with the A02.1 light chain described in Example 5 in CHO or HEK cells. Expressed. Proteins that were successfully purified from cell supernatants were then tested in a flow binding assay to cell line ARP-1. The EC ₅₀ values of the dose response curve for each antibody are listed in Table 2. All antibody-attenuated IFN fusion constructs tested bound to the CD38 positive cell line ARP-1. Heavy chain variant X02.9 (not fused to IFN) could not be easily purified, but the same variant fused to IFN (A02.9) was observed to be purified. In some cases, attenuated IFN fusion proteins could be expressed and purified when it was considered more difficult to express and / or purify equivalent monoclonal antibodies.

A BLAST search using the amino acid sequence of the A02.1 variable light chain was performed against a database of human germline immunoglobulin genes. The closest human germline variable light chain gene was IGLV5-37 ^* 01. The amino acid sequence alignment of A02.1VL and IGLV5-37 ^* 01 is shown in FIG. This alignment illustrates the 12 amino acid difference between these sequences.

  Several amino acid substitutions were made in the X02.1 variable light chain. These substitutions are shown in FIG. Co-expression of these light chain variable regions with X02.1 variable heavy chain formalized on an IgG4 constant region containing the S228P substitution was performed in CHO cells as described in Example 5.

Subsequently, antibodies purified from CHO cell supernatants were tested in a binding assay based on flow cytometry to the CD38 positive cell line ARP-1. Table 3 details the EC ₅₀ values of the dose response curves obtained for each antibody.

  Antibodies X02.96, X02.99, X02.101, X02.102, X02.103 and X02.104 bound strongly to the CD38 positive ARP-1 cell line. X02.105 was able to bind strongly to the CD38 positive H929 cell line.

  Amino acid sequence analysis of the variable heavy chain sequences of X02.1 and A02.1 identified amino acids that could potentially undergo oxidation or isomerization. These contain a potential isomerization site in D101 and a potential oxidation site in M (100C). To remove potential isomerization and oxidation sites, the following amino acid substitutions were made: D (101) E (SEQ ID NO: 30), M (100C) L (SEQ ID NO: 29) and D (101) Combination of both E and M (100C) L (SEQ ID NO: 27) (FIG. 2). Antibodies were generated using combinations of these amino acid substitutions in the variable heavy chain as shown in Table 4. The antibody heavy chain variable region was formalized with an IgG4 constant region containing the S228P substitution and coexpressed with the A02.1 light chain in CHO cells. The antibody was then purified by protein A chromatography and screened for binding to ARP-1 cells by flow cytometry. The obtained combined data is shown in Table 4.

  Antibodies X02.76 and X02.77 maintained their strong binding to the ARP-1 cell line, which eliminates potential oxidation and isomerization sites in the X02.1 and A02.1 heavy chains This indicates that the amino acid substitution for the DNA had little effect on its CD38 binding activity. The combination of these substitutions to form antibody X02.74 resulted in an antibody that was not purified using the protocol in Example 5.

  Amino acid analysis of the variable light sequence of X02.1 and A02.1 identified amino acids that could potentially undergo oxidation or deamidation. These contained a potential deamidation site in N69 and a potential oxidation site in M89. In addition, a putative N-linked glycosylation site was predicted to be present in the light chain CDR3 at position N94. The presence of N-linked glycans can cause heterogeneity in therapeutic proteins and complicate development. To eliminate these potential problems, the following point variants were synthesized: N69A (SEQ ID NO: 39), M89L (SEQ ID NO: 52) and M89I (SEQ ID NO: 51), N94T (SEQ ID NO: 48), N94Q ( SEQ ID NO: 38), G95P (SEQ ID NO: 50) and S96A (SEQ ID NO: 45) (see FIG. 3). Antibodies were generated by co-expression of heavy and light chains in CHO cells as described in Table 5 (Table 7). The antibody was purified by protein A chromatography and screened for binding to ARP-1 cells by flow cytometry. The resulting combined data is presented in Table 5.

  X02.94 bound to the CD38 positive cell line ARP-1, indicating that M89L substitution had little effect on CD38 binding activity. N94Q substitution in antibody X02.80 removed a potential N-linked glycosylation motif and had minimal effect on CD38 binding activity as measured by flow cytometry (Table 5). . Other substitutions that remove this glycosylation motif have resulted in antibodies that cannot be easily purified or antibodies that show attenuated binding to the CD38 positive cell line ARP-1. Although the potential deamidation site at position 69 was removed by substitution with alanine, this antibody (X02.81) was not easily purified.

  Other antibodies tested, including X02.1 variable heavy chain variants, are listed in Table 6. These heavy chain variable regions were formalized on an IgG4 constant region containing the S228P substitution. These heavy chains were co-expressed with A02.1 light chains in CHO cells. Antibodies were expressed and the resulting antibodies were tested in a flow cytometry based assay for binding to CD38 positive cells ARP-1. With the exception of T23K (SEQ ID NO: 21; X02.68), all variable heavy chain substitutions had minimal impact on binding to the CD38 positive cell line ARP-1 in a flow cytometry-based assay.

  Antibodies with other light chain variable region substitutions in the X02.1 sequence were also generated. These variant light chains were expressed in CHO cells as described in Example 5 in combination with X02.1 heavy chain formalized on an IgG4 constant region containing the S228P substitution. A summary of the heavy and light chains used to generate these antibody variants is listed in Table 7. Antibodies X02.83, X02.85, X02.91 and X02.82 bound strongly to the CD38 positive cell line ARP-1.

  Subsequently, substitutions that had little effect on CD38 binding activity and purification of X02 variant antibodies were generated as armed antibodies by fusion to A145D attenuated IFN-alpha 2b. X02.1 light chain substitutions were combined and the resulting variants were co-expressed with the X02.1 heavy chain point and combination variants in HEK293E cells as listed in Table 8. These antibodies were predicted primarily by potential X02.1 light chain deamidation sites, oxidation sites derived from CDR3 of the X02.1 heavy chain and in silico analysis (Epibase, Lonza, UK). The focus was on removing putative strong MHC class II binding peptides from framework region 3 of the single chain. FIG.

  The antibodies listed in Table 8 were analyzed for protein expression and binding to CD38 by surface plasmon resonance (SPR). Efficacy assays were also performed using cell culture supernatants obtained from transfected cells, and as outlined in Example 5, each of these anti-CD38 antibody-attenuated IFN fusion proteins. Relative activity was evaluated. The data obtained is listed in Table 9.

  Of the proteins tested, A02.12 was well expressed and showed efficacy in annexin V, caspase and cell proliferation assays. Replacement of N69T in antibody A02.47 did not affect expression levels or potency in annexin V or caspase assays, indicating that this deamidation site can be removed. The N69T substitution could be incorporated into other constructs described herein to remove this putative deamidation site while minimizing the loss of functional activity of the resulting antibody.

In silico immunogenicity analysis of A02.1 light chain amino acid sequence A putative immunogenic epitope was identified in the light chain variable region amino acid sequence of A02.1 using Epibase analysis software (Lonza, UK). To remove the putative immunogenic epitope, a substitution was introduced into the A02.1 variable light chain (FIG. 4). Light chain with lower putative immunogenicity in HEK293E cells with A02.12 heavy chain variable region (SEQ ID NO: 34) formalized on an IgG4 constant region containing the S228P substitution fused to A145D-attenuated IFN And co-expressed with The antibody variants produced are detailed in Table 10.

  The antibodies were analyzed for protein expression, binding to CD38 by SPR, and analyzed for potency using cell culture supernatant screening as described in Example 5. The results of these assays are detailed in Table 11. These data are expressed in annexin V, caspase and cell proliferation assays, with functional potency, by the substitution of several residues to reduce the predicted immunogenicity of the antibody-attenuated It shows that an IFN fusion protein is obtained.

Multiple amino acid substitutions result in optimization of A02.1 variants Highly optimized by combining substitutions that improve the immunogenicity, manufacturability or efficacy of the anti-CD38 antibodies described above in a single gene construct Anti-CD38 antibody and anti-CD38 antibody-attenuated IFN fusion protein were obtained. Table 12 summarizes such combinatorial substitutions and details the combinations of heavy and light chains that were tested after being co-expressed in HEK293E cells.

  Each antibody listed in Table 12 was analyzed for protein expression, binding to CD38 by SPR, and analyzed for potency using cell culture supernatants. The data obtained is listed in Table 13 (Table 15). These results show that several anti-CD38 antibody-attenuated IFN fusion proteins that are expressed in annexin V, caspase and cell proliferation assays and have functional potency by combining substitutions that are predicted to be beneficial in silico Is obtained.

Pairing of different heavy and light chain anti-CD38 antibodies To determine whether a functional anti-CD38 antibody-attenuated IFN fusion protein can be obtained, antibody X910 / 12 as described in PCT / AU2012 / 001323 -HC-L0-IFN-alpha (A145D) IgG4 heavy chain (SEQ ID NO: 110) and light chain (SEQ ID NO: 112), and antibody X913 / 15-HC-L0-IFN described in PCT / AU2012 / 001323 -Heavy chain (SEQ ID NO: 111) and light chain (SEQ ID NO: 113) derived from alpha (A145D) IgG4 were paired with each other in various combinations and with the heavy and light chains described in the Examples below. . A summary of heavy and light chain pairs is listed in Table 14.

  Each antibody produced was analyzed for protein expression, binding to CD38 by SPR and analyzed for potency using a cell culture supernatant potency assay. The results of these assays are presented in Table 15a. These data show that several anti-CD38 antibody-attenuated IFN fusion proteins expressed in annexin V, caspase and cell proliferation assays with functional and potency due to different heavy and light chain pairings from different antibodies Is obtained.

  A selection of the above anti-CD38 antibody-attenuated IFN fusion proteins was purified and analyzed for binding to CD38 positive cells in a cell-based assay. In addition, the potency assay was repeated to obtain a more accurate determination of the relative activity of each anti-CD38 antibody-attenuated IFN fusion protein. The results of these assays are listed in Table 15b.

  FIG. 5 lists the consensus variable heavy chains of related constructs of A02.1 with functional activity, and FIG. 6 lists the consensus variable light chains. Anti-CD38 antibodies X02.114, X02.115, X02.116, X02.117, X02.118, X02.119 (Figure 6), X02.120, X02.121, X02.122, X02.123, X02.124 It can further be envisaged that combinations of substitutions can be made, such as those described for X02.125, X02.126 or X02.127 (FIG. 30). Furthermore, the anti-CD38 antibody described above could be constructed as an anti-CD38 antibody-attenuated IFN fusion protein and tested for functional activity as described herein.

H929 multiple myeloma xenograft model
The in vivo efficacy of A02.1 was previously tested in the NCI-H929 sc multiple myeloma model as described in Example 5. A02.1 has been shown to have potent antitumor activity. Data is presented in PCT / AU2012 / 001323.

  The anti-tumor activity of any of the anti-CD38 antibody-attenuated IFN fusion proteins described above could be tested using the H929 multiple myeloma xenograft model.

Attenuated IFN is required for potent apoptotic activity and caspase activation in tumor cell lines Anti-CD38 antibody with potent apoptotic and caspase activation containing attenuated IFN using annexin V and caspase assays -It was shown to be dependent on attenuated IFN fusion protein (Table 16a, Figure 18). In the annexin V assay, attenuated IFN-containing proteins (A02.1 and A02.6) had a 2-fold higher activity than proteins without attenuated IFN (X02.1 and X02.6).

General method
Generation of antibodies and antibody-fusion constructs in HEK-293E cells. DNA plasmids encoding antibody constructs (antibodies and antibody-IFN-alpha 2b related constructs) were prepared using the HiSpeed Plasmid Maxi kit (Qiagen, Valencia, Calif.), Followed by commercial transfection reagents and OptiMEM medium (Invitrogen Co., Carlsbad, CA) and transfected into HEK293E cells (CNRC, Montreal, Canada) and 0.45% (w / v) D-(+)-glucose (Sigma, Castle Hill, NSW) And grown in F17 synthetic medium supplemented with 25 μg / mL geneticin (Invitrogen, Carlsbad, Calif.) And 1 × GlutaMAX (Invitrogen, Carlsbad, Calif.). Supply 5% CO ₂ and express in an incubator with shaking at 120 rpm for 6 days, then isolate culture medium and add Protein A Mab Select SuReTM agarose beads (GE Healthcare, Piscataway, NJ) Used for affinity purification to be used. Purified protein construct was purified using 0.2M Arginine HCl, PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or HiPrep 26/10 desalting column (HiTrap Desalting HiPrep 26/10 Desalting). The buffer was exchanged in 25 mM citric acid, 71.5 mM sodium hydroxide, pH 6.0. The purified protein construct was then concentrated using a 50 kDa Amicon Ultra centrifugal filter device (Millipore, Billerica, Mass.), And the protein concentration was determined by reading the absorbance at 280 nm.

Generation of antibodies and antibody-fusion constructs in CHO cells DNA plasmids encoding antibodies (antibodies and antibodies-IFN-alpha2b related constructs) are prepared using HiSpeed Plasmid Maxi Kit (Qiagen, Valencia, CA) Then, using a commercially available transfection reagent and OptiPro SFMTM medium (Invitrogen, Carlsbad, CA), transfection into CHO cells (Lonza), FreestyleTM CHO Expression Medium (Invitrogen, Carlsbad, CA). Affinity using Protein A Mab Select SuRe agarose beads (GE Healthcare, Piscataway, NJ) after supplying 6% CO ₂ and expressing in an incubator with shaking at 120 rpm for 6 days. Purified. Purified protein construct was purified using 0.2M Arginine HCl, PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or HiPrep 26/10 desalting column (HiTrap Desalting HiPrep 26/10 Desalting). The buffer was exchanged in 25 mM citric acid, 71.5 mM sodium hydroxide, pH 6.0. The purified protein construct was then concentrated using a 50 kDa Amicon Ultra centrifugal filter device (Millipore, Billerica, Mass.), And the protein concentration was determined by reading the absorbance at 280 nm.

  Anti-CD38 antibody-attenuated IFN fusion protein binding to CD38 as measured by surface plasmon resonance (SPR). The ability of anti-CD38 antibodies and anti-CD38 antibody-attenuated IFN fusion proteins to bind to human CD38, untransfected prep prepared 7: 1 with non-specific binding reducing agent (GE Healthcare, Piscataway, NJ) The cell supernatant was measured. Briefly, using Biacore (TM) 3000 or T200, Protein A, using amine coupling, CM5 research grade sensor chip flow cells (FC) 1 (FC1) and FC2 (or FC3 and FC4 ) When fixed on top, about 1500RU was obtained. FC2 (or FC4) was used as a reference throughout the experiment. Experiments were performed at 37 ° C. in HBS-P + buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% v / v Surfactant P20, pH 7.4). After regenerating both flow cells with 10 μL of 50 mM sodium hydroxide at a flow rate of 20 μl / min, 40 μL of the supernatant containing the protein was passed over FC1 (or FC3) alone. 30 μL of CD38 (10 μg / mL in running buffer) or 30 μL of running buffer was injected over FC1 and FC2 with a 5 min dissociation time. Both surfaces were regenerated twice with sodium hydroxide. Results were generated using BIAevaluation software provided with the machine. Microsoft Excel was used for the calculations. The BIAevaluation software automatically subtracted the reference sensorgram to obtain a trace amount of FC2-1 (or FC4-3) for each sample. A double reference was performed for each antibody tested by subtracting the sensorgram with CD38 injection from the sensorgram with blank running buffer injection. Protein A capture refers to response units measured from a sensorgram at a fixed time of 412.5s, which corresponds to the level of protein captured on the protein A surface. CD38 binding is a response unit measured at 507.5 s, indicating the level of CD38 bound to the sensor that captured the protein. CD38 dissociation is a response unit measured at 865.5 s, indicating the level of CD38 bound to the surface that captured the protein approximately 300 s after the dissociation phase. BIAevaluation was used to fit the sensorgram using the Langmuir 1: 1 equation to generate the equilibrium dissociation constant (KD).

Protein A HPLC
Supernatants were analyzed by Protein A HPLC using a POROS A / 20 2.1 × 30 mm Id column (Applied Biosystems) connected to an Agilent 1100 chromatography system. The column was equilibrated with PBS pH 7.4 and the protein was eluted using PBS adjusted to pH 2.2. A standard curve was generated using a known amount of monoclonal antibody in PBS. Chromatograms at wavelengths of 215 nm or 280 nm were integrated using the manufacturer's software, the area under the curve (AUC) was reported and interpolated against the generated standard curve to estimate the concentration.

Flow cytometric binding of antibodies and anti-CD38 antibody-IFN fusion proteins to human CD38 positive cell lines ARP-1 and H929 Multiple myeloma cell line ARP-1 is a myeloma from the University of Arkansas Medical Center (Little Rock, AK). It was a gift from Institute Director Bart Barlogie MD, phD. It is described in Hardin J. et al. (1994) Blood. 84: 3063-70. Multiple myeloma cell line NCI-H929 (H929) was purchased from ATCC (CRL-9068, Gazdar, Blood 67: 1542-1549, 1986).

The ability of the antibody or antibody-interferon construct to bind to the human CD38 positive myeloma cell line ARP-1 or H929 in a flow cytometry based assay was tested. ARP-1 cells or H929 cells (5 × 10 ⁵ cells, determined by trypan blue dye exclusion test), 50 μL of FACS buffer (PBS + 1% fetal calf serum, Incubated with various concentrations of each protein in FCS, 0.2M HEPES, 0.5M EDTA) or with a human IgG4 monoclonal antibody containing a protein construct of unrelated specificity. After washing the cells 3 times with FACS buffer, 50 μL FACS buffer containing goat anti-human IgG antibody (Fc specific antibody conjugated to fluorescein isothiocyanate, FITC; Sigma-Aldrich, St. Louis, MO) Incubated in for 30 minutes. After washing 3 times with FACS buffer, the cells were fixed with 50 μL of PBS containing 4% formaldehyde v / v and incubated for 16 hours at 4 ° C. in the dark. Incubated cells in suspension are diluted with an additional 150 μL FACS buffer and run on a FACS Canto II (BD Biosciences, San Diego, Calif.) Using forward scatter, side scatter and fluorescence intensity in the FITC channel. Binding was analyzed by flow cytometry. The value reported is the mean fluorescence intensity (MFI).

Targeted assay
Daudi cell proliferation assay: This assay was used to quantify the antiproliferative activity of IFN and antibody-IFN fusion protein constructs on cells displaying CD38. Daudi cells express CD38 as a cell surface associated antigen. Cell viability was measured using CellTiter-Glo® reagent from Promega (Madison, Wisconsin), catalog number G7570. This is a luminescence-based assay that determines the viability of cells in culture based on quantification of ATP. The signal intensity is proportional to the number of viable cells in the microtiter plate well. The details of the assay are as follows: Daudi cells (obtained from ATCC, Manassas, VA) were placed in a T75 flask (TPP, Trasadingen, Switzerland, catalog number 90076) in 10% fetal bovine serum (FBS; 0.5 × 10 ^{5 to} 0.8 × 10 ⁵ viable cells in RPMI1640 (Mediatech, Inc., Manassas, VA, catalog number 10-040-CV) with Hyclone, Logan, UT, catalog number SH30070.03) The cells were cultured until the desired density of / mL was reached. Cells were centrifuged at 400 × g for 5 minutes, the supernatant was discarded, and the cell pellet was harvested by resuspension in RPMI1640 + 10% FBS. Cells were then counted and the density was adjusted to 3.0 × 10 ⁵ cells / mL in RPMI 1640 + 10% FBS. Next, 50 μL of the cell suspension was aliquoted into each well of a 96-well round bottom tissue culture plate (hereinafter referred to as “experimental plate”) (TPP, catalog number 92067). On another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number 3879), the test article was serially diluted twice in RPMI 1640 + 10% FBS. 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plates were then incubated for 4 days at 37 ° C. with 5% CO ₂ . Experiment with 100 μL / well of a mixture of assay buffer supplied by the manufacturer and assay substrate (hereinafter referred to as “CellTiter-Glo® Reagent”, mixed according to manufacturer's instructions) Added to plate. The plate was shaken for 2 minutes.

  100 μL / well was then transferred from the experimental plate to a 96-well flat bottom white opaque plate (hereinafter referred to as “assay plate”; BD Biosciences, Franklin 5 Lakes, NJ, catalog number 35 3296). The contents of the assay plate were then allowed to stabilize in the dark for 15 minutes at room temperature. The luminescence was measured by reading the plate on a Victor 3V Multilabel Counter (Perkin Elmer, Waltham, MA, model number 1420-041) on the luminescence measurement channel. Results are presented as “relative luminescence units” (RLU). Data were analyzed using Prism 5 (Graphpad, San Diego, Calif.) Using non-linear regression and 3-parameter curve fitting to determine the midpoint of the curve (EC50).

  ARP-1 cell proliferation assay: This assay was used to quantify the antiproliferative activity of IFN and antibody-IFN fusion protein constructs against CD38 antigen positive cells. ARP-1 cells express CD38 as a cell surface associated antigen. Cell viability was measured using CellTiter-Glo® reagent from Promega (Madison, Wisconsin), catalog number G7570. This is a luminescence-based assay that determines the viability of cells in culture by quantifying ATP. The signal intensity is proportional to the number of viable cells in the microtiter plate well.

The details of the assay are as follows: ARP-1 cells were cultured in T175 flasks (Costar, Corning, NY Lakes, NJ, Cat.No. CLS431080) in 10% fetal calf serum (FBS; AusGeneX, Molendinar, QLD, Australia, catalog number FBS500S) was cultured in RPMI 1640 (Life Technologies, Mulgrave, VIC, catalog number 11875-093) to a preferred density of 2.0 × 10 ^{5 to} 2.0 × 10 ⁶ viable cells / mL. Cells were centrifuged at 400 × g for 5 minutes, the supernatant was discarded, and the cell pellet was harvested by resuspension in RPMI1640 + 10% FBS. Cells were then counted and the density adjusted to 2.0 × 10 ⁵ cells / mL in RPMI 1640 + 10% FBS. 50 μL of the cell suspension was then aliquoted into each well of a 96-well flat bottom white opaque plate (hereinafter referred to as “experimental plate”) (Costar, Corning, NY Lakes, NJ, catalog number CLS3917). On another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number 3799), the test article was serially diluted twice in RPMI 1640 + 10% FBS. 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plates were then incubated for 4 days at 37 ° C. with 5% CO ₂ . Each experimental plate contained the parent antibody IFN construct as a relative control.

A mixture of assay buffer supplied by the manufacturer and assay substrate [CellTiter-Glo® reagent mixed according to manufacturer's instructions] was added to the experimental plate at 100 μL / well. The plate was shaken for 2 minutes. The contents of the assay plate were then allowed to stabilize in the dark for 15 minutes at room temperature. The luminescence was measured by reading the plate on a luminescence measuring channel on a FLUOstar Galaxy plate reader (BMG Labtech, Durham, NC). Data was analyzed using Prism 5 (Graphpad, San Diego, Calif.) Using non-linear regression and 3-parameter curve fitting to determine the midpoint of the curve (EC ₅₀ ).

Annexin V assay: H929 cells were harvested by centrifuging at 400 × g for 5 minutes, discarding the supernatant, and resuspending the cell pellet in RPMI1640 + 10% FBS. Cells were then counted and the density adjusted to 1.0 × 10 ⁶ cells / mL in RPMI 1640 + 10% FBS. 50 μL of the cell suspension was then aliquoted into each well of a 96-well round bottom clear plate (hereinafter referred to as “experimental plate”; Costar, Corning, NY, catalog number CL3799). On another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number CL3799), the test article was diluted 4 times in RPMI1640 + 10% FBS to 40 nM. 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plates were then incubated for 24 hours at 37 ° C. with 5% CO ₂ . Cells are then centrifuged at 400 × g for 5 min, the supernatant discarded and resuspended in 100 μL HEPES buffer containing Annexin V-FITC (1/200) and 7-AAD (1/50) did. Cells were then incubated at room temperature for 15 min, followed by annexin by flow cytometry on FACS Canto II (BD Biosciences, San Diego, Calif.) Using forward scatter, side scatter, FITC and PerCP-Cy5.5 channels. V and 7-AAD staining were analyzed. Annexin V positive cells refer to cells that are positively stained with annexin V-FITC after treatment with 20 nM antibody construct for 24 h, and are expressed as fold changes compared to untreated cells.

  Caspase assay: Activated caspase 2, 3, 6, 7, 8, 9, 10 after treatment with test antibody, Homogeneous Caspases Assay reagent from Roche (West Sussex, UK), fluorescence, catalog number 12236869001 And measured. Details of the assay are as follows.

ARP-1 cells expressing high levels of CD38 in RPMI 1640 with 10% FBS (AusGeneX, Molendinar, QLD, Australia, catalog number FBS500S) in a T175 flask (Costar, Corning, NY, catalog number CLS431080). Life Technologies, Mulgrave, VIC, Catalog No. 11875-093) were cultured to a preferred density of 2.0 × 10 ^{5 to} 2.0 × 10 ⁶ viable cells / mL. Cells are centrifuged at 400 xg for 5 minutes, the supernatant is discarded, and the cell pellet is resuspended in RPMI 1640 phenol red free (Life Technologies, Mulgrave, VIC, catalog number 11835-030) + 10% FBS It was harvested by. Cells were then counted and the density adjusted to 2.0 × 10 ⁵ cells / mL in RPMI 1640 phenol red free + 10% FBS. 50 μL of the cell suspension was then aliquoted into each well of a 96-well flat bottom black wall clear bottom plate (hereinafter referred to as “experimental plate”; Costar, Corning, catalog number CLS3603). On another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number 3799), the test article was diluted 4 times in RPMI 1640 phenol red free + 10% FBS to 40 nM. 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plates were then incubated for 24 hours at 37 ° C. with 5% CO ₂ . The assay buffer supplied by the manufacturer was added to the substrate supplied by the manufacturer and mixed according to the manufacturer's instructions to create a “substrate solution”. 100 μL of substrate solution was then added to each well of the assay plate. The plate was shaken for 2 minutes. Plates are then incubated in the dark for 15 minutes at room temperature and finally read on a FLUOstar Galaxy plate reader (BMG Labtech, Durham, NC) with 470-500 nm excitation and 500-560 nm emission filters to obtain fluorescence. Measure and present as fold change compared to untreated cells. The caspase assay refers to caspase activation of cells after 24 h treatment with 20 nM antibody construct.

Off-target assay
iLite Gene Reporter Assay: An “off-target” iLite assay (PBL Interferon Source, Piscataway, NJ, Catalog No. 51100) was performed for the most part as described by the manufacturer with the addition of a human IgG blocking step. The iLite cell line is a "stable transfected cell line derived from a commercial premonocytic human cell line characterized by the expression of MHC class II antigens, particularly human lymphocyte antigens (HLADR), on the cell surface. As the manufacturer. This cell line contains a stably transfected luciferase gene whose expression is induced by an interferon response element (IRE) that can quantify interferon activity based on luminescence output. ILite plates supplied by the manufacturer (hereinafter referred to as assay plates) and diluent were removed from the −80 ° C. freezer and allowed to equilibrate to room temperature. Then 50 μL of diluent per well was added to the assay plate. Reporter cell vials supplied by the manufacturer were removed from the −80 ° C. freezer and thawed in a 37 ° C. water bath. A 25 μL aliquot of cells was then dispensed into each well of the assay plate. Next, 12.5 μL of 8 mg / mL human IgG diluted in RPMI 1640 + 10% FBS (Sigma Chemicals, St. Louis, MO; catalog number I4506) was added to each well. The contents were mixed and incubated for 15 minutes at 37 ° C. On another “dilution plate”, the test article was serially diluted twice in RPMI 1640 + 10% FBS. 12.5 μL of test article was then transferred from the dilution plate to the assay plate. The assay plate was then incubated for 17 hours at 37 ° C. with 5% CO ₂ . The assay buffer supplied by the manufacturer and the substrate were removed from the -80 ° C freezer and allowed to equilibrate to room temperature for 2 hours. The assay buffer supplied by the manufacturer was added to the substrate vial supplied by the manufacturer and mixed well according to the manufacturer's instructions to create a “luminescent solution”. 100 μL of luminescent solution was then added to each well of the assay plate. The plate was shaken for 2 minutes. The plates were then incubated in the dark at room temperature for 5 minutes and finally read on the luminescence measurement channel, Victor 3V Multilabel Counter, luminescence was measured and presented as RLU. Data were analyzed using Graphpad Prism 5 as described for the “on-target (Daudi) assay”. To test the anti-CD38 antibody-IFN fusion protein construct in the iLite assay, 0.25 mg / mL anti-CD38 antibody was added to the diluent supplied by the manufacturer (the same antibody clone is expressed on iLite cells) Tested as an antibody-IFN fusion protein construct to block any binding of the anti-CD38 antibody-IFN fusion protein construct to CD38).

HEK-BlueTM off-target assay: This assay is used to interferon-alpha / beta receptor (IFNAR) using the HEK-BlueTM IFN-alpha / beta cell line (InvitroGen, San Diego, CA). The ability of the antibody-IFN fusion construct to bind to) was quantified. The “off-target (HB-IFN) assay” was performed mostly as described by the manufacturer of the HEK-Blue ™ IFN-alpha / β cell line. HEK-Blue ™ IFN-alpha / β cells are specifically designed to monitor JAK-STAT pathway activation induced by type I IFN. The cells were generated by introducing human STAT2 and IRF9 genes into HEK293 cells to obtain a fully active type I IFN signaling pathway. HEK-Blue ™ IFN-alpha / β cells stably express the reporter gene secretory embryonic alkaline phosphatase (SEAP) under the control of the ISG54 promoter. ISG54 is a well-known ISG that is activated through an ISRE-dependent mechanism by type I IFNs. Upon stimulation with IFN-alpha or IFNβ, HEK-Blue ™ IFN-alpha / β cells activate the expression of the SEAP reporter gene after activating the JAK-STAT pathway. SEAP is secreted into the medium and can be quantified using the colorimetric reagent QUANTI-Blue ™. Briefly, HEK-Blue IFN-alpha / β cells (Invivogen, San Diego, CA, catalog number hkb-ifnab) were thawed and heat-inactivated DMEM medium (Mediatech, Manassas VA, catalog number 10). -013-CV) + 10% FBS (Hyclone, Logan UT, catalog number SH30070.03) (HI FBS). When cells reached 60-80% confluency, they were lifted using a Cell Stripper (Mediatech, catalog number 25-056-Cl). Cells were washed twice in DMEM + HI FBS and counted. Cells were adjusted to 3.3 × 10 ⁵ viable cells / mL in DMEM + HI FBS and 150 μL per well was aliquoted into flat bottom 96-well tissue culture plates (hereinafter “experimental plates”). 50 μL of IFN-alpha 2b or fusion protein construct diluted in DMEM + HI FBS was then added to each well. Plates were incubated for 16-24 hours at 37 ° C., 5% CO ₂ . QUANTI-Blue (Invivogen, catalog number rep-qb1) was prepared according to the manufacturer's instructions. QUANTI-Blue (150 μL) was aliquoted into each well of a flat bottom plate (hereinafter referred to as “assay plate”). Then 50 μL of supernatant from the experimental plate per well was transferred to the assay plate. The assay plate was then incubated at 37 ° C. for 1-3 hours. The absorbance of the assay plate at 630 nm was read on a model 1420-41 Victor 3V Multilabel Counter from Perkin-Elmer. Data was analyzed using Graph Pad Prism.

H929 Xenograft Model The effects of different doses of A10.38 and A10.0 anti-CD38 antibody-attenuated IFN-alpha fusion protein constructs on myeloma tumor growth were compared to non-CD38-targeted fusion protein constructs. For these comparisons, the NCI-H929 sc multiple myeloma model was used.

  Multiple myeloma cell line NCI-H929 (ATCC CRL-9068, Gazdar, Blood 67: 1542-1549, 1986) is grown subcutaneously in immunodeficient (SCID) mice.

The flank of 8-12 week old CB.17 SCID mice was injected subcutaneously with 1 × 10 ⁷ NCI-H929 tumor cells in 50% Matrigel ™. When the average tumor size reached 170-350 mm ³ , the mice were grouped into 4 cohorts of 7 mice each and treatment was started at 0 hours (T0). All treatments were given by intraperitoneal injection (ip) twice a week for 3 weeks (indicated by the bar below the graph). Except for the vehicle group, all compounds were administered at 100 μg / dose (approximately 4.5 mg / kg). Tumor volume was measured twice a week by caliper measurement. The end point was a tumor volume of 2,000 mm ³ .

  The effects of different doses of A02.6, A10.0 and A10.38 anti-CD38 antibody-attenuated IFN-alpha fusion protein constructs on myeloma tumor growth were compared to vehicle. For these comparisons, the NCI-H929 s.c. multiple myeloma model was used.

  Multiple myeloma cell line NCI-H929 (ATCC CRL-9068, Gazdar, Blood 67: 1542-1549, 1986) is grown subcutaneously in immunodeficient (SCID) mice.

The flank of 8-12 week old CB.17 SCID mice was injected subcutaneously with 1 × 10 ⁷ NCI-H929 tumor cells in 50% Matrigel. When the average tumor size reaches 90 mm ³ , the mice are grouped into 4 cohorts of 5 mice each and treatment begins at time 0 (T0). All treatments are given by intraperitoneal injection (ip) twice a week for 3 weeks (indicated by the bar below the graph). Except for the vehicle group, all compounds are administered at 100 μg / dose (approximately 4.5 mg / kg). Tumor volume is measured twice a week by caliper measurement.

Anti-CD38 antibody-attenuated IFN fusion protein containing an alternative constant region
A02.12 contains an anti-CD38 antibody-attenuated IFN fusion protein in which the constant region of the protein is HC-L0-IFN-alpha (A145D) IgG4 (SEQ ID NO: 9). The heavy chain variable region of this antibody was reformatted on an IgG1 constant region fused to A145D attenuated IFN-alpha 2b (SEQ ID NO: 10). Antibody A02.112 was obtained by co-expression of this heavy chain in HEK293E cells with the light chain of X02.107 (SEQ ID NO: 65). Comparison of antibodies A02.12 and A02.112 using a flow cytometry-based CD38 binding assay and potency assay, antibody-attenuated IFN with potent biological activity comparable to that produced using human IgG4 constant region It is shown that other antibody constant regions, such as human IgG1, that result in a fusion protein can also be used (Table 16b).

Humanization of R5D1, R5E8 and R10A2 variable regions Rat-derived anti-CD38 antibodies R5D1, R5E8 and R10A2 were described in PCT / AU2012 / 001323 and were selected for humanization. The variable regions of these antibodies were superhumanized as described in US Patent Application Publication No. 2003/0039649. Briefly, a standard structure was assigned to each rodent heavy and light chain by examination of its respective amino acid sequence. R10A2 is assigned standard structure 2-1-1 / 1-2 (V _L / V _H ), R5E8 is assigned standard structure 4-1-1 / 1-2, R5D1 is assigned standard structure 2-1-1 Assigned / 1-2. The same standard structure of human germline sequences was used as an acceptor framework for transplantation of donor CDRs. Variants of the resulting superhumanized antibody genes were also designed that contained amino acid substitutions at positions within that sequence that were likely to be important for maintaining binding activity. Various heavy chain superhumanization variable regions are shown in FIG. Various light chain superhumanized variable regions are shown in FIG.

The heavy chain variable region sequence was subcloned into the vector pEE6.4 containing the human IgG4 constant region with the S228P substitution fused to A145D attenuated IFN-alpha 2b (SEQ ID NO: 9). The light chain variable region was subcloned into vector pEE12.4 containing the human kappa constant region (SEQ ID NO: 5). Antibodies were generated by co-expression of the heavy chain in pEE6.4 and the light chain in pEE12.4 in CHO cells as previously described. Table 17 summarizes the heavy and light chain pairs used to generate 5D1-based proteins, each superhumanized. Table 18 details the heavy and light chain pairs for the generated superhumanized 5E8 based protein, but creates a superhumanized 10A2 based protein The heavy and light chain pairs used for this are listed in Table 19 (Table 23). One shot equilibrium dissociation constant (K _D ) ranking of superhumanized antibodies was performed by BIAcore ™ analysis of the resulting CHO transfection supernatant. Using this method, it was determined whether the antibody was expressed (protein A capture) and had a level of binding activity against human CD38.

  For each family of humanized antibodies -5D1, 5E8 and 10A2, some humanized heavy and light chain combinations cannot express proteins or can bind to human CD38. There wasn't. A significant number of antibodies were expressed across all three families of humanized antibodies and bound to human CD38 with equilibrium dissociation constants in the nanomolar (nM) range. A10A2.53 and A10A2.25 sharing a common light chain were selected for further optimization. A10A2.53 was renamed A10.0, and A10A2.25 was renamed A10.38.

Improved variant of A10.0
To produce a positive effect on the biophysical and in silico immunogenicity of the antibody while minimizing the effect on the functional activity of the antibody, the A10.0 antibody can be modified with variable heavy and / or light chains. Optimized by changes to the sequence.

In silico immunogenicity analysis of A10.0 heavy and light chains
An in silico immunogenicity analysis of the A10.0 heavy and light chain variable regions was generated using the Epibase software package. Several amino acid substitutions were introduced in the heavy and light chain variable regions of A10.0 to remove potential immunogenic epitopes. The amino acid sequence alignment of the generated heavy chain variable region variant aligned with the humanized heavy chain (SEQ ID NO: 156) is shown in FIG. An amino acid sequence alignment of the light chain variable region variants aligned with the humanized light chain (SEQ ID NO: 161) is shown in FIG. Details of the heavy and light chain variants co-expressed in HEK293E cells to produce the protein are summarized in Table 20.

  Each antibody generated using the heavy and light chain pairs outlined in Table 20 was evaluated for protein expression levels and binding to CD38 by SPR. In addition, potency assays were performed using cell culture supernatants to assess the relative functional activity of each of these anti-CD38 antibody-attenuated IFN fusion proteins. Table 21.

  Analysis of the amino acid sequence of the variable heavy and light chain sequences of A10.0 identified several potential deamidation sites and one potential oxidation site. A variable heavy chain substitution N98Q was prepared to remove the deamidation site from CDR3 of the heavy chain. SEQ ID NO: 197. An additional variant of the A10.0 variable light chain containing the CDR2 substitution N53Q (SEQ ID NO: 198) was created to remove this putative deamidation site. In addition, M89 in the CDR3 of the light chain is altered by amino acid substitution at this position for the combined purpose of removing this potential oxidation site and reducing the expected immunogenicity of this region of the light chain. It was. These substitutions are outlined in Table 22 along with heavy and light chain pairs that are co-expressed to generate the respective anti-CD38 antibody-attenuated IFN fusion proteins.

  Each antibody generated using the heavy and light chain pairs outlined in Table 22 was evaluated for protein expression levels and binding to CD38 by SPR. In addition, potency assays were performed using cell culture supernatants to assess the relative functional activity of each of these anti-CD38 antibody-attenuated IFN fusion proteins. Table 23.

Creating an improved variant of A10.38
A10.0 and A10.38 share a common light chain. To obtain a positive effect on the biophysical and in silico immunogenic properties of the antibody while minimizing the impact on functional activity, the optimized light chain sequence of A10.0 was Paired with antibody heavy chain. A summary of heavy chain and light chain changes and pairs is set forth in Table 24.

  Each of the above antibodies was evaluated for protein expression level and binding to CD38 by SPR. Potency assays were performed with cell culture supernatants to assess the relative functional activity of each of these anti-CD38 antibody-attenuated IFN fusion proteins. Table 25.

Attenuated IFN is required for potent apoptotic and caspase activation in tumor cell lines Relative potency of anti-CD38 antibodies A10.0 (attenuated IFN fusion) and X10.0 (no fusion) Comparisons were made using the annexin V, caspase and cell proliferation assays outlined in Example 5. The relative potency of A10.38 and X10.38 was also compared. Table 26.

  These data indicate that the strong apoptotic activity exhibited by antibodies A10.0 and A10.38 compared to X10.0 and X10.38, respectively, requires the presence of an attenuated IFN fusion. No antiproliferative activity was observed for antibodies that did not contain attenuated IFN.

  A consensus sequence alignment of heavy chain variable regions derived from proteins with functional activity is shown in FIG. A consensus sequence alignment of light chain variable regions derived from proteins with functional activity is shown in FIG. Anti-CD38 antibodies X10.60, X10.61, X10.62, X10.63, X10.64, X10.65, X10.66, X10.67, X10.68, X10.69, X10.70, X10.71 , X10.72, X10.73, X10.74, X10.75, X10.76, X10.77, X10.78, X10.79, X10.80, X10.81, X10.82, X10.83, X10 .84, X10.85, X10.86, X10.87, X10.88, X10.89, X10.90, X10.91, X10.92, X10.93, X10.94, X10.95, X10.96 , X10.97, X10.98, X10.99, X10.100, X10.101, X10.102, X10.103, X10.104, X10.105, X10.106, X10.107, X10.108, X10 .109, X10.110, X10.111, X10.112, X10.113, X10.114, X10.115, X10.116, X10.117, X10.118, X10.119, X10.120, X10.121 , X10.122, X10.123, X10.124, X10.125, X10.126, X10.127, X10.128, X10.129, X10.130, X10.131, X10.132, X10.133, X10 .134, X10.135, X10.136, X10.137, X10.138, X10.139, X10.140, X10.141, X10.142, X10.143, X10.144, X10.145, X10.146 It can be further envisioned that combinations of substitutions such as those described for X10.147 can be made (FIGS. 11, 12). In addition, the anti-CD38 antibody described above could be constructed as an anti-CD38 antibody-attenuated IFN fusion protein and tested for functional activity as described herein.

H929 multiple myeloma xenograft model
The in vivo efficacy of 10A2 variants A10.0 and A10A2.0 was evaluated in the NCI-H929 sc mouse multiple myeloma model. FIG. Both have been shown to have potent anti-tumor activity in this model. Such a model could be used to test for the anti-tumor activity of other protein constructs described herein.

Off-target activity for 10A2 variants The off-target activity of 10A2 variants A10.0, A10.38, A10A2.37 and A10A2.39 compared to the parental A10A2.0 chimeric antibody fused to wild-type and attenuated interferon 145D Evaluated in the gene assay and / or HEK Blue assay and shown in FIGS. EC ₅₀ values are provided in FIGS. 28 and 29. Off-target activity confirms the need for antibodies targeted to CD38 to restore interferon attenuation and function.

Additional in vitro potency data for A10.0 and related constructs The selection of anti-CD38 antibody-attenuated IFN fusion protein described above was purified and analyzed for binding to CD38 positive cells in a cell-based assay. In addition, the potency assay was repeated to more accurately determine the relative activity of each of these anti-CD38 antibody-attenuated IFN fusion proteins. Methods for these various assays are described in Example 5. The results for each of these assays are listed in Table 27.

Anti-CD38 antibody-attenuated IFN fusion protein with alternative constant region
A10.0 contains an anti-CD38 antibody-attenuated IFN fusion protein in which the constant region of the protein is HC-L0-IFN-alpha (A145D) IgG4 (SEQ ID NO: 9). Using gene synthesis, the constant region of this protein was replaced with HC-L0-IFN-alpha (A145D) IgG1 (SEQ ID NO: 10) and paired with the A10.0 light chain (SEQ ID NO: 161), and A10.59 Named. The protein was expressed and found to be potent in functional assays (Table 28). Although the majority of the proteins tested in the above examples were constructed on human IgG4 constant regions, these data can also be used with other antibody constant regions such as human IgG1 and the resulting antibody-attenuated IFN fusion construct Have the same strong biological activity as constructs using human IgG4 constant regions.

  Table 29 lists the variable heavy chain, variable light chain and constant region pairs for each antibody described herein. Table 30 lists the sequences used in this disclosure, AA refers to amino acids (sequence type), and DNA refers to polynucleotides (sequence type).

  The present disclosure is not limited to the embodiments described and illustrated above, but may be altered and modified within the scope of the appended claims.

Claims (364)

  A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 659, provided that the heavy chain variable region amino acid sequence does not comprise the amino acid sequence of SEQ ID NO: 13 and the light chain variable region amino acid sequence does not comprise the amino acid sequence of SEQ ID NO: 14; An isolated antibody that specifically binds to CD38, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 664.   2. The isolated antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 34.   2. The isolated antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 18.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 65.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 86.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 88.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 93.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 660.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 661.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 662.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 663.   2. The isolated antibody of claim 1, wherein the heavy chain variable region comprises CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 204 and SEQ ID NO: 222.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 233 and SEQ ID NO: 319.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 235, SEQ ID NO: 307, and SEQ ID NO: 311.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 237, SEQ ID NO: 321, and SEQ ID NO: 324. About binding to CD38-positive cells with an EC ₅₀ value of less than 150 nM, isolated antibody according to any one of claims 1 to 17. Binding to CD38-positive cells with an EC ₅₀ value of less than about 100 nM, isolated antibody according to any one of claims 1 to 17. Binding to CD38-positive cells with an EC ₅₀ value of less than about 50 nM, isolated antibody according to any one of claims 1 to 17. About binding to CD38-positive cells with an EC ₅₀ value of less than 13 nM, isolated antibody according to any one of claims 1 to 17.   The isolated antibody according to any one of claims 1 to 21, which is a monoclonal antibody.   23. The isolated antibody of any one of claims 1 to 22, which is a fully human antibody.   22. The isolated antibody according to any one of claims 1 to 21, which is a FAb.   24. The isolated antibody according to any one of claims 1 to 23 comprising a human IgG1 constant region.   26. The isolated antibody of claim 25, wherein the human IgG1 constant region comprises tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 of the constant region according to the EU numbering system.   24. The isolated antibody according to any one of claims 1 to 23 comprising a human IgG4 constant region.   The human IgG4 constant region comprises proline at position 228 according to the EU numbering system, optionally further comprising tyrosine at position 252 of the constant region according to the EU numbering system, threonine at position 254, and glutamic acid at position 256. 27. Isolated antibody according to 27.   29. The isolated antibody of any one of claims 1 to 28 fused to attenuated interferon alpha-2b having the amino acid sequence of SEQ ID NO: 649 or SEQ ID NO: 651.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 9.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 10.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 652.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 653.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 654.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 655.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 656.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 657.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 658.   30. The isolated antibody of claim 29, comprising the amino acid sequence of SEQ ID NO: 694.   40. The isolated antibody of any one of claims 1 to 39, wherein the light chain variable region comprises a kappa light chain variable region.   40. The isolated antibody of any one of claims 1 to 39, wherein the light chain variable region comprises a lambda light chain variable region.   42. A composition comprising the antibody according to any one of claims 1 to 41 and a pharmaceutically acceptable carrier.   42. An isolated polynucleotide encoding the antibody of any one of claims 1-41.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 669.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 670.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 671.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 672.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 673.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 674.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 668, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 669.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 675.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 676.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667, optionally further comprising the nucleic acid sequence of SEQ ID NO: 677.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 678.   A vector comprising the polynucleotide according to any one of claims 43 to 54.   56. A transformed cell comprising the vector of claim 55.   42. A transformed cell expressing the antibody of any one of claims 1-41.   58. The transformed cell of claim 57, which is a mammalian cell.   59. The transformed cell of claim 58, wherein the mammalian cell is a Chinese hamster ovary cell.   58. A transformed cell according to claim 57 which is a yeast cell.   58. A transformed cell according to claim 57 which is an insect cell.   An antibody according to any one of claims 29 to 39 and instructions for using said antibody in a method for inhibiting the growth of tumor cells that express the receptor for CD38 and interferon alpha-2b on the surface And a kit containing.   40. Use of the antibody of any one of claims 29 to 39 and instructions for inducing apoptosis in tumor cells expressing CD38 and interferon alpha-2b receptors on the surface thereof And a kit containing.   A method for treating a tumor comprising the antibody of any one of claims 29 to 39 and a tumor cell that expresses a receptor for CD38 and interferon alpha-2b in a subject in need thereof. A kit comprising, optionally, a pharmaceutically acceptable carrier.   30. A kit comprising the antibody of any one of claims 1 to 28 and instructions for using the antibody in a method for detecting CD38 in a tissue sample isolated from a subject.   66. The kit of claim 65, wherein the tissue sample comprises blood or bone marrow.   40. A method for treating a tumor comprising tumor cells that express a receptor for CD38 and interferon alpha-2b on a surface, wherein the antibody according to any one of claims 29 to 39 is applied to a subject having a tumor. Administering a step of administering.   68. The method of claim 67, wherein the antibody is included in a composition comprising a pharmaceutically acceptable carrier.   69. The method of claim 68, wherein the composition further comprises one or more pharmaceutically acceptable excipients.   68. The method of claim 67, wherein the administering step comprises intravenous administration.   68. The method of claim 67, wherein the subject is a non-human primate.   72. The method of claim 71, wherein the non-human primate is a cynomolgus monkey.   68. The method of claim 67, wherein the subject is a human.   68. The method of claim 67, wherein the tumor is B cell lymphoma, multiple myeloma, non-Hodgkin lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia or acute myeloid leukemia.   A step of contacting the antibody according to any one of claims 1 to 28 with a tissue sample isolated from a subject to form an antibody-CD38 complex, and detecting the complex in the tissue sample A method for detecting CD38 in a tissue sample isolated from a subject comprising the step of:   76. The method of claim 75, wherein the tissue sample comprises blood and the blood is suspected of having CD38 positive tumor cells.   77. The method of claim 76, wherein the tumor cells comprise B cell lymphoma cells, multiple myeloma cells, non-Hodgkin lymphoma cells, chronic myeloid leukemia cells, chronic lymphocytic leukemia cells or acute myeloid leukemia cells.   76. The method of claim 75, further comprising isolating a tissue sample from the subject.   79. The method of any one of claims 75 to 78, wherein the subject is a non-human primate.   80. The method of claim 79, wherein the non-human primate is a cynomolgus monkey.   79. The method of any one of claims 75 to 78, wherein the subject is a human.   An isolated antibody that specifically binds to CD38, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 665 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 666.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 165.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 166.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 167.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 179.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 180.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 156.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 197.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 152.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 161.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 182.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 184.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 185.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 188.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 198.   84. The antibody of claim 82, wherein the heavy chain variable region comprises CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 516 and SEQ ID NO: 544.   84. The antibody of claim 82, wherein the heavy chain variable region comprises CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 518, SEQ ID NO: 534, SEQ ID NO: 535, and SEQ ID NO: 536.   84. The antibody of claim 82, wherein the light chain variable region comprises CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 583, SEQ ID NO: 590, and SEQ ID NO: 696.   84. The antibody of claim 82, wherein the light chain variable region comprises CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 585, SEQ ID NO: 591, and SEQ ID NO: 605.   84. The antibody of claim 82, wherein the light chain variable region comprises CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 587 and SEQ ID NO: 594. Binding to CD38-positive cells with an EC ₅₀ value of less than about 100 nM, isolated antibody according to any one of claims 82 101. Binding to CD38-positive cells with an EC ₅₀ value of less than about 50 nM, isolated antibody according to any one of claims 82 101. About binding to CD38-positive cells with an EC ₅₀ value of less than 30 nM, isolated antibody according to any one of claims 82 101. About binding to CD38-positive cells with an EC ₅₀ value of less than 15 nM, isolated antibody according to any one of claims 82 101.   106. The isolated antibody according to any one of claims 82 to 105, which is a monoclonal antibody.   107. Isolated antibody according to any one of claims 82 to 106, wherein the antibody is a humanized antibody.   106. The isolated antibody of any one of claims 82 to 105, which is a FAb.   108. The isolated antibody of any one of claims 82 to 107, comprising a human IgG1 constant region.   110. The isolated antibody of claim 109, wherein the human IgG1 constant region comprises tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 of the constant region according to the EU numbering system.   108. The isolated antibody of any one of claims 82 to 107, comprising a human IgG4 constant region.   The human IgG4 constant region comprises proline at position 228 according to the EU numbering system, optionally further comprising tyrosine at position 252 of the constant region according to the EU numbering system, threonine at position 254, and glutamic acid at position 256. 111. Isolated antibody according to 111.   113. The isolated antibody of any one of claims 82 to 112, fused to attenuated interferon alpha-2b having the amino acid sequence of SEQ ID NO: 649 or SEQ ID NO: 651.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 9.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 10.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 652.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 653.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 654.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 655.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 656.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 657.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 658.   114. The isolated antibody of claim 113, comprising the amino acid sequence of SEQ ID NO: 694.   124. The isolated antibody of any one of claims 82 to 123, wherein the light chain variable region comprises a kappa light chain variable region.   124. The isolated antibody of any one of claims 82 to 123, wherein the light chain variable region comprises a lambda light chain variable region.   126. A composition comprising the antibody of any one of claims 82 to 125 and a pharmaceutically acceptable carrier.   126. An isolated polynucleotide encoding the antibody of any one of claims 82-125.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 680 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 679, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 681, optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 683 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 684 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 689.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 690.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 691.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 686 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 693.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 689.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 690.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 691.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 692.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 693.   144. A vector comprising the polynucleotide of any one of claims 127 to 143.   145. A transformed cell comprising the vector of claim 144.   126. A transformed cell that expresses the antibody of any one of claims 82-125.   147. The transformed cell of claim 146, which is a mammalian cell.   148. The transformed cell of claim 147, wherein the mammalian cell is a Chinese hamster ovary cell.   147. The transformed cell of claim 146, which is a yeast cell.   147. The transformed cell of claim 146, which is an insect cell.   124. Use of the antibody of any one of claims 113 to 123 and a method for inhibiting the growth of tumor cells that express the receptor for CD38 and interferon alpha-2b on the surface thereof And a kit containing.   124. Use of the antibody of any one of claims 113 to 123 and a method for inducing apoptosis in tumor cells that express CD38 and interferon alpha-2b receptors on the surface thereof And a kit containing.   124. A method for treating a tumor comprising a tumor cell that expresses a receptor for CD38 and interferon alpha-2b in a subject in need thereof, and an antibody according to any one of claims 113 to 123. A kit comprising, optionally, a pharmaceutically acceptable carrier.   113. A kit comprising the antibody according to any one of claims 82 to 112 and instructions for using the antibody in a method for detecting CD38 in a tissue sample isolated from a subject.   157. The kit of claim 154, wherein the tissue sample comprises blood or bone marrow.   124. A method for treating a tumor comprising tumor cells expressing CD38 and a receptor for interferon alpha-2b on the surface, wherein the antibody according to any one of claims 113 to 123 is applied to a subject having a tumor. A method comprising the steps of:   157. The method of claim 156, wherein the antibody is contained in a composition comprising a pharmaceutically acceptable carrier.   164. The method of claim 157, wherein the composition further comprises one or more pharmaceutically acceptable excipients.   157. The method of claim 156, wherein the administering step comprises intravenous administration.   157. The method of claim 156, wherein the subject is a non-human primate.   170. The method of claim 160, wherein the non-human primate is a cynomolgus monkey.   157. The method of claim 156, wherein the subject is a human.   157. The method of claim 156, wherein the tumor is B cell lymphoma, multiple myeloma, non-Hodgkin lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia or acute myeloid leukemia.   A step of contacting the antibody according to any one of claims 82 to 112 with a tissue sample isolated from a subject to form an antibody-CD38 complex, and detecting the complex in the tissue sample A method for detecting CD38 in a tissue sample isolated from a subject comprising the step of:   165. The method of claim 164, wherein the tissue sample comprises blood and the blood is suspected of having CD38 positive tumor cells.   166. The method of claim 165, wherein the tumor cells comprise B cell lymphoma cells, multiple myeloma cells, non-Hodgkin lymphoma cells, chronic myeloid leukemia cells, chronic lymphocytic leukemia cells or acute myeloid leukemia cells.   164. The method of claim 164, further comprising isolating a tissue sample from the subject.   168. The method of any one of claims 164 to 167, wherein the subject is a non-human primate.   171. The method of claim 168, wherein the non-human primate is a cynomolgus monkey.   168. A method according to any one of claims 164 to 167, wherein the subject is a human.   An isolated antibody that specifically binds to CD38, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 736 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 664.   172. The isolated antibody of claim 171, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 110.   172. The isolated antibody of claim 171, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 728.   172. The isolated antibody of claim 171, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 730.   172. The isolated antibody of claim 171, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 731.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 86.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 88.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 93.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 660.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 661.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 662.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 663.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 700.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 701.   172. The isolated antibody of claim 171, wherein the heavy chain variable region comprises CDR1 having the amino acid sequence of SEQ ID NO: 697.   172. The isolated antibody of claim 171, wherein the heavy chain variable region comprises CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 698 and SEQ ID NO: 737.   171. The isolated antibody of claim 171, wherein the heavy chain variable region comprises CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 699 and SEQ ID NO: 738.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 233 and SEQ ID NO: 319.   171. The isolated antibody of claim 171, wherein the light chain variable region comprises CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 235, SEQ ID NO: 307, and SEQ ID NO: 311.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 237, SEQ ID NO: 321, and SEQ ID NO: 324. Binding to CD38-positive cells with an EC ₅₀ value of less than about 100 nM, isolated antibody according to any one of claims 171 192,363, or 364. Binding to CD38-positive cells with an EC ₅₀ value of less than about 50 nM, isolated antibody according to any one of claims 171 192,363, or 364. Binding to CD38-positive cells with an EC ₅₀ value of less than about 20 nM, isolated antibody according to any one of claims 171 192,363, or 364. Binding to CD38-positive cells with an EC ₅₀ value of less than about 10 nM, isolated antibody according to any one of claims 171 192,363, or 364.   187. The isolated antibody of any one of claims 171 to 196, 363, or 364, which is a monoclonal antibody.   187. Isolated antibody according to any one of claims 171-197, 363, or 364, which is a fully human antibody.   187. Isolated antibody according to any one of claims 171 to 196, 363, or 364, which is a FAb.   361. The isolated antibody of any one of claims 171-198, 363, or 364, comprising a human IgG1 constant region.   200. The isolated antibody of claim 200, wherein the human IgG1 constant region comprises tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 of the constant region according to the EU numbering system.   361. The isolated antibody of any one of claims 171-198, 363, or 364, comprising a human IgG4 constant region.   The human IgG4 constant region comprises proline at position 228 according to the EU numbering system, optionally further comprising tyrosine at position 252 of the constant region according to the EU numbering system, threonine at position 254, and glutamic acid at position 256. 202. The isolated antibody according to 202.   364. The isolated antibody of any one of claims 171 to 203, 363, or 364 fused to attenuated interferon alpha-2b having the amino acid sequence of SEQ ID NO: 649 or SEQ ID NO: 651.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 9.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 10.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 652.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 653.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 654.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 655.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 656.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 657.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 658.   205. The isolated antibody of claim 204, comprising the amino acid sequence of SEQ ID NO: 694.   365. The isolated antibody of any one of claims 171 to 214, 363, or 364, wherein the light chain variable region comprises a kappa light chain variable region.   365. The isolated antibody of any one of claims 171-1214, 363, or 364, wherein the light chain variable region comprises a lambda light chain variable region.   137. A composition comprising the antibody of any one of claims 171 to 216, 363, or 364 and a pharmaceutically acceptable carrier.   137. An isolated polynucleotide encoding the antibody of any one of claims 171 to 216, 363, or 364.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 695, optionally further comprising the nucleic acid sequence of SEQ ID NO: 669.   218. The isolated polynucleotide of claim 218, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 695 and the nucleic acid sequence of SEQ ID NO: 669.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 732, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 702.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 733 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 702.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 734, optionally further comprising the nucleic acid sequence of SEQ ID NO: 702.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 735, optionally further comprising the nucleic acid sequence of SEQ ID NO: 702.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 732, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 703.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 733 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 703.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 734, optionally further comprising the nucleic acid sequence of SEQ ID NO: 703.   218. The isolated polynucleotide of claim 218, comprising the nucleic acid sequence of SEQ ID NO: 735, optionally further comprising the nucleic acid sequence of SEQ ID NO: 703.   A vector comprising the polynucleotide of claims 218-228.   230. A transformed cell comprising the vector of claim 229.   A transformed cell expressing the antibody of any one of claims 171 to 216, 363, or 364.   231. The transformed cell of claim 231, which is a mammalian cell.   235. The transformed cell of claim 232, wherein the mammalian cell is a Chinese hamster ovary cell.   234. The transformed cell of claim 231, which is a yeast cell.   242. The transformed cell of claim 231, which is an insect cell.   An antibody according to any one of claims 204 to 214 and instructions for using said antibody in a method for inhibiting the growth of tumor cells that express the receptor for CD38 and interferon alpha-2b on the surface And a kit containing.   An antibody according to any one of claims 204 to 214 and instructions for using said antibody in a method for inducing apoptosis in tumor cells that express the receptor for CD38 and interferon alpha-2b And a kit containing.   In a method for treating a tumor comprising the antibody according to any one of claims 204 to 214 and a tumor cell that expresses a receptor for CD38 and interferon alpha-2b in a subject in need thereof. A kit comprising, optionally, a pharmaceutically acceptable carrier.   An antibody according to any one of claims 171 to 203, 363, or 364 and instructions for using the antibody in a method for detecting CD38 in a tissue sample isolated from a subject. Including kit.   240. The kit of claim 239, wherein the tissue sample comprises blood or bone marrow.   205. A method for treating a tumor comprising tumor cells that express a receptor for CD38 and interferon alpha-2b on a surface, wherein the antibody according to any one of claims 204 to 214 is applied to a subject having a tumor. Administering a step of administering.   The method of claim 241, wherein the antibody is contained in a composition comprising a pharmaceutically acceptable carrier.   243. The method of claim 242, wherein the composition further comprises one or more pharmaceutically acceptable excipients.   242. The method of claim 241, wherein the administering step comprises intravenous administration.   242. The method of claim 241, wherein the subject is a non-human primate.   254. The method of claim 245, wherein the non-human primate is a cynomolgus monkey.   242. The method of claim 241, wherein the subject is a human.   242. The method of claim 241, wherein the tumor is B cell lymphoma, multiple myeloma, non-Hodgkin lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia or acute myeloid leukemia.   Contacting the antibody of any one of claims 171 to 203, 363, or 364 with a tissue sample isolated from a subject to form an antibody-CD38 complex; and A method for detecting CD38 in a tissue sample isolated from a subject comprising detecting the complex.   249. The method of claim 249, wherein the tissue sample comprises blood and the blood is suspected of having CD38 positive tumor cells.   253. The method of claim 250, wherein the tumor cells comprise B cell lymphoma cells, multiple myeloma cells, non-Hodgkin lymphoma cells, chronic myeloid leukemia cells, chronic lymphocytic leukemia cells or acute myeloid leukemia cells.   249. The method of claim 249, further comprising isolating a tissue sample from the subject.   253. The method according to any one of claims 249 to 252, wherein the subject is a non-human primate.   254. The method of claim 253, wherein the non-human primate is a cynomolgus monkey.   253. The method according to any one of claims 249 to 252, wherein the subject is a human.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 700.   2. The isolated antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 701.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 702.   44. The isolated polynucleotide of claim 43, comprising the nucleic acid sequence of SEQ ID NO: 667 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 703.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 720.   85. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 721.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 722.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 723.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 739.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 740.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 741.   84. The isolated antibody of claim 82, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 742.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 704.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 705.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 706.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 707.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 708.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 709.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 710.   84. The isolated antibody of claim 82, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 711.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 685, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, optionally further comprising the nucleic acid sequence of SEQ ID NO: 688.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 687, optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 712.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 713.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, optionally further comprising the nucleic acid sequence of SEQ ID NO: 714.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 715.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, optionally further comprising the nucleic acid sequence of SEQ ID NO: 716.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 717.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 718.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 724, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 725, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 726, optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 727, optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 743, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 744 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 745 and optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   128. The isolated polynucleotide of claim 127, comprising the nucleic acid sequence of SEQ ID NO: 746, and optionally further comprising the nucleic acid sequence of SEQ ID NO: 719.   172. The isolated antibody of claim 171, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 65.   172. The isolated antibody of claim 171, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 729.