JP6648171B2 - Fusion with anti-CD38 antibody and attenuated interferon alpha-2B - Google Patents

Description

REFERENCE TO SEQUENCE LISTING This application includes 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 specifically, the present disclosure relates to antibodies that specifically bind to CD38, and constructs comprising such antibodies and 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 the specification, including patents, published applications, technical articles, scholarly 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, and on a significant proportion of normal bone marrow progenitor cells. In some examples, expression of CD38 in lymphocytes may depend on the differentiation and activation status of the cells, e.g., resting T and B cells may be negative for CD38 expression, Immature and activated lymphocytes may be largely 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, resulting in tyrosine phosphorylation and activation of the cell. CD38 is also a receptor that can interact with the ligand CD31. Activation of receptors via CD31 results in intracellular events such as Ca2 ⁺ mobilization, cell activation, proliferation, differentiation and migration.

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

  Interacts with a cell surface receptor, thereby stimulating, inhibiting, or otherwise modulating a biological response (which typically includes signaling pathways inside cells carrying the receptor) A number of 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 therapeutics. Some peptide or polypeptide ligands are regulated as therapeutic products, for example, human growth hormone, insulin, interferon (IFN) -alpha 2b, IFN-alpha 2a, IFNβ, erythropoietin, G-CSF and GM-CSF Authorized by authorities.

  Although the potential of these and other ligands in therapeutic applications has been demonstrated, they may also exhibit toxicity when administered to human patients. One reason for toxicity is that many of these ligands trigger receptors on various cells, including cells other than those that mediate the desired therapeutic effect. As a result of such "off target" activity of the ligand, many ligands are not currently suitable for use as therapeutics. This is because the ligand cannot be administered at a dose high enough 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 1980's that interferons, especially IFN-alpha, can increase apoptosis and reduce the proliferation of certain cancer cells. 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 the type I IFN receptor on these cells and stimulates apoptosis, terminal differentiation or reduced proliferation. A further indirect effect of IFN-alpha on non-cancer cells is to stimulate the immune system, which can have additional anti-cancer effects by causing the immune system to reject tumors.

  These biological activities are mediated by type I interferon receptors on the surface of cancer cells and, when stimulated, initiate various signaling pathways, 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 triggers the expression of some pro-inflammatory cytokines and chemokines, resulting in toxic and detrimental effects. Such toxicity can cause severe cold-like symptoms and inhibit the administration of IFN-alpha to the subject at levels that exert maximal antiproliferative and pro-apoptotic activity against cancer cells.

  When IFN-alpha 2b is used to treat multiple sclerosis, its utility lies, at least in part, in its binding to the type I interferon receptor on myeloma cells, followed by apoptosis And / or induces reduced proliferation, thus limiting 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 the possibility of reducing tumor growth rates by chemically conjugating human IFN-alpha to tumor targeting antibodies. Described localization of the direct inhibitory activity of IFN-alpha, indicating that such conjugates have antitumor activity in a xenograft model of human cancer. Because the human IFN-alpha used in the experiments did not appreciably interact with the mouse type I IFN receptor, which could have indirect anticancer effects, the observed mechanism of anticancer activity was Attributable to the direct effect of IFN-alpha on cancer cells. Due to this lack of binding of human IFN-alpha 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, WO 01/97844 describes a direct fusion of human IFN-alpha to the C-terminus of the heavy chain of an IgG specific for the tumor antigen CD20.

  Generally, IFN can be targeted to cancer cells. While this approach may result in increased activity of IFN on cancer cells, it does not completely address the issue of unwanted activity of IFN on healthy cells. Fusion of IFN-alpha to the C-terminus of the heavy chain of IgG prolongs the half-life of IFN-alpha and can lead to undesirable adverse events. Therefore, there is a need to reduce the off-target activity of ligand-based drugs while retaining the ligand's "on-target" therapeutic effect.

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 U.S. Patent Application Publication No. 2009/0142340 U.S. Patent Application Publication No. 2009/0068175 U.S. Patent Application Publication No. 2009/0092599 U.S. Patent No. 6,602,684 U.S. Patent No. 7,326,681 U.S. Patent No. 7,388,081 PCT Publication No.WO08 / 006554 PCT application number PCT / AU2012 / 001323 U.S. 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 disclosure features new anti-CD38 antibodies and constructs comprising the anti-CD38 antibodies and attenuated IFN-alpha. Antibodies that include 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 to, for example, an attenuated form of interferon alpha to form an anti-CD38 antibody-attenuated interferon fusion construct.

  In some embodiments, the isolated antibody that specifically binds to 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 to 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 to 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, comprising the amino acid sequence of SEQ ID NO: 200, SEQ ID NO: 514 or SEQ ID NO: 697, SEQ ID NO: 202, SEQ ID NO: 516, SEQ ID NO: 544, a 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 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 Light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 585, SEQ ID NO: 591 or SEQ ID NO: 605, 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 is 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: 741, 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, SEQ ID NO: 161, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 198 or SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710 and SEQ ID NO: 711.

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

  The antibodies may be monoclonal antibodies, and are preferably fully human antibodies. Antibodies may include FAbs. Antibodies may include human IgG1 or IgG4 constant regions. The IgG1 or IgG4 constant region may comprise tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 according to the EU numbering system. The IgG4 constant region may include proline at position 228 according to the EU numbering system, where 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 include a substitution of alanine at position 145 with 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 the IgG1 or IgG4 constant region, wherein the antibody is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, sequence SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, or SEQ ID NO: 694. An antibody, such as an antibody fused to attenuated interferon alpha-2b, may be included in a composition comprising a pharmaceutically acceptable carrier.

  Provided is an isolated polynucleotide encoding the antibody and the antibody fused to attenuated interferon alpha-2b. 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, sequence 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.685, 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, Sequence 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 , SEQ ID NO: 746. A polynucleotide may include a vector. Vectors can be used, for example, to transform cells. Transformed cells containing such polynucleotides are also provided. Transformed cells may include mammalian cells, yeast cells, or insect cells.

  Stable cells expressing the antibody are also provided. The cells that express the antibody may be mammalian cells. Preferred cells are Chinese Hamster Ovary (CHO) cells.

  A kit is provided that includes an antibody fused to attenuated interferon alpha-2b. This kit comprises an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct and the use of said construct in a method for inhibiting the growth of tumor cells that express the receptor for CD38 and interferon alpha-2b. Instructions, instructions for using the construct in a method for inducing apoptosis in tumor cells that express the receptor for CD38 and interferon alpha-2b, 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, optionally comprising 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. 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 that contain cells that express the receptor for CD38 and interferon alpha-2b. In general, methods of treatment include administering to a subject having a tumor an amount of an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct that is effective to treat the tumor. The construct may include any of the constructs described or exemplified herein. The subject is preferably a mammal, more preferably a non-human primate, and most preferably a human. Tumors may include B-cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia or acute myeloid leukemia.

  Optionally, an anti-CD38 antibody fused to the 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. Generally, the method comprises the steps of contacting an antibody that specifically binds to CD38 with a tissue sample isolated from a subject, and detecting a complex of 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 cells may be CD38-positive B-cell lymphomas, multiple myeloma cells, non-Hodgkin's lymphoma cells, chronic myelogenous leukemia cells, chronic lymphocytic leukemia cells, or acute myeloid leukemia cells. The subject is preferably a mammal, more preferably a non-human primate, and most preferably a human. The method may include isolating a tissue sample from the subject. The method may further include contacting the antibody with a tissue sample that does not contain, for example, CD38-positive cells to serve as a negative control.

FIG. 3 shows an example of an anti-CD38 antibody-attenuated interferon fusion construct. FIG. 2 shows the sequence of the heavy chain variable region of X02.1, related constructs, and the most homologous germline antibody sequence. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the sequence of the heavy chain variable region of X02.1, related constructs, and the most homologous germline antibody sequence. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the sequence of X02.1, related constructs, and the light chain variable region of the most homologous germline antibody sequence. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the sequence of X02.1, related constructs, and the light chain variable region of the most homologous germline antibody sequence. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the sequence of the light chain variable region of A02.1 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 4 shows the consensus variable heavy chain sequence of A02.1 and related constructs. The boxed region contains the CDRs (as indicated) defined by the Kabat numbering system and the enhanced Chothia numbering system. CDRs defined by Kabat's numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 4 shows the consensus variable light chain sequence of A02.1 and related constructs. The boxed region contains the CDRs (as indicated) defined by the Kabat numbering system and the enhanced Chothia numbering system. CDRs defined by Kabat's numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 3 shows the sequence of the heavy chain variable region of the humanized heavy chain variable region. CDRs defined by Kabat's numbering system are underlined. FIG. 3 shows the sequence of the heavy chain variable region of the humanized heavy chain variable region. CDRs defined by Kabat's numbering system are underlined. FIG. 3 shows the sequence of the heavy chain variable region of the humanized heavy chain variable region. CDRs defined by Kabat's numbering system are underlined. FIG. 3 shows the sequence of the heavy chain variable region of the humanized light chain variable region. CDRs defined by Kabat's numbering system are underlined. FIG. 3 shows the sequence of the heavy chain variable region of the humanized light chain variable region. CDRs defined by Kabat's numbering system are underlined. FIG. 3 shows the sequence of the heavy chain variable region of the humanized light chain variable region. CDRs defined by Kabat's numbering system are underlined. FIG. 3 shows the variable heavy chains of A10.0 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 3 shows the variable heavy chains of A10.0 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the variable light chain of A10.0 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 2 shows the variable light chain of A10.0 and related constructs. CDRs defined by Kabat's numbering system are underlined. FIG. 4 shows the variable heavy chain consensus sequence of A10.0 and related constructs. The boxed region contains the CDRs (as indicated) defined by the Kabat numbering system and the enhanced Chothia numbering system. CDRs defined by Kabat's numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 4 shows the variable heavy chain consensus sequence of A10.0 and related constructs. The boxed region contains the CDRs (as indicated) defined by the Kabat numbering system and the enhanced Chothia numbering system. CDRs defined by Kabat's numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 2 shows the variable light chain consensus sequence of A10.0 and related constructs. The boxed region contains the CDRs (as indicated) defined by the Kabat numbering system and the enhanced Chothia numbering system. CDRs defined by Kabat's numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined. FIG. 2 shows the binding activity of A02.1 variant to CD38-expressing multiple myeloma cell line ARP-1 as measured by flow cytometry. Details of the assay are described in the examples herein. FIG. 2 shows the binding activity of the A02.1 variant to CD38-expressing multiple myeloma cell line NCI-H929, as measured by flow cytometry. Details of the assay are described in the examples herein. FIG. 2 shows the antiproliferative activity of A02.1 variant on multiple myeloma cell line ARP-1. The A-isotype is an antibody of irrelevant specificity fused to attenuated interferon as a control. Details of the assay are described in the examples (cell proliferation assay). FIG. 2 shows the antiproliferative activity of A02.1 variant on multiple myeloma cell line ARP-1. The A-isotype is an antibody of irrelevant specificity fused to attenuated interferon as a control. Details of the assay are described in the examples (cell proliferation assay). The antiproliferative activity of IFN-alpha2b (intron A) on multiple myeloma cell line ARP-1 compared to A02.1 and A10.0 and its corresponding unfused antibodies X02.1 and X10.0. FIG. The A-isotype is an antibody of irrelevant specificity fused to attenuated interferon as a control. Details of the assay are described in the examples (cell proliferation assay). In a multiple myeloma cell line NCI-H929 that expresses CD38 when treated with A02.1 and A10.0 and their corresponding unfused antibodies X02.1 and X10.0 for 24 hours compared to untreated controls FIG. 4 is a view showing a relative fold change in Annexin V production in Example 1. The A-isotype is an antibody of irrelevant specificity fused to attenuated interferon as a control. Details of the assay are described in the examples (Annexin V assay). FIG. 10 shows the relative fold change in caspase activation in multiple myeloma cell line H929 expressing IFN-alpha 2b (intron A) versus A02.1 and related construct CD38 compared to untreated cells. is there. Isotype 145D is an antibody of unrelated specificity fused to attenuated interferon as a control. Details of the assay are described in the examples (caspase assay). FIG. 2 shows off-target activities of IFN-alpha 2b (intron A) versus A02.6 and A02.6 [A02.6 (wt. IFN)] fused to wild-type IFN-alpha 2b against CD38-negative cells. Details of the assay are described in the examples [HEK-BLUE ™]. FIG. 4 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 an IgG4 constant region fused to attenuated IFN-alpha, while A02.112 and A10.59 contain an IgG1 constant region fused to attenuated IFN-alpha. Details of the assay are described in the examples (Annexin V / 7AAD assay). FIG. 4 shows the binding activity of the A10.0 variant to CD38-expressing multiple myeloma cell line NCI-H929, as measured by flow cytometry. Details of the assay are described in the examples herein. FIG. 3 shows caspase activation in multiple myeloma cell line H929 expressing CD38 of A10.0 and A10.38 compared to untreated cells. The A-isotype is an antibody of irrelevant specificity fused to attenuated IFN as a control. Details of the assay are described in the examples (caspase assay). FIG. 2 shows the relative fold change in caspase activation in the multiple myeloma cell line H929 expressing CD38 by the A10.0 variant compared to untreated cells. Details of the assay are described in the examples (caspase assay). FIG. 3 shows the relative fold change in Annexin V production in CD38-expressing multiple myeloma cell line H929 by A10.0 variant. Details of the assay are described in the examples (Annexin V / 7AAD assay). FIG. 4 shows the antiproliferative activity of IFN-alpha 2b (intron A) on Burkitt's lymphoma cell line Daudi compared to A02.6, A10.0, A10.38 and parental A10A2.0 chimeric antibody constructs. The A-isotype is an antibody of irrelevant specificity fused to attenuated IFN as a control. Details of the assay are described in the examples (cell proliferation assay). FIG. 2 shows the effect of humanized A10.0 versus 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. 3 shows the non-antibody antigen-targeted IFN activity of the 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. 4 shows the variable heavy chain consensus sequence of X910 / 12-HC-L0-interferon-alpha (A145D) and related sequences. The boxed region contains the CDRs (as indicated) defined by the Kabat numbering system and the enhanced Chothia numbering system. CDRs defined by Kabat's numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined.

  Various terms relating to aspects of the disclosure are used throughout the specification and claims. Unless otherwise noted, such terms should be given their ordinary meaning in the art. Other specifically defined terms should 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 altered 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 context clearly dictates otherwise.

  Anti-CD38 antibody-attenuated interferon alpha-2b fusion constructs include, but are 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. Includes any antibody described or exemplified herein that specifically binds to CD38 fused to attenuated interferon alpha-2b protein. In some embodiments, fusion of a non-mutated interferon alpha-2b protein, such as SEQ ID NO: 7, to an anti-CD38 antibody reduces the biological activity of the interferon molecule. In the present disclosure, attenuated interferon, attenuated interferon alpha-2b, IFN-alpha2b A145D, and IFN-alpha2b A145G are used interchangeably.

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

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

  It has further been found that interferon-alpha 2b can be attenuated for its biological activity mediated via interferon binding to cell surface interferon receptors by introducing certain amino acid changes in its protein sequence. Was observed. The attenuated interferon molecule specifically binds to CD38 so that the antibody can serve as a delivery vehicle for the attenuated interferon to CD38-positive cells resulting in a reduction in off-target interferon activity caused by the attenuated interferon molecule Can be fused. It was further observed that fusion of the attenuated interferon to the CD38 antibody did not significantly affect the ability of the antibody to specifically bind to CD38 on cells expressing CD38, such as cells in the body of the animal. Engineering variants of the CD38 antibody 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 the attenuated interferon.

  Thus, it is characterized by an antibody that specifically binds to CD38. It was also observed that such anti-CD38 antibodies could be used as a delivery vehicle for attenuating ligands such as interferon alpha. Without 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 thought that it is 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, especially cells that do not express CD38. By bringing the attenuated interferon close to its receptor on CD38-positive cells, the antibody will show a diminished ability to induce undesired effects on healthy cells that do not express CD38 while retaining its associated receptor. It is believed that the ability of the attenuated interferon to bind to the body can be enhanced and induce a therapeutic effect.

  Antibodies can be polyclonal, but in some embodiments, are not polyclonal. Antibodies are preferably monoclonal. The antibodies are preferably full length antibodies. Full-length antibodies generally include a variable region heavy chain and a variable region light chain. Antibodies may include derivatives or fragments or portions of antibodies that retain antigen binding specificity and preferably retain much or all of the affinity of the parent antibody molecule (eg, for CD38). For example, a derivative may include at least one variable region (either a heavy or light chain variable region). Other examples of suitable antibody derivatives and fragments include, but are not limited to, antibodies with multiepitope 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 And heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy 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 moieties. Antibody derivatives, fragments, and / or portions can be produced and expressed recombinantly by any prokaryotic or eukaryotic cell type.

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

  In a full-length antibody, 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 comprises 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 disrupted 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.

  Antibodies can 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 antibodies from any other animal species. Good. For use in human treatment, non-human-derived antibodies can be structurally altered such that they become less antigenic when administered to a human patient, such as by chimerization or humanization or superhumanization.

  In some embodiments, the antibodies are humanized antibodies. Humanized antibodies are derived from amino acids directly involved in antigen binding, e.g., the heavy chain and / or light chain, complementarity-determining regions (CDRs), in some cases, framework regions (FWRs), or portions thereof, of human origin. But the remaining amino acids in the antibody are human, or are otherwise of human origin, eg, a human antibody scaffold. A humanized antibody also has one or more residues of a human protein modified by one or more amino acid substitutions, and / or one or more non-human residues to which one or more FWR residues of the human protein correspond. Also includes antibodies replaced by A humanized antibody may also contain residues that are not found in human or non-human antibodies. The humanized antibody may be, for example, a superhumanized antibody as described in US Patent No. 7,732,578. The antibody may be a humanized chimeric antibody.

  In a highly preferred embodiment, the antibodies are fully human antibodies. Fully human antibodies are those in which the entire molecule is human, or otherwise of human origin, or comprise the same amino acid sequence as a humanized antibody. Fully 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 xenomouse technology) or other organisms transformed with genes encoding human antibodies. Nevertheless, fully human antibodies may contain amino acid residues that are not encoded by human sequences, for example, mutations introduced by random or site-specific mutations.

  The antibody may be a full-length antibody of any class, for example, IgG1, IgG2 or IgG4. The constant domains of such antibodies are preferably human. The variable regions of such antibodies may be of non-human origin, or preferably are of human origin or humanized. Antibody fragments can be used instead of full-length antibodies.

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

  In some embodiments, the antibody may include a non-immunoglobulin derived protein framework. For example, describe a four-helix bundle protein cytochrome b562 with two loops randomized to produce CDRs, selected for antigen binding (Ku & Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6655, 1995).

  Natural sequence variations may be present in the heavy and light chains and the genes encoding them, and, therefore, those of skill in the art will recognize the amino acid sequences of the antibodies described and exemplified herein, or encoding them. One can expect to find some level of mutation in the gene. These variants preferably maintain the unique binding characteristics (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 that result from the degeneracy of the genetic code, as well as not appreciably altering the properties of the encoded protein. Accordingly, such variants and homologs are considered substantially identical to one another and are included within the scope of the present disclosure. Thus, antibodies include variants with one or more amino acid substitutions, deletions, additions, or substitutions that retain the biological properties (eg, binding specificity and affinity) of the parent antibody. Variants are preferably conservative, but may be non-conservative.

  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's numbering system). . In addition, 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 the 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 FWR and CDRs were designated 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 the following: 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 be increased in length, and some amino acids may be designated by a letter after the number, according to Kabat's numbering system. This document is not limited to FWR and CDR as defined by Kabat's numbering system, but includes Chothia et al. (1987) J. Mol. Biol. 196: 901-17; Chothia et al. (1989) Nature 342: 877- 83; and / or Al-Lazikani et al. (1997) J. Mol. Biol. 273: 927-48, standard numbering system; Honnegher et al. (2001) J. Mol. Biol., 309: 657-70. Numbering system; or 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 Kabat's numbering system.

  In some particular embodiments, for any of the heavy chain CDR2 subdomains described herein according to the Kabat numbering system, the five C-terminal amino acids may not be directly involved in antigen binding; Thus, 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, according to the Kabat numbering system, for any of the light chain CDR1 subdomains described herein, the four N-terminal amino acids may not be directly involved in antigen binding, and It is understood that any one or more of these four amino acids can be replaced with another naturally occurring 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 the 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, eg, when required to be administered to a subject. Analogues of amino acids 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. And the use of other methods that impose conformational constraints on the sex molecule or its analogs.

  Antibodies may include 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. Can be The moiety comprises any chemical group or combination of groups commonly found on immunoglobulin molecules, added to the antibody by natural or otherwise recombinant expression systems, such as prokaryotic and eukaryotic expression systems. .

Examples of side chain modifications contemplated by the present disclosure, reaction with an aldehyde, then reductive alkylation by reduction with NaBH _4; amidation with methyl acetimidate; acylation with acetic anhydride; cyanate by amino groups Carbamoylation; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and lysine with pyridoxal-5-phosphate pyridoxylated, then include modification of amino groups and reduction with NaBH _4.

  The guanidine group of the arginine residue can be modified by the formation of a heterocyclic condensation product 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; formic acid oxidation to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; 4-chloromercuri Formation of mercury derivatives with benzoic acid, 4-chloromercuriphenylsulfonic acid, phenylmercuric chloride, 2-chloromercuri-4-nitrophenol and other mercury agents; modification by methods such as carbamoylation with cyanate at alkaline pH Can be.

  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 a sulfenyl halide. On the other hand, tyrosine residues can be changed by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

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

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

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

  The antibody protects the IgG from catabolism and its serum, including modifications that modulate the antibody's interaction with the neonatal Fc receptor (FcRn), a receptor that has an important role in maintaining high serum antibody concentrations. Modifications that regulate half-life and biodistribution may be included. Modifications that regulate serum half-life are described in M252Y / S254T / T256E [EU numbering system (Edelman, GM et al. (1969) Proc. Natl.Acad. Page)) (eg, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 694) in the IgG1 or IgG4 Fc region. Other substitutions may be present at positions 250 and 428 (see, e.g., U.S. Patent No. 7,217,797), and at positions 307, 380 and 434 (see, e.g., WO00 / 42072). . Examples of constant domain amino acid substitutions that regulate binding to Fc receptors and subsequent functions mediated by these receptors, such as FcRn binding and serum half-life, are described in U.S. Patent Application Publication Nos. 2009/0142340, 2009. No./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 the antibody with Fc and glycan receptors, thereby affecting antibody activity such as serum half-life. Thus, certain glycoforms that modulate a desired antibody activity can provide a therapeutic benefit. Methods for making engineered glycoforms include, but are not limited to, those described in U.S. Patent Nos. 6,602,684, 7,326,681, and 7,388,081 and PCT Publication No.WO08 / 006554. . Alternatively, the antibody sequence can be modified to remove the relevant glycoform binding site.

  The antibody can be labeled or conjugated to any chemical or biological molecule moiety. Labeled antibodies may 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. Labels / conjugates may be chemotherapeutic agents, toxins, isotopes, and other agents used to treat conditions such as killing cancer cells. 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 proteolytic cleavage or to enhance activity or stability.

The antibody preferably has a binding affinity for an epitope on CD38 that includes 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, 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, Kd is less than about 1 × 10 ^-7 M. In another embodiment, the Kd is less than about 1 × 10 ⁻⁸ M. In another embodiment, 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 another embodiment, the Kd is less than about 1 × 10 ⁻¹³ M. In other embodiments, 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 were determined by standard methods including surface plasmon resonance, such as Biacore® analysis or analysis using the Octet® Red 96 (Forte Bio) Dip-and-Read system. Point.

  Antibodies may include single-chain Fv molecules (scFv), Fab, or full-length IgG. Any such antibody may have 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 of 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 approximately the same as the binding activity (including affinity) of an antibody that does not include an amino acid change in the heavy chain, but the binding activity (including affinity). G) can be lower or higher than antibodies that do not contain amino acid changes in the heavy chain. The antibody has at least about 85%, at least about 90%, 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%. 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 the binding activity (including affinity) of an antibody that does not contain an amino acid change in the light chain, but the binding activity (including affinity) is 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 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, SEQ ID NO: 577 or SEQ ID NO: 748, and 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, the sequence 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, 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% of 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, the 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, Sequence 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. 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 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: 573, SEQ ID NO: 578, SEQ ID NO: 749 or SEQ ID NO: 750 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 Include 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: 427, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 468, Sequence 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 SEQ ID NO: 580, SEQ ID NO: 751 or SEQ ID NO: 752, and 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: 216. 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: 427, SEQ ID NO: 431, SEQ ID NO: 435, SEQ ID NO: 468, 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, 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% of the amino acid sequence of SEQ ID NO: 580, SEQ ID NO: 751 or SEQ ID NO: 752 , At least about 97%, at least about 98%, or at least about 99%. 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: 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 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, sequence 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, at least about 85%, at least about 90%, at least about 92%, It includes 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 include amino acid changes in the heavy chain framework regions (FWR1, FWR2, FWR3, and / or FWR4) retain the ability to specifically bind to CD38. The retained CD38-specific binding activity (including affinity) is preferably approximately the same as the binding activity (including affinity) of an antibody that does not include an amino acid change 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 of the heavy chain framework regions.

  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 amino acid sequences 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: 479, SEQ ID NO: 481, SEQ ID NO: 483, SEQ ID NO: 485, SEQ ID NO: 487, Sequence 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 And 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 SEQ ID NO: 471, SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 477, SEQ ID NO: 479, SEQ ID NO: 481, SEQ ID NO: 483, SEQ ID NO: 485, SEQ ID NO: 487, SEQ ID NO: 489, SEQ ID NO: 516, SEQ ID NO: 539, SEQ ID NO: SEQ ID NO: 544, SEQ ID NO: 549, 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 Amino acid sequence having at least about 92%, 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. SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 699 or SEQ ID NO: 738, and in some embodiments, the heavy chain CDR3 comprises SEQ ID NO: 204, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 223, the sequence 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 SEQ ID NO: 738 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% amino acids having sequence identity Contains an array. It is understood that antibodies that include 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 include an amino acid change in any heavy chain complementarity determining region, Its binding activity (including affinity) may be lower or higher than antibodies that do not contain amino acid changes in any of the heavy chain complementarity determining regions.

  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: 614, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 626, Sequence SEQ ID NO: 630, SEQ ID NO: 634 or SEQ ID NO: 638, and 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, Sequence 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% of 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 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, 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, Sequence 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, SEQ ID NO: 289 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: 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 at least about 90% with the amino acid sequence of SEQ ID NO: 639, at least about 90 Amino acid sequence having at least about 92%, 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: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, Sequence 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, Sequence 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: 279, 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: 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: 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: 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, Sequence 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 about the amino acid sequence of SEQ ID NO: 640. Amines 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 Comprising the amino acid sequence of 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 At least the amino acid sequence of 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%, less 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 include amino acid changes in the light chain framework regions (FWR1, FWR2, FWR3, and / or FWR4) retain the ability to specifically bind to CD38. The retained CD38-specific binding activity (including affinity) is preferably approximately the same as the binding activity (including affinity) of an antibody that does not include an amino acid change 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 of the light chain framework regions.

  In some embodiments, the light chain CDR1 comprises 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: 361, SEQ ID NO: 365, SEQ ID NO: 369, SEQ ID NO: 373, SEQ ID NO: 377, Sequence 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 Are 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, 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 85% of the amino acid sequence of SEQ ID NO: 608, or SEQ ID NO: 696. It includes amino acid sequences having 97%, at least about 98%, or at least about 99% sequence identity. 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, 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 , SEQ ID NO: 610 or SEQ ID NO: 747, and 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, SEQ ID NO: 301. 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, SEQ ID 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, 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 90% of the amino acid sequence of SEQ ID NO: 747. 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: 340, SEQ ID NO: 344, SEQ ID NO: 348, SEQ ID NO: 352, SEQ ID NO: 356, Sequence No. 360, SEQ ID No. 364, Sequence No. 368, Sequence No. 372, Sequence No. 376, Sequence No. 380, Sequence No. 384, Sequence No. 388, Sequence No. 441, Sequence No. 495, Sequence No. 587, Sequence No. 594, Sequence No. 595 Comprising the amino acid sequence of 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: 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: 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: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: At least about 85%, at least about 90%, at least about 92%, at least about 93% of the amino acid sequence of 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 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 approximately the same as the binding activity (including affinity) of an antibody that does not include an amino acid change in any light chain complementarity determining region. Its binding activity (including affinity) may be lower or higher than an antibody that does not contain an amino acid change in any of the light chain complementarity determining regions.

  In some embodiments, the antibody comprises a particular heavy and light chain pair. The heavy chain having the amino acid sequence of SEQ ID NO: 659 is paired 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 is replaced with the amino acid sequence of SEQ ID NO: 666. Or the 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 and variable light chain pairs may include pairs from the table below.

  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 a signaling pathway due to the action of the attenuated ligand on a cell surface receptor. Can be formed. These constructs are such that in the context of an antibody-ligand construct, ligand activity on antigen-negative cells is dramatically diminished, but ligand activity on antigen-positive cells is only moderately, if at all, diminished. Is based on the observation that the ligand moiety can be mutated with. Such constructs are 1, 2, 3, 4 or 5 orders of magnitude more potent against antigen positive cells compared to antigen negative cells than free ligand. In some embodiments, the antibody-attenuated ligand construct has at least 1%, at least 10%, at least 20%, at least 30%, as a non-attenuated free (i.e., not bound to antibody) ligand relative to antigen-positive cells. 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 maximal activity of the non-attenuated free (i.e., not bound to the antibody) ligand. I do. Maximal activity includes the amount of signaling activity (or downstream effects) at the high plateau portion of the dose response curve, where further increases in drug do not further increase the amount of response.

In some embodiments, the antibody fusion to the interferon ligand and the inclusion of an attenuating mutation in the interferon ligand is greater than 10-fold, preferably greater than 50-fold, as compared to the antibody without the fusion. Increase the antigen specificity index (ASI), preferably more than 100-fold, preferably more than 1000-fold, or preferably more than 10,000-fold. ASI multiplied by a fold-reducing potency of signaling activity compared to free non-mutated polypeptide ligand on target antigen-negative cells, antibody-IFN compared to free non-mutated polypeptide ligand on target antigen-positive cells Includes a fold increase potency of the signaling activity of the ligand construct. Dose ligand or antibody in the assay - refers to the concentration of ligand construct response refers to the quantitative response of a cell to a signaling activity of the particular dosage of the ligand is a numerical midpoint of dose response curves by The EC ₅₀ values Efficacy can be expressed quantitatively. Thus, typically, when measured by the same method, for example, a first compound has an EC ₅₀ (e.g., expressed in molar units) that is 10 times lower than the EC ₅₀ of a second compound on the same cell. The first compound is said to have a ten-fold higher potency when indicated to have Conversely, typically, if the first compound is shown to have an EC ₅₀ 10 times higher than the EC ₅₀ of the second compound on the same cell when measured by the same method, then the first The compound is said to have 10 times lower potency.

The antibodies are preferably capable of binding to CD38 positive cells. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 100 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 75 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 50 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 30 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 25 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 20 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 18 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 15 nM. Antibodies capable of binding to CD38-positive cells with an EC ₅₀ value of less than about 13 nM. Antibodies capable of binding 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 cells lacking cell surface expression of the CD38 antigen to which the antibody binds And 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 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). You. 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 include an amino acid change that was previously at position 168, but was now at position 145 in the truncated protein (eg, alanine 168 becomes alanine 145). In the truncated IFN-alpha 2b, alanine is preferably changed to glycine (Gly / G) (SEQ ID NO: 651) or aspartic acid (Asp / D) (SEQ ID NO: 649). Interferons with A145D alterations (SEQ ID NO: 647 or SEQ ID NO: 649) are particularly preferred as attenuating 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, for example, as an antibody-attenuated interferon construct.

  The linkage between the antibody and the interferon preferably comprises a peptide bond between the N- or C-terminus of the fusion, eg, interferon, and the N- or C-terminus of the antibody heavy or light chain. In a very preferred embodiment, there is no linker between the antibody and the interferon, and thus the antibody and the interferon are fused directly. Direct fusions without an intervening linker peptide would provide at least a measurable degree of attenuation of the interferon protein, and this attenuation would be an interferon protein such as those described or exemplified herein. It may also be additive with attenuation of the interferon protein resulting from the mutations introduced therein.

  Polynucleotide sequences encoding an antibody and its subdomains (eg, FWR and CDR) are characterized in this disclosure. Polynucleotides include, but are not limited to, RNA, DNA, cDNA, hybrids of RNA and DNA, and single, double, 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. The polynucleotide is 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: 743, SEQ ID NO: 744, SEQ ID NO: 745 or SEQ ID NO: 746 A heavy chain comprising 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. The polynucleotide is 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: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, Sequence 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, SEQ ID NO: 718 or sequence It may comprise any of the nucleic acid sequences of number 719. The polynucleotide is 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: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, Sequence 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, SEQ ID NO: 718 or sequence At least about 80%, at least about 85%, low 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% May comprise nucleic acid sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, and in some embodiments, such variants preferably comprise 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: 743, 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 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, 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 binds specifically to CD38 with an affinity approximately equal to the affinity of the antibody encoded by the parent (non-variant) polynucleotide sequence. For example, affinity can be measured according to any of the techniques described or exemplified herein, such as the techniques described in the Examples. The complement of the polynucleotide sequence and the variant polynucleotide sequence are also within the scope of the present disclosure.

  Vectors containing the polynucleotides of the present disclosure are also included within the scope of the present disclosure. The vector may be an expression vector. Thus, there is provided a recombinant expression vector containing a sequence encoding the polypeptide of interest. Expression vectors 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 transcription and translation termination.

  Expression vectors, particularly mammalian expression vectors, may have an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5 'or 3' flanking non-transcribed sequences, 5 'or 3' untranslated It may include one or more non-transcribed elements, such as sequences (such as an essential ribosome binding site), polyadenylation sites, splice donor and acceptor sites, or transcription termination sequences. Origins of replication that confer the ability to replicate in a particular host can also be incorporated.

  The vector can be used to transform any of a variety of host cells known to those of skill in the art, preferably a host cell capable of expressing the antibody. Vectors include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and baculoviruses, as well as other bacteria, eukaryotes, yeasts , And viral vectors. Suitable host cells include, but are not limited to, CHO cells, HEK293 cells, or any known or produced 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 interferon alpha ligand having one or more mutations that substantially reduces the affinity of the ligand for the interferon receptor can be used to target the mutant interferon alpha ligand to target cells displaying 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 on target antigen positive cells is maintained, but the activity of the ligand on 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 toxicity resulting from off-target ligand activity Is 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 the antiproliferative activity of IFN 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 compositions comprising an antibody of the present disclosure and an antibody-attenuated interferon construct. These compositions may 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 include at least one suitable adjuvant, such as an excipient. Pharmaceutically acceptable auxiliaries are preferred. The composition may comprise any of the antibodies described and / or exemplified herein, the 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 are preferably non-toxic to the host to which it is administered. The carrier may 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, any suitable mass or It may be present alone, or in combination, including volumes. Exemplary protein excipients include serum albumins, 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. A 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 a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric, ascorbic, gluconic, carboxylic, tartaric, succinic, acetic, or phthalic acids; Tris, tromethamine hydrochloride, or phosphate buffers . Preferred buffers for use in the compositions of the present invention are organic acid salts, such as citrate.

  Further, the compositions of the present disclosure include polyvinylpyrrolidone, ficoll (polymeric sugar), dextrate (e.g., cyclodextrin such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, antimicrobial agents, antioxidants, Inhibitors, surfactants (e.g., polysorbates such as "TWEEN (R) 20" and "TWEEN (R) 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelates A polymeric excipient / additive such as an agent (eg, EDTA) may be included.

  The compositions can also be formulated in sustained release vehicles or depot preparations. For example, the composition is formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt. can do. Liposomes and emulsions are well-known examples of suitable delivery vehicles 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 with a suitable controlled release vehicle, with an adjuvant, or as a depot preparation.

  Preparations for parenteral administration include sterile solutions injectable, sterile dry dissolvable products such as hypodermic tablets, which can be combined with solvents immediately before use, sterile injectables Suspensions, sterile dry insoluble products ready to be combined with the vehicle immediately 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 the growth of cells that express CD38 on their surface. In some embodiments, the methods for inhibiting or reducing the growth of cells that express CD38 on the surface are generally effective to inhibit or reduce the growth of cells that express CD38 and the cells. Contacting with an amount of an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct. An antibody that specifically binds to CD38 can be any of the antibodies 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. The 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. And the like, 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 can also be used, for example, to induce, facilitate, or enhance apoptosis of cells that express CD38 on their surface. In some embodiments, the method for inducing apoptosis in cells that express CD38 on the surface comprises, in general, cells expressing CD38 and an amount of an anti-CD38 antibody effective to induce apoptosis in the cell-attenuation. Contacting with an interferon alpha-2b fusion construct. An antibody that specifically binds to CD38 can be any of the antibodies 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. The 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. And the like, together with one or more adjuvants or excipients. The method can be performed in vitro, ex vivo, in vivo, or in situ.

  The 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 the surface. it can. In some embodiments, the method for treating a tumor comprising cells that express CD38 on the surface generally comprises administering to a subject in need thereof an effective amount of an anti-CD38 antibody to treat the tumor in the subject. Administering the attenuated interferon alpha-2b fusion construct. An effective treatment may include, for example, inhibiting or reducing the proliferation of CD38-positive cells in a tumor, and / or inducing apoptosis of CD38-positive cells in a tumor. An antibody that specifically binds to CD38 can be any of the antibodies described or exemplified herein. The attenuated interferon alpha 2b may comprise IFN-alpha 2b A145D or IFN-alpha 2b A145G. The 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. And the like, 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 the 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 the tumor cells.

Provided is the use of an anti-CD38 antibody or an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct in the treatment of a tumor. Methods are provided for treating tumors using an anti-CD38 antibody or an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct. 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 cancer, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, glandular sac carcinoma, adrenocortical carcinoma, idiopathic myeloid metaplasia, Alopecia, alveolar soft tissue sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia telangiectasia, 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, pediatric brain tumor, pediatric cancer, pediatric leukemia, pediatric soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, Chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, raised dermal fibrosarcoma, fibroplastic small round cell tumor, ductal carcinoma, endocrine cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, eye: Norma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, gastrointestinal carcinoid tumor, urogenital cancer, germ cell tumor, gestational trophoblastic 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 carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma , Liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelium , Metastatic cancer, oral cancer, multiple endocrine adenoma, mycosis fungoides, myelodysplastic syndrome, multiple myeloma, myeloproliferative disorder, nasal cavity cancer, nasopharyngeal cancer, nephroblastoma, neuroblast Cell tumor, neurofibromatosis, Nijmegen chromosome 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, urinary system 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's 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 include treating the subject with a retinoid, such as all-trans retinoic acid. In some preferred embodiments, wherein the antigen bound to the cell surface is CD38, the tumor or cancer is selected from multiple myeloma, non-Hodgkin's 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 combined with other drugs and / or used in addition to other cancer treatment regimens or modalities, such as radiation therapy or surgery. If the anti-CD38 antibody-attenuated interferon alpha-2b fusion construct is used in combination with a known therapeutic agent, the combinations may be sequentially (sequentially or divided over 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 the present context. The combination treatment may also be, for example, as a maintenance therapy, treatment with an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct, followed by known treatment, or treatment with a known 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 is converted to an alkylating agent (mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide cisplatin, or cisplatin, carboplatin and oxaliplatin, etc. , An antimetabolite (purine or pyrimidine analog or an antifolate such as azathioprine and mercaptopurine), anthracycline (daunorubicin, doxorubicin, epirubicin idarubicin, valrubicin, mitoxantrone, or anthracene) Cyclin analogs), plant alkaloids (vinca alkaloids or vincristine, vinblastine, vinorelbine, vindesine, taxanes such as paclitaxel or docetaxel), It can be administered in combination with a topoisomerase inhibitor (such as a type I or type II topoisomerase inhibitor), podophyllotoxin (such as etoposide or teniposide), or a tyrosine kinase inhibitor (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 can be used with steroids such as dexamethasone, proteasome inhibitors (such as bortezomib or carfilzomib), immunomodulators (such as thalidomide, lenalidomide or pomalidomide), Or other suitable such as administration of an induced chemotherapeutic agent, followed by treatment of the subject with an autologous hematopoietic stem cell transplant with or without other chemotherapeutic agents such as melphalan hydrochloride or the chemotherapeutic agents listed above. Can be administered in combination with appropriate therapy.

  For the treatment of Hodgkin's lymphoma, the anti-CD38 antibody-attenuated interferon alpha-2b fusion construct is treated with ABVD (adriamycin (doxorubicin), bleomycin, vinblastine, and decarbazine), or Stanford V (doxorubicin, bleomycin, vinblastine, vincristine, mechlorethamine, It can be administered in combination with current therapeutic approaches such as etoposide, prednisone) or BEACOPP (doxorubicin, bleomycin, vincristine, cyclophosphamide, procarbazine, etoposide, prednisone).

  In the case of non-Hodgkin's lymphoma or other lymphomas, an 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's lymphoma include Abitrexate (methotrexate), Adriamycin PFS (doxorubicin hydrochloride), Adriamycin RDF (doxorubicin hydrochloride), Ambochlorin (chlorambucil), Ambochlorin (chlorambucil), Arranon (nerarabin) , Bendamustine hydrochloride, Bexxar (tositumomab and iodine I131 tositumomab), Blenoxane (bleomycin), bleomycin, bortezomib, chlorambucil, Clafen (cyclophosphamide), cyclophosphamide, Cytoxan (cyclophosphamide), Denileukin Diftitt, DepoitCox Liposomal cytarabine), doxorubicin hydrochloride, DTIC-Dome (dacarbazine), Folex (methotrexate), Folex PFS (methotrexate), Folotyn (pralatrexate), ibritumomab tiuxetan, Istodax (romidepsin), Leukeran (chlorambucil L) (Chlorambu ), Liposomal cytarabine, Matulane (procarbazine hydrochloride), methotrexate, methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), Mozobil (prelyxafor), nelarabine, Neosar (cyclophosphamide), Ontak (denileukin diftitox), prilixaphor, pratrexate, Rituxan (rituximab), rituximab, romidepsin, tositumomab and iodine I131 tositumomab, Treanda (bendamustine hydrochloride), Velban (vinblastine sulphate), Velcade (volve) And Velsar (vinblastine sulfate), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vorinostat, Zevalin (ibritumomabtiuxetane), Zolinza (vorinostat). Examples of combination drugs used to treat non-Hodgkin's 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 an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct, and a tissue sample isolated from the 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 include 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's lymphoma cells, chronic myelogenous leukemia cells, chronic lymphocytic leukemia cells, or acute myeloid leukemia cells. The method may further include isolating the tissue sample from the subject.

  The 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 kits can be used to supply antibodies and other agents, particularly for use in diagnostics, 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, wherein the construct is optionally comprised in a composition comprising a pharmaceutically acceptable carrier. One or more methods for inhibiting or reducing cell proliferation, methods for inducing apoptosis in tumor cells expressing CD38, and / or cells expressing CD38 on the surface, And / or instructions for using the kit in a method for treating a tumor mediated thereby. Such a method may be any of the methods described or exemplified herein. The kit may include a pharmaceutically acceptable carrier. The kit may include pharmaceutically acceptable auxiliaries and / or one or more pharmaceutically acceptable excipients. In the kit, the anti-CD38 antibody can be any of the antibodies described or exemplified herein, and the attenuated interferon alpha-2b can be any of the 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 be in a sterile lyophilized form ready to be combined with a carrier immediately before 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 explain the present disclosure in more detail. They are intended to be illustrative of the present disclosure and not limiting.

Optimization of X355 / 02-HC-L0-IFN-alpha (A145D) IgG4 Other anti-CD38 antibody-attenuated IFN fusion proteins are described in PCT Application No. PCT / AU2012 / 001323. These include an antibody construct designated in this PCT application as X355 / 02-HC-L0-IFN-alpha (A145D) IgG4. As used 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 the 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 despite the fact that both antibodies share the same heavy and light chain sequences, X02 .1 does not include it.

A BLAST search against the human germline immunoglobulin gene database (Altschul SF (1997) Nucleic Acids Res. 25: 3389-3402) 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). The alignment of X02.1 VH and IGHV4-61 ^* 01 is shown in FIG. The X02.1 variable heavy chain region differs from its closest 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 be.

Some 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 (Table 2) and Table 1b (Table 3) detail the sequences of the variants tested along with their ability to bind human CD38 as 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 (Table 2) and Table 1b (Table 3).

The SPR binding of the variants detailed in Table 1a (Table 2) was evaluated separately from that of Table 1b (Table 3). The K _D (M) of the 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 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 (called A02, which when linked to IFN represents the same variant with the X02 designation. With decimal places). These heavy chain variable regions were formatted on an IgG4 constant region containing the S228P substitution fused to A145D attenuated IFN-alpha 2b and co-expressed in CHO or HEK cells with the A02.1 light chain described in Example 5. Was expressed. The successfully purified protein from the cell supernatant was 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. The 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, an attenuated IFN fusion protein could be expressed and purified if it was deemed more difficult to express and / or purify the equivalent monoclonal antibody.

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, described in FIG. This alignment illustrates a 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 the X02.1 variable heavy chain formatted on an IgG4 constant region containing an 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 (Table 5) are detailed 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 at D101 and a potential oxidation site at 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 these combinations of amino acid substitutions in the variable heavy chain as shown in Table 4. The antibody heavy chain variable region was formatted using an IgG4 constant region containing a S228P substitution and co-expressed with the A02.1 light chain in CHO cells. The antibodies were then purified by protein A chromatography and screened for binding to ARP-1 cells by flow cytometry. Table 4 (Table 6) shows the obtained binding data.

  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 Amino acid substitution 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 chain sequences of X02.1 and A02.1 identified amino acids that could potentially undergo oxidation or deamidation. These contained a potential deamidation site at N69 and a potential oxidation site at M89. In addition, a putative N-linked glycosylation site was predicted to exist within CDR3 of the light chain 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). Antibodies were purified by protein A chromatography and screened for binding to ARP-1 cells by flow cytometry. The resulting binding data is presented in Table 5 (Table 7).

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

  Other antibodies tested, including the X02.1 variable heavy chain variant, are listed in Table 6 (Table 8). These heavy chain variable regions were formalized on an IgG4 constant region containing a S228P substitution. These heavy chains were co-expressed with the A02.1 light chain in CHO cells. Antibodies were expressed and the resulting antibodies were tested for binding to CD38 positive cell ARP-1 in a flow cytometry based assay. With the exception of T23K (SEQ ID NO: 21; X02.68), all variable heavy chain substitutions had minimal effect on binding to the CD38-positive cell line ARP-1 in a flow cytometry-based assay.

  Antibodies containing 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 the X02.1 heavy chain formatted on an IgG4 constant region containing the S228P substitution. A summary of the heavy and light chains used to generate these antibody variants is provided in Table 7 (Table 9). Antibodies X02.83, X02.85, X02.91, X02.82 strongly bound to CD38-positive cell line ARP-1.

  Subsequently, substitutions with little effect on CD38 binding activity and purification of the X02 variant antibody 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 in HEK293E cells with point and combination variants of the X02.1 heavy chain, as listed in Table 8 (Table 10). These antibodies were primarily potential X02.1 light chain deamidation sites, oxidation sites from the CDR3 of the X02.1 heavy chain, and X02 predicted by in silico analysis (Epibase, Lonza, UK). The focus was on removing putative potent MHC class II binding peptides from framework region 3 of the single heavy chain. FIG.

  The antibodies listed in Table 8 were analyzed for protein expression and binding to CD38 by surface plasmon resonance (SPR). Also, potency assays were 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. The relative activity was evaluated. The data obtained is described in Table 9 (Table 11).

  Of the proteins tested, A02.12 was well expressed and showed efficacy in annexin V, caspase and cell proliferation assays. Substitution of N69T in antibody A02.47 did not affect expression levels or potency in annexin V or caspase assays, indicating that this deamidation site could be removed. The N69T substitution could be incorporated into other constructs described herein to eliminate 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 Putative immunogenic epitopes were identified in the light chain variable region amino acid sequence of A02.1 using Epibase analysis software (Lonza, UK). Substitutions were introduced in the A02.1 variable light chain to remove putative immunogenic epitopes (FIG. 4). The light chain with lower putative immunogenicity was expressed in HEK293E cells with the 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. At the same time. The antibody variants generated are detailed in Table 10.

  The antibodies were analyzed for protein expression, binding to CD38 by SPR, and for potency using cell culture supernatant screening as described in Example 5. The results of these assays are detailed in Table 11 (Table 13). These data show that annexin V, caspase and anti-CD38 antibodies with functional potency expressed in cell proliferation assays by substitution of several residues to reduce the predicted immunogenicity of the antibody-attenuated This shows that an IFN fusion protein can be obtained.

Multiple amino acid substitutions lead to optimization of A02.1 variants Highly optimized by combining substitutions that improve the immunogenicity, productivity or potency of the above anti-CD38 antibodies in a single genetic construct Anti-CD38 antibody and anti-CD38 antibody-attenuated IFN fusion protein were obtained. Table 12 summarizes such combinatorial substitutions and details the heavy and light chain combinations tested after co-expression in HEK293E cells.

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

Pairing of anti-CD38 antibodies of different heavy and light chains.To determine whether a functional anti-CD38 antibody-attenuated IFN fusion protein can be obtained, the antibody X910 / 12 according to PCT / AU2012 / 001323 -HC-L0-IFN-alpha (A145D) heavy chain (SEQ ID NO: 110) and light chain (SEQ ID NO: 112) derived from IgG4, and antibody X913 / 15-HC-L0-IFN according to PCT / AU2012 / 001323 -The heavy chain (SEQ ID NO: 111) and light chain (SEQ ID NO: 113) 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 generated was analyzed for protein expression, binding to CD38 by SPR, and 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 potency due to different heavy and light chain pairings from different antibodies Is obtained.

  A selection of the anti-CD38 antibody-attenuated IFN fusion proteins 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 obtain a more accurate determination of the relative activity of each anti-CD38 antibody-attenuated IFN fusion protein. The results of these assays are set forth in Table 15b.

  FIG. 5 lists the consensus variable heavy chains of A02.1 related constructs 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 (FIG. 6), X02.120, X02.121, X02.122, X02.123, X02.124 , X02.125, X02.126 or X02.127 (FIG. 30) can be further envisioned to be capable of making combinations of substitutions. In addition, the anti-CD38 antibodies described above could be constructed as anti-CD38 antibody-attenuated IFN fusion proteins 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.

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

Attenuated IFN is required for potent apoptotic activity and caspase activation in tumor cell lines Anti-CD38 antibody with strong apoptotic activity and caspase activation containing attenuated IFN using annexin V assay and caspase assay -It was shown to depend on the attenuated IFN fusion protein (Table 16a (Table 19), Figure 18). In the annexin V assay, the attenuated IFN-containing proteins (A02.1 and A02.6) had 2-fold higher activity than the 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 the protein constructs (antibodies and antibody-IFN-alpha2b related constructs) were prepared using a HiSpeed Plasmid Maxi kit (Qiagen, Valencia, CA), followed by a commercial transfection reagent and OptiMEM medium (Invitrogen (Carlsbad, Calif.), Transfected into HEK293E cells (CNRC, Montreal, Canada) and 0.45% (w / v) D-(+)-glucose (Sigma, Castle Hill, NSW) , 25 μg / mL of geneticin (Invitrogen, Carlsbad, Calif.), And 1 × GlutaMAX (Invitrogen, Carlsbad, Calif.). The 5% CO ₂ was supplied, after shaking expressed in an incubator for 6 days at 120 rpm, the culture medium is isolated, Protein A Mab Select SuRe (TM) agarose beads (GE Healthcare, Inc., Piscataway, NJ) and Affinity purification used. The purified protein construct was purified using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or a HiPrep 26/10 desalting column (HiTrap Desalting HiPrep 26/10 Desalting) using 0.2 M arginine HCl, 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, MA) and the protein concentration was determined by reading the absorbance at 280 nm.

Preparation of antibodies and antibody-fusion constructs in CHO cells DNA plasmids encoding protein constructs (antibody and antibody-IFN-alpha2b related constructs) are prepared using the HiSpeed Plasmid Maxi Kit (Qiagen, Valencia, CA) Then, using a commercially available transfection reagent and OptiPro SFMTM medium (Invitrogen, Carlsbad, CA), transfected into CHO cells (Lonza), and FreestyleTM CHO Expression Medium (Invitrogen, Inc. Carlsbad, CA). 10% CO ₂ supply, After shaking expressed in an incubator for 6 days at 120 rpm, the culture medium is isolated, Protein A Mab Select SuRe agarose beads (GE Healthcare, Inc., Piscataway, NJ) affinity using Purified. The purified protein construct was purified using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or a HiPrep 26/10 desalting column (HiTrap Desalting HiPrep 26/10 Desalting) using 0.2 M arginine HCl, 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, MA) 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 the anti-CD38 antibody and the anti-CD38 antibody-attenuated IFN fusion protein to bind to human CD38 was determined by using a non-specific binding reducing agent (GE Healthcare, Piscataway, NJ) in crude transfections prepared 7: 1. The measurement was performed using the supernatant of the cells thus obtained. Briefly, using Biacore® 3000 or T200, protein A is used to flow through a flow cell (FC) 1 (FC1) and FC2 (or alternatively FC3 and FC4) of a CM5 research grade sensor chip using amine coupling. ) When fixed on the 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 protein-containing supernatant 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 dissociation time of 5 minutes. Both surfaces were regenerated twice using sodium hydroxide. The results were generated using the 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 using CD38 injection from the sensorgram using blank running buffer injection. Protein A capture refers to the response units measured from the sensorgram at a fixed time of 412.5 s, which corresponds to the level of protein captured on the Protein A surface. CD38 binding is a response unit measured at 507.5 s and indicates the level of CD38 bound to the sensor that captured the protein. CD38 dissociation is a response unit measured at 865.5 s and indicates the level of CD38 bound to the surface that captured the protein approximately 300 s after the dissociation phase. Using BIAevaluation, the sensorgrams were fitted 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 x 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 215 nm or 280 nm wavelength were integrated using the manufacturer's software, the area under the curve (AUC) was reported and interpolated against a standard curve generated to estimate the concentration.

Flow cytometric binding of antibody and anti-CD38 antibody-IFN fusion protein to human CD38-positive cell lines ARP-1 and H929 Multiple myeloma cell line ARP-1 is Myeloma from the University of Arkansas Medical Center (Little Rock, AK) It was a gift from the director of the Institute, Bart Barlogie MD, phD. It is described in Hardin J. et al. (1994) Blood. 84: 3063-70. The 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, trypan blue dye exclusion determined by the test), and in the dark, on ice for 60 min, 50 [mu] L of FACS buffer in 96-well plates (PBS + 1% fetal calf serum, Incubation with various concentrations of each protein in FCS, 0.2 M HEPES, 0.5 M EDTA) or with human IgG4 monoclonal antibodies containing protein constructs of irrelevant specificity. After washing the cells three times with FACS buffer, 50 μL of FACS buffer containing goat anti-human IgG antibody (Fc-specific antibody conjugated to fluorescein isothiocyanate, FITC; Sigma-Aldrich, St. Louis, MO) For 30 minutes. After washing three times with FACS buffer, cells were fixed with 50 μL of PBS containing 4% formaldehyde v / v and incubated for 16 hours at 4 ° C. in the dark. The incubated cells in suspension are diluted with an additional 150 μL of FACS buffer and run on a FACS Canto II (BD Biosciences, San Diego, CA) using forward scatter, side scatter and fluorescence intensity in FITC channels. Analyzed for binding 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 cell viability 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) in 10% fetal bovine serum (FBS; T75 flasks (TPP, Trasadingen, Switzerland, catalog number 90076)). 0.5 × 10 ^{5 to} 0.8 × 10 ⁵ viable cells in RPMI 1640 (Mediatech, Inc., Manassas, VA, Cat.No. 10-040-CV) including Hyclone, Logan, UT, Cat.No.SH30070.03 / mL until the desired density was reached. The 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. The cells were then counted and the density adjusted to 3.0 × 10 ⁵ cells / mL in RPMI 1640 + 10% FBS. Then, 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). The test substance was serially diluted twice in RPMI 1640 + 10% FBS on another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number 3879). Then 50 μL / well was transferred from the dilution plate to the experimental plate. They were then incubated for 4 days experimental plates at 37 ° C. with 5% CO _2. Mix 100 μL / well of assay buffer supplied by the manufacturer with assay substrate [hereinafter referred to as “CellTiter-Glo® reagent”, mixed according to the manufacturer's instructions] Added to plate. The plate was shaken for 2 minutes.

  Then, 100 μL / well was 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 353296). The contents of the assay plate were then stabilized at room temperature for 15 minutes in the dark. The plate was read on a Victor 3V Multilabel Counter (Perkin Elmer, Waltham, Mass., Model number 1420-041) on the luminescence measurement channel and luminescence was measured. The results are presented as "relative light units" (RLU). The data was analyzed using Prism 5 (Graphpad, San Diego, CA) using non-linear regression and three 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 on 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 quantification of ATP. The signal intensity is proportional to the number of viable cells in the microtiter plate well.

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 bovine serum (FBS; AusGeneX, Molendinar, QLD, australia, catalog No. FBS500S) RPMI1640 (Life Technologies, Inc. including, cultured Mulgrave, VIC, until the catalog number 11875-093) in a preferred density of 2.0 × 10 ⁵ ~2.0 × 10 ⁶ viable cells / mL. The 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. The cells were then counted and the density adjusted to 2.0 × 10 ⁵ cells / mL in RPMI 1640 + 10% FBS. Then, 50 μL of the cell suspension was aliquoted into each well of a 96-well flat-bottom white opaque plate (hereinafter “experimental plate”) (Costar, Corning, NY Lakes, NJ, catalog number CLS3917). The test substance was serially diluted twice in RPMI 1640 + 10% FBS on another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number 3799). Then 50 μL / well was transferred from the dilution plate to the experimental plate. The experimental plate was then incubated at 37 ° C. with 5% CO ₂ for 4 days. 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 the 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 stabilized at room temperature for 15 minutes in the dark. The plate was read on a FLUOstar Galaxy plate reader (BMG Labtech, Durham, NC) on the luminescence measurement channel and luminescence was measured. Data was analyzed using Prism 5 (Graphpad, San Diego, Calif.) Using non-linear regression and three parameter curve fitting to determine the midpoint of the curve (EC ₅₀ ).

Annexin V assay: H929 cells were centrifuged at 400 xg for 5 minutes, the supernatant was discarded and the cell pellet was harvested by resuspension in RPMI 1640 + 10% FBS. The cells were then counted and the density adjusted to 1.0 × 10 ⁶ cells / mL in RPMI1640 + 10% FBS. Then, 50 μL of the cell suspension was aliquoted into each well of a 96-well round bottom transparent plate (hereinafter referred to as “experimental plate”; Costar, Corning, NY, catalog number CL3799). The test substance was diluted 4 times to 40 nM in RPMI 1640 + 10% FBS on another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number CL3799). Then 50 μL / well was transferred from the dilution plate to the experimental plate. The experimental plate was then incubated for 24 hours at 37 ° C. with 5% CO ₂ . The cells were then centrifuged at 400 × g for 5 min, the supernatant was discarded and resuspended in 100 μL of HEPES buffer containing Annexin V-FITC (1/200) and 7-AAD (1/50) did. The cells were then incubated at room temperature for 15 min before annexin by flow cytometry on forward scatter, side scatter, FACS Canto II (BD Biosciences, San Diego, CA) using FITC and PerCP-Cy5.5 channels. Analyzed for V and 7-AAD staining. Annexin V-positive cells refer to cells that stain positive with Annexin V-FITC after treatment with 20 nM of the 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 was treated with Homogeneous Caspases Assay reagent from Roche (West Sussex, UK) after treatment with test antibody, fluorescence, catalog number 12236869001. It measured using. The details of the assay are as follows.

ARP-1 cells expressing high levels of CD38 were cultured in T175 flasks (Costar, Corning, NY, catalog number CLS431080) with 10% FBS (AusGeneX, Molendinar, QLD, Australia, catalog number FBS500S) containing RPMI1640 ( Life Technologies, Inc., and cultured Mulgrave, VIC, until the catalog number 11875-093) in a preferred density of 2.0 × 10 ⁵ ~2.0 × 10 ⁶ viable cells / mL. Centrifuge cells at 400 xg for 5 minutes, discard supernatant and resuspend cell pellet in RPMI 1640 Phenol Red Free (Life Technologies, Mulgrave, VIC, Cat # 11835-030) + 10% FBS It was reaped. The 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 transparent bottom plate (hereinafter referred to as “experimental plate”; Costar, Corning, catalog number CLS3603). The test substance was diluted four times to 40 nM in RPMI 1640 Phenol Red Free + 10% FBS on another sterile 96-well plate (hereinafter referred to as “dilution plate”; Costar, Corning, NY, catalog number 3799). Then 50 μL / well was transferred from the dilution plate to the experimental plate. The experimental plate was 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". Then, 100 μL of the substrate solution was added to each well of the assay plate. The plate was shaken for 2 minutes. The plates were then incubated for 15 minutes at room temperature in the dark, and finally read on a FLUOstar Galaxy plate reader (BMG Labtech, Durham, NC) with a 470-500 nm excitation filter and a 500-560 nm emission filter, and read the fluorescence. Measure and present as fold change compared to untreated cells. 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, mostly with the addition of a human IgG blocking step, as described by the manufacturer. The iLite cell line is a `` stable transfected cell line derived from a commercially available promonocytic human cell line characterized by expression of MHC class II antigens, particularly human lymphocyte antigen (HLADR), on the cell surface. "As described by the manufacturer. This cell line contains a stably transfected luciferase gene, the expression of which is induced by an interferon response element (IRE) whose interferon activity can be quantified based on luminescence output. The iLite plate (hereinafter referred to as assay plate) and diluent supplied by the manufacturer 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. The vial of reporter cells supplied by the manufacturer was removed from the -80 ° C freezer and thawed in a 37 ° C water bath. A 25 μL aliquot of the 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 No. I4506) was added to each well. The contents were mixed and incubated at 37 ° C. for 15 minutes. The test substance was serially diluted twice in RPMI 1640 + 10% FBS on another “dilution plate”. Next, 12.5 μL of the test substance was transferred from the dilution plate to the assay plate. The assay plate was then incubated at 37 ° C. with 5% CO ₂ for 17 hours. Assay buffer and substrate supplied by the manufacturer 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". Then, 100 μL of the luminescence solution was added to each well of the assay plate. The plate was shaken for 2 minutes. The plates were then incubated for 5 minutes at room temperature in the dark and finally read on a luminescence measurement channel, on a Victor 3V Multilabel Counter, the luminescence was measured and presented as RLU. Data was 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 was expressed on iLite cells). (Test 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 was used to measure the interferon-alpha / beta receptor (IFNAR) using the HEK-BlueTM IFN-alpha / beta cell line (InvitroGen, San Diego, CA). ) Was quantified for its ability to bind to the antibody-IFN fusion construct. The "off-target (HB-IFN) assay" was performed as described mostly 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 created by introducing the 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, secreted embryonic alkaline phosphatase (SEAP), under the control of the ISG54 promoter. ISG54 is a well-known ISG that is activated via an ISRE-dependent mechanism by type I IFN. Upon IFN-alpha or IFNβ stimulation, HEK-Blue ™ IFN-alpha / β cells activate the JAK-STAT pathway and then SEAP reporter gene expression. SEAP is secreted into the medium and can be quantified using the colorimetric reagent QUANTI-Blue ™. Briefly, HEK-Blue IFN-alpha / beta cells (Invivogen, San Diego, CA, catalog number hkb-ifnab) were thawed and heat inactivated in 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% confluence, they were lifted using a Cell Stripper (Mediatech, Cat. No. 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 were aliquoted into flat-bottom 96-well tissue culture plates (hereinafter referred to as “experimental plates”). Then, 50 μL of IFN-alpha 2b or fusion protein construct, diluted in DMEM + HI FBS, was added to each well. Plates were incubated at 37 ° C., 5% CO ₂ for 16-24 hours. 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 per well from the experimental plate 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.

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

8-12 week old CB.17 SCID mice were injected subcutaneously in the flank with 1 × 10 ⁷ NCI-H929 tumor cells in 50% Matrigel ™. When the average tumor size reached 170～350Mm ^3, the mice were grouped into four cohorts were 7 mice, treatment was initiated at 0 hour (T0). All treatments were given by intraperitoneal injection (ip) twice a week for 3 weeks (indicated by bars below the graph). Except for the vehicle group, all compounds were administered at 100 μg / dose (about 4.5 mg / kg). Tumor volume was measured twice weekly by caliper measurement. Endpoint was the 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.

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

Anti-CD38 antibody-attenuated IFN fusion protein containing alternative constant region
A02.12 includes 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). Co-expression of this heavy chain with the light chain of X02.107 (SEQ ID NO: 65) in HEK293E cells yielded antibody A02.112. Comparison of antibodies A02.12 and A02.112 using a flow cytometry-based CD38 binding assay and potency assay shows that antibody-attenuated IFN with potent biological activity equivalent to that generated 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 are 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 canonical structure was assigned to each rodent heavy and light chain by inspection 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 / 1-2 was assigned. Human germline sequences of the same canonical structure were used as an acceptor framework for transplantation of donor CDRs. Variants of the resulting superhumanized antibody gene were also designed that contain amino acid substitutions at positions within the sequence that are likely to be important for the maintenance of avidity. Various heavy chain superhumanized 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 the vector pEE12.4 containing the human kappa constant region (SEQ ID NO: 5). Antibodies were generated in CHO cells by co-expression of the heavy chain in pEE6.4 and the light chain in pEE12.4 as previously described. Table 17 summarizes the heavy and light chain pairs used to generate each superhumanized 5D1-based protein. Table 18 details the heavy and light chain pairs for the generated superhumanized 5E8-based protein, but creates a superhumanized 10A2-based protein. The pairs of heavy and light chains used for this purpose are described in Table 19 (Table 23). One shot equilibrium dissociation constant (K _D ) ranking of the 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 to human CD38.

  For each family of humanized antibodies-5D1, 5E8, and 10A2, some humanized heavy and light chain combinations are unable to express the protein or bind human CD38. Did not. Significant numbers 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, which share 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
The A10.0 antibody was tested using a variable heavy and / or light chain to provide a positive effect on the biophysical and in silico immunogenicity of the antibody while minimizing the effect on the functional activity of the antibody Optimized by changes to sequence.

A10.0 Analysis of immunogenicity of heavy and light chains in silico
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 variants 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 by SPR for protein expression levels and binding to CD38. In addition, potency assays were performed using cell culture supernatants to evaluate 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 substituted N98Q was prepared to remove the deamidation site from the heavy chain CDR3. SEQ ID NO: 197. Additional variants of the A10.0 variable light chain containing the CDR2 substituted N53Q (SEQ ID NO: 198) were created to remove this putative deamidation site. Also, M89 within CDR3 of the light chain was altered by amino acid substitution at this position for the combined purpose of removing this potential oxidation site and reducing the predicted immunogenicity of this region of the light chain. Was. These substitutions are outlined in Table 22 with the heavy and light chain pairs co-expressed to generate the respective anti-CD38 antibody-attenuated IFN fusion protein.

  Each antibody generated using the heavy and light chain pairs outlined in Table 22 was evaluated by SPR for protein expression levels and binding to CD38. In addition, potency assays were performed using cell culture supernatants to evaluate 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 effect on functional activity, the optimized light chain sequence of A10.0 Paired with the antibody heavy chain. A summary of the heavy and light chain changes and pairs is provided in Table 24.

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

Attenuated IFN is required for potent apoptotic activity and caspase activation in tumor cell lines.The relative potency of anti-CD38 antibody 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 potencies of A10.38 and X10.38 were also compared. Table 26.

  These data indicate that the potent 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 without attenuated IFN.

  A consensus sequence alignment of the heavy chain variable region from a protein with functional activity is shown in FIG. A consensus sequence alignment of the light chain variable region from a protein with functional activity is shown in FIG. Anti-CD38 antibody 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 further be envisioned that combinations of substitutions such as those described for X10.147 can be made (FIGS. 11, 12). In addition, the anti-CD38 antibodies described above could be constructed as anti-CD38 antibody-attenuated IFN fusion proteins and tested for functional activity as described herein.

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

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 was determined by the iLite reporter. Evaluated in the genetic assay and / or HEK Blue assay and shown in FIGS. 28 and 29. EC ₅₀ values are provided in FIGS. 28 and 29. Off-target activity confirms the need for antibodies targeted to CD38 to restore attenuation and function of interferon.

Further in vitro potency data for A10.0 and related constructs A selection of the anti-CD38 antibody-attenuated IFN fusion proteins 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 of each of these assays are set forth in Table 27.

Anti-CD38 antibody-attenuated IFN fusion protein with alternative constant region
A10.0 includes 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), paired with the A10.0 light chain (SEQ ID NO: 161), and A10.59 Named. The protein was expressed and was found to be potent in functional assays (Table 28). Although most of the proteins tested in the above examples were constructed on human IgG4 constant regions, these data indicate that other antibody constant regions, such as human IgG1, can be used and the resulting antibody-attenuated IFN fusion constructs Has the same potent biological activity as the construct using the human IgG4 constant region.

  Table 29 (Table 33) lists the variable heavy, variable light 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).

  This disclosure is not limited to the embodiments described and illustrated above, but is capable of changes and modifications within the scope of the appended claims.

Claims (30)

  An antibody that specifically binds to CD38, comprising the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 665, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 666.   The heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 514, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 516, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 518, and the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 608. The antibody of claim 1, wherein the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 591, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 612.   A heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 156, and a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 185 3. The antibody of claim 1 or 2, wherein all amino acid substitutions in the heavy chain variable region or light chain variable region are outside of the CDR.   4. The antibody according to any one of claims 1 to 3, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 156, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 185.   5. The antibody according to any one of claims 1 to 4, wherein the antibody comprises a human IgG1 heavy chain constant region.   5. The antibody according to any one of claims 1 to 4, wherein the antibody comprises a human IgG4 heavy chain constant region.   7. The antibody of claim 6, wherein the human IgG4 heavy chain constant region comprises proline at position 228 according to the EU numbering system.   8. The antibody of claim 6 or 7, wherein the human IgG4 heavy chain 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.   9. The antibody according to any one of claims 1, 2 or 5 to 8, wherein the antibody is a humanized antibody.   A composition comprising the antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.   An antibody or claim according to any one of claims 1 to 9, for the treatment of B-cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia or acute myeloid leukemia. Item 11. The composition according to Item 10.   Use of the antibody according to any one of claims 1 to 9 or the composition according to claim 10 for the manufacture of a medicament for treating a tumor comprising a tumor cell that expresses CD38 on the surface. An agent for treating B-cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia or acute myeloid leukemia,
A humanized antibody that specifically binds to CD38, comprising the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 665, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 666, and attenuated. An agent comprising an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct , comprising the purified human interferon alpha-2b. The heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 514, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 516, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 518, and the light chain CDR1 comprises SEQ ID NO: 608. 14. The agent according to claim 13, wherein the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 591, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 612. The heavy chain variable region comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 156, and the light chain variable region comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 185. 15. The agent according to claim 13 or claim 14, wherein all amino acid substitutions in the heavy chain variable region or light chain variable region are outside the CDR. The agent according to any one of claims 13 to 15, wherein the antibody comprises a human IgG1 heavy chain constant region. 16. The agent according to any one of claims 13 to 15, wherein the antibody comprises a human IgG4 heavy chain constant region. 18. The agent of claim 17, wherein the human IgG4 heavy chain constant region comprises a proline at position 228 according to the EU numbering system. 19. The agent according to claim 17 or 18, wherein the human IgG4 heavy chain 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. 20. The agent according to any one of claims 13 to 19, wherein the attenuated human interferon alpha-2b comprises a substitution of alanine for aspartic acid at position 145 as compared to the amino acid sequence of SEQ ID NO: 648. 20. The agent according to any one of claims 13 to 19, wherein the attenuated human interferon alpha-2b comprises the amino acid sequence of SEQ ID NO: 647, SEQ ID NO: 649, SEQ ID NO: 650, or SEQ ID NO: 651. Use of an anti-CD38 antibody-attenuated interferon alpha-2b fusion construct for the manufacture of a medicament for treating a tumor comprising a tumor cell that expresses CD38 and a receptor for human interferon alpha-2b on its surface. ,
A humanized antibody that specifically binds to CD38, comprising the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 665, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 666, and attenuated. Use of a construct comprising the engineered human interferon alpha-2b.   The heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 514, the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 516, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 518, and the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 608. 23. The use of the construct of claim 22, wherein the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 591, and the light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 612.   The heavy chain variable region comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 156, and the light chain variable region comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 185. 24. Use of a construct according to claim 22 or claim 23, wherein all amino acid substitutions in the heavy or light chain variable region are outside of the CDRs.   25. Use of a construct according to any one of claims 22 to 24, wherein the antibody comprises a human IgGl heavy chain constant region.   25. Use of a construct according to any one of claims 22 to 24, wherein the antibody comprises a human IgG4 heavy chain constant region.   27. Use of a construct according to claim 26, wherein the human IgG4 heavy chain constant region comprises proline at position 228 according to the EU numbering system.   28. Use of a construct according to claim 26 or 27, wherein the human IgG4 heavy chain constant region comprises a tyrosine at position 252, a threonine at position 254, and glutamic acid at position 256 of the constant region according to the EU numbering system.   The construct of any one of claims 22 to 28, wherein the attenuated human interferon alpha-2b comprises an alanine to aspartic acid substitution at position 145 as compared to the amino acid sequence of SEQ ID NO: 648. use.   29. Use of a construct according to any one of claims 22 to 28, wherein the attenuated human interferon alpha-2b comprises the amino acid sequence of SEQ ID NO: 647, SEQ ID NO: 649, SEQ ID NO: 650 or SEQ ID NO: 651.