JP6853392B2 - Fusion with anti-CD38 antibody and attenuated interferon alfa-2B - Google Patents

Description

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

The present disclosure generally relates to the field of antibody engineering. More specifically, the present disclosure relates to antibodies that specifically bind to CD38, and constructs containing such antibodies with attenuated interferon-alpha ligands, and treatment methods 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, including patents, published applications, technical articles, academic articles, and gene or protein accession numbers, are cited throughout this specification. Each of these materials, in its entirety, is incorporated herein by reference in its entirety.

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, for example, with respect to CD38 expression, resting T and B cells may be negative, Most immature and activated lymphocytes may be positive. CD38 mRNA expression has been detected in non-hematopoietic organs such as the pancreas, brain, spleen and liver (Koguma, T. (1994) Biochim. Biophys. Acta 1223: 160).

CD38 is a multifunctional extra-functional enzyme 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+} migration within the cell, leading to tyrosine phosphorylation and cell activation. CD38 is also a receptor capable of interacting with the ligand CD31. CD31-mediated receptor activation ^{results in intracellular events such as Ca 2+} migration, cell activation, proliferation, differentiation and migration.

CD38 is at high levels in multiple myeloma cells, often T and B cells acute lymphoblastic leukemia, some acute myeloid leukemias, follicular center cell lymphomas and T lymphoblastic lymphomas It is expressed. CD38 is also expressed on B-cell chronic lymphoblastic leukemia (B-CLL) cells. In some cases, B-CLL patients presenting CD38 + clones are characterized by an unfavorable clinical course showing a more advanced disease stage, weaker responsiveness to chemotherapy and shorter survival. The use of antibodies against CD38 has been advocated for the treatment of CD38-expressing cancers and hematological malignancies. Therefore, it may be advantageous to provide an alternative antibody against CD38 that has the desired manufacturing, stability and immunogenicity properties.

It interacts with receptors on the cell surface, thereby stimulating, inhibiting, or otherwise regulating biological responses (usually including signaling pathways inside the cell carrying said receptor). Many peptides and polypeptide ligands that function by doing so have been reported. Examples of such ligands include peptides and polypeptide hormones, cytokines, chemokines, growth factors, and apoptosis-inducing factors.

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

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

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

These biological activities, when mediated and stimulated by type I interferon receptors on the surface of cancer cells, initiate various signaling pathways, reducing proliferation and / or inducing final differentiation or apoptosis. Bring. However, type I interferon receptors are also present on many non-cancerous cells. Activation of this receptor on non-cancerous cells by IFN-alpha causes the expression of some pre-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 a subject at levels that exert maximum antiproliferative and pre-apoptotic activity against cancer cells.

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

Published by Ozzello (Breast Cancer Research and Treatment 25: 265-76, 1993), Tumors are a means of slowing tumor growth by chemically conjugating human IFN-alpha to tumor-targeting antibodies. Described in the localization of the direct inhibitory activity of IFN-alpha, and showed that such conjugates have antitumor activity in a heterologous transplant model of human cancer. The mechanism of anticancer activity observed was that the human IFN-alpha used in the experiment did not interact imperceptibly with the mouse type I IFN receptor, which can provide indirect anticancer effects. It was due 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 antibody-IFN-alpha conjugate compared to free IFN-alpha was not assessed.

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

In general, IFN can be targeted to cancer cells. While this approach can result in increased activity of IFN on cancer cells, it does not completely address the problem of unwanted activity of IFN on healthy cells. Fusion of IFN-alpha to the C-terminus of the heavy chain of IgG can prolong the half-life of IFN alpha and lead to undesired adverse events. Therefore, it is necessary to reduce the off-target activity of ligand-based drugs while preserving the "on-target" therapeutic effect of the ligand.

WO01 / 97844 U.S. Pat. No. 7,732,578 U.S. Pat. No. 7,083,784 U.S. Pat. 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. Pat. No. 6,602,684 U.S. Pat. No. 7,326,681 U.S. Pat. No. 7,388,081 PCT publication number 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: pp. 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: pp. 3466-73 Labrin et al. (2009) Nature Biotechnology 27: 8; pp. 767-773 Altschul SF (1997) Nucleic Acids Res. 25: 3389-3402 Hardin J. et al. (1994) Blood. 84: 30 63-70 Gazdar, Blood 67: pp. 1542-1549, 1986

The disclosure features novel anti-CD38 antibodies and constructs containing anti-CD38 antibodies and attenuated IFN-alpha. Antibodies containing 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 cell surface. The antibody can be fused, for example, to an attenuated form of interferon alpha to form an anti-CD38 antibody-attenuated interferon fusion construct.

In some embodiments, 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, SEQ ID NO: 202, SEQ ID NO: 516, SEQ ID NO: containing the amino acid sequence of SEQ ID NO: 200, SEQ ID NO: 514 or SEQ ID NO: 697. Heavy chain CDR2 containing the amino acid sequence of 544, 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, SEQ ID NO: 235, SEQ ID NO: 307, SEQ ID NO: 311 containing the heavy chain CDR3 containing 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. , A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 585, SEQ ID NO: 591 or SEQ ID NO: 605, a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 237, SEQ ID NO: 321, SEQ ID NO: 324, SEQ ID NO: 587 or SEQ ID NO: 594. It 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, Amino acid sequences of 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 comprises 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: Includes the amino acid sequences of 709, SEQ ID NO: 710, SEQ ID NO: 711.

The antibody is preferably capable of binding to CD38 positive cells. The antibody can bind to CD38 positive cells with an EC50 value of less than about 100 nM. 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 antibody may be a monoclonal antibody, preferably a fully human antibody. The antibody may include FAb. The antibody may include a human IgG1 constant region or a human IgG4 constant region. The IgG1 or IgG4 constant region may contain 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 contain proline at position 228 according to the EU numbering system, and 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 alfa-2b. Interferon alfa-2b may include substitution of alanine at position 145 with glycine or aspartic acid, such as interferon alfa-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-terminal of the IgG1 or IgG4 constant region, and the antibodies are SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: It may include the amino acid sequence of No. 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, or SEQ ID NO: 694. Antibodies, such as antibodies fused to attenuated interferon alfa-2b, may be included in compositions containing a pharmaceutically acceptable carrier.

An isolated polynucleotide encoding an antibody and an antibody fused to attenuated interferon alfa-2b is provided. The polynucleotides are SEQ ID NO: 667, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 675, SEQ ID NO: 676, or SEQ ID NO: 677, SEQ ID NO: 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, SEQ ID NO: Number 718, SEQ ID NO: 719, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745 , The nucleic acid sequence of SEQ ID NO: 746 may be included. The polynucleotide may include a vector. The vector 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 cell expressing the antibody may be a mammalian cell. Preferred cells are Chinese hamster ovary (CHO) cells.

A kit containing an antibody fused to attenuated interferon alfa-2b is provided. This kit is for the use of anti-CD38 antibody-attenuated interferon alpha-2b fusion constructs and the constructs in methods for inhibiting the growth of tumor cells that express receptors for CD38 and interferon alpha-2b on their surface. Instructions, instructions for using the construct in methods for inducing apoptosis in tumor cells that express receptors for CD38 and interferon alpha-2b on the surface, CD38 and interferon alpha-in subjects who require it. Includes instructions for using the construct in methods for treating tumors containing cells expressing the receptor of 2b on the surface, and optionally includes pharmaceutically acceptable carriers. Kits containing anti-CD38 antibodies are provided, such kits are described for using anti-CD38 antibodies and said antibodies in methods for detecting CD38-positive tumor cells in tissue samples isolated from a subject. The antibody may optionally be fused to an attenuated interferon alfa-2b protein.

The anti-CD38 antibody-attenuated interferon alfa-2b fusion construct can be used as a therapy in the treatment of tumors containing cells expressing the receptors for CD38 and interferon alfa-2b on the surface. Generally, the treatment method comprises administering to the subject with the tumor an effective amount of an anti-CD38 antibody-attenuated interferon alfa-2b fusion construct to treat the tumor. The construct may include any construct described or exemplified herein. The subject is preferably a mammal, more preferably a non-human primat, 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 myelogenous leukemia.

Optionally, an anti-CD38 antibody fused to an attenuated interferon alfa-2b protein can be used in a method for detecting CD38 or CD38 positive tumor cells in tissue samples isolated from a subject. In general, the method involves contacting an antibody that specifically binds to CD38 with a tissue sample isolated from a subject, and detecting a complex of the antibody and CD38 in the tissue sample or CD38-positive cells. including. Tissue samples may be known or suspected of having CD38-positive tumor cells. Tissue may include blood or bone marrow. CD38-positive tumor cells may be CD38-positive B-cell lymphoma, multiple myeloma cells, non-Hodgkin's lymphoma cells, chronic myelogenous leukemia cells, chronic lymphocytic leukemia cells, or acute myelogenous leukemia cells. The subject is preferably a mammal, more preferably a non-human primat, and most preferably a human. The method may include the step of isolating the tissue sample from the subject. The method may further include contacting the antibody with, for example, a tissue sample free of CD38-positive cells serving as a negative control.

FIG. 5 shows an example of an anti-CD38 antibody-attenuated interferon fusion construct. It is a figure which shows the sequence of the heavy chain variable region of X02.1, the related construct, and the most homologous germline antibody sequence. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the sequence of the heavy chain variable region of X02.1, the related construct, and the most homologous germline antibody sequence. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the sequence of the light chain variable region of X02.1, a related construct, and the most homologous germline antibody sequence. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the sequence of the light chain variable region of X02.1, a related construct, and the most homologous germline antibody sequence. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the sequence of the light chain variable region of A02.1. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the sequence of the light chain variable region of A02.1. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the sequence of the light chain variable region of A02.1. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the sequence of the light chain variable region of A02.1. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the consensus variable heavy chain arrangement of A02.1. The enclosed area contains CDRs (as shown) as defined by Kabat's numbering system and enhanced Chothia's numbering system. The CDRs defined by Kabat's numbering system are shown in bold. The CDRs defined by the enhanced Chothia numbering system are underlined. It is a figure which shows the consensus variable light chain sequence of A02.1. The enclosed area contains CDRs (as shown) as defined by Kabat's numbering system and enhanced Chothia's numbering system. The CDRs defined by Kabat's numbering system are shown in bold. The CDRs defined by the enhanced Chothia numbering system are underlined. It is a figure which shows the arrangement of the heavy chain variable region of a humanized heavy chain variable region. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the arrangement of the heavy chain variable region of a humanized heavy chain variable region. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the arrangement of the heavy chain variable region of a humanized heavy chain variable region. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the arrangement of the heavy chain variable region of a humanized light chain variable region. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the arrangement of the heavy chain variable region of a humanized light chain variable region. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the arrangement of the heavy chain variable region of a humanized light chain variable region. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the variable heavy chain of A10.0 and the related construct. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the variable heavy chain of A10.0 and the related construct. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the variable light chain of A10.0 and the related construct. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the variable light chain of A10.0 and the related construct. The CDRs defined by Kabat's numbering system are underlined. It is a figure which shows the variable heavy chain consensus sequence of A10.0 and the related construct. The enclosed area contains CDRs (as shown) as defined by Kabat's numbering system and enhanced Chothia's numbering system. The CDRs defined by Kabat's numbering system are shown in bold. The CDRs defined by the enhanced Chothia numbering system are underlined. It is a figure which shows the variable heavy chain consensus sequence of A10.0 and the related construct. The enclosed area contains CDRs (as shown) as defined by Kabat's numbering system and enhanced Chothia's numbering system. The CDRs defined by Kabat's numbering system are shown in bold. The CDRs defined by the enhanced Chothia numbering system are underlined. It is a figure which shows the variable light chain consensus sequence of A10.0 and the related construct. The enclosed area contains CDRs (as shown) as defined by Kabat's numbering system and enhanced Chothia's numbering system. The CDRs defined by Kabat's numbering system are shown in bold. The CDRs defined by the enhanced Chothia numbering system are underlined. It is a figure which shows the binding activity of the A02.1 variant to the multiple myeloma cell line ARP-1 which expresses CD38 measured by the flow cytometry. Details of the assay are described in the examples herein. It is a figure which shows the binding activity of the A02.1 variant to the multiple myeloma cell line NCI-H929 which expresses CD38 measured by the flow cytometry. Details of the assay are described in the examples herein. It is a figure which shows the antiproliferative activity of A02.1 variant on multiple myeloma cell line ARP-1. The A-isotype is an antibody of unrelated specificity fused with attenuated interferon as a control. Details of the assay are described in Examples (Cell Proliferation Assay). It is a figure which shows the antiproliferative activity of A02.1 variant on multiple myeloma cell line ARP-1. The A-isotype is an antibody of unrelated specificity fused with attenuated interferon as a control. Details of the assay are described in Examples (Cell Proliferation Assay). The antiproliferative activity of IFN-alpha 2b (intron A) on multiple myeloma cell line ARP-1 compared to A02.1 and A10.0 and their corresponding non-fused antibodies X02.1 and X10.0. It is a figure which shows. The A-isotype is an antibody of unrelated specificity fused with attenuated interferon as a control. Details of the assay are described in Examples (Cell Proliferation Assay). In multiple myeloma cell line NCI-H929 expressing 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. It is a figure which shows the relative multiple change of the annexin V production in. The A-isotype is an antibody of unrelated specificity fused with attenuated interferon as a control. Details of the assay are described in Examples (Annexin V Assay). In the figure showing the relative multiple changes in caspase activation in multiple myeloma cell line H929 expressing IFN-alpha 2b (intron A) vs. A02.1 and related construct CD38 compared to untreated cells. is there. Isotype 145D is an antibody of unrelated specificity fused with attenuated interferon as a control. Details of the assay are described in the Examples (Caspase Assay). FIG. 5 shows off-target activity of IFN-alpha 2b (intron A) vs. 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 Examples [HEK-BLUE ™]. The figure shows the relative multiple changes in annexin V production in the CD38-expressing multiple myeloma cell line H929 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, whereas A02.112 and A10.59 contain an IgG1 constant region fused to attenuated IFN-alpha. Details of the assay are described in Examples (Annexin V / 7AAD Assay). It is a figure which shows the binding activity of the A10.0 variant to the multiple myeloma cell line NCI-H929 which expresses CD38 measured by the flow cytometry. Details of the assay are described in the examples herein. FIG. 5 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). It is a figure which shows the relative multiple change of the caspase activation in the multiple myeloma cell line H929 which expresses CD38 by the A10.0 variant compared with the untreated cell. Details of the assay are described in the Examples (Caspase Assay). It is a figure which shows the relative multiple change of the production of annexin V in the multiple myeloma cell line H929 which expresses CD38 by the A10.0 variant. Details of the assay are described in Examples (Annexin V / 7AAD Assay). It is a figure which shows the antiproliferative activity of IFN-alpha 2b (intron A) compared with A02.6, A10.0, A10.38 and parent A10A2.0 chimeric antibody constructs on Burkitt lymphoma cell line Daudi. The A-isotype is an antibody of irrelevant specificity fused to attenuated IFN as a control. Details of the assay are described in Examples (Cell Proliferation Assay). FIG. 5 shows the effect of humanized A10.0 vs. parental A10A2.0 chimeric antibody-attenuated interferon constructs on the growth of subcutaneous H929 myeloma tumors in SCID mice. Bars labeled "Treatment Phase" indicate the duration of treatment with the compound. It is a figure which shows the non-antibody antigen targeting 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 the parent A10A2.0 chimeric antibody [A10A2.0 chimera (wt.IFN)] fused to the A10.0 variant and wild-type IFN-alpha 2b. It is a figure. Details of the assay are described in the Examples [“Off-Target Assay”-HEK-BLUE ™]. It is a figure which shows the variable heavy chain consensus sequence of X910 / 12-HC-L0-interferon-alpha (A145D) and the related sequence. The enclosed area contains CDRs (as shown) as defined by Kabat's numbering system and enhanced Chothia's numbering system. The CDRs defined by Kabat's numbering system are shown in bold. The CDRs defined by the enhanced Chothia numbering system are underlined.

Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms should be given their usual meaning in the industry, unless otherwise noted. 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 are preferred, including companion mammals and livestock mammals, as well as rodents such as mice, rabbits, and rats, and other rodents. Non-human primates such as cynomolgus monkeys are more preferred, and humans are very preferred.

Molecules such as antibodies are "isolated" if they have been altered and / or removed from their natural environment by human hands.

As used herein, the singular forms "a", "an" and "the" include a plurality of referents unless otherwise explicitly stated.

Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs include, but are not limited to, interferon alfa-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 an attenuated interferon alfa-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 attenuates the biological activity of the interferon molecule. In the present disclosure, attenuated interferon, attenuated interferon alpha-2b, IFN-alpha 2b A145D, and IFN-alpha 2b A145G are used interchangeably.

Specificity need not be absolute, but may be a relative term indicating 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 antibody-IFN-alpha fusion protein constructs 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-fold specificity for antigen-positive cells as 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.

Furthermore, by introducing certain amino acid changes in its protein sequence, interferon-alpha 2b can be attenuated for its biological activity mediated by interferon that binds to cell surface interferon receptors. Observed. The attenuated interferon molecule is specifically bound to CD38 so that the antibody can serve as a delivery vehicle for attenuated interferon to CD38-positive cells where the reduction in off-target interferon activity caused by the attenuated interferon molecule is obtained. Can be fused to the antibody to be used. It was further observed that fusion of attenuated interferon to 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 an animal. Variants of the CD38 antibody are engineered so that the antibody has reduced immunogenicity and enhanced stability and half-life without significantly losing the antibody's specificity or affinity for the CD38 antigen. Can be expressed. These variant antibodies can be fused to attenuated interferon.

Therefore, 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 medium for attenuated ligands such as interferon alpha. Although not intended to be limited to any particular theory or mechanism of action, the antibody directs interferon alpha to bind them to CD38-positive cells, which interact with its receptor. It is thought that it can be done. 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 those that do not express CD38. By bringing attenuated interferon closer to its receptor on CD38-positive cells, the antibody exhibits its associated receptor while showing a reduced ability to induce unwanted effects on healthy cells that do not express CD38. It is thought that it can enhance the ability of attenuated interferon to bind to the body and induce therapeutic effects.

Antibodies may be polyclonal, but in some embodiments they are not. The antibody is preferably monoclonal. The antibody is preferably a full length antibody. Full-length antibodies generally include variable region heavy chains and variable region light chains. The antibody may comprise a derivative or fragment or portion of the antibody that retains antigen binding specificity and preferably retains much or all of the affinity of the parent antibody molecule (eg, for CD38). For example, the derivative may contain at least one variable region (either heavy chain or light chain variable region). Other examples of suitable antibody derivatives and fragments include, but are not limited to, antibodies with multiemetic specificity, bispecific antibodies, multispecific antibodies, diabodies, single-stranded molecules, and Fabs. F (Ab') 2, Fd, Fabc, and Fv molecules, single-stranded (Sc) antibodies, single-stranded Fv antibodies (scFv), individual antibody light chains, individual antibody heavy chains, antibody chains and other molecules Examples include fusions with, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy chain and one light chain, and other multimers. The single-strand Fv antibody may be multivalent. Any antibody isotype can be used to produce antibody derivatives, fragments, and moieties. Antibody derivatives, fragments, and / or moieties can be recombinantly produced and expressed in 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 the IFN-alpha is attenuated IFN-alpha 2b, then the isolated antibody is an anti-CD38 antibody-attenuated IFN-alpha fusion protein, or, as used herein, an anti-CD38 antibody-attenuated IFN-alpha fusion protein. Also called anti-CD38 antibody-attenuated IFN-alpha fusion construct.

In a full-length antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains 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 contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FWR or FR). Each VH and VL contains 3 CDRs and 4 FWRs arranged in the following order from amino terminus to carboxy terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. Typically, antibody binding properties are less likely to be disturbed by changes to FWR sequences than to changes to CDR sequences. The immunoglobulin molecule may be of any type (eg, IgG, IgE, IgM, IgD, IgA and Igγ), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

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

In some embodiments, the antibody is a humanized antibody. Humanized antibodies are of human origin in amino acids that are directly involved in antigen binding, such as the complementarity determining regions (CDRs), in some cases, framework regions (FWRs), or parts of heavy and / or light chains. Although not of human origin, the remaining amino acids in the antibody are human or otherwise of human origin, eg, human antibody scaffolding. Humanized antibodies are also non-human residues in which one or more residues of the human protein have been modified by one or more amino acid substitutions and / or one or more FWR residues of the human protein correspond. Also includes antibodies replaced by. Humanized antibodies may also contain residues not found in human or non-human antibodies. The humanized antibody may be, for example, a superhumanized antibody as described in US Pat. No. 7,732,578. The antibody may be a humanized chimeric antibody.

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

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

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

In some embodiments, the antibody may comprise a non-immunoglobulin-derived protein framework. For example, we describe a 4-helix bundle protein cytochrome b562 with two loops randomized to produce CDRs, selected for antigen binding, (Ku & Schutz, Proc. Natl. Acad. Sci. USA 92: pp. 6552-6556, 1995).

Natural sequence mutations may be present in the heavy and light chains as well as in the genes encoding them, and thus those skilled in the art will encode the amino acid sequences of the antibodies described and exemplified herein, or them. It can be expected to find some level of mutation in the gene. These variants preferably maintain the unique binding properties of the parent antibody (eg, specificity and affinity). Such conjectures are in part due to the degeneracy of the genetic code, as well as the known evolutionary success of conservative amino acid sequence mutations that do not change the properties of the encoded protein in a perceptible manner. Therefore, such variants and homologues are considered to be substantially identical to each other and are included within the scope of the present disclosure. Thus, an antibody comprises a variant having one or more amino acid substitutions, deletions, additions, or substitutions that retain the biological properties of the parent antibody (eg, binding specificity and binding affinity). Variants are preferably conservative, but may be non-conservative.

Amino acid positions assigned to CDRs and FWRs can be defined according to the 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, the amino acid positions assigned to CDRs and FWRs can be defined according to the enhanced Chothia numbering scheme (http://www.bioinfo.org.uk/mdex.html). The heavy chain constant region of an antibody can be defined by the EU numbering system (Edelman, GM et al. (1969) Proc. Natl. Acad. USA, 63, pp. 78-85).

According to Kabat's numbering system, VH FWR and CDR are 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), VL FWR and CDR are: Residues 1-23 (FWR1), 24-34 (CDR1), 35-49 ( It is positioned as 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, according to Kabat's numbering system, some amino acids can be specified by letters after the numbers. This specification is not limited to FWRs and CDRs defined by Kabat's numbering system, but 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: Standard numbering system on pp. 927-48; Honnegher et al. (2001) J. Mol. Biol., 309: 657-70. Numbering systems; or include any numbering system, such as the IMGT system discussed on pages 206-11 of Giudicelli et al. (1997) Nucleic Acids Res. In some embodiments, the CDR is defined according to Kabat's numbering system.

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

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

The antibody may contain post-translational modifications or moieties that may 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. Be done. The portion comprises any chemical group or combination of groups commonly found on immunoglobulin molecules, either naturally or otherwise added to the antibody by a recombinant expression system such as a prokaryotic and eukaryotic expression system. ..

Examples of side chain modifications contemplated by the present disclosure include reaction with aldehydes, followed _{by reductive alkylation by reduction with NaBH 4} ; amidation with methylacetimide; acylation with anhydrous acetic acid; amino groups with cyanate. Carbamoylation; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic acid anhydride; and lysine with pyridoxal-5-phosphate Modification of amino groups such as pyridoxylation followed _{by reduction with NaBH 4 can be mentioned.}

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

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

Carboxmethylation of sulfhydryl groups with iodoacetic acid or iodoacetamide; Performic acid oxidation to cysteine acid; Formation of mixed disulfides with other thiol compounds; Reactions with maleimide, maleic anhydride or other substituted maleimides; 4-chloromercly Formation of mercury derivatives with benzoic acid, 4-chloromercriphenylsulfonic acid, phenylmercury chloride, 2-chloromercri-4-nitrophenol and other mercury agents; modification by methods such as carbamoylation with cyanate at alkaline pH. Can be done.

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

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

_{Crosslinkers, such as homobifunctional crosslinkers such as bifunctional imide esters with (CH 2} ) n spacer groups n = 1 to n = 6, glutaaldehyde, N-hydroxysuccinimide esters, and usually N-hydroxy. 3D of antibodies and constructs using heterobifunctional reagents containing an amino-reactive moiety such as succinimide and another group-specific reactive moiety such as maleimide or dithio moiety (SH) or carbodiimide (COOH). The conformation can be stabilized.

The antibody may be affinity matured or may contain amino acid changes that reduce immunogenicity, for example by removing the expected MHC class II binding motif. The therapeutic utility of the antibodies described herein includes antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cellular cytotoxicity (CDC), serum half-life, biodistribution and binding to Fc receptors or these. It can be further enhanced by adjusting its functional features, such as a combination of any of the above. This regulation can be achieved by protein engineering, glycan engineering or chemical methods. It may be advantageous to increase or decrease any of these activities, depending on the therapeutic application required. Examples of glycan engineering used the Potelligent® method described in Shinkawa T. et al. (2003) J. Biol. Chem. 278: pp. 3466-73.

Antibodies are their serum, including modifications that regulate antibody interaction with the neonatal Fc receptor (FcRn), a receptor that protects IgG from catalysis and plays 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 include M252Y / S254T / T256E [EU Numbering System (Edelman, GM et al. (1969) Proc. Natl. Acad. USA 63, 78-85, as described in US Pat. No. 7,083,784. Numbered by (page)] may be present in the Fc region of IgG1 or IgG4, such as triple substitutions (eg, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 694). Other substitutions may be present at positions 250 and 428 (see, eg, US Pat. No. 7,217,797), and at positions 307, 380, and 434 (see, eg, WO 00/42072). .. Examples of constant domain amino acid substitutions that regulate subsequent functions mediated by these receptors, such as binding to Fc receptors and FcRn binding and serum half-life, are described in US Patent Application Publication Nos. 2009/0142340, 2009. It is described in / 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; pp. 767-773).

Glycans linked to antibody molecules are known to affect the interaction of antibodies with Fc and glycan receptors, thereby affecting antibody activity such as serum half-life. Thus, certain sugar types that regulate the desired antibody activity can provide therapeutic benefits. Methods for creating engineered sugar molds include, but are not limited to, those described in US Pat. Nos. 6,602,684, 7,326,681, and 7,388,081 and PCT Publication No. WO 08/006554. .. Alternatively, the antibody sequence can be modified to remove the associated sugar-type binding site.

The antibody can be labeled or conjugated to any chemical or biological molecular 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 protein cleavage or to enhance activity or stability.

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

The antibody may include a single chain Fv molecule (scFv), Fab, or full length IgG. Any such antibody is with the amino acid sequence of SEQ ID NO: 659 or SEQ ID NO: 665 or SEQ ID NO: 736, provided that the heavy chain containing the amino acid sequence of SEQ ID NO: 659 or a variant thereof does not contain the amino acid sequence of SEQ ID NO: 13. An amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity. It may contain a heavy chain having. It is understood that antibodies containing amino acid changes in the heavy chain retain the ability to specifically bind to CD38. The retained CD38-specific binding activity (including affinity) is preferably about the same as the binding activity (including affinity) of an antibody that does not contain amino acid changes in the heavy chain, but its binding activity (affinity). (Including sex) may be lower or higher than antibodies that do not contain amino acid changes in the heavy chain. The antibody is at least about 85%, at least about 90% of the amino acid sequence of SEQ ID NO: 664 or SEQ ID NO: 666, provided that the light chain containing the amino acid sequence of SEQ ID NO: 664 or a variant thereof does not contain 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% may include a light chain having an amino acid sequence having sequence identity. It is understood that antibodies containing 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 amino acid changes in the light chain, but its binding activity (affinity). (Including sex) 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 has 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, SEQ ID NO: 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 is SEQ ID NO: 199, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 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, Sequence At least about 85%, at least about 90%, at least about 92%, at least about 93% with the amino acid sequence of 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. Includes amino acid sequences having 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. 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, SEQ ID NO: In some embodiments, it comprises the amino acid sequence of SEQ ID NO: 543, SEQ ID NO: 548, SEQ ID NO: 553, SEQ ID NO: 558, SEQ ID NO: 563, SEQ ID NO: 568, SEQ ID NO: 573, SEQ ID NO: 578, SEQ ID NO: 749 or SEQ ID NO: 750. The 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, SEQ ID NO: 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, at least about 85%, at least about 90%, at least about 92 with the amino acid sequence of SEQ ID NO: 749 or SEQ ID NO: 750. %, 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% contains amino acid sequences having 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, SEQ ID NO: 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 is SEQ ID NO: 203, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 216, SEQ ID NO: 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 and SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 540, SEQ ID NO: 545, SEQ ID NO: 550, SEQ ID NO: At least about 85%, at least about 90%, at least about 92% of the amino acid sequence of 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. 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. In some embodiments, the heavy chain FWR4 has 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 is SEQ ID NO: 205, SEQ ID NO: 395, SEQ ID NO: 519, SEQ ID NO: 541, SEQ ID NO: 546, SEQ ID NO: At least about 85%, at least about 90%, at least about 92% with the amino acid sequence of 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, 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 containing 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 about the same as the binding activity (including affinity) of an antibody that does not contain amino acid changes in any heavy chain framework region. Its binding activity (including affinity) may be lower or higher than that of an antibody that does not contain amino acid changes in any heavy chain framework region.

In some embodiments, the heavy chain CDR1 is 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%, Alternatively, it contains an amino acid sequence having at least about 99% sequence identity. In some embodiments, the heavy chain CDR2 comprises SEQ ID NO: 202, SEQ ID NO: 392, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 406, SEQ ID NO: 410, SEQ ID NO: 414, SEQ ID NO: 418, SEQ ID NO: 422, SEQ ID NO: 426. , SEQ ID NO: 430, SEQ ID NO: 434, SEQ ID NO: 467, SEQ ID NO: 471, SEQ ID NO: 473, SEQ ID NO: 475, SEQ ID NO: 477, SEQ ID NO: 479, SEQ ID NO: 481, SEQ ID NO: 483, SEQ ID NO: 485, SEQ ID NO: 487, SEQ ID NO: No. 489, SEQ ID NO: 516, SEQ ID NO: 539, SEQ ID NO: 544, SEQ ID NO: 549, SEQ ID NO: 554, SEQ ID NO: 559, SEQ ID NO: 564, SEQ ID NO: 569, SEQ ID NO: 574, SEQ ID NO: 579, SEQ ID NO: 698 or SEQ ID NO: 737. In some embodiments, the heavy chain CDR2 comprises SEQ ID NO: 202, SEQ ID NO: 392, SEQ ID NO: 398, SEQ ID NO: 402, SEQ ID NO: 406, SEQ ID NO: 410, SEQ ID NO: 414, SEQ ID NO: 418, SEQ ID NO: Number 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: 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: At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% with the amino acid sequence of number 698 or SEQ ID NO: 737. , Includes an amino acid sequence having at least about 98%, or at least about 99% sequence identity. In some embodiments, the heavy chain CDR3 has 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 is SEQ ID NO: 204, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: At least about 85%, at least about 85% of the amino acid sequence of 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 with sequence identity Contains an array. It is understood that antibodies containing amino acid changes in the heavy chain complementarity determining regions (CDR1, CDR2, and / or CDR3) retain the ability to specifically bind to CD38. The retained CD38-specific binding activity (including affinity) is preferably about the same as the binding activity (including affinity) of an antibody that does not contain amino acid changes in any heavy chain complementarity determining region. , Its binding activity (including affinity) may be lower or higher than an antibody that does not contain amino acid changes in any heavy chain complementarity determining region.

In some embodiments, the light chain FWR1 comprises SEQ ID NO: 232, SEQ ID NO: 247, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 436, SEQ ID NO: 443, SEQ ID NO: 447, SEQ ID NO: 451 and 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, SEQ ID NO: Containing the amino acid sequence of SEQ ID NO: 630, SEQ ID NO: 634 or SEQ ID NO: 638, in some embodiments, the light chain FWR1 is 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: At least about 85%, at least about 90%, at least about 92%, at least about 93% with the amino acid sequence of number 614, SEQ ID NO: 618, SEQ ID NO: 622, SEQ ID NO: 626, SEQ ID NO: 630, SEQ ID NO: 634 or SEQ ID NO: 638. Includes amino acid sequences having 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. 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 and 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: 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: Number 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, At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% with the amino acid sequence of number 635 or SEQ ID NO: 639. , Includes an amino acid sequence having at least about 98%, or at least about 99% sequence identity. In some embodiments, the light chain FWR3 contains 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: Number 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 323, SEQ ID NO: 327 , SEQ ID NO: 331, SEQ ID NO: 335, SEQ ID NO: 339, SEQ ID NO: 343, SEQ ID NO: 347, SEQ ID NO: 351, SEQ ID NO: 355, SEQ ID NO: 359, SEQ ID NO: 363, SEQ ID NO: 367, SEQ ID NO: 371, SEQ ID NO: 375, SEQ ID NO: 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, 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. In some embodiments, the light chain FWR3 contains 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: Number 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 323, SEQ ID NO: 327 , SEQ ID NO: 331, SEQ ID NO: 335, SEQ ID NO: 339, SEQ ID NO: 343, SEQ ID NO: 347, SEQ ID NO: 351, SEQ ID NO: 355, SEQ ID NO: 359, SEQ ID NO: 363, SEQ ID NO: 367, SEQ ID NO: 371, SEQ ID NO: 375, SEQ ID NO: Number 3 79, 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, At least about the amino acid sequence of 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 SEQ ID NO: 640. 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 at least about 99% sequences Contains amino acid sequences with identity. In some embodiments, the light chain FWR4 comprises SEQ ID NO: 238, SEQ ID NO: 442, SEQ ID NO: 446, SEQ ID NO: 450, SEQ ID NO: 454, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 496, SEQ ID NO: 500, SEQ ID NO: 504. , SEQ ID NO: 508, SEQ ID NO: 512, SEQ ID NO: 588, SEQ ID NO: 613, SEQ ID NO: 617, SEQ ID NO: 621, SEQ ID NO: 625, SEQ ID NO: 629, SEQ ID NO: 633, SEQ ID NO: 637 or SEQ ID NO: 641. In some embodiments, the light chain FWR4 comprises SEQ ID NO: 238, SEQ ID NO: 442, SEQ ID NO: 446, SEQ ID NO: 450, SEQ ID NO: 454, SEQ ID NO: 458, SEQ ID NO: 462, SEQ ID NO: 496, SEQ ID NO: 500, SEQ ID NO: 504. , SEQ ID NO: 508, SEQ ID NO: 512, SEQ ID NO: 588, SEQ ID NO: 613, SEQ ID NO: 617, SEQ ID NO: 621, SEQ ID NO: 625, SEQ ID NO: 629, SEQ ID NO: 633, SEQ ID NO: 637 or at least the amino acid sequence of SEQ ID NO: 641. 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 at least about 99% Includes amino acid sequences with sequence identity. It is understood that antibodies containing 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 about the same as that of an antibody that does not contain amino acid changes in any light chain framework region (including affinity), although Its binding activity (including affinity) may be lower or higher than that of an antibody that does not contain amino acid changes in any light chain framework region.

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, Array Containing the amino acid sequence of SEQ ID NO: 381, SEQ ID NO: 385, SEQ ID NO: 437, SEQ ID NO: 491, SEQ ID NO: 583, SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 608, or SEQ ID NO: 696, in some embodiments light chain CDR1. Is SEQ ID NO: 233, SEQ ID NO: 250, SEQ ID NO: 525, SEQ ID NO: 255, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 319, SEQ ID NO: 322, SEQ ID NO: 325, SEQ ID NO: 329, SEQ ID NO: 333, SEQ ID NO: 337, SEQ ID NO: 341, SEQ ID NO: 345, SEQ ID NO: 349, SEQ ID NO: 353, SEQ ID NO: 357, SEQ ID NO: 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: At least about 85%, at least about 90%, at least about 92%, at least about 93% with the amino acid sequence of 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. Includes amino acid sequences having 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. 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, SEQ ID NO: 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: 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: At least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94 with the amino acid sequence of No. 493, SEQ ID NO: 585, SEQ ID NO: 591, SEQ ID NO: 605, SEQ ID NO: 610 or SEQ ID NO: 747. %, At least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% contains an amino acid sequence having 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, SEQ ID NO: Number 360, SEQ ID NO: 364, SEQ ID NO: 368, SEQ ID NO: 372, SEQ ID NO: 376, SEQ ID NO: 380, SEQ ID NO: 384, SEQ ID NO: 388, SEQ ID NO: 441, SEQ ID NO: 495, SEQ ID NO: 587, SEQ ID NO: 594, SEQ ID NO: 595 , SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 606 or SEQ ID NO: 612. In some embodiments, the light chain CDR3 comprises SEQ ID NO: 237, SEQ ID NO: 244, SEQ ID NO: 251, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 268, SEQ ID NO: 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: Number 360, SEQ ID NO: 364, SEQ ID NO: 368, SEQ ID NO: 372, SEQ ID NO: 376, SEQ ID NO: 380, SEQ ID NO: 384, SEQ ID NO: 388, SEQ ID NO: 441, SEQ ID NO: 495, SEQ ID NO: 587, SEQ ID NO: 594, SEQ ID NO: 595 , SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 606 or at least the amino acid sequence of SEQ ID NO: 612. 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 at least about 99% Includes amino acid sequences with sequence identity. It is understood that antibodies containing amino acid changes in the light chain complementarity determining regions (CDR1, CDR2, and / or CDR3) retain the ability to specifically bind to CD38. The retained CD38-specific binding activity (including affinity) is preferably about the same as the binding activity (including affinity) of an antibody that does not contain amino acid changes in any light chain complementarity determining region. , Its binding activity (including affinity) may be lower or higher than an antibody that does not contain amino acid changes in any light chain complementarity determining region.

In some embodiments, the antibody comprises a particular heavy and light chain pair. 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 paired with the amino acid sequence of SEQ ID NO: 666. It can be paired with any light chain that has, or a heavy chain that has the amino acid sequence of SEQ ID NO: 736 can be paired with any light chain that has the amino acid sequence of SEQ ID NO: 664.

The variable heavy chain and variable light chain pair may include a pair derived from the table below.

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

In some embodiments, the fusion of the antibody to the interferon ligand and the inclusion of the attenuated mutation in the interferon ligand are greater than 10-fold, preferably greater than 50-fold, as compared to the antibody without the fusion. Increases the antigen specificity index (ASI), preferably greater than 100-fold, preferably greater than 1000-fold, or preferably greater than 10,000-fold. ASI is an antibody-IFN compared to a free non-mutated polypeptide ligand on a target antigen-positive cell, multiplied by a multiple reduction effect of signaling activity compared to a free non-mutated polypeptide ligand on a target antigen-negative cell. Includes the potency of increasing 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 The efficacy can be expressed quantitatively. Thus, typically, when measured by the same method, for example, the first compound is _{expressed in EC 50} (eg, molar concentration units) that is 10 times lower than _{the EC 50 of the second compound on the same cell.} The first compound is said to be 10 times more potent if it is shown to have. Conversely, typically when measured by the same method, when the first compound shown to have a 10-fold higher EC ₅₀ than EC ₅₀ for the second compound on the same cell, the first Compounds are said to have 10-fold lower potency.

The antibody is 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.

An antibody-linked interferon 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. Includes changes in its amino acid sequence, including. A highly preferred variant of interferon alpha comprises an amino acid change at position 168 of the interferon alfa 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 converted to glycine (Gly / G) (SEQ ID NO: 650) or aspartic acid (Asp / D) (SEQ ID NO: 647). To. 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 human IgG1 or human IgG4 heavy chain constant domain. The truncated IFN-alpha 2b does not have the 23 N-terminal amino acids of SEQ ID NO: 7 (Met1 to Gly23 are deleted) and the truncated IFN-alpha 2b contains the amino acid sequence of SEQ ID NO: 648. .. The truncated IFN-alpha 2b may also contain an amino acid change that was previously at position 168 but is now at position 145 in the truncated protein (eg, alanine 168 becomes alanine 145). In the truncated IFN-alpha 2b, alanine is preferably converted to glycine (Gly / G) (SEQ ID NO: 651) or aspartic acid (Asp / D) (SEQ ID NO: 649). Interferon with A145D alteration (SEQ ID NO: 647 or SEQ ID NO: 649) is particularly preferred as an attenuated ligand fused to the antibodies of the present disclosure. Any of these point-mutated, attenuated versions of IFN-alpha can be linked to any of the antibodies described herein, eg, as an antibody-attenuated interferon construct.

The linkage between the antibody and interferon preferably comprises a peptide bond between the fusion, eg, the N-terminus of interferon and the N-terminus of the heavy or light chain of the antibody. In a highly preferred embodiment, there is no linker between the antibody and interferon, thus the antibody and interferon are directly fused. A direct fusion without the interlingering linker peptide is believed to provide at least measurable attenuation of the interferon protein, and this attenuation is described or exemplified herein for the interferon protein. It is also thought to be additive with the attenuation of the interferon protein resulting from the mutation introduced into it.

The polynucleotide sequences encoding the antibody and its subdomains (eg, FWR and CDR) are characterized in the present disclosure. Polynucleotides include, but are not limited to, RNA, DNA, cDNA, RNA-DNA hybrids, and RNA, DNA, or single-stranded, double-stranded, or triple-stranded hybrids thereof.

In some embodiments, the polynucleotide encodes the heavy chain of an antibody that specifically binds to an epitope on CD38. The polynucleotides are SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO:, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 695. , SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745 or SEQ ID NO: 746. A heavy chain containing the amino acid sequence may be encoded. The polynucleotides are 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 either the amino acid sequence of SEQ ID NO: 718 or SEQ ID NO: 719 may be encoded. The polynucleotides are SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO:, 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: 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 SEQ ID NO: It may contain any of the nucleic acid sequences of number 719. The polynucleotides are SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO:, 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: 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 SEQ ID NO: At least about 80%, at least about 85%, 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% with any of the numbers 719 , 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% of nucleic acid sequences having sequence identity. In that embodiment, such variants are preferably 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: 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, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, Sequence 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 , It encodes the same amino acid encoded by the polynucleotide sequence of SEQ ID NO: 717, SEQ ID NO: 718 or SEQ ID NO: 719. Preferably, the antibody encoded by the polynucleotide variant specifically binds to CD38 with an affinity approximately equal to that of the antibody encoded by the parent (non-variant) polynucleotide sequence. Affinity can be measured according to any technique described or exemplified herein, for example, the technique described in the Examples. Polynucleotide sequences and complements of variant polynucleotide sequences 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, a recombinant expression vector containing a sequence encoding the polypeptide of interest is provided. The expression vector may contain one or more additional sequences, such as, but not limited to, regulatory sequences, selectable markers, purification tags, or polyadenylation signals. Such regulatory elements may include transcriptional promoters, enhancers, mRNA ribosome binding sites, or sequences that control transcription and translation termination.

Expression vectors, especially mammalian expression vectors, are replication origins, suitable promoters and enhancers linked to the gene to be expressed, other 5'or 3'flanking non-transcriptional sequences, 5'or 3'untranslated. It may contain one or more non-transcriptional elements such as sequences (such as essential ribosome binding sites), polyadenylation sites, splice donor and acceptor sites, or transcription termination sequences. It is also possible to incorporate an origin of replication that imparts the ability to replicate in a particular host.

The vector can be used to transform any various array of host cells well known to those of skill in the art, preferably host cells 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 generated stable eukaryotic cell line, including bacterial, 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 antiferon alpha ligand having one or more mutations that substantially reduces the ligand's affinity for the interferon receptor targets the mutant interferon alpha ligand against target cells that represent the corresponding antigen of the antibody. It was observed according to the present disclosure that when linked to the CD38 antibody, the activity of the ligand against target antigen-positive cells is maintained, but the activity of the ligand against non-target antigen-negative cells is substantially reduced. Net results are much more potent in activating its receptor on antigen-positive target cells compared to antigen-negative non-target cells, which provide a means for reducing the toxicity resulting from off-target ligand activity. It 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 IFN antiproliferative activity against CD38-positive tumor cells with high potency, while exhibiting much lower potency against CD38-negative non-tumor cells in the body.

The present disclosure also provides a composition comprising the antibodies of the present disclosure and an antibody-attenuated interferon construct. These compositions are, but are not limited to, one or more diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or other suitable carriers and / or It may further comprise at least one suitable auxiliary agent, such as an excipient. Pharmaceutically acceptable auxiliaries are preferred. The composition may include any antibody described and / or exemplified herein, an antibody-attenuated interferon construct, and an acceptable carrier, such as a pharmaceutically acceptable carrier. Suitable carriers include any medium 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, brine, or alcohol, or an aqueous solution such as a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or saline buffer. The carrier may contain a formulation agent such as a suspending agent, a stabilizer and / or a dispersant.

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

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

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

Further, the compositions of the present disclosure include polyvinylpyrrolidone, ficol (polymeric sugar), dextrin (eg, cyclodextrin such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, antimicrobial agents, antioxidants, charged. Inhibitors, surfactants (eg, polysorbates such as "TWEEN® 20" and "TWEEN® 80"), lipids (eg, phospholipids, fatty acids), steroids (eg, cholesterol), and chelates. It may contain a polymeric excipient / additive such as an agent (eg, EDTA).

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

The composition can be formulated for administration to the 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 medium, either with an adjuvant or as a depot formulation.

Preparations for parenteral administration include sterile dry soluble products that are ready to be combined with a solvent immediately prior to use, such as sterile solutions ready for injection, subcutaneous injection tablets, and sterile ready to be injected. Examples include suspensions, sterile dry insoluble products that are ready to be combined with the medium immediately prior to use, and sterile emulsions.

Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs can be used, for example, to inhibit, reduce, reduce, block, or prevent the growth of cells expressing CD38 on the surface. In some embodiments, methods for inhibiting or reducing the growth of cells expressing CD38 on the surface are generally effective in inhibiting or reducing the growth of cells expressing CD38 and the cells. Includes contact with a significant amount of anti-CD38 antibody-attenuated interferon alfa-2b fusion construct. The antibody that specifically binds to CD38 may be any antibody described or exemplified herein. The attenuated interferon alpha 2b may include 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. Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs are, for example, pharmaceutically acceptable carriers and optionally any such carrier, adjunct, or excipient described or exemplified herein. May be included in the composition with one or more auxiliaries or excipients such as. The method can be performed in vitro, ex vivo, in vivo, or in situ.

Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs can also be used, for example, to induce, facilitate, or enhance apoptosis in cells expressing CD38 on the surface. In some embodiments, methods for inducing apoptosis in cells that express CD38 on the surface generally include CD38-expressing cells and an amount of anti-CD38 antibody-attenuated that is effective in inducing apoptosis in the cells. Includes contact with interferon alfa-2b fusion constructs. The antibody that specifically binds to CD38 may be any antibody described or exemplified herein. The attenuated interferon alpha 2b may include 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. Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs are, for example, pharmaceutically acceptable carriers and optionally any such carrier, adjunct, or excipient described or exemplified herein. May be included in the composition with one or more auxiliaries or excipients such as. The method can be performed in vitro, ex vivo, in vivo, or in situ.

Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs can also be used to treat subjects containing cells expressing CD38 on the surface and / or having tumors mediated by it, at least in part. it can. In some embodiments, methods for treating tumors containing cells that express CD38 on the surface generally include an amount of anti-CD38 antibody that is effective in treating the tumor in the subject in need of it. Includes the step of administering an attenuated interferon alfa-2b fusion construct. Effective treatment may include, for example, inhibiting or reducing the proliferation of CD38-positive cells in the tumor and / or inducing apoptosis of the CD38-positive cells in the tumor. The antibody that specifically binds to CD38 may be any antibody described or exemplified herein. The attenuated interferon alpha 2b may include IFN-alpha 2b A145D or IFN-alpha 2b A145G. Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs are, for example, pharmaceutically acceptable carriers and optionally any such carrier, adjunct, or excipient described or exemplified herein. May be included in the composition with one or more auxiliaries or excipients such as.

Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs or compositions containing such constructs can be administered to the tumor by administering the construct of the composition to the blood. Anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs or compositions containing such constructs can be administered such that the constructs diffuse through the bloodstream and / or into tumor cells. The construct may be internalized by tumor cells.

The use of anti-CD38 antibody or anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs in the treatment of tumors is provided. A method for treating a tumor using an anti-CD38 antibody or an anti-CD38 antibody-attenuated interferon alfa-2b fusion construct is provided. Any anti-CD38 antibody or anti-CD38 antibody-attenuated interferon alfa-2b fusion construct described or exemplified herein can be used. Tumors that can be treated include, but are not limited to, AIDS-related cancers, acoustic neuromas, acute lymphocytic leukemias, acute myeloid leukemias, adenocarcinomas, adrenocortical cancers, idiopathic myelination, Alopecia, follicular soft sarcoma, anal cancer, angiosarcoma, malregenerative anemia, stellate cell tumor, capillary diastolic dyskinesia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestine Cancer, brain stem glioma, brain tumor and CNS tumor, breast cancer, CNS tumor, cartinoid 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, elevated cutaneous fibrosarcoma, fibrogenic small round cell tumor, ductal carcinoma, endocrine cancer, endometrial cancer, coat tumor, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinal blastoma, oviduct cancer, fanconi anemia, fibrosarcoma, bile sac cancer, gastric cancer, gastrointestinal cancer, gastrointestinal tract Cartinoid tumor, urogenital cancer, embryonic cell tumor, gestational chorionic villus disease, glioma, gynecological cancer, hematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histocyte proliferation Disease, Hodgkin's disease, human papillomavirus, spore-forming miraculous, hypercalcemia, hypopharyngeal cancer, intraocular melanoma, islet cell cancer, capsicum sarcoma, kidney cancer, Langerhans cell histocytoproliferative disease, laryngeal cancer, smoothing Myoma, leukemia, Lee Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, male breast cancer, malignant labdoid tumor of the kidney, myeloma, melanoma, Merkel cell carcinoma, mesenteric tumor, metastatic cancer, oral cancer, multiple endocrine adenomas, mycobacterial sarcoma, myelodystrophy syndrome, multiple myeloma, myeloproliferative disorder, nasal cavity cancer, nasopharyngeal cancer , Renal blastoma, Neuroblastoma, Neurofibromatosis, Naimihen chromosomal instability syndrome, Non-melanoma skin cancer, Non-small cell lung cancer (NSCL)
C), eye cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cavity cancer, parathyroid cancer, parotid gland Cancer, Penile Cancer, Peripheral Neuroectodermal Tumor, Hydrus Cancer, True Erythrocytosis, Prostate Cancer, Rare Cancers and Related Disorders, Renal Cell Cancer, Retinal Memoroma, Lacterial Myoma, Rotomund Thomson syndrome, salivary adenocarcinoma, sarcoma, nerve sheath tumor, cesarly syndrome, skin cancer, small cell lung cancer (SCLC), small bowel cancer, soft tissue sarcoma, spinal cord tumor, squamous epithelial cancer (skin), gastric cancer (stomach cancer) , Luminous sarcoma, testicular cancer, thoracic adenocarcinoma, thyroid cancer, transition epithelial cancer (bladder), transition epithelial cancer (renal tract / urinary tract), trohoblast cancer, urinary tract cancer, urinary system cancer, uroplakin (uroplakin), uterine sarcoma, uterine cancer, vaginal cancer, genital cancer, Waldenstrem macroglobulinemia and Wilms tumor. 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 where 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 myelogenous leukemia. be able to.

The anti-CD38 antibody-attenuated interferon alfa-2b fusion construct can be used in combination with other drugs and / or in addition to other cancer treatment regimens or modality such as radiation therapy or surgery. When the anti-CD38 antibody-attenuated interferon alfa-2b fusion construct is used in combination with a known therapeutic agent, the combination may be used in sequence (continuously or in divided periods without treatment), simultaneously or simultaneously. It can be administered as a mixture. In the case of cancer, there are several known anti-cancer agents that can be used in this context. Combination treatments also include, for example, as maintenance therapy, treatment with anti-CD38 antibody-attenuated interferon alfa-2b fusion construct, followed by treatment with known or known agents, followed by anti-CD38 antibody-attenuated interferon alpha-. It is also intended to include treatment with 2b fusion constructs. For example, in the treatment of cancer, anti-CD38 antibody-attenuated interferon alpha-2b fusion constructs can be used with alkylating agents (mechloretamine, cyclophosphamide, chlorambusyl, ifosphamide cisplatin, or cisplatin, carboplatin and oxaliplatin, etc. Platinum-containing alkylating agents, etc.), metabolic antagonists (such as purines or pyrimidine analogs or antifolic agents such as azathiopurine and mercaptopurine), anthracyclines (daunorubicin, doxorubicin, epirubicin, idalbisin, balrubicin, mitoxanthrone, or anthracyclines) Cycline analogs, etc.), plant alkaloids (such as binca alkaloids or vincristine, vinblastine, binolerubin, bindecin, taxan such as paclitaxel or docetaxel), topoisomerase inhibitors (such as type I or type II topoisomerase inhibitors), podophylrotoxins (etopocid) Alternatively, it is contemplated that it can be administered in combination with a tyrosine kinase inhibitor (such as imatinib mesylate, nirotinib, or dasatinib).

For the treatment of multiple myeloma, anti-CD38 antibody-attenuated interferon alfa-2b fusion constructs, steroids such as dexamethasone, proteasome inhibitors (such as bortezomib or calfilzomib), immunomodulators (such as salidamide, lenalidomide or pomalidemid), Alternatively, administration of an inducible chemotherapeutic agent, followed by other chemotherapeutic agents such as melphalan hydrochloride or other suitable treatments such as treatment of the subject by autologous hematopoietic stem cell transplantation with or without the chemotherapeutic agents listed above. It can be administered in combination with various therapies.

For the treatment of hodgkin lymphoma, anti-CD38 antibody-attenuated interferon alpha-2b fusion construct, ABVD (adriamycin (doxorubicin), bleomycin, vinblastine, and decarbazine), or Stanford V (doxorubicin, bleomycin, vinblastine, vincristine, mechloretamine, mechroletamine, It can be administered in combination with current therapeutic approaches such as etopocid (prednison) or BEACOPP (doxorubicin, bleomycin, vincristine, cyclophosphamide, procarbazine, etopocid, prednison).

For non-Hodgkin's lymphoma or other lymphomas, an anti-CD38 antibody-attenuated interferon alfa-2b fusion construct can be administered in combination with current therapeutic approaches. Examples of drugs approved for non-hosphamide lymphoma include Abitrexate, Adriamycin PFS, Adriamycin RDF, Ambochlorin, Ambochlorin, Arranon. , Bendamstin Hydrochloride, Bexxar (Tocitumomab and Iedone I131 Toshitumomab), Blenoxane (Blenoxane), Breomycin, Voltezomib, Chlorambusil, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Denileukin Diftitox, DepoC Liposomal citarabin), doxorubicin hydrochloride, DTIC-Dome (dacarbazine), Folex (methotrexate), Folex PFS (methotrexate), Folotyn (pralatrexate), ibritsumomabutiuxetane, Istodax (lomidepsin), Leukeran (chlorambucil) (Chlorambusil), Liposomal Citarabin, Maturane (Procarbazine Hydrochloride), Methotrexate, Methotrexate LPF (Methtrexate), Mexate (Mettrexate), Mexate-AQ (Mettrexate), Mozobil (Prelixafor), Nerarabin, Neosar (Cyclophosphamide) , Ontak, Prelixafol, Pralatrexate, Rituxan, Rituximab, Lomidepsin, Toshitumomab and Iedone I131 Toshitumomab, Treanda (Bendamstin hydrochloride), Velban (Bimbrastin Sulfate), Velban ), As well as Velsar, binblastin sulfate, Vincasar PFS, bincristin sulfate, bolinostat, Zevalin (ibritsumomabutiuxetan), Zolinza (bolinostat). Examples of combination drugs used to treat non-hodgkin lymphoma include CHOP (C = cyclophosphamide, H = doxorubicin hydrochloride (hydroxydaunomycin), O = vincristine sulfate (Oncovin), P = prednison). COPP (C = cyclophosphamide, O = vincristine sulfate (Oncovin), P = procarbazine hydrochloride, P = prednison); CVP (C = cyclophosphamide, V = vincristine sulfate, P = prednison); EPOCH [E = etopocid, P = prednison, O = vincristine sulfate (Oncovin), C = cyclophosphamide, H = doxorubicin hydrochloride (hydroxydaunomycin)]; ICE (I = ifosphamide, C = carboplatin, E = etopocid ) And R-CHOP (R = rituximab, C = cyclophosphamide, H = doxorubicin hydrochloride (hydroxydaunomycin), O = vincristine sulfate (Oncovin), P = prednison).

The anti-CD38 antibody, or anti-CD38 antibody-attenuated interferon alfa-2b fusion construct, can be used to detect CD38-positive cells, such as CD38-positive tumor cells. In some embodiments, these constructs are brought into contact with an anti-CD38 antibody, or anti-CD38 antibody-attenuated interferon alfa-2b fusion construct, with a tissue sample isolated from the subject, and the antibody 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 the step of detecting a complex of constructs 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 myelogenous leukemia cells. The method may further include the step of isolating the tissue sample from the subject.

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

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

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

The following examples are provided to illustrate the disclosure in more detail. They are intended to illustrate, but not to limit, this disclosure.

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 named X355 / 02-HC-L0-IFN-alpha (A145D) IgG4 in this PCT application. As used herein, X355 / 02-HC-L0-IFN-alpha (A145D) IgG4 has been renamed to 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 formalized on the human IgG4 constant region (SEQ ID NO: 3) containing the S228P substitution (EU numbering) and its variable heavy chain A02.1 (SEQ ID NO: 13). ) And co-expressed. This antibody is referred to herein as X02.1, where A02.1 contains a fusion with IFN-alpha 2b, although both antibodies share the same heavy and light chain sequences, X02. .1 does not include it.

A BLAST search for a database of human germline immunoglobulin genes (Altschul SF (1997) Nucleic Acids Res. 25: 3389-3402) was performed using the variable heavy chain amino acid sequence 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 IGH V4-61 ^* 01 is shown in Figure 2. The X02.1 variable heavy chain region differs by 8 amino acids from its closest germline amino acid sequence. To reduce the immunogenicity of the X02.1 heavy chain variable region, germline amino acid residue substitutions should be generated at residues different from the germline sequence and the resulting antibody variants tested for anti-CD38 antibody binding activity. Can be done.

Several heavy chain variants of the X02.1 parent sequence are detailed in Figure 2. These heavy chain variable regions were formalized on the IgG4 S228P constant region and co-expressed with the A02.1 light chain. Table 1a and Table 1b detail the sequences of variants tested with their ability to bind human CD38 when evaluated using flow cytometry and surface plasmon resonance (SPR). Is. Briefly, antibody chains were transiently co-expressed in CHO cells and purified by the protein A chromatography described in Example 5. Variants were evaluated using the flow binding assay described in Example 5. Also EC ₅₀ for the obtained dose response curves for each antibody are set forth in Table 1a (Table 2) and Table 1b (Table 3).

The SPR bindings of the variants detailed in Table 1a (Table 2) were evaluated separately from those in Table 1b (Table 3). Of the parent antibody X02.1 K _D (M) was in the range of 2.7 × 10 ^-8 ~3.78 × 10 ^-10 In SPR binding experiments. Flow cytometric binding experiments showed that the antibodies X02.8, X02.11, X02.69 and X02.71 strongly bind to the CD38 positive cell line ARP-1.

Antibodies with the above amino acid substitutions were subsequently searched for in the context of fusion proteins by conjugation to attenuated IFN-alpha 2b (called A02 and, when linked to IFN, represent the same variant with the X02 designation. Has numbers after the decimal point). These heavy chain variable regions were formalized on an IgG4 constant region containing an S228P substitution fused to A145D attenuated IFN-alpha 2b and simultaneously in CHO or HEK cells with the A02.1 light chain described in Example 5. It was expressed. Proteins successfully purified from cell supernatants were then tested in a flow binding assay to cell line ARP-1. EC ₅₀ values of the dose-response curves for each antibody are set forth in Table 2 (Table 4). 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 readily 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 equivalent monoclonal antibody expression and / or purification would be considered more difficult.

A02.1 BLAST search using the amino acid sequence of the variable light chain was performed on the database of human germline immunoglobulin genes. The closest human germline variable light chain gene was IGLV5-37 ^* 01. The amino acid sequence alignments of A02.1VL and IGLV5-37 ^* 01 are shown in Figure 3. This alignment illustrates the 12 amino acid differences between these sequences.

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

Then, the antibody purified from the CHO cell supernatant was tested in a flow cytometry-based binding assay 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.

The antibodies X02.96, X02.99, X02.101, X02.102, X02.103 and X02.104 strongly bound 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 A021 identified amino acids that could potentially be oxidized or isomerized. These contain a potential isomerization site in D101 and a potential oxidation site in M (100C). To eliminate 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). A combination of both E and M (100C) L (SEQ ID NO: 27) (Fig. 2). Antibodies were made using these amino acid substitution combinations in variable heavy chains as shown in Table 4 (Table 6). The antibody heavy chain variable region was formalized with an IgG4 constant region containing the S228P substitution and co-expressed in CHO cells with the A02.1 light chain. The antibody was then purified by protein A chromatography and screened for binding to ARP-1 cells by flow cytometry. The obtained join data is shown in Table 4.

Antibodies X02.76 and X02.77 maintained their strong binding to the ARP-1 cell line, which eliminates potential oxidation and isomerization sites in the X02.1 and A02.1 heavy chains. It shows that the amino acid substitution for the cell 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 A021 identified amino acids that could potentially be oxidized or deamidated. These contained a potential deamidation site in N69 and a potential oxidation site in M89. In addition, a putative N-linked glycosylation site was predicted to be present in 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 Figure 3). Antibodies were prepared by co-expression of heavy and light chains in CHO cells as shown in Table 5. Antibodies were purified by protein A chromatography and screened for binding to ARP-1 cells by flow cytometry. The resulting join data is presented in Table 5.

X02.94 bound to the CD38-positive cell line ARP-1, indicating that M89L substitution had little effect on CD38-binding activity. N94Q substitution in antibody X02.80 removed the potential N-binding glycosylation motif 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 readily purified or that showed attenuated binding to the CD38 positive cell line ARP-1. The potential deamidation site at position 69 was removed by substitution with alanine, but this antibody (X02.81) was not readily purified.

Other antibodies tested, including the X02.1 variable heavy chain variant, are listed in Table 6. These heavy chain variable regions were formalized on an IgG4 constant region containing S228P substitutions. These heavy chains were co-expressed with the A02.1 light chain in CHO cells. The antibody was expressed and the resulting antibody was tested in a flow cytometry-based assay for binding to CD38-positive cells ARP-1. With the exception of T23K (SEQ ID NO: 21; X02.68), all variable heavy chain substitutions had minimal 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 combined with X02.1 heavy chains formalized on an IgG4 constant region containing S228P substitutions and expressed in CHO cells as described in Example 5. A summary of the heavy and light chains used to generate these antibody variants is given in Table 7. Antibodies X02.83, X02.85, X02.91, X02.82 strongly bound to the CD38 positive cell line ARP-1.

Substitutions that had little effect on CD38 binding activity and purification of the X02 variant antibody were subsequently generated as armed antibodies by fusion with A145D attenuated IFN-alpha 2b. The X02.1 light chain substitutions were combined and the resulting variants were co-expressed in HEK293E cells with the X02.1 heavy chain point and combination variants as listed in Table 8 (Table 10). These antibodies were predominantly predicted by potential X02.1 light chain deamidation sites, CDR3 derived oxidation sites of the X02.1 heavy chain and in silico analysis (Epibase, Lonza, UK). The focus was on removing putatively potent MHC class II-binding peptides from single-stranded framework region 3. Figure 4.

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

Of the proteins tested, A02.12 was well expressed and showed efficacy in the Annexin V, caspase and cell proliferation assays. Substitution of N69T in antibody A02.47 did not affect expression levels or potency in the Annexin V or caspase assay, indicating that this deamidation site can be removed. N69T substitutions 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 the A02.1 light chain amino acid sequence A putative immunogenicity epitope was identified in the light chain variable region amino acid sequence of A02.1 using Epibase analysis software (Lonza, UK). Substitutions were introduced into the A02.1 variable light chain to remove the putative immunogenic epitope (Fig. 4). A light chain with lower putative immunogenicity in HEK293E cells with an A02.12 heavy chain variable region (SEQ ID NO: 34) formalized on an IgG4 constant region containing an S228P substitution fused to A145D-attenuated IFN. Simultaneously expressed in. The antibody variants produced are detailed in Table 10.

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

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

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

Anti-CD38 antibody pairing of different heavy and light chains A functional anti-CD38 antibody-antibody X910/12 described in PCT / AU2012 / 001323 to determine if an attenuated IFN fusion protein can be obtained. -HC-L0-IFN-Alpha (A145D) IgG4-derived heavy and light chains (SEQ ID NO: 110) and light chains (SEQ ID NO: 112), as well as the antibody X913 / 15-HC-L0-IFN described in PCT / AU2012 / 001323. -Heavy chains (SEQ ID NO: 111) and light chains (SEQ ID NO: 113) derived from alpha (A145D) IgG4 were paired with each other in various combinations and with the heavy and light chains described in the Examples below. .. An overview of heavy and light chain pairs is given in Table 14.

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

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

FIG. 5 enumerates the consensus variable heavy chains of A02.1 related constructs with functional activity, and FIG. 6 enumerates the consensus variable light chains. Anti-CD38 antibody 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), it can be further envisioned that substitution combinations can be made. In addition, the anti-CD38 antibody described above could be constructed as an anti-CD38 antibody-attenuated IFN fusion protein and tested for functional activity as described herein.

H929 multiple myeloma xenotransplantation model
The in vivo efficacy of A02.1 was pre-tested in an NCI-H929 sc multiple myeloma model as described in Example 5. A02.1 has been shown to have strong antitumor activity. The data are presented in PCT / AU2012 / 001323.

The H929 multiple myeloma xenograft model could be used to test the antitumor activity of any of the above anti-CD38 antibody-attenuated IFN fusion proteins.

Anti-CD38 antibody containing attenuated IFN with potent apoptotic activity and caspase activation using the Anexin V and caspase assays, which require attenuated IFN for potent apoptotic activity and caspase activation in tumor cell lines -It has been shown to be dependent on attenuated IFN fusion proteins (Table 16a (Table 19), Figure 18). In the Annexin V assay, attenuated IFN-containing proteins (A02.1 and A02.6) had twice as much activity as proteins without attenuated IFN (X02.1 and X02.6).

General method
Generation of antibodies and antibody-fusion constructs in HEK-293E cells. DNA plasmids (antibodies and antibody-IFN-alpha 2b-related constructs) encoding protein constructs were prepared using the HiSpeed plasmid Maxi kit (Qiagen, Valencia, CA), followed by commercially available transfection reagents and OptiMEM medium (Invitrogen). Transfected into HEK293E cells (CNRC, Montreal, Canada) using Company, Carlsbad, CA) and 0.45% (w / v) of D- (+)-glucose (Sigma, Castle Hill, NSW). , 25 μg / mL of geneticin (Invitrogen, Carlsbad, CA), and 1x GlutaMAX (Invitrogen, Carlsbad, CA) were added to the cells for growth in F17 synthetic medium. After supplying 5% CO ₂ and expressing in an incubator for 6 days with shaking at 120 rpm, the culture medium was isolated and protein A Mab Select SuRe ™ agarose beads (GE Healthcare, Piscataway, NJ) were used. Affinity purification used. Purified protein constructs using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or HiPrep 26/10 desalting column (HiTrap Desalting HiPrep 26/10 Desalting), 0.2M arginine HCl, Buffer exchange was performed 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 centrifuge filter (Millipore, Billerica, MA) and then the protein concentration was determined by reading the absorbance at 280 nm.

Generation of antibody and antibody-fusion constructs in CHO cells DNA plasmids (antibodies and antibody-IFN-alpha 2b-related constructs) encoding protein constructs were prepared using the HiSpeed Gibraltar Maxi Kit (Qiagen, Valencia, CA). Then, using a commercially available transfection reagent and OptiPro SFM ™ medium (Invitrogen, Carlsbad, CA), the plasmid was transfected into CHO cells (Lonza), and Freestyle ™ CHO Expression Medium (Invitrogen), It was grown in Carlsbad, CA). After supplying 10% CO ₂ and expressing in an incubator for 6 days with shaking at 120 rpm, the culture medium is isolated and affinity using Protein A Mab Select SuRe agarose beads (GE Healthcare, Piscataway, NJ). It was refined. Purified protein constructs using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, NJ) or HiPrep 26/10 desalting column (HiTrap Desalting HiPrep 26/10 Desalting), 0.2M arginine HCl, Buffer exchange was performed 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 centrifuge filter (Millipore, Billerica, MA) and then 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). Unpurified transfection prepared 7: 1 with non-specific binding reducing agents (GE Healthcare, Piscataway, NJ) for the ability of anti-CD38 and anti-CD38 antibody-attenuated IFN fusion proteins to bind human CD38. It was measured using the supernatant of the cells. Briefly, protein A using Biacore ™ 3000 or T200, flow cells (FC) 1 (FC1) and FC2 (or FC3 and FC4) of CM5 study grade sensor chips using amine coupling. ) When fixed on, about 1500 RU was obtained. FC2 (or FC4) was used as a reference throughout the experiment. Experiments were performed in HBS-P + buffer (0.01M HEPES, 0.15M NaCl, 0.005% v / v Surfactant P20, pH 7.4) at 37 ° C. 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) only. 30 μL of CD38 (10 μg / mL in running buffer) or 30 μL of running buffer was injected onto FC1 and FC2 with a dissociation time of 5 minutes. Both surfaces were regenerated twice with sodium hydroxide. Results were generated using the BIA evaluation software provided with the machine. Microsoft Excel was used for the calculation. BIAevaluation software automatically subtracted the reference sensorgrams to give trace amounts of FC2-1 (or FC4-3) for each sample. Double reference was performed for each antibody tested by subtracting the sensorgram with CD38 injection from the sensorgram with blank running buffer injection. Protein A capture refers to the response unit measured from the sensorgram at a fixed time point 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 507.5s 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, indicating the level of CD38 bound to the surface on which the protein was captured approximately 300 s after the dissociation phase. Using BIAevaluation, sensorgrams were fitted using the Langmuir 1: 1 equation to generate the equilibrium dissociation constant (KD).

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

Flow cytometric binding of antibodies to human CD38-positive cell lines ARP-1 and H929 and anti-CD38 antibody-IFN fusion protein Multiple myeloma cell line ARP-1 is Myeloma from the University of Arkansas Medical Center (Little Rock, AK). It was a gift from Institute directors Bart Barlogie MD, phD. It is described in Hardin J. et al. (1994) Blood. 84:30, pp. 63-70. 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 was tested in a flow cytometry-based assay. ARP-1 cells or H929 cells (5 x 10 ⁵ cells, determined by trypan blue pigment elimination test) in a dark room, on ice for 60 minutes, in a 96-well plate, 50 μL of FACS buffer (PBS + 1% bovine fetal serum, Incubated with various concentrations of each protein in FCS, 0.2M HEPES, 0.5M EDTA) or with a human IgG4 monoclonal antibody containing a protein construct of irrelevant specificity. 50 μL FACS buffer containing goat anti-human IgG antibody (Fc-specific antibody conjugated to fluorescein isothiocyanate, FITC; Sigma-Aldrich, St. Louis, MO) after washing cells 3 times with FACS buffer Incubated in for 30 minutes. After washing 3 times with FACS buffer, cells were fixed in 50 μL PBS containing 4% formaldehyde v / v and incubated in a dark room at 4 ° C. for 16 hours. Incubated cells in suspension are diluted with an additional 150 μL FACS buffer and used on FACS Canto II (BD Biosciences, San Diego, CA) with forward, lateral and fluorescence intensities in FITC channels. Binding was analyzed by flow cytometry. The value reported is the average fluorescence intensity (MFI).

Target 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-related antigen. Cell viability was measured using CellTiter-Glo® reagent from Promega (Madison, Wisconsin), Catalog No. G7570. This is a luminescence-based assay that determines the viability of cells in culture based on the quantification of ATP. 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 T75 flasks (TPP, Trasadingen, Switzerland, Catalog No. 90076) in 10% fetal bovine serum (FBS; ^{0.5 × 10 5 to} 0.8 × 10 ⁵ living cells in RPMI 1640 (Mediatech, Inc., Manassas, VA, Catalog No. 10-040-CV) containing Hyclone, Logan, UT, Catalog No. SH30070.03) The cells were cultured to a preferable density of / mL. Cells were centrifuged at 400 xg for 5 minutes, the supernatant was discarded and the cell pellet was harvested by resuspending in RPMI 1640 + 10% FBS. Cells were then counted and the density ^{adjusted to 3.0 × 10 5} cells / mL in RPMI 1640 + 10% FBS. A 50 μL cell suspension was then aliquoted into each well of a 96-well round bottom tissue culture plate (hereinafter referred to as the “experimental plate”) (TPP, Catalog No. 92067). The test substance was serially diluted twice in RPMI 1640 + 10% FBS on another sterile 96-well plate (hereinafter referred to as the "dilution plate"; Costar, Corning, NY, Catalog No. 3879). 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plate was then _{incubated with 5% CO 2 at} 37 ° C. for 4 days. Experiments with a mixture of assay buffer supplied by the manufacturer and assay substrate [hereinafter referred to as "CellTiter-Glo® Reagent", mixed according to the manufacturer's instructions] at 100 μL / well. Added to the plate. The plate was shaken for 2 minutes.

100 μL / well was then transferred from the experimental plate to a 96-well flat-bottomed white opaque plate (hereinafter referred to as the “assay plate”; BD Biosciences, Franklin 5 Lakes, NJ, Catalog No. 35 3296). The contents of the assay plate were then stabilized in a dark room for 15 minutes at room temperature. The luminescence was measured by reading the plate on a Victor 3V Multilabel Counter (Perkin Elmer, Waltham, MA, model number 1420-041) on the luminescence measurement channel. The results are presented as "relative emission units" (RLU). Data were analyzed using Prism 5 (Graphpad, San Diego, CA) using non-linear regression and 3-parameter curve fitting to determine the midpoint of the curve (EC50).

ARP-1 Cell Proliferation Assay: This assay was used to quantify the antiproliferative activity of IFN and antibody-IFN fusion protein constructs against CD38 antigen-positive cells. ARP-1 cells express CD38 as a cell surface-related antigen. Cell viability was measured using CellTiter-Glo® reagent from Promega (Madison, Wisconsin), Catalog No. G7570. This is a luminescence-based assay that determines the viability of cells in culture by quantifying ATP. 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 in a T175 flask (Costar, Corning, NY Lakes, NJ, Catalog No. CLS431080) with 10% fetal bovine serum (FBS; AusGeneX, Molendinar, QLD,). It was cultured in RPMI1640 (Life Technologies, Mulgrave, VIC, Catalog No. 11875-093) containing Australia, Catalog No. FBS500S) to ^{a preferred density of 2.0 × 10 5 to} 2.0 × 10 ⁶ live cells / mL. Cells were centrifuged at 400 xg for 5 minutes, the supernatant was discarded and the cell pellet was harvested by resuspending in RPMI 1640 + 10% FBS. Cells were then counted and the density ^{adjusted to 2.0 × 10 5} cells / mL in RPMI 1640 + 10% FBS. A 50 μL cell suspension was then aliquoted into each well of a 96-well flat-bottomed white opaque plate (hereinafter referred to as the “experimental plate”) (Costar, Corning, NY Lakes, NJ, Catalog No. CLS3917). The test substance was serially diluted twice in RPMI 1640 + 10% FBS on another sterile 96-well plate (hereinafter referred to as the "dilution plate"; Costar, Corning, NY, Catalog No. 3799). 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plate was then _{incubated with 5% CO 2 at} 37 ° C. 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 in a dark room for 15 minutes at room temperature. The luminescence was measured by reading the plate on the FLUOstar Galaxy plate reader (BMG Labtech, Durham, NC) on the luminescence measurement channel. Data were analyzed using Prism 5 (Graphpad, San Diego, CA) using non-linear regression and 3-parameter curve fitting to determine the midpoint of the curve (EC ₅₀ ).

Annexin V Assay: H929 cells were harvested by centrifuging at 400 xg for 5 minutes, discarding the supernatant and resuspending the cell pellet in RPMI 1640 + 10% FBS. Cells were then counted and the density ^{adjusted to 1.0 × 10 6} cells / mL in RPMI 1640 + 10% FBS. A 50 μL cell suspension was then aliquoted into each well of a 96-well round bottom clear plate (hereinafter referred to as the “experimental plate”; Costar, Corning, NY, Catalog No. CL3799). On another sterile 96-well plate (hereinafter referred to as the "dilution plate"; Costar, Corning, NY, Catalog No. CL3799), the test substance was diluted to 40 nM four times in RPMI1640 + 10% FBS. 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plate was then _{incubated with 5% CO 2 at} 37 ° C. for 24 hours. The cells were then centrifuged at 400 xg for 5 min, the supernatant was discarded and resuspended in 100 μL HEPES buffer containing Annexin V-FITC (1/200) and 7-AAD (1/50). did. The cells were then incubated for 15 min at room temperature and then annexin by forward scatter, lateral scatter, flow cytometry on FACS Canto II (BD Biosciences, San Diego, CA) using FITC and PerCP-Cy 5.5 channels. V and 7-AAD staining was analyzed. Annexin V-positive cells refer to cells that are positively stained with annexin V-FITC after being treated with a 20 nM antibody construct for 24 hours and are expressed as multiple changes compared to untreated cells.

Caspase Assay: Activated caspases 2, 3, 6, 7, 8, 9, 10 with Homogeneous Caspases Assay Reagent, Fluorescence, Catalog No. 12236869001 from Roche (West Sussex, UK) after treatment with test antibody. Measured using. Details of the assay are as follows.

RPMI1640 (AusGeneX, Molendinar, QLD, Victoria, Catalog No. FBS500S) containing ARP-1 cells expressing high levels of CD38 in a T175 flask (Costar, Corning, NY, Catalog No. CLS431080). Cultivated in Life Technologies, Mulgrave, VIC, Catalog No. 11875-093) to ^{a preferred density of 2.0 × 10 5 to} 2.0 × 10 ⁶ live cells / mL. Centrifuge cells at 400 xg for 5 minutes, discard supernatant and resuspend cell pellet in RPMI1640 Phenol Red Free (Life Technologies, Mulgrave, VIC, Catalog No. 11835-030) + 10% FBS. It was harvested by. ^{Cells were then counted and the density adjusted to 2.0 × 10 5} cells / mL in RPMI 1640 phenol red-free + 10% FBS. A 50 μL cell suspension was then aliquoted into each well of a 96-well flat-bottomed black-walled transparent bottom plate (hereinafter referred to as the “experimental plate”; Costar, Corning, Catalog No. CLS3603). On another sterile 96-well plate (hereinafter referred to as the "dilution plate"; Costar, Corning, NY, Catalog No. 3799), the test substance was diluted to 40 nM four times in RPMI1640 phenol red-free + 10% FBS. 50 μL / well was then transferred from the dilution plate to the experimental plate. The experimental plate was then _{incubated with 5% CO 2 at} 37 ° C. for 24 hours. The assay buffer supplied by the manufacturer was added to the substrate supplied by the manufacturer and mixed according to the manufacturer's instructions to create a "substrate solution". 100 μL of substrate solution was then added to each well of the assay plate. The plate was shaken for 2 minutes. The plate is then incubated in a dark room at room temperature for 15 minutes and finally read on a FLUOstar Galaxy plate reader (BMG Labtech, Durham, NC) with an excitation filter at 470-500 nm and an emission filter at 500-560 nm for fluorescence. It is measured and presented as a multiple change compared to untreated cells. The caspase assay refers to cell caspase activation after 24 hours of treatment with a 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 with the addition of a human IgG blocking step, mostly as described by the manufacturer. The iLite cell line is a stable transfected cell line derived from a commercially available premonocyte human cell line characterized by the expression of MHC class II antigens, in particular human lymphocyte antigen (HLADR), on the cell surface. Is described by the manufacturer. This cell line contains a stably transfected luciferase gene whose expression is induced by an interferon response element (IRE), which can quantify interferon activity based on luminescence output. The iLite plate (hereinafter referred to as the assay plate) and diluent supplied by the manufacturer were taken out of the freezer at -80 ° C and equilibrated to room temperature. Then 50 μL of diluent per well was added to the assay plate. Vials of reporter cells supplied by the manufacturer were removed from the -80 ° C freezer and thawed in a 37 ° C water bath. A 25 μL aliquot of cells was then dispensed into each well of the assay plate. Next, 12.5 μL of 8 mg / mL human IgG diluted in RPMI 1640 + 10% FBS (Sigma Chemicals, St. Louis, MO; Catalog No. I4506) was added to each well. The contents were mixed and incubated at 37 ° C for 15 minutes. On a separate "dilution plate", the test substance was serially diluted twice in RPMI 1640 + 10% FBS. 12.5 μL of the test substance was then transferred from the dilution plate to the assay plate. The assay plate was then _{incubated with 5% CO 2 at} 37 ° C. for 17 hours. The assay buffer supplied by the manufacturer and the substrate were removed from the -80 ° C freezer and equilibrated to room temperature for 2 hours. The assay buffer supplied by the manufacturer was added to the substrate vial supplied by the manufacturer and mixed well according to the manufacturer's instructions to create a "luminescent solution". 100 μL of luminescent solution was then added to each well of the assay plate. The plate was shaken for 2 minutes. The plate was then incubated in a dark room at room temperature for 5 minutes and finally read on a luminescence measurement channel on a Victor 3V Multilabel Counter, the luminescence was measured and presented as an RLU. Data were analyzed using Graphpad Prism 5 as described for the "on-target (Daudi) assay". To test the anti-CD38 antibody-IFN fusion protein construct in the iLite assay, 0.25 mg / mL anti-CD38 antibody was added to the diluent supplied by the manufacturer (the same antibody clone is expressed on iLite cells). Tested as an antibody-IFN fusion protein construct to block any binding of the anti-CD38 antibody-IFN fusion protein construct to CD38).

HEK-Blue ™ Off-Target Assay: This assay is used with the HEK-Blue ™ IFN-alpha / β cell line (Invitrogen, San Diego, CA) to interferon-alpha / β receptor (IFNAR). ) Quantified the ability of the antibody-IFN fusion construct. The "off-target (HB-IFN) assay" was performed mostly as described by the manufacturer of the HEK-Blue ™ IFN-alpha / β cell line. HEK-Blue ™ IFN-alpha / β cells are specifically designed to monitor activation of the JAK-STAT pathway induced by type I IFN. These cells were generated 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 secretory 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 stimulation with IFN-alpha or IFNβ, HEK-Blue ™ IFN-alpha / β cells activate the JAK-STAT pathway and then activate the expression of the SEAP reporter gene. SEAP is secreted into the medium and can be quantified using the colorimetric reagent QUANTI-Blue ™. Briefly, HEK-Blue IFN-alpha / β cells (Invivogen, San Diego, CA, Catalog No. hkb-ifnab) are thawed and heat-inactivated DMEM medium (Mediatech, Manassas VA, Catalog No. 10). -013-CV) + 10% FBS (Hyclone, Logan UT, Catalog No. SH30070.03) (HI FBS). When the cells reached a density of 60-80%, they were lifted using a Cell Stripper (Mediatech, Catalog No. 25-056-Cl). Cells were washed twice in DMEM + HI FBS and counted. ^{Cells were adjusted to 3.3 × 10 5} live cells / mL in DMEM + HI FBS and 150 μL per well was aliquoted into a flat-bottomed 96-well tissue culture plate (hereinafter referred to as “experimental plate”). Then 50 μL of IFN-alpha 2b or fusion protein construct diluted in DMEM + HI FBS was added to each well. The plates were incubated at 37 ° C. at 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 the “assay plate”). 50 μL of supernatant per well from the experimental plate was then 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 were 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 with non-CD38-targeted fusion protein constructs. For these comparisons, the NCI-H929 sc multiple myeloma model was used.

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

The flanks of 8- to 12-week-old CB.17 SCID mice ^{were subcutaneously injected with 1 × 10 7} NCI-H929 tumor cells in 50% Matrigel ™. When the mean tumor size ^{reached 170-350 mm 3} , mice were grouped into 4 cohorts of 7 mice each and treatment was initiated at 0 hours (T0). All treatments were given by intraperitoneal injection (ip) twice weekly for 3 weeks (indicated by the bar at the bottom of 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 a week by caliper measurement. The end point was a tumor volume of ^{2,000 mm 3.}

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

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

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

Anti-CD38 antibody-attenuated IFN fusion protein containing an alternative constant region
A02.12 contains an anti-CD38 antibody-attenuated IFN fusion protein in which the constant region of the protein is HC-L0-IFN-alpha (A145D) IgG4 (SEQ ID NO: 9). The heavy chain variable region of this antibody was reformulated on an IgG1 constant region fused to A145D attenuated IFN-alpha 2b (SEQ ID NO: 10). Co-expression of this heavy chain in HEK293E cells with the light chain of X02.107 (SEQ ID NO: 65) gave antibody A02.112. By comparing antibodies A02.12 and A02.112 using the flow cytometry-based CD38 binding assay and efficacy assay, antibodies with as strong biological activity as those made using the human IgG4 constant region-attenuated IFN It has been shown that other antibody constant regions, such as human IgG1, that result in fusion proteins can also be used (Table 16b).

Humanized R5D1, R5E8 and R10A2 Variable Regions Rat-derived anti-CD38 antibodies R5D1, R5E8 and R10A2 have been 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, standard structures were assigned to each rodent heavy and light chain by examination of their respective amino acid sequences. R10A2 is assigned the standard structure 2-1-1 / 1-2 (V _L / V _H ), R5E8 is assigned the standard structure 4-1-1 / 1-2, and R5D1 is assigned the standard structure 2-1-1. Assigned / 1-2. Human germline sequences of the same standard structure were used as an acceptor framework for transplantation of donor CDRs. We also designed a variant of the resulting superhumanized antibody gene that contains an amino acid substitution at a position within its sequence that is considered likely to be important for maintaining binding activity. 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 vector pEE6.4 containing a human IgG4 constant region with 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 by co-expression of the heavy chain in pEE6.4 and the light chain in pEE12.4 in CHO cells as previously described. Table 17 summarizes the heavy and light chain pairs used to produce the superhumanized 5D1-based proteins, respectively. Table 18 details the heavy and light chain pairs for the superhumanized 5E8-based protein produced, but creates a superhumanized 10A2-based protein. The pairs of heavy and light chains used for this are shown in Table 19 (Table 23). One-shot equilibrium dissociation constant (K _D ) rankings of superhumanized antibodies were 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 can bind to human CD38. There wasn't. 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 concentration (nM) range. A10A2.53 and A10A2.25, which share a common light chain, were selected for further optimization. A10A2.53 was renamed to A10.0 and A10A2.25 was renamed to A10.38.

Improved variant of A10.0
Variable heavy chain and / or light chain to have a positive effect on the biophysical and in silico immunogenicity of the antibody while minimizing the effect of the A10.0 antibody on the functional activity of the antibody. Optimized by changes to the sequence.

A10.0 Analysis of heavy and light chain in silico immunogenicity
A10.0 heavy and light chain variable region in silico immunogenicity analyzes were made using the Epibase software package. Several amino acid substitutions were introduced into the heavy and light chain variable regions of A10.0 to remove potential immunogenic epitopes. The amino acid sequence alignment of the generated heavy chain variable region variant aligned with the humanized heavy chain (SEQ ID NO: 156) is shown in FIG. The amino acid sequence alignment of the light chain variable region variant 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 generate proteins are summarized in Table 20.

Each antibody prepared using the heavy and light chain pairs outlined in Table 20 was evaluated for protein expression levels and binding to CD38 by SPR. In addition, cell culture supernatants were used to perform efficacy assays to assess the relative functional activity of each of these anti-CD38 antibody-attenuated IFN fusion proteins. Table 21 (Table 25).

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

Each antibody prepared using the heavy and light chain pairs outlined in Table 22 was evaluated for protein expression levels and binding to CD38 by SPR. In addition, cell culture supernatants were used to perform efficacy assays to assess the relative functional activity of each of these anti-CD38 antibody-attenuated IFN fusion proteins. Table 23 (Table 27).

Creating an improved variant of A10.38
A10.0 and A10.38 share a common light chain. An optimized light chain sequence of A10.0, A10.38, to obtain a positive effect on the biophysical properties and in silico immunogenicity properties of the antibody while minimizing its effect on functional activity. It was paired with the heavy chain of the antibody. A summary of heavy and light chain changes and pairs is given 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 assess the relative functional activity of each of these anti-CD38 antibody-attenuated IFN fusion proteins. Table 25 (Table 29).

The relative efficacy of anti-CD38 antibody A10.0 (attenuated IFN fusion) and X10.0 (no fusion), which requires attenuated IFN for potent apoptotic activity and caspase activation in tumor cell lines, Comparisons were made using the annexin V, caspase and cell proliferation assays outlined in Example 5. The relative efficacy of A10.38 and X10.38 was also compared. Table 26 (Table 30).

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 that did not contain attenuated IFN.

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

H929 multiple myeloma xenograft model
The in vivo efficacy of 10A2 variants A10.0 and A10A2.0 was evaluated in the NCI-H929 sc mouse multiple myeloma model. Figure 27. Both have been shown to have strong antitumor activity in this model. Such a model could be used to test the antitumor activity of other protein constructs described herein.

Off-target activity for 10A2 variants The off-target activity of 10A2 variants A10.0, A10.38, A10A2.37 and A10A2.39 compared to the parent A10A2.0 chimeric antibody fused to wild-type and attenuated interferon 145D, iLite Reporter Evaluated in genetic and / or HEK Blue assays and shown in FIGS. 28 and 29. Providing an EC ₅₀ value in FIGS. 28 and 29. Off-target activity confirms the need for antibodies targeted to CD38 to attenuate and restore interferon function.

Further in vitro efficacy data for A10.0 and related constructs A selection of the above anti-CD38 antibody-attenuated IFN fusion proteins was purified and analyzed for binding to CD38-positive cells in a cell-based assay. In addition, efficacy assays were 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 shown in Table 27.

Anti-CD38 antibody with alternative constant region-attenuated IFN fusion protein
A10.0 comprises an anti-CD38 antibody-attenuated IFN fusion protein in which the constant region of the protein is HC-L0-IFN-alpha (A145D) IgG4 (SEQ ID NO: 9). Using gene synthesis, the constant region of this protein was replaced with HC-L0-IFN-alpha (A145D) IgG1 (SEQ ID NO: 10) and paired with A10.0 light chain (SEQ ID NO: 161) to A10.59. I named it. The protein was expressed and found to be potent in the functional assay (Table 28). Although most of the proteins tested in the above examples were constructed on human IgG4 constant regions, these data can also use other antibody constant regions such as human IgG1 to obtain antibody-attenuated IFN fusion constructs. Has been shown to have as strong biological activity as constructs using the human IgG4 constant region.

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

The present disclosure is not limited to the embodiments described and exemplified above, but modifications and modifications may be made within the scope of the appended claims.

Claims (15)

A polynucleotide comprising a nucleic acid sequence encoding an antibody that specifically binds to CD38, wherein the antibody is the light chain complementarity determining regions 1 (CDR1), CDR2, and CDR3 of SEQ ID NO: 665 and light of SEQ ID NO: 666. A polynucleotide comprising the strands CDR1, CDR2, and CDR3, wherein the CDRs are based on the Kabat numbering system. The nucleic acid sequence includes a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 514, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 516, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 518, and a light chain containing the amino acid sequence of SEQ ID NO: 608. The polynucleotide according to claim 1, which encodes an antibody comprising chain CDR1, light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 591, and light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 612. The nucleic acid sequence contains a heavy chain variable region containing an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 156, and a light chain containing an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 185. The polynucleotide according to claim 1 or 2, wherein an antibody comprising a chain variable region is encoded, and all amino acid substitutions in the heavy chain variable region or the light chain variable region are present outside the CDR. The invention according to any one of claims 1 to 3, wherein the nucleic acid sequence encodes an antibody containing a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 156 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 185. Polynucleotide. The polynucleotide according to any one of claims 1 to 4, wherein the nucleic acid sequence encodes an antibody containing a human IgG1 heavy chain constant region. The polynucleotide according to any one of claims 1 to 4, wherein the nucleic acid sequence encodes an antibody containing a human IgG4 heavy chain constant region. The polynucleotide according to claim 6, wherein the human IgG4 heavy chain constant region contains proline at position 228 according to the EU numbering system. The polynucleotide according to claim 6 or 7, wherein the human IgG4 heavy chain constant region contains tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 according to the EU numbering system. The polynucleotide according to any one of claims 1, 2 or 5 to 8, wherein the nucleic acid sequence encodes a humanized antibody. The polynucleotide according to any one of claims 1 to 9, which comprises a nucleic acid sequence containing SEQ ID NO: 685 encoding a heavy chain variable region and a nucleic acid sequence containing SEQ ID NO: 691 encoding a light chain variable region. The polynucleotide according to any one of claims 1 to 10, wherein the nucleic acid sequence encodes an attenuated human interferon alpha-2b fused to the antibody. The polynucleotide according to claim 11, wherein the nucleic acid sequence encodes an attenuated human interferon alfa-2b comprising a substitution of alanine for aspartic acid at position 145 with respect to the amino acid sequence of SEQ ID NO: 648. The polynucleotide according to claim 11, wherein the nucleic acid sequence encodes an attenuated human interferon alfa-2b comprising the amino acid sequence of SEQ ID NO: 647, SEQ ID NO: 649, SEQ ID NO: 650, or SEQ ID NO: 651. A vector comprising the polynucleotide according to any one of claims 1 to 13. In vitro transformed mammalian cells comprising the vector of claim 14.

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