CD38 antibody variants and uses thereof

Abstract

The present invention relates to antibody variants comprising one or more mutations in the Fc region, in particular anti-CD38 antibodies comprising mutations in one or more amino acid residues corresponding to E430, E345 and S440 in a human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index.

Description

Technical Field

[0001] Antibody variants, particularly anti-CD38 antibody variants, comprising one or more mutations in the Fc region. Background Art

[0002] CD38 is a type II transmembrane glycoprotein that is commonly found on hematopoietic cells and is at low levels in solid tissues. The expression of CD38 in hematopoietic cells depends on the differentiation and activation state of the cells. Lineage-committed hematopoietic cells express this protein, which is lost by mature cells and expressed again on activated lymphocytes. CD38 is also expressed on B cells, whereby plasma cells express particularly high levels of CD38. Approximately 80% of resting NK cells and monocytes express CD38 at lower levels, as do various other blood cell types, including lymph node germinal center lymphoblasts, follicular cells, dendritic cells, erythrocytes, and platelets (Lee and Aarhus 1993; Zocchi, Franco et al. 1993; Malavasi, Funaro et al. 1994; Ramaschi, Torti et al. 1996). With respect to solid tissues, CD38 is expressed by intraepithelial cells and lamina propria lymphocytes in the intestine, Purkinje cells and neurofibrillary tangles in the brain, epithelial cells in the prostate, beta cells in the pancreas, osteoclasts in the skeleton, retinal cells in the eye, and the sarcolemma of smooth and striated muscle.

[0003] CD38 is expressed in a large number of hematological malignancies. Expression has been observed specifically in malignant cells of multiple myeloma (MM) (Lin, Owens et al. 2004) and chronic lymphocytic leukemia (CLL) (Damle 1999), and has also been reported in Waldenstrom's macroglobulinemia (Konoplev, Medeiros et al. 2005), primary systemic amyloidosis (Perfetti, Bellotti et al. 1994), mantle cell lymphoma (Parry-Jones, Matutes et al. 2007), acute lymphoblastic leukemia (Keyhani, Huh et al. 2000), acute myeloid leukemia (Marinov, Koubek et al. 1993; Keyhani, Huh et al. 2000), NK cell leukemia (Suzuki, Suzumiya et al. 2004), NK / T cell lymphoma (Wang, Wang et al. 2015), and plasma cell leukemia (van de Donk, Lokhorst et al. 2012).

[0004] Other diseases in which CD38 expression may be implicated include, for example, lung bronchial epithelial carcinoma, breast cancer (derived from malignant proliferation of the epithelial lining in the mammary ducts and lobules), pancreatic tumors derived from beta cells (insulinomas), tumors derived from the epithelium in the intestine (such as adenocarcinomas and squamous cell carcinomas), carcinomas in the prostate, seminoma in the testes, ovarian cancer, and neuroblastoma. Other publications have also suggested a role for CD38 in autoimmunity, such as Graves' disease and thyroiditis (Antonelli, Fallahi et al., 2001), type 1 and type 2 diabetes (Mallone and Perin, 2006), and inflammation of airway smooth muscle cells during asthma (Deshpande, White et al., 2005). In addition, CD38 expression has been associated with HIV infection (Kestens, Vanham et al., 1992; Ho, Hultin et al., 1993).

[0005] CD38 is a multifunctional protein. The functions attributed to CD38 include both receptor mediation in adhesion and signaling events, as well as (extracellular) enzymatic activity. As an extracellular enzyme, CD38 uses NAD + It serves as a substrate for the formation of cyclic ADP-ribose (cADPR) and ADPR, as well as nicotinamide and nicotinic acid-adenine dinucleotide phosphate (NAADP). cADPR has been shown to act as a Ca transporter from the endoplasmic reticulum. 2+ The second messenger of mobilization.

[0006] Several anti-CD38 antibodies are described in the literature, e.g., WO 2006 / 099875 A1, WO 2008037257 A2, WO 2011 / 154453 A1, WO 2007 / 042309 A1, WO 2008 / 047242 A1, WO 2012 / 092612 A1, Cotner, Hemler et al. 1981; Ausiello, Urbani et al. 2000; Lande, Urbani et al. 2002; de Weers, Tai et al. 2011; Deckert, Wetzel et al. 2014; Raab, Goldschmidt et al. 2015; Eissler, Filosto et al. 2018; Roepcke, Plock et al. 2018; and Schooten 2018.

[0007] Antibodies to CD38 can affect tumor cells expressing CD38 through one or more of the following mechanisms: complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), programmed cell death, trogocytosis, elimination of immunosuppressive cells, and modulation of enzymatic activity (van de Donk, Janmaat et al. 2016; Krejcik, Casneuf et al. 2016; Krejcik, Frerichs et al. 2017; Chatterjee, Daenthanasanmak et al. 2018; van de Donk 2018). However, as proposed in 2014, CD38 antibodies that can induce effective CDC, ADCC, and ADCP, as well as effectively inhibit CD38 enzymatic activity, have not yet been described (Lammerts van Bueren, Jakobs et al. 2014).

[0008] Optimization of effector function can improve the effectiveness of therapeutic antibodies for treating cancer or other diseases, for example, by improving the ability of the antibody to elicit an immune response to cells expressing the antigen. Such efforts are described, for example, in WO 2013 / 004842 A2; WO 2014 / 108198 A1; WO 2018 / 031258 A1; Dall'Acqua, Cook et al. 2006; Moore, Chen et al. 2010; Desjarlais and Lazar 2011; Kaneko and Niwa 2011; Song, Myojo et al. 2014; Brezski and Georgiou 2016; Sondermann and Szymkowski 2016; Zhang, Armstrong et al. 2017; Wang, Mathieu et al. 2018.

[0009] Despite these and other efforts in the art, however, there remains a need for CD38 therapeutic antibodies with modulatory efficacy. Summary of the Invention

[0010] The present invention relates to variants of CD38 antibody C, in particular variants having one or more mutations in the Fc region, wherein at least one of these mutations is in a residue corresponding to E430, E345 or S440 in the human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index.

[0011] Thus, in one aspect, the present invention relates to an antibody variant that binds to human CD38, the antibody variant comprising

[0012] (a) an antigen-binding region comprising a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VLCDR3 having the sequence shown in SEQ ID NO: 7, and

[0013] (b) a variant Fc region comprising a mutation in one or more amino acid residues selected from the group consisting of amino acid residues corresponding to E430, E345 and S440 in a human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index.

[0014] In one aspect, the present invention relates to an antibody variant that binds to human CD38, the antibody variant comprising

[0015] (a) a heavy chain comprising a VH region comprising a VH CDR1 having the sequence as shown in SEQ ID NO: 2, a VH CDR2 having the sequence as shown in SEQ ID NO: 3, and a VH CDR3 having the sequence as shown in SEQ ID NO: 4, and a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, wherein the amino acid residues are numbered according to the EU index;

[0016] (b) A light chain comprising a VL region comprising a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7.

[0017] In one aspect, the present invention relates to an antibody variant that binds to human CD38, the antibody variant comprising

[0018] (a) a heavy chain comprising a VH region comprising SEQ ID NO: 1, and a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, wherein the amino acid residue numbering is according to the EU index, and

[0019] (b) A light chain comprising a VL comprising SEQ ID NO:5.

[0020] In one aspect, the invention relates to an isolated nucleic acid encoding an antibody variant according to any aspect or embodiment herein.

[0021] In one aspect, the invention relates to expression vectors comprising such nucleic acids.

[0022] In one aspect, the invention relates to a recombinant host cell that produces an antibody variant according to any aspect or embodiment herein.

[0023] In one aspect, the invention relates to a method of producing an antibody variant according to any aspect or embodiment herein, comprising culturing such a recombinant host cell in a culture medium and under conditions suitable for production of the antibody variant.

[0024] In one aspect, the invention relates to a method for increasing the effector function of a parent antibody, said parent antibody comprising an Fc region and an antigen binding region that binds CD38, said method comprising introducing mutations in one or more amino acid residues selected from the group consisting of E430, E345 and S440 in the Fc region corresponding to a human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index;

[0025] wherein the antigen binding region comprises a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7.

[0026] In some embodiments of the aspects described herein, the mutation in one or more amino acid residues is selected from E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W, such as, for example, E430G.

[0027] In one aspect, the present invention relates to a method for producing a variant of a parent antibody comprising an Fc region and an antigen binding region that binds to CD38, wherein the variant has increased effector function compared to the parent antibody, the method comprising

[0028] (a) introducing mutations in one or more amino acid residues selected from the group consisting of E430, E345 and S440 in the Fc region corresponding to a human IgG1 heavy chain into the Fc region to obtain a variant antibody,

[0029] (b) selecting any variant antibodies that have increased effector function compared to the parent antibody, and

[0030] (c) producing the variant antibody in a recombinant host cell,

[0031] wherein the antigen binding region comprises a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7.

[0032] In one aspect, the invention relates to antibodies obtained or obtainable by such methods.

[0033] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody variant as defined in any aspect or embodiment herein and a pharmaceutically acceptable carrier.

[0034] In one aspect, the invention relates to an antibody variant according to any aspect or embodiment herein for use as a medicament.

[0035] In one aspect, the invention relates to an antibody variant according to any aspect or embodiment herein for use in treating a disease involving CD38 expressing cells.

[0036] In one aspect, the invention relates to an antibody variant according to any aspect or embodiment herein for use in inducing a CDC response against a tumor comprising CD38 expressing cells.

[0037] In one aspect, the invention relates to an antibody variant according to any aspect or embodiment herein for use in treating or preventing cancer in a subject comprising cells expressing human CD38.

[0038] In one aspect, the invention relates to an antibody variant according to any aspect or embodiment herein for use in treating or preventing rheumatoid arthritis.

[0039] In one aspect, the invention relates to a method for treating a disease comprising CD38 expressing cells, comprising administering to a patient in need thereof an antibody variant according to any aspect or embodiment herein, optionally wherein the antibody variant or pharmaceutical composition is administered in a therapeutically effective amount and / or for a time sufficient to treat the disease.

[0040] These and other aspects and embodiments of the invention are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1Shown is an amino acid sequence alignment of human IgG1m(a), IgG1m(f), IgG2, IgG3, and IgG4 Fc segments corresponding to residues P247 to K447 in the human IgG1 heavy chain using Clustal 2.1 software, wherein the amino acid residues are numbered according to the EU index as set forth in Kabat. The amino acid sequences shown correspond to residues 130 to 330 in the heavy chain constant region of allotypic variants of human IgG1 designated as IgG1m(za) (SEQ ID NO:64; UniProt Accession No. P01857), IgG1m(f) (SEQ ID NO:65), IgG1m(z) (SEQ ID NO:66), IgG1m(a) (SEQ ID NO:67), and IgG1m(x) (SEQ ID NO:68); residues 126 to 326 of the IgG2 heavy chain constant region (SEQ ID NO:79; UniProt Accession No. P01859); residues 177 to 377 of the IgG3 heavy chain constant region (SEQ ID NO:80; UniProt Accession No. P01860), and residues 127 to 327 of the IgG4 heavy chain constant region (SEQ ID NO:81; UniProt Accession No. P01861).

[0042] Figure 2 Binding of CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G and IgG1-C-E430G to CD38-expressing NALM16 cells is shown compared to CD38 antibodies IgG1-A, IgG1-B, IgG1-C and an isotype control antibody. For more details, see Example 2.

[0043] Figure 3 shows the binding of CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G to CD38 expressed on cynomolgus monkey PBMCs (A) or Daudi cells expressing high copy numbers of human CD38 (B) compared to an isotype control antibody. For more details, see Example 2.

[0044] Figure 4Shown are the percentage lysis of Ramos (A), Daudi (B), Wien-133 (C), NALM-16 (D), REH (E), RS4; 11 (F), U266 (G) and RC-K8 (H) tumor cell lines induced by CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G and IgG1-C-E430G in a CDC assay compared to CD38 antibodies IgG1-A, IgG1-B and IgG1-C. For more details, see Example 3.

[0045] Figure 5 Shown are the effects of CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G on the number of viable NK cells (A), T cells (B), and B cells (C) in a CDC assay performed on whole blood compared to CD38 antibodies IgG1-A, IgG1-B, and IgG1-C. For more details, see Example 3.

[0046] Figure 6 Shown are the percentages of Daudi cell lysis induced by CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G in a chromium release ADCC assay compared to CD38 antibodies IgG1-A, IgG1-B, IgG1-C, and an isotype control antibody. For more details, see Example 4.

[0047] Figure 7 Shown are dose-dependent FcγRIIIa cross-linking in an ADCC reporter assay for CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G compared to CD38 antibodies IgG1-A, IgG1-B, IgG1-C, and an isotype control antibody. For more details, see Example 4.

[0048] Figure 8 The ADCC assay of CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G against PKH-29 is shown in comparison with CD38 antibodies IgG1-A, IgG1-B, IgG1-C, and an isotype control antibody. pos 、CD14 pos and CD19 neg Effect of macrophage percentage. See Example 5 for more details.

[0049] Figure 9Shown are the percentages of lysis of Ramos (A), Daudi (B, C), Wien-133 (D, E) and NALM-16 (F, G) tumor cell lines induced by CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G and IgG1-C-E430G in apoptosis assays with (C, E, G) or without (A, B, D, F) Fc cross-linking antibodies compared to CD38 antibodies IgG1-A, IgG1-B, IgG1-C and an isotype control antibody. For more details, see Example 6.

[0050] Figure 10 The enzymatic activity of CD38 is shown.

[0051] Figure 11 Shown are the effects of CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G on the cyclase activity of HisCD38 (A), Daudi cells (B), and Wien-133 cells (C), as reflected by % NDG conversion over time, compared to CD38 antibodies IgG1-A, IgG1-B, IgG1-C, and an isotype control antibody.

[0052] Figure 12 Shown are the effects of CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G on CD38 expression on Daudi cells after 45 minutes of co-culture with macrophages, compared to CD38 antibodies IgG1-A, IgG1-B, IgG1-C, and an isotype control antibody. Macrophages were from donor A (A, B) or donor B (B, D), and antibody-opsonized cells were tested for CD38 expression (A, B) or human IgG staining (C, D).

[0053] Figure 13 Shown are the effects of CD38 antibody variants IgG1-B-E430G and IgG1-C-E430G compared to IgG1-B on CD38 expression on T regulatory cells with or without PBMCs.

[0054] Figure 14 Shown are the percentages of lysis of different B cell tumor cell lines induced by CD38 antibody variants IgG1-A-E430G (closed triangles), IgG1-B-E430G (closed circles) and IgG1-C-E430G (closed squares) in a CDC assay compared to CD38 antibody IgG1-B (open circles) and an isotype control antibody (open diamonds). For more details, see Example 3.

[0055] Figure 15 A summary of some of the EC50 values depicted in Table 4 is shown. Shown are the EC50 values for CDC induced by antibodies IgG1-B, IgG1-B-E430G, and IgG1-C-E430G against 20 different B-cell tumor cell lines. Each square, triangle, or circle represents a different B-cell tumor cell line. EC50 values obtained with AML cell lines are not included because IgG1-B-E430G was not tested against AML cell lines.

[0056] Figure 16 Shown are the percentages of lysis of different AML tumor cell lines induced by the CD38 antibody variant IgG1-C-E430G (filled circles) in a CDC assay compared to the CD38 antibody IgG1-B (open circles) and an isotype control antibody (filled squares). For more details, see Example 3.

[0057] Figure 17 Shown are the percentages of lysis of T regulatory cells induced by the CD38 antibody variants IgG1-B-E430G (filled circles) and IgG1-C-E430G (filled squares) in a CDC assay compared to the CD38 antibody IgG1-B (open circles). For more details, see Example 3.

[0058] Figure 18 The percentage of lysis of Daudi, Wien-133, Granta 519 and MEC-2 cells induced by CD38 antibody variants IgG1-B-E430G, IgG1-C-E430G in a chromium release ADCC assay is shown compared to CD38 antibodies IgG-B, IgG1-C and IgG1-b12-E430G. For more details, see Example 4.

[0059] Figure 19 Shown are dose-dependent FcγRIIIa crosslinking of CD38 antibody variants IgG1-A-E430G, IgG1-B-E430G, and IgG1-C-E430G in an ADCC reporter assay with T regulatory cells compared to CD38 antibodies IgG1-A, IgG1-B, IgG1-C, and an isotype control antibody. For more details, see Example 4.

[0060] Figure 20 Shown are the tumor sizes (mm) in mice treated with CD38 antibody variant IgG1-C-E430G or PBS (negative control). 3 ). See Example 9 for more details.

[0061] Figure 21The assay setup for measuring cytokinesis is shown. 1) Daudi cells are labeled with PKH-26 (membrane stain) and cell trace violet (cytoplasmic stain) and opsonized with CD38 antibody. 2) Labeled Daudi cells and macrophages are co-incubated at 37°C for 2 hours to allow macrophage attachment. 3) Cell membrane transfer or cytokinesis from Daudi cells to macrophages. 4) Disengagement of macrophage-Daudi interactions and degradation of Daudi cell membranes in macrophages. For more details, see Example 8.

[0062] Figure 22 Shown are complement-mediated cytotoxicity by IgG1-C-E430G or Darzalex® in bone marrow mononuclear cells from three newly diagnosed MM patients (A, B, and D) and one relapsed / refractory MM patient (C). Detailed Description of the Invention

[0064] In describing the embodiments of the present invention, specific terminology will be employed for the sake of clarity. However, the present invention is not intended to be limited to the specific terminology so selected, and it should be understood that each specific term includes all technical equivalents that operate in a similar manner to achieve a similar purpose.

[0065] definition

[0066] As used herein, the term "CD38" generally refers to human CD38 (UniProtKB - P28907 (CD38_HUMAN)) having the sequence set forth in SEQ ID NO: 38, but may also refer to variants, isoforms, and orthologs thereof unless inconsistent with the context. Variants of human CD38 having S274, Q272R, T237A, or D202G mutations are described in WO 2006 / 099875 A1 and WO 2011 / 154453 A1.

[0067] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all of which are potentially interconnected by disulfide bonds. The structure of immunoglobulins has been fully characterized. See, for example, Chapter 7 of Fundamental Immunology (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). In short, each heavy chain is generally composed of a heavy chain variable (VH) region and a heavy chain constant (CH) region. The CH region is generally composed of three domains, CH1, CH2, and CH3. The heavy chains are generally interconnected via disulfide bonds in the so-called "hinge region." Each light chain is generally composed of a light chain variable (VL) region and a light chain constant region, the latter generally consisting of a domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability also called complementarity determining regions (CDRs) (or hypervariable regions that can be hypervariable in the form of loops defined by sequence and / or structure), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)).

[0068] Unless otherwise indicated or contradicted by context, CDR sequences herein are identified according to IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999; 27: 209-212, and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also the internet http address www.imgt.org / ).

[0069] Unless otherwise indicated or contradicted by the context, references to amino acid positions in the CH or Fc region / Fc domain herein are based on EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1):78-85; Kabat et al., Sequences of proteins of immunological interest. 5th ed. - 1991 NIH Publication No. 91-3242). However, alternatively, amino acid residues in the CH of another isotype other than human IgG1 may be referred to by the corresponding amino acid position in the wild-type human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index. Specifically, the amino acid residues may be numbered as follows: Figure 1 , i.e., by (a) comparing the amino acid sequence of a non-IgG1 constant region (or a segment thereof) with the amino acid sequence of a human IgG1 heavy chain (or a segment thereof), wherein the amino acid residues are numbered according to the EU index, and (b) identifying those amino acid positions in the IgG1 heavy chain to which the amino acid residues are compared. Accordingly, the position of such amino acid residues may be referred to herein as "an amino acid residue at a position corresponding to ...," followed by the amino acid positions in the wild-type human IgG1 heavy chain numbered according to the EU index. When one or more of a plurality of different amino acid positions are mentioned, this may be referred to herein as "a mutation in one or more amino acid residues at a position selected from the group consisting of ...," "a mutation in one or more amino acid residues at a position corresponding to ...," or simply "a mutation in one or more amino acid residues selected from the group consisting of ...," followed by two or more amino acid positions (e.g., E430, E345, and S440) in the wild-type human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index.

[0070] As used herein, the term "hinge region" is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to EU numbering.

[0071] As used herein, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be of any other subtype as described herein.

[0072] As used herein, the term "CH3 region" or "CH3 domain" is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be of any other subtype as described herein.

[0073] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof that has the ability to specifically bind to an antigen. The antibodies of the present invention comprise the Fc domain and the antigen-binding region of an immunoglobulin. Antibodies generally contain two CH2-CH3 regions and a connecting region, such as a hinge region, such as at least one Fc domain. Therefore, the antibodies of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region or "Fc" region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. As used herein, unless otherwise contradicted by the context, the Fc region of an immunoglobulin typically contains at least the CH2 domain and the CH3 domain of the immunoglobulin CH, and may comprise a connecting region, such as a hinge region. The Fc region is generally in a dimerized form via, for example, a disulfide bridge connecting two hinge regions and / or non-covalent interactions between two CH3 regions. The dimer can be a homodimer (wherein the two Fc region monomer amino acid sequences are identical), or a heterodimer (wherein the two Fc region monomer amino acid sequences are different in one or more amino acids). Preferably, the dimer is a homodimer. As is well known in the art, the Fc region fragment of a full-length antibody can be generated, for example, by digesting the full-length antibody with papain. In addition to the Fc region and the antigen-binding region, an antibody as defined herein can further include one or both of the immunoglobulin CH1 region and the CL region. The antibody can also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody that has specificity for at least two different, typically non-overlapping, epitopes. Such epitopes can be on the same or different targets. If the epitope is on different targets, such targets can be on the same cell or different cells or cell types. As indicated above, unless otherwise indicated or clearly contradictory to the context, the term antibody herein includes fragments of antibodies that comprise at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments can be provided by any known technology, such as enzymatic cleavage, peptide synthesis, and recombinant expression technology. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" include, but are not limited to, monovalent antibodies (described by Genmab in WO2007059782); heavy chain antibodies, consisting of only two heavy chains and naturally occurring in, for example, camelids (e.g., Hamers-Casterman (1993) Nature 363:446);ThioMab (Roche, WO2011069104), a chain exchange engineering domain (SEED or Seedbody), which is an asymmetric and bispecific antibody-like molecule (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792), an asymmetric scaffold (AzymetricScaffold) (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), Xmab (Xencor), a dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); a dual domain diabody (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone / Eli Lilly), Knobs-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); Bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545), DuetMab (MedImmune, US2014 / 0348839), Electrostatically manipulated antibody formats (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2);Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus, WO2013157953), dual targeting domain antibodies (GSK / Domantis), two-in-one antibodies or dual-action Fabs recognizing two targets (Genentech, NovImmune, Adimab), cross-linked Mabs (Karmanos Cancer Center), covalently fused mAbs (AIMM), CovX bodies (CovX / Pfizer), FynomAb (Covagen / Janssen ilag), DutaMab (Dutalys / Roche), iMab (MedImmune), IgG-like bispecifics (ImClone / Eli Lilly, Shen, J. et al. J Immunol Methods, 2007. 318(1-2): 65-74), TIG bodies, DIG bodies and PIG bodies (Pharmabcine), dual affinity retargeting molecules (Fc-DART or Ig-DART from Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538), BEAT (Glenmark), Zybodies (Zyngenia), methods using common light chains (Crucell / Merus, US7262028) or common heavy chains (κλ bodies from NovImmune, WO2012023053), and fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc region, such as scFv fusions, such as BsAb from ZymoGenetics / BMS, Biogen HERCULES (US007951918) of Idec, SCORPIONS of Emergent BioSolutions / Trubion and Zymogenetics / BMS, Ts2Ab (MedImmune / AZ (Dimasi, N. et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusions of Genentech / Roche, scFv fusions of Novartis, scFv fusions of Immunomedics, scFv fusions of Changzhou Adam Biotech Inc (CN 102250246), TvAb of Roche (WO 2012025525, WO 2012025530), mAb of f-Star;2 (WO2008 / 003116) and dual scFv fusions. It should be understood that, unless otherwise indicated, the term antibody includes monoclonal antibodies (e.g., human monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, monospecific antibodies (e.g., bivalent monospecific antibodies), bispecific antibodies, antibodies of any isotype and / or allotype; for example, antibody mixtures (recombinant polyclonal) generated by the technology developed by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, and fusion proteins as described in WO2014 / 031646. Although these different antibody fragments and forms are generally included in the meaning of antibodies, they are collectively and independently unique features of the present invention, showing different biological properties and utilities.

[0074] As used herein, a "CD38 antibody" or "anti-CD38 antibody" is an antibody that specifically binds to the antigen CD38.

[0075] As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by in vitro random mutagenesis or site-specific mutagenesis or by in vivo somatic mutations). However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted onto human framework sequences.

[0076] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to preparations of Ab molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an Ab displaying a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs can be produced by hybridomas comprising non-human animals derived from transgenic or transchromosomal animals, such as transgenic mice, having a genome comprising a human heavy chain transgenic reservoir and a light chain transgenic reservoir, wherein the B cells are rearranged to produce functional human antibodies and fused with immortalized cells.

[0077] As used herein, "isotype" refers to the immunoglobulin class encoded by the heavy chain constant region gene, which includes, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgA2, IgE and IgM, as well as any of their allotypes, such as IgG1m(z), IgG1m(a), IgG1m(x), IgG1m(f), and mixed allotypes thereof, such as IgG1m(za), IgG1m(zax), IgG1m(fa), etc. (see, for example, de Lange, Experimental and Clinical Immunogenetics 1989; 6(1):7–17).

[0078] Furthermore, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain. The term "mixed isotype" is used herein to refer to an Fc region of an immunoglobulin generated by combining the structural features of one isotype with similar regions from another isotype, thereby generating a hybrid isotype. A mixed isotype can comprise an Fc region having a sequence consisting of two or more isotypes selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM, thereby generating a combination such as IgG1 / IgG3, IgG1 / IgG4, IgG2 / IgG3, IgG2 / IgG4, or IgG1 / IgA.

[0079] The term "full length antibody" when used herein refers to an antibody (e.g., a parent or variant antibody) that contains all heavy and light chain constant and variable domains corresponding to those normally found in a wild-type antibody of the isotype in question.

[0080] As used herein, a "full-length bivalent, monospecific monoclonal antibody" refers to a bivalent, monospecific antibody (e.g., a parent or variant antibody) formed by a pair of identical HCs and a pair of identical LCs, whose constant and variable domains correspond to those typically found in antibodies of the particular isotype in question.

[0081] As used herein, the terms "antigen-binding region," "antigen binding region," "binding region," or antigen binding domain refer to the region of an antibody that is capable of binding to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which can be further subdivided into regions of high variability (or hypervariable regions that can be hypervariable in the form of loops defined by sequence and / or structure) also known as complementarity determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). An antigen can be any molecule, such as a polypeptide, present on a cell.

[0082] As used herein, the term "target" refers to the molecule to which the antigen-binding region of an antibody binds. Targets include any antigen against which an antibody is raised. With respect to antibodies, the terms "antigen" and "target" are used interchangeably and have the same meaning and purpose with respect to any aspect or embodiment of the present invention.

[0083] The term "epitope" means a protein determinant that is capable of specific binding to an antibody variable domain. An epitope is typically composed of surface groupings of molecules such as amino acids, sugar side chains, or combinations thereof, and typically has specific three-dimensional structural characteristics and specific charge characteristics. The difference between a conformational epitope and a non-conformational epitope is that the binding to the former, rather than the latter, is lost in the presence of a denaturing solvent. An epitope can include amino acid residues that are directly involved in the binding (also referred to as the immunodominant component of the epitope) as well as other amino acid residues that are not directly involved in the binding.

[0084] As used herein, " variant " refers to a protein or polypeptide sequence that is different from a parent or reference sequence in one or more amino acid residues. Variant can, for example, have at least 80%, such as 90%, or 95%, or 97%, or 98%, or 99% sequence identity with a parent or reference sequence. Additionally or alternatively, variant can differ from a parent or reference sequence by 12 or less, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, such as replacement, insertion or deletion of amino acid residues. Accordingly, " variant antibody " or " antibody variant " used interchangeably herein refer to antibodies that are different in one or more amino acid residues, such as in the antigen binding region, Fc region or both, compared to a parent or reference antibody. Likewise, a "variant Fc region" or "Fc region variant" refers to an Fc region that differs in one or more amino acid residues compared to a parent or reference Fc region, optionally differing from the parent or reference Fc region amino acid sequence by 12 or fewer, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations, e.g., substitutions, insertions, or deletions of amino acid residues. The parent or reference Fc region is typically that of a human wild-type antibody, which may be of a specific isotype depending on the context. The variant Fc region may be in a dimeric form, either as a homodimer or a heterodimer, e.g., wherein one of the amino acid sequences of the dimerizing Fc region comprises a mutation and the other is identical to the parent or reference wild-type amino acid sequence. Examples of wild-type (typically parent or reference sequence) IgG CH and variant IgG constant region amino acid sequences are set forth in Table 1, which comprise Fc region amino acid sequences.

[0085] In the context of the present invention, conservative substitutions can be defined as substitutions within the following amino acid classes:

[0086] - Acidic residues: Asp (D) and Glu (E)

[0087] - Basic residues: Lys(K), Arg(R) and His(H)

[0088] - Hydrophilic non-charged residues: Ser (S), Thr (T), Asn (N) and Gln (Q)

[0089] - Aliphatic uncharged residues: Gly(G), Ala(A), Val(V), Leu(L) and Ile(I)

[0090] - Non-polar, non-charged residues: Cys (C), Met (M), and Pro (P)

[0091] - Aromatic residues: Phe(F), Tyr(Y) and Trp(W)

[0092] Alternative conservative amino acid residue substitution categories:

[0093] 1. AST

[0094] 2. DE

[0095] 3. NQ

[0096] 4. RK

[0097] 5. ILM

[0098] 6. FYW

[0099] Alternative physical and functional classification of amino acid residues:

[0100] - Residues containing alcohol groups: S and T

[0101] - Aliphatic residues: I, L, V and M

[0102] - Cycloalkenyl related residues: F, H, W and Y

[0103] - Hydrophobic residues: A, C, F, G, H, I, L, M, R, T, V, W, and Y

[0104] - Negatively charged residues: D and E

[0105] - Polar residues: C, D, E, H, K, N, Q, R, S and T

[0106] - Positively charged residues: H, K, and R

[0107] - Small residues: A, C, D, G, N, P, S, T, and V

[0108] - Very small residues: A, G, and S

[0109] - Residues involved in turn formation: A, C, D, E, G, H, K, N, Q, R, S, P, and T

[0110] - Flexible residues: Q, T, K, S, G, N, D, E, and R

[0111] As used herein, "sequence identity" refers to the percentage of identity between two sequences (i.e., percentage homology = # of identical positions / total # of positions x 100) as a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which gaps need to be introduced for optimal alignment of the two sequences. The percentage of identity between two nucleotide or amino acid sequences can, for example, be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970). Other tools for sequence alignment are publicly available on the internet and include, but are not limited to, Clustal Omega and EMBOSS Needle on the EMBL-EBI website www.ebi.ac.uk Typically, the default settings can be used.

[0112] In the context of the present invention, unless otherwise indicated, the following notation is used to describe mutations: the name of the amino acid that is mutated, followed by the position number of the mutation, followed by the content of the mutation. Thus, if the mutation is a substitution, the name of the amino acid that replaces the previous amino acid is included, if the amino acid is deleted, it is indicated by an "*", and if the mutation is an addition, the added amino acid is included after the original amino acid. Amino acid names can be single-letter or three-letter codes. Thus, for example: the substitution of glutamic acid at position 430 by glycine is referred to as E430G, the substitution of glutamic acid at position 430 by any amino acid is referred to as E430X, the deletion of glutamic acid at position 430 is referred to as E430*, and the addition of a proline after the glutamic acid at position E430 is referred to as E430EP.

[0113] As used herein, "immunosuppressive cells" refer to immune cells that can suppress the immune response in a subject, for example, by suppressing the activity of effector T cells and / or inhibiting T cell proliferation. Examples of such immunosuppressive cells include, but are not limited to, regulatory T cells (Treg), regulatory B cells (Breg), and myeloid-derived suppressor cells (MDSC). There are also immunosuppressive NK cells, NKT cells, macrophages, and antigen presenting cells (APC). An example of a phenotype of immunosuppressive NK cells is CD56. bright CD16 - .

[0114] "Regulatory T cells" or "'Tregs" or "Treg" refers to T lymphocytes that regulate the activity of other T cells and / or other immune cells, typically by suppressing their activity. Examples of Treg phenotypes are CD3 + CD4 + CD25 + CD127 dim Tregs may further express Foxp3. It should be understood that Tregs may not be completely restricted to this phenotype.

[0115] "Effector T cells" or "Teffs" or "Teff" refer to T lymphocytes that carry out immune response functions, such as killing tumor cells and / or activating anti-tumor immune responses that can lead to the elimination of tumor cells from the body. Examples of Teff phenotypes include CD3 + CD4 + and CD3 + CD8 + Teffs may secrete, contain or express markers such as IFNγ, granzyme B and ICOS. It should be understood that Teffs may not be completely restricted to these phenotypes.

[0116] "Myeloid-derived suppressor cells" or "MDSCs" or "MDSC" refers to a specific cell population of the hematopoietic lineage that expresses the macrophage / monocyte marker CD11b and the granulocyte marker Gr-1 / Ly-6G. An example of an MDSC phenotype is CD11b + HLA-DR - CD14 - CD33 + CD15 +MDSCs also typically show low or undetectable expression of the mature antigen-presenting cell markers MHC class II and F480. MDSCs are immature cells of the myeloid lineage and may further differentiate into other cell types, such as macrophages, neutrophils, dendritic cells, monocytes, or granulocytes. MDSCs can naturally occur in normal adult bone marrow in humans and animals, or in normal hematopoietic sites such as the spleen.

[0117] "Regulatory B cells" or "Breg" or "Bregs" refer to B lymphocytes that suppress immune responses. An example of a Breg phenotype is CD19 + CD24 + CD38 + Bregs can suppress immune responses by inhibiting T cell proliferation mediated by IL-10 secreted by Bregs. It is understood that other Breg subsets exist and are described in, for example, Ding et al. (2015) Human Immunology 76: 615-621.

[0118] As used herein, the term "effector cell" refers to an immune cell related to the effector phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils. Some effector cells express Fc receptors (FcR) or complement receptors and perform specific immune functions. In some embodiments, effector cells such as natural killer cells can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells and Kupffer cells expressing FcR, relate to the specific killing of target cells and / or present antigens to other components of the immune system, or are combined with cells presenting antigens. In some embodiments, ADCC can be further enhanced by the classical complement activation driven by antibodies, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands of complement receptors (CR) such as CR3 expressed on myeloid cells. Complement fragments can promote enhanced Fc receptor-mediated ADCC by the recognition of CR on effector cells. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products may promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can engulf target antigens, target particles or target cells, which may depend on antibody binding and mediation of FcγR expressed by effector cells. The expression of specific FcR or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, it has been found that the expression of FcγRI is upregulated by interferon γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of cells carrying FcγRI against targets. Effector cells can engulf target antigens or engulf or lyse target cells. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products can promote phagocytosis directly by effector cells or indirectly by enhancing antibody-mediated phagocytosis.

[0119] As used herein, the term "Fc effector function" is intended to refer to functions that are a consequence of binding of a polypeptide or antibody to its target (e.g., an antigen) on a cell membrane, wherein the Fc effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-γ receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) complement receptor binding mediated by antibodies with opsonizing antibodies, (x) opsonization, (xi) cytotoxicity, and (xii) any combination of (i) to (xi).

[0120] As used herein, the term "complement activation" refers to the activation of the classical complement pathway, which is initiated by the binding of a macromolecular complex called C1 to an antibody-antigen complex on the surface. C1 is a complex composed of six recognition proteins, C1q, and a heterotetramer, C1r2C1s2, of a serine protease. C1 is the first protein complex in the early events of the classical complement cascade, which involves a series of cleavage reactions that begin with the cleavage of C4 into C4a and C4b and the cleavage of C2 into C2a and C2b. C4b is deposited and, together with C2a, forms an enzymatically active convertase called C3 convertase, which cleaves the complement component C3 into C3b and C3a, forming the C5 convertase. This C5 convertase breaks down C5 in C5a and C5b, and the last component is deposited on the membrane and, in turn, triggers the late events of complement activation, in which the terminal complement components C5b, C6, C7, C8, and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores in the cell membrane, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation can be measured using C1q efficacy, CDC kinetics CDC assays (as described in WO2013 / 004842, WO2014 / 108198), or by the method described in Beurskens et al., J Immunol April 1, 2012, Vol. 188, No. 7, 3532-3541. Cellular deposition of C3b and C4b ( C3b and C4b Cell deposition) was assessed.

[0121] As used herein, the term "complement dependent cytotoxicity" (CDC) is intended to refer to the process of antibody-mediated complement activation, which results in lysis of cells to which the antibody is bound, and without being bound by theory, is believed to be the result of pores in the membrane that are created by the assembly of the so-called membrane attack complex (MAC). Suitable assays for evaluating CDC are known in the art and include, for example, in vitro assays in which normal human serum is used as a source of complement, as described in Example 3. A non-limiting example of an assay for determining the maximum lysis of CD38 expressing cells, or EC50 value, as mediated by a CD38 antibody, may include the following steps:

[0122] (a) Approximately 100,000 CD38-expressing cells / well were plated in 40 µL of culture medium supplemented with 0.2% BSA in a multi-well plate.

[0123] (b) Cells were preincubated with 40 μL of serially diluted CD38 antibody (0.0002-10 μg / mL) for 20 min;

[0124] (c) incubating each well with 20% pooled normal human serum at 37°C for 45 minutes;

[0125] (d) Viability dye was added, and the percentage of cell lysis was measured on a flow cytometer;

[0126] (e) Nonlinear regression was used to determine maximum lysis and / or calculate EC50 values.

[0127] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" ("ADCC") is intended to refer to a mechanism by which antibody-coated target cells are killed by cells expressing Fc receptors that recognize the constant region of the bound antibody. Suitable assays for evaluating ADCC are known in the art and include, for example, the assay described in Example 4. Non-limiting examples of assays for determining ADCC of CD38-expressing cells, such as mediated by CD38 antibodies, can include the assays described below. 51 Steps for Cr release assay or reporter assay.

[0128] use 51 Cr release assay to determine ADCC

[0129] (a) Approximately 5,000 cells were plated in 50 µL of culture medium supplemented with 0.2% BSA in a multiwell plate. 51 Cr-labeled CD38-expressing cells (e.g., Daudi cells) / well;

[0130] (b) Cells were preincubated with 50 μL of serially diluted CD38 antibody (0.0002-10 μg / mL) for 15 min;

[0131] (c) incubating each well with 500,000 freshly isolated peripheral blood mononuclear cells (PBMCs) / well at 37°C for 4 hours;

[0132] (d) Measure the amount of γ in 75 μL of the supernatant on a gamma counter. 51 Cr release;

[0133] (e) The percentage of cell lysis was calculated as (cpm sample - cpm spontaneous lysis) / (cpm maximum lysis - cpm spontaneous lysis), where cpm is counts per minute.

[0134] Determination of ADCC using a reporter assay

[0135] (a) approximately 5,000 CD38-expressing cells (e.g., Daudi cells) are plated in 10 μL of standard culture medium (e.g., RPMI 1640) supplemented with 25% low IgG serum in multiwell plates suitable for optical reading (e.g., 384-well OptiPlates from PerkinElmer Inc.);

[0136] (b) Each well was incubated with 10 μL of engineered Jurkat cells stably expressing the FcγRIIIa receptor, the V158 (high affinity) variant, and a NFAT response element driving firefly luciferase expression as effector cells and 10 μL of serially diluted CD38 antibody (0.0002-10 μg / mL) at 37°C for 6 hours;

[0137] (c) Each well was incubated with 30 μL of luciferase substrate for 5 minutes at RT and luminescence was measured.

[0138] As used herein, the term "antibody-dependent cellular phagocytosis" ("ADCP") is intended to refer to a mechanism by which antibody-coated target cells are eliminated by internalization via phagocytes. The internalized antibody-coated target cells are contained in vesicles called phagosomes, which then fuse with one or more lysosomes to form phagolysosomes. Suitable assays for evaluating ADCP are known in the art and include, for example, in vitro cytotoxicity assays and video microscopy with macrophages as effector cells, as described by van Bij et al. in Journal of Hepatology, Vol. 53, No. 4, October 2010, pp. 677-685, and in vitro cytotoxicity assays as described in Example 5. A non-limiting example of an assay for determining ADCP of CD38-expressing cells, as mediated by a CD38 antibody, may include the following steps:

[0139] (a) Freshly isolated monocytes were differentiated into macrophages by 5 days of incubation in medium containing GM-CSF.

[0140] (b) Approximately 100,000 macrophages were plated per well in a multi-well plate in dendritic cell culture medium with GM-CSF.

[0141] (c) adding 20,000 CD38-expressing cells (e.g., Daudi cells) opsonized with CD38 antibodies / well for 45 minutes at 37°C, wherein the cells are labeled with a universal fluorescent membrane dye;

[0142] (d) The percentage of CD14-positive, CD19-negative, membrane dye-positive macrophages was measured on a flow cytometer.

[0143] As used herein, "cytosis" refers to a process characterized by the transfer of cell surface molecules from donor cells to recipient cells (e.g., effector cells). Common recipient cells include T cells and B cells, monocytes / macrophages, dendritic cells, neutrophils, and NK cells. The transfer of cell surface molecules (e.g., CD38) mediated by cytosis from donor cells to recipient cells may also result in the transfer of antibody-antigen complexes from donor cells to recipient cells, i.e., antibody-antigen complexes in which antibodies are bound to cell surface molecules. In particular, when the recipient cells are effector cells expressing Fc-γ receptors (FcγRs), a specialized form of cytosis may occur; typically, after FcγRs bind to the Fc region of the antibody, these recipient cells can ingest and internalize donor cell-associated immune complexes, which are composed of specific antibodies bound to target antigens on the donor cells. Suitable assays for assessing cytosis are known in the art and include, for example, assays in Example 8. Non-limiting examples of assays for determining the cytosis of CD38-expressing cells as mediated by CD38 antibodies include the following:

[0144] Cytokinesis (Daudi cells):

[0145] (a') Freshly isolated monocytes were differentiated into macrophages using GM-CSF for 5 days;

[0146] (b') Approximately 100,000 macrophages were plated per well in dendritic cell culture medium with GM-CSF.

[0147] (c') Approximately 20,000 CD38 antibody-opsonized Daudi cells / well were added and labeled with a universal fluorescent membrane dye at 37°C for 45 minutes;

[0148] (d') CD38 expression on Daudi cells was measured on a flow cytometer, where a decrease in CD38 on CD38 antibody-opsonized Daudi cells compared to controls indicates cytotoxicity.

[0149] Tregs:

[0150] (a) Plate approximately 500,000 freshly isolated PBMCs / well in cell culture medium at 37°C overnight.

[0151] (b) adding approximately 100,000 CD38 antibody-opsonized Tregs / well at 37°C overnight (O / N), wherein the Tregs are labeled with a universal fluorescent intracellular amine dye; and

[0152] (c) CD38 expression on Tregs was measured on a flow cytometer, where decreased CD38 on CD38 antibody-opsonized Tregs compared to controls indicates cytotoxicity.

[0153] A control can be selected by a skilled artisan based on the specific purpose of the study or assay in question. However, non-limiting examples of controls include (i) the absence of any antibody and (ii) an isotype control antibody. An example of an isotype control antibody is antibody b12, which has the VH and VL sequences described in Table 1. In some embodiments where it is desired to assess the cytotoxicity effects of antibody variants as described herein, a control can be (iii) a parent or reference antibody with a different antigen binding region and / or a different Fc region.

[0154] In some embodiments, in step (b), Tregs are labeled with a general fluorescent membrane dye in addition to or instead of the fluorescent intracellular amine dye.

[0155] In some embodiments, in steps (d') and (c) of the cytotoxicity assay outlined above, the reduction of CD38 antibodies on donor cells can also be measured. For example, in cases where the CD38 antibody is a human IgG (huIgG) antibody, a secondary antibody can be used to detect huIgG.

[0156] In addition to Daudi cells (ATCC CCL-213), tumor cells suitable for the first assay include, but are not limited to, those listed in Table 2, particularly those with high CD38 expression.

[0157] In addition to Tregs, suitable CD38-expressing cells for the second assay include immune cells, such as NK cells, B cells, T cells, and monocytes, and tumor cells listed in Table 2, particularly those with low CD38 expression levels.

[0158] As used herein, the term "vector" is intended to refer to a nucleic acid molecule that can induce transcription of a nucleic acid segment connected to a vector. One type of vector is a "plasmid," which is a circular double-stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid segment can be connected to a viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell and thereby replicated together with the host genome. In addition, certain vectors can guide the expression of genes to which they are operably connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention contemplates expression vectors of this type, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), that function in an equivalent manner.

[0159] As used herein, the term "recombinant host cell" (or simply, "host cell") is intended to refer to a cell into which one or more expression vectors have been introduced. For example, both the HC and LC of the antibody variants as described herein can be encoded by the same expression vector, and the host cell can be transfected with the expression vector. Alternatively, the HC and LC of the antibody variants as described herein can be encoded by different expression vectors, and the host cell can be co-transfected with the expression vector. It should be understood that the term "host cell" is intended to refer not only to the specific subject cell, but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli ( E. coli ) and other eukaryotic hosts, such as plant cells and fungi.

[0160] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express Ab or target antigen, such as CHO cells, PER.C6, NSO cells, HEK-293 cells, plant cells, or fungi, including yeast cells.

[0161] The term "treatment" refers to the administration of an effective amount of a therapeutically active antibody variant of the invention for the purpose of alleviating, ameliorating, preventing or eradicating (curing) the symptoms or disease state.

[0162] The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at the dosages and for the necessary time periods. A therapeutically effective amount of an antibody can vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects of the antibody variant outweigh any toxic or detrimental effects.

[0163] Specific embodiments of the present invention

[0164] As described above, the present invention relates to antibodies that are variants of anti-CD38 antibody C, particularly those comprising a variant Fc region comprising a mutation in one or more amino acid residues selected from those corresponding to E430, E345 and S440 in a human IgG1 heavy chain.

[0165] As shown in Example 3, after introduction of the E430G mutation, CDC was enhanced for all three tested CD38 IgG1 antibodies—A, B, and C. However, surprisingly, the magnitude of the CDC enhancement varied among the antibody clones tested. Without the E430G mutation, IgG1-B was already a good CDC inducer, while IgG1-C and IgG1-A induced moderate and no CDC, respectively. However, after introduction of the E430G mutation, IgG1-C-E430G induced more potent CDC compared to IgG1-B-E430G. In particular, the EC50 value of IgG1-C-E430G was lower than that of IgG1-B-E430G in tumor cells and T regulatory cells with lower CD38 expression levels.

[0166] In addition, antibody variants according to the present invention may also exhibit ADCC. For example, as shown in Example 4, 51 IgG1-C achieved a higher maximum lysis percentage compared to IgG1-B in the Cr release assay and increased FcγRIIIa binding compared to IgG1-B in the ADCC reporter assay. For all three antibodies, the introduction of the E430G mutation reduced 51 IgG1-C-E430G induced the maximum percentage of lysis in the Cr release assay and FcγRIIIa binding in the ADCC reporter assay. 51 Similar maximal percentage lysis compared to IgG1-B-E430G and IgG1-A-E430G in the Cr release assay, and similar FcγRIIIa binding in the ADCC reporter assay.

[0167] Furthermore, the ability of anti-CD38 antibodies to inhibit CD38 cyclase activity can be retained in the form of antibody variants according to the present invention. For example, as shown in Example 7, IgG1-C-E430G exhibited stronger inhibition of CD38 cyclase activity compared to IgG1-B-E430G, with the former resulting in about 40% inhibition and the latter resulting in about 25% inhibition. Without being limited by theory, stronger inhibition of CD38 cyclase activity may reduce the production of cADPR, which is a regulator of Ca from the cytoplasm. 2+ Mobilization of potent second messengers, which in turn may lead to reduced Ca 2+ Mobilization and signaling of downstream pathways that control various biological processes, such as proliferation and insulin secretion, are reduced. Without being limited by theory, greater inhibition of CD38 cyclase activity may therefore affect, for example, reducing the ability of immunosuppressive cells to suppress the immune response.

[0168] Other functions that can be modulated include cytotoxicity. Specifically, CD38 expression on Daudi cells was significantly reduced by co-culturing with macrophages and CD38 antibodies; however, the reduction in CD38 expression was strongest with the E430G mutated antibody (Example 8). Surprisingly, CD38 expression on T regulatory cells co-cultured with PBMCs was reduced only after incubation with the E430G mutated CD38 antibody; no reduction in CD38 expression was found when T regulatory cells were incubated with antibody B. Without being limited by theory, the ability of the antibody variants according to the present invention to induce cytotoxicity of non-cancerous immune cells expressing CD38, particularly immunosuppressive cells, may lead to an increased immune response against tumor cells in cancer patients, regardless of whether the tumor cells express CD38.

[0169] Antibody variants of the invention may also be able to kill tumor cells in vivo, as shown in Example 9, where twice-weekly doses of IgG1-C-E430G reduced tumor growth in two of the five tested DLBCL PDX models with the highest CD38 mRNA expression.

[0170] Thus, in one aspect, the present invention provides an antibody variant that binds to human CD38, comprising an antigen-binding region comprising the VH and VLCDRs of antibody C as shown in SEQ ID NO: 2 (VH-3003-C_CDR1), SEQ ID NO: 3 (VH-3003-C_CDR2), SEQ ID NO: 4 (VH-3003-C_CDR3), SEQ ID NO: 6 (VL-3003-C_CDR1), AAS (VL-3003-C_CDR2) and SEQ ID NO: 7 (VL-3003-C_CDR3) in Table 1, and a variant Fc region comprising a mutation in one or more amino acid residues selected from the group consisting of E430, E345 and S440 corresponding to a human IgG1 heavy chain.

[0171] In one embodiment, an antibody variant that binds to human CD38 comprises

[0172] (a) an antigen-binding region comprising a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VLCDR3 having the sequence shown in SEQ ID NO: 7, and

[0173] (b) a variant Fc region comprising a mutation in one or more amino acid residues selected from the group consisting of amino acid residues corresponding to E430, E345 and S440 in a human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index.

[0174] In further embodiments, antibody variants may also or alternatively be characterized by specific amino acid sequences or specific mutations in the antigen binding region or Fc region, and / or their ability to induce effector function or modulate CD38 enzymatic activity. These are further described below.

[0175] Antigen binding region and variable region

[0176] The antigen binding region comprises one or more antibody variable domains that allow specific binding to CD38, such as a VH region and a VL region. Similarly, the heavy chain and light chain comprise a VH and a VL region, respectively. Hereinafter, reference to the sequence in the antigen binding region can be similarly applied to the sequence of the heavy chain and / or light chain of the variant antibody according to the present invention. Advantageously, the CDR, VH region and / or VL region are similar or identical to those of Antibody C, as shown in Table 1.

[0177] In a preferred embodiment, the antigen binding region, and / or the heavy and / or light chain comprises the CDRs of antibody C as shown in SEQ ID NO: 2 (VH-3003-C_CDR1), SEQ ID NO: 3 (VH-3003-C_CDR2), SEQ ID NO: 4 (VH-3003-C_CDR3), SEQ ID NO: 6 (VL-3003-C_CDR1), AAS (VL-3003-C_CDR2) and SEQ ID NO: 7 (VL-3003-C_CDR3). In another preferred embodiment, the VH and VL sequences are those of antibody C, i.e., the VH region comprises the sequence of SEQ ID NO: 1 (VH-3003-C) and the VL region comprises the sequence of SEQ ID NO: 5 (VL-3003-C).

[0178] However, it is well known in the art that mutations in the VH and VL of an antibody can be prepared to, for example, increase the affinity of the antibody to its target antigen, reduce its potential immunogenicity, and / or increase the productive rate of the antibody expressed by the host cell. Accordingly, in some embodiments, variants comprising the CDR, VH and / or VL sequences of antibody C, particularly antibodies of the functional variants in the VL and / or VH district of antibody C are also contemplated. Compared with parent VH and / or VL sequences, functional variants can be different, for example, in one or more amino acids in one or more CDRs, but still allow the antigen-binding region to retain at least a basic ratio (at least about 50%, 60%, 70%, 80%, 90%, 95% or higher) of the affinity and / or specificity of the parent antibody. Typically, such functional variants retain significant sequence identity with the parent sequence. Exemplary variants include those that differ from the respective parent VH or VL regions by 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, such as substitutions, insertions or deletions of amino acid residues. Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of the parent sequence primarily by conservative amino acid substitutions; for example, 12, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions in the variant may be conservative. In some cases, antibodies comprising variants of the VH and / or VL of antibody C may be associated with greater affinity and / or specificity than the parent antibody. For the purposes of the present invention, particularly preferred are VH and / or VL variants that allow the antibody to retain or improve affinity and specificity in its binding to CD38.

[0179] For example, WO 2011 / 154453 A1 discloses CD38 antibodies comprising suitable variant CDR, VH and VL region amino acid sequences, wherein the amino acid residues at certain positions differ from those in the CDR, VH and VL of Antibody C as shown in Table 1. Thus, these positions represent candidate positions at which mutations in the CDR, VH and VL sequences can be made while retaining or improving the affinity and specificity of the antibody in its binding to CD38. In particular, positions in the VH and VL CDRs that can be mutated in functional variants of the VH and VL of Antibody C are indicated in SEQ ID NOs: 40 to 43.

[0180] Thus, in some embodiments, one or more specific mutations are made in the CDRs as shown in SEQ ID NOs: 40 to 43, i.e., any functional variant of the VH and / or VL region comprises a mutation in the CDRs as shown in one or more of SEQ ID NOs: 40 (VH CDR1), SEQ ID NOs: 41 (VH CDR2), SEQ ID NOs: 42 (VH CDR3), and SEQ ID NOs: 44 (VL CDR3). The VH and VL regions of such antibody variants may optionally maintain the original framework regions of antibody C. In a specific embodiment, the antigen binding region comprises the CDRs as shown in SEQ ID NO: 40 wherein Xi is S (VHCDR1), SEQ ID NO: 41 wherein Xi is R, X2 is K, X3 is A (VH CDR2), SEQ ID NO: 42 wherein Xi is A, X2 is D, and X3 is V (VH CDR3), SEQ ID NO: 43 (VL CDR1), AAS (VL CDR2), and SEQ ID NO: 44 wherein Xi is S (VL CDR3). In a specific embodiment, the antigen binding region comprises the CDRs as shown in SEQ ID NO: 40 wherein Xi is R (VH CDR1), SEQ ID NO: 41 wherein Xi is V, X2 is K, X3 is T (VH CDR2), SEQ ID NO: 42 wherein Xi is T, X2 is A, and X3 is F (VH CDR3), SEQ ID NO: 43 (VL CDR1), AAS (VL CDR2), and SEQ ID NO: 44 wherein Xi is N (VL CDR3). In a specific embodiment, the antigen binding region comprises the CDRs as shown in SEQ ID NO: 40 wherein Xi is S (VH CDR1), SEQ ID NO: 41 wherein Xi is R, X2 is K, X3 is T (VH CDR2), SEQ ID NO: 42 wherein Xi is A, X2 is D, and X3 is V (VH CDR3), SEQ ID NO: 43 (VL CDR1), AAS (VL CDR2), and SEQ ID NO: 44 wherein Xi is S (VL CDR3).In a specific embodiment, the antigen binding region comprises the CDRs as shown in SEQ ID NO: 40 wherein Xi is R (VH CDR1), SEQ ID NO: 41 wherein Xi is V, X2 is K, X3 is V (VH CDR2), SEQ ID NO: 42 wherein Xi is T, X2 is A, and X3 is F (VH CDR3), SEQ ID NO: 43 (VLCDR1), AAS (VL CDR2), and SEQ ID NO: 44 wherein Xi is N (VL CDR3).

[0181] In some embodiments, no mutations are made in the CDRs, i.e., any functional variants of the VH and / or VL regions retain the CDR sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6, AAS, SEQ ID NO: 7, which represent the VH CDR1-3 or VL CDR1-3 sequences of antibody C, respectively.

[0182] In one embodiment, the VH region comprises SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, such as 90% or 95% or 97% or 98% or 99% identical. For example, the VH may differ from SEQ ID NO: 1 by 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations, such as substitutions, insertions, or deletions of amino acid residues. In one embodiment, the VH region differs from SEQ ID NO: 1 by only 12 or fewer, such as 5 or fewer, such as 5, 4, 3, 2, or 1 amino acid substitutions. The amino acid substitutions may, for example, be conservative amino acid substitutions as described elsewhere herein. In a specific embodiment, no mutations are made in the VH CDRs, i.e., any variant VH retains the C CDR sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0183] In one embodiment, the VL region comprises SEQ ID NO: 5, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 5, such as 90% or 95% or 97% or 98% or 99% identical. For example, the VL may differ from SEQ ID NO: 5 by 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations, such as substitutions, insertions, or deletions of amino acid residues. In one embodiment, the VL region differs from SEQ ID NO: 5 by only 12 or fewer, such as 5 or fewer, such as 5, 4, 3, 2, or 1 amino acid substitutions. The amino acid substitutions may, for example, be conservative amino acid substitutions as described elsewhere herein. In a specific embodiment, no mutations are made in the VL CDRs, i.e., any variant VH retains the C CDR sequences set forth in SEQ ID NO: 6, AAS, or SEQ ID NO: 7.

[0184] In one embodiment, the antibody variant comprises a VH region comprising the sequence of SEQ ID NO:1, and a VL region comprising the sequence of SEQ ID NO:5.

[0185] Variant Fc and CH regions

[0186] Mutations in the amino acid residues at positions corresponding to E430, E345, and S440 in the human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index, can improve the ability of the antibody to induce CDC (see, e.g., Example 3). Without being bound by theory, it is believed that by substituting one or more amino acids at these positions, oligomerization of the antibody can be stimulated, thereby modulating effector functions such as increasing C1q binding, complement activation, CDC, ADCP, internalization, or other related functions that may provide in vivo efficacy.

[0187] The present invention relates to variant antibodies comprising an antigen binding region and a variant Fc region.

[0188] In certain embodiments, the antibody variant that binds to human CD38 comprises

[0189] (a) a heavy chain comprising a VH region comprising a VH CDR1 having the sequence as shown in SEQ ID NO: 2, a VH CDR2 having the sequence as shown in SEQ ID NO: 3, and a VH CDR3 having the sequence as shown in SEQ ID NO: 4, and a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, wherein the amino acid residues are numbered according to the EU index;

[0190] (b) A light chain comprising a VL region comprising a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7.

[0191] In certain other embodiments, the antibody variant that binds to human CD38 comprises

[0192] (a) a heavy chain comprising a VH region comprising SEQ ID NO: 1, and a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, the amino acid residues being numbered according to the EU index, and

[0193] (b) A light chain comprising a VL region comprising SEQ ID NO:5.

[0194] The variant antibodies of the present invention comprise a variant Fc region or human IgG1 CH region comprising a mutation in one or more of E430, E345 and S440. Hereinafter, references to mutations in the Fc region may similarly apply to mutations in the human IgG1 CH region.

[0195] As described herein, the amino acid position to be mutated in the Fc region can be given relative to (i.e., "corresponds to") its position in a naturally occurring (wild-type) human IgG1 heavy chain when numbered according to the EU index. Thus, if the parent Fc region already contains one or more mutations, and / or if the parent Fc region is, for example, an IgG2, IgG3 or IgG4 Fc region, the amino acid position corresponding to an amino acid residue in a human IgG1 heavy chain numbered according to the EU index, such as, for example, E430, can be determined by alignment. Specifically, the parent Fc region is aligned with the wild-type human IgG1 heavy chain sequence in order to identify the residue in the position corresponding to E430 in the human IgG1 heavy chain sequence. Any wild-type human IgG1 constant region amino acid sequence can be used for this purpose, including any of the different human IgG1 allotypes shown in Table 1. This is described in more detail in the accompanying drawings. Figure 1 As shown in Figure 1 Shown is an alignment between two different human IgG1 allotypes - IgG1m(f) and IgG1m(a) - and wild-type human IgG2, IgG3, and IgG4, specifically corresponding to the segment from residues P247 to K447 in the human IgG1 heavy chain, where the amino acid residues are numbered according to the EU index.

[0196] Accordingly, in the remainder of this section and elsewhere herein, unless otherwise indicated or contradicted by context, references to amino acid positions are to those corresponding to amino acid residues in a wild-type human IgG heavy chain, where the amino acid residues are numbered according to the EU index:

[0197] In separate and specific embodiments, the variant Fc region and / or human IgG1 CH region comprises a mutation in: only one of E430, E345, and S440; both E430 and E345; both E430 and S440; both E345 and S440; or all of E430, E345, and S440. In some embodiments, the variant Fc region and / or human IgG1 CH region comprises a mutation in: only one of E430, E345, and S440; both E430 and E345; both E430 and S440; both E345 and S440; or all of E430, E345, and S440, with the proviso that any mutation in S440 is S440W or S440Y. In other separate and specific embodiments, the mutation is an amino acid substitution. In one embodiment, the mutation is an amino acid substitution among: only one of E430X, E345X, and S440X; both E430X and E345X; both E430X and S440X; both E345X and S440X; or all of E430X, E345X, and S440X, preferably with the proviso that any mutation in S440X is S440Y or S440W. More preferably, the E430X, E345X, and S440X mutations are individually selected from E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W.

[0198] In one embodiment, the mutation in one or more amino acid residues is selected from E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W.

[0199] In a preferred embodiment, the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E345K, E430S and E345Q.

[0200] In one embodiment, the mutation is in the amino acid residue corresponding to E430, such as the amino acid substitution E430X, for example, selected from those corresponding to E430G, E430S, E430F, or E430T. In a preferred embodiment, the mutation in one or more amino acid residues comprises E430G. In another preferred embodiment, the mutation in one or more amino acid residues comprises E430S, optionally wherein no mutation is made in the amino acid residues corresponding to E345 and S440. In a particularly preferred embodiment, the mutation in one or more amino acid residues consists of E430G, i.e., no mutation is made in the amino acid residues corresponding to E345 and S440.

[0201] In one embodiment, the mutation is in the amino acid residue corresponding to E345, such as the amino acid substitution E345X, for example selected from those corresponding to E345K, E345Q, E345R and E345Y. In a preferred embodiment, the mutation in the one or more amino acid residues comprises E345K. In another preferred embodiment, the mutation in the one or more amino acid residues comprises E345Q, optionally wherein no mutation is made in the amino acid residues corresponding to E430 and S440. In a particularly preferred embodiment, the mutation in the one or more amino acid residues consists of E345K, i.e., no mutation is made in the amino acid residues corresponding to E430 and S440.

[0202] In one embodiment, the mutation is in the amino acid residue corresponding to S440, such as the amino acid substitution S440X, typically selected from those corresponding to S440Y and S440W. In a preferred embodiment, the mutation in one or more amino acid residues comprises S440W, optionally wherein no mutation is made in the amino acid residues corresponding to E430 and E345. In a preferred embodiment, the mutation in one or more amino acid residues comprises S440Y, optionally wherein no mutation is made in the amino acid residues corresponding to E430 and E345.

[0203] Preferably, the antibody variant comprises a variant Fc district according to any one of the aforementioned sections, the variant Fc district being a variant of the human IgG Fc district selected from human IgG1, IgG2, IgG3 and IgG4 Fc districts. That is, the mutation corresponding to one or more amino acid residues in E430, E345 and S440 is prepared in a parent Fc district, the parent Fc district being a human IgG Fc district selected from IgG1, IgG2, IgG3 and IgG4 Fc districts. Preferably, the parent Fc district is a naturally occurring (wild type) human IgG Fc district, such as human wild type IgG1, IgG2, IgG3 or IgG4 Fc district, or a mixed isotype thereof. Therefore, except for the mutation (selected from one or more amino acid residues corresponding to E430, E345 and S440), the variant Fc district can be a human IgG1, IgG2, IgG3 or IgG4 isotype, or a mixed isotype thereof.

[0204] In one embodiment, the parent Fc region and / or human IgG1 CH region is a wild-type human IgG1 isotype.

[0205] Thus, except for the mutation (in one or more amino acid residues selected from those corresponding to E430, E345 and S440), the variant Fc region may be a human IgG1 Fc region.

[0206] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(f) isotype.

[0207] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(z) isotype.

[0208] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(a) isotype.

[0209] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(x) isotype.

[0210] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is a human wild-type IgG1 of mixed allotype, such as IgG1m(za), IgG1m(zax), IgG1m(fa), and the like.

[0211] Thus, except for the mutations (in one or more amino acid residues selected from those corresponding to E430, E345 and S440), the variant Fc region and / or human IgG1 CH region may be of the human IgG1m(f), IgG1m(a), IgG1m(x), IgG1m(z) allotype, or a mixed allotype of any two or more thereof.

[0212] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of human wild-type IgG1m(za) isotype.

[0213] In a specific embodiment, the parent Fc region is a human wild-type IgG2 isotype.

[0214] In a specific embodiment, the parent Fc region is a human wild-type IgG3 isotype.

[0215] In a specific embodiment, the parent Fc region is a human wild-type IgG4 isotype.

[0216] CH region amino acid sequences for specific examples of wild-type human IgG isotypes and IgG1 allotypes are set forth in Table 1. In some embodiments, the parent Fc region comprises a CH2-CH3 or, optionally, a hinge-CH2-CH3 segment of such a wild-type CH region amino acid sequence.

[0217] Thus, in a specific embodiment, the parent Fc region is a human wild-type IgG1 isotype comprising amino acid residues corresponding to 231-447 in a human IgG1 heavy chain according to EU numbering. For example, the parent Fc region may comprise amino acid residues 114 to 330 (direct numbering) selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23. In a specific embodiment, the parent Fc region is a human wild-type IgG1 isotype comprising amino acid residues corresponding to 216-447 in a human IgG1 heavy chain according to EU numbering. For example, the parent Fc region may comprise amino acid residues 99 to 330 (direct numbering) selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23. As described elsewhere herein, for the production of therapeutic antibodies, the C-terminal amino acid K447 may sometimes be deleted or removed. Thus, the parent Fc region may comprise amino acid residues 114 to 329 (direct numbering) or amino acid residues 99 to 329 (direct numbering) of SEQ ID NO: 45.

[0218] In a specific embodiment, the variant Fc region is a variant of the human wild-type IgG1 isotype comprising amino acid residues corresponding to 231-447 in a human IgG1 heavy chain according to EU numbering. For example, the variant Fc region may comprise amino acid residues 114 to 330 (direct numbering) of a sequence selected from SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33. In another embodiment, the variant Fc region may comprise amino acid residues 114 to 329 (direct numbering) of SEQ ID NO: 46.

[0219] In a specific embodiment, the variant Fc region is a variant of the human wild-type IgG1 isotype comprising amino acid residues corresponding to 216-447 in a human IgG1 heavy chain according to EU numbering. For example, the variant Fc region may comprise amino acid residues 99 to 330 (direct numbering) of a sequence selected from SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33. In another embodiment, the variant Fc region may comprise amino acid residues 99 to 329 (direct numbering) of SEQ ID NO: 46.

[0220] Thus, the present invention can be applied to antibody molecules having a human IgG1 heavy chain, for example, a human IgG1 heavy chain comprising a human IgG1 CH region amino acid sequence comprising SEQ ID NO: 19 (IgGm(za). Thus, except for the mutation, the human IgG1 CH region may comprise the sequence of SEQ ID NO: 19.

[0221] The present invention can also be applied to antibody molecules having a human IgG1 heavy chain, for example, a human IgG1 heavy chain comprising a human IgG1 CH region amino acid sequence comprising SEQ ID NO: 20 (IgGm(f)) or SEQ ID NO: 45. Thus, the human IgG1 CH region can comprise the sequence of SEQ ID NO: 20, except for the mutation. In another embodiment, the human IgG1 CH region can comprise the sequence of SEQ ID NO: 45, except for the mutation.

[0222] The present invention can also be applied to antibody molecules having a human IgG1 heavy chain, for example, a human IgG1 heavy chain comprising a human IgG1 CH region amino acid sequence comprising SEQ ID NO: 21 (IgGm(z)). Thus, in addition to the mutation, the human IgG1 CH region can comprise the sequence of SEQ ID NO: 21.

[0223] The present invention can also be applied to antibody molecules having a human IgG1 heavy chain, for example, a human IgG1 heavy chain comprising a human IgG1 CH region amino acid sequence comprising SEQ ID NO: 22 (IgGm(a)). Thus, in addition to the mutation, the human IgG1 CH region can comprise the sequence of SEQ ID NO: 22.

[0224] The present invention can also be applied to antibody molecules having a human IgG1 heavy chain, for example, a human IgG1 heavy chain comprising a human IgG1 CH region amino acid sequence comprising SEQ ID NO: 23 (IgG1m(x)). Thus, in addition to the mutation, the human IgG1 CH region can comprise the sequence of SEQ ID NO: 23.

[0225] In other separate and specific embodiments, the human IgG1 CH region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:24 to SEQ ID NO:33 and SEQ ID NO:45.

[0226] In a specific embodiment, the human IgG1 CH region comprises SEQ ID NO:24 (IgG1m(f)-E430G) or SEQ ID NO:46, optionally wherein the light chain comprises a CL comprising SEQ ID NO:37.

[0227] In a specific embodiment, the antibody variant is a monospecific antibody comprising two HCs that are identical in amino acid sequence, and two LCs that are identical in amino acid sequence.

[0228] The present invention can also be applied to antibody molecules having a human IgG2 heavy chain, for example, a human IgG2 heavy chain comprising the amino acid sequence of the human IgG2 CH region comprising SEQ ID NO: 34.

[0229] The present invention can also be applied to antibody molecules having a human IgG3 heavy chain, for example, a human IgG3 heavy chain comprising a human IgG3 CH region amino acid sequence comprising SEQ ID NO: 35.

[0230] The present invention can also be applied to antibody molecules having a human IgG4 heavy chain, for example, a human IgG4 heavy chain comprising the amino acid sequence of the human IgG4 CH region comprising SEQ ID NO: 36.

[0231] However, for the antibody variants disclosed herein, variant Fc regions comprising one or more further mutations, i.e., mutations in one or more other amino acid residues other than those corresponding to E430, E345, and S440 in a human IgG1 heavy chain when numbered according to the EU index, are also contemplated. Additionally or alternatively, the Fc region may be of mixed isotype, e.g., wherein different CH regions are derived from different IgG isotypes. Accordingly, as described in more detail below, the parent Fc region may already comprise one or more further mutations compared to such a wild-type (naturally occurring) human IgG Fc region, or may be of mixed isotype.

[0232] In one embodiment, the parent Fc-region into which a mutation selected from one or more amino acid residues corresponding to E430, E345 and S440 is introduced is a human IgG Fc-region comprising one or more further mutations compared to a wild-type human IgG1, IgG2, IgG3 and IgG4 Fc-region as shown in, e.g., one of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36. Expressed alternatively, a variant Fc region comprising a mutation in E430, E345, and / or S440 may also differ from a reference Fc region, e.g., a reference wild-type human IgG1, IgG2, IgG3, and IgG4 Fc region, in one or more further mutations, e.g., a reference wild-type human IgG1, IgG2, IgG3, and IgG4 Fc region as set forth in one of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. For example, the variant Fc region may differ from the wild-type Fc region by 12 or fewer, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, e.g., a substitution, insertion, or deletion of an amino acid residue, in addition to a mutation in one or more amino acid residues selected from those corresponding to E430, E345, and S440. For example, the C-terminal amino acid Lys (K) at position 447 (Eu numbering) may be deleted. Some host cells used to produce antibodies may contain enzymes capable of removing Lys at position 447, and such removal may not be homogeneous. Therefore, therapeutic antibodies can be produced without a C-terminal Lys(K) to increase product homogeneity. Methods for producing antibodies without a C-terminal Lys(K) are well known to those skilled in the art and include genetic engineering of nucleic acids expressing the antibodies, enzymatic methods, and the use of specific host cells. Thus, for example, a parent Fc region can comprise the sequence shown in SEQ ID NO: 45.

[0233] Preferably, any such one or more further mutations do not reduce the ability of the antibodies disclosed herein to induce CDC and / or ADCC, i.e., antibodies comprising mutations in one or more amino acid residues selected from the group consisting of E430, E345, and S440 corresponding to human IgG1 heavy chains. More preferably, any such one or more further mutations do not reduce the ability of the antibody to induce CDC. Most preferably, any such one or more further mutations do not reduce the ability of the antibody to induce either CDC and ADCC. Candidates for one or more further mutations can be tested, for example, in CDC or ADCC assays as disclosed herein, for example, in Examples 3 and 4. For example, CDC for antibodies as described herein, such as IgG1-C-E430G, can be tested in an assay in Example 3 or an assay as described in the next section (or similar assays), with or without specific candidates for one or more further mutations, to ascertain the effect of the candidate further mutations on the ability of the antibody to induce CDC. Likewise, ADCC of an antibody as described herein, e.g., IgG1-C-E430G, can be tested in the assay in Example 4 or as described in the next section (or similar assays) with and without specific candidates for one or more further mutations to ascertain the effect of the candidate further mutation on the ability of the antibody to induce ADCC.

[0234] Preferably, in antibody variants comprising two HCs and two LCs, the Fc regions in the first HC and the second HC are identical, such that the Fc regions in the dimerized form are homodimers.

[0235] However, in some embodiments, in antibody variants comprising two HCs and two LCs, the Fc region in the first HC may differ from the Fc region in the second HC in one or more amino acids, such that the Fc region in the dimerized form is a heterodimer. For example, a mutation in one or more amino acid residues selected from the group consisting of E430, E345, and S440 corresponding to an IgG1 heavy chain, where the amino acid residues are numbered according to the EU index, may be present in only one of the Fc regions. Accordingly, in some embodiments, one Fc region may be SEQ ID NO:45, or a wild-type human IgG Fc region selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, while the other Fc region may be identical except for the mutation in one or more amino acid residues selected from the group consisting of E430, E345, and S440 corresponding to an IgG1 heavy chain.

[0236] In one embodiment, the antibody variant according to any aspect or embodiment herein is a human antibody except for the mutations.

[0237] In one embodiment, the antibody variant according to any aspect or embodiment herein is a full length antibody, eg, a human full length antibody, except for the mutations.

[0238] In one embodiment, the antibody variant according to any aspect or embodiment herein is a bivalent antibody, eg, a human bivalent antibody, eg, a human bivalent full length antibody, except for the mutations.

[0239] In one embodiment, the antibody variant according to any aspect or embodiment herein is a monoclonal antibody, eg, a human monoclonal antibody, eg, a human bivalent monoclonal antibody, eg, a human bivalent full-length monoclonal antibody, except for the mutations.

[0240] In a preferred embodiment, the antibody variant according to any aspect or embodiment herein is, in addition to the mutations, an IgG1 antibody, e.g., a full-length IgG1 antibody, e.g., a human full-length IgG1 antibody, optionally a human monoclonal full-length bivalent IgG1,κ antibody, e.g., a human monoclonal full-length bivalent IgG1m(f),κ antibody.

[0241] The antibody variants according to the present invention are advantageously bivalent monospecific forms comprising two antigen-binding regions that bind to the same epitope. However, bispecific forms in which one of the antigen-binding regions binds to a different epitope are also contemplated. Thus, unless otherwise contradicted by the context, the antibody variants according to any aspect or embodiment herein may be monospecific antibodies or bispecific antibodies.

[0242] Thus, in one embodiment, the antibody variant according to any aspect or embodiment herein is, except for the mutations, a monospecific antibody, e.g., a human monospecific antibody, e.g., a human full-length monospecific antibody, e.g., a human full-length monospecific bivalent monoclonal antibody, e.g., a human full-length bivalent monospecific monoclonal antibody.

[0243] In another embodiment, the antibody variant according to any aspect or embodiment herein is, in addition to the mutations, a bispecific antibody, such as a full length bispecific antibody, optionally a full length bispecific and bivalent IgG1,κ antibody.

[0244] Functional regulation

[0245] The antibody variants according to any aspect or embodiment herein can typically induce one or more, preferably all, of CDC, ADCC, ADCP, apoptosis in the presence of an Fc cross-linking agent (but not in the absence of an Fc cross-linking agent), cytotoxicity, or any combination thereof, of target cells expressing human CD38, typically in the presence of complement cells and effector cells.

[0246] The antibody variants according to any aspect or embodiment herein can generally modulate the enzymatic activity of CD38.

[0247] In a further embodiment, the antibody variant according to any aspect or embodiment herein may induce one or more of CDC, ADCC, ADCP, apoptosis in the presence of an Fc cross-linker (but not in its absence), cytotoxicity, and modulation of the enzymatic activity of CD38, or any combination thereof.

[0248] Complement-dependent cytotoxicity (CDC):

[0249] In one embodiment, the antibody variants disclosed herein induce CDC. In particular, when bound to CD38 on the surface of, for example, CD38-expressing cells or cell membranes, the antibody variants of the present invention can mediate increased CDC compared to a control. The control can be, for example, a reference antibody whose amino acid sequence (typically heavy and light chain amino acid sequences) is identical to that of the antibody variant, except for one or more mutations in E430, E345, and / or S440 in the variant antibody. Alternatively, the control can be a reference antibody whose amino acid sequence (typically heavy and light chain amino acid sequences) is identical to that of the antibody variant, except for different VH and VL sequences. Such reference antibodies can, for example, instead have the VH and VL sequences of antibody B or A, as shown in Table 1. Preferably, the VH and VL sequences of the reference antibody are those of antibody B. Alternatively, the reference antibody can be an antibody that binds to the same target but has a different amino acid sequence. Alternatively, the control can be an isotype control antibody, for example, such that the VH and VL sequences are those of antibody b12 as shown in Table 1.

[0250] Accordingly, in one embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher CDC against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of antibody C, i.e., SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and CH and CL region sequences that are identical to those of the antibody variant except for one or more mutations in E430, E345 and / or S440.

[0251] In another embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher CDC against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody C, i.e., SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and the CH and CL region sequences of SEQ ID NO: 20 (IgGm(f)) and SEQ ID NO: 37 (κ), respectively.

[0252] In another embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher CDC against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody B, i.e., SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and CH and CL region sequences equivalent to those of the antibody variant.

[0253] In another embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher CDC against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody A, i.e., SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and CH and CL region sequences equivalent to those of the antibody variant.

[0254] In another embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher CDC against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of antibody b12, i.e., SEQ ID NO: 12 and SEQ ID NO: 16, respectively, and CH and CL region sequences equivalent to those of the antibody variant.

[0255] In a specific embodiment, the CDC response is described as maximum lysis, wherein a higher maximum lysis reflects increased CDC. In a specific embodiment, the CDC response is described as EC50 (concentration at which half of the maximum lysis is observed), wherein a lower EC50 indicates increased CDC. In a specific embodiment, the target cell expressing CD38 is a tumor cell, such as a lymphoma cell. Non-limiting examples of lymphoma target cells include (commercial sources are indicated in parentheses):

[0256] - Daudi cells (ATCC CCL-213);

[0257] - Ramos cells (ATCC CRL-1596);

[0258] - REH cells (DSMZ ACC 22);

[0259] - Wien-133 cells (BioAnaLab, Oxford, UK);

[0260] - RS4;11 cells (DSMZ ACC 508);

[0261] - NALM-16 (DSMZ ACC 680);

[0262] - U266 (ATCC TIB-196);

[0263] - RC-K8 (DSMZ ACC 561);

[0264] - SU-DHL-8;

[0265] - Oci-Ly-7;

[0266] - Oci-Ly-19;

[0267] - Oci-Ly-18;

[0268] - Raji;

[0269] - DOHH-2;

[0270] - SU-DHL-4;

[0271] - WSU-DLCL-2;

[0272] - Z-138;

[0273] - JVM-13;

[0274] - Jeko-1;

[0275] - 697;

[0276] - Granta 519;

[0277] - DB;

[0278] - Pfeiffer.

[0279] The target cell expressing CD38 can also be an AML cell, for example, selected from but not limited to one of the following: THP1, monomac6, Oci-AML3, KG-1, ML2, U937, Nomo-1, AML-193, MEGAL, MOLM13, HL-60 and Oci-M1.

[0280] In another specific embodiment, the target cell expressing CD38 is a tumor cell, such as a lymphoma cell or a myeloma cell, wherein the approximate mean number of CD38 molecules per cell, optionally when determined as described in Example 1, is in one of the following ranges:

[0281] - 150,000-250,000, for example, about 200,000;

[0282] - 200,000-300,000, for example, about 260,000;

[0283] - 80,000-180,000, for example about 130,000;

[0284] - 50,000-150,000, for example about 100,000;

[0285] - 40,000-120,000, for example about 80,000;

[0286] - 30,000-70,000, for example about 50,000;

[0287] - 10,000-20,000, for example, about 15,000;

[0288] - 5,000-15,000, for example, about 10,000.

[0289] In one embodiment, the antibody variant according to any aspect or embodiment as disclosed herein induces increased CDC against target cells expressing CD38 compared to a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody B, i.e., SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and CH and CL region sequences equivalent to those of the antibody variant, wherein the CDC response is an EC50, and the CD38-expressing target cells are selected from the group consisting of NALM-16 (DSMZ ACC 680), U266 (ATCC TIB-196), and RC-K8 (DSMZ ACC 561).

[0290] In a preferred embodiment, the antibody variant according to any aspect or embodiment as disclosed herein induces increased CDC against target cells expressing CD38 compared to a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody C, i.e., SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and the CH and CL region sequences of SEQ ID NO: 20 (IgGm(f)) and SEQ ID NO: 37 (κ), respectively, wherein the CDC response is maximal lysis, and the CD38-expressing target cells are selected from Daudi cells (ATCC CCL-213) and Ramos cells (ATCC CRL-1596). The antibody variant may in particular result in at least 50%, such as at least 60% or at least 70% greater maximal lysis than the reference antibody.

[0291] Any in vitro or in vivo method or assay known to those skilled in the art and suitable for assessing the ability of an antibody, such as an IgG antibody, to induce CDC against target cells expressing CD38 may be used. Preferably, the assay comprises, in relevant part, the steps of the CDC assay described in Example 3.

[0292] A non-limiting example of an assay for determining the maximum lysis of CD38 expressing cells, as mediated by a CD38 antibody, or an EC50 value, may include the following steps:

[0293] (a) Approximately 100,000 CD38-expressing cells / well were plated in 40 µL of culture medium supplemented with 0.2% BSA in a multi-well plate.

[0294] (b) Cells were preincubated with 40 μL of serially diluted CD38 antibody (0.0002-10 μg / mL) for 20 min;

[0295] (c) incubating each well with 20% pooled normal human serum at 37° C. for 45 minutes;

[0296] (d) Viability dye was added, and the percentage of cell lysis was measured on a flow cytometer;

[0297] (e) Nonlinear regression was used to determine maximum lysis and / or calculate EC50 values.

[0298] Tumor cells suitable for this assay include, but are not limited to, those listed in Table 2, such as Daudi cells (ATCC CCL-213).

[0299] In certain embodiments, the antibody variants induce CDC against Daudi cells (ATCC No. CCL-213) or Ramos cells (ATCC No. CRL-1596) resulting in at least 50%, such as at least 60%, such as at least 70% greater maximal lysis than that obtained with a reference antibody that differs only in the absence of a mutation in one or more amino acid residues selected from the group consisting of E430, E435, and S440 corresponding to a human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index. In one embodiment, the reference antibody comprises the VH and VL region sequences of Antibody C, i.e., SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and the CH and CL region sequences of SEQ ID NO: 20 (IgGm(f)) and SEQ ID NO: 37 (κ), respectively.

[0300] Antibody-dependent cell-mediated cytotoxicity (ADCC):

[0301] In one embodiment, the antibody variants according to any aspect or embodiment herein induce ADCC. In some embodiments, when bound to CD38 on the surface of, for example, a CD38-expressing cell or cell membrane, the antibody variants of the present invention can mediate ADCC. Compared to the same antibody without the E430G mutation, it was found that the anti-CD38 antibody comprising the E430G mutation induced slightly lower levels of ADCC. When bound to CD38 on the surface of, for example, a CD38-expressing cell or cell membrane, the antibody variants of the present invention can mediate higher ADCC than the control, wherein the control can be, for example, a reference antibody having an amino acid sequence (typically a heavy chain and light chain amino acid sequence) identical to the antibody variant, except for different VH and VL sequences. Such reference antibodies can, for example, instead have the VH and VL sequences of antibody B or A, as shown in Table 1. Preferably, the VH and VL sequences of the reference antibody are those of antibody B. Alternatively, the control can be an isotype control antibody, for example, such that the VH and VL sequences are those of antibody b12 as shown in Table 1.

[0302] Accordingly, in one embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher ADCC against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody B, i.e., SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and CH and CL region sequences equivalent to those of the antibody variant. In a specific embodiment, the ADCC response is maximal lysis, wherein higher maximal lysis reflects higher ADCC. In a specific embodiment, the ADCC response is assessed in an assay that determines FcγRIIIa binding, wherein higher binding indicates higher ADCC. In a specific embodiment, the target cell expressing CD38 is a tumor cell. Non-limiting examples of target cells include Daudi, Wien-133, Granta 519, MEC-2, and the tumor cell lines listed in Table 2.

[0303] In one embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher ADCC against CD38-expressing Daudi cells compared to a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody B, i.e., SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and CH and CL region sequences equivalent to those of the antibody variant, optionally wherein the ADCC response is maximal lysis or FcγRIIIa binding.

[0304] In one embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher ADCC against CD38-expressing Daudi cells compared to a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of antibody b12, i.e., SEQ ID NO: 12 and SEQ ID NO: 16, respectively, and CH and CL region sequences equivalent to the antibody variant, optionally wherein the ADCC response is maximal lysis or FcγRIIIa binding.

[0305] Any in vitro or in vivo method or assay known to those skilled in the art and suitable for assessing the ability of an antibody, such as an IgG antibody, to induce ADCC against target cells expressing CD38 may be used. Preferably, the assay includes, in relevant part, the assay described in Example 4. 51 Cr release antibody-dependent cellular cytotoxicity assay, or ADCC reporter bioassay. Non-limiting examples of assays for determining ADCC of CD38 expressing cells, such as mediated by CD38 antibodies, may include the following: 51 Steps for Cr release assay or reporter assay.

[0306] use 51 Cr release determines ADCC:

[0307] (a) Approximately 50,000 cells were plated in 50 µL of culture medium supplemented with 0.2% BSA in a multiwell plate. 51 Cr-labeled CD38-expressing cells (e.g., Daudi cells) / well;

[0308] (b) Cells were preincubated with 50 μL of serially diluted CD38 antibody (0.0002-10 μg / mL) for 15 min;

[0309] (c) incubating each well with 500,000 freshly isolated peripheral blood mononuclear cells (PBMCs) / well at 37°C for 4 hours;

[0310] (d) Measure the amount of γ in 75 μL of the supernatant on a gamma counter. 51 Cr release;

[0311] (e) The percentage of cell lysis was calculated as (cpm sample - cpm spontaneous lysis) / (cpm maximum lysis - cpm spontaneous lysis), where cpm is counts per minute.

[0312] ADCC was determined using a reporter assay:

[0313] (a) Plate approximately 5,000 Daudi cells in 10 μL of standard culture medium (e.g., RPMI 1640) supplemented with 25% low IgG serum in multiwell plates suitable for optical reading (e.g., 384-well OptiPlates from PerkinElmer Inc.);

[0314] (b) Each well was incubated with 10 μL of engineered Jurkat cells stably expressing the FcγRIIIa receptor, the V158 (high affinity) variant, and a NFAT response element driving firefly luciferase expression as effector cells and 10 μL of serially diluted CD38 antibody (0.0002-10 μg / mL) at 37°C for 6 hours;

[0315] (c) Each well was incubated with 30 μL of luciferase substrate at RT for 5 minutes and luminescence was measured.

[0316] Antibody-dependent cellular phagocytosis (ADCP):

[0317] In one embodiment, the antibody variants according to any aspect or embodiment herein induce ADCP. In some embodiments, when bound to, for example, CD38 on the surface of a CD38-expressing cell or cell membrane, the antibody variants of the invention can mediate ADCP. When bound to, for example, CD38 on the surface of a CD38-expressing cell or cell membrane, the antibody variants of the invention can mediate higher ADCP than a control, wherein the control is an isotype control antibody, for example, such that the VH and VL sequences are those of antibody b12 as shown in Table 1.

[0318] Accordingly, in one embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces higher ADCP against target cells expressing CD38 than a reference antibody, wherein the reference antibody differs from the antibody variant only by one or more mutations in E430, E345, and / or S440 in the variant antibody. In an alternative embodiment, the reference antibody comprises the VH and VL region sequences of antibody b12, i.e., SEQ ID NO: 12 and SEQ ID NO: 16, respectively, and CH and CL region sequences equivalent to those of the antibody variant.

[0319] In a specific embodiment, the target cell expressing CD38 is a tumor cell, such as a myeloma or lymphoma cell. Non-limiting examples of target cells that are tumor cells include those listed in Table 2.

[0320] Any in vitro or in vivo method or assay known to those skilled in the art and suitable for evaluating the ability of an antibody, such as an IgG antibody, to induce ADCP against a target cell expressing CD38 may be used. Preferably, the assay comprises, in relevant part, the steps of the macrophage-based ADCP assay described in Example 5. In particular, an assay for determining ADCP of CD38-expressing cells, as mediated by a CD38 antibody, may comprise the steps set forth below:

[0321] ADCP:

[0322] (a) Freshly isolated monocytes were differentiated into macrophages by 5 days of incubation in medium containing GM-CSF.

[0323] (b) Approximately 100,000 macrophages were plated per well in a multi-well plate in dendritic cell culture medium with GM-CSF.

[0324] (c) adding 20,000 CD38-expressing cells (e.g., Daudi cells) opsonized with CD38 antibodies / well for 45 minutes at 37°C, wherein the cells are labeled with a universal fluorescent membrane dye;

[0325] (d) The percentage of CD14-positive, CD19-negative, membrane dye-positive macrophages was measured on a flow cytometer.

[0326] Apoptosis:

[0327] In one embodiment, the antibody variants for use according to the present invention may not induce apoptosis in the absence of an Fc cross-linking agent. In a further embodiment, the antibody variants may induce apoptosis in the presence of an Fc cross-linking agent, but not in the absence of an Fc cross-linking agent.

[0328] In one embodiment, the Fc cross-linker is an antibody.

[0329] In one embodiment, apoptosis can be determined as described in Example 6.

[0330] Cell chewing effect:

[0331] In one embodiment, antibody variants as disclosed herein induce cytokine production, such as CD38 cytokine production from donor cells expressing CD38 to recipient cells. Common recipient cells include T cells and B cells, monocytes / macrophages, dendritic cells, neutrophils and NK cells. Preferably, recipient cells are lymphocytes expressing Fc-γ-(Fcγ)-receptors, such as macrophages or PBMCs. In particular, compared to the control, the antibody variants of the present invention can mediate increased cytokine production. The control can be, for example, a reference antibody having an amino acid sequence (typically heavy chain and light chain amino acid sequence) identical to that of the antibody variant, except for one or more mutations in E430, E345 and / or S440 in the variant antibody. In another embodiment, the control is a reference antibody having an amino acid sequence (typically heavy chain and light chain amino acid sequence) identical to that of the antibody variant, except for different VH and VL sequences. For example, the control can be an isotype control antibody, for example, such that the VH and VL sequences are those of the antibody b12 as shown in Table 1.

[0332] Suitable assays for assessing cytotoxicity are known in the art and include, for example, the assay in Example 8. Non-limiting examples of assays for determining cytotoxicity of CD38 expressing cells, as mediated by CD38 antibodies, include the following:

[0333] Cytokinesis (Daudi cells):

[0334] (a') Freshly isolated monocytes were differentiated into macrophages using GM-CSF for 5 days;

[0335] (b') Approximately 100,000 macrophages were plated per well in dendritic cell culture medium with GM-CSF.

[0336] (c') Approximately 20,000 CD38 antibody-opsonized Daudi cells / well were added and labeled with a universal fluorescent membrane dye at 37°C for 45 minutes;

[0337] (d') CD38 expression on Daudi cells was measured on a flow cytometer, where a decrease in CD38 on CD38 antibody-opsonized Daudi cells compared to controls indicates cytotoxicity.

[0338] Tregs:

[0339] (a) Plate approximately 500,000 freshly isolated PBMCs / well in cell culture medium at 37°C overnight.

[0340] (b) adding approximately 100,000 CD38 antibody-opsonized Tregs / well at 37°C overnight (O / N), wherein the Tregs are labeled with a universal fluorescent intracellular amine dye; and

[0341] (c) CD38 expression on Tregs was measured on a flow cytometer, where decreased CD38 on CD38 antibody-opsonized Tregs compared to controls indicates cytotoxicity.

[0342] In addition to Daudi cells (ATCC CCL-213), tumor cells suitable for the first assay include, but are not limited to, those listed in Table 2, particularly those with high CD38 expression. In addition, in addition to Tregs, suitable CD38-expressing cells for the second assay include immune cells, such as NK cells, B cells, T cells, and monocytes, as well as the tumor cells listed in Table 2, particularly those with low CD38 expression levels.

[0343] Accordingly, in one embodiment, the antibody variant according to any aspect or embodiment disclosed herein induces a higher level of cytotoxicity against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of antibody C, i.e., SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and CH and CL region sequences that are identical to those of the antibody variant except for one or more mutations in E430, E345 and / or S440.

[0344] In some embodiments, the antibody variant according to any aspect or embodiment disclosed herein induces a higher level of cytotoxicity against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody B, i.e., SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and CH and CL region sequences that are equivalent to those of the antibody variant.

[0345] In some embodiments, the antibody variant according to any aspect or embodiment disclosed herein induces a higher level of cytotoxicity against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody A, i.e., SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and CH and CL region sequences that are equivalent to those of the antibody variant.

[0346] In some embodiments, the antibody variant according to any aspect or embodiment disclosed herein induces a higher level of cytotoxicity against target cells expressing CD38 than a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of antibody b12, i.e., SEQ ID NO: 12 and SEQ ID NO: 16, respectively, and CH and CL region sequences equivalent to those of the antibody variant.

[0347] Regulation of CD38 enzyme activity

[0348] The antibody variants according to any aspect or embodiment herein can generally modulate one or more enzymatic activities of human CD38. In one embodiment, the antibody variants disclosed herein have an inhibitory effect on CD38 cyclase activity compared to a control, e.g., an isotype control antibody, e.g., antibody b12. For example, the antibody variants can have an inhibitory effect on the cyclase activity of CD38 expressed by cells, e.g., tumor cells, and / or on isolated CD38, e.g., a soluble fragment of CD38 (e.g., SEQ ID NO: 39).

[0349] Any in vitro or in vivo method or assay known to those skilled in the art and suitable for evaluating the ability of an anti-CD38 antibody to inhibit CD38 cyclase activity may be used. Suitable assays for testing CD38 cyclase activity are described, for example, in WO 2006 / 099875 A1 and WO 2011 / 154453 A1. Preferably, the method includes, in relevant part, the steps of the specific assay described in Example 6, using nicotinamide guanine dinucleotide sodium salt (NGD) as a substrate for CD38 to test cyclase activity. Non-fluorescent NGD is cyclized by CD38 to a fluorescent analog of cADPR, cyclic GDP-ribose (see, for example, Comb, Chem High Throughput Screen. 2003 Jun; 6(4): 367-79A). Non-limiting examples of assays include the following steps for determining inhibition of CD38 cyclase activity:

[0350] (a) 200,000 Daudi or Wien133 cells were seeded in 100 μL of 20 mM Tris-HCl per well; or 0.6 μg / mL His-tagged soluble CD38 (SEQ ID NO: 39) was seeded in 100 μL of 20 mM Tris-HCl per well in a multi-well plate.

[0351] (b) 1 µg / mL CD38 antibody and 80 µM NGD were added to each well;

[0352] (c) measuring fluorescence until a steady state is reached (e.g., 5, 10, or 30 minutes); and

[0353] (d) Determine the percent inhibition compared to a control (e.g., wells incubated with an isotype control antibody).

[0354] In one embodiment, the antibody variant is capable of inhibiting the cyclase activity of CD38, specifically the maximum percentage of NGD conversion, by at least about 40%, such as at least about 50%, such as at least about 60%, such as from about 40% to about 60%, in such an assay compared to a control (typically CD38 cyclase activity in the presence of an isotype control antibody). For example, the isotype control antibody may comprise the VH and VL region sequences of antibody b12, i.e., SEQ ID NO: 12 and SEQ ID NO: 16, respectively, and CH and CL region sequences equivalent to those of the antibody variant. In a specific embodiment, the assay utilizes hisCD38 (SEQ ID NO: 39) for determining cyclase activity.

[0355] In some embodiments, the antibody variant according to any aspect or embodiment disclosed herein has increased (i.e., more potent) inhibition of CD38 cyclase activity compared to a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of antibody B, i.e., SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and CH and CL region sequences equivalent to those of the antibody variant.

[0356] In some embodiments, the antibody variant according to any aspect or embodiment disclosed herein has increased (i.e., more potent) inhibition of CD38 cyclase activity compared to a reference antibody, wherein the reference antibody comprises the VH and VL region sequences of Antibody A, i.e., SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and CH and CL region sequences equivalent to those of the antibody variant.

[0357] Furthermore, in some embodiments, the antibody variants described herein induce apoptosis in CD38 expressing cells in the presence of an Fc cross-linking antibody, but do not induce apoptosis in the absence of an Fc cross-linking antibody. These functionalities can be measured in an assay that includes, in relevant part, the apoptosis assay steps described in Example 6. In one embodiment, the apoptosis assay can include the following steps:

[0358] (a) 100,000 CD38-expressing tumor cells were plated per well in 100 µL of culture medium supplemented with 0.2% BSA.

[0359] (b) Each well was incubated O / N at 37°C with serially diluted CD38 antibody (0.0002-10 μg / mL) and 10 μg / mL goat anti-human IgG1;

[0360] (c) The percentage of dead cells was measured on a flow cytometer.

[0361] Conjugate

[0362] In one aspect, the invention relates to antibody variants conjugated to a drug, cytotoxic agent, toxin, radiolabel or radioisotope.

[0363] In one embodiment, antibody variants are provided that include one or more radiolabeled amino acids. Radiolabeled variants can be used for in vitro diagnostic purposes, in vivo diagnostic purposes, therapeutic purposes, or a combination thereof. Non-limiting examples of radiolabeled antibodies include 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 125 I. 131 I and 186 Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, for example, Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2nd ed., Chafner and Longo, eds., Lippincott Raven (1996)), and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (USRE 35,500), US 5,648,471, and US 5,697,902. For example, radioisotopes of halogens such as iodine or bromine can be conjugated by the chloramine-T method.

[0364] In one embodiment, the antibody variants of the present invention are conjugated to a radioisotope or a chelate containing a radioisotope. For example, the variant can be conjugated to a chelating agent linker, such as DOTA, DTPA, or tiuxetan, which allows the antibody to be complexed with the radioisotope. The variant can also or alternatively contain or be conjugated to one or more radiolabeled amino acids, or other radiolabeled molecules. Radiolabeled variants can be used for both diagnostic and therapeutic purposes. In one embodiment, the variants of the present invention are conjugated to an α-emitter. Non-limiting examples of α-emitting radioisotopes include 213 Bs, 225 Ac and 227 Th.

[0365] In one embodiment, the antibody variant is attached to a chelator linker, such as tiuxetan, which allows conjugation of the antibody variant to a radioisotope.

[0366] Nucleic Acids

[0367] Antibodies are well known as therapeutic agents that can be used to treat various diseases. Another method for administering an antibody to a subject in need thereof comprises administering a nucleic acid encoding the antibody or a combination of nucleic acids for expressing the antibody in vivo.

[0368] Therefore, in one aspect, the present invention also relates to a nucleic acid encoding the heavy chain of an antibody variant according to the present invention, wherein the heavy chain comprises a VH region comprising a VH CDR1 having the sequence as shown in SEQ ID NO: 2, a VH CDR2 having the sequence as shown in SEQ ID NO: 3, a VH CDR3 having the sequence as shown in SEQ ID NO: 4, and a human IgG1 CH region having a mutation in one or more of E430, E345 and S440, the amino acid residues being numbered according to the EU index.

[0369] In one aspect, the present invention also relates to a nucleic acid or a combination of nucleic acids encoding an antibody variant according to the invention.

[0370] In some embodiments, the invention relates to a nucleic acid or combination of nucleic acids encoding an antibody variant comprising:

[0371] a) an antigen binding region comprising a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VLCDR3 having the sequence shown in SEQ ID NO: 7, and

[0372] b) a variant Fc region comprising a mutation in one or more amino acid residues selected from the group consisting of amino acid residues corresponding to E430, E345 and S440 in a human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index.

[0373] In one embodiment, the antibody variants of the present invention are encoded by one nucleic acid. Thus, the nucleotide sequence encoding the antibody variants of the present invention is present in one nucleic acid or the same nucleic acid molecule.

[0374] In another embodiment, the antibody variants of the present invention are encoded by a nucleic acid combination, typically two nucleic acids. In one embodiment, the nucleic acid combination includes a nucleic acid encoding the heavy chain of the antibody variant and a nucleic acid encoding the light chain of the antibody variant.

[0375] In some embodiments, the invention relates to a nucleic acid or combination of nucleic acids encoding an antibody variant comprising:

[0376] a) a heavy chain comprising a VH region comprising a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, and a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, wherein the amino acid residues are numbered according to the EU index;

[0377] b) a light chain comprising a VL region comprising a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7.

[0378] In one embodiment, the antibody variants of the present invention are encoded by one nucleic acid. Thus, the nucleotide sequence encoding the antibody variants of the present invention is present in one nucleic acid or the same nucleic acid molecule.

[0379] In another embodiment, the antibody variants of the present invention are encoded by a nucleic acid combination, typically two nucleic acids. In one embodiment, the nucleic acid combination includes a nucleic acid encoding the heavy chain of the antibody variant and a nucleic acid encoding the light chain of the antibody variant.

[0380] As described above, nucleic acids can be used as a means of delivering therapeutic proteins, such as antibodies, to a subject in need thereof.

[0381] In some embodiments, the nucleic acid can be deoxyribonucleic acid (DNA). DNA suitable for expressing therapeutic proteins (such as antibodies) in vivo and methods for preparing the DNA are well known to those skilled in the art, and include but are not limited to the methods described by Patel A et al., 2018, Cell Reports 25, 1982-1993.

[0382] In some embodiments, the nucleic acid can be a ribonucleic acid (RNA), such as a messenger RNA (mRNA). In some embodiments, the mRNA can comprise only naturally occurring nucleotides. In some embodiments, the mRNA can comprise modified nucleotides, wherein modified refers to that the nucleotides are chemically different from naturally occurring nucleotides. In some embodiments, the mRNA can comprise both naturally occurring nucleotides and modified nucleotides.

[0383] Different nucleic acids suitable for expressing therapeutic proteins, such as antibodies, in a subject are well known to those skilled in the art. For example, mRNA suitable for expressing therapeutic antibodies in a subject often comprises an open reading frame (ORF) flanked by untranslated regions (UTRs) comprising specific sequences and 5' and 3' ends formed by a cap structure and a poly (A) tail (see, e.g., Schlake et al., 2019, Molecular Therapy, Vol. 27, No. 4, April).

[0384] Examples of methods for optimizing RNA and RNA molecules, such as mRNA, suitable for in vivo expression include, but are not limited to, those described in US9,254,311; US9,221,891; US20160185840 and EP3118224.

[0385] Naked nucleic acids administered to subjects for in vivo expression are susceptible to degradation and / or immunogenic responses in subjects. In addition, for in vivo expression of antibodies encoded by nucleic acids, the nucleic acids are typically administered in a form suitable for nucleic acid entry into the cells of the subject. There are different methods for delivering nucleic acids for in vivo expression, and include two methods involving mechanical and chemical means. For example, such methods may involve electroporation or tattooing of nucleic acids onto the skin (Patel et al., 2018, Cell Reports 25, 1982–1993). Other methods suitable for administering nucleic acids to subjects involve administering nucleic acids with suitable preparations. Therefore, the present invention also relates to delivery vehicles comprising nucleic acids of the present invention.

[0386] In some embodiments, the delivery vehicle may comprise a nucleic acid encoding a heavy chain of an antibody variant according to the present invention. Thus, in one embodiment, the nucleic acid may encode a heavy chain comprising a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, wherein the VH region comprises a VH CDR1 having a sequence as shown in SEQ ID NO: 2, a VH CDR2 having a sequence as shown in SEQ ID NO: 3, and a VH CDR3 having a sequence as shown in SEQ ID NO: 4, wherein the amino acid residues are numbered according to the EU index.

[0387] In some embodiments, the present invention also relates to a delivery vehicle comprising a nucleic acid encoding a light chain of an antibody variant according to the present invention. Thus, in one embodiment, the nucleic acid can encode a light chain comprising a VL region comprising a VL CDR1 having a sequence as shown in SEQ ID NO: 6, a VL CDR2 having a sequence AAS, and a VL CDR3 having a sequence as shown in SEQ ID NO: 7.

[0388] The present invention also relates to a mixture of delivery vehicles, comprising a delivery vehicle comprising a nucleic acid encoding the heavy chain of an antibody variant according to the present invention and a delivery vehicle comprising a nucleic acid encoding the light chain of an antibody variant according to the present invention. Thus, in one embodiment, the mixture of delivery vehicles comprises a delivery vehicle comprising a nucleic acid encoding a heavy chain and a delivery vehicle comprising a nucleic acid encoding a light chain, wherein the heavy chain comprises a VH region and a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, wherein the VH region comprises a VH CDR1 having a sequence as shown in SEQ ID NO: 2, a VH CDR2 having a sequence as shown in SEQ ID NO: 3, and a VH CDR3 having a sequence as shown in SEQ ID NO: 4, wherein the amino acid residues are numbered according to the EU index; and the light chain comprises a VL region comprising a VL CDR1 having a sequence as shown in SEQ ID NO: 6, a VL CDR2 having a sequence AAS, and a VL CDR3 having a sequence as shown in SEQ ID NO: 7.

[0389] In some embodiments, the delivery vehicle comprises a nucleic acid encoding the heavy chain and a nucleic acid encoding the light chain or a combination of nucleic acids of an antibody variant according to the invention.

[0390] Thus, in one embodiment, the delivery vehicle may comprise nucleic acids encoding a heavy chain comprising a VH region comprising a VH CDR1 having the sequence as shown in SEQ ID NO: 2, a VH CDR2 having the sequence as shown in SEQ ID NO: 3, and a VH CDR3 having the sequence as shown in SEQ ID NO: 4, wherein the amino acid residues are numbered according to the EU index, and a human IgG1 CH region having a mutation in one or more of E430, E345, and S440, and a light chain comprising a VL region comprising a VL CDR1 having the sequence as shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence as shown in SEQ ID NO: 7. Thus, the nucleic acid sequences encoding the heavy and light chains of the antibody variants according to the invention are present in one (same) nucleic acid molecule.

[0391] In another embodiment, the delivery vehicle may comprise a nucleic acid encoding a heavy chain comprising a VH region comprising a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, and a VH CDR3 having the sequence shown in SEQ ID NO: 4, wherein the amino acid residues are numbered according to the EU index, and a nucleic acid encoding a light chain comprising a VL region comprising a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7. Thus, the nucleic acid sequences encoding the heavy and light chains of the antibody variants according to the present invention are present on separate or different nucleic acid molecules.

[0392] In some embodiments, the delivery vehicle can be a lipid formulation. The lipid of the formulation can be a particle, such as a lipid nanoparticle (LNP). The nucleic acid or nucleic acid combination of the present invention can be encapsulated in the particle, such as the LNP.

[0393] Different lipid formulations suitable for administering nucleic acid to a subject for in vivo expression are well known to those skilled in the art. For example, the lipid formulation can typically comprise a lipid, an ionizable amino lipid, a PEG-lipid, cholesterol, or any combination thereof.

[0394] Various forms and preparation methods suitable for administering nucleic acid to a subject for expressing the lipid formulations of therapeutic antibodies are well known in the art. Examples of such lipid formulations include, but are not limited to, the lipid formulations described in US20180170866 (Arcturus), EP 2391343 (Arbutus), WO 2018 / 006052 (Protiva), WO2014152774 (Shire HumanGenetics), EP 2 972 360 (Translate Bio), US10195156 (Moderna) and US20190022247 (Acuitas).

[0395] Generation of variant antibodies

[0396] In another aspect, the present invention also relates to a method of increasing at least one effector function of an antibody comprising the CDR, VH and / or VL amino acid sequences of antibody C, comprising introducing into the antibody mutations in one or more amino acid residues corresponding to E430, E345 and S440 in the Fc region of a human IgG1 heavy chain, said amino acid residues being numbered according to the EU index.

[0397] Thus, in certain embodiments, there is provided a method of increasing the effector function of a parent antibody comprising an Fc region and an antigen binding region that binds CD38, the method comprising introducing into the Fc region a mutation in one or more amino acid residues selected from the group consisting of E430, E345 and S440 in the Fc region corresponding to a human IgG1 heavy chain, wherein the amino acid residues are numbered according to the EU index; and

[0398] wherein the antigen binding region comprises a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7.

[0399] In certain other embodiments, a method is provided for producing a variant of a parent antibody comprising an Fc region and an antigen binding region, optionally wherein the variant has increased effector function compared to the parent antibody, the method comprising:

[0400] (a) introducing mutations into the Fc region in one or more amino acid residues selected from the group consisting of E430, E345 and S440 corresponding to the Fc region of a human IgG1 heavy chain to obtain a variant antibody,

[0401] (b) selecting any variant antibodies that have increased effector function compared to the parent antibody, and

[0402] (c) producing the variant antibody in a recombinant host cell,

[0403] wherein the antigen binding region comprises a VH CDR1 having the sequence shown in SEQ ID NO: 2, a VH CDR2 having the sequence shown in SEQ ID NO: 3, a VH CDR3 having the sequence shown in SEQ ID NO: 4, a VL CDR1 having the sequence shown in SEQ ID NO: 6, a VL CDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO: 7.

[0404] In one embodiment of any of the aforementioned methods, the effector function is CDC.

[0405] In one embodiment of any of the aforementioned methods, the effector function is cytotoxicity.

[0406] In one embodiment of any of the aforementioned methods, the effector functions are CDC and cytotoxicity.

[0407] In one embodiment of any of the aforementioned methods, the mutation in the one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W. For example, the mutation in the one or more amino acid residues may comprise or consist of E430G.

[0408] In one embodiment of any of the aforementioned methods, in addition to the mutation, the Fc region of the parent antibody is a human IgG1, IgG2, IgG3, or IgG4 Fc region, or a mixture of isotypes thereof. Optionally, the Fc region comprises one of the sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 45, and SEQ ID NO: 36. In a specific embodiment, the Fc region of the parent antibody is a human IgG1 Fc region. For example, the parent antibody can be a full-length human IgG1 antibody, optionally a human monoclonal full-length bivalent IgG1, kappa antibody. Additionally, the parent antibody can be a monospecific or bispecific antibody, e.g., a monospecific antibody.

[0409] While the Fc region of the parent antibody is typically a naturally occurring (wild-type) sequence, in some embodiments, the Fc region of the parent antibody comprises one or more further mutations, as described elsewhere herein.

[0410] The present invention also relates to antibodies obtained or obtainable according to any of the above methods.

[0411] The present invention also provides isolated nucleic acids and vectors encoding antibody variants according to any one of the aspects and embodiments described herein, as well as vectors and expression systems encoding the variants. Suitable nucleic acid constructs, vectors, and expression systems for antibodies and variants thereof are known in the art and include, but are not limited to, those described in the Examples. In embodiments where the variant antibody comprises an HC and a LC, the HC and LC are separate polypeptides rather than being contained in a single polypeptide (e.g., as in an scFv-Fc fusion protein), and the nucleotide sequences encoding the heavy and light chains may be present in the same or different nucleic acids or vectors.

[0412] In one aspect, the present invention relates to a nucleic acid or expression vector comprising

[0413] (i) a nucleotide sequence encoding the heavy chain sequence of an antibody variant according to any one of the embodiments disclosed herein;

[0414] (ii) a nucleotide sequence encoding the light chain sequence of an antibody variant according to any one of the embodiments disclosed herein; or

[0415] (iii) Both (i) and (ii).

[0416] In one aspect, the present invention relates to a nucleic acid or expression vector comprising a nucleotide sequence encoding the heavy chain sequence of an antibody variant according to any one of the embodiments disclosed herein.

[0417] In one aspect, the present invention relates to a nucleic acid sequence or expression vector comprising a nucleotide sequence encoding the heavy chain sequence and the light chain sequence of an antibody variant according to any one of the embodiments disclosed herein.

[0418] In one aspect, the invention relates to a combination of a first nucleic acid and a second nucleic acid, or a combination of a first expression vector and a second expression vector, optionally in the same host cell, wherein the first comprises a nucleotide sequence according to (i) and the second comprises a nucleotide sequence according to (ii).

[0419] In the context of the present invention, the expression vector may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (containing a nucleic acid sequence of a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid is contained in a naked DNA or RNA vector comprising, for example, a linear expression element (as described, for example, in Sykes and Johnston, Nat Biotech 17, 35559 (1997)), a compact nucleic acid vector (as described, for example, in US 6,077,835 and / or WO 00 / 70087), a plasmid vector such as pBR322, pUC 19 / 18 or pUC 118 / 119, a "midge" minimal size nucleic acid vector (as described, for example, in Schakowski et al., Mol Ther 3, 793 800 (2001)), or as a precipitated nucleic acid vector construct, for example, a CaPO4 precipitated construct (as described, for example, in WO200046147, Benvenisty and Reshef, PNAS USA 83, 955155 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0420] In one embodiment, the vector is suitable for expressing the antibody variant in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989), pET vectors (Novagen, Madison WI) etc.).

[0421] The expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al., eds. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516 544 (1987)).

[0422] The expression vector may also or alternatively be a vector suitable for expression in mammalian cells, such as a vector comprising glutamine synthetase as a selectable marker, such as that described in Bebbington (1992) Biotechnology (NY) 10: 169-175.

[0423] Nucleic acid and / or vector can also comprise the nucleic acid sequence of coding secretion / localization sequence, and it can target polypeptide such as nascent polypeptide chain to periplasmic space or enter into cell culture medium.Such sequence is known in the art, and comprises secretion leader sequence or signal peptide.

[0424] The expression vector can comprise any suitable promoter, enhancer and other expression promoting element, or be related thereto.The example of this type of element comprises strong expression promoter (for example people CMV IE promoter / enhancer, and RSV, SV40, SL33, MMTV and HIV LTR promoter), effective poly (A) terminator sequence, the replication origin of the plasmid product in Escherichia coli, as the antibiotic resistance gene of selectable marker and / or convenient cloning site (for example, polylinker).With constitutive promoter for example CMV IE contrary, nucleic acid can also comprise inducible promoter.

[0425] In one embodiment, expression vectors encoding antibody variants can be localized in and / or delivered to a host cell or host animal via a viral vector.

[0426] The present invention also provides recombinant host cells that produce antibody variants as disclosed herein, optionally wherein the host cell comprises nucleic acid or vector isolated according to the present invention. Typically, the host cell has been transformed or transfected with nucleic acid or vector. The recombinant host cell claimed can be, for example, a eukaryotic cell, a prokaryotic cell, or a microbial cell, such as a transfectoma. In a specific embodiment, the host cell is a eukaryotic cell. In a specific embodiment, the host cell is a prokaryotic cell. In some embodiments, the antibody is a heavy chain antibody. However, in most embodiments, the antibody variant will contain both heavy and light chains, and therefore the host cell expresses both the constructs encoding heavy and light chains on the same or different vectors.

[0427] In one embodiment, the host cell comprises a first nucleic acid construct and a second nucleic acid construct stably integrated into the cell genome, wherein the first encoding antibody variants disclosed herein is a heavy chain, and the second encoding light chain. In another embodiment, the invention provides a nucleic acid comprising non-integrated, such as a plasmid, a clay, a phagemid or a linear expression element cell, wherein the non-integrated nucleic acid comprises the first nucleic acid construct and the second nucleic acid construct as specified above.

[0428] In one embodiment, the host cell is a cell capable of Asn-linked glycosylation of proteins, such as a eukaryotic cell, such as a mammalian cell, such as a human cell. In a further embodiment, the host cell is a non-human cell that has been genetically engineered to produce glycoproteins with human-like or human glycosylation. Examples of such cells are genetically modified Pichia pastoris ( Pichia pastoris )(Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318-325), and genetically modified duckweed ( Lemna minor )(Cox et al., Nature Biotechnology 12 (2006) 1591-1597).

[0429] In one embodiment, the host cell is one that is incapable of efficiently removing the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 of Liu et al. (2008) J Pharm Sci 97:2426 (incorporated herein by reference) lists a number of such antibody production systems, such as Sp2 / 0, NS / 0, or transgenic mammary gland (goat), in which only partial removal of the C-terminal lysine is achieved. In one embodiment, the host cell is one that has altered glycosylation machinery. Such cells have been described in the art and can be used as host cells in which to express variants of the present invention, thereby producing antibodies with altered glycosylation. See, for example, Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, and EP1176195; WO03 / 035835; and WO99 / 54342. Additional methods for generating engineered glycoforms are known in the art and include, but are not limited to, those described in Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US 6602684, WO 00 / 61739 A1; WO 01 / 292246 A1; WO 02 / 311140 A1; WO 02 / 30954 A1; Potelligent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49, and those described in WO2018 / 114877, WO2018 / 114878 and WO2018 / 114879.

[0430] In an even further aspect, the invention relates to a transgenic non-human animal or plant comprising nucleic acid encoding one or two sets of human heavy chains and human light chains, wherein said animal or plant produces an antibody variant as disclosed herein.

[0431] In one embodiment, a method of producing an antibody variant as disclosed herein is provided, comprising culturing a recombinant host cell in a culture medium and under conditions suitable for production of the antibody variant, and optionally purifying or isolating the antibody variant from the culture medium.

[0432] In one embodiment, there is provided an antibody obtained or obtainable by the above method.

[0433] Compositions and kits of parts

[0434] The present invention further relates to a composition comprising an antibody variant according to the invention, a nucleic acid according to the invention, an expression vector according to the invention or a host cell according to the invention.

[0435] In a further embodiment, the composition according to the present invention is a pharmaceutical composition, which generally comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition contains an antibody variant as defined in any aspect or embodiment disclosed herein, or an expression vector as defined in any aspect or embodiment disclosed herein.

[0436] In yet a further embodiment, the present invention relates to a pharmaceutical composition comprising:

[0437] - an antibody variant as defined in any of the aspects and embodiments disclosed herein, and

[0438] - A pharmaceutically acceptable carrier.

[0439] Pharmaceutical compositions can be formulated according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutical compositions of the invention can, for example, include diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0440] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, etc., which are physiologically compatible with the antibody variants of the present invention. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloid solutions, tragacanth gum and injectable organic esters such as ethyl oleate, and / or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For example, by using a coating material such as lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant, suitable fluidity can be maintained.

[0441] The pharmaceutical composition may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0442] The pharmaceutical compositions can also include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0443] The pharmaceutical composition can also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which can enhance the shelf life or effectiveness of the pharmaceutical composition. The pharmaceutical composition of the present invention can be prepared with a carrier that protects the antibody from rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Such carriers can include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, alone or with wax, or other materials well known in the art. Methods for preparing such preparations are generally known to those skilled in the art.

[0444] Sterile injectable solutions can be prepared by mixing the desired amount of the active compound with, for example, one of the ingredients listed above or a combination thereof in an appropriate solvent, followed by sterilization microfiltration, as needed. Typically, dispersions are prepared by mixing the active compound into a sterile vehicle containing a basic dispersion medium and, for example, other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile-filtered solution thereof.

[0445] Actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon various pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound to be employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient to be treated, and similar factors well known in the medical arts.

[0446] The pharmaceutical composition can be administered by any suitable route and mode. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0447] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0448] The present invention also relates to a kit-of-parts comprising an antibody variant according to the invention, or a composition comprising an antibody variant according to the invention, for simultaneous, separate or sequential use in therapy, optionally wherein the kit-of-parts contains more than one dose of the antibody variant.

[0449] In one embodiment, a kit of parts comprises the antibody variant or composition, eg, in one or more containers, eg, vials.

[0450] In one embodiment, a kit-of-parts comprises an antibody variant or composition, eg, for simultaneous, separate or sequential use in therapy.

[0451] Therapeutic applications

[0452] The antibody variants of the present invention have numerous therapeutic uses related to the treatment of diseases and conditions involving cells expressing CD38, such as tumor cells or immune cells expressing CD38. For example, the antibody variants can be administered, for example, in vitro or ex vivo to cells in culture, or in vivo to human subjects, to treat or prevent various conditions and diseases. As used herein, the term "subject" is intended to include humans and non-human animals that can benefit from or respond to the antibodies. Subjects can, for example, include human patients suffering from a disease or condition that can be corrected or ameliorated by modulating CD38 function, such as enzymatic activity, and / or inducing lysis of CD38-expressing cells, and / or eliminating / reducing the number of CD38-expressing cells, and / or reducing the amount of CD38 on the cell membrane. Accordingly, the antibody variants can be used to elicit one or more of the following biological activities in vivo or in vitro: CDC of CD38-expressing cells in the presence of complement; inhibition of CD38 cyclase activity; phagocytosis or ADCC of CD38-expressing cells in the presence of human effector cells; and cytotoxicity of CD38-expressing cells, such as tumor cells or immune cells.

[0453] Thus, in one aspect, the present invention relates to an antibody variant according to the invention, a nucleic acid or a combination of nucleic acids according to the invention, a delivery vehicle according to the invention, an expression vector according to the invention, a host cell according to the invention, a composition according to the invention or a pharmaceutical composition according to the invention for use as a medicament.

[0454] In one aspect, the present invention relates to the use of an antibody variant according to the invention, a nucleic acid or a combination of nucleic acids according to the invention, a delivery vehicle according to the invention, an expression vector according to the invention, a host cell according to the invention, a composition according to the invention or a pharmaceutical composition according to the invention for the preparation of a medicament for treating or preventing a disease or disorder.

[0455] In one aspect, the present invention relates to an antibody variant according to the invention, a nucleic acid or a combination of nucleic acids according to the invention, a delivery vehicle according to the invention, an expression vector according to the invention, a host cell according to the invention, a composition according to the invention or a pharmaceutical composition according to the invention for use in treating or preventing a disease or disorder, such as for treating or preventing a disease or disorder involving CD38-expressing cells, such as for treating a disease involving CD38-expressing cells. In one aspect, the present invention relates to an antibody variant according to the invention, a nucleic acid according to the invention, an expression vector according to the invention, a host cell according to the invention, a composition according to the invention or a pharmaceutical composition according to the invention for inducing a CDC response against a tumor comprising CD38-expressing cells.

[0456] In one aspect, the present invention relates to a method of treating a disease or disorder comprising administering to a subject in need thereof an antibody variant according to the invention, a nucleic acid or a combination of nucleic acids according to the invention, a delivery vehicle according to the invention, an expression vector according to the invention, a host cell according to the invention, a composition according to the invention or a pharmaceutical composition according to the invention.

[0457] In one aspect, the invention relates to an antibody variant according to any aspect or embodiment for use as a medicament.

[0458] In one aspect, the invention relates to the use of an antibody variant according to any aspect or embodiment in the preparation of a medicament for the treatment or prevention of a disease or disorder.

[0459] In one aspect, the invention relates to an antibody variant according to any aspect or embodiment for use in treating or preventing a disease or disorder.

[0460] In one aspect, the invention relates to a method of treating a disease or disorder comprising administering to a subject in need thereof an antibody variant according to any aspect or embodiment, typically in a therapeutically effective amount and / or for a time sufficient to treat the disease or disorder.

[0461] In one aspect, the invention relates to a pharmaceutical composition comprising the antibody variant according to any aspect or embodiment for use as a medicament.

[0462] In one aspect, the invention relates to a pharmaceutical composition comprising the antibody variant according to any aspect or embodiment for use in treating or preventing a disease or disorder.

[0463] In one aspect, the invention relates to a method of treating a disease or disorder comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody variant according to any aspect or embodiment, typically in a therapeutically effective amount and / or for a time sufficient to treat the disease or disorder.

[0464] In one aspect, the invention relates to a method of treating a disease or condition comprising the steps of:

[0465] - selection of subjects with the disease or condition, and

[0466] - The antibody variant according to any aspect or embodiment, or a pharmaceutical composition comprising the antibody variant, is typically administered to a subject in a therapeutically effective amount and / or for a time sufficient to treat the disease or disorder.

[0467] In one embodiment, the disease or disorder involving CD38-expressing cells is cancer, i.e., a neoplastic disorder, e.g., a disorder characterized by the presence of tumor cells or immune cells expressing CD38, including, e.g., hematological cancers such as B-cell lymphomas, plasma cell malignancies, T / NK-cell lymphomas, myeloid malignancies, and solid tumor malignancies.

[0468] In some embodiments, the disease or disorder is cancer involving tumor cells expressing CD38.

[0469] In some embodiments, the disease or disorder is cancer involving immunosuppressive cells expressing CD38, eg, non-cancerous immunosuppressive cells expressing CD38.

[0470] In some embodiments, the disease or disorder is cancer involving both tumor cells and immunosuppressive cells expressing CD38.

[0471] In some embodiments, the disease or disorder is cancer involving immunosuppressive cells that express CD38 and tumor cells that do not express CD38.

[0472] In yet other embodiments, the disease or disorder is an inflammatory and / or autoimmune disease or disorder involving CD38 expressing cells.

[0473] In yet other embodiments, the disease or disorder is a metabolic disorder involving CD38 expressing cells.

[0474] Hematological cancers:

[0475] In one aspect, the disease or disorder is a hematological cancer. Examples of such hematological cancers include B cell lymphomas / leukemias, including precursor B cell lymphoblastic leukemia / lymphomas and B cell non-Hodgkin's lymphomas; acute promyelocytic leukemia, acute lymphoblastic leukemia and mature B cell neoplasms, such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell acute lymphocytic leukemia, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, and leukemia. The following are the main types of B-cell lymphoma: MCL, follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT, nodal, and splenic), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, plasmacytoma, plasma cell myeloma, plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, plasma cell leukemia, and anaplastic large cell lymphoma (ALCL).

[0476] Examples of B-cell non-Hodgkin's lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain diseases (including gamma, mu and alpha diseases), lymphomas induced by treatment with immunosuppressants, such as cyclosporine-induced lymphoma and methotrexate-induced lymphoma.

[0477] In one embodiment of the invention, the disorder involving CD38 expressing cells is Hodgkin's lymphoma.

[0478] Other examples of disorders involving CD38-expressing cells include malignancies derived from T cells and NK cells, including mature T cell and NK cell neoplasms, including T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia / lymphoma, extranodal NK / T cell lymphoma, nasal type, enteropathy type T cell lymphoma, hepatosplenic T cell lymphoma, subcutaneous panniculitis-like T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders (primary cutaneous anaplastic large cell lymphoma C-ALCL, lymphomatoid papules, marginal lesions), angioimmunoblastic T cell lymphoma, unspecified peripheral T cell lymphoma and anaplastic large cell lymphoma.

[0479] Examples of malignancies derived from myeloid cells include acute myeloid leukemias, including acute promyelocytic leukemia, and chronic myeloproliferative disorders, including chronic myeloid leukemia.

[0480] In some embodiments, the hematological cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL).

[0481] In some embodiments, the cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (adult) (AML), acute lymphoblastic leukemia (ALL), and follicular lymphoma (FL).

[0482] In some embodiments, the cancer is multiple myeloma (MM).

[0483] In some embodiments, the cancer is chronic lymphocytic leukemia (CLL).

[0484] In some embodiments, the cancer is mantle cell lymphoma (MCL).

[0485] In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0486] In some embodiments, the cancer is follicular lymphoma (FL).

[0487] In some embodiments, the cancer is acute myeloid leukemia (adult) (AML).

[0488] In some embodiments, the cancer is acute lymphoblastic leukemia (ALL).

[0489] Solid tumor malignancies:

[0490] In one aspect, the disease or condition is cancer comprising a solid tumor. That is, the patient suffering from cancer has a solid tumor.

[0491] Examples of solid tumors include, but are not limited to, melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary tract cancer, endometrial cancer, cervical cancer, lung adenocarcinoma, renal cell carcinoma (RCC) (e.g., clear cell renal carcinoma or papillary renal cell carcinoma), mesothelioma, nasopharyngeal carcinoma (NPC), esophageal cancer, or gastrointestinal cancer, or metastatic lesions of any of them.

[0492] In a preferred embodiment, solid tumors are from cancers containing immunosuppressive cells such as Tregs and expressing CD38. T regulatory cells (Tregs) can have the high expression of CD38, and compared with the Tregs with medium CD38 expression, the Tregs with high CD38 expression are more immunosuppressive (Krejcik J. et al. Blood 2016 128:384-394). Accordingly, without being limited by theory, the antibody variants of the present invention reduce the ability of the CD38 amount expressed on Tregs via cytokinesis, particularly allowing the treatment of solid tumors wherein Tregs express CD38 in patients. When the CD38 expression on Tregs is statistically significant compared with the control, such as using well-known methods, the expression detected with anti-CD38 antibodies is relative to the expression detected with isotype control antibodies, and Tregs express CD38. This can be, for example, by obtaining a biological sample, such as a blood sample, a bone marrow sample or a tumor biopsy is tested.

[0493] Thus, in one aspect, the invention relates to an antibody variant according to any aspect or embodiment, or a pharmaceutical composition comprising the antibody variant, for use in treating or preventing a solid tumor in a subject comprising CD38-expressing Tregs.

[0494] In another aspect, the invention relates to a method of treating a solid tumor in a subject comprising Tregs expressing CD38, the method comprising administering to the subject an antibody variant according to any aspect or embodiment, or a pharmaceutical composition comprising the antibody variant, typically in a therapeutically effective amount and / or for a time sufficient to treat the disease or disorder.

[0495] In some embodiments, the solid tumor is melanoma.

[0496] In some embodiments, the solid tumor is lung cancer.

[0497] In some embodiments, the solid tumor is squamous non-small cell lung cancer (NSCLC).

[0498] In some embodiments, the solid tumor is non-squamous NSCLC.

[0499] In some embodiments, the solid tumor is colorectal cancer.

[0500] In some embodiments, the solid tumor is prostate cancer.

[0501] In some embodiments, the solid tumor is castration-resistant prostate cancer.

[0502] In some embodiments, the solid tumor is stomach cancer.

[0503] In some embodiments, the solid tumor is ovarian cancer.

[0504] In some embodiments, the solid tumor is gastric cancer.

[0505] In some embodiments, the solid tumor is liver cancer.

[0506] In some embodiments, the solid tumor is pancreatic cancer.

[0507] In some embodiments, the solid tumor is thyroid cancer.

[0508] In some embodiments, the solid tumor is head and neck squamous cell carcinoma.

[0509] In some embodiments, the solid tumor is esophageal or gastrointestinal cancer.

[0510] In some embodiments, the solid tumor is breast cancer.

[0511] In some embodiments, the solid tumor is fallopian tube cancer.

[0512] In some embodiments, the solid tumor is brain cancer.

[0513] In some embodiments, the solid tumor is urethral cancer.

[0514] In some embodiments, the solid tumor is a genitourinary tract cancer.

[0515] In some embodiments, the solid tumor is endometrial cancer.

[0516] In some embodiments, the solid tumor is cervical cancer.

[0517] In some embodiments, the tumor cells of a solid tumor lack detectable expression of CD38. When CD38 expression on tumor cells isolated from a solid tumor is statistically insignificant compared to a control, for example, expression detected with an anti-CD38 antibody relative to expression detected with an isotype control antibody using well-known methods, the tumor cells of a solid tumor lack detectable expression of CD38. This can be tested, for example, by obtaining a biological sample, such as a biopsy, from the tumor.

[0518] In some embodiments, the cancer is in a patient comprising T regulatory cells expressing CD38.

[0519] In specific embodiments, the antibody variant is administered in a therapeutically effective amount and / or for a period of time sufficient to treat the cancer.

[0520] Metabolic disorders:

[0521] In one aspect, the disease or disorder is a metabolic disorder. That is, the patient suffers from a metabolic disorder.

[0522] In some embodiments, the metabolic disorder is amyloidosis. Amyloidosis is a broad range of diseases defined by the presence of insoluble protein deposits in tissues. Its diagnosis is based on histological findings. In a further embodiment, the amyloidosis can be AL amyloidosis.

[0523] patient:

[0524] The antibody variants of the present invention can be used to treat or prevent a disease or condition in a subject who has received at least one prior therapy for the same disease or condition with one or more compounds, wherein the one or more compounds are different from the antibody variants of the present invention. In one embodiment, the disease or condition can be any disease or condition described herein; for example, a cancer, an inflammatory and / or autoimmune disease or condition involving CD38-expressing cells, or a metabolic disorder involving CD38-expressing cells.

[0525] For example, in some embodiments, the antibody variants of the present invention can be used to treat or prevent a disease or condition in a subject who has received prior treatment with a proteasome inhibitor (PI) and / or an immunomodulatory drug (IMiD). Examples of proteasome inhibitors include, but are not limited to, bortezomib, carfilzomib, and ixazomib. Examples of IMiDs include, but are not limited to, thalidomide, lenalidomide, and pomalidomide. In a further embodiment, the disease or condition can be a cancer or tumor, such as multiple myeloma, mantle cell lymphoma, or myelodysplastic syndrome (MDS). Thus, the subject can be a cancer patient, such as a multiple myeloma, mantle cell lymphoma, or myelodysplastic syndrome (MDS) patient.

[0526] The antibody variants of the present invention can be used to treat or prevent a disease or condition in a subject who has not received any prior treatment with an anti-CD38 antibody. Typically, such subjects or patients are referred to as patients who are naive to treatment with an anti-CD38 antibody. In one embodiment, the anti-CD38 antibody is daratumumab; that is, the subject or patient has not received any prior treatment with daratumumab. Thus, in one embodiment, the subject or patient is a subject / patient being treated with daratumumab for the first time. According to any aspect or embodiment disclosed herein, the disease or condition can be a cancer or tumor or a metabolic disease, such as amyloidosis.

[0527] The present invention also provides antibody variants for use in treating or preventing a disease or disorder in a subject who has received at least one prior therapy comprising a CD38 antibody.

[0528] The present invention also provides antibody variants for treating cancer patients who have received at least one prior therapy comprising a CD38 antibody. The present invention also provides antibody variants for treating patients with metabolic diseases such as amyloidosis who have received at least one prior therapy comprising a CD38 antibody. Such prior therapy may be one or more cycles of a planned treatment program comprising a CD38 antibody, such as one or more planned cycles of a CD38 antibody as a single agent therapy or combination therapy, and a series of treatments administered in a planned manner. In one embodiment, the prior therapy is a CD38 antibody monotherapy. In one embodiment, the prior therapy is a combination therapy comprising a CD38 antibody. For example, the prior therapy may be a CD38 antibody in combination with a proteasome inhibitor (PI) and an immunomodulator. In some embodiments, the CD38 antibody is daratumumab.

[0529] In some aspects, the cancer patient may also be one in whom administration of daratumumab as monotherapy has a limited effect.

[0530] In some aspects, cancer can be characterized as a cancer that is "refractory" or "relapsed" to a previous therapy. In a further embodiment, the previous therapy may include one or more of a PI, an IMiD, and a CD38 antibody, for example, wherein the CD38 antibody is daratumumab. Typically, this indicates that the previous therapy has achieved less than a complete response (CR), for example, cancer has not responded to a single or combined therapy with a CD38 antibody, or cancer has progressed within a predetermined time period after the end of the CD38 antibody therapy. Examples of such combination therapies include, but are not limited to, a combination of a CD38 antibody and a PI or IMiD, or a combination of a PI and an IMiD. Similarly, this may indicate that the previous therapy has achieved less than a complete response (CR), for example, cancer has not responded to a PI, an IMiD, or a combined therapy thereof, or cancer has progressed within a predetermined time period after the end of the therapy. Technicians can determine whether cancer is refractory to a previous therapy based on knowledge known in the art (including guidelines available for each cancer).

[0531] For example, in multiple myeloma, the guidelines published by Rajkumar, Harousseau et al. on behalf of the International Myeloma Workshop Consensus Panel can be followed. Consensus recommendations for the uniform reporting of clinical trials: report of the International Myeloma Workshop Consensus Panel ,Bloo d 2011；117:4691- 4695 , to identify refractory and relapsed disease:

[0532] Refractory Myeloma can be defined as disease that has not responded to initial or salvage therapy, or has progressed within 60 days of the last line of therapy. Nonresponsive disease is defined as failure to achieve a minimal response, or development of progressive disease (PD) on treatment. There can be two categories of refractory myeloma: "relapsed and refractory myeloma" and "primary refractory myeloma":

[0533] Relapsed and refractory Myeloma can be defined as disease that has not responded to salvage therapy or has progressed within 60 days of the last therapy in patients who have achieved a minimal response (MR) or who had improved at some previous time point and then progressed in their disease course.

[0534] Primary refractoryMyeloma can be defined as a disease that is unresponsive in patients who have never achieved a minimal response or improvement to any therapy. This includes patients who have never achieved a MR or improvement, in whom there is no significant change in the M protein and no evidence of clinical progression, as well as primary refractory disease, PD (in which patients meet the criteria for true PD). When reporting treatment efficacy for primary refractory patients, the efficacy in these two subgroups ("no response-non-progressive" and "progressive") should be specified separately.

[0535] Relapsed myeloma It can be defined as a previously treated myeloma that progressed and required initiation of salvage therapy but did not meet the criteria for the “primary refractory myeloma” or “relapsed and refractory myeloma” categories.

[0536] For details on specific responses (CR, PR, etc.) in multiple myeloma and how to detect them, see Rajkumar, Harousseau et al., 2011 (supra).

[0537] Accordingly, in some embodiments, the antibody variants according to any aspect or embodiment herein, or pharmaceutical compositions comprising the antibody variants, are used to treat a cancer that is refractory to a previous treatment comprising one or more of a PI, an IMiD, and a CD38 antibody. In one embodiment, the previous treatment comprises a CD38 antibody. In a specific embodiment, the cancer is identified as a refractory cancer prior to use.

[0538] In another embodiment, a method for treating cancer in a subject is provided, comprising the steps of:

[0539] (i) identifying the subject as refractory to prior therapy comprising one or more of a PI, an IMiD, and a CD38 antibody, and

[0540] (ii) administering to the subject a therapeutically effective amount of the antibody variant according to any aspect or embodiment herein, or a pharmaceutical composition comprising the antibody variant.

[0541] In one embodiment, the prior treatment comprised a CD38 antibody.

[0542] In another embodiment, a method for treating a cancer that is refractory to a previous treatment in a subject, wherein the previous treatment comprises one or more of a PI, an IMiD, and a CD38 antibody, is provided, the method comprising administering to the subject a therapeutically effective amount of an antibody variant according to any aspect or embodiment herein, or a pharmaceutical composition comprising the antibody variant. In one embodiment, the previous treatment comprises a CD38 antibody.

[0543] In some embodiments, the PI is selected from bortezomib, carfilzomib, and ixazomib.

[0544] In some embodiments, the IMiD is selected from thalidomide, lenalidomide, and pomalidomide.

[0545] In some embodiments, the CD38 antibody is daratumumab.

[0546] In some embodiments, the antibody variants according to any aspect or embodiment herein, or pharmaceutical compositions comprising the antibody variants, are used to treat cancer that has relapsed after a prior treatment comprising one or more of a PI, an IMiD, and a CD38 antibody. In one embodiment, the prior treatment comprises a CD38 antibody. In a specific embodiment, the cancer is identified as recurrent prior to use.

[0547] In another embodiment, a method for treating cancer in a subject is provided, comprising the steps of:

[0548] (i) identifying the subject as having relapsed after prior treatment comprising one or more of a PI, an IMiD, and a CD38 antibody, and

[0549] (ii) administering to the subject a therapeutically effective amount of the antibody variant according to any aspect or embodiment herein, or a pharmaceutical composition comprising the antibody variant.

[0550] In one embodiment, the prior treatment comprised a CD38 antibody.

[0551] In another embodiment, a method for treating a cancer that has recurred after a prior treatment in a subject is provided, wherein the prior treatment comprises one or more of a PI, an IMiD, and a CD38 antibody, the method comprising administering to the subject a therapeutically effective amount of an antibody variant according to any aspect or embodiment herein, or a pharmaceutical composition comprising the antibody variant. In one embodiment, the prior treatment comprises a CD38 antibody.

[0552] In some embodiments, the PI is selected from bortezomib, carfilzomib, and ixazomib.

[0553] In some embodiments, the IMiD is selected from thalidomide, lenalidomide, and pomalidomide.

[0554] In some embodiments, the CD38 antibody is daratumumab.

[0555] In specific embodiments, the antibody variants according to the invention are administered in a therapeutically effective amount and / or for a period of time sufficient to treat a refractory or recurrent cancer.

[0556] In some embodiments, the refractory or relapsed cancer is a hematological cancer.

[0557] In some embodiments, the refractory or relapsed cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (adult) (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL).

[0558] In some embodiments, the refractory or relapsed cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma (FL).

[0559] In some embodiments, the refractory or relapsed cancer is multiple myeloma (MM).

[0560] In some embodiments, the refractory or relapsed cancer is chronic lymphocytic leukemia (CLL).

[0561] In some embodiments, the refractory or relapsed cancer is mantle cell lymphoma (MCL).

[0562] In some embodiments, the refractory or relapsed cancer is diffuse large B-cell lymphoma (DLBCL).

[0563] In some embodiments, the refractory or relapsed cancer is follicular lymphoma (FL).

[0564] In some embodiments, the refractory or recurrent cancer is a solid tumor. In some embodiments, the refractory or recurrent cancer is selected from melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethra cancer, genitourinary tract cancer, endometrial cancer, cervical cancer.

[0565] In some embodiments, the refractory or relapsed cancer is melanoma.

[0566] In some embodiments, the refractory or relapsed cancer is lung cancer.

[0567] In some embodiments, the refractory or relapsed cancer is squamous non-small cell lung cancer (NSCLC).

[0568] In some embodiments, the refractory or relapsed cancer is non-squamous NSCLC.

[0569] In some embodiments, the refractory or relapsed cancer is colorectal cancer.

[0570] In some embodiments, the refractory or relapsed cancer is prostate cancer.

[0571] In some embodiments, the refractory or relapsed cancer is castration-resistant prostate cancer.

[0572] In some embodiments, the refractory or relapsed cancer is stomach cancer.

[0573] In some embodiments, the refractory or relapsed cancer is ovarian cancer.

[0574] In some embodiments, the refractory or relapsed cancer is gastric cancer.

[0575] In some embodiments, the refractory or relapsed cancer is liver cancer.

[0576] In some embodiments, the refractory or relapsed cancer is pancreatic cancer.

[0577] In some embodiments, the refractory or relapsed cancer is thyroid cancer.

[0578] In some embodiments, the refractory or relapsed cancer is head and neck squamous cell carcinoma.

[0579] In some embodiments, the refractory or relapsed cancer is esophageal or gastrointestinal cancer.

[0580] In some embodiments, the refractory or relapsed cancer is breast cancer.

[0581] In some embodiments, the refractory or relapsed cancer is fallopian tube cancer.

[0582] In some embodiments, the refractory or relapsed cancer is brain cancer.

[0583] In some embodiments, the refractory or relapsed cancer is urethral cancer.

[0584] In some embodiments, the refractory or relapsed cancer is a genitourinary tract cancer.

[0585] In some embodiments, the refractory or relapsed cancer is endometrial cancer.

[0586] In some embodiments, the refractory or relapsed cancer is cervical cancer.

[0587] Autoimmune and inflammatory diseases and conditions:

[0588] In another embodiment of the invention, the disorder involving CD38 expressing cells is an immune disorder in which B cells, macrophages, plasma cells, monocytes and T cells expressing CD38 are involved, such as inflammatory and / or autoimmune diseases. Examples of immune disorders involving B cells, plasma cells, monocytes and T cells expressing CD38 include autoimmune disorders such as psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile onset diabetes, Reiter's disease, Behcet's disease, immune complex disease, The present invention relates to the treatment of thrombocytopenia.Nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omenn syndrome, chronic renal failure, acute infectious mononucleosis, multiple sclerosis, HIV and herpes virus related diseases. Further example is severe acute respiratory distress syndrome and chorioretinitis (choreoretinitis). In addition, other diseases and conditions are also included, such as those caused or mediated by B cells infected by viruses such as Epstein-Barr virus (EBV).

[0589] In one embodiment, the disorder involving CD38 expressing cells is rheumatoid arthritis.

[0590] Further examples of inflammatory, immune and / or autoimmune disorders in which autoantibodies and / or excessive B and T lymphocyte activity predominate and which may be treated according to the present invention include the following: vasculitis and other vascular disorders, such as microscopic polyangiitis, Churg-Schönlein syndrome and other ANCA-associated vasculitis, polyarteritis nodosa, primary cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, Kawasaki disease, Takayasu's arteritis, giant cell arthritis, Henoch-Schonlein purpura, primary or isolated cerebral angiitis, erythema nodosum, thromboangiitis obliterans, thrombotic thrombocytopenic purpura (including hemolytic uremic syndrome), and secondary vasculitis, including cutaneous leukocytoclastic vasculitis (e.g., secondary to type B virus infection). further examples are erythema nodosum, allergic vasculitis, panniculitis, Weck's disease, hyperglobulinemic purpura, and Buerger's disease; skin disorders such as contact dermatitis, linear IgA dermatosis, vitiligo, pyoderma gangrenosum, epidermolysis bullosa acquisitive, pemphigus vulgaris (including cicatricial pemphigoid and bullous pemphigoid), alopecia areata (including alopecia universalis and totalis), dermatitis herpetiformis, erythema multiforme, and chronic autoimmune urticaria (including angioedema and urticarial vasculitis); immune-mediated cytopenias such as autoimmune neutropenia and pure Red cell aplasia; connective tissue disorders such as CNS lupus, discoid lupus erythematosus, CREST syndrome, mixed connective tissue disease, polymyositis / dermatomyositis, inclusion body myositis, secondary amyloidosis, cryoglobulinemia type I and II, fibromyalgia, phospholipid antibody syndrome, secondary hemophilia, relapsing polychondritis, sarcoidosis, stiff-man syndrome and rheumatic fever; further examples are eosinophilic fasciitis; arthritis (arthritide), such as ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease and SAPHO syndrome; further examples are sacroiliitis, reactive arthritis, Still's disease and gout; hematological disorders such as aplastic anemia, primary hemolytic anemias secondary to CLL or systemic lupus erythematosus; POEMS syndrome, pernicious anemia, and Waldenstrom's purpura; further examples are agranulocytosis, autoimmune neutropenia, Franklin's disease, Seligman's disease, gamma heavy chain disease, paraneoplastic syndromes secondary to thymoma and lymphoma, and factor VIII inhibitor formation; endocrinopathy, such as polyendocrinopathy and Addison's disease; further examples are autoimmune hypoglycemia, autoimmune hypothyroidism, insulin autoimmune syndrome, Quervain's thyroiditis, and insulin resistance mediated by insulin receptor antibodies;Hepato-gastrointestinal disorders, such as celiac disease, Whipple's disease, primary biliary cirrhosis, chronic active hepatitis and primary sclerosing cholangitis; further examples are autoimmune gastritis; renal disorders, such as rapidly progressive glomerulonephritis, poststreptococcal glomerulonephritis, Goodpasture's syndrome, membranous glomerulonephritis and cryoglobulinemic nephritis; further examples are minimal change disease; nervous system disorders, such as autoimmune neuropathies, mononeuritis multiplex, Lambert-Eaton myasthenic syndrome, Sydenham's chorea, tabes dorsalis and Guillain-Barré syndrome; further examples are myelopathy / tropical spastic paresis, myasthenia gravis, Acute inflammatory demyelinating polyneuropathy and chronic inflammatory demyelinating polyneuropathy; multiple sclerosis; cardiac and pulmonary disorders, such as COPD, fibrosing alveolitis, bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis, Löffler syndrome, myocarditis and pericarditis; further examples are hypersensitivity pneumonitis and paraneoplastic syndromes secondary to lung cancer; allergic disorders, such as bronchial asthma and hyper-IgE syndrome; further examples are amaurosis fugax; ophthalmological disorders, such as idiopathic chorioretinitis; infectious diseases, such as parvovirus B infection (including hands-and-socks syndrome); gynecological disorders, such as recurrent miscarriage, recurrent pregnancy loss and intrauterine growth retardation; further examples are paraneoplastic syndromes secondary to gynecological tumors; male reproductive system disorders, such as paraneoplastic syndromes secondary to testicular tumors; and transplant-derived disorders, such as allograft and xenograft rejection, and graft-versus-host disease.

[0591] In one embodiment, the disease or condition is rheumatoid arthritis.

[0592] Dosage regimens and combinations

[0593] The dosage regimen in the above-described methods of treatment and uses is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0594] The effective dosage and dosage regimen of the antibody variants depends on the disease or condition to be treated and can be determined by one skilled in the art.An exemplary, non-limiting range for a therapeutically effective amount of the antibody variants of the invention is about 0.001-30 mg / kg.

[0595] The antibody variants may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence while the cancer is in remission.

[0596] The antibody variants may also be administered in combination therapy, ie, in combination with other therapeutic agents or treatment modalities relevant to the disease or condition to be treated.

[0597] Accordingly, in one embodiment, the antibody variants are used in combination with one or more further therapeutic agents, such as chemotherapeutic agents, anti-inflammatory agents, or immunosuppressive and / or immunomodulatory agents, such as another therapeutic antibody. Such combined administration can be simultaneous, separate, or sequential. For simultaneous administration, the agents can be administered as a single composition or as separate compositions, as appropriate.

[0598] The antibody variants may also be used in combination with radiation therapy and / or surgery and / or autologous or allogeneic peripheral stem cell or bone marrow transplantation.

[0599] Diagnostic applications

[0600] In further aspects, diagnostic compositions and uses comprising the antibody variants according to any aspect or embodiment are also contemplated, e.g., for diseases involving CD38 expressing cells as listed above. The antibody variants can be labeled, e.g., with a radioactive agent (as described elsewhere herein) or a radiopaque agent. In one embodiment, the diagnostic composition is a companion diagnostic for screening and selecting patients who will benefit from treatment with the antibody variant.

[0601] In one embodiment, the invention relates to the use of an antibody variant, composition or kit of parts according to any aspect or embodiment herein for use in a diagnostic method.

[0602] In one embodiment, the invention relates to a diagnostic method comprising administering a polypeptide, antibody, composition or kit of parts according to any aspect or embodiment herein to at least a part of the body of a human or other mammal.

[0603] In another embodiment, the invention relates to the use of an antibody variant, composition or kit of parts according to any aspect or embodiment herein for imaging at least a part of the body of a human or other mammal.

[0604] In another embodiment, the invention relates to a method for imaging at least a portion of a human or other mammalian body comprising administering a variant, composition or kit of parts according to any aspect or embodiment described herein.

[0605] Table 1 - Amino acid and nucleic acid sequences

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615] Example

[0616] The present invention is further illustrated by the following examples, which should not be construed as limiting.

[0617] Example 1 - Antibodies and cell lines

[0618] Antibody expression constructs

[0619] To express the human and humanized antibodies used herein, variable heavy (VH) and variable light (VL) chain sequences were prepared by gene synthesis (GeneArt GeneSynthesis; ThermoFisher Scientific) and cloned into pcDNA3.3 expression vectors (ThermoFisher Scientific) containing the constant region of a human IgG heavy chain (HC) (constant region human IgG1m(f)HC: SEQ ID NO: 20) and / or the constant region of a human kappa light chain (LC): SEQ ID NO: 37. Desired mutations were introduced by gene synthesis. The CD38 antibody variants in the present application have VH and VL sequences derived from the following previously described CD38 antibodies: IgG1-A (WO 2006 / 099875 A1, WO 2008 / 037257 A2, WO 2011 / 154453 A1; VH: SEQ ID NO: 10; VL: SEQ ID NO: 11), IgG1-B (WO 2006 / 099875 A1, WO 2008 / 037257 A2, WO 2011 / 154453 A1; VH: SEQ ID NO: 8; VL: SEQ ID NO: 9), and IgG1-C (WO 2011 / 154453 A1; VH: SEQ ID NO: 1; VL: SEQ ID NO: 5). Human IgG1 antibody b12, an HIV gpl20-specific antibody, was used as a negative control in some experiments (Barbas et al., J Mol Biol. 1993 Apr 5; 230(3):812-23; VH: SEQ ID NO: 12; VL: SEQ ID NO: 16).

[0620] Transient expression of antibody constructs

[0621] Essentially as described by Vink et al. (Vink et al., 2014 Methods 65(1):5-10), a mixture of plasmid DNA encoding both the heavy and light chains of the antibody was transiently transfected in Expi293F cells (Gibco, catalog number A14635) using 293fectin (Life Technologies). The antibody concentration in the supernatant was measured by absorbance at 280 nm. The antibody-containing supernatant was used directly for in vitro assays or the antibody was purified as described below.

[0622] Antibody purification and quality assessment

[0623] The antibodies were purified by protein A affinity chromatography. The culture supernatant was filtered through a 0.20 μM dead-end filter and loaded onto a 5 mL MabSelect SuRe column (GE Healthcare), washed and eluted with 0.02 M sodium citrate-NaOH, pH 3. Immediately after purification, the eluate was loaded onto a HiPrep Desalting column (GE Healthcare), and the antibody buffer was exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer (B.Braun or Thermo Fisher). After buffer exchange, the sample was sterile filtered through a 0.2 μm dead-end filter. The purified protein was analyzed by a variety of bioanalytical assays, including capillary electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (CE-SDS) and high performance size exclusion chromatography (HP-SEC). Concentration was measured by absorbance at 280 nm. The purified antibodies were stored at 2-8°C.

[0624] The cell lines used in the examples are described below in Table 2. The average number of CD38 and CD59 molecules per cell was determined by quantitative flow cytometry (Qifi, DAKO).

[0625] Table 2: Overview of cell lines and expression of CD38 and CD59

[0626]

[0627] ABC = antibody bound per cell

[0628] The origin / source of the cell lines are as follows:

[0629] Cell lines: source: Daudi ATCC; CCL-213 Ramos ATCC;CRL-1596 Wien-133 BioAnaLab, Oxford, UK NALM-16 DSMZ;ACC 680 U266 ATCC;TIB-196 RC-K8 DSMZ;ACC 561

[0630] Example 2 - Binding of CD38 Antibodies and Variants to Human and Cynomolgus Monkey CD38 Expressed on Cell Surfaces

[0631] Binding to CD38 expressed on the cell surface of Daudi and NALM16 cells and PBMCs from cynomolgus monkeys was determined by flow cytometry. Cells resuspended in RPMI containing 0.2% BSA were seeded at 100,000 cells / well in polystyrene 96-well round-bottom plates (Greiner bio-one) and incubated at 300×. g, centrifuged at 4 ° C for 3 minutes. Serial dilutions of CD38 or control antibodies (with 3x serial dilutions of 0.005-10 μg / mL final antibody concentration) were added, and the cells were incubated at 4 ° C for 30 minutes. Using FACS buffer (PBS / 0.1% BSA / 0.01% sodium azide), the plate was washed / centrifuged twice. Next, the cells were incubated at 4 ° C for 30 minutes with R-phycoerythrin (PE) conjugated goat anti-human IgG F (ab') 2 (Jackson) or FITC-conjugated goat anti-human IgG (Southern Biotech) for analyzing cynomolgus monkey PBMCs at 1 / 100 dilution. The cells were washed / centrifuged twice using FACS buffer, resuspended in FACS buffer, and analyzed by measuring the mean fluorescence intensity using FACS_Fortessa (BD). Binding curves were generated using nonlinear regression (sigmoidal dose response with variable slope) analysis within GraphPad Prism V6.04 software (GraphPad Software).

[0632] Figure 2 The CD38 antibodies IgG1-B, IgG1-C, and IgG1-A were shown to bind to NALM16 cells expressing CD38 in a dose-dependent manner. The introduction of the hexamerization-enhancing E430G mutation into these antibodies did not affect binding.

[0633] Figure 3 shows that the CD38 antibody IgG1-A-E430G, but not IgG1-B-E430G and IgG1-C-E430G, binds dose-dependently to CD38 expressed on cynomolgus monkey PBMCs (A). Gating based on FSC and SSC is depicted, and the average binding to CD38 expressed on cynomolgus monkey B, T, and NK cells is depicted. As a positive control, binding to Daudi cells expressing high copy numbers of human CD38 is also depicted (B).

[0634] Example 3 - Complement-dependent cytotoxicity (CDC) by CD38 antibodies with the E430G mutation

[0635] CDC on tumor cell lines

[0636] Daudi, Wien133, Ramos, NALM16, U266, and RC-K8 cells were resuspended in RPMI containing 0.2% BSA and cultured at 1×10 5The density of individual cells / well (40 μL / well) was spread onto a polystyrene 96-well round-bottom plate (Greiner bio-one). CD38 antibody, its variant and isotype control Ab were serially diluted (with 0.0002-10 μg / mL final antibody concentration of 3x serial dilutions), and 40 μL diluted Ab was added to each well. Cells and Ab were pre-incubated at room temperature for 20 minutes. After this, 20 μL of normal human serum (Sanquin) combined was added to each well and incubated for another 45 minutes at 37°C. Thereafter, the plate was centrifuged (3 minutes, 1200 rpm), and the supernatant was discarded. The cell pellet was resuspended in FACS buffer (Life Technologies) supplemented with 0.25 μM topro-3 iodide, and lysis was detected by measuring the topro-3 iodine-positive cell percentage on FACS_Fortessa (BD). CDC is described as percentage lysis. Data shown are N = 3 (Daudi and NALM16), N = 2 (Wien133 and U266 cells), or N = 1 (RC-K8 and Ramos). Isotype control antibodies were included only for Daudi and Wien133 cells.

[0637] Figure 4 It was demonstrated that CD38 antibodies B, C, and A, which do not contain the E430G mutation, induced ~85, ~50, and 0 percent lysis of Ramos and Daudi cells, respectively. No significant lysis was observed with these CD38 antibodies for any of the other cell lines tested. Introduction of the E430G mutation into these CD38 antibodies resulted in higher CDC activity at significantly lower antibody concentrations. All three antibodies with the E430G mutation induced up to 100% lysis of Ramos and Daudi cells. Furthermore, with respect to cell lines with lower CD38 expression, CD38 antibodies with the E430G mutation were able to induce maximal (Wien133) or partial (NALM16 and U266) CDC, whereas CD38 antibodies without the E430G mutation did not induce CDC. These results demonstrate that CD38 Abs with the E430G mutation induce stronger CDC than CD38 antibodies without the E430G mutation and require less CD38 expression. In tumor cells with lower CD38 expression levels (NALM-16, RS4;11, and REH), IgG1-C-E430G showed lower EC50 values compared to IgG1-B-E430G.

[0638] Table 3 EC50 values for lysis.

[0639] Some cell lines were tested only once (Ramos, RS4;11, REH)

[0640] Ramos Daudi Wien-133 NALM-16 U266 RS4;11 REH B 0.126 0.183 0.199 - - - - B-E430G 0.019 0.018 0.013 0.075 - 0.243 0.054 C 0.158 0.250 0.193 - - - - C-E430G 0.014 0.019 0.015 0.022 0.052 0.056 0.017 A - - - - - - - A-E430G 0.133 0.206 0.271 - - - -

[0641] The CDC assay described above was repeated using a number of additional tumor cell lines derived from B cell tumors, including DLBCL, Burkitt's lymphoma, FL, MCL, B-ALL, CLL, or MM, and antibodies IgG1-B, IgG1-B-E430G, IgG1-C-E430G, IgG1-A-E430G, and an isotype control antibody. The percent lysis was plotted against the antibody concentration, and the maximum percent lysis and EC50 values were calculated using Graphpad Prism (GraphPad Software, Inc; Version 8.1.0) software and are shown in Table 4. The results are also shown in Table 4. Figure 14 middle.

[0642] Figure 14 It was demonstrated that the wild-type CD38 mAb IgG1-B induced lysis of high CD38 expressing cell lines (SU-DHL-8, Oci-Ly-7, Oci-Ly-19, Ramos, Daudi, Oci-Ly-18, and Raji), but not of any other cell lines expressing fewer CD38 molecules on the membrane. Introduction of the E430G mutation in IgG1-B resulted in higher CDC activity at significantly lower Ab concentrations for cell lines already sensitive to wild-type IgG1-B, and in lysis of additional cell lines with lower CD38 copy numbers that were insensitive to IgG1-B-induced CDC (e.g., DOHH2, SU-DHL-4, WSU-DLCL2, Z-138, JVM-13, REH, Jeko-1, Wien-133, 697, RS4;11, NALM-16, and JVM-3). Some cell lines with very low CD38 expression (RC-K8 and Pfeiffer) or very high CD59 expression (DB and Granta-519) did not show lysis when exposed to IgG1-B and IgG1-B-E430G. For almost all cell lines tested, IgG1-C-E430G induced cell lysis at lower antibody concentrations compared to IgG1-B-E430G, while IgG1-A-E430G induced lysis at much higher Ab concentrations. This is also reflected by the higher EC50 values for IgG1-A-E430G in Table 4. This confirms that the E430G mutant CD38 mAb induces more potent CDC than the wild-type CD38 antibody and induces CDC on tumor cells with lower CD38 expression levels, where the wild-type CD38 antibody does not induce CDC. In addition, the potency of the E430G mutant CD38 antibody to induce CDC may vary between different CD38-targeting antibody clones.

[0643] Figure 15 A summary of some of the EC50 values depicted in Table 4 is shown. Shown are the EC50 values for CDC induced by antibodies IgG1-B, IgG1-B-E430G, and IgG1-C-E430G against 20 different B-cell tumor cell lines. Each square, triangle, or circle represents a different B-cell tumor cell line. EC50 values obtained with AML cell lines are not included because IgG1-B-E430G was not tested against AML cell lines.

[0644] Selected acute myeloid leukemia (AML) cell lines were also evaluated by CDC of IgG1-C-E430G ( Figure 16 ). It was performed as described above for the B cell tumor cell line, the only difference being the tumor cell line.

[0645] Figure 16 We confirmed that CDC was induced by IgG1-C-E430G in all CD38-expressing AML cell lines, whereas no CDC was observed in CD38-negative AML cell lines. CDC by IgG1-C-E430G occurred at much lower EC50 values compared to IgG1-B, while maximum cell lysis was higher for IgG1-C-E430G compared to IgG1-B (Table 4).

[0646] Table 4 Maximum lysis and EC50 values of lysis

[0647]

[0648]

[0649] CDC induction by wild-type and E430G mutant CD38 antibodies was also determined using T regulatory cells. T regulatory cells were generated as described in Example 8 (cytotoxicity of CD38 from T regulatory cells) and tested in the CDC assay as described above for tumor cell lines. The percentage of lysis is shown in Table 1 along with the EC50 values. Figure 17 middle.

[0650] Figure 17 It was confirmed that IgG1-B hardly induced lysis of T regulatory cells; whereas IgG1-B-E430G and IgG1-C-E430G induced lysis of T regulatory cells, with IgG1-C-E430G showing a lower EC50 value compared to IgG1-B-E430G.

[0651] CDC in whole blood

[0652] Whole blood from healthy donors was collected in hirudin tubes to prevent coagulation without interfering with physiological calcium levels (essential for CDC). 50 μL / well was plated onto 96-well flat-bottom tissue culture plates (Greiner bio-one). CD38 antibodies, their variants, and control Abs were serially diluted in RPMI containing 0.2% BSA (with 5x serial dilutions of 0.016-10 μg / mL final antibody concentration), and 50 μL of diluted Ab was added to each well and incubated overnight at 37°C. As a positive control for CDC of B cells, CD20 Ab IgG1-7D8 was tested with and without 60 μg / mL eculizumab blocking CDC. Cells were transferred to polystyrene 96-well round-bottom plates (Greiner bio-one, centrifuged), centrifuged (3 minutes, 1200 rpm), and each well was washed once with 150 μL PBS (B.Braun). The cell pellet was resuspended in 80 μL PBS with a 1000x dilution of amine-reactive viability dye (BD) and incubated for 30 minutes at 4° C. Next, the cells were washed with 150 μL PBS and incubated with 80 μL PBS containing a lymphocyte phenotyping antibody cocktail (mouse anti-human CD3-EF450 [OKT3, ebioscience] at 1:200, mouse anti-human CD19-BV711 [HIB19, Biolegend] at 1:50, and mouse anti-human CD56-PE / CF594 [NCAM16.2, BD] at 1:100) for 30 minutes at 4° C. The cells were washed with 150 μL PBS and incubated with 150 μL erythrocyte lysis buffer (10 mM KHCO3 [Sigma], 0.01 mM EDTA [Fluka], 155 mM NH4Cl [Sigma] dissolved in 1 L H2O [B.Braun] and adjusted to pH 7.2) at 4°C for 10 minutes. The cells were washed with 150 μL FACS buffer, resuspended in 100 μL FACS buffer, and analyzed on a FACS_Fortessa (BD). Figure 5 Live NK cells (CD56 阳性 、CD3 阴性 and amine-reactive vibrant dyes 阴性 ), T cells (CD3 阳性 and amine-reactive vibrant dyes 阴性 ), and B cells (CD19 阳性 and amine-reactive vibrant dyes 阴性 Data from 1 representative donor out of 5 tested are shown.

[0653] Figure 5 The CD38 antibody containing the E430G mutation was shown to induce minimal CDC in healthy blood lymphocytes. The positive control CD20 Ab IgG1-7D8 demonstrated specific CDC in CD20-positive B cells, which was completely blocked by the CDC inhibitor eculizumab. Wild-type IgG1 CD38 antibody did not induce CDC in B, T, and NK cells. Following incubation with clones B and C containing the E430G mutation (approximately 40% NK cell lysis at the highest concentration of IgG1-B-E430G), some CDC was observed for NK cells, but not for B and T cells.

[0654] Overall, these results indicate that the CD38 antibody with the E430G mutation has broad CDC activity against a panel of tumor cell lines with variable CD38 expression. The CD38 antibody with the E430G mutation was also tested against lymphocytes obtained from healthy donors and was shown to induce only up to 40% NK cell lysis. NK cells expressed an average of 15,000 CD38 molecules / cell, which is similar to the MM cell line U266. Both cell types were equally sensitive to CDC by the CD38 antibody with the E430G mutation, indicating that CDC by the CD38 antibody with the E430G mutation is associated with CD38 expression. Without being limited by theory, based on these data, it is believed that the threshold for CDC by the CD38 antibody with the E430G mutation is approximately 15,000 CD38 molecules / cell. While most B-cell tumor cell lines express relatively high levels of CD38, ranging from 15,000 to 400,000 CD38 molecules / cell, healthy lymphocytes express only 2,000-15,000 CD38 molecules / cell, making these cells less susceptible to CDC by antibodies against the E430G mutation.

[0655] Example 4 - Antibody-dependent cellular cytotoxicity (ADCC) by CD38 antibodies with the E430G mutation

[0656] The ability of the E430G mutant CD38 antibody to induce antibody-dependent cellular cytotoxicity (ADCC) was determined by chromium release assay. Daudi cells (5 × 10 6 cells / mL), to which 100 μCi 51 Cr (chromium-51; PerkinElmer). The cells were incubated in a water bath at 37°C for 1 hour while shaking. After washing the cells (twice in PBS, 1500 rpm, 5 minutes), the cells were resuspended in culture medium and counted by trypan blue exclusion. The cells were diluted to 1×10 5Cells were plated at a density of 10 cells / mL and then pipetted into a 96-well round-bottom microtiter plate (Greiner Bio-One), and 50 μL of a concentration series of CD38 or isotype control antibodies diluted in culture medium (0.005-10 μg / mL final concentration in 3-fold dilutions) were added. Cells were pre-incubated with Ab for 15 minutes at room temperature (RT).

[0657] In parallel, peripheral blood mononuclear cells (PBMCs) from healthy volunteers (Sanquin) were isolated from 45 mL of freshly drawn heparinized blood (buffy coat) using lymphocyte separation medium (Bio Whittaker) according to the manufacturer's instructions. After resuspension in the culture medium, cells were counted by trypan blue exclusion and diluted to 1×10 7 The density of cells / mL.

[0658] After pre-incubation of target cells with Ab, 50 μL of effector cells were added, resulting in an effector cell to target cell ratio of 100:1. The cells were incubated for 4 hours at 37°C and 5% CO2. To determine maximum lysis, 50 μL of 51 Cr-labeled Daudi cells (5,000 cells) were incubated with 100 µL of 5% Triton-X100; to determine spontaneous lysis (background lysis), 5,000 cells were incubated with 100 µL of 5% Triton-X100. 51 Cr-labeled Daudi cells were incubated in 150 μL of culture medium without any antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5,000 Daudi cells with 500,000 PBMCs in the absence of antibody. The plate was centrifuged (1200 rpm, 10 minutes) and 75 μL of the supernatant was transferred to a micronic tube, after which the released Cr was counted using a gamma counter. 51 Cr. The percentage of antibody-mediated lysis was calculated as follows:

[0659] % specific lysis = (cpm sample - cpm spontaneous lysis) / (cpm maximum lysis - cpm spontaneous lysis), where cpm is counts per minute.

[0660] Figure 6 All CD38 Abs were shown to be able to induce lysis of Daudi, as indicated by the increased lysis seen with the CD38 Abs compared to the isotype control (IgG1-b12-E430G). Cell lysis was noted at the lowest antibody concentration, suggesting that the antibodies should be further diluted to observe a dose-dependent effect. CD38 antibodies containing the E430G mutation showed lower maximal lysis compared to the wild-type antibody.

[0661] The above chromium release assay was repeated using peripheral blood mononuclear cells (effector cells) from different healthy volunteers, the following target cells: Daudi, Wien-133, Granta 519 and MEC-2, and the antibodies IgG1-B-E430G, IgG1-B, IgG1-C-E430G, IgG1-C and IgG1-b12-E430G. The results are shown in Figure 18 middle.

[0662] Figure 18 All CD38 Abs were shown to be able to induce lysis of Daudi, Wien-133, Granta 519, and MEC-2 cells, as indicated by the increased lysis seen for the CD38 Abs compared to the isotype control (IgG1-b12-E430G). In most cases, dose-dependent target cell lysis was seen, but some variation was observed between different PBMC donors.

[0663] The ability of the CD38 antibody to induce ADCC was further evaluated using a luminescent ADCC reporter bioassay (Promega, catalog # G7018) that detects FcɣRIIIa (CD16) cross-linking as a surrogate for ADCC. As effector cells, the kit provides Jurkat human T cells that have been engineered to stably express the high-affinity FcγRIIIa (V158) and a nuclear factor of activated T cells (NFAT) response element that drives firefly luciferase expression. Briefly, Daudi or T regulatory cells (5,000 cells / well) were seeded into 384-well white Optiplates (Perkin Elmer) in ADCC assay buffer [RPMI-1640 medium supplemented with 3.5% low IgG serum [(Lonza, catalog # BE12-115F)] and incubated for 6 hours at 37°C / 5% CO2 in a total volume of 30 μL containing an antibody concentration series (0.5-250 ng / mL final concentration in 3.5-fold dilutions) and thawed ADCC bioassay effector cells. After the plates were adjusted to room temperature (RT) for 15 minutes, 30 μL of Bio Glo luciferase assay reagent was added and the plates were incubated at RT for 5 minutes. Luciferase production was quantified by luminescence readout on an EnVision Multilabel Reader (Perkin Elmer). Background levels were determined from wells to which only target cells and antibodies (no effector cells) were added. As negative controls, wells containing target and effector cells only (no antibody) were used.

[0664] Figure 7Results obtained with Daudi cells are shown, showing that CD38 antibodies are highly effective in inducing dose-dependent FcγRIIIa cross-linking, as determined in a reporter assay. CD38 antibodies containing the E430G mutation showed lower maximal cross-linking compared to the respective wild-type antibodies, consistent with the results obtained for the chromium release assay.

[0665] Figure 19 Results obtained with T regulatory cells are shown, which show that CD38 antibodies are highly effective in inducing dose-dependent FcγRIIIa cross-linking as determined in a reporter assay. CD38 antibodies containing the E430G mutation showed lower maximal cross-linking compared to the respective wild-type antibodies.

[0666] Example 5 - Antibody-dependent cellular phagocytosis (ADCP) by CD38 antibodies with the E430G mutation

[0667] The ability of the CD38 antibody with the E430G mutation to induce antibody-dependent cellular phagocytosis was adapted from Overdijk M.B. et al. mAbs 7:2,311-320. Macrophages were obtained by isolating PBMCs from healthy volunteers (Sanquin) using lymphocyte separation medium (BioWhittaker) according to the manufacturer's instructions. Monocytes were isolated from PBMCs via negative selection using the Dynabeads Untouched Human Monocyte Isolation Kit (Invitrogen). The isolated monocytes were cultured in serum-free dendritic cell culture medium (CellGenix GmbH) supplemented with 50 ng / mL GM-CSF (Invitrogen) for 3 days and then in serum-free dendritic cell culture medium supplemented with 100 ng / mL GM-CSF for 2 days to induce macrophage differentiation. Differentiated macrophages were dissociated using Versene (Life Technologies) and cell scraping and characterized by flow cytometry staining with CD1a-FITC (BD), CD14-PE / Cy7 (BD), CD40-APC / H7 (BD), CD80-APC (Miltenyi biotec), CD83-PE (BD), and CD86-PerCP-Cy5.5 (Biolegend). Macrophages were seeded at 100,000 cells / well into 96-well flat-bottom culture plates (Greiner bio-one) and allowed to adhere overnight at 37°C in serum-free dendritic cell culture medium supplemented with 100 ng / mL GM-CSF.

[0668] Target cells (Daudi) were labeled with PKH-26 (Sigma) according to the manufacturer's instructions, opsonized with 10 μg / mL CD38 antibody (30 minutes at 4°C), washed three times with FACS buffer, and added to macrophages at an effector: target (E:T) ratio of 5:1. The plate was briefly spun at 300 rpm to bring the effector and target cells into close proximity and incubated at 37°C for 45 minutes. Next, macrophages were collected using Versene and stained with CD14-BV605 (biolegend) and CD19-BV711 (biolegend). Phagocytosis was depicted as the percentage of CD14-positive macrophages that were also positive for PKH-26 but negative for CD19 (to exclude macrophages that attached only to Daudi cells), as measured on a flow cytometer (BD).

[0669] Figure 8 All CD38 Abs were shown to be able to induce ADCP in Daudi cells, as shown by the PKH-29 inhibition observed for CD38 Abs compared to isotype controls (IgG1-b12 and IgG1-b12-E430G). 阳性 、CD14 阳性 and CD19 阴性 Depending on the donor used, CD38 antibodies containing the E430G mutation showed a higher percentage of PKH-29 compared to wild-type antibodies. 阳性 、CD14 阳性 and CD19 阴性 Macrophages, indicating that CD38-Ab-mediated phagocytosis can be increased by introducing the E430G mutation.

[0670] Example 6 - Induction of apoptosis in tumor cell lines by CD38 antibodies

[0671] Apoptosis induction by CD38 antibodies was investigated by overnight incubation of tumor cell lines with CD38 antibodies, followed by live / dead analysis on a flow cytometer. Cells resuspended in RPMI containing 0.2% BSA were seeded into 96-well flat-bottom tissue culture plates (Greiner bio-one) at 100,000 cells / well. In the absence or presence of 10 μg / mL goat anti-human IgG1 (Jackson), serial dilutions of CD38 or control antibodies (final antibody concentration of 0.01-10 μg / mL at 4x serial dilutions) were added to provide additional Fc cross-linking. The cells were incubated overnight at 37°C, washed / centrifuged twice using FACS buffer (PBS / 0.1% BSA / 0.01% sodium azide), and resuspended in FACS buffer (Life Technologies) supplemented with 1:4000 dilution of Topro-3-iodine. Cell viability was analyzed on a FACS_Fortessa (BD) and depicted as the percentage of apoptotic (topro-3-iodine positive) cells.

[0672] Figure 9 It was shown that wild-type and E430G mutant CD38 antibodies alone did not induce apoptosis, but the addition of Fc-crosslinking antibodies led to approximately 30% apoptosis. No difference was observed between wild-type and E430G mutant CD38 antibodies.

[0673] Example 7 - Inhibition of CD38 enzymatic activity in the absence of PBMCs

[0674] Inhibition of CD38 cyclase activity

[0675] CD38 is an extracellular enzyme that converts NAD into cADPR and ADPR. These activities depend on the presence of H2O. When there is H2O, NAD is converted into ADPR (glycosyl hydrolase activity), and cADPR is converted into ADPR (hydrolase activity). About 95% of NAD is converted into ADPR by (glycosyl) hydrolase activity. In the absence of H2O, CD38 uses its cyclase activity to convert NAD into cADPR. In order to measure the inhibition of CD38 enzymatic activity, an NAD derivative that becomes fluorescent after being processed by CD38 is used.

[0676] Figure 10 The enzymatic activity of CD38 is shown.

[0677] First, nicotinamide guanine dinucleotide sodium salt phosphodiesterase (NGD, Sigma) was used as a substrate for CD38 to measure the inhibition of CD38 cyclase activity. As sources of CD38, tumor cell lines with different CD38 expression levels and recombinant his-tagged CD38 extracellular domain (hisCD38) were used. Tumor cells (Daudi and Wien133) were harvested and washed with 20 mM Tris-HCl. The cells were resuspended in 20 mM Tris-HCl and 200,000 cells / well were seeded into 96-well white opaque plates (PerkinElmer) at 100 μL / well. HisCD38 was seeded into 100 μL / well of 20 mM Tris-HCl at 0.6 μg / mL. CD38 antibody was diluted to 100µg / mL in 20 mM Tris-HCL, and 10 µL was added to the cells and hisCD38 (final concentration was 9 µg / mL) and incubated for 20 minutes at room temperature. Control wells were incubated with b12 antibody instead of CD38 antibody, or without antibody at all. Next, 10 µL (80 µM) of NGD diluted in 20 mM Tris-HCL was added to the plate, and fluorescence was immediately measured on an Envision multilabel reader (PerkinElmer) using excitation 340 nm and emission 430 nm. Figure 11 Fluorescence was measured at the indicated time points until a steady state was reached to track the conversion of NGD in real time. For hisCD38, fluorescence was measured every 3 minutes for a total of 27 minutes; for Daudi cells, fluorescence was measured after 5, 15, 30, 60, 120, and 185 minutes, and for Wien133, fluorescence was measured after 5, 15, 30, 60, 150, 220, 300, and 360 minutes. Inhibition of CD38 cyclase activity is depicted as percent inhibition compared to a control, where the control is a sample with hisCD38 and NGD but no Ab. One representative experiment is depicted for each condition tested.

[0678] Figure 11 A demonstrates the rapid turnover of NGD by hisCD38 cyclase activity. Conversion is complete after approximately 9 minutes. The maximum percentage of NGD turnover is reduced by ~25% in the presence of CD38 Ab B and ~50% in the presence of CD38 Ab C, whereas CD38 Ab A has no effect on the total turnover of NGD. Inhibition of CD38 cyclase activity is not affected by the presence of the E430G mutation. Figure 11Similar results are seen in B and 11C, where NGD conversion by CD38 present on Daudi and Wien133 cells was measured. The kinetics of NGD conversion were a bit slower on Daudi and especially Wien133 cells, which is likely associated with the presence of fewer CD38 molecules. However, a 25% inhibition of CD38 cyclase activity was induced by Ab B (~25% inhibition) and a ~40% inhibition of CD38 cyclase activity was induced by Ab C, while Ab A showed no effect. Wild-type antibody and E430G mutant antibody showed similar results, indicating that the E430G mutation does not affect antibody-mediated inhibition of CD38 cyclase activity.

[0679] Example 8 - Antibody-dependent cytotoxicity by CD38 antibodies with the E430G mutation

[0680] Cytotoxicity of CD38 antibodies against E430G mutation on Daudi cells:

[0681] The ability of CD38 antibodies with E430G mutation to induce cytotoxicity to Daudi cells was assessed. Macrophages were obtained by isolating PBMCs from healthy volunteers (Sanquin) using lymphocyte separation medium (Bio Whittaker) according to the manufacturer's instructions. Monocytes were isolated from PBMCs via negative selection using Dynabeads Untouched Human Monocyte Isolation Kit (Invitrogen). The isolated monocytes were cultured for 3 days in serum-free dendritic cell culture medium (CellGenix GmbH) supplemented with 50 ng / mL GM-CSF (Invitrogen) and subsequently cultured for 2 days in serum-free dendritic cell culture medium supplemented with 100 ng / mL GM-CSF to induce macrophage differentiation. Differentiated macrophages were dissociated using Versene (Life Technologies) and cell scraping and characterized by flow cytometry staining with CD1a-FITC (BD), CD14-PE / Cy7 (BD), CD40-APC / H7 (BD), CD80-APC (Miltenyi biotec), CD83-PE (BD), and CD86-PerCP-Cy5.5 (Biolegend). Macrophages were seeded at 100,000 cells / well into 96-well flat-bottom culture plates (Greiner bio-one) and allowed to adhere overnight at 37°C in serum-free dendritic cell culture medium supplemented with 100 ng / mL GM-CSF.

[0682] Target cells (Daudi) were labeled with PKH-26 (Sigma) according to the manufacturer's instructions, opsonized with 10 μg / mL LCD38 antibody (30 minutes at 4°C), washed three times with FACS buffer, and added to macrophages at an effector: target (E:T) ratio of 5: 1. The plate was briefly spun at 300 rpm to bring the effector and target cells into close proximity and incubated at 37°C for 45 minutes.

[0683] Figure 21 The assay setup for measuring cytotoxicity is shown.

[0684] CD38 expression and human IgG staining were determined on Daudi cells by incubation with FITC-conjugated CD38 clone A and goat anti-human IgG-FITC (Southern Biotech), respectively. CD38 clone A was used to stain CD38 because this Ab recognizes a non-overlapping epitope on CD38 compared to clones B and C.

[0685] Figure 12 The results show that after 45 minutes of co-culture with macrophages and CD38 antibodies, CD38 expression on Daudi cells was significantly reduced. The reduction in CD38 expression was greatest with the E430G mutant CD38 antibody. The same trend was seen for human IgG staining on antibody-opsonized Daudi cells.

[0686] Cytotoxicity of T regulatory cells by CD38 antibodies targeting the E430G mutation:

[0687] Compared with Tregs with medium CD38 expression, T regulatory cells (Tregs) with high CD38 expression are more immunosuppressive (Krejcik J. et al. Blood 2016 128:384-394). Therefore, strategies to reduce CD38 expression on Tregs may reduce the immunosuppressive effects of these cells. We investigated whether CD38 antibodies with E430G mutations can reduce CD38 expression on Tregs by cytotoxicity. According to the manufacturer's instructions, lymphocyte separation medium (BioWhittaker) was used to isolate Tregs from PBMCs from healthy volunteers (Sanquin). CD4 + T cells were then enriched for CD4 Tregs using a Treg isolation kit (Miltenyi) according to the manufacturer's instructions. + CD25 + T regulatory cells. Subsequently, Tregs were divided into 5x10 4Cells were expanded at 5 × 10 cells / mL in serum-free dendritic cell culture medium supplemented with 5% human serum (Sigma), 1000 U / mL IL-2 (peprotech), 100 ng / mL rapamycin (Sigma), and CD3 / CD28-coated beads (Gibco) at a bead:cell ratio of 4:1 at 37°C for 20 days. Every 3 to 4 days, the cell density was adjusted to 5 × 10 cells / mL using serum-free dendritic cell culture medium supplemented with 1000 U / mL IL-2 and 100 ng / mL rapamycin. 5 T regulatory phenotype was followed over time using flow cytometry staining with the following antibodies: CCR7-BV785 (Biolegend), CD62L-FITC (BD), CD4-APC / efluor780 (e-biosciences), CD25-PerCP / Cy5 (Biolegend), Foxp3-PE / CF594 (BD), CTLA4-efluor660 (e-biosciences), CD127-PE / CY7, and CD38-GV605 (Biolegend).

[0688] To evaluate Ab-induced CD38 cytotoxicity from Tregs, Tregs (target cells) were co-cultured with PBMCs (effector cells) and CD38 expression was monitored on Tregs. Briefly: PBMCs were isolated from buffy coats (Sanquin) using lymphocyte separation medium (Bio Whittaker) according to the manufacturer's instructions and cultured at 5×10 5 The cells were plated at a density of 10 cells / well in RPMI-1640 medium (Lonza) supplemented with 0.2% BSA and cultured for 3 days to allow monocytes to adhere. Tregs were labeled with 0.25 μM CellTrace far red (CTFR) according to the manufacturer's instructions and pre-incubated with E430G mutant CD38 Ab for 10 minutes at 37°C. Tregs were washed and 1×10 5 Each Ab-opsonized cell / well was transferred to a plate containing PBMCs. PBMCs and Tregs were briefly spun at 300 rpm to bring the cells into close proximity and incubated at 37°C for 23 hours. CD38 cytotoxicity was measured by flow cytometry using FITC-conjugated CD38 clone A to analyze CD38 expression in CTFR-positive Tregs.

[0689] Figure 13The results show that CD38 expression on T regulatory cells is reduced after incubation with E430G mutant CD38 antibodies and PBMCs. In the absence of PBMCs, no reduction in CD38 expression on T regulatory cells was observed, strongly suggesting cytotoxicity. Furthermore, in the presence of PBMCs, IgG1-B did not induce CD38 cytotoxicity, while a strong reduction in CD38 expression was induced by E430G mutant B and C. This suggests that E430G mutant CD38 antibodies induce enhanced cytotoxicity of CD38.

[0690] Example 9: Antibody C against E430G-mutated CD38 in a patient-derived diffuse large B-cell lymphoma model Tumor activity

[0691] Patient-derived diffuse large B-cell lymphoma (DLBCL) cells were inoculated subcutaneously into CB17.SCID mice, and the mice were transplanted when tumors reached approximately 150–250 mm. 3 Antibody treatment was started when the mean volume of the tumor was ≥ 1.5 (5 mg / kg IgG1-C-E430G, intravenous injection twice weekly; PBS was used as a negative control). Tumor volume was measured in two dimensions using a caliper and expressed in mm using the following formula: 3 Denote volume: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L), and Figure 20 The responses were plotted over time in Figure 2. Each treatment group consisted of one mouse. To calculate the response value, the following formula was used: (tumor volume of IgG1-C-E430G-treated mice on day X - tumor volume of IgG1-C-E430G-treated mice on day 0) / (tumor volume of control mice on day X - tumor volume of control mice on day 0)

[0692] X = the last day of the period between day 7 and day 25 when both animals were alive and tumor measurements were performed.

[0693] The response values as well as CD38 mRNA expression are depicted in Table 5. The model with the highest CD38 mRNA level also showed the best response. This can also be seen from Figure 20 Thus, twice-weekly dosing of IgG1-C-E430G reduced tumor growth in two of the five tested DLBCL PDX models, the ones with the highest CD38 mRNA expression.

[0694] Table 5. Summary of CD38 mRNA expression and calculated response values for five DLBCL PDX models. Low response values indicate tumor regression.

[0695] Model CD38 (determined by RNASeq: log2(TPM value + 1)) Response (∆T / ∆C) Response to the calculation of days; Ly12638 6,427 -11% 15 Ly11212 6,066 -2% 11 Ly13976 6,017 54% 13 Ly13693 4,796 58% 22 Ly14862 0 83% 11

[0696] Example 10: IgG1-C-E430G induces potent cytokines in bone marrow mononuclear cells from newly diagnosed MM patients Complement-mediated cytotoxicity

[0697] Bone marrow mononuclear cells (BM-MNCs) were isolated from whole bone marrow aspirates from three newly diagnosed MM patients and one relapsed / refractory MM patient by Ficoll-Hypaque density gradient and frozen at -80°C until use. On the day of use, BM-MNCs were thawed, viable cells were counted and plated in 96-well plates. The cells were incubated with serial dilutions (0.01–10 μg / mL) of IgG1-C-E430G or Darzalex® for 15 minutes at room temperature on a plate shaker. As a negative control, cells were not treated or incubated with 10 μg / mL IgG1-b12. As a source of complement, 20% normal human serum was added 45 minutes before FACS measurement, using flow cytometric beads as a constant to determine the absolute number of cells. To determine the total percentage of lysis, untreated control wells were used as control values. The percentage of multiple myeloma cell lysis relative to the control was determined using the following formula:

[0698] % cell lysis = (1-(number of viable cells in antibody-treated samples / number of viable cells in untreated controls) x 100%

[0699] Figure 22 A and B show that IgG1-C-E430G induced higher levels of lysis in two BM-MNC samples from newly diagnosed MM patients compared to Darzalex®. The maximum lysis induced by IgG1-C-E430G was in the range of 84-90%, compared to the 31-55% range induced by Darzalex®. In the other two BM-MNC samples, one was from a relapsed / refractory MM patient ( Figure 22 C), and one from a newly diagnosed MM patient ( Figure 22 D), no CDC induction was noted with IgG-C-E430G or Darzalex® ( Figure 22 C and D).

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Claims

1. An antibody variant that binds to human CD38, the antibody variant comprising (a) an antigen binding region comprising a VHCDR1 having a sequence as set forth in SEQ ID NO: 2, a VH CDR2 having a sequence as set forth in SEQ ID NO: 3, a VH CDR3 having a sequence as set forth in SEQ ID NO: 4, a VLCDR1 having a sequence as set forth in SEQ ID NO: 6, a VLCDR2 having a sequence of AAS, and a VLCDR3 having a sequence as set forth in SEQ ID NO: 7, and (b) a variant Fc region, wherein the mutation in the human IgG1 heavy chain is E430G, wherein the amino acid residues of the human IgG1 heavy chain are numbered according to the EU index, and wherein the variant Fc region comprises or consists of the following sequence: Amino acid residues 114 to 330 of SEQ ID NO. 24, which correspond to amino acid residues 231-447 in a human IgG1 heavy chain according to EU numbering; or amino acid residues 114 to 329 of SEQ ID NO. 46, which correspond to amino acid residues 231-447 in a human IgG1 heavy chain according to EU numbering and the C-terminal amino acid K447 is deleted. 2 . The antibody variant according to claim 1 , comprising a variable heavy chain (VH) region comprising SEQ ID NO:

1. 3 .

3. The antibody variant according to claim 1, comprising a variable light chain (VL) region comprising SEQ ID NO:

5. The antibody variant according to claim 1 , comprising a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO:

5.

5. The antibody variant according to claim 1, which is a bivalent antibody.

6. The antibody variant according to claim 1, which is a full-length antibody.

7. The antibody variant according to claim 1, wherein the antibody variant is a fully human antibody.

8. The antibody variant according to claim 1, which is a monoclonal antibody.

9. The antibody variant according to claim 1, wherein the antibody variant is an IgG1 antibody.

10. The antibody variant according to claim 1, wherein the antibody variant is a fully human monoclonal full-length bivalent IgG1m(f),κ antibody.

11. An antibody variant that binds to human CD38, the antibody variant comprising (a) a heavy chain comprising a VH region and a human IgG1 CH region, the VH region comprising a VHCDR1 as shown in SEQ ID NO: 2, a VH CDR2 as shown in SEQ ID NO: 3, and a VH CDR3 as shown in SEQ ID NO: 4, wherein the mutation in the human IgG1 CH region is E430G, wherein the amino acid residues in the heavy chain are numbered according to the EU index, and wherein the mutated human IgG1 CH region is as shown in the amino acid sequence of SEQ ID NO: 24 or 46; (b) A light chain comprising a VL region comprising a VLCDR1 having the sequence shown in SEQ ID NO: 6, a VLCDR2 having the sequence AAS, and a VL CDR3 having the sequence shown in SEQ ID NO:

7.

12. An antibody variant that binds to human CD38, the antibody variant comprising (a) a heavy chain comprising a VH region as shown in SEQ ID NO: 1 and a human IgG1 CH region, wherein the mutation in the human IgG1 CH region is E430G, wherein the amino acid residues in the heavy chain are numbered according to the EU index, and wherein the mutated human IgG1 CH region is as shown in the amino acid sequence of SEQ ID NO: 24 or 46, and (b) A light chain comprising the VL region shown in SEQ ID NO:

5.

13. The antibody variant according to claim 1, 11 or 12, wherein the mutated human IgG1 CH region is represented by the amino acid sequence of SEQ ID NO:

46.

14. The antibody variant according to claim 1, 11 or 12, wherein the mutated human IgG1 CH region is represented by the amino acid sequence of SEQ ID NO:

24.

15. The antibody variant according to claim 1, 11 or 12, wherein the light chain comprises a CL comprising SEQ ID NO:

37.

16. The antibody variant according to claim 1, 11 or 12, comprising a variable heavy chain (VH) region as shown in SEQ ID NO: 1, a variable light chain (VL) region as shown in SEQ ID NO: 5, a CH region as shown in SEQ ID NO: 46 and a CL region as shown in SEQ ID NO:

37.

17. The antibody variant according to claim 11 or 12, which is a bivalent antibody.

18. The antibody variant according to claim 1, 11 or 12, wherein the antibody variant is a monospecific antibody.

19. The antibody variant according to claim 1, 11 or 12, wherein the antibody variant is a bispecific antibody.

20. The antibody variant according to claim 1, 11 or 12, which has an inhibitory effect on the cyclase activity of human CD38.

21. The antibody variant according to claim 20, which inhibits the cyclase activity of human CD38 by at least 40%.

22. The antibody variant according to claim 21, wherein the inhibition of cyclase activity is determined by an assay comprising the steps of: (a) In a multi-well plate, 200,000 Daudi or Wien133 cells were seeded in 100 μL of 20 mM Tris-HCl per well; or 0.6 μg / mL of His-tagged soluble human CD38 as shown in SEQ ID NO: 39 was seeded in 100 μL of 20 mM Tris-HCl per well; (b) 1 μg / mL LCD38 antibody and 80 μM nicotinamide guanine dinucleotide sodium salt were added to each well; (c) measuring fluorescence until a steady state is reached; and (d) The percent inhibition compared to the control was determined.

23. The antibody variant according to claim 1, 11 or 12, which induces apoptosis in the presence of an Fc cross-linking antibody but does not induce apoptosis in the absence of the Fc cross-linking antibody.

24. The antibody variant according to claim 1, 11 or 12, wherein the antibody variant induces CDC, ADCC, antibody-dependent cellular phagocytosis (ADCP), cytokinesis or any combination thereof of cells expressing human CD38.

25. The antibody variant according to claim 1, 11 or 12, wherein the antibody variant induces CDC of cells expressing human CD38.

26. The antibody variant according to claim 1, 11 or 12, wherein the antibody variant induces CDC against Daudi cells (ATCC No. CCL-213) or Ramos cells (ATCC No. CRL-1596) resulting in a maximum lysis that is at least 50% higher than the maximum lysis obtained with a reference antibody variant that differs only in the absence of a mutation in the Fc region.

27. The antibody variant according to claim 26, wherein the CDC is determined by an assay comprising the steps of: (a) 100,000 CD38-expressing cells / well were seeded in 40 μL of culture medium supplemented with 0.2% BSA in a multiwell plate; (b) Preincubate cells with 40 μL of serially diluted CD38 antibodies ranging from 0.0002 to 10 μg / mL for 20 minutes; (c) incubating each well with 20% pooled normal human serum at 37° C. for 45 minutes; (d) Viability dye was added, and the percentage of cell lysis was measured on a flow cytometer; (e) Nonlinear regression was used to determine maximum lysis.

28. The antibody variant according to claim 1, 11 or 12, wherein the antibody variant is conjugated to a cytotoxic agent, a radioisotope or a drug.

29. A nucleic acid encoding an antibody variant according to any one of claims 1, 11 and 12.

30. A nucleic acid combination encoding an antibody variant according to any one of claims 1, 11 and 12.

31. An expression vector comprising the nucleic acid of claim 29.

32. An expression vector combination comprising the nucleic acid combination of claim 30.

33. A delivery vehicle comprising the nucleic acid according to claim 29 or the nucleic acid combination according to claim 30.

34. The delivery vehicle according to claim 33, wherein the delivery vehicle is a particle.

35. The delivery vehicle according to claim 34, wherein the particle is a lipid nanoparticle (LNP).

36. The delivery vehicle according to claim 35, wherein the LNP comprises a lipid.

37. The delivery vehicle according to claim 35, wherein the LNP comprises an ionizable amino lipid, a PEG-lipid, cholesterol, or any combination thereof.

38. A recombinant host cell that produces an antibody variant as defined in any one of claims 1, 11 and 12, wherein the host cell comprises the nucleic acid of claim 29, the nucleic acid combination of claim 30, the expression vector of claim 31 or the expression vector combination of claim 32.

39. The recombinant host cell according to claim 38, which is a eukaryotic or prokaryotic cell.

40. A method of producing the antibody variant of any one of claims 1, 11 and 12, comprising culturing the recombinant host cell of claim 38 in a culture medium under conditions suitable for production of the antibody variant, and purifying or isolating the antibody variant from the culture medium.

41. A method of increasing at least one effector function of a parent antibody, said parent antibody comprising an Fc region and an antigen binding region that binds CD38, said method comprising introducing a mutation into the Fc region of human IgG1, said mutation being E430G, wherein the amino acid residues of the heavy chain of the human IgG1 are numbered according to the EU index; wherein the antigen binding region comprises a VH CDR1 having a sequence as shown in SEQ ID NO: 2, a VH CDR2 having a sequence as shown in SEQ ID NO: 3, a VH CDR3 having a sequence as shown in SEQ ID NO: 4, a VLCDR1 having a sequence as shown in SEQ ID NO: 6, a VLCDR2 having a sequence of AAS, and a VLCDR3 having a sequence as shown in SEQ ID NO:

7.

42. The method according to claim 41, wherein the antigen binding region comprises a VH region as shown in the sequence of SEQ ID NO: 1 and a VL region as shown in the sequence of SEQ ID NO:

5.

43. The method according to claim 41, wherein the effector function is CDC, cytotoxicity or both.

44. The method according to claim 41, wherein the parent antibody is a fully human monoclonal full-length bivalent IgG1,κ antibody.

45. The method according to claim 41, wherein the parent antibody is a monospecific or bispecific antibody.

46. A composition comprising the antibody variant according to any one of claims 1, 11 and 12, the nucleic acid of claim 29, the nucleic acid combination of claim 30, the expression vector of claim 31, the expression vector combination of claim 32, the delivery vehicle of claim 33 or the host cell of claim 38.

47. A pharmaceutical composition comprising the antibody variant according to any one of claims 1, 11 and 12, the nucleic acid of claim 29, the nucleic acid combination of claim 30, the expression vector of claim 31, the expression vector combination of claim 32, the delivery vehicle of claim 33 and a pharmaceutically acceptable carrier.

48. Use of an antibody variant according to any one of claims 1, 11 and 12 in the preparation of a medicament for treating or preventing cancer in a subject comprising cells expressing human CD38, wherein the cancer is a hematological cancer selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), mantle cell lymphoma, follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL).

49. Use of the antibody variant according to claim 48, wherein the cancer is refractory to a previous therapy comprising a CD38 antibody.

50. Use of the antibody variant according to claim 48, wherein the cancer is a cancer that relapsed after a previous therapy comprising a CD38 antibody.

51. The use according to claim 49 or 50, wherein the CD38 antibody is daratumumab.

52. The use according to claim 48, wherein the cancer is MM.

53. The use according to claim 48, wherein the cancer is CLL.

54. The use according to claim 48, wherein the cancer is mantle cell lymphoma.

55. Use according to claim 48, wherein the cancer is DLBCL.

56. Use according to claim 48, wherein the cancer is FL.

57. Use according to claim 48, wherein the cancer is acute myeloid leukemia (AML).

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