Anti-CD38 antibody

Abstract

The present disclosure relates to anti-CD38 binding domains characterized by their CDR sequences. CD38-TM1, CD38-TM2, and CD38-TM5 bind to non-overlapping epitope residues on human CD38 compared to daratumumab. CD38-TM3 and CD38-TM4 bind to epitopes on human CD38 that partially overlap with daratumumab.

Description

[0001] I. Cross-Reference to Related Applications

[0002] This application claims priority under 35 USC §119 to U.S. Provisional Application No. 62 / 795,855, filed on January 23, 2019, the contents of which are expressly incorporated by reference in their entirety.

[0003] II. Reference to a "Sequence Listing", Table or Computer Program Listing Appendix Submitted on a CD-ROM

[0004] The present invention incorporates by reference a copy of the sequence listing text submitted herewith, which was created on January 21, 2020, named 101588-5011-WO_ST25.txt, and is 76KB in size. Background Art

[0005] CD38, also known as cyclic ADP ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 is a member of a group of related membrane-bound or soluble enzymes that include CD157 and the Aplysia ADPR cyclase. Enzymes in this family have the unique ability to convert NAD into cyclic ADP ribose or nicotinic acid-adenine dinucleotide phosphate.

[0006] In addition, CD38 has been reported to be involved in Ca 2+ CD38 is involved in signal transduction via tyrosine phosphorylation of many signaling molecules, including phospholipase Cγ, ZAP-70, syk, and c-cbl. Based on these observations, CD38 is proposed to be an important signaling molecule in the maturation and activation of lymphoid cells during normal development.

[0007] CD38 is found to be expressed on the surface of many immune cells, including B cells, plasma cells, CD4+T cells, CD8+T cells, NK cells, NKT cells, mature dendritic cells (DCs), and activated monocytes. In hematopoietic cells, CD38 is found to be involved in functional effects such as lymphocyte proliferation, cytokine release, regulation of B cell and myeloid cell development and survival, and induction of dendritic cell maturation ( Figure 1 ).

[0008] The putative natural ligand for CD38 is CD31 (PECAM-1; platelet endothelial cell adhesion molecule 1), a 130 kD member of the immunoglobulin superfamily expressed on the surface of circulating platelets, neutrophils, monocytes, and naive B-lymphocytes. Functionally, CD31 is thought to act as an adhesion molecule, and the interaction of CD38 with CD31 may play a role in promoting the survival of leukemic cells.

[0009] CD38 is upregulated in many hematopoietic malignancies and cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL) and chronic myeloid leukemia (CML). CD38 has therefore been used as a prognostic marker for leukemia.

[0010] Despite recent advances in the discovery and development of anticancer agents, many cancer forms involving CD38-expressing tumors still have a poor prognosis. Thus, there is a need for improved methods for treating such cancer forms, and for the development of anti-CD38 antibodies with improved properties, despite the antibodies already on the market. Summary of the invention

[0011] The present invention relates to compositions comprising novel anti-CD38 antigen binding domains.

[0012] In one aspect, the composition comprises an anti-CD38 antigen binding domain, which contains a heavy chain variable domain (VH), wherein the heavy chain variable domain comprises a vhCDR1 having an amino acid sequence of SEQ ID NO: 2, a vhCDR2 having an amino acid sequence of SEQ ID NO: 3, and a vhCDR3 having an amino acid sequence of SEQ ID NO: 4; and a light chain variable domain (VL), wherein the light chain variable domain comprises a vlCDR1 having an amino acid sequence of SEQ ID NO: 6, a vlCDR2 having an amino acid sequence of SEQ ID NO: 7, and a vlCDR3 having an amino acid sequence of SEQ ID NO: 8.

[0013] In some embodiments, the composition comprises an anti-CD38 antigen binding domain comprising a heavy chain variable domain having an amino acid sequence identical to SEQ ID NO: 1 and a light chain variable domain having an amino acid sequence identical to SEQ ID NO: 5. In some embodiments, the composition comprises an anti-CD38 antigen binding domain comprising a heavy chain variable domain having an amino acid sequence identical to SEQ ID NO: 1 and a light chain variable domain having an amino acid sequence identical to SEQ ID NO: 25.

[0014] In another aspect, the composition comprises an anti-CD38 antigen binding domain, which contains a heavy chain variable domain (VH), wherein the heavy chain variable domain comprises a vhCDR1 having an amino acid sequence of SEQ ID NO: 10, a vhCDR2 having an amino acid sequence of SEQ ID NO: 11, and a vhCDR3 having an amino acid sequence of SEQ ID NO: 12; and a light chain variable domain (VL), wherein the light chain variable domain comprises a vlCDR1 having an amino acid sequence of SEQ ID NO: 14, a vlCDR2 having an amino acid sequence of SEQ ID NO: 15, and a vlCDR3 having an amino acid sequence of SEQ ID NO: 16.

[0015] In some embodiments, the composition comprises an anti-CD38 antigen binding domain comprising a heavy chain variable domain having an amino acid sequence identical to SEQ ID NO:9 and a light chain variable domain having an amino acid sequence identical to SEQ ID NO:13.

[0016] In another aspect, the composition comprises an anti-CD38 antigen binding domain, which contains a heavy chain variable domain (VH), wherein the heavy chain variable domain comprises a vhCDR1 having an amino acid sequence of SEQ ID NO: 18, a vhCDR2 having an amino acid sequence of SEQ ID NO: 19, and a vhCDR3 having an amino acid sequence of SEQ ID NO: 20; and a light chain variable domain (VL), wherein the light chain variable domain comprises a vlCDR1 having an amino acid sequence of SEQ ID NO: 22, a vlCDR2 having an amino acid sequence of SEQ ID NO: 23, and a vlCDR3 having an amino acid sequence of SEQ ID NO: 24.

[0017] In some embodiments, the composition comprises an anti-CD38 antigen binding domain comprising a heavy chain variable domain having an amino acid sequence identical to SEQ ID NO:17 and a light chain variable domain having an amino acid sequence identical to SEQ ID NO:21.

[0018] In another aspect, the composition comprises an anti-CD38 antigen binding domain, which contains a heavy chain variable domain (VH), wherein the heavy chain variable domain comprises a vhCDR1 having an amino acid sequence of SEQ ID NO: 54, a vhCDR2 having an amino acid sequence of SEQ ID NO: 55, and a vhCDR3 having an amino acid sequence of SEQ ID NO: 56; and a light chain variable domain (VL), wherein the light chain variable domain comprises a vlCDR1 having an amino acid sequence of SEQ ID NO: 58, a vlCDR2 having an amino acid sequence of SEQ ID NO: 59, and a vlCDR3 having an amino acid sequence of SEQ ID NO: 60.

[0019] In some embodiments, the composition comprises an anti-CD38 antigen binding domain comprising a heavy chain variable domain having an amino acid sequence identical to SEQ ID NO:53 and a light chain variable domain having an amino acid sequence identical to SEQ ID NO:57.

[0020] In some embodiments, the composition comprising an anti-CD38 antigen binding domain as described herein comprises a heavy chain variable domain and a light chain variable domain on a single polypeptide. In some embodiments, a single polypeptide comprises a scFv linker, a heavy chain variable domain, and a light chain variable domain in an orientation from the N-terminus to the C-terminus of a VH-scFv linker-VL or a VL-scFv linker-VH.

[0021] In some embodiments, the composition comprising an anti-CD38 antigen binding domain as described herein comprises a first polypeptide comprising a heavy chain variable domain and a second polypeptide comprising a light chain variable domain.

[0022] In some embodiments, the composition comprising an anti-CD38 antigen binding domain as described herein is an antibody comprising a heavy chain containing a heavy chain variable domain and a light chain containing a light chain variable domain. In some embodiments, the antibody contains a heavy chain, the heavy chain comprising a heavy chain variable domain and a heavy chain constant domain, the heavy chain constant domain is selected from the heavy chain constant domains of human IgG1, IgG2 and IgG4 and variants thereof. In some embodiments, the antibody contains a heavy chain, the heavy chain comprising a heavy chain variable domain and a heavy chain constant domain of human IgG1 or its variants. In some embodiments, the antibody contains a heavy chain, the heavy chain comprising a heavy chain variable domain and a heavy chain constant domain, the heavy chain constant domain is selected from the heavy chain constant domain of human IgG1 variants with eliminated FcγR binding. In some embodiments, the antibody contains a heavy chain, the heavy chain comprising a heavy chain variable domain and a heavy chain constant domain, the heavy chain constant domain is selected from the heavy chain constant domain of human IgG4 variants with S228P amino acid substitution.

[0023] The present invention further relates to a nucleic acid composition encoding a heavy chain variable domain and a light chain variable domain. In some embodiments, the nucleic acid composition comprises a first nucleic acid encoding a heavy chain variable domain and a second nucleic acid encoding a heavy chain variable domain. In some embodiments, the nucleic acid composition comprises a single nucleic acid encoding a heavy chain variable domain and a light chain variable domain.

[0024] Another aspect of the present invention relates to an expression vector composition comprising any of the nucleic acid compositions described herein; and a host cell comprising any of the expression vector compositions described herein. In some embodiments, the expression vector composition comprises a first expression vector comprising the first nucleic acid and a second expression vector comprising the second nucleic acid described herein. In some embodiments, the expression vector composition comprises the nucleic acid composition comprising a single nucleic acid encoding a heavy chain variable domain and a light chain variable domain.

[0025] The present invention further relates to a method for preparing any of the compositions containing the anti-CD38 antigen binding domain described herein, comprising culturing the host cell under conditions that express the anti-CD38 antigen binding domain, and recovering the composition.

[0026] The present invention also includes a method of treating multiple myeloma using an effective amount of any of the compositions containing the anti-CD38 antigen binding domain described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Describe the CD38 expression spectrum of lymphoid lineage cells, and the asterisk indicates high CD38 expression. CD38 expression is identified on pro-B cells (CD34+CD19+CD20), activated B cells (CD19+CD20+), plasma cells (CD138+CD19-CD20-), activated CD4+ and CD8+T cells, NKT cells (CD3+CD56+) and NK cells (CD56+CD16+). In addition, CD38 expression is found on lymphoid progenitor cells (CD34+CD45RA+CD10+CD19-) but not on lymphoid stem cells. In addition, increased CD38 expression is observed on mature DC and activated monocytes.

[0028] Figure 2A-2B Binding of anti-CD38 binding clones to purified human CD38 as determined by ELISA is shown. Each anti-CD38 binding clone was formatted as a scFv and then fused to the Shiga toxin A subunit (scFv-SLTA fusion format).

[0029] Figure 3A-3B Binding of anti-CD38 binding clones to purified cynomolgus CD38 as determined by ELISA is shown. Each anti-CD38 binding clone was formatted as a scFv and then fused to the Shiga toxin A subunit.

[0030] Figure 4Key contact residues on the CD38 extracellular domain for the anti-CD38 binding clone CD38TM4 (F216, L262, C119, L124, C201, L230, C254, C275) and the anti-CD38 monoclonal antibody daratumumab (S274) are shown.

[0031] Figure 5A-5B Binding of anti-CD38 binding clones (in scFv-SLTA fusion format) to CD38-expressing MOLP-8 cells in the presence of anti-CD38 antibodies daratumumab (Dara), HB-7, AT13-5 and OKT-10 and several fusion proteins is shown. Reference molecule #1 (CD38TR1) targeting CD38 is also in the form of a scFv-SLTA fusion protein. Figure 5C Competitive binding of anti-CD38 binding clones including CD38-targeting reference molecule #1 (CD38TR1) in scFv-SLTA fusion format to CD38-expressing MOLP-8 cells is shown. All signals were detected by flow cytometry. Figure 5D is a table summarizing the binding of anti-CD38 binding clones in scFv-SLTA fusion protein format (top row) in the presence of anti-CD38 binding clones in scFv format alone (first column from the left).

[0032] Figures 6A-6G The sequences of the heavy and light chain variable domains and CDRs (underlined in full domains) of CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 are depicted.

[0033] Figure 7 The sequence of the CD38 target antigen of the present invention is shown.

[0034] Figure 8 Some linker sequences are depicted.

[0035] Fig. 9 The sequences of some Shiga toxin proteins fused to the anti-CD38 binding domain as used in the Examples are depicted.

[0036] Fig. 10A and Fig. 10BA number of IgG heavy chain constant domain "backbones" based on human IgG1, IgG2, and IgG4 (including some standard variants) are depicted, as well as sequences of light chain constant kappa and light chain constant lambda sequences. As will be appreciated by those skilled in the art, and as described below, any of these backbones can be combined with the heavy and light chain variable domains of CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6, and CD38TR1.

[0037] Fig.11A and 11B The purification of CD38TM3-SLTA and CD38TM4-SLTA by IMAC column and Protein L (Pro-L) column are shown respectively. The MW mark indicates the position of the molecular weight marker. Fig. 11C Shown is the replacement of the first 21 amino acids (underlined) of CD38TM3 with the first 22 amino acids (underlined) of CD38TR1 to derive CD38TM4.

[0038] Fig.12 Shown is an X-ray structure illustration of a diabody formed by two identical scFvs, each scFv comprising VH-GGGGS-VL from N-terminus to C-terminus, wherein VH and VL are from CD38TM4. The VH of one scFv chain (chain A) is complexed with the VL of the other scFv chain (chain B) to form a CD38 binding domain. DETAILED DESCRIPTION

[0039] A. Overview

[0040] Increased expression of CD38 is associated with a variety of hematopoietic diseases. Such diseases include, but are not limited to: multiple myeloma, chronic lymphocytic leukemia, B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia (including B-cell acute lymphocytic leukemia), Waldenstrom's macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic leukemia / myeloid leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, NK cell leukemia and plasma cell leukemia. Therefore, CD38 provides an effective target for the treatment of hematopoietic diseases. Several anti-CD38 antibodies are in clinical trials for the treatment of CD38-related cancers. Therefore, antibodies to CD38 can be used to treat and diagnose cancer.

[0041] The present invention provides anti-CD38 binding domains that can bind to human and cynomolgus monkey CD38 with high affinity. In addition, the anti-CD38 binding domains disclosed herein can bind to CD38 in the presence of daratumumab, and thus provide advantages in clinical applications that some of the existing anti-CD38 antibodies have not seen in clinical testing.

[0042] B. Definition

[0043] In order that the present application may be more fully understood, several definitions are set forth below. Such definitions are intended to encompass grammatical equivalents.

[0044] "Elimination" means in this article the reduction or removal of activity. In some embodiments, it is useful to remove activity from the constant domain of an antibody. Thus, for example, "eliminating FcγR binding" means that less than 50% of the Fc region amino acid variants begin to bind to FcγR compared to the Fc region not comprising a specific variant, wherein less than 70%-80%-90%-95%-98% of the activity loss is preferred, and in general, the activity is lower than the detectable binding level in the Biacore assay. For example, one elimination variant in the IgG1 constant region is the N297A variant, which removes the native glycosylation site and significantly reduces FcγRIIIa binding, and thereby reduces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0045] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" means a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa results in increased ADCC activity.

[0046] By "antigen binding domain" or "ABD" herein is meant a set of six complementary determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind to a target antigen as described herein. Thus, an "anti-CD38 antigen binding domain" binds to the CD38 antigen as outlined herein. As known in the art, these CDRs are typically present as a first set of variable heavy chain CDRs (vhCDRs or VHCDRs) and a second set of light chain variable CDRs (vlCDRs or VLCDRs), each set comprising three CDRs: vhCDR1, vhCD R2, vhCDR3 belonging to the heavy chain and vlCDR1, vlCDR2 and vlCDR3 belonging to the light chain. As understood in the art, the CDRs are separated by framework regions in each of the heavy and light chain variable regions: for the light chain variable region, these are (VL)FR1-vlCDR1-(VL)FR2-vlCDR2-(VL)FR3-vlCDR3-(VL)FR4, and for the heavy chain variable region, these are (VH)FR1-vhCDR1-(VH)FR2-vhCDR2-(VH)FR3-vhCDR3-(VH)FR4. The antigen binding domains of the invention can be embodied in a variety of formats, for example, as Fab, Fv and scFv. In the "Fab" format, the set of 6 CDRs is contributed by two different polypeptide sequences, i.e., a heavy chain variable region (vh or VH; comprising vhCDR1, vhCDR2, and vhCDR3) and a light chain variable region (vl or VL; comprising vlCDR1, vlCDR2, and vlCDR3), wherein the C-terminus of VH is connected to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of VL is connected to the N-terminus of the light chain constant domain (and thus forms a light chain). The heavy chain variable region and the light chain variable region together form an Fv, as described herein, as outlined herein, which may be scFv or Fab. Thus, in some cases, the six CDRs of the antigen binding domain are contributed by VH and VL. In the scFv format, VH and VL are typically covalently linked to a single polypeptide sequence using a linker as outlined herein, which may be (starting from the N-terminus) VH-linker-VL or VL-linker-VH.

[0047] "Joint" means in this article the domain joint that two protein domains are connected together, such as for scFv and / or other proteins and protein fusion structures.Usually, there are many suitable joints that can be used, including traditional peptide bonds, and the suitable joint is produced by recombinant technology, and the recombinant technology allows to recombinantly connect two domains with enough length and flexibility to allow each domain to retain its biological function.Joint peptide can mainly include the following amino acid residues: Gly, Ser, Ala or Thr.Joint peptide should have a certain length, and the length is enough to connect two molecules in a way so that the molecule presents correct conformation relative to each other, thereby making the molecule retain required activity.In one embodiment, the length of the joint is about 1 to 50 amino acid, and preferably the length is about 1 to 30 amino acid.In one embodiment, a length of 1 to 20 amino acid whose joint can be used, wherein about 5 to about 10 amino acid can be used in some embodiments. Useful linkers include glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1 (typically 3 to 4); glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of non-protein polymers (including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol) can be used as linkers. Other linker sequences may include any sequence of a CL / CH1 domain of any length, but not all residues of the CL / CH1 domain; for example, the first 5 to 12 amino acid residues of the CL / CH1 domain. The linker may also be derived from an immunoglobulin light chain, such as Cκ or Cλ. The linker may be derived from an immunoglobulin heavy chain of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. The linker sequence can also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), hinge region derived sequences, and other natural sequences from other proteins. Although any suitable linker can be used, many embodiments use glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1 (and typically 2 to 3 to 4 to 5). "ScFv linkers" typically include these glycine-serine polymers, such as Figure 8 In general, the scFv linker is sufficiently long to allow the VL and VH domains to properly associate to form an antigen binding domain (ABD) on a single polypeptide.

[0048] The term "antibody" is used in the broadest sense and includes, for example, complete immunoglobulins or antigen binding portions. Traditional antibody structural units generally include tetramers. Each tetramer is generally composed of two pairs of identical polypeptide chains, each pair having a "light" chain (generally having a molecular weight of about 25kDa) and a "heavy" chain (generally having a molecular weight of about 50-70kDa). Human light chains are classified as κ and λ light chains. The present invention relates to antibodies or antibody fragments (antibody monomers) generally based on IgG classes with several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. In general, IgG1, IgG2 and IgG4 are more frequently used than IgG3. It should be noted that IgG1 has different allotypes due to polymorphisms at 356 (D or E) and 358 (L or M). The sequences shown herein use 356D / 358M allotypes, but other allotypes are included herein. That is, any sequence comprising an IgG1 Fc domain encompassed herein may have the 356E / 358L, thereby replacing the 356D / 358M allotype.

[0049] Thus, "isotype" as used herein means any subclass of immunoglobulins defined by the chemical and antigenic characteristics of its constant regions.

[0050] The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition, which is generally referred to in the art and herein as an "Fv domain" or "Fv region". In the variable region, three loops are assembled for each of the V domains with heavy and light chains to form an antigen binding site. Each of the loops is called a complementary determining region (hereinafter referred to as a "CDR"), in which the amino acid sequence varies most significantly. "Variable" refers to the fact that the sequence of certain segments of the variable region is widely different between antibodies. The variability within the variable region is not evenly distributed. In fact, the V region consists of relatively invariant elongated regions called framework regions (FRs) with 15-30 amino acids, separated by highly variable shorter regions called "hypervariable regions" that are each 9-15 amino acids long or longer.

[0051] Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs, which are arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0052] The hypervariable region generally encompasses about amino acid residues 24-34 (LCDR1; "L" for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and amino acid residues approximately 31-35B (HCDR1; "H" for heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or those residues that form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2) in the light chain variable region.

[00136] In the invention, the CDRs of the invention are described below.

[0053] As will be appreciated by those skilled in the art, the exact numbering and placement of CDRs may differ between different numbering systems. However, it will be appreciated that disclosures of heavy chain variable sequences and / or light chain variable sequences include disclosures of associated (intrinsic) CDRs. Thus, disclosures of each heavy chain variable region are disclosures of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and disclosures of each light chain variable region are disclosures of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0054] A useful comparison of CDR numbering is made as follows, see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):

[0055] Table 1

[0056]

[0057] Throughout this specification, the Kabat numbering system is generally used when referring to residues in the variable domain (approximately, residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), and the EU numbering system is generally used for the Fc region (e.g., Kabat et al., (1991) supra).

[0058] The present invention provides a large number of different CDR sets. In this case, a "complete CDR set" includes three light chain variable CDRs and three heavy chain variable CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. Accordingly, these can be part of a larger light chain variable domain or a heavy chain variable domain. In addition, as more fully outlined herein, when heavy and light chains are used (e.g., when Fab is used), the heavy chain variable domain and the light chain variable domain can be on separate polypeptide chains, or on a single polypeptide chain in the case of an scFv sequence.

[0059] The CDRs contribute to the formation of the antigen binding site, or more specifically, epitope binding site, of an antibody.

[0060] "Epitope" refers to a determinant cluster that interacts with a specific antigen binding site in the variable region of an antibody molecule, called a paratope. Epitopes are groups of molecules such as amino acids or sugar side chains, and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. An epitope may contain amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen binding peptide; in other words, the amino acid residues are within the footprint of the specific antigen binding peptide. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids in different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. The difference between conformational and non-conformational epitopes may be that in the presence of a denaturing solvent, the binding to the former is lost without losing the binding to the latter.

[0061] "Modification" herein means an amino acid substitution, insertion and / or deletion in a polypeptide sequence, or a change to a portion chemically attached to a protein. For example, the modification can be a carbohydrate or PEG structure attached to a change in the protein. "Amino acid modification" herein means an amino acid substitution, insertion and / or deletion in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are always directed to amino acids encoded by DNA, such as the 20 amino acids with codons in DNA and RNA.

[0062] "Amino acid substitution" or "substitution" herein means that the amino acid at a specific position in the parent polypeptide sequence is replaced by another amino acid. In particular, in some embodiments, the substitution is for an amino acid that is not naturally present at a specific position, or is not naturally present in an organism or in any organism. For example, substitution N297A refers to a variant polypeptide in which the asparagine at position 297 is replaced by alanine, in this case an Fc variant. For the sake of clarity, a protein that is engineered to change the nucleic acid coding sequence but does not change the starting amino acid (e.g., CGG (encoding arginine) is converted to CGA (still encoding arginine) to increase the expression level of the host organism) is not an "amino acid substitution"; that is, although a new gene encoding the same protein is generated, if the protein has the same amino acid at its starting specific position, then this is not an amino acid substitution.

[0063] As used herein, "amino acid insertion" or "insertion" means adding an amino acid sequence at a specific position in a parent polypeptide sequence. For example, -233E or 233E indicates that glutamic acid is inserted after position 233 and before position 234. In addition, -233ADE or A233ADE indicates that AlaAspGlu is inserted after position 233 and before position 234.

[0064] As used herein, "amino acid deletion" or "deletion" means removing an amino acid sequence at a specific position in a parent polypeptide sequence. For example, E233- or E233#, E233() or E233del indicates the deletion of glutamic acid at position 233. In addition, EDA233- or EDA233# indicates the deletion of the sequence GluAspAla starting from position 233.

[0065] As used herein, "variant protein" or "protein variant" or "variant" means a protein that is different from the protein of the parent protein by at least one amino acid modification. Protein variants can refer to the protein itself, a composition comprising the protein, or an amino sequence encoding the protein. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example, about 1 to about 70 amino acid modifications compared to the parent body, and preferably about 1 to about 5 amino acid modifications. As described below, in some embodiments, the parent polypeptide (e.g., Fc parent polypeptide) is a human wild-type sequence, such as the Fc region from IgG1, IgG2, IgG3 or IgG4, but the human sequence with variants can also be used as a "parent polypeptide". The protein variant sequence herein preferably has at least about 80% identity with the parent protein sequence, and most preferably has at least about 90% identity, more preferably has at least about 95%-98%-99% identity. Variant protein can refer to the variant protein itself, a composition comprising a protein variant, or a DNA sequence encoding the variant protein.

[0066] Thus, by "antibody variant" or "variant antibody" as used herein is meant an antibody that differs from a parent antibody by at least one amino acid modification, and by "IgG variant" or "variant IgG" as used herein is meant an antibody that differs from a parent IgG (again, in many cases a human IgG sequence) by at least one amino acid modification. By "immunoglobulin variant" or "variant immunoglobulin" as used herein is meant an immunoglobulin sequence that differs from the sequence of a parent immunoglobulin sequence by at least one amino acid modification. By "Fc variant" or "variant Fc" as used herein is meant a protein that comprises amino acid modifications in the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. Thus, for example, S241P or S228P is a hinge variant having a substituted proline at position 228 relative to a parent IgG4 hinge polypeptide, wherein the numbering S228P is according to the EU index and S241P is the Kabat numbering. Similarly, M252Y / S254T / T256E defines an Fc variant having substitutions M252Y, S254T and T256E relative to a parent Fc polypeptide (these mutations increase the binding of the Fc domain to the FcRn receptor, thereby increasing the half-life of the molecule). The identity of the wild-type amino acid may be uncertain, in which case the aforementioned variant is referred to as 252Y / 254T / 256E. It should be noted that the order in which the substitutions are provided is arbitrary, that is, for example, 252Y / 254T / 256E is the same Fc variant as 254T / 252Y / 256E, etc. For all positions discussed in the present invention related to antibodies, unless otherwise indicated, the amino acid position numbering is performed according to Kabat for variable region numbering, and is performed according to the EU index for constant region, including Fc region. The EU index, or the EU index as in the Kabat or EU numbering scheme, refers to the EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications may be additions, deletions or substitutions. Substitutions may include naturally occurring amino acids, and in some cases include synthetic amino acids.Examples include: U.S. Pat. No. 6,586,207; WO 98 / 48032; WO 03 / 073238; US2004-0214988A1; WO 05 / 35727A2; WO 05 / 74524A2; JW Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Chin and P.G. Schultz, (2002), ChemBioChem 11:1135-1137; JW Chin et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang and P.G. Schultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entirety.

[0067] As used herein, "protein" means at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides and peptides. Peptide groups include naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e., "analogs", such as peptoids (see Simon et al., PNAS USA 89 (20): 9367 (1992), which is incorporated by reference in its entirety). Amino acids can be naturally occurring or synthetic (e.g., amino acids not encoded by DNA); as is understood by those skilled in the art. For example, for the purposes of the present invention, homophenylalanine, citrulline, ornithine and norleucine are considered synthetic amino acids, and amino acids in both D- and L- (R or S) conformations can be utilized. Variants of the invention may include modifications including the use of synthetic amino acids incorporated using techniques such as those developed by Schultz and coworkers, including but not limited to the methods described in Cropp and Shultz, 2004, Trends Genet. 20(12):625-30; Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71; Zhang et al., 2003, 303(5656):371-3; and Chin et al., 2003, Science 301(5635):964-7, all of which are incorporated by reference in their entirety. In addition, polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, pegylation, cyclic permutations, cyclizations, linkers to other molecules, fusions to proteins or protein domains, and addition of peptide tags or labels.

[0068] As used herein, "residue" means a position in a protein and its associated amino acid identity. For example, asparagine 297 (also referred to as Asn297 or N297) is the residue at position 297 in the human antibody IgG1, which, incidentally, is the position where the Fc region is glycosylated. Removal of glycosylation eliminates FcγRIIIa binding, resulting in loss of ADCC activity.

[0069] As used herein, "Fab" or "Fab region" means a polypeptide comprising the VH, CH1, VL and CL immunoglobulin domains. Fab can refer to this region alone, or to this region in the context of a full-length antibody or antibody fragment.

[0070] As used herein, "Fv" or "Fv fragment" or "Fv region" means a polypeptide comprising the VL and VH domains of a single antibody. As will be appreciated by those skilled in the art, these polypeptides are composed of two domains, a heavy chain variable domain and a light chain variable domain.

[0071] "Single-chain Fv" or "scFv" means herein a heavy chain variable domain covalently linked to a light chain variable domain to form an scFv or scFv domain, typically using an scFv linker as described herein. The scFv domain can be in any orientation from N-terminus to C-terminus (vh-linker-vl or vl-linker-vh). In general, the linker is an scFv linker as known in the art and discussed above.

[0072] As used herein, "IgG subclass modification" or "isotype modification" means an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid in a different aligned IgG isotype. For example, since IgG1 contains a tyrosine at EU position 296, and IgG2 contains a phenylalanine, a F296Y substitution in IgG2 is considered an IgG subclass modification. Similarly, since IgG1 has a proline at position 241, and IgG4 has a serine there, an IgG4 molecule with S241P is considered an IgG subclass modification. It should be noted that subclass modifications are considered amino acid substitutions herein.

[0073] As used herein, "non-naturally occurring modification" means a non-isotypic amino acid modification. For example, since none of the IgGs contain asparagine at position 297, the substitution N297A in IgG1, IgG2, IgG3 or IgG4 (or a hybrid thereof) is considered a non-naturally occurring modification.

[0074] As used herein, "amino acid" and "amino acid identity" means one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0075] As used herein, "effector function" means the biochemical events resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0076] As used herein, "parent polypeptide" means the starting polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. The parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or an amino acid sequence encoding the parent polypeptide. Thus, as used herein, "parent immunoglobulin" means an unmodified immunoglobulin polypeptide that is modified to produce a variant, and as used herein, "parent antibody" means an unmodified antibody that is modified to produce a variant antibody. It should be noted that "parent antibody" includes known commercially available recombinantly produced antibodies as outlined below.

[0077] As used herein, "Fc" or "Fc region" or "Fc domain" means a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain (e.g., CH1), and in some cases excluding part of the hinge. For IgG, the Fc domain comprises the immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the lower hinge region between CH1 (Cγ1) and CH2 (Cγ2). Although the boundaries of the Fc region may vary, the generally defined human IgG heavy chain Fc region includes residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index as in Kabat. Thus, the "CH" domain in the context of IgG is as follows: "CH1" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat. Thus, an "Fc domain" includes a -CH2-CH3 domain, and optionally a hinge domain (hinge-CH2-CH3). In some embodiments, as described more fully below, the Fc region is amino acid modified, e.g., to alter binding to one or more FcγR receptors or FcRn receptors.

[0078] As used herein, "Fcγ receptor," "FcγR," or "FcγR (FcgammaR)" means any member of the protein family that binds the Fc region of an IgG antibody and is encoded by an FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. In some cases, as outlined herein, binding to one or more FcγR receptors is reduced or eliminated. For example, reduced binding to FcγRIIIa will reduce ADCC, and in some cases, reduced binding to both FcγRIIIa and FcγRIIb is desirable.

[0079] As used herein, "FcRn" or "neonatal Fc receptor" means a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by an FcRn gene. FcRn can be from any organism, including but not limited to: humans, mice, rats, rabbits and monkeys. As is known in the art, a functional FcRn protein comprises two polypeptides, which are often referred to as a heavy chain and a light chain. The light chain is beta-2 microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated herein, FcRn or FcRn protein refers to a complex of FcRn heavy chain and beta-2 microglobulin. As described herein, binding to the FcRn receptor is desirable, and in some cases, Fc variants can be introduced to increase binding to the FcRn receptor.

[0080] "Heavy chain constant region" means herein the CH1-hinge-CH2-CH3 portion of a human IgG antibody. A number of suitable heavy chain constant regions are shown in FIG. 10 .

[0081] By "light chain constant region" is meant the CL domain from either kappa or lambda, the sequence of which is shown in FIG10 .

[0082] As used herein, "position" means a position in a protein sequence. Positions may be numbered sequentially, or according to an established format, such as the EU index numbering for antibody numbering.

[0083] As used herein, "target antigen" means a molecule that is specifically bound by the variable region of a given antibody. In this case, as defined below, the target antigen of interest herein is a CD38 protein, typically human CD38 and optionally cynomolgus CD38. Thus, an "anti-CD38 binding domain" is an antigen binding domain (ABD) whose antigen is CD38.

[0084] As used herein, "target cell" means a cell that expresses a target antigen.

[0085] As used herein, "variable domain" means a region of an immunoglobulin comprising one or more Ig domains, which are substantially encoded by any one of the Vκ (V.kappa), Vλ (V.lamda) and / or VH genes constituting the κ, λ and heavy chain immunoglobulin loci, respectively. Thus, a "heavy chain variable domain" comprises (VH) FR1-vhCDR1-(VH) FR2-vhC DR2-(VH) FR3-vhCDR3-(VH) FR4, and a "light chain variable domain" comprises (VL) FR1-vlCDR1-(VL) FR2-vlCDR2-(VL) FR3-vlCDR3-(VL) FR4.

[0086] "Wild type or WT" herein means an amino acid sequence or nucleotide sequence that occurs in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0087] The antibodies of the present invention are generally isolated or recombinant antibodies. "Isolated" when used to describe the various polypeptides disclosed herein means a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. "Recombinant" means that the antibody is produced using recombinant nucleic acid technology in an exogenous host cell.

[0088] "Specific binding" or "binds specifically to" or is "specific for" a particular antigen or epitope means binding that is distinct from nonspecific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0089] The term "Kassoc" or "Ka" as used herein is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. D" is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The KD value of an antibody can be determined using well-established methods in the art. In some embodiments, the method for determining the KD of an antibody is by using surface plasmon resonance, for example, by using a biosensor system such as In some embodiments, the K D Determined by biolayer interferometry. In some embodiments, K D Measured via antigen expressing cells using flow cytometry. In some embodiments, K D The value is measured when the antigen is immobilized. D The K value is measured when the antibody (e.g., parent mouse antibody, chimeric antibody, or humanized antibody variant) is immobilized. In certain embodiments, K D The value is measured in a bivalent binding mode. In other embodiments, K D The value is measured in monovalent binding mode. Specific binding to a particular antigen or epitope can be determined, for example, by the K D is at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, at least about 10 -13 M, at least about 10 -14 M antibody display. Generally, antibodies that specifically bind to antigens have a K D The K of the reference molecule relative to the antigen or epitope D 20 times, 50 times, 100 times, 500 times, 1000 times, 5,000 times, 10,000 times or more larger.

[0090] "Percentage (%) of amino acid sequence identity" relative to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular (parent) sequence, after aligning the sequences and (if necessary) introducing gaps to achieve the maximum percentage of sequence identity and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. A particular program is the ALIGN-2 program outlined in paragraphs

[0279] to

[0280] of U.S. Publication No. 20160244525, which is incorporated herein by reference. Another approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981). This algorithm can be applied to amino acid sequences using a scoring matrix, which is developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff, ed., 5th Supplement, 3:353-358, National Biomedical Research Foundation, Washington, DC, USA and normalized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986).

[0091] An implementation example of this algorithm for determining sequence identity percentage is provided by the "BestFit" practical application of Genetics Computer Group (Madison, WI). The default parameters of the method are described in Wisconsin Sequence Analysis Package Program Manual, the 8th edition (1995) (available from Genetics Computer Group, Madison, WI). Another method for establishing identity percentage in the context of the present invention is to use the MPSRCH program package owned by Edinburgh University (University of Edinburgh), which is developed by John F.Collins and Shane S.Sturrok, and issued by IntelliGenetics, Inc. (MountainView, CA). According to this set of software packages, Smith-Waterman algorithm can be used, wherein default parameters are used for scoring tables (for example, gap opening penalty is 12, gap extension penalty is 1 and gap is 6). According to the data generated, the "matching" value reflects "sequence identity". Other suitable programs for calculating identity or similarity percentage between sequences are well known in the art, for example, another comparison program is BLAST, which is used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = double strand; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; screening = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Spupdate + PIR. Detailed information on these programs can be found on the Internet address of http: / / before blast.ncbi.nlm.nih.gov / Blast.cgi.

[0092] The degree of identity between an amino acid sequence of the invention ("inventive sequence") and a parental amino acid sequence is calculated as the number of exact matches in the alignment of the two sequences divided by the length of the "inventive sequence" or the length of the parental sequence, whichever is the shortest. The result is expressed as a percentage of identity.

[0093] The terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, or reducing the likelihood of a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or side effects attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, especially a human, and includes: (a) preventing the onset of disease in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., curbing the development or progression of the disease; and (c) alleviating the disease, i.e., causing regression of the disease and / or alleviating one or more symptoms of the disease. "Treatment" is also intended to cover the delivery of an agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" covers the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, such as in the case of a vaccine.

[0094] An "effective amount" or "therapeutically effective amount" of a composition includes an amount of the composition sufficient to provide a beneficial effect to a subject to which the composition is administered. An "effective amount" of a delivery vehicle includes an amount sufficient to effectively bind or deliver the composition.

[0095] The term "nucleic acid" includes RNA or DNA molecules having more than one nucleotide in any form, including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term "nucleotide sequence" includes the order of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of a nucleic acid.

[0096] As used herein, the term "promoter" includes a DNA sequence operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. The promoter is usually located upstream of the nucleic acid sequence to be transcribed and provides a site for the specific binding of RNA polymerase and other transcription factors.

[0097] "Vector" is capable of transferring a gene sequence to a target cell. In general, "vector construct", "expression vector" and "gene transfer vector" mean any nucleic acid construct capable of directing the expression of a target gene and transferring a gene sequence to a target cell, which can be accomplished by integrating all or part of a genomic vector, or a vector that is transiently or heritably maintained as an extrachromosomal element. Therefore, the term includes cloning and expression vehicles, as well as integration vectors.

[0098] As used herein, the term "regulatory element" includes nucleotide sequences that control some aspects of nucleic acid sequence expression. Examples of regulator elements illustratively include enhancers, internal ribosome entry sites (IRES), introns, replication origins, polyadenylation signals (pA), promoters, enhancers, transcription termination sequences and upstream regulatory domains, which contribute to the replication, transcription and / or post-transcriptional processing of nucleic acid sequences. In many cases, regulator elements can also include cis-regulatory DNA elements and transposable elements (TE). Those of ordinary skill in the art can only select and use these and other regulator elements in expression constructs by routine experiments. Expression constructs can use genetic recombination methods to produce or use the method known to produce synthetically.

[0099] "Control elements" or "control sequences" are nucleotide sequences involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including replication, repetition, transcription, splicing, translation, or degradation of the polynucleotide. Regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region that is capable of binding RNA polymerase under certain conditions and initiates transcription of a coding region that is usually located downstream (in the 3' direction) of the promoter.

[0100] C. CD38 Protein and Anti-CD38 Binding Domain

[0101] 1. CD38 protein

[0102] The present invention provides an anti-CD38 binding domain that specifically binds to human and cynomolgus CD38 proteins. The amino acid sequences of human and cynomolgus CD38 proteins are shown as RefSeq access identifiers NP_001766.2 (SEQ ID NO: 28) and NP_001274206.1 (SEQ ID NO: 30), respectively, wherein the ECD sequences (SEQ ID NO: 29 and SEQ ID NO: 31, respectively) are shown in Figure 7 middle.

[0103] Thus, as used herein, the term "CD38" or "CD38 protein" or "CD38 polypeptide" may optionally include any such protein, variant, conjugate or fragment thereof, including but not limited to known or wild-type CD38 as described herein, as well as any naturally occurring splice variant, amino acid variant or isoform, and in particular the extracellular domain (ECD) fragment of CD38. That is, the ABD of the present invention generally binds to the ECD domain of both human and cynomolgus CD38 proteins.

[0104] 2. Anti-CD38 binding domain

[0105] The present invention provides many anti-CD38 binding domains in different forms or orientations. The specific CDRs of the anti-CD38 binding domains are described below. As described above, the exact numbering and placement of the CDRs may be different between different numbering systems. However, it should be understood that the disclosures of the heavy chain variable region and / or the light chain variable region include the disclosures of the associated (intrinsic) CDRs. Therefore, the disclosures of each heavy chain variable region are the disclosures of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosures of each light chain variable region are the disclosures of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0106] The present disclosure provides novel anti-CD38 binding domains. Such antigen binding domains can bind to human and cynomolgus CD38 proteins. FIG. 6A to FIG. 6G The amino acid sequences of four different anti-CD38 ABDs that can bind to both human and cynomolgus CD38 are shown. In some embodiments, the heavy chain variable region and the light chain variable region are arranged in a Fab format, as described below, which is optionally included in a full-length antibody. In some embodiments, the heavy chain variable region and the light chain variable region are fused together to form a scFv as generally outlined herein.

[0107] Also included herein are anti-CD38 ABDs having amino acid modifications in one or more of the CDR and / or framework regions.

[0108] As outlined herein, in some embodiments, a set of 6 CDRs may have 0, 1, 2, 3, 4, 5, or 6 amino acid modifications (where amino acid substitutions are found to be particularly useful). That is, a CDR may have an amino acid modification (e.g., 1, 2, 3, 4, 5, or 6 amino acid modifications from the set of CDRs (that is, a CDR may be modified as long as the total number of changes in the set of 6 CDRs is less than 6 amino acid modifications, which changes any combination of CDRs; for example, there may be one change in vlCDR1, two changes in vhCDR2, no changes in vhCDR3, etc.)). In some embodiments, amino acid substitutions in vhCDR3 are less preferred or are to be avoided. In some cases, the binding affinity to one or both of human and cynomolgus CD38 may be increased, while in other embodiments, the binding affinity may be decreased. In these embodiments, binding to human and cynomolgus CD38 is retained. Suitable assays for testing whether an anti-CD38 antigen binding domain comprises modifications compared to the VH and VL sequences outlined herein are known in the art, such as a Biacore assay or the binding assays outlined in Examples 1, 2 or 3.

[0109] In some embodiments, the anti-CD38 ABDs outlined herein may also have amino acid modifications in the framework regions of either or both of the heavy chain variable and light chain variable framework regions (again, amino acid substitutions have been found to be particularly useful), as long as the framework (excluding the CDRs) retains at least about 80%, 85%, or 90% identity to the human germline sequence. Thus, for example, the same CDRs as described herein may be combined with different framework sequences from human germline sequences, as long as the framework regions retain at least 80%, 85%, or 90% identity to the human germline sequence.

[0110] In another aspect, the present invention further provides an anti-CD38 binding domain comprising a variant of the heavy chain variable region and the light chain variable region listed above. The heavy chain variable region can be at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the "VH" sequence herein, and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes. The light chain variable region may be at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the "VL" sequence herein, and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes. In these embodiments, the invention includes these variants as long as the antigen binding domain still binds to human and cynomolgus CD38. Suitable assays for testing whether the anti-CD38 antigen binding domain contains mutations compared to the VH and VL sequences outlined herein are known in the art, such as Biacore assays or those in Examples 1, 2 and 3.

[0111] In some embodiments, the anti-CD38 binding domain is CD38TM1 and includes: a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:9; and a light chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:9; NO:13 is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence.

[0112] In some embodiments, the anti-CD38 binding domain is CD38TM1 and includes: vhCDR1 comprising SEQ ID NO: 10, vhCDR2 comprising SEQ ID NO: 11, vhCDR3 comprising SEQ ID NO: 12, vlCDR1 comprising SEQ ID NO: 14, vlCDR2 comprising SEQ ID NO: 15, and vlCDR3 comprising SEQ ID NO: 16. In some embodiments, one or more of these 6 CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, any single CDR comprises no more than 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0113] In some embodiments, the anti-CD38 binding domain of CD38TM1 has a VH domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:9.

[0114] In some embodiments, the anti-CD38 binding domain of CD38TM1 has a VL domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:13.

[0115] In some embodiments, the anti-CD38 binding domain of CD38TM1 has a VH domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:9, and a VL domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:13.

[0116] In some embodiments, the anti-CD38 binding domain is CD38TM1 and has a VH comprising SEQ ID NO:9 and a VL comprising SEQ ID NO:13.

[0117] In some embodiments, the anti-CD38 binding domain is CD38TM2 and includes: a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17; and a light chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17; NO:21 is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence.

[0118] In some embodiments, the anti-CD38 binding domain is CD38TM2 and includes: vhCDR1 comprising SEQ ID NO: 18, vhCDR2 comprising SEQ ID NO: 19, vhCDR3 comprising SEQ ID NO: 20, vlCDR1 comprising SEQ ID NO: 22, vlCDR2 comprising SEQ ID NO: 23, and vlCDR3 comprising SEQ ID NO: 24. In some embodiments, one or more of these 6 CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR comprises no more than 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0119] In some embodiments, the anti-CD38 binding domain of CD38TM2 has a VH domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:17.

[0120] In some embodiments, the anti-CD38 binding domain of CD38TM1 has a VL domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:21.

[0121] In some embodiments, the anti-CD38 binding domain of CD38TM2 has a VH domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:17, and a VL domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:21.

[0122] In some embodiments, the anti-CD38 binding domain is CD38TM2 and has a VH comprising SEQ ID NO:17 and a VL comprising SEQ ID NO:21.

[0123] In some embodiments, the anti-CD38 binding domain is CD38TM3 and includes: a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1; and a light chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1; NO:5 is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence.

[0124] In some embodiments, the anti-CD38 binding domain is CD38TM3 and includes: vhCDR1 comprising SEQ ID NO: 2, vhCDR2 comprising SEQ ID NO: 3, vhCDR3 comprising SEQ ID NO: 4, vlCDR1 comprising SEQ ID NO: 6, vlCDR2 comprising SEQ ID NO: 7, and vlCDR3 comprising SEQ ID NO: 8. In some embodiments, one or more of these 6 CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR comprises 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0125] In some embodiments, the anti-CD38 binding domain of CD38TM3 has a VH domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:1.

[0126] In some embodiments, the anti-CD38 binding domain of CD38TM3 has a VL domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:5.

[0127] In some embodiments, the anti-CD38 binding domain of CD38TM3 has a VH domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:1, and a VL domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:5.

[0128] In some embodiments, the anti-CD38 binding domain is CD38TM3 and has a VH comprising SEQ ID NO:1 and a VL comprising SEQ ID NO:5.

[0129] In some embodiments, the anti-CD38 binding domain is CD38TM4 in the present disclosure, comprising: a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 1; and a light chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 1; NO:25 is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence.

[0130] In some embodiments, the anti-CD38 binding domain is CD38TM4 and includes: vhCDR1 comprising SEQ ID NO: 2, vhCDR2 comprising SEQ ID NO: 3, vhCDR3 comprising SEQ ID NO: 4, vlCDR1 comprising SEQ ID NO: 6, vlCDR2 comprising SEQ ID NO: 7, and vlCDR3 comprising SEQ ID NO: 8. In some embodiments, one or more of these 6 CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR comprises 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0131] In some embodiments, the anti-CD38 binding domain of CD38TM4 has a VH domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:1.

[0132] In some embodiments, the anti-CD38 binding domain of CD38TM1 has a VL domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:25.

[0133] In some embodiments, the anti-CD38 binding domain of CD38TM4 has a VH domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:1, and a VL domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:25.

[0134] In some embodiments, the anti-CD38 binding domain is CD38TM4 and has a VH comprising SEQ ID NO:1 and a VL comprising SEQ ID NO:25.

[0135] In some embodiments, the anti-CD38 binding domain in the present disclosure is CD38TM5, comprising: a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 53; and a light chain variable region having an amino acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 53; NO:57 is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence.

[0136] In some embodiments, the anti-CD38 binding domain is CD38TM5 and includes: vhCDR1 comprising SEQ ID NO: 54, vhCDR2 comprising SEQ ID NO: 55, vhCDR3 comprising SEQ ID NO: 56, vlCDR1 comprising SEQ ID NO: 58, vlCDR2 comprising SEQ ID NO: 59, and vlCDR3 comprising SEQ ID NO: 60. In some embodiments, one or more of these 6 CDRs have 1, 2, 3, 4, or 5 amino acid modifications. In other embodiments, a single CDR comprises 1 or 2 amino acid substitutions, and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0137] In some embodiments, the anti-CD38 binding domain of CD38TM5 has a VH domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:53.

[0138] In some embodiments, the anti-CD38 binding domain of CD38TM5 has a VL domain with no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:57.

[0139] In some embodiments, the anti-CD38 binding domain of CD38TM5 has a VH domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:53, and a VL domain having no more than 1, 2, 3, 4, or 5 amino acid changes in SEQ ID NO:57.

[0140] In some embodiments, the anti-CD38 binding domain is CD38TM5 and has a VH comprising SEQ ID NO:53 and a VL comprising SEQ ID NO:57.

[0141] In addition to sequence variants in the heavy and light chain variable regions and / or CDRs of each anti-CD38 binding domain described herein, changes in one or more framework regions of one or more heavy chain variable regions and / or light chain variable regions can also be made. In some embodiments, changes are made to framework regions that maintain at least 80%, 85%, 90% or 95% identity to the framework region sequences described in Table 1, while keeping the 6 CDRs unchanged and retaining binding to human and / or cynomolgus CD38.

[0142] In some embodiments, both the framework regions and the six CDRs are altered while retaining binding of the anti-CD38 binding domain to human and / or cynomolgus CD38. In these embodiments, the CDRs may have amino acid modifications (e.g., 1, 2, 3, 4, or 5 amino acid modifications from a set of six CDRs (that is, a CDR may be modified as long as the total number of changes in the set of six CDRs is less than six amino acid modifications, which changes any combination of CDRs; for example, there may be one change in vlCDR1, two changes in vhCDR2, no changes in vhCDR3, etc.)).

[0143] The epitopes of the anti-CD38 binding domains are mapped as outlined below. The present invention includes not only the antigen binding domains and antibodies listed herein, but also those that compete for binding with the epitopes bound by the listed antigen binding domains, such as CD38TM1, CD38TM2, CD38TM3, CD38TM4, and CD38TM5. Antigen binding domains that recognize the same epitope can be validated in a simple competitive immunoassay, thereby showing the ability of one antigen binding domain (or an antibody comprising such an antigen binding domain) to prevent another antigen binding domain (or an antibody comprising such an antigen binding domain) from binding to the target antigen CD38, such as "merging". Competitive binding studies can be performed as known in the art, which are typically performed using SPR / Binding assays, as well as ELISA and cell-based assays were performed.

[0144] D. Characteristics of the Anti-CD38 Binding Domain

[0145] The present invention provides novel anti-CD38 binding domains. The anti-CD38 binding domains specifically bind to human and cynomolgus CD38, preferably the ECD of human and cynomolgus CD38. Binding to human and cynomolgus CD38 is useful because cynomolgus animals can be used as models for human subjects to help determine, estimate and understand the in vivo effects and behaviors of molecules targeting CD38, such as, for example, with respect to pharmacokinetics, pharmacodynamics and toxicology; wherein the model can have target binding and target cell killing by molecules that can be translated into relevant species.

[0146] In some embodiments, the anti-CD38 binding domains described herein bind to human and cynomolgus CD38 with high affinity. D The value can be measured under antigen fixation or under antibody fixation. D Values ​​can also be measured in monovalent or bivalent binding mode. For example, the K of the antigen binding domain binding to human CD38 when formatted as IgG1 and measured by flow cytometry D The value can be 1×10 -6 M or below, 5×10 -7 M or below, 2.5×10 -7 M or less, 1×10 -7 M or below, 5×10 -8 M or less, 1×10 -8 M or less, 1×10 -9 M or less or 1×10 -10 M or less. K of the antigen-binding domain binding to cynomolgus monkey CD38 D The value can be 1×10 -6 M or below, 5×10 -7 M or below, 2.5×10-7 M or less, 1×10 -7 M or below, 5×10 -8 M or below, 2.5×10 -8 M or less, 1×10 -8 M or below, 5×10 -9 M or less, 1×10 -9 M or below, 5×10 -10 M or less or 1×10 -10 M or less. In some embodiments, the K of the antigen binding domain that binds to human CD38 is D The values ​​range from 0.1 nM to 1 μM, 0.25 nM to 500 nM, 0.5 nM to 250 nM, 1 nM to 100 nM, or 1.5 nM to 50 nM. In some embodiments, the K of an antigen binding domain that binds to cynomolgus monkey CD38 is D Values ​​range from 0.1 nM–1 μM, 0.25 nM–500 nM, 0.5 nM–250 nM, 1 nM–100 nM, or 2 nM–50 nM.

[0147] In addition, the anti-CD38 binding domains provided in the present disclosure can bind to various amino acids in epitopes on human CD38 ECD. In some embodiments, the anti-CD38 binding domain binds to R78 and / or D81. In some embodiments, the anti-CD38 binding domain binds to F216, L262, L124, C201, L230, C254 and / or S274. In some embodiments, the anti-CD38 binding domains described herein bind to an epitope on CD38 that does not overlap with daratumumab. In some embodiments, the anti-CD38 binding domains described herein bind to an epitope on CD38 that partially overlaps with daratumumab. In some embodiments, the anti-CD38 binding domains described herein are capable of binding to human and / or cynomolgus monkey CD38 in the presence of daratumumab.

[0148] In some embodiments, one or more of the anti-CD38 binding domains disclosed herein are included in a composition, such as an antibody, which can be used to induce complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) in cells with increased CD38 expression, such as multiple myeloma cells.

[0149] E. Composition of anti-CD38 binding domains

[0150] As described herein, the anti-CD38 binding domains of the invention can be used in different formats, for example as a scFv or as a Fab contained in a traditional tetrameric antibody.

[0151] 1. scFv containing anti-CD38 binding domain

[0152] In some embodiments, the composition comprises an scFv comprising an anti-CD38 binding domain as described herein. scFvs bind to human and cynomolgus CD38 and comprise a heavy chain variable region (VH) and a light chain variable region (VL) connected by a scFv linker. VL and VH can be in any orientation, such as "VH-scFv linker-VL" or "VL-scFv linker-VH" from N-terminus to C-terminus.

[0153] Although any suitable linker can be used, many embodiments use glycine-serine polymers, including, for example, a 15-residue (Gly4Ser)3 peptide (SEQ ID NO: 32). Suitable scFv linkers that can be used to form non-covalent multivalent structures include, for example, GGS (SEQ ID NO: 33), GGGS (SEQ ID NO: 34), GGGGS (SEQ ID NO: 35), GGGGSGGG (SEQ ID NO: 35), GGGGSGGG (SEQ ID NO: 36), GGSGGGG (SEQ ID NO: 37), and GSTSGGGSGGGSGGGGSS (SEQ ID NO: 38) or any peptide sequence that allows the heavy chain variable region and the light chain variable region to be recombinantly connected with sufficient length and flexibility to allow each domain to retain its biological function.

[0154] In the present invention, variable length joints can be used. In some embodiments, a joint of 1 to 50 amino acids in length is used. In some embodiments, a joint of 3 to 12 amino acids in length is used, and the resulting scFv monomer tends to form a polymer or oligomer (e.g., double antibody, three antibodies and four antibodies) due to self-association, wherein most of the forms are dimers. In some embodiments, a joint of 5 amino acids in length is used, such as GGGGS (SEQ ID NO: 35), and double antibodies can be formed by ABD. In some embodiments, a joint of more than 12 (e.g., 13, 14, 15, 16, 17, 18, 19 or 20) amino acids in length is used, and the resulting scFv mainly forms a monomer, and only a few parts experience spontaneous multimerization. In some embodiments, a joint of 1 to 3 amino acids is used, which promotes scFv multimerization into a higher order structure larger than the dimer form, such as a mixture of trimers and / or trimers and tetramers.

[0155] The present invention also includes the multimerization of scFv. It can be controlled by covalent or non-covalent molecular engineering strategies, for example, covalent strategies involving single-chain tandem arrangements, covalent strategies involving cysteine-mediated, disulfide-stabilized multimers, and / or non-covalent strategies involving dimerization domains, linker selection, and / or variable domain order. Multiple strategies (e.g., linker-related non-covalent multimerization and covalent disulfide bond stabilization) can be combined to produce scFv multimers (see, e.g., Lu D et al., J Immunol Methods 279: 219-32 (2003)).

[0156] In some embodiments, the present invention provides an ABD comprising a heavy chain variable domain and a light chain variable domain, including a VH and a VL domain selected from the group consisting of CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, and CD38TM6. In some embodiments, the ABD is in the form of a scFv. The VH and VL domains are connected by a 5-amino acid scFv linker, such as GGGGS (SEQ ID NO: 35) to form a "VH-scFv linker-VL" or "VL-scFv linker-VH" from the N-terminus to the C-terminus. Diabodies can be formed by such ABDs. An illustration of an exemplary scFv's X-ray structure is shown in Fig.12 This scFv comprises VH and VL from CD38TM4 and the scFv linker GGGGS (SEQ ID NO: 35) and forms a diabody structure.

[0157] 2. Antibodies containing anti-CD38 binding domains

[0158] In some embodiments, the composition of the present invention is a traditional tetrameric antibody, which comprises a heavy chain variable domain and a light chain variable domain of the present invention forming an ABD, including CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5 and CD38TM6. In general, these VH and VL pairs are added to the heavy chain constant domains (CH1-hinge-CH2-CH3) of human and variant human IgG1, IgG2 and IgG4, whose sequences are shown in Figure 10, and the light chain constant domains (CL) of λ or κ, also shown in Figure 10. In some embodiments, the present invention provides a composition comprising Fab, which comprises an anti-CD38 binding domain as described herein. The C-terminus of the heavy chain variable region (comprising vhCDR1, vhCDR2 and vhCDR3) is connected to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of the light chain variable region (comprising vlCDR1, vlCDR2 and vlCDR3) is connected to the N-terminus of the light chain constant domain. Either the constant lambda or kappa domains may be used in the present invention, as may the variants of the CH1 domain and light chain constant domains described herein.

[0159] Suitable heavy chain constant domains include, but are not limited to, those depicted in FIG10 .

[0160] In some embodiments, the antibody comprises a heavy chain having VH and VL of CD38TM1 (VH-CH1-hinge-CH2-CH3). In these embodiments, the heavy chain constant domain may be from human and variant IgG1, IgG2, and IgG4 as shown in FIG. 10 .

[0161] In some embodiments, VH (SEQ ID NO:9) is formed with a heavy chain constant region selected from the group consisting of SEQ ID NO:39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and VL (SEQ ID NO:13) is formed with a light chain constant domain selected from SEQ ID NO:51 and 52.

[0162] In some embodiments, the antibody comprises a heavy chain having VH and VL of CD38TM2 (VH-CH1-hinge-CH2-CH3). In these embodiments, the heavy chain constant domain may be from human and variant IgG1, IgG2, and IgG4 as shown in FIG. 10 .

[0163] In some embodiments, VH (SEQ ID NO: 17) is formed with a heavy chain constant region selected from the group consisting of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and VL (SEQ ID NO: 21) is formed with a light chain constant domain selected from SEQ ID NO: 51 and 52.

[0164] In some embodiments, the antibody comprises a heavy chain having VH and VL of CD38TM3 (VH-CH1-hinge-CH2-CH3). In these embodiments, the heavy chain constant domain may be from human and variant IgG1, IgG2, and IgG4 as shown in FIG. 10 .

[0165] In some embodiments, VH (SEQ ID NO: 1) is formed with a heavy chain constant region selected from the group consisting of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and VL (SEQ ID NO: 5) is formed with a light chain constant domain selected from SEQ ID NO: 51 and 52.

[0166] In some embodiments, the antibody comprises a heavy chain having VH and VL of CD38TM4 (VH-CH1-hinge-CH2-CH3). In these embodiments, the heavy chain constant domain may be from human and variant IgG1, IgG2, and IgG4 as shown in FIG. 10 .

[0167] In some embodiments, VH (SEQ ID NO: 1) is formed with a heavy chain constant region selected from the group consisting of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and VL (SEQ ID NO: 25) is formed with a light chain constant domain selected from SEQ ID NO: 51 and 52.

[0168] In some embodiments, the antibody comprises a heavy chain having VH and VL of CD38TM5 (VH-CH1-hinge-CH2-CH3). In these embodiments, the heavy chain constant domain may be from human and variant IgG1, IgG2, and IgG4 as shown in FIG. 10 .

[0169] In some embodiments, VH (SEQ ID NO:53) is formed with a heavy chain constant region selected from the group consisting of SEQ ID NO:39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and VL (SEQ ID NO:57) is formed with a light chain constant domain selected from SEQ ID NO:51 and 52.

[0170] As described below, additional substitutions may be introduced in the Fc region of an antibody.

[0171] 3. Included in fusion protein

[0172] In some embodiments, the anti-CD38 binding domains of the invention may be included in fusion proteins, such as those generally described in U.S. Application Nos. 14 / 774,130, 14 / 965,882, 15 / 114,487, 15 / 114,474, 15 / 125,126, 15 / 125,142, and 15 / 317,892; and U.S. Provisional Application Nos. 62 / 795,633, 62 / 945,107, and 62 / 945,106, such as Fig. 9 As shown in .

[0173] 4. Antibody Engineering

[0174] Anti-CD38 binding domains and compositions comprising such anti-CD38 binding domains can be modified or engineered to alter the amino acid sequence by amino acid substitutions. As discussed herein, amino acid substitutions can be made to alter the affinity of the CDR for a protein (e.g., human and / or cynomolgus CD38, including increasing and decreasing binding), as well as to alter additional functional properties of antigen binding domains and antibodies.

[0175] In addition, in some embodiments, the Fc region of the antibody can be engineered to include modification, typically in order to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc gamma receptor binding and / or antigen-dependent cellular toxicity. In addition, the antigen-binding domains and antibodies according to at least some embodiments of the present invention can be chemically modified (e.g., one or more chemical moieties can be connected to the antigen-binding domains and the antibody) or modified to change its glycosylation, or to change one or more functional properties of the antigen-binding domains and the antibody. Such embodiments are further described below. The residue numbering in the Fc region is the numbering of the EU index of Kabat.

[0176] In one embodiment, the hinge region is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This method is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, e.g., to facilitate assembly of the light and heavy chains, or to increase or decrease the stability of the antibody.

[0177] In another embodiment, antibodies can be modified to eliminate in vivo Fab arm exchange, particularly when using IgG4 constant domains. Specifically, this process involves exchanging IgG4 half molecules (one heavy chain plus one light chain) between other IgG4 antibodies, thereby effectively generating functionally monovalent bispecific antibodies. Mutations in the heavy chain hinge region and constant domains can eliminate this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105: 9-19). As described herein, mutations with specific uses in the present invention are S241P (Kabat numbering, S228P EU numbering) in the case of IgG4 constant domains. IgG4 can be used in the present invention because it has no significant effector function and is therefore used to block the binding of receptors to their ligands without consuming cells.

[0178] In some embodiments, amino acid substitutions may be made in the Fc region, typically to change binding to FcγR receptors. There are many useful Fc substitutions that may be made to change binding to one or more FcγR receptors. Substitutions that result in increased binding as well as reduced binding may be useful. For example, it is known that increased binding to FcγRIIIa typically results in increased ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize antibodies bound to target cells and subsequently cause lysis of target cells. Similarly, in some cases, reduced binding to FcγRIIb (inhibitory receptors) may also be beneficial. Amino acid substitutions that may be used in the present invention include those listed in U.S. Serial No. 11 / 124,620 (particularly FIG. 41 ) and U.S. Pat. No. 6,737,056, both of which are expressly incorporated herein by reference in their entirety and specifically with respect to the variants disclosed therein.

[0179] In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors and / or to increase FcRn binding by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290 0, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This method is further described in PCT Publication WO 00 / 42072 by Presta. In addition, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped, and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcγRIII. In addition, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A. In addition, mutations such as M252Y / S254T / T256E or M428L / N434S can improve binding to FcRn and increase antibody circulation half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

[0180] In addition, the antibodies of the present invention are modified to increase their biological half-life. Various methods are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in Ward's U.S. Patent No. 6,277,375. Alternatively, in order to increase the biological half-life, the antibody can be changed in the CH1 or CL region to include a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in Presta et al. U.S. Patent Nos. 5,869,046 and 6,121,022. Other mutations that increase serum half-life are disclosed in U.S. Patent Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A, 434S and 428L / 434S.

[0181] In another embodiment, the glycosylation of the antibody is modified. For example, an antibody without glycosylation can be prepared (i.e., the antibody lacks glycosylation). Glycosylation can be changed, for example, to increase the affinity of the antibody for the antigen or to reduce effector functions, such as ADCC. Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites (e.g., N297) within the antibody sequence. For example, one or more amino acid substitutions can be performed, which result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site, wherein alanine substitutions are useful in some embodiments.

[0182] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as low fucosylated antibodies with reduced fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved by, for example, expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art, and can be used as host cells, expressing recombinant antibodies according to at least some embodiments of the present invention therein, thereby producing antibodies with altered glycosylation. See, for example, U.S. Patent Publication No. 20040110704 and WO 2003 / 035835.

[0183] Another modification of the antibodies herein contemplated by the invention is pegylation or the addition of other water-soluble moieties, typically polymers, e.g., to increase half-life. Antibodies may be pegylated, e.g., to increase the biological (e.g., serum) half-life of antibodies known in the art.

[0184] In addition to substitutions made to alter binding affinity to FcγR and / or FcRn and / or to increase in vivo serum half-life, additional antibody modifications may be made, as described in further detail below.

[0185] In some cases, affinity maturation is complete. Amino acid modifications in CDRs are sometimes referred to as "affinity maturation". An "affinity matured" antigen binding domain or antibody is an antibody that has one or more changes in one or more CDRs to improve the affinity of the antigen binding domain or antibody to the antigen compared to an antibody that does not have those changes. In some cases, it may be necessary to reduce the affinity of the antibody to its antigen. Affinity maturation can be performed to increase the binding affinity of the antigen binding domain or antibody to the antigen, increasing by at least about 10% to 50-100-150% or more, or 1 to 5 times, compared to the "parent" antibody. Preferred affinity matured antigen binding domains or antibodies have nanomolar or even picomolar affinity for the antigen. Affinity matured antibodies are produced by known procedures.

[0186] In some embodiments, one or more amino acid modifications may be made in one or more of the CDRs of the CD38 binding domains of the invention. Generally, only 1 or 2 or 3 amino acids are substituted in any single CDR, and usually, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes are made within a set of 6 CDRs (e.g., vhCDR1-3 and vlCDR1-3). However, it should be understood that any combination of no substitution, 1, 2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitutions.

[0187] Alternatively, "silent" amino acid modifications, e.g., modifications that do not significantly alter the affinity of the antigen-binding domain or antibody for the antigen, may be made in one or more of the CDRs of the antigen-binding domains of the invention. These modifications may be made for a number of reasons, including optimizing expression (e.g., modifications that may be made to nucleic acids encoding antigen-binding domains or antibodies of the invention).

[0188] F. Nucleic Acids Encoding Antibodies

[0189] Also provided are nucleic acid compositions encoding anti-CD38 binding domains and compositions comprising such antigen binding domains, as well as expression vectors containing the nucleic acids and host cells transformed with the nucleic acid and / or expression vector compositions. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, the protein sequences described herein may be encoded by any number of possible nucleic acid sequences.

[0190] The nucleic acid composition encoding the composition comprising the anti-CD38 binding domain will depend on the form of the composition. Traditional tetrameric antibodies comprising two heavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. As known in the art, they can be placed in a single expression vector or two expression vectors, transformed into a host cell, where they are expressed to form the antibodies of the present invention. In some embodiments, for example when a scFv construct is used, a single nucleic acid encoding a heavy chain variable region-joint-light chain variable region is generally used, which can be inserted into an expression vector for transformation into a host cell. The nucleic acid can be placed in an expression vector containing appropriate transcription and translation control sequences, including but not limited to signal and secretion sequences, regulatory sequences, promoters, replication origins, selection genes, and the like.

[0191] Preferred mammalian host cells for expressing recombinant antibodies according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells), PER.C6, HEK293, and other cells known in the art.

[0192] To generate scFv genes, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, such as the amino acid sequence GGGGS and other sequences discussed herein, so that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL and VH regions are connected by a flexible linker.

[0193] G. Preparation

[0194] The therapeutic composition used in the practice of the above method can be formulated into a pharmaceutical composition, and the pharmaceutical composition comprises a carrier suitable for the required delivery method. Suitable carriers include, when combined with the therapeutic composition, any material that retains the therapeutic function of the therapeutic composition and is usually unresponsive to the patient's immune system. Examples include, but are not limited to, any carrier in many standard drug carriers, such as sterile phosphate buffered saline solution, antibacterial water and similar carriers (generally referring to Remington's Pharmaceutical Sciences, the 16th edition, A.Osal. compiles, 1980). Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the dosage and concentration used and may include a buffer.

[0195] Pharmaceutical compositions comprising the anti-CD38 binding domains or antibodies of the invention can be formulated for any suitable route and mode of administration, including but not limited to intravenous infusion.

[0196] For intravenous infusion, in some embodiments, a pharmaceutical composition comprising an anti-CD38 binding domain or antibody of the invention is formulated in an aqueous buffer solution containing a cryoprotectant and a surfactant. In some embodiments, a pharmaceutical composition comprising an anti-CD38 binding domain or antibody of the invention is formulated in an aqueous sodium citrate buffer solution containing sucrose as a cryoprotectant and polysorbate 80 as a surfactant and maintained at pH 4.8-5.2. In some embodiments, a pharmaceutical composition comprising an anti-CD38 binding domain or antibody of the invention is formulated in 20 mM sodium citrate buffer pH 5.0 containing 200 mM sucrose and 0.02% volume / volume polysorbate 80. An exemplary formulation is listed below:

[0197]

[0198] The formulations comprising the pharmaceutical compositions of this invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In such form, the composition is divided into unit doses containing appropriate quantities of the active ingredient.

[0199] In a preferred embodiment, the dosage and frequency of administration are selected so as to be therapeutically or prophylactically effective. As is known in the art, adjustments may have to be made for rates of protein degradation, systemic versus local delivery, and new protease synthesis, as well as age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition, and will be able to be determined by a person skilled in the art using routine experience.

[0200] H. Methods Using Antibodies

[0201] The anti-CD38 binding domains of the invention and compositions comprising such anti-CD38 binding domains can be used in a variety of diagnostic and therapeutic applications.

[0202] 1. Diagnostic Use

[0203] The anti-CD38 binding domains of the invention and compositions comprising such anti-CD38 binding domains can be used for in vitro or in vivo diagnosis of cancers expressing CD38, including imaging of tumors that overexpress CD38.

[0204] Typically, diagnosis can be accomplished in several ways. In one embodiment, a tissue from a patient, such as a biopsy sample, is contacted with a generally labeled anti-CD38 antibody so that the antibody binds to endogenous CD38. The signal level from the label is compared with the signal level of normal non-cancerous tissue from the same patient or reference sample to determine whether there is a cancer expressing CD38. The biopsy sample can be from a solid tumor, a blood sample (for lymphomas and leukemias, such as multiple myeloma, ALL, T-cell lymphoma, etc.). Typically, in this embodiment, the anti-CD38 antibody is labeled, for example, with a fluorophore or other optical marker, which is detected using a fluorometer or other optical detection system known in the art.

[0205] In another embodiment, a labeled secondary antibody is contacted with the sample, for example, anti-human IgG antibodies from different mammals (mouse, rat, rabbit, goat, etc.) are used to form a sandwich assay known in the art. Alternatively, the anti-CD38 antibody can be directly labeled (i.e., biotin) and can be detected by a secondary antibody raised in the art against the labeling agent.

[0206] Once overexpression of CD38 is observed, treatment can be performed by administering an anti-CD38 antigen binding domain or a composition comprising an anti-CD38 binding domain according to the invention as outlined herein.

[0207] In other embodiments, in vivo diagnosis is performed. Typically, in this embodiment, the anti-CD38 antibody (including antibody fragments) is injected into the patient and imaged. For example, in this embodiment, the antibody is typically labeled with an optical marker or MRI marker, such as a gadolinium chelate, a radioactive label of the mAb (including fragments).

[0208] Particularly useful antibodies for diagnostics include, but are not limited to, the listed antibodies, or antibodies utilizing CDRs with variant sequences, or antibodies that compete for binding with any of the antibodies described herein.

[0209] In many embodiments, the diagnostic antibody is labeled. "Label" herein means that one or more elements, isotopes or chemical compounds are attached to the antibody disclosed herein to achieve detection in a screening or diagnostic procedure. In general, labels are divided into several categories: a) immunolabeling, which can be an antigenic epitope incorporated as a fusion partner recognized by the antibody, b) isotopic labeling, which can be a radioactive or heavy isotope, c) small molecule labeling, which can include fluorescent and colorimetric dyes, or molecules such as biotin that can perform other labeling methods, and d) labels such as particles (including bubbles for ultrasound labeling) or paramagnetic labels that allow body imaging. As known in the art, labels can be incorporated into any position in the antibody and can be incorporated in vitro or in vivo during protein expression.

[0210] Diagnosis can be performed in vivo by administering diagnostic antibodies that allow whole body imaging as described below, or in vitro on a sample removed from a patient. A "sample" in this context includes many things, including but not limited to body fluids (including but not limited to blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen) and tissue samples such as biopsies obtained from relevant tissues.

[0211] In addition, as described below, information about CD38 protein expression levels can be used to determine which antibodies should be administered to a patient.

[0212] 2. Disease treatment

[0213] The anti-CD38 binding domains of the present invention and compositions comprising such anti-CD38 binding domains are particularly useful in treating diseases, disorders and / or conditions that may be mediated, regulated or otherwise affected by CD38 overexpression in a subject or human patient in need thereof. Treatment comprises administering to the subject or patient a therapeutically effective amount of a composition comprising an anti-CD38 binding domain of the present invention. In certain embodiments of the present invention, a composition comprising an anti-CD38 binding domain of the present invention is used to treat or prevent cancer or immune disorders associated with CD38 overexpression. In some embodiments, the disease, disorder or condition to be treated using this method of the present invention is a hematopoietic cancer containing CD38 overexpressing cells, such as multiple myeloma, and more generally, a condition associated with loss of growth control of cells expressing CD38. In addition to multiple myeloma, CD38 is upregulated in many other hematopoietic malignancies and cell lines derived from various hematopoietic malignancies, such as non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL) and chronic myeloid leukemia (CML).

[0214] In some embodiments, the anti-CD38 binding domains of the present invention and / or compositions comprising such anti-CD38 binding domains are used in combination with existing anti-CD38 antibodies, such as daratumumab, for the treatment of cancer or immune disorders associated with CD38 overexpression, such as multiple myeloma. In some embodiments, the anti-CD38 binding domains of the present invention and / or compositions comprising such anti-CD38 binding domains are used to treat patients associated with CD38 overexpression, such as multiple myeloma patients, who have been exposed to anti-CD38 antibodies, such as, for example, daratumumab. In some embodiments, the anti-CD38 binding domains of the present invention and / or compositions comprising such anti-CD38 binding domains are used to treat patients associated with CD38 overexpression, such as multiple myeloma patients, who have experienced treatment failure with existing anti-CD38 antibodies, such as daratumumab. In some embodiments, anti-CD38 binding domains and / or compositions comprising such anti-CD38 binding domains are used in combination with existing anti-CD38 antibodies to treat diseases associated with overexpression of CD38, such as multiple myeloma, wherein the anti-CD38 binding domain binds to an epitope on CD38 that is different from the epitope bound by the existing anti-CD38 antibodies.

[0215] VI. Examples

[0216] Having generally described the invention, the invention will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0217] A. Example 1: Anti-CD38 antigen binding clones bind to human and cynomolgus monkey CD38

[0218] 1. Anti-CD38 antigen binding clones bind to cells expressing CD38

[0219] Anti-CD38 antigen binding clones are formatted as human IgG1, and their combination with CD38 protein is determined. Human multiple myeloma cells MOLP-8 expressing human CD38 and cells expressing recombinant cynomolgus monkey CD38 are incubated with anti-CD38 antibodies or only two anti-controls at a concentration of 100nM. The combination of anti-human IgG two anti-detection antibodies and CD38 is detected using fluorophore-conjugated anti-human IgG two anti-detection antibodies. Cells are then analyzed by flow cytometry, and the mean fluorescence intensity (MFI) of anti-CD38 antibodies is used to calculate background multiples (FOB) compared with only two anti-controls. Anti-CD38 binding clones are combined with human and cynomolgus monkey CD38, and exemplary data from clones CD38TM3, CD38TM1 and CD38TM2, CD38TM5 and CD38TM6 are shown in Table 2.

[0220] Table 2

[0221] clone Human CD38(FOB) Cynomolgus monkey CD38 (FOB) CD38TM3 212.2 528.2 CD38TM1 397.1 1920.1 CD38TM2 39.6 271.6 CD38TM5 550.1 104.5 CD38TM6 104.1 176.1

[0222] 2. Binding of anti-CD38 antigen binding clones to purified CD38

[0223] The anti-CD38 antigen binding clones were also tested for binding to purified human and cynomolgus CD38 molecules. The anti-CD38 antigen binding clones were formatted into scFvs, which were then fused to the Shiga toxin A subunit to generate molecules targeting CD38. For each anti-CD38 scFv, the heavy chain variable region (VH) was fused to the N-terminus or C-terminus of the light chain variable region (VL) via a GGGGS linker. Also included in the test were the reference anti-CD38 binding clone fused to the Shiga toxin A subunit (reference molecule targeting CD38 #1-SLTA, or CD38TR1-SLTA), and the improved anti-CD38 binding clone CD38TM4 fused to the Shiga toxin A subunit, which contained amino acid substitutions in the light chain framework region of CD38TM3, enabling purification by protein L chromatography. The amino acid sequences of the anti-CD38 / Shiga toxin fusion proteins tested were: SEQ ID NO:40 (CD38TM1-SLTA), SEQ ID NO:41 (CD38TM2-SLTA), SEQ ID NO:42 (CD38TM3-SLTA), SEQ ID NO:43 (CD38TM4-SLTA) and SEQ ID NO:44 (CD38TR1-SLTA).

[0224] Briefly, Nunc maxisorp plates were coated with recombinant human or cynomolgus CD38 in PBS at 4°C overnight. The wells were washed and blocked, then incubated with serially diluted CD38-targeting molecules. CD38-targeting molecules bound to the wells were detected by a two-step method, including a mouse monoclonal antibody detecting the Shiga toxin domain, followed by an anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody. HRP activity was detected by Ultra TMB ELISA Substrate (Thermo Fisher), and absorbance was read at 450 nM.

[0225] The anti-CD38 antigen binding clones CD38TM1, CD38TM2, CD38TM3, and CD38TM4 bind to both human and cynomolgus CD38. In contrast, CD38TR1 binds only to human CD38. The binding of each anti-CD38 binding clone was tested in the form of a scFv fusion with the Shiga toxin A subunit (SETA), as Figures 2A-3B and Figure 3A-3BCD38TM4 is derived from CD38TM3 and retains the CD38 binding ability of CD38TM3. The binding affinity of the anti-CD38 antigen binding clones to the scFv fusion form of SETA was calculated and shown in Table 3.

[0226] Table 3.

[0227]

[0228] B. Example 2: Affinity determination of anti-CD38 binding clones

[0229] Cell binding affinity assay is carried out to quantitatively bind the binding affinity of anti-CD38 binding clones to cells expressing CD38. The MOLP-8 cells expressing human CD38 are suspended in 1% FBS / PBS with a viable cell concentration of about 2 million cells / mL. The anti-CD38 binding clones formatted as IgG1 are serially diluted across wells (2 times dilution from 102nM to 24.95pM) on 6-well plates in PBS. The last well of each titration contains only PBS. Then the cell suspension and additional PBS are added to each well so that each well contains a final volume of 300 μL and about 100,000 cells. The plate is placed in a plate shaker at 4°C for 5 hours, and then the cells in each well are washed 3 times at 4°C using PBS. Then 200 μl of 99 nM Cy5 goat anti-human IgG Fc specific polyclonal antibody (Jackson ImmunoResearch Laboratories, #109-175-008) was added to each well, and the plate was shaken for 30 minutes at 4°C. The cells were washed again twice with PBS at 4°C and then analyzed by FACSCanto TM The mean fluorescence intensity (MFI) of 5000 events was measured for each well containing a unique antibody concentration, and the MFI curve as a function of antibody concentration was nonlinearly fitted using Scientist 3.0 software to estimate the antigen binding affinity K using the following equation: D :F=p[(K D +LT+n(M))-{(K D +LT+n(M))2-4n(M)(LT)}1 / 2] / 2+B

[0230] where F is the MFI, LT is the total antibody binding site concentration equivalent to 2 times the concentration of antibody molecules, p is the proportionality constant associating any fluorescence unit with bound antibody, M is the cell concentration in molar concentration and is given as 0.553 fM based on 100,000 cells in 300 μl, n is the number of receptors per well, B is the background signal, and K D = equilibrium dissociation constant.

[0231] For each antibody titration curve, K was obtained when P, n, B, and KD were free to float in a nonlinear analysis. D Estimated value. For a detailed derivation of the above equation, see Drake and Klakamp (2007), "A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants," J. Immunol. Methods 318: 157-62, which is incorporated herein by reference. Table 4 lists the measured K values ​​of anti-CD38 antibody clones. D Nonlinear curve fitting was performed using antibody binding site concentration (2× molecular concentration).

[0232] Table 4

[0233] clone <![CDATA[K D (M)]]> CD38TM3 1.9E-09 CD38TM1 3.2E-08 CD38TM2 3.4E-08 CD38TM5 3.4E-08 CD38TM6 4.6E-09

[0234] C. Epitope mapping of anti-CD38 antigen binding clones

[0235] In order to identify the contact residues on the CD38 extracellular domain (ECD) by anti-CD38 antigen binding clones, epitope mapping was performed by Integral Molecular. Epitope mapping was performed by shotgun mutagenesis, which is a standard assay setting with alanine scanning and additional mutations to change amino acids from human residues to cynomolgus monkey residues as appropriate. In short, wild-type or mutated human CD38 ECD was expressed on the cell surface of HEK293 cells, and then the ability of the anti-CD38 antigen binding clones of interest to bind to CD38 ECD when certain amino acids were mutated was compared by flow cytometry with the binding of the same anti-CD38 antigen binding clones to wild-type CD38 ECD. A series of control molecules that also target CD38 were used to control mutations that affect the expression or general folding of the molecule. Table 5 shows the binding to surface-expressed CD38 ECD proteins with specific mutations as a percentage of binding to wild-type CD38. Values ​​marked in bold indicate amino acids that are critical for binding and surface accessibility of the anti-CD38 antigen binding clones (based on the published CD38 structure).

[0236] Table 5.

[0237]

[0238]

[0239] Anti-CD38 antigen binding clones CD38TM1 and CD38TM2 share the same key contact residues (R78 and D81) on human CD38. The key residues of CD38TM5 on human CD38 include R78, H79 and D81 (data not shown). The key residues of CD38TM3 on human CD38 include F216, L262, L124, C201, L230, C254 and S275. The key residues of CD38TM4 on human CD38 include F216, L262, C201, L230, C254 and S275. Figure 4 The positions of key residues for CD38TM3 are shown, including the surface accessible contact residues F216, L262 shown in red; the key structural residues L124, C230 shown in purple, and the key structural residues C119 / C201 and LC254 / C275 involved in the disulfide bond pair shown in grey.

[0240] CD38TM1, CD38TM2, and CD38TM5 bind to non-overlapping epitope residues on human CD38 compared to daratumumab. CD38TM6 binds to overlapping epitope residues on human CD38 with daratumumab. CD38TM3 and CD38TM4 bind to epitopes on human CD38 that partially overlap with CD38TR1 and daratumumab. CD38TR1 binds to epitopes on human CD38 that include F216, L262, Q272, and F273. Daratumumab binds to epitopes on human CD38 that include F274 and F275.

[0241] Clones CD38TM4 and / or CD38TM3 bind to an epitope on human CD38 that partially overlaps with the reference molecule CD38TR1 and possibly didaratumumab. Figure 4 The positions of key residues for CD38TM4 on human CD38 are shown, including the surface accessible contact residues F216, L262 shown in red; the key structural residues L124, C201 shown in purple, and the key structural residues C119 / C201 and LC254 / C275 involved in the disulfide bond pair shown in gray.

[0242] The binding of anti-CD38 antigen binding clones to CD38 in the presence of existing CD38 antibodies was tested, including daratumumab, HB-7 (Santa Cruz, catalog number: sc-18858), AT13-5 (Thermo Fisher, catalog number: MA5-16578) and OKT-10 (Sigma, catalog number: 87021903). As described above, CD38TM2, CD38TM1 and CD38TM3 were formatted as scFv and fused with Shiga toxin A subunit. MOLP-8 cells were incubated for 70 minutes with 20 μg / ml of existing anti-CD38 antibodies (~130nM) or control molecules (Shiga toxin A subunit alone). Then 0.5 μg / ml of anti-CD38 / Shiga toxin fusion molecules (~5nM) were added and incubated with cells for 48 hours. Antibody binding to CD38 was detected using a primary antibody recognizing the A subunit of Shiga toxin and a FITC-conjugated anti-IgG secondary antibody followed by flow cytometry. The mean fluorescence intensity (MFI) of the anti-CD38 antigen-binding clones was calculated after subtracting the signal from the secondary antibody-only control.

[0243] like Figure 5A and 5B As shown, CD38TM1 and CD38TM2 in the form of scFv SLTA fusion proteins can bind to CD38 in the presence of daratumumab, HB-7, AT13-5, and OKT-10, indicating that they bind to an epitope on CD38 that is different from the epitope bound by daratumumab, HB-7, AT13-5, and OKT-10. CD38TM3 in the form of scFv SLTA fusion protein can bind to CD38 in the presence of OKT-10, but not in the presence of daratumumab, HB-7, and AT13-5, indicating that the epitope bound by CD38TM3 to CD38 is different from the epitope bound by OKT-10, and that CD38TM3 shares or partially shares an epitope on CD38 with daratumumab, HB-7, and AT13-5.

[0244] Competitive binding of anti-CD38 antigen binding clones with CD38 was also tested. As described above, CD38TM2, CD38TM1 and CD38TM3 were formatted as scFv and fused with Shiga toxin A subunit. MOLP-8 cells were incubated for 70 minutes with 20 μg / ml of anti-CD38 binding clones (~400nM) or control molecules (Shiga toxin A subunit only). Then 0.5 μg / ml of anti-CD38 antigen binding clones in the form of scFv SLTA fusion proteins were added and incubated with cells for 48 hours. CD38 binding was detected by flow cytometry using the primary antibody and FITC-conjugated anti-IgG secondary antibody of Shiga toxin A subunit. The mean fluorescence intensity (MFI) of the anti-CD38 antigen binding clones was calculated after subtracting the signal from the secondary antibody control only, and was plotted as the percentage of the MFI measured from the single Shiga toxin A subunit as a control.

[0245] like Figure 5C and 5D As shown, CD38TM1 and CD38TM2 bind to CD38 in a competitive manner, which is consistent with the fact that they bind to the same epitope on CD38. CD38TM3 binds to CD38 in a semi-competitive manner with CD38TM1 and CD38TM2.

[0246] D. Induction of CDC and ADCC by anti-CD38 antibodies

[0247] Anti-CD38 binding clones were formatted as human IgG1 and tested for their ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0248] For CDC assay, MOLP-8 cells were seeded at a density of 10,000 cells per well in 50 μL complete medium (RPMI supplemented with 10% fetal bovine serum) in a black 96-well flat-bottom tissue culture plate. 50 μL of anti-CD38 antibody (9 μg / ml), control IgG1 antibody (9 μg / ml), or culture medium alone was added to each well and incubated at room temperature (RT) for 10 minutes. In addition to the control wells, different amounts (2-15 μL) of purified rabbit complement (Cat# CL 3441 Cedarlane Laboratories, Canada) were added to each well. After incubation at 37°C for one hour, the plate was allowed to reach room temperature and 100 μL of cell titration CytoTox Glo was added to each well. TM Reagent (Promega G7571 / G7573), shake the plate for 5 to 7 minutes, and Luminescence was read on a (Perkin Elmer) luminescence plate reader. Tested conditions: cells alone; cells + complement; cells + IgG1 control + complement; cells + antibody + complement. %CDC was calculated using the following equation: 100-(RLUT / RLUC) × 100), where RLUT is the relative luminescence unit of the test sample and RLUc is the relative luminescence unit of the complement-only sample. The assay was performed in triplicate and statistical analysis was performed using PRISM software.

[0249] To measure ADCC, MOLP-8 cells were used as target cells. TM Separate and separate peripheral blood mononuclear cells (PBMC) as effector cells. Dilute the sample 1:3 with 2% FBS in PBS. 15 mL Ficoll-Plaque TM (GE Healthcare) was gently layered under the 35 mL diluted sample and centrifuged at 1800 rpm (brake) for 25 minutes. The turbid interphase containing PBMCs was collected, washed three times in 2% FBS / PBS, and frozen in 10% DMSO / FBS at 50×10 6 Before use, thaw the frozen aliquot of PBMCs and aliquot at 2 × 10 6 Cells / mL were cultured overnight in 10% FBS / RPMI + 5ng / mL recombinant human IL2 (R&D systems #202-IL).

[0250] For ADCC assays, all steps were performed in complete medium. 5000 target cells were plated per well in a 96-well plate, and 50 μL of anti-CD38 antibody (10 μg / ml), control IgG1 (10 μg / ml), or medium alone was added to each well. 50 μL of human effector PBMCs were then added to the wells at a ratio of 1:25 to 1:50 for target: effector (T:E) cells. The plate was briefly centrifuged at 800 rpm for 30 seconds to bring all cells into close proximity. After incubation for 4 hours at 37°C, the plate was centrifuged at 1100 rpm for 5 minutes, and 100 μL of supernatant was transferred to a white plate. 100 μL of CytoTox Glo was added to the supernatant. TM Reagent (Promega cat# G9292), and then the plate was shaken for 20-30 minutes at room temperature (RT). Luminescence was read on a luminescence plate reader (Perkin Elmer), and the specific lysis percentage was calculated using the following equation: (RLUT / RLUE / T) / (RLUL / RLUE / T)×100, where RLUT is the relative luminescence unit of the test sample, RLUE / T is the relative luminescence unit of the sample containing target cells and effector cells alone, and RLUL is the relative luminescence unit of cells lysed with Triton X-100. The assay was performed in triplicate, and statistical analysis was performed using PRISM software.

[0251] The percentages of CDC and ADCC induced by the anti-CD38 antibody clones are shown in Table 5. All anti-CD38 antibody clones were able to induce CDC and ADCC on cells expressing CD38.

[0252] Table 6

[0253]

[0254] E. Derivation of CD38TM4 from CD38TM3

[0255] CD38TM3 VH and VL did not bind to standard monoclonal antibody purification resins (Protein A or Protein L) and were purified using His-tag and IMAC columns ( Fig.11A Schematic diagram showing the purification of CD38TM3 in the form of scFv-SLTA fusion protein as an example). For easier purification, the light chain of CD38TM3, which is a lambda light chain, was modified by framework mutations to enable it to bind to protein L, thereby achieving affinity purification without an additional tag. Fig. 11C As shown, the first 21 amino acids of the CD38TM3 VL domain were replaced with the first 22 amino acids of the VL domain of CD38TR1, which is the kappa light chain from which the VL domain of CD38TM4 was derived. The resulting CD38TM4 was able to bind to protein L and was therefore purified by a protein L column ( Fig. 11B Purification of CD38TM4 in the form of a scFv-SLTA fusion protein is shown as an example). Sequence Listing <110> Millennium Pharmaceuticals, Inc. <120> Anti-CD38 antibody <130> 101588-5011-WO <160> 68 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Pro Ser Thr Gly Phe Trp Ser Gly Asn Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 2 Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 3 Ile Tyr Pro Gly Asp Ser Asp Thr 1 5 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 4 Ala Arg Gly Pro Ser Thr Gly Phe Trp Ser Gly Asn Tyr Phe Asp Tyr 1 5 10 15 <210> 5 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 5 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Phe Tyr Pro Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala 35 40 45 Leu Ile Tyr Ala Thr Asn Asn Lys Tyr Ser Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Ser Arg Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Val Tyr Tyr Asp Gly 85 90 95 Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 6 Thr Gly Ala Val Thr Ser Gly Phe Tyr 1 5 <210> 7 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 7 Ala Thr Asn 1 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 8 Leu Val Tyr Tyr Asp Gly Ala Trp 1 5 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu His Ser Asn Tyr Phe Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 10 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 11 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 12 Ala Arg Glu His Ser Asn Tyr Phe Tyr Gly Met Asp Val 1 5 10 <210> 13 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 13 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Gly Ser Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 14 Ser Ser Asn Ile Gly Ser Asn Tyr 1 5 <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 15 Gly Asn Ser 1 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 16 Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Gly 1 5 10 <210> 17 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Glu Thr Ser Phe Gly Leu Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 18 Gly Phe Thr Phe Ser Ser Tyr Trp 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 19 Ile Ser Gly Ser Gly Gly Gly Thr 1 5 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 20 Ala Arg Glu Gly Glu Thr Ser Phe Gly Leu Asp Val 1 5 10 <210> 21 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 21 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Ser Val Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> twenty two Ser Ser Asn Ile Gly Gly Asn Tyr 1 5 <210> twenty three <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> twenty three Arg Asn Asn 1 <210> twenty four <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> twenty four Gln Ser Tyr Asp Ser Ser Leu Ser Val Ser 1 5 10 <210> 25 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser 20 25 30 Gly Phe Tyr Pro Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg 35 40 45 Ala Leu Ile Tyr Ala Thr Asn Asn Lys Tyr Ser Trp Thr Pro Ala Arg 50 55 60 Phe Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Ser Arg 65 70 75 80 Val Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Val Tyr Tyr Asp 85 90 95 Gly Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 26 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> CD38-targeted reference molecule <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Asn Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Ser Met Ile Thr Thr Gly Phe Val Met Asp Ser Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser 115 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CD38-targeting reference molecule <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Asn Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> CD38 Homo sapiens <400> 28 Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys 1 5 10 15 Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val 20 25 30 Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 40 45 Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu 50 55 60 Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val 65 70 75 80 Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys 85 90 95 His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu 100 105 110 Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser Arg Ile 115 120 125 Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr 130 135 140 Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys 145 150 155 160 Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp 165 170 175 Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val 180 185 190 Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu 195 200 205 Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser 210 215 220 Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu Glu Ala 225 230 235 240 Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu Cys Gln Asp 245 250 255 Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile Gln 260 265 270 Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285 Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile 290 295 300 <210> 29 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> CD38 Homo sapiens extracellular domain <400> 29 Val Pro Arg Trp Arg Gln Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg 1 5 10 15 Phe Pro Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Ile His 20 25 30 Pro Glu Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys 35 40 45 Gly Ala Phe Ile Ser Lys His Pro Cys Asn Ile Thr Glu Glu Asp Tyr 50 55 60 Gln Pro Leu Met Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Ile 65 70 75 80 Leu Leu Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val 85 90 95 Gln Arg Asp Met Phe Thr Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala 100 105 110 Asp Asp Leu Thr Trp Cys Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr 115 120 125 Gln Ser Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser 130 135 140 Val Phe Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp 145 150 155 160 Val Val His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys 165 170 175 Asn Ser Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys 180 185 190 Val Gln Thr Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser 195 200 205 Arg Asp Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile 210 215 220 Ser Lys Arg Asn Ile Gln Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp 225 230 235 240 Lys Phe Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser 245 250 255 Glu Ile <210> 30 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> CD38 Long-tailed Macaque <400> 30 Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys 1 5 10 15 Arg Leu Ser Arg Arg Ala Gln Val Cys Leu Gly Val Cys Leu Leu Val 20 25 30 Leu Leu Ile Leu Val Val Val Val Ala Val Val Leu Pro Arg Trp Arg 35 40 45 Gln Gln Trp Ser Gly Ser Gly Thr Thr Ser Arg Phe Pro Glu Thr Val 50 55 60 Leu Ala Arg Cys Val Lys Tyr Thr Glu Val His Pro Glu Met Arg His 65 70 75 80 Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser 85 90 95 Lys Tyr Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Val Lys 100 105 110 Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Thr Leu Leu Trp Ser Arg 115 120 125 Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe 130 135 140 Thr Leu Glu Asp Met Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp 145 150 155 160 Cys Gly Glu Phe Asn Thr Phe Glu Ile Asn Tyr Gln Ser Cys Pro Asp 165 170 175 Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr 180 185 190 Val Ser Arg Arg Phe Ala Glu Thr Ala Cys Gly Val Val His Val Met 195 200 205 Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly 210 215 220 Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Ala Leu Glu 225 230 235 240 Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu Cys Gln 245 250 255 Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile 260 265 270 Arg Phe Phe Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys 275 280 285 Val Lys Asn Pro Glu Asp Ser Ser Cys Leu Ser Gly Ile 290 295 300 <210> 31 <211> 258 <212> PRT <213> Artificial Sequence <220> <223> CD38 long-tailed macaque extracellular domain <400> 31 Leu Pro Arg Trp Arg Gln Gln Trp Ser Gly Ser Gly Thr Thr Ser Arg 1 5 10 15 Phe Pro Glu Thr Val Leu Ala Arg Cys Val Lys Tyr Thr Glu Val His 20 25 30 Pro Glu Met Arg His Val Asp Cys Gln Ser Val Trp Asp Ala Phe Lys 35 40 45 Gly Ala Phe Ile Ser Lys Tyr Pro Cys Asn Ile Thr Glu Glu Asp Tyr 50 55 60 Gln Pro Leu Val Lys Leu Gly Thr Gln Thr Val Pro Cys Asn Lys Thr 65 70 75 80 Leu Leu Trp Ser Arg Ile Lys Asp Leu Ala His Gln Phe Thr Gln Val 85 90 95 Gln Arg Asp Met Phe Thr Leu Glu Asp Met Leu Leu Gly Tyr Leu Ala 100 105 110 Asp Asp Leu Thr Trp Cys Gly Glu Phe Asn Thr Phe Glu Ile Asn Tyr 115 120 125 Gln Ser Cys Pro Asp Trp Arg Lys Asp Cys Ser Asn Asn Pro Val Ser 130 135 140 Val Phe Trp Lys Thr Val Ser Arg Arg Phe Ala Glu Thr Ala Cys Gly 145 150 155 160 Val Val His Val Met Leu Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys 165 170 175 Asn Ser Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys 180 185 190 Val Gln Ala Leu Glu Ala Trp Val Ile His Gly Gly Arg Glu Asp Ser 195 200 205 Arg Asp Leu Cys Gln Asp Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile 210 215 220 Ser Lys Arg Asn Ile Arg Phe Phe Cys Lys Asn Ile Tyr Arg Pro Asp 225 230 235 240 Lys Phe Leu Gln Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Leu Ser 245 250 255 Gly Ile <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 32 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 33 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 33 Gly Gly Ser 1 <210> 34 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 34 Gly Gly Gly Ser 1 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 35 Gly Gly Gly Gly Ser 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 36 Gly Gly Gly Gly Ser Gly Gly Gly 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 37 Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 38 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Linker sequence <400> 38 Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Ser <210> 39 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 constant region <400> 39 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 40 <211> 526 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety - SLTA <400> 40 Met Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 1 5 10 15 Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile 20 25 30 Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ile Gly Asp 35 40 45 Asn Leu Phe Ala Val Asp Ile Leu Gly Phe Asp Phe Thr Leu Gly Arg 50 55 60 Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 65 70 75 80 Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 85 90 95 Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Ala Asp 100 105 110 Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly 115 120 125 Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 130 135 140 Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 145 150 155 160 Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 165 170 175 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Ala Ser Tyr Val 180 185 190 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 195 200 205 Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg 210 215 220 Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile 225 230 235 240 Leu Asn Ser His His His Ala Ser Ala Val Ala Ala Glu Phe Pro Lys 245 250 255 Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala Pro Gln Ser Val Leu 260 265 270 Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile 275 280 285 Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp 290 295 300 Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn 305 310 315 320 Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 325 330 335 Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu 340 345 350 Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Gly 355 360 365 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly 370 375 380 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 385 390 395 400 Gly Gly Gly Gly Ser Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 405 410 415 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 420 425 430 Phe Thr Phe Ser Asp Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly 435 440 445 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 450 455 460 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 465 470 475 480 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 485 490 495 Thr Ala Val Tyr Tyr Cys Ala Arg Glu His Ser Asn Tyr Phe Tyr Gly 500 505 510 Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 515 520 525 <210> 41 <211> 503 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety - SLTA <400> 41 Met Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 1 5 10 15 Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile 20 25 30 Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ile Gly Asp 35 40 45 Asn Leu Phe Ala Val Asp Ile Leu Gly Phe Asp Phe Thr Leu Gly Arg 50 55 60 Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 65 70 75 80 Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 85 90 95 Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Ala Asp 100 105 110 Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly 115 120 125 Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 130 135 140 Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 145 150 155 160 Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 165 170 175 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Ala Ser Tyr Val 180 185 190 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 195 200 205 Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg 210 215 220 Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile 225 230 235 240 Leu Asn Ser His His His Ala Ser Ala Val Ala Ala Glu Phe Pro Lys 245 250 255 Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala Pro Gln Ser Val Leu 260 265 270 Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile 275 280 285 Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn Tyr Val Tyr Trp 290 295 300 Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asn 305 310 315 320 Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 325 330 335 Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu 340 345 350 Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu Ser Val Ser Val 355 360 365 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser 370 375 380 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 385 390 395 400 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 405 410 415 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 420 425 430 Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Phe Tyr Ala Asp Ser Val 435 440 445 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 450 455 460 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 465 470 475 480 Ala Arg Glu Gly Glu Thr Ser Phe Gly Leu Asp Val Trp Gly Gln Gly 485 490 495 Thr Leu Val Thr Val Ser Ser 500 <210> 42 <211> 506 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety - SLTA <400> 42 Met Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 1 5 10 15 Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile 20 25 30 Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ile Gly Asp 35 40 45 Asn Leu Phe Ala Val Asp Ile Leu Gly Phe Asp Phe Thr Leu Gly Arg 50 55 60 Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 65 70 75 80 Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 85 90 95 Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Ala Asp 100 105 110 Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly 115 120 125 Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 130 135 140 Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 145 150 155 160 Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 165 170 175 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Ala Ser Tyr Val 180 185 190 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 195 200 205 Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg 210 215 220 Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile 225 230 235 240 Leu Asn Ser His His His Ala Ser Ala Val Ala Ala Glu Phe Pro Lys 245 250 255 Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala Pro Gln Val Gln Leu 260 265 270 Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile 275 280 285 Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp 290 295 300 Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr 305 310 315 320 Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val 325 330 335 Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser 340 345 350 Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Pro 355 360 365 Ser Thr Gly Phe Trp Ser Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly 370 375 380 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Thr Val Val 385 390 395 400 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Glu Thr Val Thr Leu 405 410 415 Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Phe Tyr Pro Asn 420 425 430 Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile Tyr Ala 435 440 445 Thr Asn Asn Lys Tyr Ser Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu 450 455 460 Leu Gly Asp Lys Ala Ala Leu Thr Leu Ser Arg Val Gln Pro Glu Asp 465 470 475 480 Glu Ala Asp Tyr Tyr Cys Leu Val Tyr Tyr Asp Gly Ala Trp Val Phe 485 490 495 Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 500 505 <210> 43 <211> 507 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety - SLTA <400> 43 Met Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 1 5 10 15 Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile 20 25 30 Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ile Gly Asp 35 40 45 Asn Leu Phe Ala Val Asp Ile Leu Gly Phe Asp Phe Thr Leu Gly Arg 50 55 60 Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 65 70 75 80 Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 85 90 95 Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Ala Asp 100 105 110 Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly 115 120 125 Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 130 135 140 Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 145 150 155 160 Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 165 170 175 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Ala Ser Tyr Val 180 185 190 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 195 200 205 Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg 210 215 220 Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile 225 230 235 240 Leu Asn Ser His His His Ala Ser Ala Val Ala Ala Glu Phe Pro Lys 245 250 255 Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala Pro Gln Val Gln Leu 260 265 270 Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile 275 280 285 Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp 290 295 300 Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr 305 310 315 320 Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val 325 330 335 Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser 340 345 350 Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Pro 355 360 365 Ser Thr Gly Phe Trp Ser Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly 370 375 380 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 385 390 395 400 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 405 410 415 Ile Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Phe Tyr Pro 420 425 430 Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Ala Leu Ile Tyr 435 440 445 Ala Thr Asn Asn Lys Tyr Ser Trp Thr Pro Ala Arg Phe Ser Gly Ser 450 455 460 Leu Leu Gly Asp Lys Ala Ala Leu Thr Leu Ser Arg Val Gln Pro Glu 465 470 475 480 Asp Glu Ala Asp Tyr Tyr Cys Leu Val Tyr Tyr Asp Gly Ala Trp Val 485 490 495 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 500 505 <210> 44 <211> 499 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety - SLTA <400> 44 Met Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 1 5 10 15 Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gln Thr Ile 20 25 30 Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp 35 40 45 Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp Pro Glu Glu Gly Arg 50 55 60 Phe Asn Asn Leu Arg Leu Ile Val Glu Arg Asn Asn Leu Tyr Val Thr 65 70 75 80 Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr Arg Phe Ala Asp Phe 85 90 95 Ser His Val Thr Phe Pro Gly Thr Thr Ala Val Thr Leu Ser Gly Asp 100 105 110 Ser Ser Tyr Thr Thr Leu Gln Arg Val Ala Gly Ile Ser Arg Thr Gly 115 120 125 Met Gln Ile Asn Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met 130 135 140 Ser His Ser Gly Thr Ser Leu Thr Gln Ser Val Ala Arg Ala Met Leu 145 150 155 160 Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln 165 170 175 Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Tyr Val 180 185 190 Met Thr Ala Glu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Leu Ser 195 200 205 Ser Val Leu Pro Asp Tyr His Gly Gln Asp Ser Val Arg Val Gly Arg 210 215 220 Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Leu Ile 225 230 235 240 Leu Asn Cys His His His Ala Ser Ala Val Ala Ala Glu Phe Pro Lys 245 250 255 Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala Pro Asp Ile Gln Met 260 265 270 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 275 280 285 Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg Leu Thr Trp Tyr 290 295 300 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Ser Gly Ala Thr 305 310 315 320 Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 325 330 335 Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 340 345 350 Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Asn Pro Tyr Thr Phe Gly Gln 355 360 365 Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gln Val Gln Leu 370 375 380 Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu 385 390 395 400 Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp 405 410 415 Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Val Met Trp 420 425 430 Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met Ser Arg Leu Asn 435 440 445 Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Arg Leu Ser Ser 450 455 460 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys Ser Met Ile 465 470 475 480 Thr Thr Gly Phe Val Met Asp Ser Trp Gly Gln Gly Ser Leu Val Thr 485 490 495 Val Ser Ser <210> 45 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 D265A Constant Region <400> 45 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 46 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 N297A Constant Region <400> 46 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 47 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Human IgG2 Constant Region <400> 47 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 48 <211> 377 <212> PRT <213> Artificial Sequence <220> <223> Human IgG3 constant region <400> 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 49 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Human IgG4 constant region (wild type) <400> 49 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325 <210> 50 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Human IgG4 constant region (S241P hinge mutant) <400> 50 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325 <210> 51 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> human kappa light chain <400> 51 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 52 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Human lambda light chain <400> 52 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 53 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 53 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gly Pro Tyr Tyr Leu Tyr Gly Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 54 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 55 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 55 Ile Ser Ser Ser Ser Ser Tyr Ile 1 5 <210> 56 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 56 Ala Thr Glu Gly Pro Tyr Tyr Leu Tyr Gly Phe Asp Ile 1 5 10 <210> 57 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 57 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn Thr Leu 85 90 95 Ser Gly Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 58 Ser Ser Asn Ile Gly Ser Asn Tyr 1 5 <210> 59 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 59 Gly Asn Ser 1 <210> 60 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 60 Gln Ser Tyr Asp Asn Thr Leu Ser Gly Val 1 5 10 <210> 61 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 61 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Leu Phe His Asp Ser Ser Gly Tyr Tyr Phe Gly His 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 62 Gly Phe Thr Phe Asp Asp Tyr Gly 1 5 <210> 63 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 63 Ile Asn Trp Asn Gly Gly Ser Thr 1 5 <210> 64 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 64 Ala Arg Gly Gly Leu Phe His Asp Ser Ser Gly Tyr Tyr Phe Gly His 1 5 10 15 <210> 65 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 65 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Ser 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Asp Asn Leu 85 90 95 Ser Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 66 Ser Ser Asn Ile Gly Asn Ser Tyr 1 5 <210> 67 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 67 Arg Asn Asn 1 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CD38 targeting moiety <400> 68 Ser Ala Trp Asp Asp Asn Leu Ser Val 1 5

Claims

1. A composition comprising an anti-CD38 antigen binding domain, wherein the antigen binding domain comprises: a) a heavy chain variable domain VH comprising: i) VHCDR1 consisting of the sequence of SEQ ID NO: 2; ii) VHCDR2 consisting of the sequence of SEQ ID NO: 3; and iii) a VHCDR3 consisting of the sequence of SEQ ID NO: 4; and b) a light chain variable domain VL comprising: i) VLCDR1 consisting of the sequence of SEQ ID NO: 6; ii) VLCDR2 consisting of the sequence of SEQ ID NO: 7; and iii) VLCDR3 consisting of the sequence of SEQ ID NO:

8.

2. The composition of claim 1, wherein the VH comprises the sequence of SEQ ID NO: 1 and the VL comprises the sequence of SEQ ID NO:

25.

3. The composition of claim 1, wherein the VH comprises the sequence of SEQ ID NO: 1 and the VL comprises the sequence of SEQ ID NO:

5.

4. A composition comprising an anti-CD38 antigen binding domain, wherein the anti-CD38 antigen binding domain comprises: a) a heavy chain variable domain VH comprising: i) VHCDR1 consisting of the sequence of SEQ ID NO: 10; ii) VHCDR2 consisting of the sequence of SEQ ID NO: 11; and iii) a VHCDR3 consisting of the sequence of SEQ ID NO: 12; and b) a light chain variable domain VL comprising: i) VLCDR1 consisting of the sequence of SEQ ID NO: 14; ii) VLCDR2 consisting of the sequence of SEQ ID NO: 15; and iii) VLCDR3 consisting of the sequence of SEQ ID NO:

16.

5. The composition of claim 4, wherein the VH comprises the sequence of SEQ ID NO:9 and the VL comprises the sequence of SEQ ID NO:

13.

6. A composition comprising an anti-CD38 antigen binding domain, wherein the anti-CD38 antigen binding domain comprises: a) a heavy chain variable domain VH comprising: i) VHCDR1 consisting of the sequence of SEQ ID NO: 18; ii) VHCDR2 consisting of the sequence of SEQ ID NO: 19; and iii) a VHCDR3 consisting of the sequence of SEQ ID NO: 20; and b) a light chain variable domain VL comprising: i) VLCDR1 consisting of the sequence of SEQ ID NO:22; ii) VLCDR2 consisting of the sequence of SEQ ID NO: 23; and iii) VLCDR3 consisting of the sequence of SEQ ID NO:

24.

7. The composition of claim 6, wherein the VH comprises the sequence of SEQ ID NO: 17 and the VL comprises the sequence of SEQ ID NO:

21.

8. A composition comprising an anti-CD38 antigen binding domain, wherein the anti-CD38 antigen binding domain comprises: a) a heavy chain variable domain VH comprising: i) VHCDR1 consisting of the sequence of SEQ ID NO: 54; ii) VHCDR2 consisting of the sequence of SEQ ID NO:55; and iii) a VHCDR3 consisting of the sequence of SEQ ID NO: 56; and b) a light chain variable domain VL comprising: i) VLCDR1 consisting of the sequence of SEQ ID NO:58; ii) VLCDR2 consisting of the sequence of SEQ ID NO:59; and iii) VLCDR3 consisting of the sequence of SEQ ID NO:

60.

9. The composition of claim 8, wherein the VH comprises the sequence of SEQ ID NO:53 and the VL comprises the sequence of SEQ ID NO:

57.

10. The composition of any one of claims 1-9, wherein the VH and the VL are in a single polypeptide.

11. The composition of claim 10, wherein the polypeptide comprises a scFv linker and the polypeptide has an N-terminus to C-terminus orientation of VH-scFv linker-VL.

12. The composition of claim 10, wherein the polypeptide comprises a scFv linker and the polypeptide has an N-terminus to C-terminus orientation of VL-scFv linker-VH.

13. The composition of any one of claims 1 to 9, wherein the composition comprises a first polypeptide and a second polypeptide, the first polypeptide comprising the VH, and the second polypeptide comprising the VL.

14. The composition according to any one of claims 1 to 9, wherein the composition is an antibody comprising: a) a heavy chain comprising said VH; and b) a light chain comprising said VL.

15. The composition of claim 14, wherein the heavy chain comprises the heavy chain variable domain and a heavy chain constant domain, and the heavy chain constant domain is selected from the group consisting of heavy chain constant domains of human IgG1, IgG2 and IgG4 or variants thereof.

16. The composition of claim 15, wherein the heavy chain constant domain is a heavy chain constant domain of human IgG1.

17. The composition of claim 16, wherein the heavy chain constant domain is a variant heavy chain constant domain of human IgG1.

18. The composition of claim 17, wherein the variant heavy chain constant domain of human IgG1 has abolished FcγR binding.

19. The composition of claim 15, wherein the heavy chain constant domain is a variant of a human IgG4 heavy chain constant domain comprising a S228P amino acid substitution compared to a wild-type heavy chain constant domain of human IgG4.

20. A nucleic acid composition encoding the composition of claim 13, wherein the nucleic acid composition comprises: a) a first nucleic acid encoding said first polypeptide; and b) a second nucleic acid encoding the second polypeptide.

21. A nucleic acid composition comprising a nucleic acid encoding the composition of claim 10.

22. An expression vector composition comprising the nucleic acid of claim 21.

23. An expression vector composition comprising the first nucleic acid and the second nucleic acid of claim 20.

24. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid of claim 20; and b) a second expression vector comprising the second nucleic acid of claim 20.

25. A host cell comprising the expression vector composition according to any one of claims 22 to 24.

26. A method for preparing a composition comprising an anti-CD38 antigen binding domain, comprising culturing the host cell of claim 25 under conditions where the composition comprising an anti-CD38 binding domain is expressed, and recovering the composition.

27. Use of a composition according to any one of claims 1 to 19 in the preparation of a medicament for treating multiple myeloma.

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