Anti-CD38 binding domain

Abstract

To provide anti-CD38 binding domains, to provide compositions comprising anti-CD38 binding domains, to provide nucleic acids encoding anti-CD38 binding domains, and to provide methods of using anti-CD38 binding domains or compositions to treat multiple myeloma.SOLUTION: A composition comprises an anti-CD38 antigen-binding domain, and the anti-CD38 antigen-binding domain comprises a variable heavy domain (VH) comprising a vhCDR1 having a specific amino acid sequence, a vhCDR2 having a specific amino acid sequence, and a vhCDR3 having a specific amino acid sequence, and a variable light domain (VL) comprising a vlCDR1 having a specific amino acid sequence, a vlCDR2 having a specific amino acid sequence, and a vlCDR3 having a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 62 / 795,855, filed January 23, 2019, the contents of which are expressly incorporated by reference in their entirety.

[0002] II. REFERENCE TO AN ACCOMPANYING "SEQUENCE LISTING", TABLE, OR COMPUTER PROGRAM SUBMITTED ON A COMPACT DISC A 76 kilobyte text copy of the sequence listing entitled 101588-5011-WO_ST25.txt (created on January 21, 2020) is submitted herewith, and is incorporated herein by reference. [Background technology]

[0003] III. Background of the Invention CD38 is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain, also known as cyclic ADP-ribose hydrolase. CD38 is a member of a group of related membrane-bound or soluble enzymes, including CD157 and Aplysia ADPR cyclase. Enzymes of this family have the unique ability to convert NAD to cyclic ADP-ribose or nicotinic acid adenine dinucleotide phosphate.

[0004] In addition, CD38 inhibits Ca 2+ It has been reported that CD38 is involved in the recruitment and signal transduction by inducing tyrosine phosphorylation of a number of signaling molecules, including phospholipase Cγ, ZAP-70, syk, and c-cbl. Based on these findings, it has been proposed that CD38 is an important signaling molecule in the maturation and activation of lymphoid cells during normal development of such cells.

[0005] CD38 has been shown 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 has been shown to be involved in functional effects such as lymphocyte proliferation, cytokine release, control of B cell and myeloid cell development and survival, and induction of dendritic cell maturation (Figure 1).

[0006] 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 interaction of CD38 with CD31 may result in pro-survival effects for leukemic cells.

[0007] 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-cell and T-cell 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). Therefore, CD38 is used as a prognostic marker for leukemia.

[0008] Despite recent advances in the discovery and development of anti-cancer drugs, many forms of cancer associated with CD38-expressing tumors remain associated with poor prognosis. Thus, there is a need for improved methods of treating such forms of cancer, and a need to develop anti-CD38 antibodies with improved characteristics, despite the existence of antibodies on the market. Summary of the Invention [Means for solving the problem]

[0009] IV. BRIEF SUMMARY OF THE INVETION The present invention relates to compositions comprising novel anti-CD38 antigen-binding domains.

[0010] In one aspect, a composition comprises an anti-CD38 antigen binding domain comprising a variable heavy domain (VH) comprising a vhCDR1 having the amino acid sequence of SEQ ID NO: 2, a vhCDR2 having the amino acid sequence of SEQ ID NO: 3, and a vhCDR3 having the amino acid sequence of SEQ ID NO: 4, and a variable light domain (VL) comprising a vlCDR1 having the amino acid sequence of SEQ ID NO: 6, a vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and a vlCDR3 having the amino acid sequence of SEQ ID NO: 8.

[0011] In some embodiments, the composition comprises an anti-CD38 antigen binding domain, which comprises a variable heavy domain having an amino acid sequence identical to SEQ ID NO: 1 and a variable light domain having an amino acid sequence identical to SEQ ID NO: 5. In some embodiments, the composition comprises an anti-CD38 antigen binding domain, which comprises a variable heavy domain having an amino acid sequence identical to SEQ ID NO: 1 and a variable light domain having an amino acid sequence identical to SEQ ID NO: 25.

[0012] In another aspect, a composition comprises an anti-CD38 antigen binding domain, the anti-CD38 antigen binding domain comprising a variable heavy domain (VH) comprising a vhCDR1 having the amino acid sequence of SEQ ID NO: 10, a vhCDR2 having the amino acid sequence of SEQ ID NO: 11, and a vhCDR3 having the amino acid sequence of SEQ ID NO: 12, and a variable light domain (VL) comprising a vlCDR1 having the amino acid sequence of SEQ ID NO: 14, a vlCDR2 having the amino acid sequence of SEQ ID NO: 15, and a vlCDR3 having the amino acid sequence of SEQ ID NO: 16.

[0013] In some embodiments, the composition comprises an anti-CD38 antigen binding domain, which comprises a variable heavy domain having an amino acid sequence identical to SEQ ID NO:9 and a variable light domain having an amino acid sequence identical to SEQ ID NO:13.

[0014] In another embodiment, a composition comprises an anti-CD38 antigen binding domain, the anti-CD38 antigen binding domain comprising a variable heavy domain (VH) comprising a vhCDR1 having the amino acid sequence of SEQ ID NO: 18, a vhCDR2 having the amino acid sequence of SEQ ID NO: 19, and a vhCDR3 having the amino acid sequence of SEQ ID NO: 20, and a variable light domain (VL) comprising a vlCDR1 having the amino acid sequence of SEQ ID NO: 22, a vlCDR2 having the amino acid sequence of SEQ ID NO: 23, and a vlCDR3 having the amino acid sequence of SEQ ID NO: 24.

[0015] In some embodiments, the composition comprises an anti-CD38 antigen binding domain, which comprises a variable heavy domain having an amino acid sequence identical to SEQ ID NO: 17 and a variable light domain having an amino acid sequence identical to SEQ ID NO: 21.

[0016] In another embodiment, a composition comprises an anti-CD38 antigen binding domain, the anti-CD38 antigen binding domain comprising a variable heavy domain (VH) comprising a vhCDR1 having the amino acid sequence of SEQ ID NO: 54, a vhCDR2 having the amino acid sequence of SEQ ID NO: 55, and a vhCDR3 having the amino acid sequence of SEQ ID NO: 56, and a variable light domain (VL) comprising a vlCDR1 having the amino acid sequence of SEQ ID NO: 58, a vlCDR2 having the amino acid sequence of SEQ ID NO: 59, and a vlCDR3 having the amino acid sequence of SEQ ID NO: 60.

[0017] In some embodiments, the composition comprises an anti-CD38 antigen binding domain, which comprises a variable heavy domain having an amino acid sequence identical to SEQ ID NO:53 and a variable light domain having an amino acid sequence identical to SEQ ID NO:57.

[0018] In some embodiments, the compositions comprising an anti-CD38 antigen binding domain described herein comprise a variable heavy domain and a variable light domain on a single polypeptide, hi some embodiments, the single polypeptide comprises an scFv linker, a variable heavy domain, and a variable light domain in an N-terminal to C-terminal orientation of VH-scFv linker-VL or VL-scFv linker-VH.

[0019] In some embodiments, the composition comprising an anti-CD38 antigen binding domain described herein comprises a first polypeptide comprising a variable heavy domain and a second polypeptide comprising a variable light domain.

[0020] In some embodiments, the composition comprising an anti-CD38 antigen binding domain described herein is an antibody, the antibody comprising a heavy chain comprising a variable heavy domain and a light chain comprising a variable light domain. In some embodiments, the antibody comprises a heavy chain comprising a variable heavy domain and a heavy constant domain selected from the heavy constant domains of human IgG1, IgG2, and IgG4, and variants thereof. In some embodiments, the antibody comprises a heavy chain comprising a variable heavy domain and a heavy constant domain of human IgG1 or a variant thereof. In some embodiments, the antibody comprises a heavy chain comprising a variable heavy domain and a heavy constant domain selected from the heavy constant domain of a human IgG1 variant truncated for FcγR binding. In some embodiments, the antibody comprises a heavy chain comprising a variable heavy domain and a heavy constant domain selected from the heavy constant domain of a human IgG4 variant with a S228P amino acid substitution.

[0021] The present invention also relates to nucleic acid compositions encoding variable heavy and variable light domains. In some embodiments, the nucleic acid composition comprises a first nucleic acid encoding a variable heavy domain and a second nucleic acid encoding a variable heavy domain. In some embodiments, the nucleic acid composition comprises a single nucleic acid encoding the variable heavy and variable light domains.

[0022] Another aspect of the invention relates to an expression vector composition comprising any one of the nucleic acid compositions described herein, and a host cell comprising any one 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 variable heavy domain and a variable light domain.

[0023] The invention further relates to a method of making any of the compositions comprising an anti-CD38 antigen binding domain described herein, comprising culturing the host cell under conditions in which the anti-CD38 antigen binding domain is expressed, and recovering the composition.

[0024] Also included in the invention is a method of treating multiple myeloma using an effective amount of any of the compositions comprising an anti-CD38 antigen binding domain described herein. [Brief description of the drawings]

[0025] [Figure 1] Figure 1 shows the CD38 expression profile on lymphoid cells, with stars indicating high CD38 expression. CD38 expression has been 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+). CD38 expression is also seen on lymphoid progenitor cells (CD34+CD45RA+CD10+CD19-), but not on lymphoid stem cells. Increased CD38 expression is also seen on mature DCs and activated monocytes. [Diagram 2] Binding of anti-CD38 binding clones to purified human CD38 by ELISA assay is shown. Each anti-CD38 binding clone was formatted into an scFv and then fused to the Shiga toxin A subunit (scFv-SLTA fusion format). [Diagram 3] Binding of anti-CD38 binding clones to purified cynomolgus monkey CD38 by ELISA assay is shown. Each anti-CD38 binding clone was formatted into an scFv and then fused to Shiga toxin A subunit. [Figure 4] Key contact residues on the CD38 extracellular domain are shown for the anti-CD38 binding clone CD38TM4 (F216, L262, C119, L124, C201, L230, C254, C275) and the anti-CD38 monoclonal antibody daratumumab (S274). [Figure 5A] Binding of anti-CD38 binding clones (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, as well as several proteins, is shown. Reference molecule #1 (CD38TR1) targeting CD38 is also in the scFv-SLTA fusion protein format. [Figure 5B] Binding of anti-CD38 binding clones (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, as well as several proteins, is shown. Reference molecule #1 (CD38TR1) targeting CD38 is also in the scFv-SLTA fusion protein format. [Figure 5C] Competitive binding of anti-CD38 binding clones, including reference molecule #1 (CD38TR1) targeting CD38 in scFv-SLTA fusion format, to CD38-expressing MOLP-8 cells. All signals were detected by flow cytometry. [Figure 5D] 1 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 antibody clones (scFv only format) (first left column). [Figure 6A]The sequences of the CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 chain variable heavy and variable light domains and CDRs (full domains underlined) are shown. [Figure 6B] The sequences of the CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 chain variable heavy and variable light domains and CDRs (full domains underlined) are shown. [Figure 6C] The sequences of the CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 chain variable heavy and variable light domains and CDRs (full domains underlined) are shown. [Figure 6D] The sequences of the CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 chain variable heavy and variable light domains and CDRs (full domains underlined) are shown. [Figure 6E] The sequences of the CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 chain variable heavy and variable light domains and CDRs (full domains underlined) are shown. [Figure 6F] The sequences of the CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 chain variable heavy and variable light domains and CDRs (full domains underlined) are shown. [Figure 6G] The sequences of the CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1 chain variable heavy and variable light domains and CDRs (full domains underlined) are shown. [Figure 7] 1 shows the sequence of the CD38 target antigen of the present invention. [Figure 8] Some linker sequences are shown. [Figure 9-1] 1 shows the sequences of several Shiga toxin proteins fused to anti-CD38 binding domains used in the Examples. [Figure 9-2] 1 shows the sequences of several Shiga toxin proteins fused to anti-CD38 binding domains used in the Examples. [Figure 10A] A number of IgG heavy constant domain "scaffolds" based on human IgG1, IgG2 and IgG4, including several standard variants, as well as constant light kappa and constant light lambda sequences are shown. As will be appreciated by those of skill in the art and as described below, any of these scaffolds can be combined with the variable heavy and variable light domains of CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1. [Figure 10B] A number of IgG heavy constant domain "scaffolds" based on human IgG1, IgG2 and IgG4, including several standard variants, as well as constant light kappa and constant light lambda sequences are shown. As will be appreciated by those of skill in the art and as described below, any of these scaffolds can be combined with the variable heavy and variable light domains of CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1. [Figure 11A] Purification of CD38TM3-SLTA and CD38TM4-SLTA by IMAC and Protein L (Pro-L) columns, respectively, is shown. MW markers indicate where the molecular weight markers are located. [Figure 11B] Purification of CD38TM3-SLTA and CD38TM4-SLTA by IMAC and Protein L (Pro-L) columns, respectively, is shown. MW markers indicate where the molecular weight markers are located. [Figure 11C] The first 21 amino acids of CD38TM3 (underlined) are replaced by the first 22 amino acids of CD38TR1 (underlined) to derive CD38TM4. [Figure 12]Shown is a representation of the X-ray structure of a diabody (VH and VL are derived from CD38TM4) formed by two identical scFvs (each containing, from N- to C-terminus, VH-GGGGS-VL). The VH of one scFv chain (chain A) complexes with the VL of the other scFv (chain B) to form the CD38 binding domain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] V. Detailed Description of the Invention A. Overview Increased expression of CD38 is associated with various diseases of hematopoietic origin. Such diseases include, but are not limited to, multiple myeloma, chronic lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, acute lymphoblastic leukemia including B-cell acute lymphocytic leukemia, Waldenstrom's macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, prolymphocytic / myeloid leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, NK cell leukemia and plasma cell leukemia. Thus, CD38 provides a useful target in the treatment of diseases of the hematopoietic system. Several anti-CD38 antibodies are in clinical trials for the treatment of CD38-related cancers. Thus, antibodies against CD38 are useful for treating and diagnosing cancer.

[0027] The present invention provides anti-CD38 binding domains capable of binding to human and cynomolgus CD38 with high affinity. Furthermore, the anti-CD38 binding domains disclosed herein are capable of binding to CD38 in the presence of daratumumab, thus providing advantages in clinical applications not seen with some of the existing anti-CD38 antibodies in clinical trials.

[0028] B. Definition In order that this application may be more fully understood, several definitions are set forth below. Such definitions are meant to encompass all grammatical equivalents.

[0029] "Ablation" in this specification means reducing or eliminating an activity. In some embodiments, it is useful to remove an activity from the constant domain of an antibody. Thus, for example, "eliminating FcγR binding" means that an Fc region amino acid variant has less than 50% starting binding to FcγR compared to an Fc region that does not contain the particular variant, with less than 70-80-90-95-98% loss of activity being preferred, and usually the activity is below the level of detectable binding in a Biacore assay. For example, one ablation variant in the IgG1 constant region is the N297A variant, which removes the native glycosylation site and significantly reduces FcγRIIIa binding, thereby reducing antibody-dependent cell-mediated cytotoxicity (ADCC).

[0030] "ADCC" or "antibody-dependent cell-mediated cytotoxicity" as used herein refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognize bound antibody on target cells and subsequently cause lysis of the target cells. ADCC correlates with binding to FcγRIIIa; increased binding to FcγRIIIa leads to increased ADCC activity.

[0031] An "antigen binding domain" or "ABD" herein refers to a set of six complementarity determining regions (CDRs) that, when presented as part of a polypeptide sequence, specifically binds to a target antigen as discussed herein. Thus, an "anti-CD38 antigen binding domain" binds to the CD38 antigen as outlined herein. As known in the art, these CDRs are usually provided as a first set of variable heavy CDRs (vhCDRs or VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), each of which comprises three CDRs: vhCDR1, vhCDR2, vhCDR3 for the heavy chain and vlCDR1, vlCDR2 and vlCDR3 for the light chain. As understood in the art, the CDRs are separated by framework regions in each of the heavy and light variable regions: for the light variable region, these are (VL)FR1-vlCDR1-(VL)FR2-vlCDR2-(VL)FR3-vlCDR3-(VL)FR4, and for the heavy variable region, these are (VH)FR1-vhCDR1-(VH)FR2-vhCDR2-(VH)FR3-vhCDR3-(VH)FR4. The antigen binding domains of the present invention can be embodied in multiple formats, for example, Fab, Fv and scFv. In the "Fab" format, the set of six CDRs is provided by two different polypeptide sequences, namely a heavy variable region (vh or VH; containing vhCDR1, vhCDR2 and vhCDR3) and a light variable region (vl or VL; containing vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the VH being linked to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the VL being linked to the N-terminus of the constant light domain (thereby forming the light chain). The heavy and light variable regions together form an Fv, which can be either an sdFv or a Fab, as outlined herein. Thus, in some cases, the six CDRs of the antigen-binding domain are provided by the VH and VL. In the scFv format, the VH and VL are covalently linked into a single polypeptide sequence, usually via the use of a linker as outlined herein, which can be either VH-linker-VL or VL-linker-VH (starting from the N-terminus).

[0032] By "linker" herein is meant a domain linker that joins two protein domains together and is used, for example, in scFv and / or other proteins and protein fusion constructs. There are a number of suitable linkers that can be used, including traditional peptide bonds, which are usually produced by recombinant techniques that allow recombinant joining of the two domains, and have sufficient length and flexibility to allow each domain to retain its biological function. Linker peptides may primarily include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a suitable length to link two molecules such that they assume the correct configuration relative to each other to retain the desired activity. In one embodiment, the linker is about 1-50 amino acids in length, preferably about 1-30 amino acids in length. In one embodiment, a linker of 1-20 amino acids in length may be used, with some embodiments utilizing about 5 to about 10 amino acids. Useful linkers include, for example, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, including (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1 (usually 3-4), and other flexible linkers. Alternatively, a variety of non-proteinaceous polymers can be utilized as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Other linker sequences can include any sequence of any length of the CL / CH1 domain that is not all residues of the CL / CH1 domain; for example, the first 5-12 amino acid residues of the CL / CH1 domain. Linkers can also be derived from immunoglobulin light chains, such as Cκ or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (eg, TCR, FcR, KIR), sequences from hinge regions, and other naturally occurring sequences from other proteins.While any suitable linker may be used, many embodiments utilize glycine-serine polymers including, for example, (GS), (GSGGS), (GGGGS), and (GGGS), where (GGGS) is an integer of at least 1 (usually 2 to 3 to 4 to 5). "scFv linkers" typically include these glycine-serine polymers, such as those shown in Figure 8. Typically, scFv linkers are long enough to allow the VL and VH domains to properly associate to form an antigen binding domain (ABD) on a single polypeptide.

[0033] The term "antibody" is used in the broadest sense and includes, for example, intact immunoglobulins or antigen-binding domains. Traditional antibody structural units typically include a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention is directed to antibodies or antibody fragments (antibody monomers) generally based on the IgG class, which has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. Typically, IgG1, IgG2, and IgG4 are used more frequently than IgG3. It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M). The sequences shown herein use the 356D / 358M allotype, but other allotypes are included herein. That is, any sequence that includes the Fc domain of IgG1 is included herein and may have the 356E / 358L replacing the 356D / 358M allotype.

[0034] Thus, "isotype" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[0035] The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that contributes to antigen recognition, which is commonly referred to in the art and herein as an "Fv domain" or "Fv region." In the variable region, three loops for each of the heavy and light chain V domains come together to form the antigen-binding site. Each of the loops is referred to as a complementarity determining region (hereinafter referred to as a "CDR") where amino acid sequence variation is most pronounced. "Variable" refers to the fact that certain segments of the variable region differ widely in sequence among antibodies. The variability within the variable region is not uniformly distributed. Instead, the V regions consist of relatively invariant stretches called framework regions (FR) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions," each of which is 9-15 amino acids long or longer.

[0036] Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0037] The hypervariable regions typically consist of approximately amino acid residues 24-34 (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region and approximately amino acid residues 31-35B (HCDR1; "H" indicates 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 which form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Particular CDRs of the invention are described below.

[0038] As will be appreciated by those skilled in the art, the exact numbering and arrangement of the CDRs may vary between different numbering systems. However, it should be understood that the disclosure of a variable heavy and / or variable light sequence includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2 and vhCDR3), and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3).

[0039] A useful comparison of CDR numbering is given below, see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003): [Table 1]

[0040] Throughout this specification, when referring to residues in the variable domains (approximately residues 1-107 for light chain variable regions and residues 1-113 for heavy chain variable regions), the Kabat numbering system is generally used; for the Fc region, the EU numbering system is used (e.g., Kabat et al., supra (1991)).

[0041] The present invention provides a number of different CDR sets. In this case, a "complete CDR set" includes three variable light CDRs and three variable heavy CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These can be part of a larger variable light domain or variable heavy domain, respectfully. Furthermore, as more fully outlined herein, the variable heavy and variable light domains can be on separate polypeptide chains when heavy and light chains are used (e.g., when Fabs are used) or on a single polypeptide chain in the case of scFv sequences.

[0042] The CDRs contribute to forming the antigen-binding site, or more specifically, the epitope-binding site of an antibody.

[0043] "Epitope" refers to a determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule known as the paratope. An epitope is a grouping of molecules, such as amino acids or sugar side chains, that usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. An epitope may include 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 the specific antigen-binding peptide. In other words, the amino acid residues are present within the footprint of the specific antigen-binding peptide. Epitopes can be either conformational or linear. Conformational epitopes are created by amino acids that are spatially juxtaposed with different segments of a linear polypeptide chain. Linear epitopes are created by adjacent amino acid residues within a polypeptide chain. Conformational and nonconformational epitopes can be distinguished in that the binding to conformational epitopes, but not to nonconformational epitopes, is lost in the presence of denaturing solvents.

[0044] As used herein, "modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or a change to a moiety chemically attached to a protein. For example, a modification can be an altered carbohydrate or PEG structure attached to a protein. As used herein, "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications are always to amino acids encoded by DNA, e.g., the 20 amino acids that have codons in DNA and RNA.

[0045] As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid with a different amino acid at a particular position in a parent polypeptide sequence. In particular, in some embodiments, the substitution is for an amino acid that does not naturally occur at the particular position and does not naturally occur in the organism or any organism. For example, the substitution N297A refers to a mutant polypeptide, in this case an Fc mutant, in which the asparagine at position 297 is replaced with an alanine. For clarity, a protein that has been modified to change the nucleic acid coding sequence but does not change the starting amino acid (e.g., replacing CGG (which codes for arginine) with CGA (which also codes for arginine) to increase expression levels in the host organism) is not an "amino acid substitution", i.e., it is not an amino acid substitution if the protein has the same amino acid at the starting particular position, regardless of the creation of a new gene that codes for the same protein.

[0046] As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 233E represents the insertion of glutamic acid after position 233 and before position 234. Additionally, -233ADE or A233ADE represents the insertion of AlaAspGlu after position 233 and before position 234.

[0047] As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233- or E233#, E233(), or E233del represents the deletion of a glutamic acid at position 233. Additionally, EDA233- or EDA233# represents the deletion of the sequence GluAspAla starting at position 233.

[0048] As used herein, "mutant protein" or "protein variant" or "variant" refers to a protein that differs from the amino acid of a parent protein by at least one amino acid modification. A protein variant can refer to the protein itself, a composition comprising the protein, or an amino acid sequence encoding the protein. Preferably, a protein variant has at least one amino acid modification compared to the parent protein, e.g., about 1 to about 70 amino acid modifications, and preferably about 1 to about 5 amino acid modifications, compared to the parent. As described below, in some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, is a human wild-type sequence, such as an Fc region from IgG1, IgG2, IgG3, or IgG4, while a human sequence comprising a variant can also serve as a "parent polypeptide". A protein variant sequence herein has at least about 80% identity to the parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95 to 98 to 99% identity. A mutant protein can refer to the mutant protein itself, a composition comprising the protein variant, or a DNA sequence encoding the protein variant.

[0049] Thus, as used herein, "antibody variant" or "variant antibody" refers to an antibody that differs from a parent antibody by at least one amino acid modification, and as used herein, "IgG variant" or "variant IgG" refers to an antibody that differs from a parent IgG (again, often a human IgG sequence) by at least one amino acid modification. As used herein, "immunoglobulin variant" or "variant immunoglobulin" refers to an immunoglobulin sequence that differs from an amino acid of the parent immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" refers to a protein that comprises an amino acid modification in the Fc domain. The Fc variants of the present invention are defined according to the amino acid modification that constitutes the Fc variant of the present invention. Thus, for example, S241P or S228P is a hinge variant with a substituted proline at position 228 compared to the parent IgG4 hinge polypeptide, where the numbering S228P is according to the EU index and S241P is the Kabat numbering. Similarly, M252Y / S254T / T256E defines an Fc variant having the substitutions M252Y, S254T, and T256E compared to the parent Fc polypeptide (these mutations increase the binding of the Fc domain to the FcRn receptor, thus increasing the half-life of the molecule). The identity of the wild-type amino acids may not be specified, in which case the variant is referred to as 252Y / 254T / 256E. It should be noted that the order in which the substitutions are made is arbitrary, i.e., for example, 252Y / 254T / 256E is the same Fc variant as 254T / 252Y / 256E, and so on. For all positions discussed in the present invention relating to antibodies, unless otherwise specified, the numbering of the amino acid positions is according to Kabat for the numbering of the variable regions and according to the EU index for the constant regions, including the Fc region. The EU index, or EU index in the Kabat or EU numbering scheme, refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications can be additions, deletions, or substitutions.Substitutions can include naturally occurring amino acids, and, in some cases, synthetic amino acids. Examples include U.S. Pat. No. 6,586,207; WO 98 / 48032; WO 03 / 073238; U.S. Pat. No. 2004-0214988A1; WO 05 / 35727A2; WO 05 / 74524A2, JW Hin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Hin, & PG Schultz, (2002), ChemBioChem 11:1135-1137; JW Hin, et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang, & PG Schultz, (2002), Chem. 1-10, which are incorporated by reference in their entireties.

[0050] As used herein, "protein" means at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups include naturally occurring amino acids and peptide bonds, or "analogs," such as synthetic peptidomimetic structures, i.e., peptoids (see Simon et al., PNAS USA 89(20):9367 (1992), which is incorporated by reference in its entirety). Amino acids can be either naturally occurring or synthetic (e.g., not DNA-encoded amino acids), as will be understood by those of skill in the art. For example, homophenylalanine, citrulline, ornithine, and norleucine are considered synthetic amino acids for the purposes of the present invention, and both D- and L- (R- or S-) configurations of amino acids can be utilized. Variants of the present invention can be prepared, for example, as described in Cropp & Shultz, 2004, Trends, which is incorporated by reference in its entirety. 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. Additionally, polypeptides can include synthetic derivatization at one or more side chains or termini, glycosylation, PEGylation, circular permutations, cyclization, linkers to other molecules, fusions to proteins or protein domains, and addition of peptide tags or labels.

[0051] As used herein, "residue" refers to the position in the protein and its associated amino acid identity. For example, asparagine 297 (also called Asn297 or N297) is the residue at position 297 of human antibody IgG1, which happens to be the position where the Fc region is glycosylated. Removal of glycosylation abolishes FcγRIIIa binding and eliminates ADCC activity.

[0052] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region alone or in the context of a full-length antibody or antibody fragment.

[0053] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide comprising the VL and VH domains of a single antibody, which, as will be appreciated by those skilled in the art, is made up of two domains, a variable heavy domain and a variable light domain.

[0054] By "single chain Fv" or "scFv" herein is meant a variable heavy domain covalently linked to a variable light domain to form an scFv or scFv domain, generally using an scFv linker as discussed herein. The scFv domain may be arranged in either N-terminal to C-terminal orientation (vh-linker-vl or vl-linker-vh). Generally, the linker is a scFv linker, as generally known in the art and as discussed above.

[0055] As used herein, "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different aligned IgG isotype. For example, an F296Y substitution in IgG2 is considered an IgG subclass modification, since IgG1 contains a tyrosine at EU position 296 and IgG2 a phenylalanine. Similarly, an IgG4 molecule with S241P is considered an IgG subclass modification, since IgG1 has a proline at position 241 and IgG4 has a serine. Note that subclass modifications are considered amino acid substitutions herein.

[0056] As used herein, a "non-naturally occurring modification" refers to a non-isotypic amino acid modification. For example, the substitution N297A in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification, since no IgG contains an asparagine at position 297.

[0057] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0058] As used herein, "effector function" refers to a biochemical event that results 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.

[0059] As used herein, "parent polypeptide" refers to a starting polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a naturally occurring polypeptide or a variant or engineered version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence that encodes it. Thus, as used herein, "parent immunoglobulin" refers to an unmodified immunoglobulin polypeptide that is modified to generate a variant, and as used herein, "parent antibody" refers to an unmodified antibody that is modified to generate a variant antibody. Note that "parent antibody" includes known commercially available recombinantly produced antibodies, as outlined below.

[0060] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1) and optionally a portion of the hinge. In the case of an IgG, the Fc domain comprises 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 human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, this numbering according to the EU index as in Kabat. Thus, the "CH" domains in the context of an IgG are 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 per Kabat, and "CH3" refers to positions 341-447 according to the EU index as per Kabat. Thus, an "Fc domain" comprises a -CH2-CH3 domain, and optionally a hinge domain (hinge-CH2-CH3). In some embodiments, amino acid modifications are made to the Fc region to, for example, alter binding to one or more FcγR or FcRn receptors, as described in more detail below.

[0061] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that binds to the IgG antibody Fc region and are encoded by the FcγR genes. 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, fully incorporated by reference), as well as any undiscovered human FcγR or FcγR isoforms or allotypes. In some cases, as outlined herein, binding to one or more FcγR receptors is reduced or eliminated. For example, reducing binding to FcγRIIIa reduces ADCC, and in some cases, it is desirable to reduce binding to FcγRIIIa and FcγRIIb.

[0062] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to an IgG antibody Fc region and is at least partially encoded by the FcRn gene. FcRn can be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. As is known in the art, a functional FcRn protein comprises two polypeptides, often referred to as heavy and light chains. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of FcRn heavy chain and beta-2-microglobulin. As discussed herein, binding to the FcRn receptor is desirable, and in some cases, Fc variants may be introduced to increase binding to the FcRn receptor.

[0063] By "heavy constant region" herein is meant the CH1-hinge-CH2-CH3 portion of a human IgG antibody. A number of suitable heavy constant regions are shown in FIG.

[0064] By "light constant region" is meant the CL domain from kappa or lambda, the sequences of which are shown in FIG.

[0065] As used herein, "position" refers to a location in a sequence of a protein. Positions may be numbered sequentially or according to established formats, such as the EU index for antibody numbering.

[0066] As used herein, "target antigen" refers to a molecule that is specifically bound by the variable region of a given antibody. In this case, the target antigen of interest herein is the CD38 protein, typically human CD38, and optionally cynomolgus CD38, as defined below. Thus, an "anti-CD38 binding domain" is an antigen binding domain (ABD) in which the antigen is CD38.

[0067] As used herein, "target cell" means a cell that expresses a target antigen.

[0068] As used herein, a "variable domain" refers to a region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the Vκ (V.kappa), Vλ (V.lambda), and / or VH genes that constitute the kappa, lambda, and heavy chain immunoglobulin loci, respectively. Thus, a "variable heavy domain" comprises (VH)FR1-vhCDR1-(VH)FR2-vhCDR2-(VH)FR3-vhCDR3-(VH)FR4, and a "variable light domain" comprises (VL)FR1-vlCDR1-(VL)FR2-vlCDR2-(VL)FR3-vlCDR3-(VL)FR4.

[0069] By "wild type or WT" herein is meant an amino acid or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0070] The antibodies of the present invention are generally isolated or recombinant. "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. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. "Recombinant" means that the antibody is produced using recombinant nucleic acid technology in an exogenous host cell.

[0071] "Specific binding" or "specifically binds" or "specific" for a particular antigen or epitope means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally 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.

[0072] As used herein, the term "Kassoc" or "Ka" 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 The term "dissociation constant" refers to the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). D Values ​​can be determined using methods well established in the art. In some embodiments, the K D A method for determining the K of an antibody is by using surface plasmon resonance, for example, by using a biosensor system such as a BIACORE® system. In some embodiments, the K of an antibody D is determined by biolayer interferometry. In some embodiments, K D is measured using flow cytometry with antigen-expressing cells. D In another embodiment, the K value is measured using immobilized antigen. D The K value is measured with an immobilized antibody (e.g., a parent mouse antibody, a chimeric antibody, or a humanized antibody variant). D The K value is measured in bivalent binding mode. D Values ​​are measured in monovalent binding mode. Specific binding to a particular antigen or epitope is, for example, 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 K against an antigen or epitope of M DTypically, an antibody that specifically binds to an antigen has a K that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or greater for a control molecule compared to the antigen or epitope. D has.

[0073] "Percent (%) amino acid sequence identity" with respect to protein sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular (parent) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the algorithm, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. One specific program is the ALIGN-2 program, as outlined in paragraphs

[0279] to

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

[0074] An example of the implementation of this algorithm to determine the percent identity of sequences is given by the Genetics Computer Group (Madison, WI) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, WI). In the context of the present invention, another method for establishing percent identity is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this set of packages, the Smith-Waterman algorithm can be used, where default parameters are used in the score table (e.g., gap open penalty of 12, gap extension penalty of 1, and gap of 6). From the resulting data, the "Match" value reflects "sequence identity". Other suitable programs for calculating the percent identity or similarity between sequences are commonly known in the art, for example, another algorithm program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; discrimination=HIGH SCORE;database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http: / / in front of blast.ncbi.nlm.nih.gov / Blast.cgi.

[0075] The degree of identity between an amino acid sequence of the present invention ("sequence of the present invention") and a parent amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "sequence of the present invention" or the length of the parent sequence, whichever is the shortest. The result is expressed as a percentage identity.

[0076] The terms "treatment", "treating", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents or reduces the disease or its symptoms, and / or it may be therapeutic in that it partially or completely cures the disease and / or side effects caused by the disease. "Treatment", as used herein, covers any treatment of disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who has not yet been diagnosed with the disease but who may be susceptible; (b) inhibiting the disease, i.e., arresting its onset or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to impart a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of generating an immune response or conferring immunity in the absence of a disease state, for example in the case of a vaccine.

[0077] An "effective amount" or "therapeutically effective amount" of a composition includes an amount of the composition that is sufficient to impart 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.

[0078] The term "nucleic acid" includes RNA or DNA molecules having more than one nucleotide in any form, including single-stranded, double-stranded, oligonucleotides or polynucleotides. The term "nucleotide sequence" includes the ordering of nucleotides in an oligonucleotide or polynucleotide in the single-stranded form of a nucleic acid.

[0079] The term "promoter," as used herein, includes a DNA sequence operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. Promoters are generally positioned upstream of the nucleic acid sequence to be transcribed and provide sites for specific binding by RNA polymerase and other transcription factors.

[0080] A "vector" is capable of introducing a gene sequence into a target cell. Typically, "vector construct", "expression vector" and "gene transfer vector" refer to any nucleic acid construct capable of inducing expression of a gene of interest, which can introduce a gene sequence into a target cell, and can be achieved by genomic integration of all or part of the vector, or by transient or genetic maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning and expression vehicles, as well as integrating vectors.

[0081] As used herein, the term "regulatory element" includes nucleotide sequences that control some aspects of the expression of a nucleic acid sequence. Examples of regulatory elements include, illustratively, enhancers, internal ribosome entry sites (IRES), introns, origins of replication, polyadenylation signals (pA), promoters, enhancers, transcription termination sequences, and upstream regulatory domains, which contribute to the replication, transcription, and / or post-translational processing of nucleic acid sequences. In some cases, regulatory elements may also include cis-regulatory DNA elements and transposition elements (TEs). Those skilled in the art can select these and other regulatory elements for use in expression constructs using only routine experimentation. Expression constructs can be made synthetically using recombinant approaches or well-known methods.

[0082] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including the replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, rate, or specificity of the process and can 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 initiating transcription of a coding region that is usually located downstream (3' direction) of the promoter.

[0083] C. CD38 Protein and Anti-CD38 Binding Domains 1.CD38 Protein The present invention provides anti-CD38 binding domains that specifically bind to human and cynomolgus CD38 proteins, the amino acid sequences of which are shown in RefSeq accession identifiers NP_001766.2 (SEQ ID NO: 28) and NP_001274206.1 (SEQ ID NO: 30), respectively, and the ECD sequences (SEQ ID NO: 29 and SEQ ID NO: 31, respectively) are shown in FIG.

[0084] Thus, as used herein, the term "CD38" or "CD38 protein" or "CD38 polypeptide" can include any such protein, variant, conjugate, or fragment thereof, including, but not limited to, the known or wild-type CD38 described herein, as well as any naturally occurring splice variants, amino acid variants, or isoforms, particularly 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.

[0085] 2. Anti-CD38 Binding Domain The present invention provides a number of anti-CD38 binding domains in different formats or orientations. The specific CDRs of the anti-CD38 binding domains are described below. As mentioned above, the exact numbering and arrangement of the CDRs may vary between different numbering systems. However, it should be understood that the disclosure of the heavy variable region and / or the light variable region includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each heavy variable region is the disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2 and vhCDR3), and the disclosure of each light variable region is the disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3).

[0086] The present disclosure provides novel anti-CD38 binding domains. Such antigen binding domains can bind to human and cynomolgus CD38 proteins. Figures 6A-6G show the amino acid sequences of four different anti-CD38 ABDs that can bind to both human and cynomolgus CD38. In some embodiments, the heavy and light chain variable regions are arranged in a Fab format, which is optionally included in a full-length antibody, as discussed below. In some embodiments, the heavy and light chain variable regions are fused together to form a scFv, as generally outlined herein.

[0087] Also included herein are anti-CD38 ABDs with amino acid modifications in one or more of the CDRs and / or framework regions.

[0088] As outlined herein, in some embodiments the set of six CDRs may have 0, 1, 2, 3, 4, 5 or 6 amino acid modifications (amino acid substitutions find particular use). That is, the CDRs may have amino acid modifications in the set of CDRs (e.g., 1, 2, 3, 4, 5 or 6 amino acid mutations (i.e., the CDRs may be modified as long as the total number of changes in the set of 6 CDRs is less than 6 amino acid modifications, and any combination of CDRs may be altered, e.g., there may be one change in vlCDR1, two changes in vhCDR2, no change in vhCDR3, etc.). In some embodiments, amino acid substitutions in vhCDR3 are less preferred or avoided. In some cases, binding affinity to either or both human and cynomolgus CD38 may be increased, while in other embodiments, binding affinity may be decreased. In these embodiments, binding to human and cynomolgus CD38 is retained. A suitable assay to test whether an anti-CD38 antigen binding domain containing modifications compared to the VH and VL sequences outlined herein is known in the art, such as a Biacore assay or the binding assays outlined in Examples 1, 2 or 3.

[0089] In some embodiments, the anti-CD38 ABDs outlined herein may also have amino acid modifications in either or both of the variable heavy and variable light framework regions (again, amino acid substitutions have found particular use), so long as the frameworks (excluding the CDRs) retain at least about 80, 85, or 90% identity to the human germline sequences. Thus, for example, the same CDRs described herein can be combined with different framework sequences from human germline sequences, so long as the framework regions retain at least about 80, 85, or 90% identity to the human germline sequences.

[0090] In other embodiments, the present invention further provides an anti-CD38 binding domain comprising a variant of the heavy and light chain variable regions described above. The heavy 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 "VH" sequences herein and / or may 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" sequences herein and / or may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid changes. In these embodiments, the invention includes these variants, so long as the antigen binding domain binds to human and cynomolgus CD38. Suitable assays to test whether anti-CD38 antigen binding domains containing mutations are known in the art compared to the VH and VL sequences outlined herein, such as Biacore assays or binding assays outlined in Examples 1, 2, or 3.

[0091] In some embodiments, the anti-CD38 binding domain is CD38TM1 and comprises a heavy chain variable region having an amino acid sequence 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 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:13.

[0092] In some embodiments, the anti-CD38 binding domain is CD38TM1 and comprises 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 such six CDRs have one, two, three, four or five amino acid modifications. In further embodiments, any single CDR comprises no more than one or two amino acid substitutions and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0093] 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.

[0094] 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.

[0095] 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 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO:13.

[0096] In some embodiments, the anti-CD38 binding domain is CD38TM1 and has a VH having SEQ ID NO:9 and a VL having SEQ ID NO:13.

[0097] In some embodiments, the anti-CD38 binding domain is CD38TM2 and comprises a heavy chain variable region having an amino acid sequence 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 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:21.

[0098] In some embodiments, the anti-CD38 binding domain is CD38TM2 and comprises 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 such six CDRs have one, two, three, four or five amino acid modifications. In further embodiments, a single CDR comprises no more than one or two amino acid substitutions and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0099] 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.

[0100] 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.

[0101] 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 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO:21.

[0102] In some embodiments, the anti-CD38 binding domain is CD38TM2 and has a VH having SEQ ID NO:17 and a VL having SEQ ID NO:21.

[0103] In some embodiments, the anti-CD38 binding domain is CD38TM3 and comprises a heavy chain variable region having an amino acid sequence 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 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:5.

[0104] In some embodiments, the anti-CD38 binding domain is CD38TM3 and comprises 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 such six CDRs have one, two, three, four or five amino acid modifications. In further embodiments, a single CDR comprises one or two amino acid substitutions and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0105] 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.

[0106] 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.

[0107] 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 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO:5.

[0108] In some embodiments, the anti-CD38 binding domain is CD38TM3 and has a VH having SEQ ID NO:1 and a VL having SEQ ID NO:5.

[0109] In some embodiments, the anti-CD38 binding domain is CD38TM4 in the present disclosure and comprises a heavy chain variable region having an amino acid sequence 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 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:25.

[0110] In some embodiments, the anti-CD38 binding domain is CD38TM4 and comprises 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 such six CDRs have one, two, three, four or five amino acid modifications. In further embodiments, a single CDR comprises one or two amino acid substitutions and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0111] 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.

[0112] 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.

[0113] 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 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO:25.

[0114] In some embodiments, the anti-CD38 binding domain is CD38TM4 and has a VH having SEQ ID NO:1 and a VL having SEQ ID NO:25.

[0115] In some embodiments, the anti-CD38 binding domain is CD38TM5 in the present disclosure and comprises a heavy chain variable region having an amino acid sequence 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 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:57.

[0116] In some embodiments, the anti-CD38 binding domain is CD38TM5 and comprises 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 such six CDRs have one, two, three, four or five amino acid modifications. In further embodiments, a single CDR comprises one or two amino acid substitutions and the modified anti-CD38 antigen binding domain retains binding to human and / or cynomolgus CD38.

[0117] 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.

[0118] 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.

[0119] 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 and a VL domain with no more than 1, 2, 3, 4 or 5 amino acid changes in SEQ ID NO:57.

[0120] In some embodiments, the anti-CD38 binding domain is CD38TM5 and has a VH having SEQ ID NO:53 and a VL having SEQ ID NO:57.

[0121] In addition to the sequence variants described herein in the heavy and light chain variable regions and / or CDRs for each anti-CD38 binding domain, changes may be made in the framework region(s) of the heavy and / or light variable region(s). In some embodiments, variations are made in the framework regions that retain at least 80, 85, 90 or 95% identity to the framework region sequences set forth in Table 1, while leaving the six CDRs unchanged and retaining binding to human and / or cynomolgus CD38.

[0122] In some embodiments, variations are made in both the framework regions and the six CDRs while retaining the 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 in the set of six CDRs, i.e., the CDRs may be modified, as long as the total number of changes in the set of six CDRs is less than six amino acid modifications, and any combination of CDRs may be changed, e.g., there may be one change in vlCDR1, two changes in vhCDR2, none in vhCDR3, etc.).

[0123] The epitopes of 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 to 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 verified in a simple competitive immunoassay that shows the ability of one antigen binding domain (or an antibody that contains such an antigen binding domain) to block the binding of another antigen binding domain (or an antibody that contains such an antigen binding domain) to the target antigen CD38, such as "binning". Competitive binding tests can be generally performed using SPR / Biacore® binding assays as well as ELISA and cell-based assays, as known in the art.

[0124] D. Characterization of the Anti-CD38 Binding Domain The present invention provides a novel anti-CD38 binding domain.The anti-CD38 binding domain specifically binds to the ECD of human and cynomolgus CD38, preferably human and cynomolgus CD38.Binding to both human and cynomolgus CD38 is useful because cynomolgus animals can be used as a model of human subjects to help determine, predict and understand the in vivo effects and behavior of molecules that target CD38, for example, in terms of pharmacokinetics, pharmacodynamics and toxicology, where said model can have target binding and target cell killing by molecules that can be translated to related species.

[0125] In some embodiments, the anti-CD38 binding domains described herein bind to human and cynomolgus CD38 with high affinity. D The K value can be measured in either the antigen-fixed or antibody-fixed state. D Values ​​can also be measured in monovalent or bivalent binding formats. For example, the K of an antigen-binding domain that binds to human CD38 when formatted as IgG1 and measured by flow cytometry is DThe value is 1 x 10 -6 M or less, 5×10 -7 M or less, 2.5×10 -7 M or less, 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 1×10 -9 M or less or 1×10 -10 The K of the antigen-binding domain that binds to cynomolgus monkey CD38 can be equal to or less than M. D The value is 1 x 10 -6 M or less, 5×10 -7 M or less, 2.5×10 -7 M or less, 1×10 -7 M or less, 5×10 -8 M or less, 2.5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less or 1×10 -10 In some embodiments, the K of the antigen-binding domain that binds to human CD38 may be equal to or less than 1 M. D The value is in the range of 0.1 nM-1 μM, 0.25 nM-500 nM, 0.5 nM-250 nM, 1 nM-100 nM, or 1.5 nM-50 nM. In some embodiments, the K D Values ​​are in the range of 0.1 nM-1 μM, 0.25 nM-500 nM, 0.5 nM-250 nM, 1 nM-100 nM or 2 nM-50 nM.

[0126] Furthermore, 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 epitopes on CD38 that do not overlap with daratumumab. In some embodiments, the anti-CD38 binding domains described herein bind to epitopes on CD38 that overlap with daratumumab. In some embodiments, the anti-CD38 binding domains described herein can bind to human and / or cynomolgus CD38 in the presence of daratumumab.

[0127] In some embodiments, one or more of the anti-CD38 binding domains disclosed herein are comprised in compositions, e.g., antibodies, that can be used to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in cells with elevated expression of CD38, e.g., multiple myeloma cells.

[0128] E. Composition of Anti-CD38 Binding Domains As outlined herein, the anti-CD38 binding domains of the invention can be used in different formats, for example as scFv or as Fab contained in a conventional tetrameric antibody.

[0129] 1. scFv containing anti-CD38 binding domain In some embodiments, the composition comprises an scFv comprising an anti-CD38 binding domain as described herein. The scFv binds human and cynomolgus CD38 and comprises a heavy chain variable region (VH) and a light chain variable region (VL) linked by an scFv linker. The VL and VH can be in either the "VH-scFv linker-VL" or "VL-scFv linker-VH" orientation, e.g., from N-terminus to C-terminus.

[0130] While any suitable linker can be used, many embodiments utilize a glycine-serine polymer, for example, comprising the 15 residue (Gly4Ser)3 peptide (SEQ ID NO: 32). Suitable scFv linkers that may 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: 36), GGSGGGG (SEQ ID NO: 37), and GSTSGGGSGGGSGGGGSS (SEQ ID NO: 38), or any peptide sequence that allows recombinant combination of heavy and light chain variable regions with sufficient length and flexibility to allow each domain to retain its biological function.

[0131] Linkers of variable length can be used in the present invention. In some embodiments, linkers of 1 to 50 amino acids are used. In some embodiments, linkers of 3 to 12 amino acids are used, and the resulting scFv monomers tend to form multimers or oligomers (e.g., diabodies, triabodies, and tetrabodies) due to self-association, with the majority of forms being dimeric. In some embodiments, linkers of 5 amino acids are used, such as GGGGS (SEQ ID NO: 35), and diabodies can be formed from ABD. In some embodiments, linkers of more than 12 amino acids (e.g., 13, 14, 15, 16, 17, 18, 19, or 20) are used, and the resulting scFvs form primarily monomers, with only a small fraction undergoing spontaneous multimerization. In some embodiments, linkers of 1 to 3 amino acids are used, and these promote multimerization of scFvs into higher order structures beyond the dimeric form, such as trimers and / or mixtures of trimers and tetramers.

[0132] The multimerization of scFv is also included in the present invention. This can be controlled by molecular engineering strategies, either covalent or non-covalent, for example, covalent strategies including single-chain tandem arrangements, covalent strategies including cysteine-mediated disulfide bond stabilized multimers, and / or non-covalent strategies including 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 generate scFv multimers (see, for example, Lu D et al., J Immunol Methods 279:219-32 (2003)).

[0133] In some embodiments, the present invention provides an ABD comprising variable heavy and variable light domains, including VH and VL domains selected from the group consisting of CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5 and CD38TM6. In some embodiments, the ABD is in the format of a scFv. The VH and VL domains are linked by a 5 amino acid scFv linker, e.g., 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 from such ABDs. A diagram of the X-ray structure of an exemplary scFv is shown in FIG. 12. This scFv comprises VH and VL from CD38TM4 and the scFv linker GGGGS (SEQ ID NO: 35) to form a diabody structure.

[0134] 2. Antibodies containing anti-CD38 binding domains In some embodiments, the compositions of the invention are conventional tetrameric antibodies comprising the variable heavy and variable light domains of the invention forming an ABD, including CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5 and CD38TM6. Typically, these VH and VL pairs are attached to the heavy chain constant domains (CH1-hinge-CH2-CH3) of human and mutant human IgG1, IgG2 and IgG4, the sequences of which are shown in FIG. 10, and the light chain constant domains (CL) of lambda or kappa are also shown in FIG. 10. In some embodiments, the invention provides compositions comprising Fabs comprising the anti-CD38 binding domains described herein. The C-terminus of the heavy chain variable region (including vhCDR1, vhCDR2 and vhCDR3) is bound to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of the light chain variable region (including vlCDR1, vlCDR2 and vlCDR3) is bound 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 well as variants of the CH1 domain and light chain constant domains described herein.

[0135] Suitable heavy chain constant domains include, but are not limited to, those shown in FIG.

[0136] In some embodiments, the antibody comprises a heavy chain having a VH and VL of CD38TM1 (VH-CH1-hinge-CH2-CH3). In these embodiments, the constant heavy domains can be derived from human and mutant IgG1, IgG2 and IgG4, as shown in FIG.

[0137] In some embodiments, the VH (SEQ ID NO: 9) is made up of a heavy constant region selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO: 13) is made up of a light constant domain selected from SEQ ID NOs: 51 and 52.

[0138] In some embodiments, the antibody comprises a heavy chain having a VH and VL of CD38TM2 (VH-CH1-hinge-CH2-CH3). In these embodiments, the constant heavy domains can be derived from human and mutant IgG1, IgG2 and IgG4, as shown in FIG.

[0139] In some embodiments, the VH (SEQ ID NO: 17) is made up of a heavy constant region selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO: 21) is made up of a constant light domain selected from SEQ ID NOs: 51 and 52.

[0140] In some embodiments, the antibody comprises a heavy chain having a VH and VL of CD38TM3 (VH-CH1-hinge-CH2-CH3). In these embodiments, the constant heavy domains may be derived from human and mutant IgG1, IgG2 and IgG4, as shown in FIG.

[0141] In some embodiments, the VH (SEQ ID NO: 1) is made up of a heavy constant region selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO: 5) is made up of a constant light domain selected from SEQ ID NOs: 51 and 52.

[0142] In some embodiments, the antibody comprises a heavy chain having a VH and VL of CD38TM4 (VH-CH1-hinge-CH2-CH3). In these embodiments, the constant heavy domains may be derived from human and mutant IgG1, IgG2 and IgG4, as shown in FIG.

[0143] In some embodiments, the VH (SEQ ID NO: 1) is made up of a heavy 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 the VL (SEQ ID NO: 25) is made up of a constant light domain selected from SEQ ID NO: 51 and 52.

[0144] In some embodiments, the antibody comprises a heavy chain having a VH and VL of CD38TM5 (VH-CH1-hinge-CH2-CH3). In these embodiments, the constant heavy domains may be derived from human and mutant IgG1, IgG2 and IgG4, as shown in FIG.

[0145] In some embodiments, the VH (SEQ ID NO:53) is made up of a heavy constant region selected from the group consisting of SEQ ID NOs:39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO:57) is made up of a light constant domain selected from SEQ ID NOs:51 and 52.

[0146] Further substitutions may be introduced into the Fc region of the antibody as outlined below.

[0147] 3. Inclusion in a fusion protein In some embodiments, the anti-CD38 binding domains of the invention may be comprised in a fusion protein as generally described in U.S. Patent 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, e.g., as shown in FIG. 9.

[0148] 4.Antibody engineering Anti-CD38 binding domains and compositions comprising such anti-CD38 binding domains can be modified or engineered to change the amino acid sequence by amino acid substitutions. As discussed herein, amino acid substitutions can be made to change the affinity of the CDRs to the protein (e.g., human and / or cynomolgus CD38, including both increased and decreased binding), as well as to change further functional properties of the antigen binding domain and the antibody.

[0149] Furthermore, in some embodiments, the Fc region of the antibody may typically be engineered to include modifications to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fcγ receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, the antigen-binding domains and antibodies according to at least some embodiments of the present invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antigen-binding domains and antibodies), or modified to change their glycosylation, or to alter one or more functional properties of the antigen-binding domains and antibodies. Such embodiments are further described below. The numbering of the residues in the Fc region is that of the EU index of Kabat.

[0150] In one embodiment, the hinge region is modified such that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased.This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al.The number of cysteine ​​residues in the hinge region of CH1 is changed, for example, to facilitate assembly of light and heavy chains or to increase or decrease the stability of the antibody.

[0151] In yet other embodiments, antibodies can be modified to abolish in vivo Fab arm exchange, especially when IgG4 constant domains are used. Specifically, this process involves the exchange of IgG4 half molecules (one heavy chain + one light chain) between other IgG4 antibodies, effectively resulting in a bispecific antibody that is functionally monovalent. Mutations to the hinge region and constant domain of the heavy chain can abolish this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19). As outlined herein, a mutation that finds particular use in the present invention is S241P (Kabat numbering, S228P EU numbering) in the context of the IgG4 constant domain. IgG4 finds use in the present invention because it has no significant effector function and is therefore used to block receptor binding to its ligand without cell depletion.

[0152] In some embodiments, amino acid substitutions can be made in the Fc region to generally alter binding to FcγR receptors. There are many useful Fc substitutions that can be made to alter binding to one or more FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, increased binding to FcγRIIIa generally results in increased ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells. Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can also be beneficial in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Patent Application Publication No. 11 / 124,620 (particularly FIG. 41) and U.S. Patent No. 6,737,056, both of which are expressly incorporated herein by reference in their entirety, particularly for the variations disclosed therein.

[0153] In yet other examples, the Fc region is modified by modifying one or more amino acids at the following positions 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: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 277, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, No. 8,280,283,285,286,289,290,292,293,294,295,296,298,301,303,305,307,309,312,315,320,322,324,326,327,329,330,331,333,334,335,337,338,340,360,373,376,378,382,388,389,398,414,416,419,430,434,435,437,438 or 439. This approach is described further in PCT International Application Publication No. 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and mutants with improved binding have been described (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 FcyRIII. Additionally, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A. Furthermore, mutations such as M252Y / S254T / T256E or M428L / N434S improve binding to FcRn and increase antibody circulating half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

[0154] Furthermore, the antibody of the present invention is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US Patent No. 6,277,375 by Ward. Alternatively, to increase biological half-life, the antibody can be altered in the CH1 or CL region to include a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in US Patent Nos. 5,869,046 and 6,121,022 by Presta et al. Further mutations to increase serum half-life are disclosed in US Patent Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A, 434S and 428L / 434S.

[0155] In yet other embodiments, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, 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 accomplished, for example, by modifying one or more sites of glycosylation within the antibody sequence, for example, N297. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site, and alanine substitutions have found use in some embodiments.

[0156] Additionally or alternatively, antibodies can be made with altered types of glycosylation, for example, hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies according to at least some embodiments of the invention, thereby producing antibodies with altered glycosylation. See, for example, US Patent Publication No. 20040110704 and WO 2003 / 035835.

[0157] Another modification of the antibodies herein that is contemplated by the invention is PEGylation or the addition of other water-soluble moieties, typically polymers, e.g., to increase half-life. Antibodies can be PEGylated, as known in the art, to, e.g., increase the biological (e.g., serum) half-life of the antibody.

[0158] In addition to substitutions made to alter binding affinity to FcγR and / or FcRn and / or increase serum half-life in vivo, further antibody modifications can be made, as described in more detail below.

[0159] In some cases, affinity maturation is performed. Amino acid modification of CDRs may also be referred to as "affinity maturation". An "affinity matured" antigen binding domain or antibody is one that has one or more change(s) in one or more CDRs that results in an improved affinity of the antigen binding domain or antibody for the antigen compared to the parent that does not have those change(s). In some cases, it may be desirable to reduce the affinity of the antibody for the antigen. Affinity maturation can be performed to increase the binding affinity of the antigen binding domain or antibody for the antigen by at least about 10% to 50-100-150% or more, or 1 to 5 fold, compared to the "parent" antibody. Preferred affinity matured antigen binding domains or antibodies will have nanomolar or even picomolar affinities for the antigen. Affinity matured antibodies are produced by known procedures.

[0160] In some embodiments, one or more amino acid modifications are made in one or more of the CDRs of the CD38 binding domain of the present invention. Generally, only 1, or 2, or 3 amino acids are substituted in any single CDR, and generally, within a set of 6 CDRs (e.g., vhCDR1-3 and vlCDR1-3), no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 changes are made. However, it should be understood that in any CDR, any combination of none, 1, 2, or 3 substitutions can be made independently and optionally combined with any other substitutions.

[0161] Alternatively, amino acid modifications in one or more of the CDRs of the CD38 binding domain of the invention can be made that are "silent", e.g., do not significantly alter the affinity of the antigen binding domain or antibody for the antigen. These can be made for a number of reasons, including to optimize expression (as can be made for nucleic acids encoding antigen binding domains or antibodies of the invention).

[0162] F. Nucleic Acid Encoding Antibodies Also provided are nucleic acid compositions encoding anti-CD38 binding domains and compositions comprising such antigen binding domains, as well as expression vectors comprising the nucleic acids, and host cells transformed with the nucleic acid and / or expression vector compositions. As will be appreciated by one of skill in the art, the protein sequences provided herein can be encoded by any number of possible nucleic acid sequences due to the degeneracy of the genetic code.

[0163] The nucleic acid composition encoding the composition comprising the anti-CD38 binding domain depends on the format of the composition. A conventional tetrameric antibody comprising two heavy chains and two light chains is encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be placed in a single expression vector or two expression vectors transformed into a host cell, as known in the art, and expressed to form the antibody of the present invention. In some embodiments, for example when an scFv construct is used, a single nucleic acid encoding the heavy chain variable region-linker-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 transcriptional and translational control sequences, including but not limited to signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.

[0164] Preferred mammalian host cells for expressing recombinant antibodies according to at least some embodiments of the present invention include Chinese Hamster Ovary (CHO cells), PER.C 6, HEK293 and others known in the art.

[0165] To generate an scFv gene, the DNA fragments encoding the VH and VL are operably linked to other fragments encoding a flexible linker, e.g., encoding the amino acid sequence GGGGS and others discussed herein, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions being linked by the flexible linker.

[0166] G. Preparations The therapeutic compositions used to practice the aforementioned methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the therapeutic function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers, such as sterile phosphate buffered saline, bacteriostatic water, etc. (generally, see Remington's Pharmaceutical Sciences 16). th (See, eg, A. Osal., Ed., 1980.) Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers.

[0167] Pharmaceutical compositions comprising an anti-CD38 binding domain or antibody of the invention may be formulated for any suitable route and means of administration, including but not limited to intravenous infusion.

[0168] For intravenous infusion, in some embodiments, pharmaceutical compositions comprising anti-CD38 binding domains or antibodies of the invention are formulated in an aqueous buffer containing a cryoprotectant and a surfactant. In some embodiments, pharmaceutical compositions comprising anti-CD38 binding domains or antibodies of the invention are 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, pharmaceutical compositions comprising anti-CD38 binding domains or antibodies of the invention are formulated in 20 mM sodium citrate buffer (pH 5.0) containing 200 mM sucrose and 0.02% v / v polysorbate 80. Exemplary formulations are listed below: [Table A]

[0169] The formulations of the pharmaceutical compositions of the present 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 component.

[0170] The dosage and frequency of administration are selected in preferred embodiments to be therapeutically or prophylactically effective.As is known in the art, adjustments may be required for the rate of proteolysis, 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 symptoms, which can be ascertained by routine experimentation by those skilled in the art.

[0171] H. How to Use Antibodies The anti-CD38 binding domains of the invention and compositions comprising such anti-CD38 binding domains can be used in a number of diagnostic and therapeutic applications.

[0172] 1. Diagnostic Use The anti-CD38 binding domains of the invention and compositions comprising such anti-CD38 binding domains may find use in the in vitro or in vivo diagnosis of CD38-expressing cancers, including the imaging of tumours which overexpress CD38.

[0173] Generally, diagnosis can be performed in several ways. In one embodiment, tissue from a patient, such as a biopsy sample, is contacted with an anti-CD38 antibody, typically labeled, so that the antibody binds to endogenous CD38. The level of signal from the label is compared to that of normal non-cancerous tissue from either the same patient or a reference sample to determine the presence or absence of CD38-expressing cancer. 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.). Generally, in this embodiment, the anti-CD38 antibody is labeled with a fluorophore or other optical label, which is detected, for example, using a fluorometer or other optical detection system known in the art.

[0174] In other embodiments, a labeled secondary antibody is contacted with the sample using, for example, an anti-human IgG antibody from a different mammal (mouse, rat, rabbit, goat, etc.) to form a sandwich assay as known in the art. Alternatively, the anti-CD38 antibody can be directly labeled (i.e., biotin) and detection can be performed by a secondary antibody to a labeling agent as known in the art.

[0175] If overexpression of CD38 is found, treatment can proceed by administration of an anti-CD38 antigen binding domain or a composition comprising an anti-CD38 binding domain according to the invention, as outlined herein.

[0176] In other embodiments, in vivo diagnosis is performed. Generally, in this embodiment, anti-CD38 antibodies (including antibody fragments) are injected into a patient and imaging is performed. In this embodiment, for example, the antibody is generally labeled with an optical label or MRI label, such as a gadolinium chelate, a radioactive label of the mAb (including fragments).

[0177] Particularly useful antibodies for diagnostic use include, but are not limited to, the antibodies listed, or antibodies that utilize CDRs with variant sequences, or antibodies that compete for binding with any of the antibodies described herein.

[0178] In many embodiments, the diagnostic antibody is labeled. As used herein, "label" means that the antibody disclosed herein has one or more elements, isotopes or chemical compounds attached to it to allow detection in screening or diagnostic procedures. Generally, labels are divided into several classes: a) immunolabels, which can be epitopes incorporated as fusion partners recognized by antibodies; b) isotopic labels, which can be radioactive or heavy isotopes; c) small molecule labels, which can include molecules such as biotin that allow fluorescent and colorimetric dyes, or other labeling methods; and d) labels such as particles (including gas bubbles for ultrasound labeling) or paramagnetic labels that allow body imaging. Labels may be incorporated into the antibody at any position, and may be incorporated in vitro or in vivo during protein expression, as known in the art.

[0179] Diagnosis can be performed in vivo by administration of diagnostic antibodies that allow for whole body imaging as described below, or in vitro on a sample removed from the patient. A "sample" in this context includes any number of body fluids, including but not limited to blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat and semen, as well as tissue samples, such as results from a biopsy of a relevant tissue.

[0180] Furthermore, as outlined below, information regarding protein expression levels of CD38 can be used to determine which antibodies should be administered to a patient.

[0181] 2. Disease Treatment The anti-CD38 binding domains of the invention and compositions comprising such anti-CD38 binding domains find particular use in treating diseases, disorders and / or conditions that can be mediated, regulated or otherwise affected by CD38 overexpression in a subject or human patient in need thereof. Treatment involves administering to the subject or patient a therapeutically effective amount of a composition comprising an anti-CD38 binding domain of the invention. Among particular embodiments of the invention are compositions comprising an anti-CD38 binding domain of the invention for treating or preventing cancer or immune disorders associated with CD38 overexpression. In some embodiments, the disease, disorder or condition treated using this method of the invention is a hematopoietic cancer involving CD38-overexpressing cells, such as multiple myeloma, or more generally, a condition associated with loss of growth control in CD38-expressing cells. Apart from multiple myeloma, CD38 is also upregulated in many other hematopoietic malignancies, as well as in 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).

[0182] In some embodiments, the anti-CD38 binding domains of the invention and / or compositions comprising such anti-CD38 binding domains are used in combination with an existing anti-CD38 antibody, such as daratumumab, to treat cancer or immune disorders associated with CD38 overexpression, such as multiple myeloma. In some embodiments, the anti-CD38 binding domains of the 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 previously exposed to an anti-CD38 antibody, such as daratumumab. In some embodiments, the anti-CD38 binding domains of the 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 failed treatment with an existing anti-CD38 antibody, 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 CD38 overexpression, such as multiple myeloma, where the anti-CD38 binding domain binds to an epitope on CD38 that is different from the epitope bound by the existing anti-CD38 antibody.

[0183] V. Working Examples Having now generally described the invention, it will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0184] A. Example 1: Anti-CD38 antigen-binding clones bind to human and cynomolgus CD38 1. Anti-CD38 antigen-binding clones bind to cells expressing CD38 Anti-CD38 antigen-binding clones were formatted into human IgG1 and their binding to CD38 protein was assayed. Human multiple myeloma cells MOLP-8 expressing human CD38 and cells expressing recombinant cynomolgus monkey CD38 were incubated with anti-CD38 antibody or secondary antibody only control at a concentration of 100 nM. Antibody binding to CD38 was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were then analyzed by flow cytometry and the mean fluorescence intensity (MFI) of the anti-CD38 antibody was used to calculate the fold over background (FOB) compared to the secondary antibody only control. Anti-CD38 binding clones bind to both human and cynomolgus monkey CD38, and exemplary data from clones CD38TM3, CD38TM1 and CD38TM2, CD38TM5 and CD38TM6 are shown in Table 2. [Table 2]

[0185] 2. Anti-CD38 antigen-binding clones bind to purified CD38 The binding of anti-CD38 antigen-binding clones to purified human and cynomolgus CD38 molecules was also tested. Anti-CD38 antigen-binding clones were formatted into scFvs and then fused to Shiga toxin A subunits to generate molecules targeted to CD38. For each anti-CD38 scFv, the heavy chain variable region (VH) was fused to either the N-terminus or C-terminus of the light chain variable region (VL) via a GGGGS linker. Reference anti-CD38 binding clones fused to Shiga toxin A subunits (CD38 targeting reference molecule number 1-SLTA or CD38TR1-SLTA) were also included in the study, as well as a modified anti-CD38 binding clone CD38TM4 fused to Shiga toxin A subunits, which contains amino acid substitutions in the light chain framework region of CD38TM3, allowing purification by protein L chromatography. The amino acid sequences of the anti-CD38 / Shiga toxin fusion proteins tested are 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).

[0186] Briefly, Nunc maxisorp plates were coated with recombinant human or cynomolgus CD38 in PBS overnight at 4°C. Wells were washed, blocked, and then incubated with serial dilutions of CD38-targeting molecules. Well-bound CD38-targeting molecules were detected by a two-step method involving a mouse monoclonal antibody detecting a 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.

[0187] 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 format of ScFV fusion to Shiga toxin A subunit (SLTA) as shown in Figures 2A-3B, 3A-3B. CD38TM4 retains the CD38 binding capacity of CD38TM3 from which it is derived. The binding affinities of the anti-CD38 antigen-binding clones were calculated and are shown in Table 3. [Table 3]

[0188] B. Example 2: Affinity Determination of Anti-CD38 Binding Clones A cell binding affinity assay was performed to quantify the binding affinity of anti-CD38 binding clones to CD38-expressing cells. MOLP-8 cells expressing human CD38 were suspended in 1% FBS / PBS at a viable cell concentration of approximately 2 million cells / mL. Anti-CD38 binding clones formatted as IgG1 were serially diluted in PBS between wells on a 6-well plate (2-fold dilutions from 102 nM to 24.95 pM). The last well of each titration contained only PBS. Cell suspension and additional PBS were then added to each well so that each well contained a harvest volume of 300 μL and approximately 100,000 cells. The plate was placed on a plate shaker at 4° C. for 5 hours, after which the cells in each well were washed three times at 4° C. with PBS. Then, 200 μl of 99 nM Cy 5 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 twice again with PBS at 4° C. and then analyzed by a FACSCanto™ II HTS flow cytometer. For each well containing a unique antibody concentration, the mean fluorescence intensity (MFI) of 5000 events was measured and a plot of MFI as a function of antibody concentration was nonlinearly fitted using Scientist 3.0 software using the following equation to determine the antigen binding affinity, K D was estimated as: F = p [(K D +LT+n(M))-{(K D +LT+n(M))2-4n(M)(LT)}1 / 2] / 2+B where F is the MFI, LT is the total antibody binding site concentration, which is equal to twice the antibody molecule concentration, p is the proportionality constant relating any fluorescence unit to bound antibody, M is the cell concentration in molar terms, expressed as 0.553 fM based on 100,000 cells in 300 μl, n is the number of receptors per cell, B is the background signal, and K D is the equilibrium dissociation constant.

[0189] For each antibody titration curve, P, n, B and K Dwas allowed to vary freely in a nonlinear analysis to obtain an estimate of KD. For a detailed derivation of the above equation, see Drake and Klakamp (2007), "A rigorous multiple independent binding site model for determining cell-based equivalence dissociation constants," J. Immunol. Methods 318:157-62, which is incorporated herein by reference. Table 4 shows the KD measured for anti-CD38 antibody clones. D The antibody binding site concentration (2× molecule concentration) was used for nonlinear curve fitting. [Table 4]

[0190] C. Epitope mapping of anti-CD38 antigen-binding clones Epitope mapping was performed by Integral Molecular to identify contact residues on the CD38 extracellular domain (ECD) by anti-CD38 antigen-binding clones. Epitopes were mapped by shotgun mutagenesis, a standard assay setup using alanine scanning and additional mutations to change amino acids from human to cynomolgus residues as needed. Briefly, wild-type or mutant human CD38ECD was expressed on the cell surface of HEK293 cells, and then the ability of the anti-CD38 antigen-binding clone of interest to bind CD38ECD when specific amino acids were mutated was compared to the binding of the same anti-CD38 antigen-binding clone to wild-type CD38ECD by flow cytometry. The assay was controlled for mutations affecting the expression or general folding of the molecule by a series of control molecules that also target CD38. 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 (based on the published CD38 structure) important for binding and surface accessibility of anti-CD38 antigen-binding clones. [Table 5]

[0191] Anti-CD38 antigen-binding clones CD38TM1 and CD38TM2 share the same critical contact residues (R78 and D81) on human CD38. Critical residues of CD38TM5 on human CD38 include R78, H79 and D81 (data not shown). Critical residues of CD38TM3 on human CD38 include F216, L262, L124, C201, L230, C254 and S275. Critical residues of CD38TM4 on human CD38 include F216, L262, C201, L230, C254 and S275. FIG. 4 shows the locations of key residues in CD38TM3, including surface accessible contact residues F216, L262 shown in red, key structural residues L124, C230 shown in purple, and key structural residues C119 / C201 and LC254 / C275 involved in disulfide pairs shown in grey.

[0192] CD38TM1, CD38TM2 and CD38TM5 bind to non-overlapping epitope residues on human CD38 compared to daratumumab. CD38TM6 binds to epitope residues on human CD38 that overlap 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.

[0193] Clones CD38TM4 and / or CD38TM3 bind to epitopes on human CD38 that partially overlap with the reference molecule CD38TR1 and possibly daratumumab. Figure 4 shows the location of key residues of CD38TM4 on human CD38 (surface accessible residues F216, L262 shown in red; key structural residues L124, C201 shown in purple, and key structural residues involved in disulfide pairs C119 / C201 and LC254 / C275 shown in grey).

[0194] Binding of anti-CD38 antigen-binding clones to CD38 in the presence of pre-existing CD38 antibodies including Daratumumab, HB-7 (Santa Cruz, Catalog No.: sc-18858), AT 13-5 (Thermo Fisher, Catalog No.: MA 5-16578) and OKT-10 (Sigma, Catalog No.: 87021903) was tested. CD38TM2, CD38TM1 and CD38TM3 were formatted into scFvs as described above and fused to Shiga toxin A subunit. MOLP-8 cells were incubated with 20 μg / ml of pre-existing anti-CD38 antibodies (~130 nM) or control molecule (Shiga toxin A subunit alone) for 70 minutes. 0.5 μg / ml of anti-CD38 / Shiga toxin fusion molecule (~5 nM) was then added and incubated with the cells for 48 hours. Antibody binding to CD38 was detected using a primary antibody recognizing the Shiga toxin A subunit and a FITC-conjugated anti-IgG secondary antibody, followed by flow cytometry. The mean fluorescence intensity (MFI) of anti-CD38 antigen-binding clones was calculated after subtraction of the signal from the secondary antibody only control.

[0195] As shown in Figures 5A and 5B, CD38TM1 and CD38TM2 in the scFv-SLTA fusion protein format can bind to CD38 in the presence of daratumumab, HB-7, AT 13-5, and OKT-10, indicating that they bind to an epitope on CD38 that is different from the epitope(s) bound by daratumumab, HB-7, AT 13-5, and OKT-10 in the scFv-SLTA fusion protein format. CD38TM3 can bind to CD38 in the presence of OKT-10, but not in the presence of daratumumab, HB-7, and AT 13-5, indicating that CD38TM3 binds to an epitope on CD38 that 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 AT 13-5.

[0196] Competitive binding of anti-CD38 antigen-binding clones to CD38 was also tested. CD38TM2, CD38TM1 and CD38TM3 were formatted into scFvs as described above and fused to Shiga toxin A subunit. MOLP-8 cells were incubated with 20 μg / ml of anti-CD38 / Shiga toxin fusion molecule (approximately 400 nM) or control molecule (Shiga toxin A subunit alone) for 70 minutes. Then, 0.5 μg / ml of anti-CD38 in scFv-SLTA fusion protein format was added and incubated with the cells for 48 hours. CD38 binding was detected by a primary antibody recognizing Shiga toxin A subunit and a FITC-conjugated anti-IgG secondary antibody followed by flow cytometry. The mean fluorescence intensity (MFI) of anti-CD38 antigen-binding clones was calculated after subtracting the signal from the secondary antibody only control and plotted as a percentage of the MFI measured from Shiga toxin A subunit alone as a control. As shown in Figures 5C and 5D, CD38TM1 and CD38TM2 bind to CD38 in a competitive manner, consistent with the fact that they bind to the same epitope on CD38. CD38TM3 binds to CD38 semi-competitively with CD38TM1 and CD38TM2.

[0197] D. Induction of CDC and ADCC by anti-CD38 antibodies 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).

[0198] For the CDC assay, MOLP-8 cells were seeded at a density of 10,000 cells per well in 50 μL of complete medium (RPMI supplemented with 10% fetal bovine serum) in black 96-well flat-bottom tissue culture plates. 50 μL of anti-CD38 antibody (9 μg / ml), control IgG1 antibody (9 μg / ml), or medium alone was added to each well and incubated for 10 min at room temperature (RT). Various amounts (2-15 μL) of purified rabbit complement (catalog no. CL 3441 Cedarlane Laboratories, Canada) were added to each well except for the control wells. After 1 hour incubation at 37°C, plates were brought to room temperature, 100 μL of Cell Titer CytoTox Glo™ Reagent (Promega G7571 / G7573) was added to each well, plates were shaken for 5-7 minutes, and luminescence was read on an EnVision® (Perkin Elmer) luminescence plate reader. Conditions tested were: cells alone; cells + complement; cells + IgG1 control + complement; cells + antibody + complement. %CDC was calculated using the following formula: 100-(RLUT / RLUC) x 100), where RLUT is the relative luminescence units of the test sample and RLUc is the relative luminescence units of the sample containing complement alone. Assays were performed in triplicate and statistical analysis was performed using PRISM software.

[0199] For measuring ADCC, MOLP-8 cells were used as target cells. Peripheral blood mononuclear cells (PBMCs) were isolated as effector cells by Ficoll-Plaque™ separation from buffy coats or LRS obtained from Stanford Blood Center (Palo Alto, CA). Samples were diluted 1:3 with 2% FBS in PBS. 15 mL of Ficoll-Plaque™ (GE Healthcare) was gently layered under 35 mL of diluted sample and centrifuged at 1800 rpm (brake off) for 25 min. The turbid interface containing PBMCs was collected, washed three times with 2% FBS / PBS, and aliquoted at 50 × 10 per aliquot in 10% DMSO / FBS. 6Prior to use, frozen aliquots of PBMCs were thawed and resuspended in 2×10 PBS in 10% FBS / RPMI + 5 ng / mL recombinant human IL 2 (R&D systems #202-IL). 6 cells / mL and cultured overnight.

[0200] For the ADCC assay, all steps were performed in complete medium. 5000 target cells per well were seeded 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 4 hours of incubation at 37°C, the plate was centrifuged at 1100 rpm for 5 minutes, and 100 μL of the supernatant was transferred to a white plate. 100 μL of CytoTox Glo™ reagent (Promega catalog number G 9292) was added to the supernatant, and the plate was then shaken at room temperature for 20-30 minutes. Luminescence was read on an EnVision® (Perkin Elmer) luminescence plate reader and the percentage of specific lysis was calculated using the following formula: (RLUT / RLUE / T) / (RLUL / RLUE / T)×100, where RLUT is the relative luminescence units of the test sample, RLUE / T is the relative luminescence units of the sample containing target and effector cells alone, and RLUL is the relative luminescence units of cells lysed with Triton X-100. Assays were performed in triplicate and statistical analysis was performed using PRISM software.

[0201] The percentages of CDC and ADCC induced by the anti-CD38 antibody clones are shown in Table 6. All anti-CD38 antibody clones can induce CDC and ADCC against CD38-expressing cells. [Table 6]

[0202] E. Induction of CD38TM4 from CD38TM3 The VH and VL of CD38TM3 do not bind to standard monoclonal antibody purification resins (protein A or protein L) and were purified using His-tag and IMAC columns (Figure 11A shows the purification of CD38TM3 in scFv-SLTA fusion protein format as an example). To make purification easier, the light chain of CD38TM3, a lambda light chain, was modified by framework mutations to be able to bind to protein L, allowing affinity purification without additional tags. As shown in Figure 11C, the first 21 amino acids of the CD38TM3 VL domain were replaced with the first 22 amino acids of the VL domain of CD38TR1, a kappa light chain, to derive the VL domain of CD38TM4. The resulting CD38TM4 was able to bind to protein L and was therefore purified by protein L column (Figure 11B shows the purification of CD38TM4 in scFv-SLTA fusion protein format as an example). The present invention provides, for example, the following items. (Item 1) a) a variable heavy domain (VH) comprising: i) VHCDR1 comprising the sequence of SEQ ID NO: 2, ii) a VHCDR2 comprising the sequence of SEQ ID NO:3, and iii) a VH CDR3 comprising the sequence of SEQ ID NO: 4; b) a variable light domain (VL) comprising: i) VLCDR1 comprising the sequence SEQ ID NO:6, ii) a VLCDR2 comprising the sequence of SEQ ID NO: 7, and iii) VLCDR3 comprising the sequence of SEQ ID NO:8; 4. A composition comprising an anti-CD38 antigen binding domain comprising: (Item 2) The composition of item 1, wherein the VH comprises the sequence of SEQ ID NO: 1 and the VL comprises the sequence of SEQ ID NO: 25. (Item 3) The composition of item 1, wherein the VH comprises the sequence of SEQ ID NO: 1 and the VL comprises the sequence of SEQ ID NO: 5. (Item 4) a) a variable heavy domain (VH) comprising: i) VHCDR1 comprising the sequence of SEQ ID NO: 10, ii) a VHCDR2 comprising the sequence of SEQ ID NO: 11, and iii) a VH CDR3 comprising the sequence of SEQ ID NO: 12; b) a variable light domain (VL) comprising: i) VLCDR1 comprising the sequence SEQ ID NO: 14, ii) VLCDR2 comprising the sequence of SEQ ID NO: 15, and iii) VLCDR3 comprising the sequence of SEQ ID NO: 16; 2. A composition comprising an anti-CD38 antigen binding domain, comprising: (Item 5) 5. The composition of item 4, wherein the VH comprises the sequence of SEQ ID NO: 9 and the VL comprises the sequence of SEQ ID NO: 13. (Item 6) a) a variable heavy domain (VH) comprising: i) VHCDR1 comprising the sequence of SEQ ID NO: 18, ii) a VHCDR2 comprising the sequence of SEQ ID NO: 19, and iii) a VH CDR3 comprising the sequence of SEQ ID NO: 20; b) a variable light domain (VL) comprising: i) VLCDR1 comprising the sequence of SEQ ID NO: 22, ii) VLCDR2 comprising the sequence of SEQ ID NO: 23, and iii) VLCDR3 comprising the sequence of SEQ ID NO: 24; 2. A composition comprising an anti-CD38 antigen binding domain, comprising: (Item 7) 7. The composition of item 6, wherein the VH comprises the sequence of SEQ ID NO: 17 and the VL comprises the sequence of SEQ ID NO: 21. (Item 8) a) a variable heavy domain (VH) comprising: i) VHCDR1 comprising the sequence of SEQ ID NO: 54, ii) a VHCDR2 comprising the sequence of SEQ ID NO: 55, and iii) a VH CDR3 comprising the sequence of SEQ ID NO: 56; b) a variable light domain (VL) comprising: i) VLCDR1 comprising the sequence of SEQ ID NO: 58, ii) VLCDR2 comprising the sequence of SEQ ID NO: 59, and iii) VLCDR3 comprising the sequence of SEQ ID NO: 60; 2. A composition comprising an anti-CD38 antigen binding domain comprising: (Item 9) The composition of item 8, wherein the VH comprises the sequence of SEQ ID NO: 53 and the VL comprises the sequence of SEQ ID NO: 57. (Item 10) The composition of any one of the preceding claims, wherein the VH and the VL are in a single polypeptide. (Item 11) 11. The composition of claim 10, wherein the polypeptide comprises an scFv linker and the polypeptide has an N-terminus to C-terminus orientation of VH-scFv linker-VL. (Item 12) The composition of claim 10, wherein the polypeptide comprises an scFv linker and the polypeptide has an N-terminus to C-terminus orientation of VL-scFv linker-VH. (Item 13) 10. The composition according to any one of items 1 to 9, wherein the composition comprises a first polypeptide comprising the VH and a second polypeptide comprising the VL. (Item 14) The composition, a) a heavy chain comprising said VH; b) a light chain comprising said VL; 10. The composition according to any one of items 1 to 9, wherein the composition is an antibody comprising: (Item 15) 15. The composition according to item 14, wherein the heavy chain comprises the variable heavy domain and a heavy constant domain selected from the group consisting of heavy constant domains derived from human IgG1, IgG2 and IgG4 or variants thereof. (Item 16) 16. The composition of claim 15, wherein the heavy constant domain is the heavy constant domain of human IgG1. (Item 17) 17. The composition of claim 16, wherein the heavy constant domain is a variant of the heavy constant domain of human IgG1. (Item 18) 18. The composition of claim 17, wherein the mutant of the human IgG1 heavy constant domain is truncated for FcγR binding. (Item 19) 16. The composition of claim 15, wherein the heavy constant domain is a variant of the heavy constant domain of human IgG4 comprising an S228P amino acid substitution compared to the wild-type heavy constant domain of human IgG4. (Item 20) a) a first nucleic acid encoding a first polypeptide; b) a second nucleic acid encoding a second polypeptide; 16. A nucleic acid composition encoding the composition of item 15. (Item 21) 11. A nucleic acid composition comprising a nucleic acid encoding the composition of claim 10. (Item 22) 22. An expression vector composition comprising the nucleic acid according to item 21. (Item 23) 21. An expression vector composition comprising the first nucleic acid and the second nucleic acid according to item 20. (Item 24) a) a first expression vector comprising the first nucleic acid according to item 20; a) a second expression vector comprising the second nucleic acid according to item 20; 2. An expression vector composition comprising: (Item 25) 25. A host cell comprising the expression vector composition according to any one of items 22 to 24. (Item 26) 1. A method of making a composition comprising an anti-CD38 antigen binding domain, comprising: Culturing the host cell according to item 25 under conditions in which the composition comprising the anti-CD38 antigen binding domain is expressed; and Recovering said composition. The method comprising: (Item 27) A method for treating multiple myeloma, comprising administering an effective amount of the composition according to any one of items 1 to 19 to a subject in need of such treatment.

Claims

[Claim 1] The invention described in this specification.

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