WO2022271987A1 - Anti-cd38 antibodies and epitopes of same - Google Patents

Abstract

Binding compounds, such as multispecific antibodies binding to CD38, are disclosed, along with methods of making such binding compounds, compositions, including pharmaceutical compositions, comprising such binding compounds, and their various uses.

Description

ANTI-CD38 ANTIBODIES AND EPITOPES OF SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of the filing date of U.S. Provisional Patent Application Serial No. 63/214,261, filed on June 23, 2021, the disclosure of which is incorporated by reference herein in its entirety. This application also claims priority benefit of the filing date of U.S. Provisional Patent Application Serial No. 63/311,888, filed on February 18, 2022, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention concerns binding compounds, such multispecific antibodies binding to CD38. Aspects of the invention relate to anti-CD38 heavy chain antibodies, combinations, including synergistic combinations, of anti-CD38 heavy chain antibodies targeting different epitopes on CD38, multispecific anti-CD38 heavy chain antibodies that bind to more than one epitope on CD38, as well as methods of making such binding compounds, compositions, including pharmaceutical compositions, comprising such binding compounds, and their various uses.

BACKGROUND OF THE INVENTION

CD38 Ectoenzvme

[0003] The CD38 ectoenzyme is a membrane protein that has its catalytic site on the outside of the membrane in the extracellular compartment. This cell surface protein facilitates many functions and is found on a wide variety of cells, such as immune cells, endothelial cells, and neuronal tissue cells.

[0004] CD38, also known as ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1, is a single-pass type

II transmembrane protein with ectoenzymatic activities. Using NAD(P) as a substrate, it catalyzes the formation of several products: cyclic ADP-ribose (cADPR); ADP-ribose (ADPR); nicotinic acid adenine dinucleotide phosphate (NAADP); nicotinic acid (NA); ADP-ribose-2’-phosphate (ADPRP) (see, e.g. H. C. Lee, Mo/. Med., 2006, 12: 317-323). CD38 can also use Nicotinamide Mononucleotide (NMN) as a substrate and convert it to nicotinamide and R5P (Liu et ak, “Covalent and noncovalent intermediates of an NAD utilizing enzyme, human CD38.” Chem Biol 15(10): 1068-78.

[0005] CD38 is expressed predominantly on immune cells, including plasma cells, activated effector T cells, antigen-presenting cells, smooth muscle cells in the lung, Multiple Myeloma (MM) cells, B cell lymphoma, B cell leukemia cells, T cell lymphoma cells, breast cancer cells, myeloid derived suppressor cells, B regulatory cells, and T regulatory cells. CD38 on immune cells interacts with CD31/PECAM-1 expressed by endothelial cells and other cell lineages. This interaction promotes leukocyte proliferation, migration, T cell activation, and monocyte-derived DC maturation.

[0006] Antibodies binding to CD38 are described, for example, in Deckert et ak, Clin. Cancer Res., 2014, 20(17):4574-83 and US Patent Nos. 8,153,765; 8,263,746; 8,362,211; 8,926,969; 9,187,565; 9,193,799; 9,249,226; and 9,676,869.

[0007] Daratumumab, an antibody specific for human CD38, was approved for human use in 2015 for the treatment of Multiple Myeloma (reviewed in Shallis et ak, Cancer Immunol. Immunother. 2017, 66(6):697-703). Another antibody against CD38, Isatuximab (SAR650984), is in clinical trials for the treatment of Multiple Myeloma. (See, e.g., Deckert et ak, Clin Cencer Res, 2014, 20(17):4574-83; Martin et ak, Blood, 2015, 126:509; Martin et ak, Blood, 2017, 129:3294-3303). The epitope to which Isatuximab binds is decribed in the literature (e.g., Jutta Deckert, Marie-Cecile Wetzel, Laura M. Bartle, et ak, Clin Cancer Res 2014; 20:4574-4583, the disclosure of which is incorporated by reference herein in its entirety). These antibodies induce potent complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and indirect apoptosis of tumor cells. Isatuximab also blocks the cyclase and hydrolase enzymatic activities of CD38 and induces direct apoptosis of tumor cells.

[0008] Examples of allosteric modulation of proteins by antibodies are human growth hormone, integrins, and beta-glactosidase (L. P. Roguin & L. A. Retegui, 2003, Scand. J. Immunol. 58(4):387- 394). These examples show modulation of ligand-receptor interactions by single antibodies targeting different epitopes. One example of a bispecific antibody targeting two epitopes on a single molecule is against c-MET or hepatocyte growth factor receptor (HGFR) (DaSilva, J., Abstract 34: A MET x MET bispecific antibody that induces receptor degradation potently inhibits the growth of MET-addicted tumor xenografts. AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC).

Heavy Chain Antibodies

[0009] In a conventional IgG antibody, the association of the heavy chain and light chain is due in part to a hydrophobic interaction between the light chain constant region and the CHI constant domain of the heavy chain. There are additional residues in the heavy chain framework 2 (FR2) and framework 4 (FR4) regions that also contribute to this hydrophobic interaction between the heavy and light chains.

[0010] It is known, however, that sera of camelids (sub-order Tylopoda, which includes camels, dromedaries and llamas) contain a major type of antibodies composed solely of paired H-chains (heavy- chain only antibodies, heavy -chain antibodies, or UniAbs™). The UniAbs™ of Camelidae ( Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) have a unique structure consisting of a single variable domain (VHH), a hinge region and two constant domains (CH2 and CH3), which are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first domain of the constant region (CHI), which is present in the genome, but is spliced out during mRNA processing. The absence of the CHI domain explains the absence of the light chain in the UniAbs™, since this domain is the anchoring place for the constant domain of the light chain. Such UniAbs™ naturally evolved to confer antigen-binding specificity and high affinity by three CDRs from conventional antibodies, or fragments thereof (Muy ermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish, such as sharks, have also evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0011] The ability of heavy chain-only antibodies devoid of light chain to bind antigen was established in the 1960s (Jaton et al. (1968) Biochemistry , 7, 4185-4195). Heavy chain immuno globulin physically separated from light chain retained 80% of antigen-binding activity relative to the tetrameric antibody. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that removal of the CHI domain from a rearranged mouse m gene results in the production of a heavy chain-only antibody, devoid of light chain, in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.

[0012] Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBSLett. 414, 521-526 (1997)).

[0013] Mice in which the l (lambda) light (L) chain locus and/or the l and k (kappa) L chain loci have been functionally silenced, and antibodies produced by such mice, are described in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain-only antibodies in mice and rats has been reported, for example, in W02006008548; U.S. Application Publication No. 20100122358; Nguyen et al., 2003, Immunology, 109(1), 93-101; Briiggemann et al., Crit. Rev. Immunol., 2006, 26(5):377-90; and Zou et al., 2007 , J Exp Med, 204(13): 3271-3283. The production of knockout rats via embryo microinjections of zinc-finger nucleases is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain-only antibodies and transgenic rodents comprising a heterologous heavy chain locus producing such antibodies are described in U. S. Patent Nos. 8,883,150 and 9,365,655. CAR-T structures comprising single-domain antibodies as a binding (targeting) domain are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386.0 SUMMARY OF THE INVENTION

[0014] Aspects of the invention include multispecific antibodies that bind to two different epitopes on a CD38 protein, comprising: a first binding unit that binds to a first epitope on the CD38 protein; and a second binding unit that binds to a second epitope on the CD38 protein.

[0015] In some embodiments, the first binding unit comprises a heavy chain variable region paired with a light chain variable region. In some embodiments, the light chain variable region is a fixed light chain variable region. In some embodiments, the first binding unit comprises a heavy chain-only variable region. In some embodiments, the first binding unit lacks a light chain. In some embodiments, the heavy chain-only variable region is in a monovalent or bivalent configuration.

[0016] In some embodiments, the first binding unit competes for binding to the first epitope with an anti-CD38 heavy chain-only antibody comprising a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 11, and a CDR3 sequence of SEQ ID NO: 22.

[0017] In some embodiments, the first binding unit comprises a CDR3 sequence having at least 41% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 47% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 52% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 58% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 64% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 70% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 76% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 82% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 88% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having at least 94% identity to SEQ ID NO: 22. In some embodiments, the first binding unit comprises a CDR3 sequence having 100% identity to SEQ ID NO: 22.

[0018] In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 72% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 75% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 78% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 81% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 84% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 87% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 90% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 93% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 96% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22. In some embodiments, the first binding unit comprises a full set of CDRs 1, 2, and 3 having 100% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22

[0019] In some embodiments, the first binding unit comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 28-71. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising SEQ ID NO: 28. In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 88% identity to SEQ ID NO: 28. In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 95% identity to SEQ ID NO: 28. In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 96% identity to SEQ ID NO: 28. In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 97% identity to SEQ ID NO: 28. In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 98% identity to SEQ ID NO: 28. In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 99% identity to SEQ ID NO: 28.

[0020] In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising one or more amino acid residues selected from the group consisting of: S30, S31, Y32, R45, W47, D53, K58, Y59, Y60, A61, D62, K65, D99, R100, G101, T102, M103, R104, V105, V106, V107, Y108, D109, T110, LI 11, and W114. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues S30, S31 and Y32. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues K58, Y59, Y60, A61, and D62. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues D99, R100, G101, T102, Ml 03, R104, V105, V106, V107, Y108, D109, T110, and LI 11. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising one or more amino acid residues selected from the group consisting of: S31, D53, Y60, K65, D99, T102, M103, Y108, D109, T110, and LI 11. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues T102 and Ml 03. In some embodiments, the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues Y108, D109, T110, and LI 11.

[0021] In some embodiments, the second binding unit comprises a heavy chain variable region paired with a light chain variable region. In some embodiments, the light chain variable region is a fixed light chain variable region. In some embodiments, the second binding unit comprises a heavy chain-only variable region. In some embodiments, the second binding unit lacks a light chain. In some embodiments, the heavy chain-only variable region is in a monovalent or bivalent configuration.

[0022] In some embodiments, the second binding unit competes for binding to the second epitope with an anti-CD38 heavy chain-only antibody comprising a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 72, a CDR2 sequence of SEQ ID NO: 86, and a CDR3 sequence of SEQ ID NO: 94.

[0023] In some embodiments, the second binding unit comprises a CDR3 sequence having at least 45% identity to SEQ ID NO: 94. In some embodiments, the second binding unit comprises a CDR3 sequence having at least 54% identity to SEQ ID NO: 94. In some embodiments, the second binding unit comprises a CDR3 sequence having at least 63% identity to SEQ ID NO: 94. In some embodiments, the second binding unit comprises a CDR3 sequence having at least 72% identity to SEQ ID NO: 94. In some embodiments, the second binding unit comprises a CDR3 sequence having at least 81% identity to SEQ ID NO: 94. In some embodiments, the second binding unit comprises a CDR3 sequence having at least 90% identity to SEQ ID NO: 94. In some embodiments, the second binding unit comprises a CDR3 sequence having 100% identity to SEQ ID NO: 94.

[0024] In some embodiments, the second binding unit comprises a full set of CDRs 1, 2, and 3 having at least 74% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. In some embodiments, the second binding unit comprises a full set of CDRs 1, 2, and 3 having at least 77% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. In some embodiments, the second binding unit comprises a full set of CDRs 1, 2, and 3 having at least 81% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. In some embodiments, the second binding unit comprises a full set of CDRs 1, 2, and 3 having at least 85% identity to a full set of CDRs

1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. In some embodiments, the second binding unit comprises a full set of CDRs 1, 2, and 3 having at least 88% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. In some embodiments, the second binding unit comprises a full set of CDRs 1, 2, and 3 having at least 92% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. In some embodiments, the second binding unit comprises a full set of CDRs 1,

2, and 3 having at least 96% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. In some embodiments, the second binding unit comprises a full set of CDRs 1, 2, and 3 having 100% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94. [0025] In some embodiments, the second binding unit comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 96-135. In some embodiments, the second binding unit comprises a heavy chain variable region sequence comprising SEQ ID NO: 96. In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 83% identity to SEQ ID NO: 96. In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 92% identity to SEQ ID NO: 96. In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 94% identity to SEQ ID NO: 96. In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 95% identity to SEQ ID NO: 96. In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 96% identity to SEQ ID NO: 96. In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 97% identity to SEQ ID NO: 96. In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 98% identity to SEQ ID NO: 96. In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 99% identity to SEQ ID NO: 96.

[0026] In some embodiments, the second binding unit competes with Isatuximab for binding to the second epitope on the CD38 protein.

[0027] In some embodiments, the CD38 protein is a human CD38 protein (SEQ ID NO: 136).

[0028] In some embodiments, the first epitope is a conformational epitope comprising two or more amino acid residues selected from the group consisting of amino acid residues 120, 135, 139, 142, 202, 203, 205, 236, 237, 239, 241, 252, 254, 255, 272-279, 284, 287, 288, 291-294, 296, 297, 299 and 300 of SEQ ID NO: 136. In some embodiments, the first epitope is a conformational epitope comprising amino acid residues 120, 135, 139, 142, 202, 203, 205, 236, 237, 239, 241, 252, 254, 255, 272-279, 284, 287, 288, 291-294, 296, 297, 299 and 300 of SEQ ID NO: 136.

[0029] In some embodiments, the second epitope is a conformational epitope comprising two or more amino acid residues selected from the group consisting of amino acid residues 188-201, 262-264, and 275-284 of SEQ ID NO: 136. In some embodiments, the second epitope is a conformational epitope comprising amino acid residues 188-201, 262-264, and 275-284 of SEQ ID NO: 136.

[0030] In some embodiments, an anti-CD38 multispecific antibody further comprises at least one heavy chain constant region sequence in the absence of a CHI sequence. In some embodiments, an anti- CD38 multispecific antibody further comprises two heavy chain constant region sequences that each lack a CHI sequence.

[0031] In some embodiments, an anti-CD38 multispecific antibody is bispecific. In some embodiments, an anti-CD38 multispecific antibody is a three chain antibody-like molecule (TCA) comprising a first binding unit comprising a heavy chain variable region and a light chain variable region, and a second binding unit comprising a heavy chain-only variable region, in a monovalent or bivalent configuration.

[0032] Aspects of the invention include pharmaceutical compositions comprising a multispecifc antibody as described herein. Aspects of the invention include polynucleotides encoding a multispecifrc antibody as described herein. Aspects of the invention include vectors comprising the polynucleotides as described herein. Aspects of the invention include cells comprising the vectors as described herein.

[0033] Aspects of the invention include methods of producing a multispecific antibody as described herein, the methods comprising growing a cell as described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell and/or a cell culture medium in which the cell is grown.

[0034] Aspects of the invention include methods of making a multispecific antibody as described herein, the methods comprising immunizing a UniRat animal with a CD38 protein and identifying CD38 protein-binding heavy chain sequences.

[0035] Aspects of the invention include methods for the treatment of a disease or disorder characterized by expression of CD38, comprising administering to a subject with said disease or disorder a multispecifrc antibody or a pharmaceutical composition as described herein.

[0036] In some embodiments, the disease or disorder is characterized by a hydrolase enzymatic activity of CD38, a cyclase enzymatic activity of CD38, or a combination thereof. In some embodiments, the disease or disorder is a telomere shortening disease. In some embodiments, the telomere shortening disease is accelerated aging. In some embodiments, the telomere shortening disease is aplastic anemia. In some embodiments, the telomere shortening disease is dyskeratosis congenita. In some embodiments, the telomere shortening disease is Franconi’s anemia. In some embodiments, the telomere shortening disease is idiopathic pulmonary fibrosis. In some embodiments, the disease or disorder is an inflammatory disease. In some embodiments, the inflammatory disease is ulcerative colitis. In some embodiments, the inflammatory disease is graft versus host disease (GvHD). In some embodiments, the GvHD is acute GvHD. In some embodiments, the GvHD is chronic GvHD. In some embodiments, the GvHD is transplant-associated GvHD. In some embodiments, the inflammatory disease is acute kidney injury. In some embodiments, the disease or disorder is fibrosis-associated disorder. In some embodiments, the fibrosis-associated disorder is scleroderma. In some embodiments, the disease or disorder is a metabolic syndrome. In some embodiments, the metabolic syndrome is type II diabetes mellitus (T2DM). In some embodiments, the metabolic syndrome is obesity. In some embodiments, the metabolic syndrome is systemic inflammation. In some embodiments, the disease or disorder is doxorubicin-induced toxicity. In some embodiments, the doxorubicin-induced toxicity comprising cardiotoxicity. In some embodiments, the disease or disorder is an organ transplantation-associated disease or disorder. In some embodiments, the organ transplantation-associated disease or disorder is a skin transplantation-associated disease or disorder. In some embodiments, the organ transplantation- associated disease or disorder is a kidney transplantation-associated disease or disorder. In some embodiments, the disease or disorder is a cardiovascular disease or disorder. In some embodiments, the cardiovascular disease or disorder comprises heart failure.

[0037] Aspects of the invention include methods of treating a disease or disorder characterized by reduced sirtuin activity, the methods comprising administering to a subject with the disease or disorder a multispecific antibody or a pharmaceutical composition as described herein. In some embodiments, the methods further comprise administering nicotinamide mononucleotide (NMN) to the subject. In some embodiments, the disease or disorder is a metabolic disease or disorder. In some embodiments, the disease or disorder is a cardiovascular disease or disorder. In some embodiments, the disease or disorder is a neurode generative disease or disorder. In some embodiments, the disease or disorder is a cancer.

[0038] Aspects of the invention include methods of increasing nicotinamide adenine dinucleotide (NAD+) concentration in a cell, the methods comprising contacting the cell with a multispecific antibody or a pharmaceutical composition as described herein. In some embodiments, the methods further comprise contacting the cell with NMN.

[0039] Aspects of the invention include methods of increasing sirtuin activity in a cell, the methods comprising contacting the cell with a multispecific antibody or a pharmaceutical composition as described herein. In some embodiments, the methods further comprise contacting the cell with NMN.

[0040] Aspects of the invention include methods of treating a disease or disorder characterized by expression of CD38, the methods comprising administering to a subject with the disease or disorder a multispecific antibody or a pharmaceutical composition as described herein. In some embodiments, the disease or disorder is characterized by a hydrolase enzymatic activity of CD38, a cyclase enzymatic activity of CD38, or a combination thereof. In some embodiments, administering the multispecific antibody to the subject results in an inhibition of CD38 enzymatic activity.

[0041] Aspects of the invention include methods of increasing nicotinamide adenine dinucleotide (NAD+) concentration in a cell without depleting or activating CD38 expressing cells, the methods comprising contacting the cell with a multispecific antibody as described herein, or a pharmaceutical composition as described herein.

[0042] Aspects of the invention include multispecific antibodies as described herein, which inhibit CD38 enzymatic activity without directly or indirectly lysing CD38+ cells.

[0043] These and further aspects will be further explained in the rest of the disclosure, including the Examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a table providing amino acid sequence information for members of the FI 1 A family.

[0045] FIG. 2 is a table providing amino acid sequence information for members of the FI 1 A family.

[0046] FIG. 3 is a table providing amino acid sequence information for members of the F12A family.

[0047] FIG. 4 is a table providing amino acid sequence information for members of the F12A family.

[0048] FIG. 5 provides sequence information for additional amino acid sequences in the application.

[0049] FIG. 6 shows a schematic representation of two bivalent (Panels C and D) and two tetravalent

(Panels A and B) UniAb™ formats in accordance with embodiments of the invention.

[0050] FIG. 7 is a graph showing epitope binning curves with CD38 for antibody clones 309157 and 330304.

[0051] FIG. 8 is a graph showing epitope binning curves with CD38 for antibody clone 309157 and Daratumumab.

[0052] FIG. 9 is a graph showing epitope binning curves with CD38 for antibody clone 330304 and Isatuximab.

[0053] FIG. 10 is a table showing F11A family VH sequences with percent CD38 inhibition and percent sequence identity to the sequence of Clone ID No. 309157.

[0054] FIG. 11 is a table showing F12A family VH sequences with percent CD38 inhibition and percent sequence identity to the sequence of Clone ID No. 330304.

[0055] FIG. 12 is a graph showing an entropy plot of FI 1 A family members.

[0056] FIG. 13 is a graph showing an entropy plot of F12A family members.

[0057] FIG. 14 is graph showing a bar plot of neg_logl0_p_values for FI 1 A family members.

[0058] FIG. 15 is graph showing a bar plot of neg_logl0_p_values for F12A family members.

[0059] FIG. 16 is a table showing statistically significant positions and corresponding possible amino acids at the position for FI 1 A family members.

[0060] FIG. 17 is a table showing statistically significant positions and corresponding possible amino acids at the position for F12A family members.

[0061] FIG. 18 is a table summarizing X-ray data and a refinement summary of diffraction data for a CD38-F11 A complex.

[0062] FIG. 19 is a table summarizing surface area analysis data by PISA (CCP4).

[0063] FIG. 20 is a sequence summary chart that shows interfacing residues of FI 1 A, Daratumumab and Isatuximab on the human CD38 ECD.

[0064] FIG. 21, panels A-C, provide A) a frequency distribution plot of percent inhibition values of anti-CD38 binders (373 total) that were screened for CD38 inhibition. CHO-HuCD38 cells were treated with antibodies for 15 min at RT. Following incubation, sNAD+ substrate was added to each well and fluorescence was immediately analyzed using a microplate reader for 30 min. B) UniAbs CD38 F11 A and CD38 F12A were tested for CD38 inhibition in a dose curve. Percent inhibition by the combination of both UniAbs was compared to the individual UniAbs. C) schematic illustrations of generation of TNB-738 by pairing CD38 F11A and CD38 F12A into bispecific format using knob-into-hole technology on a silenced IgG4 Fc.

[0065] FIG. 22, panels A-E, show TNB-738 binds to CD38 and inhibits CD38 enzyme activity. A) On-target cell binding of TNB-738 was assessed by flow cytometry using Daudi, Ramos, and CHO- HuCD38 cells. B) Off-target cell binding of TNB-738 was assessed by flow cytometry using 293F, CHO, K562, and HL-60 cells. C) Cells were incubated with antibodies at 37°C for 2 hours and capping was determined using an immunofluorescent microscope. In total, 100 cells were counted. D) TNB- 738-mediated CD38 inhibition was evaluated on Daudi, Ramos, and CHO cells and (E) recombinant CD38 protein.

[0066] FIG. 23, panels A-D, show TNB-738-mediated inhibition of CD38 ectoenzyme activity increases NAD+ levels and SIRT1 activity downstream. Jurkat (A) or Ramos (B) cells were treated with a dilution series of TNB-738 or isotype control in the presence or absence of 50 nM NMN for 24 h at 37°C. Following incubation, NAD+ levels were measured using the Cell Biolabs NAD+ assay kit. SIRT1 activity was measured using the SIRT-Glo kit (Promega) in Jurkat (C) or Ramos (D) cells following treatment with TNB-738 or isotype control in the presence of absence of 50 nM NMN.

[0067] FIG. 24, panels A-D, show TNB-738 neither lyses CD38-expressing cells nor promotes the growth of CD38-expressing tumors. A) Daudi and Ramos cells were incubated with increasing concentrations of TNB-738 or daratumumab in the presence of 5% rabbit complement serum for 45 min at 37°C. Cell viability was measured using Cell Titer Glo 2.0. B) NK cells were isolated from PBMCs and incubated with Daudi or Ramos cells and increasing concentrations of TNB-738 or daratumumab for 4 h at 37°C. Tumor killing was evaluated by LDH release. C) Daudi or Ramos cells were treated with TNB-738 or isatuximab for 24 h at 37°C. After incubation, the cells were stained with 7-AAD and Annexin V. Cell viability was analyzed by flow cytometry. D) A549 tumor spheroids were treated with TNB-738, isotype control, or EGF every 2-3 days 5 total treatments. (Di) Tumor area was measured using a microscope camera and ImageJ Shape Descriptor Plugin. (Dii) Representative images of the spheroids on Day 10 are shown. (Dili) NAD+ concentration in the tumors was quantified using the Cell Biolabs NAD+ assay kit.

[0068] FIG. 25 provides a crystal structure of the F11A/CD38 complex in the epitope mapping of FI 1 A on CD38. CD38 ECD is shown in blue with the active site in pink. UniDab FI 1A is highlighted yellow.

[0069] FIG. 26 is a table showing data collection and refinement statistics in the epitope mapping of F11A on CD38. [0070] FIG. 27, panels A-C, provide results of insilico epitope mapping of F12A on CD38. A) Results of insilico prediction of F12A on CD38 using MabTope. B) Table summarizing the predicted peptides with suggested mutant residues. C) Binding of F12A antibody to wildtype and mutated CD38. The percent PE+APC+ cells as well as the mean APC signal were collected from 4 independent experiments. The amount of PE+APC+ cells were normalized to the total PE+ cells. The results are expressed in mean ± sem of the maximal response. One star (*) indicate significant statistical difference at p<0.05, two stars (**) atp<0.01.

[0071] FIG. 28 is a schematic representation of intracellular NAD+ boosting by TNB-738-mediated CD38 inhibition.

[0072] FIG. 29 provides a comparison of CD38 epitopes of daratumumab, F11A, isatuximab, and FI 2 A. A) Epitope on CD38 of daratumumab (i) in yellow, FI 1A (ii) in green, and isatuximab (iii) in pink. B) Peptides predicted by MabSilico for F12A epitope (green-CD38_12m2, grey- CD38_12m4). The residues of the active site of CD38 are represented by ball and stick model.

[0073] FIG. 30 is a table providing a comparison of the functional properties of TNB-738, daratumumab, and isatuximab.

[0074] FIG. 31 , panels A and B, are graphs showing results of evaluation of pharmacokinetics of TNB- 738 in mice (A) and cynomolgus monkeys (B) following single dose administration of TNB-738 at 1 or 10 mg/kg. Concentrations of TNB-738 in serum were measured using AlphaLISA (Perkin Elmer).

[0075] FIG. 32, panels A and B, show results of evaluation of SIRT3 and PARP activity following incubation of Daudi or Ramos cells with TNB-738. A) SIRT3 activity was evaluated by incubating Daudi or Ramos cells with increasing concentrations of TNB-738 or isotype control followed by measurement of SIRT3 activity using the SIRT-Glo kit. B) PARP activity was measured by staining for poly-ADP ribose by flow cytometry after incubation of TNB-738 with Ramos or Daudi cells in the presence or absence of NMN.

[0076] FIG. 33, panels A and B, show the results of an assessment of TNB-738 internalization. A) Representative images of Daudi cells incubated with 10 pg/mL TNB-738 or daratumumab for 0, 30, 60, or 120 min at 37°C, labelled with DAPI (1st column), Texas Red Phalloidin (2nd column), antihuman IgG FITC (3rd column) and anti-CD98 Alexa647 (4th column). Merged images of all stains are shown in the 5th column. White arrows show capping. B) Histograms of Daudi cells stained with isotype control, daratumumab, or TNB-738 coupled to a pH-sensitive fluoroprobe (pHrodo iFL, ThermoFisher).

[0077] FIG. 34, panels A and B, show results of evaluation of TNB-738 and daratumumab binding on immune cell subsets. A) Percentage of CD38 staining on different hPBMC populations stained with TNB-738 (in grey) or daratumumab (in black). Results represent the mean of 3 independent experiments. B) Histograms of isotype control, daratumumab, or TNB-738 staining on B cells, monocytes, and NR cells.

[0078] FIG. 35 shows the effects of TNB-738 on immune cell activation. Representative dot plot of CD25 and IFNy staining on hPBMC incubated 16 hours without antibody (left dot plot), with anti-CD3 and anti-CD28 (middle dot plot), or TNB-738 (5 pg/mL) (right dot plot) gated on CD4+FoxP3- cells.

[0079] FIG. 36, panels 1-6, provides competition data by surface plasmon resonance (SPR). 1) Epitope binning curves with F11A (1), F12A (2), isatuximab (3), and daratumumab (4) stacked on sensor, then bound to 400 nM of huCD38 followed by a second injection that contained separately each antibody as well as the huCD38 at 400 nM. Sensogram (5) represents the amine coupling of huCD38 to the sensor followed by injection of each antibody separately. Sensogram (6) shows binding to uncoupled sensors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et ak, 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et ak, eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et ak, ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et ak, 2001); kiarlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0081] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0082] Unless indicated otherwise, antibody residues herein are numbered according to the Rabat numbering system ( e.g ., Rabat et al, Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0083] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0084] All references cited throughout the disclosure, including patent applications and publications, are incorporated by reference herein in their entirety.

I. Definitions

[0085] By “comprising” it is meant that the recited elements are required in the composition/method/kit, but other elements may be included to form the composition/method/kit etc. within the scope of the claim.

[0086] By “consisting essentially of’, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention.

[0087] By “consisting of’, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim.

[0088] The terms “binding compound” and “binding composition” as used interchangeably herein refer to a molecular entity having binding affinity to one or more binding targets. Binding compounds in accordance with embodiments of the invention can include, without limitation, antibodies, antigen binding domains of antibodies, antigen-binding fragments of antibodies, antibody-like molecules, heavy -chain antibodies (e.g., UniAbs™), ligands, receptors, and the like.

[0089] The term “antibody” is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain only antibodies, three chain antibodies, single chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0090] The term antibody may reference a full-length heavy chain, a full length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that comprises an antigen-binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. Light chains of the subject antibodies can be kappa light chains (Vkappa) or lambda light chains (Vlambda). The immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin. [0091] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-113 of the heavy chain) ( e.g ., Kabat et al, Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies mean residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies mean residue numbering by the EU numbering system.

[0092] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies in accordance with the present invention can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be made via recombinant protein production methods (see, e.g., U.S. Patent No. 4,816,567), for example.

[0093] The term “variable”, as used in connection with antibodies, refers to the fact that certain portions of the antibody variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a b-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the b-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC). [0094] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; 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 from a “hypervariable loop” residues 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, “CDR” means a complementary determining region of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region/CDR residues as herein defined.

[0095] Exemplary CDR designations are shown herein, however, one of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989; 342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181:6230-6235; Almagro “Identification of differences in the specificity -determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is herein specifically incorporated by reference.

[0096] The terms “heavy chain-only antibody,” and “heavy-chain antibody” are used interchangeably herein and refer, in the broadest sense, to antibodies lacking the light chain of a conventional antibody. The terms specifically include, without limitation, homodimeric antibodies comprising the VH antigen binding domain and the CH2 and CH3 constant domains, in the absence of the CHI domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NAR comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR) and functional fragments thereof; and soluble single domain antibodies (sUniDabs™). In one embodiment, a heavy chain-only antibody is composed of the variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, the heavy chain- only antibody is composed of an antigen-binding domain, at least part of a hinge region and CH2 and CH3 domains, the absence of a CHI domain. In another embodiment, the heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH2 domain. In a further embodiment, the heavy chain-only antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH3 domain. Heavy chain-only antibodies in which the CH2 and/or CH3 domain is truncated are also included herein. In a further embodiment, the heavy chain is composed of an antigen binding domain, and at least one CH (CHI, CH2, CH3, or CH4) domain but no hinge region. In a further embodiment the heavy chain is composed of an antigen binding domain, at least one CH (CHI, CH2, CH3, or CH4) domain, and at least a portion of a hinge region. The heavy chain-only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded or otherwise, covalently or non-covalently, attached with each other. The heavy chain-only antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclass, are also included herein. In a particular embodiment, the heavy -chain antibody is of the IgGl, IgG2, IgG3, or IgG4 subtype, in particular the IgGl or IgG4 subtype. In one embodiment, the heavy -chain antibody is of the IgG4 subtype, wherein one or more of the CH domains are modified to alter an effector function of the antibody. In one embodiment, the heavy -chain antibody is of the IgGl subtype, wherein one or more of the CH domains are modified to alter an effector function of the antibody. Modifications of CH domains that alter effector function are further described herein. Non-limiting examples of heavy -chain antibodies are described, for example, in W02018/039180, the disclosure of which is incorporated herein by reference in its entirety.

[0097] In one embodiment, the heavy chain-only antibodies herein are used as a binding (targeting) domain of a chimeric antigen receptor (CAR). The definition specifically includes human heavy chain- only antibodies produced by human immunoglobulin transgenic rats (UniRat™), called UniAbs™. The variable regions (VH) of UniAbs™ are called UniDabs™, and are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with multi-specificity, increased potency and extended half-life. Since the homodimeric UniAbs™ lack a light chain and thus a VL domain, the antigen is recognized by one single domain, i.e., the variable domain (antigen-binding domain) of the heavy chain of a heavy -chain antibody (VH).

[0098] An “antibody-drug conjugate” (ADC) or “immunoconjugate” means an antibody, or antigen binding fragment thereof, conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0099] An “intact antibody chain” as used herein is one comprising a full length variable region and a full length constant region (Fc). An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CF) and heavy chain constant domains, CHI, hinge, CH2 and CH3 for secreted IgG. Other isotypes, such as IgM or IgA may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody -dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors. Constant region variants include those that alter the effector profde, binding to Fc receptors, and the like.

[0100] Depending on the amino acid sequence of the Fc (constant domain) of their heavy chains, antibodies and various antigen-binding proteins can be provided as different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains that correspond to the different classes of antibodies may be referenced as a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called k (kappa) and l (lambda), based on the amino acid sequences of their constant domains. Antibodies in accordance with embodiments of the invention can comprise kappa light chain sequences or lambda light chain sequences.

[0101] A “functional Fc region” possesses an “effector function” of a native-sequence Fc region. Non limiting examples of effector functions include Clq binding; CDC; Fc-receptor binding; ADCC; ADCP; down-regulation of cell-surface receptors (e.g., B-cell receptor), etc. Such effector functions generally require the Fc region to interact with a receptor, e.g., the FcyRI; FcyRIIA; FcyRIIBl; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors, and the low affinity FcRn receptor; and can be assessed using various assays known in the art. A “dead” or “silenced” Fc is one that has been mutated to retain activity with respect to, for example, prolonging serum half-life, but which does not activate a high affinity Fc receptor, or which has a reduced affinity to an Fc receptor.

[0102] A “native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include, for example, a native-sequence human IgGl Fc region (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc region, as well as naturally occurring variants thereof. [0103] A “variant Fc region” comprises an amino acid sequence that differs from that of a native- sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native-sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native-sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0104] Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement Clq binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution into human IgGl or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. etal., 1999 Eur J Immunol 29(8):2613-24; and Shields RL. etal., 2001. J Biol Chem. 276(9):6591- 604). The human IgGl amino acid sequence (UniProtKB No. P01857) is provided herein as SEQ ID NO: 137. The human IgG4 amino acid sequence (UniProtKB No. P01861) is provided herein as SEQ ID NO: 138. Silenced IgGl is described, for example, in Boesch, A.W., et al., “Highly parallel characterization of IgG Fc binding interactions.” MAbs, 2014. 6(4): p. 915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0105] Other Fc variants are possible, including, without limitation, one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N- terminal end of a native Fc, or a methionine residue is added thereto. Thus, in some embodiments, one or more Fc portions of a binding compound can comprise one or more mutations in the hinge region to eliminate disulfide bonding. In yet another embodiment, the hinge region of an Fc can be removed entirely. In still another embodiment, a binding compound can comprise an Fc variant.

[0106] Further, an Fc variant can be constructed to remove or substantially reduce effector functions by substituting (mutating), deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, and not limitation, a deletion may occur in a complement-binding site, such as a Clq-binding site. Techniques for preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like. [0107] In some embodiments, a binding compound (e.g., an antibody) comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which can optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, a binding compound (e.g., an antibody) comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which can optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 “knobs-in-holes” mutations described herein can be utilized in any suitable manner so as to place a “knob” on a first heavy chain constant region of a first monomer of a binding compound dimer, and a “hole” on a second heavy chain constant region of a second monomer of the binding compound dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the binding compound.

[0108] In some embodiments, a binding compound comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation. This collection of mutations can be introduced into an IgG4 heavy chain sequence to reduce effector function activity of the resulting antibody or binding compound, and can be used interchangeably with the knobs-in-holes mutations described herein. For example, in some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, as well as a T366W mutation (knob). In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, and an L235A mutation, as well as a T366S mutation, an L368A mutation, and a Y407V mutation (hole).

[0109] The term “Fc-region-comprising antibody” refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, an antibody having an Fc region according to this invention can comprise an antibody with or without K447.

[0110] The Fc may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or may be in an aglycosylated or deglycosylated form. The increase, decrease, removal or other modification of the sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method or by expressing it in a genetically engineered production cell line. Such cell lines can include microorganisms, e.g., Pichia Pastoris, and mammalian cell lines, e.g. CHO cells, that naturally express glycosylating enzymes. Further, microorganisms or cells can be engineered to express glycosylating enzymes, or can be rendered unable to express glycosylation enzymes (See e.g., Hamilton, et al., Science, 313:1441 (2006); Kanda, et al, J. Biotechnology, 130:300 (2007); Kitagawa, et al., J. Biol. Chem., 269 (27): 17872 (1994); Ujita-Lee et al., J. Biol. Chem., 264 (23): 13848 (1989); Imai-Nishiya, et al, BMC Biotechnology 7:84 (2007); and WO 07/055916). As one example of a cell engineered to have altered sialylation activity, the alpha-2, 6-sialyltransferase 1 gene has been engineered into Chinese Hamster Ovary cells and into sf9 cells. Antibodies expressed by these engineered cells are thus sialylated by the exogenous gene product. A further method for obtaining Fc molecules having a modified amount of sugar residues compared to a plurality of native molecules includes separating said plurality of molecules into glycosylated and non-glycosylated fractions, for example, using lectin affinity chromatography (See, e.g., WO 07/117505). The presence of particular glycosylation moieties has been shown to alter the function of immunoglobulins. For example, the removal of sugar chains from an Fc molecule results in a sharp decrease in binding affinity to the Clq part of the first complement component Cl and a decrease or loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been correlated with anti-inflammatory activity (See, e.g., Kaneko, et al, Science 313:760 (2006)), whereas removal of fucose from the IgG leads to enhanced ADCC activity (See, e.g., Shoj-Hosaka, et al, J. Biochem., 140:777 (2006)).

[0111] In alternative embodiments, binding coumpounds of the invention may have an Fc sequence with enhanced effector functions, e.g., by increasing their binding capacities to FcyRIIIA and increasing ADCC activity. For example, fucose attached to the A-linkcd glycan at Asn-297 of Fc sterically hinders the interaction of Fc with FcyRIIIA, and removal of fucose by glyco-engineering can increase the binding to FcyRIIIA, which translates into >50-fold higher ADCC activity compared with wild type IgGl controls. Protein engineering, through amino acid mutations in the Fc portion of IgGl, has generated multiple variants that increase the affinity of Fc binding to FcyRIIIA. Notably, the triple alanine mutant S298A E333 A K334A displays 2-fold increase binding to FcyRIIIA and ADCC function. S239D/I332E (2X) and S239D/I332E/A330L (3X) variants have a significant increase in binding affinity to FcyRIIIA and augmentation of ADCC capacity in vitro and in vivo. Other Fc variants identified by yeast display also showed the improved binding to FcyRIIIA and enhanced tumor cell killing in mouse xenograft models. See, e.g., Liu et al. (2014) JBC 289(6):3571-90, herein specifically incorporated by reference.

[0112] The term “Fc-region-comprising antibody” refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering the nucleic acid encoding the antibody. Accordingly, an antibody having an Fc region according to this invention can comprise an antibody with or without K447. [0113] “Humanized” forms of non-human (e.g., rodent) antibodies, including single chain antibodies, are chimeric antibodies (including single chain antibodies) that contain minimal sequence derived from non-human immuno globulin. See, e.g., Jones et al, (1986) Nature 321:522-525; Chothia et al (1989) Nature 342:877; Riechmann et al (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al (1993) J. Immunol 151, 2623-2632; Werther et al (1996) J. Immunol. Methods 157:4986-4995; and Presta et al (2001) Thromb. Haemost. 85:379-389. For further details, see U.S. Pat. Nos. 5,225,539; 6,548,640; 6,982,321; 5,585,089; 5,693,761; 6,407,213; Jones et al (1986) Nature, 321:522-525; and Riechmann et al (1988) Nature 332:323-329.

[0114] Aspects of the invention include binding compounds having multi-specific configurations, which include, without limitation, bispecific, trispecific, etc. A large variety of methods and protein configurations are known and used in bispecific monoclonal antibodies (BsMAB), tri-specific antibodies, etc.

[0115] Aspects of the invention include antibodies comprising a heavy chain-only variable region in a monovalent or bivalent configuration. As used herein, the term “monovalent configuration” as used in reference to a heavy chain-only variable region domain means that only one heavy chain-only variable region domain is present, having a single binding site (see, e.g., FIG. 6, Panel D, right side of depicted molecule). In contrast, the term “bivalent configuration” as used in reference to a heavy chain-only variable region domain means that two heavy chain-only variable region domains are present (each having a single binding site), and are connected by a linker sequence (see, e.g., FIG. 6, Panel B, either side of depicted molecule). Non-limiting examples of linker sequences are discussed further herein, and include, without limitation, GS linker sequences of various lengths. When a heavy chain-only variable region is in a bivalent configuration, each of the two heavy chain-only variable region domains can have binding affinity to the same antigen, or to different antigens (e.g., to different epitopes on the same protein; to two different proteins, etc.). However, unless specifically noted otherwise, a heavy chain- only variable region denoted as being in a “bivalent configuration” is understood to contain two identical heavy chain-only variable region domains, connected by a linker sequence, wherein each of the two identical heavy chain-only variable region domains have binding affinity to the same target antigen.

[0116] Various methods for the production of multivalent artificial antibodies have been developed by recombinantly fusing variable domains of two or more antibodies. In some embodiments, a first and a second antigen-binding domain on a polypeptide are connected by a polypeptide linker. One non limiting example of such a polypeptide linker is a GS linker, having an amino acid sequence of four glycine residues, followed by one serine residue, and wherein the sequence is repeated n times, where n is an integer ranging from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 139) (n=l) and GGGGSGGGGS (SEQ ID NO: 140) (n=2). Other suitable linkers can also be used, and are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013 October 15; 65(10): 1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0117] The term “bispecific three-chain antibody like molecule” or “TCA” is used herein to refer to antibody-like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen-binding region and at least one CH domain. This heavy chain/light chain pair has binding specificity for a first antigen. In some embodiments, a TCA comprises a light chain polypeptide subunit comprising a CDR1 sequence of SEQ ID NO: 49, a CDR2 sequence of SEQ ID NO: 50, and a CDR3 sequence of SEQ ID NO: 51 , in a human light chain framework. In some embodiments, the human light chain framework is a human kappa (Vkappa) or a human lambda (Vlambda) framework. In some embodiments, a TCA comprises a light chain polypeptide subunit comprising a light chain variable region (VL) comprising a sequence having at least about 80%, 85%, 90%, 95%, or 99% identity to the sequence of SEQ ID NO: 52. In some embodiments, a TCA comprises a light chain polypeptide subunit that comprises the sequence of SEQ ID NO: 52. In some embodiments, a TCA comprises a light chain polypeptide subunit that comprises a light chain constant region (CL). In some embodiments, the light chain constant region is a human kappa light chain constant region or a human lambda light chain constant region. In some embodiments, a TCA comprises a light chain polypeptide subunit comprising a full length light chain comprising a sequence having at least about 80%, 85%, 90%, 95%, or 99% identity to the sequence of SEQ ID NO: 48. In some embodiments, a TCA comprises a light chain polypeptide subunit that comprises the sequence of SEQ ID NO: 48. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy -chain only antibody comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and an antigen binding domain that binds an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain. Parts of such variable region may be encoded by V_H and/or V_L gene segments, D and J_H gene segments, or J_L gene segments. The variable region may be encoded by rearranged V_HDJ_H, V_LDJ_h, V_HJ_L, or V_LJ_L gene segments.

[0118] A TCA binding compound makes use of a “heavy chain only antibody” or “heavy chain antibody” or “heavy chain polypeptide” which, as used herein, mean a single chain antibody comprising heavy chain constant regions CH2 and/or CH3 and/or CH4 but no CHI domain. In one embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region and CH2 and CH3 domains. In another embodiment, the heavy chain antibody is composed of an antigen binding domain, at least part of a hinge region and a CH2 domain. In a further embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least part of a hinge region and a CH3 domain. Heavy chain antibodies in which the CH2 and/or CH3 domain is truncated are also included herein. In a further embodiment the heavy chain is composed of an antigen binding domain, and at least one CH (CHI, CH2, CH3, or CH4) domain but no hinge region. The heavy chain only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded or otherwise covalently or non- covalently attached with each other, and can optionally include an asymmetric interface between two or more of the CH domains to facilitate proper pairing between polypeptide chains. The heavy -chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclass, are also included herein. In a particular embodiment, the heavy chain antibody is of the IgGl, IgG2, IgG3, orIgG4 subtype, in particular the IgGl subtype or the IgG4 subtype. Non-limiting examples of a TCA binding compound are described in, for example, WO2017/223111 and W02018/052503, the disclosures of which are incorporated herein by reference in their entirety.

[0119] Heavy-chain antibodies constitute about one fourth of the IgG antibodies produced by the camelids, e.g., camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but are devoid of light chains. As a consequence, the variable antigen binding part is referred to as the VHH domain and it represents the smallest naturally occurring, intact, antigen-binding site, being only around 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies termed VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0120] The term “interface”, as used herein, is used to refer to a region, which comprises those “contact” amino acid residues (or other non-amino acid groups such as, for example, carbohydrate groups,) in a first heavy chain constant region which interact with one or more “contact” amino acid residues (or other non-amino acid groups) in a second heavy chain constant region.

[0121 ] The term “asymmetric interface” is used to refer to an interface (as hereinabove defined) formed between two polypeptide chains, such as a first and a second heavy chain constant region and/or between a heavy chain constant region and its matching light chain, wherein the contact residues in the first and the second chains are different by design, comprising complementary contact residues. The asymmetric interface can be created by, e.g., knobs/holes interactions and/or salt bridges coupling (charge swaps) and/or other techniques known in the art.

[0122] A “cavity” or “hole” refers to at least one amino acid side chain which is recessed from the interface of the second polypeptide and therefore accommodates a corresponding protuberance (“knob”) on the adjacent interface of the first polypeptide. The cavity (hole) may exist in the original interface or may be introduced synthetically (e.g., by altering a nucleic acid encoding the interface residue). Normally, a nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the second polypeptide. The preferred import residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T), valine (V) and glycine (G). Most preferred amino acid residues are serine, alanine or threonine, most preferably alanine. In one preferred embodiment, the original residue for the formation of the protuberance has a large side chain volume, such as tyrosine (Y), arginine (R), phenylalanine (F) or tryptophan (W). Asymmetric interfaces are described in detail, for example, in Xu et ak, “Production of bispecific antibodies in ‘knobs-into-holes’ using a cell-free expression system”, MAbs. 2015, 7(l):231-42, the disclosure of which is incorporated by reference herein in its entirety.

[0123] The term “CD38” as used herein refers to a single-pass type II transmembrane protein with ectoenzymatic activities, also known as ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1. The term “CD38” includes a CD38 protein of any human or non-human animal species, and specifically includes human CD38 as well as CD38 of non-human mammals.

[0124] The term “human CD38” as used herein includes any variants, isoforms and species homologs of human CD38 (UniProt P28907; SEQ ID NO: 136), regardless of its source or mode of preparation. Thus, “human CD38” includes human CD38 naturally expressed by cells, and CD38 expressed on cells transfected with the human CD38 gene.

[0125] The terms “anti-CD38 heavy chain-only antibody,” “CD38 heavy chain-only antibody,” “anti- CD38 heavy-chain antibody” and “CD38 heavy-chain antibody” are used herein interchangeably to refer to a heavy chain-only antibody as hereinabove defined, immunospecifically binding to CD38, including human CD38, as hereinabove defined. The definition includes, without limitation, human heavy chain antibodies produced by transgenic animals, such as transgenic rats or transgenic mice expressing human immunoglobulin, including UniRats™ producing human anti-CD38 UniAb™ antibodies, as hereinabove defined. [0126] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.

[0127] An “isolated” binding compound (such as an isolated antibody) is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the binding compound, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In preferred embodiments, the binding compound will be purified (1) to greater than 95% by weight of binding compound as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated binding compound includes the binding compound in situ within recombinant cells, since at least one component of the binding compound’s natural environment will not be present. Ordinarily, however, isolated binding compound will be prepared by at least one purification step.

[0128] Binding compounds in accordance with embodiments of the invention include multi-specific binding compounds. Multi-specific binding compounds have more than one binding specificity. The term “multi-specific” specifically includes “bispecific” and “trispecific,” as well as higher-order independent specific binding affinities, such as higher-order polyepitopic specificity, as well as tetravalent binding compounds and antigen-binding fragments of binding compounds (e.g., antibodies and antibody fragments). “Multi-specific” binding compounds specifically include antibodies comprising a combination of different binding entities as well as antibodies comprising more than one of the same binding entity. The terms “multi-specific antibody,” “multi-specific heavy chain-only antibody,” “multi-specific heavy-chain antibody,” and “multi-specific UniAb™” are used herein in the broadest sense and cover all antibodies with more than one binding specificity. The multi-specific heavy chain anti-CD38 antibodies of the present invention specifically include antibodies immunospecifically binding to two or more epitopes on a CD38 protein, such as a human CD38. In some embodiments, a multispecific binding compound can bind to two non-overlapping epitopes on a CD38 protein. In some embodiments, a multispecific binding compound can bind to two epitopes on a CD38 protein that are at least partially overlapping, meaning that the two epitopes share at least one amino acid residue in common.

[0129] An “epitope” is the site on the surface of an antigen molecule to which an antigen-binding region of a binding compound binds. Generally, an antigen has several or many different epitopes, and reacts with many different binding compounds (e.g., many different antibodies). The term specifically includes linear epitopes and conformational epitopes.

[0130] “Epitope mapping” is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure.

[0131] “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As noted above, the present invention specifically includes anti-CD38 heavy-chain antibodies with polyepitopic specificities, i.e., anti-CD38 heavy-chain antibodies binding to two or more epitopes on a CD38 protein, such as a human CD38. The term “non overlapping epitope(s)” or “non-competitive epitope(s)” of an antigen is defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies, but not the other member. Pairs of antibodies, or antigen-binding regions targeting the same antigen on a multi-specific antibody, recognizing non-overlapping epitopes, do not compete for binding to that antigen and are able to bind that antigen simultaneously.

[0132] A binding compound binds “essentially the same epitope” as a reference binding compound (e.g., a reference antibody), when the binding compound and the reference antibody recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody. Usually, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.

[0133] The term “compete” as used herein with respect to a binding compound (e.g., an antibody) and a reference binding compound (e.g., a reference antibody) means that the binding compound causes about a 15-100% reduction in the binding of the reference binding compound to the target antigen, as determined by standard techniques, such as by the competition binding assays described herein.

[0134] The term “competition group” as used herein refers to two or more binding compounds (e.g., a first and a second antibody) that bind to the same target antigen (or epitope) and that compete with the members of the competition group for binding to the target antigen. Members of the same competition group compete with one another for binding to a target antigen, but do not necessarily have the same functional activity.

[0135] The term “valent” as used herein refers to a specified number of binding sites in an antibody molecule or binding compound.

[0136] A “multi-valent” binding compound has two or more binding sites. Thus, the terms “bivalent”, “trivalent”, and “tetravalent” refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, a bispecifc antibody according to the invention is at least bivalent and may be trivalent, tetravalent, or otherwise multi-valent. A large variety of methods and protein configurations are known and used for the preparation of bispecific monoclonal antibodies (BsMAB), tri-specific antibodies, and the like.

[0137] The term “chimeric antigen receptor” or “CAR” is used herein in the broadest sense to refer to an engineered receptor, which grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) to membrane-spanning and intracellular-signaling domains. Typically, the receptor is used to graft the specificity of a monoclonal antibody onto a T cell to create a chimeric antigen receptor (CAR). (Dai et ak, J Natl Cancer Inst, 2016; 108(7):djv439; and Jackson et ak, Nature Reviews Clinical Oncology, 2016; 13:370-383.).

[0138] The term “human antibody” is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term “human antibody” specifically includes heavy chain-only antibodies having human heavy chain variable region sequences, produced by transgenic animals, such as transgenic rats or mice, in particular UniAbs™ produced by UniRats™, as defined above.

[0139] By a “chimeric antibody” or a “chimeric immunoglobulin” is meant an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, e.g., a transgenic antibody comprising a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human Fc-regions or artificial Fc-regions, and human idiotypes. Such immunoglobulins can be isolated from animals of the invention that have been engineered to produce such chimeric antibodies.

[0140] As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and carry out specific immune functions. In some embodiments, an effector cell, such as a natural killer cell, is capable of inducing antibody- dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages, which express FcR, are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, an effector cell may phagocytose a target antigen or target cell.

[0141] “Human effector cells” are leukocytes which express receptors such as T cell receptors or FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with NK cells being preferred. The effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.

[0142] The term “immune cell” is used herein in the broadest sense, including, without limitation, cells of myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0143] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody -dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

[0144] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0145] “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed.

[0146] “Binding affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound.

[0147] As used herein, the “Kd” or “Kd value” refers to a dissociation constant determined by BioLayer Interferometry, using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, anti-mouse Fc sensors are loaded with mouse-Fc fused antigen and then dipped into antibody-containing wells to measure concentration dependent association rates (kon). Antibody dissociation rates (koff) are measured in the final step, where the sensors are dipped into wells containing buffer only. The Kd is the ratio of koff/kon. (For further details see, Concepcion, J, et ak, Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0148] The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0149] A “therapeutically effective amount” is intended for an amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a “therapeutically effective amount” is an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with a disease or which improves resistance to a disorder.

[0150] A “sirtuin” refers to a member of a class of proteins that possess either mono-ADP- ribosyltransferase or deacylase activity, including deacetylase, desuccinylase, demalonylase, demyristoylase and depalmitoylase activity. Sirtuins are generally involved with cellular processes such as aging, transcription, apoptosis, inflammation, stress resistance, energy efficiency, and alertness during low-calorie situations. Satoh et ak, The Journal of Neuroscience . 30 (30): 10220-32 (July 2010). As used herein, “sirtuin” includes all sirtuin subtypes, including, without limitation, all mammalian sirtuins (SIRT1-7), which occupy different subcellular compartments: SIRT1, SIRT6 and SIRT7 are predominantly found in the nucleus, SIRT2 in the cytoplasm, and SIRT3, SIRT4 and SIRT5 in the mitochondria. Ye et al., Oncotarget (Review). 8 (1): 1845-1859 (January 2017).

[0151] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated. In an embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having cancer, and/or individuals with autoimmune diseases, and the like. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mouse, rat, etc.

[0152] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.

[0153] The terms “synergy” and “synergistic” as used herein refer to a combination of two or more individual components (e.g., two or more heavy -chain antibodies) that are together more effective at achieving a particular result (e.g., a reduction in hydrolase activity) as compared to the results achieved when the two or more individual components are used separately. For example, a synergistic combination of two or more hydrolase blocking heavy-chain antibodies is more effective at inhibiting hydrolase activity than either of the individual hydrolase blocking heavy-chain antibodies when used separately. Similarly, a synergistic therapeutic combination is more effective than the effects of the two or more single agents that make up the therapeutic combination. A determination of a synergistic interaction between two or more single agents in a therapeutic combination can be based on results obtained from various assays known in the art. The results of these assays can be analyzed using the Chou and Talalay combination method and Dose-Effect Analysis with CalcuSyn software in order to obtain a Combination Index “Cl” (Chou and Talalay (1984) Adv. Enzyme Regul. 22:27-55). A combination therapy may provide “synergy” and prove “synergistic”, i.e., the effect achieved when the active ingredients used together is greater than the effects that result from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, dining alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially in time.

[0154] A “sterile” formulation is aseptic or free or essentially free from all living microorganisms and their spores. A “frozen” formulation is one at a temperature below 0 °C.

[0155] A “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993), for example. Stability can be measured at a selected temperature for a selected time period. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy -terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomeriation), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

II. Detailed Description

[0156] The invention is based, at least in part, on the finding that binding compounds, such as heavy- chain antibodies, that have binding specificity to one or more epitopes on an ectoenzyme can be used to inhibit enzymatic activity of a target ectoenzyme, and thereby treat various diseases or disorders that are characterized by ectoenzyme activity. The invention is also based, at least in part, on the finding that binding compounds, or combinations thereof, that have binding specificity for at least two epitopes on an ectoenzyme (e.g., multispecific, e.g., bispecific binding compounds) work synergistically to modulate (e.g., inhibit) enzymatic activity of the target ectoenzyme. Aspects of the invention therefore relate to binding compounds, including, without limitation, monospecific binding compounds having binding specificity for a single target (e.g., a single epitope on an ectoenzyme), as well as multispecific (e.g., bispecific) binding compounds having binding specificity for at least two targets (e.g., a first and a second epitope on an ectoenzyme). Aspects of the invention also relate to therapeutic combinations of the binding compounds described herein, as well as methods of making and using such binding compounds.

Ectoenzymes

[0157] Ectoenzymes are a diverse group of membrane proteins having catalytic sites outside the plasma membrane. Many ectoenzymes are found on leukocytes and endothelial cells, where they play multiple biological roles. Apart from the extracellular catalytic activity that is common to all, ectoenzymes are a diverse class of molecules that are involved in very different types of enzymatic reactions. Different ectoenzymes can modulate each step of leukocyte-endothelial contact interactions, as well as subsequent cell migration in tissues. Ectoenzymes include, without limitation, CD38, CD10, CD13, CD26, CD39, CD73, CD156b, CD156c, CD157, CD203, VAP1, ART2, and MT1-MMP.

[0158] The ectoenzyme CD38 belongs to the family of nucleotide-metabolizing enzymes which, in addition to recycling nucleotides, generate compounds that control cellular homeostasis and metabolism. The catalytic activity of CD38 is required for various physiological processes, including insulin secretion, muscarinic Ca²⁺ signaling in pancreatic acinar cells, neutrophil chemotaxis, dendritic cell trafficking, oxytocin secretion, and in the development of diet-induced obesity. See, Vaisitti et ak, Laeukemia, 2015, 29: 356-368, and the references cited therein. CD38 has bifunctional ecto-enzymatic cyclase as well as hydrolase activity. CD38 is expressed in a variety of malignancies, including chronic lymphocytic leukemia (CLL). CD38 has been shown to identity a particularly aggressive form of CLL, and is considered a negative prognostic marker, predicting a shorter overall survival of patients with this aggressive variant of CLL. See, Malavasi et ak, 2011, Blood, 118:3470-3478, and Vaisitti, 2015, supra.

[0159] CD38 is also expressed on solid tumors, and is overexpressed on tumor cells of PD 1 -refractory non-small cell lung cancer patients (SNCLC) (Chen et ak, Cancer Discov, 8(9): 1156-75). CD38 possibly plays a role in other solid tumors that are resistant to immune checkpoint blockade, such pancreatic tumors, renal cell carcinoma, melanoma, colo-rectal carcinoma, and others.

Anti-CD38 Binding Compounds

[0160] Aspects of the invention include binding compounds having binding affinity to an ectoenzyme, such as CD38. The binding compounds can include, without limitation, a variety of antibody-like molecules, such as those depicted in FIG. 6. In some embodiments, a binding compound includes a variable domain of an antibody having binding affintity to a particular epitope on an ectoenzyme. In some embodiments, a binding compound includes at least one antigen-binding domain of a heavy -chain antibody having binding affinity to a particular epitope. In certain embodiments, a binding compound includes two or more antigen-binding domains, wherein one antigen-binding domain has binding affinity to a first epitope, and one antigen-binding domain has binding affinity to a second epitope. In certain embodiments, the epitopes are non-overlapping epitopes. In certain embodiments, the epitopes are partially overlapping epitopes that share at leat one amino acid residue in common with one another. The binding compounds described herein provide a number of benefits that contribute to utility as clinically therapeutic agent(s). The binding compounds include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity.

[0161] Aspects of the invention include heavy-chain antibodies that bind to human CD38. The antibodies comprise a set of CDR sequences as defined herein and shown in FIGS. 1 and 3, and are exemplified by the provided heavy chain variable region (VH) sequences set forth in FIGS. 2 and 4. The antibodies provide a number of benefits that contribute to utility as clinically therapeutic agent(s). The antibodies include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity.

[0162] A suitable binding compound may be selected from those provided herein for development and therapeutic or other use, including, without limitation, use as a bispecific binding compound, e.g., as shown in FIG. 6, or a tri-specific antibody, or part of a CAR-T structure.

[0163] Determination of affinity for a candidate protein can be performed using methods known in the art, such as Biacore measurements. Binding compounds described herein may have an affinity for CD38 with a Kd of from about 10⁶ to around about 10ⁿ, including without limitation: from about 10⁶ to around about 10¹⁰; from about 10⁶ to around about 10⁹; from about 10⁶ to around about 10⁸; from about 10⁸ to around about 10ⁿ; from about 10⁸ to around about 10¹⁰; from about 10⁸ to around about 10⁹; from about 10⁹ to around about 10ⁿ; from about 10⁹ to around about 10¹⁰; or any value within these ranges. The affinity selection may be confirmed with a biological assessment for modulating, e.g., blocking, a CD38 biological activity, such as hydrolase activity, including in vitro assays, pre-clinical models, and clinical trials, as well as assessment of potential toxicity.

[0164] The binding compounds described herein are not cross-reactive with the CD38 protein of Cynomolgus macaque, but can be engineered to provide cross-reactivity with the CD38 protein of Cynomolgus macaque, or with the CD38 of any other animal species, if desired.

[0165] The CD38-specific binding compounds herein comprise an antigen-binding domain, comprising CDR1, CDR2 and CDR3 sequences in a human VH framework. The CDR sequences may be situated, as an example, in the region of around amino acid residues 26-35; 53-59; and 98-117 for CDR1, CDR2 and CDR3, respectively, of the provided exemplary variable region sequences set forth in the figures. It will be understood by one of ordinary skill in the art that the CDR sequences may be in different positions if a different framework sequence is selected, although generally the order of the sequences will remain the same.

[0166] Representative CDR1, CDR2 and CDR3 sequences are shown in FIGS. 1 and 3. [0167] In some embodiments, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising a CDR1 sequence of any one of SEQ ID NOs: 1- 9. In one particular embodiment, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 1.

[0168] In some embodiments, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising a CDR2 sequence of any one of SEQ ID NOs: 11-21. In one particular embodiment, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising a CDR2 sequence of SEQ ID NO: 11.

[0169] In some embodiments, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising a CDR3 sequence of any one of SEQ ID NOs: 22-26. In one particular embodiment, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising a CDR3 sequence of SEQ ID NO: 26.

[0170] In some embodiments, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising a CDR1 sequence of any one of SEQ ID NOs: 72-85. In one particular embodiment, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 72.

[0171] In some embodiments, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising a CDR2 sequence of any one of SEQ ID NOs: 86-93. In one particular embodiment, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising a CDR2 sequence of SEQ ID NO: 86

[0172] In some embodiments, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising a CDR3 sequence of any one of SEQ ID NOs: 94-95. In one particular embodiment, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising a CDR3 sequence of SEQ ID NO: 94.

[0173] In a further embodiment, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 1; the CDR2 sequence of SEQ ID NO: 11 ; and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 72; the CDR2 sequence of SEQ ID NO: 86; and the CDR3 sequence of SEQ ID NO: 94.

[0174] In a further embodiment, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising a sequence of any one of SEQ ID NOs: 28-71. In a further embodiment, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising a sequence of any one of SEQ ID NOs: 96-135. In one embodiment, an anti-CD38 antibody of the invention comprises a first binding unit comprising a heavy chain variable region comprising the SEQ ID NO: 28. In one embodiment, an anti-CD38 antibody of the invention comprises a second binding unit comprising a heavy chain variable region comprising the SEQ ID NO: 96.

[0175] In some embodiments, an anti-CD38 antibody preferably comprises a first binding unit comprising a heavy chain variable region (VH) in which the CDR3 sequence has greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,

75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in FIG. 1, and binds to a first epitope on CD38.

[0176] In some embodiments, an anti-CD38 antibody preferably comprises a second binding unit comprising a heavy chain variable region (VH) in which the CDR3 sequence has greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,

75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in FIG. 3, and binds to a second epitope on CD38.

[0177] In some embodiments, an anti-CD38 antibody preferably comprises a first binding unit comprising a heavy chain variable region (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in FIG. 1, and binds to a first epitope CD38.

[0178] In some embodiments, an anti-CD38 antibody preferably comprises a second binding unit comprising a heavy chain variable region (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in FIG. 3, and binds to a second epitope CD38. [0179] In some embodiments, an anti-CD38 antibody comprises a first binding unit comprising a heavy chain variable region sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the heavy chain variable region sequences provided in FIG. 2, and binds to a first epitope on CD38.

[0180] In some embodiments, an anti-CD38 antibody comprises a second binding unit comprising a heavy chain variable region sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the heavy chain variable region sequences provided in FIG. 4, and binds to a second epitope on CD38.

[0181] Aspects of the invention include anti-CD38 antibodies comprising a first binding unit that competes for binding to a first epitope (referred to herein as the FI 1 A epitope) on a CD38 protein (e.g, a human CD38 protein (SEQ ID NO: 136)) with a member of the F11A family of antibodies whose sequences are provided in FIGS. 1-2. In some embodiments, the first epitope is a conformational epitope comprising two or more amino acid residues selected from the group consisting of amino acid residues 120, 135, 139, 142, 202, 203, 205, 236, 237, 239, 241, 252, 254, 255, 272-279, 284, 287, 288, 291-294, 296, 297, 299 and 300 of SEQ ID NO: 136. In some embodiments, the first epitope is a conformational epitope comprising amino acid residues 120, 135, 139, 142, 202, 203, 205, 236, 237, 239, 241, 252, 254, 255, 272-279, 284, 287, 288, 291-294, 296, 297, 299 and 300 of SEQ ID NO: 136.

[0182] Aspects of the invention include anti-CD38 antibodies comprising a second binding unit that competes for binding to a second epitope (referred to herein as the F12A epitope) on a CD38 protein (e.g, a human CD38 protein (SEQ ID NO: 136)) with a member of the F12A family of antibodies whose sequences are provided in FIGS. 3-4. In some embodiments, the second epitope is a conformational epitope comprising two or more amino acid residues selected from the group consisting of amino acid residues 188-201, 262-264, and 275-284 of SEQ ID NO: 136. In some embodiments, the second epitope is a conformational epitope comprising amino acid residues 188-201, 262-264, and 275-284 of SEQ ID NO: 136. In some embodiments, the second binding unit competes for binding to the second epitope with Isatuximab.

[0183] Additional anti-CD38 antibody binding sequences and their properties are described in PCT Application No. PCT/US2020/066088, the disclosure of which is incorporated by reference herein in its entirety.

[0184] In some embodiments, an anti-CD38 antibody of the invention comprises a first binding unit that binds to the F11A epitope, comprising one or more amino acid residues that modulate CD38 enzymatic activity (e.g., inhibit CD38 enzymatic activity). For example, in some embodiments, an anti- CD38 antibody in accordance with embodiments of the invention comprises a first binding unit comprising one or more amino acid residues selected from the group: S31, G33, H35, E44, R45, A49, V50, D53, D54, S56, N57 and K58. As provided in FIG. 16, these amino acid residues, when present in an antibody sequence that binds to the FI 1 A epitope, reduce CD38 enzymatic activity.

[0185] In some embodiments, an anti-CD38 antibody of the invention comprises a second binding unit that binds to the F12A epitope, comprising one or more amino acid residues that modulate CD38 enzymatic activity (e.g., inhibit CD38 enzymatic activity). For example, in some embodiments, an anti- CD38 antibody in accordance with embodiments of the invention comprises a first binding unit comprising one or more amino acid residues selected from the group: G16, S31, S32, W33, S35, V37, A49, N50, K52, Q53, D54, E57, K58, D59, V61, and E89. As provided in FIG. 17, these amino acid residues, when present in an antibody sequence that binds to the F12A epitope, reduce CD38 enzymatic activity.

[0186] In some embodiments, bispecific or multispecific binding compounds are provided, which may have any of the configurations discussed herein, including, without limitation, a bispecific, bivalent heavy -chain antibody comprising two non-identical polypeptide subunits that are associated with one another via an asymmetric interface; a bispecific, tetravent heavy-chain antibody comprising two identical polypeptide subunits, each containing a first and a second antigen-binding domain; a bispecific, tetravalent heavy-chain antibody comprising two identical heavy chain polypeptide subunits and two identical light chain polypeptide subunits; or a bispecific three-chain antibody-like molecule, comprising a first heavy chain polypeptide subunit, a first light chain polypeptide subunit, and a second heavy chain polypeptide subunit.

[0187] In some embodiments, a bispecific antibody can comprise a heavy chain/light chain pair that has binding specificity for a first antigen, and a heavy chain from a heavy chain-only antibody, comprising an Fc portion comprising CH2 and/or CH3 and/or CH4 domains, in the absence of a CHI domain, and an antigen binding domain that binds an epitope of a second antigen or a different epitope of the first antigen (e.g., a second epitope on a CD38 protein).

[0188] In some embodiments, where a protein of the invention is a bispecific antibody, one arm of the antibody (one binding unit, or binding moiety) binds to a first epitope on CD38, while the other arm of the antibody binds to a second epitope on CD38.

[0189] Various formats of bispecific binding compounds are within the ambit of the invention, including, without limitation, single chain polypeptides, two chain polypeptides, three chain polypeptides, four chain polypeptides, and multiples thereof.

[0190] In some embodiments, a binding compound includes a first and a second polypeptide, i.e., a first and a second polypeptide subunit, wherein each polypeptide comprises an antigen-binding domain of a heavy-chain antibody. In some embodiments, each of the first and second polypeptides further includes a hinge region, or at least a portion of a hinge region, which can facilitate formation of at least one disulfide bond between the first and second polypeptides. In some embodiments, each of the first and second polypeptides further includes at least one heavy chain constant region (CH) domain, such as a CH2 domain, and/or a CH3 domain, and/or a CH4 domain. In certain embodiments, the CH domain lacks a CHI domain. The antigen-binding domain of each of the first and second polypeptides can incorporate any of the CDR sequences and/or variable region sequences described herein in order to impart antigen-binding capability on the binding compound. As such, in certain embodiments, each polypeptide in the binding compound can include an antigen-binding domain that has binding specificity to the same epitope, or to different epitopes (e.g., a first and a second epitope on CD38 protein).

[0191] In some embodiments, a binding compound comprises a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence comprising an S228P mutation, an F234A mutation, an L235 A mutation, and a T366W mutation (knob), and a second heavy chain constant region sequence comprising an S228P mutation, an F234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, and a Y407V mutation (hole). This variant, or modified, IgG4 Fc domain prevents unwanted Fab exchange, reduces effector function of the antibody, and also facilitates heterodimerization of the heavy chain polypeptide subunits to form the binding compound (e.g., a bispecific antibody).

[0192] A non-limiting example of a binding compound in accordance with embodiments of the invention is depicted in FIG. 6, Panel C. In the depicted embodiment, the binding compound is a bispecific, bivalent heavy-chain antibody that comprises a first polypeptide comprising an antigen binding domain of a heavy-chain antibody, at least a portion of a hinge region, a CH domain comprising a CH2 and a CH3 domain (and lacking a CHI domain), and a second polypeptide comprising an antigen-binding domain of a heavy -chain antibody, at least a portion of a hinge region, and a CH domain comprising a CH2 and a CH3 domain (and lacking a CHI domain). The depicted embodiment includes an asymmetric interface between the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide, and at least one disulfide bond in the hinge region that connects the first and second polypeptides to form the binding compound. Asymmetric interfaces in accordaince with embodiments of the invention are further described herein.

[0193] In some embodiments, a binding compound includes a first and a second polypeptide, i.e., a first and a second polypeptide subunit, wherein each polypeptide comprises two antigen binding domains. In some embodiments, each of the first and second polypeptides further includes a hinge region, or at least a portion of a hinge region, which can facilitate formation of at least one disulfide bond between the first and second polypeptides. In some embodiments, each of the first and second polypeptides further includes at least one heavy chain constant region (CH) domain, such as a CH2 domain, and/or a CH3 domain, and/or a CH4 domain. In certain embodiments, the CH domain lacks a CHI domain. The antigen-binding domain of each of the first and second polypeptides can incorporate any of the CDR sequences and/or variable region sequences described herein in order to impart antigen binding capability on the binding compound. As such, in certain embodiments, each polypeptide in the binding compound can include two antigen-binding domains, having binding specificity to the same epitope, or to different epitopes (e.g., a first and a second epitope on a CD38 protein).

[0194] A non-limiting example of a binding compound in accordance with embodiments of the invention is depicted in FIG. 6, Panel B. In the depicted embodiment, the binding compound is a bispecific, tetravalent binding compound that comprises a first polypeptide comprising two antigen binding domains, one with binding specificity to a first epitope and one with binding specificity to a second, non-overlapping epitope, at least a portion of a hinge region, a CH domain comprising a CH2 and a CH3 domain (and lacking a CHI domain), and a second polypeptide comprising two antigen binding domains, one with binding specificity to the first epitope and one with binding specificity to the second epitope, at least a portion of a hinge region, a CH domain comprising a CH2 and a CH3 domain (and lacking a CHI domain). The depicted embodiment includes at least one disulfide bond in the hinge region that connects the first and second polypeptides to form the binding compound.

[0195] In some embodiments, the first and second antigen-binding domains on each polypeptide are connected by a polypeptide linker. One non-limiting example of a polypeptide linker that can connect the first and second antigen-binding domains is a GS linker, such as the G4S linker having the amino acid sequence GGGGS (SEQ ID NO: 139). Other suitable linkers can also be used, and are described, for example, in Chen et ak, Adv Drug Deliv Rev. 2013 October 15; 65(10: 1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0196] In some embodiments, a binding compound includes a first and a second heavy chain polypeptide, i.e., first and second heavy chain polypeptide subunits, as well as a first and a second light chain polypeptide, i.e., first and second light chain polypeptide subunits. In some embodiments, each of the heavy chain polypeptides comprises an antigen-binding domain of a heavy -chain antibody. In some embodiments, each of the heavy chain polypeptides further includes a hinge region, or at least a portion of a hinge region, which can facilitate formation of at least one disulfide bond between the first and second heavy chain polypeptides. In some embodiments, each of the first and second heavy chain polypeptides further includes at least one heavy chain constant region (CH) domain, such as a CH2 domain, and/or a CH3 domain, and/or a CH4 domain. In certain embodiments, the CH domain includes a CHI domain. The antigen-binding domain of each of the first and second heavy chain polypeptides can incorporate any of the CDR sequences and/or variable region sequences described herein in order to impart antigen-binding capability on the binding compound.

[0197] In some embodiments, each of the light chain polypeptides comprises an antigen-binding domain of a heavy-chain antibody. In some embodiments, each of the light chain polypeptides further includes a light chain constant region (CL) domain. The antigen-binding domain of each of the first and second light chain polypeptides can incorporate any of the CDR sequences and/or variable region sequences described herein in order to impart antigen-binding capability on the binding compound. Additionally, the CHI domains on the heavy chain polypeptides and the CL domains on the light chain polypeptides can each include at least one cysteine residue that facilitates formation of a disulfide bond that connects each light chain polypeptide to one of the heavy chain polypeptides.

[0198] A non-limiting example of a binding compound in accordance with embodiments of the invention is depicted in FIG. 6, Panel A. In the depicted embodiment, the binding compound is a bispecific, tetravalent binding compound comprising two heavy chain polypeptides and two light chain polypeptides. Each heavy chain polypeptide comprises an antigen-binding domain with binding specificity to a first epitope, a CHI domain, at least a portion of a hinge region, a CH2 domain and a CH3 domain. The depicted embodiment includes at least one disulfide bond in the hinge region that connects the first and second heavy chain polypeptides. Each light chain polypeptide comprises an antigen-binding domain with binding specificity to a second epitope, and a CL domain. The depicted embodiment includes at least one disulfide bond between the CL and CHI domains that connects the first and second heavy chain polypeptides to the first and second light chain polypeptides to form the binding compound.

[0199] A non-limiting example of a binding compound in accordance with embodiments of the invention is depicted in FIG. 6, Panel D. In the depicted embodiment, the binding compound is a bispecific, bivalent binding compound comprising three polypeptides (two heavy chain polypeptides and one light chain polypeptide). The first heavy chain polypeptite subunit and the light chain polypeptide subunit together form a binding unit having binding affinity to a first epitope, and the second heavy chain polypeptide comprises a heavy chain-only variable region having binding affinity to a second epitope. In some embodiments, the second polypeptide subunit comprises a single heavy chain-only variable region domain (monovalent configuration). In some embodiments, the second polypeptide subunit comprises two heavy chain-only variable regions (bivalent configuration), connected by a linker. The first heavy chain polypeptide comprises an antigen-binding domain with binding specificity to a first epitope, a CHI domain, at least a portion of a hinge region, a CH2 domain and a CH3 domain. The depicted embodiment includes at least one disulfide bond in the hinge region that connects the first and second heavy chain polypeptides. The light chain polypeptide comprises an antigen-binding domain with binding specificity to the first epitope, and a CL domain.

[0200] Aspects of the invention include combinations (e.g., therapeutic combinations) of two or more binding compounds described herein. In some embodiments, a therapeutic combination comprises a first binding compound that has binding specificity for a first epitope on CD38, and a second binding compound that has binding specificity for a second epitope on CD38. Therapeutic combinations in accordance with embodiments of the invention can comprise two or more of the binding compound described herein, or can comprise one or more of the binding compounds described herein, as well as one or more binding compounds known in the art, e.g., one or more second antibodies that bind to CD38.

[0201] For example, Isatuximab (SAR650984), which is an antibody in clinical trials for the treatment of Multiple Myeloma, induces potent complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and indirect apoptosis of tumor cells. Isatuximab also blocks the cyclase and hydrolase enzymatic activities of CD38 and induces direct apoptosis of tumor cells. Aspects of the invention include therapeutic combinations that include one or more of the binding compounds described herein, as well as isatuximab. The heavy chain variable region sequence of isatuximab is provided in SEQ ID NO: 141, and the light chain variable region sequence of isatuximab is provided in SEQ ID NO: 142. Isatuximab is described, for example, in Deckert, J., et ak, “SAR650984, a novel humanized CD38-targeting antibody, demonstrates potent antitumor activity in models of multiple myeloma and other CD38+ hematologic malignancies.” Clin Cancer Res, 2014. 20(17): p. 4574-83, the disclosure of which is incorporated herein by reference in its entirety.

[0202] Daratumumab, an antibody specific for human CD38, was approved for human use in 2015 for the treatment of Multiple Myeloma (reviewed in Shallis et ak, Cancer Immunol. Immunothen, 2017, 66(6):697-703). Aspects of the invention include therapeutic combinations that include one or more of the binding compounds described herein, as well as daratumumab.

Preparation of anti-ectoenzvme binding compounds

[0203] The binding compounds of the present invention can be prepared by methods known in the art. In a preferred embodiment, the binding compounds herein are produced by transgenic animals, including transgenic mice and rats, preferably rats, in which the endogenous immunoglobulin genes are knocked out or disabled. In a preferred embodiment, the binding compounds herein are produced in UniRat™. UniRat™ have their endogenous immunoglobulin genes silenced and use a human immunoglobulin heavy-chain translocus to express a diverse, naturally optimized repertoire of fully human heavy-chain antibodies. While endogenous immunoglobulin loci in rats can be knocked out or silenced using a variety of technologies, in UniRat™ the zinc-finger (endo)nuclease (ZNF) technology was used to inactivate the endogenous rat heavy chain J-locus, light chain CK locus and light chain Cl locus. ZNF constructs for microinjection into oocytes can produce IgH and IgL knock out (KO) lines. For details see, e.g., Geurts et ak, 2009, Science 325:433. Characterization of Ig heavy chain knockout rats has been reported by Menoret et ak, 2010, Eur. J. Immunol. 40:2932-2941. Advantages of the ZNF technology are that non-homologous end joining to silence a gene or locus via deletions up to several kb can also provide a target site for homologous integration (Cui et ak, 2011, Nat Biotechnol 29:64-67). Human heavy-chain antibodies produced in UniRat™ are called UniAbs™ and can bind epitopes that cannot be attacked with conventional antibodies. Their high specificity, affinity, and small size make them ideal for mono- and poly-specific applications.

[0204] In addition to UniAbs™, specifically included herein are heavy chain-only antibodies lacking the camelid VHH framework and mutations, and their functional VH regions. Such heavy chain-only antibodies can, for example, be produced in transgenic rats or mice which comprise fully human heavy chain-only gene loci as described, e.g., in W02006/008548, but other transgenic mammals, such as rabbit, guinea pig, rat can also be used, rats and mice being preferred. Heavy chain-only antibodies, including their VHH or VH functional fragments, can also be produced by recombinant DNA technology, by expression of the encoding nucleic acid(s) in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), E. coli or yeast.

[0205] Domains of heavy chain-only antibodies combine advantages of antibodies and small molecule drugs: can be mono- or multi-valent; have low toxicity; and are cost-effective to manufacture. Due to their small size, these domains are easy to administer, including oral or topical administration, are characterized by high stability, including gastrointestinal stability; and their half-life can be tailored to the desired use or indication. In addition, VH and VHH domains of heavy -chain antibodies can be manufactured in a cost-effective manner.

[0206] In a particular embodiment, the heavy chain antibodies of the present invention, including UniAbs™, have the native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Rabat numbering system), substituted by another amino acid residue, which is capable of disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position. Such hydrophobic patches are normally buried in the interface with the antibody light chain constant region but become surface exposed in heavy -chain antibodies and are, at least partially, responsible for the unwanted aggregation and light chain association of heavy -chain antibodies. The substituted amino acid residue preferably is charged, and more preferably is positively charged, such as lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment, the heavy chain-only antibodies derived from the transgenic animals contain a Trp to Arg mutation at position 101. The resultant heavy -chain antibodies preferably have high antigen binding affinity and solubility under physiological conditions in the absence of aggregation.

[0207] In certain embodiments, a binding compound is an anti-ectoenzyme heavy chain antibody that binds to CD38. In a preferred embodiment, the anti-CD38 heavy chain antibodies are UniAbs™.

[0208] As part of the present invention, human IgG heavy chain anti-CD38 antibody families with unique CDR3 sequences from UniRat™ animals (UniAb™) were identified that bind human CD38 in ELISA (recombinant CD38 extracellular domain) protein and cell-binding assays. Heavy chain variable region (VH) sequences comprising two sequence families (FI 1, F12, see FIGS. 1-4) are positive for human CD38 protein binding and/or for binding to CD38+ cells, and are all negative for binding to cells that do not express CD38. UniAbs™ from these three sequence families fall into two broad synergistic groups based on the ability to inhibit the hydrolase function of CD38.

[0209] Combinations of two or more UniAbs™ binding to distinct, non-overlapping epitopes induce potent CDC activity and direct apoptosis, whereas the same UniAbs™, when administered alone, do not induce either of these effector functions. Combinations of UniAbs™ also inhibited enzymatic activities more potently than the individual UniAbs™ when administered alone. In other words, in certain embodiments, a combination of two different binding compounds (e.g., a therapeutic combination) of the present invention results in one or more synergistic results (e.g., synergistic CDC activity, synergistic enzymatic modulation activity, e.g., synergistic hydrolase blocking activity).

[0210] Binding compounds in accordance with embodiments of the invention bind to CD38-positive Burkitfs lymphoma cell line Ramos, and are cross-reactive with the CD38 protein of Cynomolgus macaque. In addition, they can be engineered to provide cross-reactivity with the CD38 protein of any animal species, if desired.

[0211] Binding compounds in accordance with embodiments of the invention may have an affinity for

CD38 with a Kd of from from about 1(U to around about 10ⁿ, including without limitation: from about 10⁶ to around about 10¹⁰; from about 10⁶ to around about 10⁹; from about 10⁶ to around about 10⁸; from about 10⁸ to around about 10ⁿ; from about 10⁸ to around about 10¹⁰; from about 10⁸ to around about 10⁹; from about 10⁹ to around about 10ⁿ; from about 10⁹ to around about 10¹⁰; or any value within these ranges. The affinity selection may be confirmed with a biological assessment for modulating, e.g. blocking, a CD38 biological activity, including in vitro assays, pre-clinical models, and clinical trials, as well as assessment of potential toxicity.

[0212] Binding compounds in accordance with embodiments of the invention which bind to two or more epitopes on an ectoenzyme target, including but not limited to anti-CD38 heavy chain antibodies, e.g., UniAbs™ can be identified by competition binding assays, such as enzyme-linked immunoassays (ELISA assays) or flow cytometric competitive binding assays. For example, one can use competition between known antibodies binding to the target antigen and the antibody of interest. By using this approach, one can divide a set of antibodies into those that compete with the reference antibody and those that do not. The non-competing antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized, the antigen is bound, and a second, labeled (e.g., biotinylated) antibody is tested in an ELISA assay for ability to bind the captured antigen. This can be performed also by using surface plasmon resonance (SPR) platforms, including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR imager (Ibis technologies B.V.), as well as on biolayer interferometry platforms, such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details see the Examples section below.

[0213] Typically, a binding compound (e.g., an antibody) competes with a reference binding compound (e.g., a reference antibody) if it causes about 15-100% reduction in the binding of the reference antibody to the target antigen, as determined by standard techniques, such as by the competition binding assays described herein. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher.

Antibody Drug Conjugates (ADCs)

[0214] Aspects of the invention include immunoconjugates, or antibody-drug conjugates (ADC), comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In another aspect, the invention further provides methods of using the immunoconjugates. In one aspect, an immunoconjugate comprises any of the above anti-CD38 antibodies covalently attached to a cytotoxic agent or a detectable agent. ADCs are described, for example, in US Patent No. 8,362,213, the disclosure of which is incorporated by reference herein in its entirety.

[0215] The use of ADCs for the local delivery of cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit tumor cells in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5 :543- 549; Wu et al (2005) Nature Biotechnology 23(9): 1137-1146; Payne, G. (2003) Cancer Cell 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al (ed.s), pp. 475-506). Efforts to improve the therapeutic index, i.e., maximal efficacy and minimal toxicity of ADC have focused on the selectivity of polyclonal (Rowland et al (1986) Cancer Immunol. Immunother., 21:183-87) and monoclonal antibodies (mAbs) as well as drug-linking and drug-releasing properties (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549). Drug moieties used in ADCs include bacterial protein toxins such as diphtheria toxin, plant protein toxins such as ricin, small molecules such as auristatins, geldanamycin (Mandler et al (2000) J. of the Nat. Cancer Inst. 92(19): 1573-1581 ; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342), daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al (1986) supra). The drug moieties may affect cytotoxic and cytostatic mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0216] The auristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin (WO 02/088172), have been conjugated as drug moieties to: (i) chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas); (ii) cACIO which is specific to CD30 on hematological malignancies (Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773; Doronina et al (2003) Nature Biotechnology 21(7):778-784; Francisco et al (2003) Blood 102(4): 1458- 1465; US 2004/0018194; (iii) anti-CD20 antibodies such as rituxan (WO 04/032828) for the treatment of CD20-expressing cancers and immune disorders; (iv) anti-EphB2R antibody 2H9 for treatment of colorectal cancer (Mao et al (2004) Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskar et al (2003) Cancer Res. 63 :6387-6394); (vi) trastuzumab (HERCEPTIN®, US 2005/0238649), and (vi) anti-CD30 antibodies (WO 03/043583). Variants of auristatin E are disclosed in U.S. Pat. No. 5,767,237 and U.S. Pat. No. 6,124,431. Monomethyl auristatin E conjugated to monoclonal antibodies are disclosed in Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004. Auristatin analogs MMAE and MMAF have been conjugated to various antibodies (US 2005/0238649).

[0217] Conventional means of attaching, i.e., linking through covalent bonds, a drug moiety to an antibody generally leads to a heterogeneous mixture of molecules where the drug moieties are attached at a number of sites on the antibody. For example, cytotoxic drugs have typically been conjugated to antibodies through the often-numerous lysine residues of an antibody, generating a heterogeneous antibody-drug conjugate mixture. Depending on reaction conditions, the heterogeneous mixture typically contains a distribution of antibodies with from 0 to about 8, or more, attached drug moieties. In addition, within each subgroup of conjugates with a particular integer ratio of drug moieties to antibody, is a potentially heterogeneous mixture where the drug moiety is attached at various sites on the antibody. Analytical and preparative methods may be inadequate to separate and characterize the antibody-drug conjugate species molecules within the heterogeneous mixture resulting from a conjugation reaction. Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Their reactivities with linker reagents and drug-linker intermediates are dependent on factors such as pH, concentration, salt concentration, and co-solvents. Furthermore, the multistep conjugation process may be non-reproducible due to difficulties in controlling the reaction conditions and characterizing reactants and intermediates.

[0218] Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and less nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Extracellular proteins generally do not have free thiols (Garman, 1997, Non- Radioactive Labelling: A Practical Approach, Academic Press, London, at page 55). Antibody cysteine thiol groups are generally more reactive, i.e., more nucleophilic, towards electrophilic conjugation reagents than antibody amine or hydroxyl groups. Cysteine residues have been introduced into proteins by genetic engineering techniques to form covalent attachments to ligands or to form new intramolecular disulfide bonds (Better et al (1994) J. Biol. Chem. 13:9644-9650; Bernhard et al (1994) Bioconjugate Chem. 5:126-132; Greenwood et al (1994) Therapeutic Immunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci. USA 96:4862-4867; Kanno et al (2000) J. of Biotechnology, 76:207-214; Chmura et al (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; U.S. Pat. No. 6,248,564). However, engineering in cysteine thiol groups by the mutation of various amino acid residues of a protein to cysteine amino acids is potentially problematic, particularly in the case of unpaired (free Cys) residues or those which are relatively accessible for reaction or oxidation. In concentrated solutions of the protein, whether in the periplasm of E. coli, culture supernatants, or partially or completely purified protein, unpaired Cys residues on the surface of the protein can pair and oxidize to form intermolecular disulfides, and hence protein dimers or multimers. Disulfide dimer formation renders the new Cys unreactive for conjugation to a drug, ligand, or other label furthermore, if the protein oxidatively forms an intramolecular disulfide bond between the newly engineered Cys and an existing Cys residue, both Cys thiol groups are unavailable for active site participation and interactions furthermore, the protein may be rendered inactive or non-specific, by misfolding or loss of tertiary structure (Zhang et al (2002) Anal. Biochem. 311:1-9).

[0219] Cysteine -engineered antibodies have been designed as Lab antibody fragments (thioLab) and expressed as full-length, IgG monoclonal (thioMab) antibodies (Junutula, J. R. et al. (2008) J Immunol Methods 332:41-52; US 2007/0092940, the contents of which are incorporated by reference). ThioLab and ThioMab antibodies have been conjugated through linkers at the newly introduced cysteine thiols with thiol-reactive linker reagents and drug-linker reagents to prepare antibody drug conjugates (Thio ADC).

Cellular Internalization

[0220] In some embodiments, the antibodies or antibody-drug conjugates of the invention, once bound to a binding target (e.g., CD38), internalize into cells, where internalization is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% or more, in comparison to one or more control antibodies as described herein. In some embodiments, aspects of the methods described herein involve internalizing an antibody or antibody -drug conjugate within a cell to achieve a desired effect, e.g., to deliver a cytotoxic or a cytostatic agent to the cell.

Pharmaceutical Compositions

[0221] It is another aspect of the present invention to provide pharmaceutical compositions comprising one or more binding compounds of the present invention in admixture with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

[0222] In one embodiment, a pharmaceutical composition comprises two or more heavy-chain antibodies binding to two different epitopes on an ectoenzyme, such as, for example, CD38. In a preferred embodiment, the pharmaceutical compositions comprise synergistic combinations of two or more heavy-chain antibodies binding to two different epitopes of an ectoenzyme, such a, for example, CD38.

[0223] In another embodiment, a pharmaceutical composition comprises a multi-specific (including bispecific) heavy -chain antibody with binding specificity for two or more epitopes on an ectoenzyme, such as, for example, CD38. In a preferred embodiment, a pharmaceutical composition comprises a multi-specific (including bispecific) heavy-chain antibody with binding specificity to two or more epitopes on an ectoenzyme, e.g., CD38, having synergistically improved properties relative to any of the monospecific antibodies binding to the same epitopes.

[0224] Pharmaceutical compositions of the binding compounds used in accordance with the present invention are prepared for storage by mixing proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; saltforming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0225] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). The formulation depends on the route of administration chosen. The binding compounds herein can be administered by intravenous injection or infusion or subcutaneously. For injection administration, the binding compounds herein can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection. The solution can contain carriers, excipients, or stabilizers as discussed above. Alternatively, binding compounds can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0226] Anti-CD38 antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy chain antibodies, including UniAbs™, of the present invention. Subcutaneous antibody formulations are described, for example, in US 20160355591 and US 20160166689.

Articles of Manufacture

[0227] Aspects of the invention include articles of manufacture, or “kits”, containing one or more binding compounds of the invention that are useful for the treatment of the diseases and disorders described herein. In one embodiment, a kit comprises a container comprising an anti-CD38 binding compound as described herein. The kit may further comprise a label or package insert, on or associated with the container. The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container may be formed from a variety of materials such as glass or plastic. The container may hold one or more anti-CD38 binding compounds as described herein, or a formulation thereof, e.g., a combination formulation of two or more anti-CD38 binding compounds, which is effective for treating a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the condition of choice, such as a cancer or an immunological disorder. Alternatively, or additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate -buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, and syringes.

[0228] The kit may further comprise directions for the administration of one or more binding compounds and, if present, a combination formulation thereof. For example, if the kit comprises a first pharmaceutical composition comprising a first anti-CD38 binding compound and a second pharmaceutical composition comprising a second anti-CD38 binding compound, the kit may further comprise directions for the simultaneous, sequential or separate administration of the first and second pharmaceutical compositions to a patient in need thereof. Where a kit comprises two or more compositions, the kit may comprise a container for containing the separate compositions, such as a divided bottle or a divided foil packet, however, the separate compositions may also be contained within a single, undivided container. A kit can comprise directions for the administration of the separate components, or for the administration a combined formulation thereof.

Methods of Use

[0229] The binding compounds described herein, which bind to two different epitopes on an ectoenzyme, combinations, including synergistic combinations, of such binding compounds, multi specific antibodies with binding specificities to two or more different epitopes on an ectoenzyme, and pharmaceutical compositions comprising such antibodies and antibody combinations, can be used to target diseases and conditions characterized by the expression of the target ectoenzyme.

[0230] In various embodiments, the ectoenzyme is selected from the group consisting of CD10, CD13, CD26, CD38, CD39, CD73, CD156b, CD156c, CD157, CD203, VAP1, ART2, and MT1-MMP.

[0231] In a particular embodiment, the ectoenzyme is CD38.

[0232] CD38 is a 46-kDa type II transmembrane glycoprotein with a short 20-aa N-terminal cytoplasmic tail and a long 256-aa extracellular domain (Malavasi et al Immunol. Today, 1994, 15:95- 97). Due to its high level of expression in a number of hematological malignancies, including multiple myeloma (MM), non-Hodgkin’s lymphoma (reviewed in Shallis et al., Cancer Immunol. Immunother., 2017, 66(6):697-703), B-cell chronic lymphocylic leukemia (CLL) (Vaisitti et al., Leukemia, 2015, 29”356-368), B-cell acute lymphoblastic leukemia (ALL), an dT-cell ALL, CD38 is a promising target for antibody-based therapeutics to treat hematological malignancies. CD38 has also be implicated as a key actor in age-related nicotinamide adenine dinucleotide (NAD) decline, and it has been suggested that CD38 inhibition, combined with NAD precursors may serve as a potential therapy for metabolic dysfunction and age-related diseases (see, e.g., Camacho-Pereira et al., Cell Metabolism 2016, 23:1127- 1139). CD38 has also been described as being involved in the development of airway hyperresponsiveness, a hallmark feature of asthma, and has been suggested as a target to treat such conditions.

[0233] Nicotinamide adenine dinucleotide (NAD+) metabolism plays a critical role in many inflammatory disorders, including metabolic diseases and Alzheimer's disease. NAD is a major coenzyme in bioenergetic processes and its cleavage by several enzymes, including CD38, is key to many biological processes such as cell metabolism, inflammatory responses and cell death (Chini et al., Trends Pharmacol Sci, 39(4):424-36.

[0234] The NAD cleaving enzyme, CD38, promotes intestinal inflammation in animal models. CD38 is a multifunctional ectoenzyme involved in the degradation of NAD+ and the production of cellactivating metabolites such as adenosine diphosphate ribose (ADPR) and cyclic ADPR (cADPR). CD38 is mainly expressed on hematopoietic cells, such as T cells, B cells, and macrophages. Immune cells upregulate expression of CD38 after activation and differentiation. Based on animal studies, it appears that immune responses of both T cells, macrophages and neutrophils are modulated by CD38. High- level CD38 expression and its associated ectoenzymatic functions seem to enhance the development of inflammatory diseases. In contrast, CD38 deficiency, and concomitant increased NAD concentrations, reduces recruitment of cells to inflamed sites and reduces production of pro-inflammatory cytokines (Schneider et al., PLos One, 10(5): e0126007 (2015); Gerner et al., Gut, 06 September 2017, doi: 10.1136/gutjnl-2017-314241; Garcia-Rodriguez et al., Sci Rep, 8(1): 3357 (2018)). In autoimmune models, CD38-/- mice show ameliorated development of disease, less joint inflammation in a collagen- induced arthritis model and less inflammation of the gut in a dextran sulfate sodium (DSS) colitis model (Garcia-Rodriguez et al., Sci Rep, 8(1): 3357 (2018)). All these results combined support the hypothesis that colonic inflammation leads to a decrease in NAD levels in cells via activation of CD38. The subsequent NAD decline would decrease the activity of the NAD-dependent deacetylases (sirtuins) that are known to have anti-inflammatory and tissue protective effects.

[0235] Monoclonal antibodies against CD38 have been shown to be highly efficacious in the treatment of Multiple Myeloma (MM), however, they are not suitable for the treatment of IBD. Currently, four monoclonal antibodies are in clinical trials for the treatment of CD38+ malignancies. The most advanced is Daratumumab (Janssen Biotech) which was approved for human use by the FDA for the treatment of MM in 2015. All three anti-CD38 monoclonals antibodies in clinical trials for MM show similar favorable safety and efficacy profiles (van de Donk, et al., Blood 2017, blood-2017-06-740944; doi: https://doi.org/10.1182/blood-2017-06-740944). One monoclonal antibody (TAK-079) is in clinical trials for the treatment of auto-immune diseases including Systemic Lupus Erythematosis (SLE) and rheumatoid arthritis. Besides plasma cells, anti-CD38 monoclonal antibodies deplete other CD38+ cells in the spleen and blood, including all NK cells and ~50% of monocytes, T cells and B cells. Critical regulatory immune cells, such as Treg cells and Myeloid Derived Suppressor Cells (MDSC), are depleted in MM patients after treatment with anti-CD38 monoclonal antibodies, and expansion of effector T cells is observed (Krejcik, et al., Blood, 128(3): 384-94 (2016)). In all likelihood, expansion of anti-tumor effector T cells contributes to the effectiveness of anti-CD38 mAbs in MM. However, removing important regulatory immune cells in auto-immune diseases could lead to exacerbation of disease.

[0236] Inhibition of enzyme function of CD38 could be a safe and effective approach to treating inflammatory disorders. Several small molecule inhibitors, including one with strong potency (Kd-5nM, Haffner et al 2015) of CD38 have been developed (Haffner et al., J Med. Chem, 58(8): 3548-71 (2015)). This compound elevated NAD levels in tissues of mice 6 hours post-injection, indicating that inhibition of CD38 leads to higher intracellular NAD in mice. However, CD38 is also expressed in the brain and plays a role in behavior, so that such molecules have significant risk of toxicity. In contrast to small molecule compounds, antibodies cannot cross the blood-brain barrier, and generally have superior target specificity compared to small molecules and thus should have a significantly better safety profile. Inflammatory diseases include Multiple Sclerosis, Systemic Lupus Erythematosus, rheumatoid arthritis, Graft versus Host disease, etc.

[0237] Antibodies in clinical trials were selected on the basis of cytolysis and poorly inhibit biological functions of CD38, but modulation of these functions may also be relevant for cancer therapies. Recent papers by Chatterjee et al. and Chen et al. established that the CD38-NAD+ axis is important in preclinical models of lung cancer and melanoma. These studies indicate that high levels of NAD+, negatively regulated by CD38, preserve effector T cell (Teff) functionality.

[0238] The binding compounds described herein, including heavy chain-only anti-CD38 antibodies, antibody combinations, multi-specific antibodies, and pharmaceutical compositions herein can be used to target diseases and conditions characterized by the expression or overexpression of CD38, including, without limitation, the conditions and diseases listed herein.

[0239] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat telomere shortening diseases, including, but not limited to, accelerated aging, aplastic anemia, dyskeratosis congenita, Franconi’s anemia, or idiopathic pulmonary fibrosis.

[0240] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat inflammatory diseases, including, but not limited to, ulcerative colitis, graft v. host disease (GvHD), including acute, chronic and transplant-associated GvHD, or acute kidney injury.

[0241] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat fibrosis-associated disorders, including, but not limited to, scleroderma. [0242] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat metabolic syndromes, including, but not limited to, type II diabetes mellitus (T2DM), obesity, or systemic inflammation.

[0243] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat diseases or disorders characterized by reduced sirtuin activity, including, but not limited to, metabolic, cardiovascular, or neurodegenerative diseases or disorders, or cancer.

[0244] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat diseases or disorders characterized by doxorubicin-induced toxicity, including, but not limited to, cardiotoxicity.

[0245] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat organ transplantation-associated diseases or disorders, including, but not limited to, skin transplantation-associated diseases or disorders, or kidney transplantation-associated diseases or disorders.

[0246] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat cardiovascular diseases or disorders, including, but not limited to, cardiovascular disorders involving heart failure.

[0247] In one aspect, the CD38 binding compounds and pharmaceutical compositions herein can be used to treat a disease or disorder characterized by expression of CD38, including, but not limited to, a disease or disorder characterized by a hydrolase enzymatic activity of CD38, a cyclase enzymatic activity of CD38, or a combination thereof. In some embodiments, administering a CD38 binding compound as described herein to a subject results in an inhibition of CD38 enzymatic activity in the subject. In some embodiments, administering a CD38 binding compound as described herein to a subject results in an inhibition of CD38 enzymatic activity in the subject without inducing, directly or indirectly, lysis of CD38+ cells. In some embodiments, administering a CD38 binding compound as described herein to a subject results in an inhibition of CD38 enzymatic activity in the subject without depleting or activating CD38-expressing cells.

[0248] In certain embodiments, aspects of the methods involve administering nicotinamide mononucleotide (NMN) to a subject in combination with one or more CD38 binding compounds or pharmaceutical compositions. As is the case for the binding compounds described herein, NMN can also be administered to a subject in any suitable manner, including, but not limited to, oral administration, parenteral administration (i.e., injection), etc.

[0249] The CD38 binding compounds and pharmaceutical compositions herein can also be used to modulate (e.g., increase) the concentration of nicotinamide adenine dinucleotide (NAD+) in a cell by contacting the cell with the CD38 binding compound. In some embodiments, the methods further involve contacting the cell with NMN, or otherwise exposing the cell to NMN, to further modulate (e.g., further increase) the NAD+ concentration.

[0250] The CD38 binding compounds and pharmaceutical compositions herein can also be used to modulate (e.g., increase) sirtuin activity in a cell by contacting the cell with the CD38 binding compound. In some embodiments, the methods further involve contacting the cell with NMN to enhance the increase in sirtuin activity.

[0251] Effective doses of the compositions of the present invention for the treatment of disease vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is a human or another animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals may also be treated, e.g., companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy.

[0252] Dosage levels of the subject binding compounds, as well as NMN, can be readily determined by the ordinarily skilled clinician, and can be modified as required, e.g., as required to modify a subject's response to therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

[0253] In some embodiments, the therapeutic dosage the agent may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once every two weeks or once a month or once every 3 to 6 months. Therapeutic entities of the present invention are usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, therapeutic entities of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0254] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, directly or after reconstitution of solid (e.g., lyophilized) compositions. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0255] Toxicity of the binding compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD₅o (the dose lethal to 50% of the population) or the LDioo (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in humans. The dosage of the binding compounds described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0256] The compositions for administration will commonly comprise a binding compound of the invention dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et ak, eds., 1996)).

[0257] Also within the scope of the invention are articles of manufacture, or “kits” (as described above) comprising the active agents and formulations thereof, of the invention and instructions for use. The kit can further contain a least one additional reagent, e.g. a chemotherapeutic drug, etc. Kits typically include a label indicating the intended use of the contents of the kit. The term ‘Tabel” includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0258] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. EXAMPLES

Materials and Methods

[0259] The following materials and methods were utilized to carry out the examples described below. Cloning. Expression and Purification of Antibodies

[0260] Anti-CD38 heavy chain only antibodies (UniAbs) were generated using genetically engineered transgenic rats that express fully human IgG antibodies (UniRat) together with an NGS-based antibody discovery pipeline (TeneoSeek). UniRats were immunized with recombinant human CD38 protein fused to a his-tag (R&D Systems, Minneapolis, Minnesota, USA) for up to 8 weeks using either Titermax/Ribi or CFA/IFA adjuvant. Draining lymph nodes from all animals were then harvested, and total RNA was collected. cDNA samples containing the full heavy chain variable domain (VH) underwent next-generation sequencing using the MiSeq platform (Illumina, San Diego, California, USA) with 2x300 paired-end reads. Data from all animals were analyzed, and the most frequent 373 VH sequences were selected for cloning followed by expression in HEK 293 cells.

[0261] All the antibodies were expressed with ExpiCHO cells using a high titer protocol as described by the manufacturer (Thermo Fischer Scientific, #A29133). Clarified harvests were purified over protein A (MabSelect Sure, Cytiva). Neutralized protein A eluate was concentrated and polished by gel filtration (Superdex 200 Increase 10/30 GL) to remove aggregates. All antibodies were formulated in 20 mM Citrate, 100 mM NaCl pH 6.2 Flow Cytometry

[0262] Cells were combined with antibodies in flow buffer in a 96-well plate (IX PBS + 1% BSA + 0.1% NaN3) and incubated at 4°C in the dark for 30 minutes. The plate was then washed 2x with flow cytometry buffer and incubated in a 1:100 dilution of goat anti -human IgG PE secondary antibody. Following incubation, the cells were washed 2x with flow cytometry buffer, resuspended in flow buffer, and analyzed on the FACSCelesta system (BD Biosciences).

CD38 Hydrolase Inhibition Assay

[0263] CD38+ cells were incubated with antibodies in hydrolase assay buffer (40 mM Tris, 0.25 M

Sucrose, 0.8 mg/mL BSA, pH 7.4) in a black, flat-bottom 96-well plate for 15 min at room temperature. Following incubation, nicotinamide l,N6-ethenoadenine dinucleotide (sNAD+), an enzymatically active derivative of NAD+ that produces a fluorescence signal hydrolysis, was added to each well at a final concentration of 150 mM. Fluorescence (ex/em: 300/410) was measured over time using a SpectraMax i3x multi-mode plate reader. The enzymatic activity of CD38 (as determined by V0) was measured over the range of antibody concentrations. Percent inhibition was calculated as follows: 100- [100*(V0 (test article)/ V0 (untreated))]. Capping Experiment

[0264] Daudi cells were incubated with 10 mg/mL TNB-738 or daratumumab for 0, 30, 60, or 120 min at 37°C then fixed and permeabilized (FIX & PERM™ Cell Permeabilization Kit, ThermoFisher, #GAS003). Cells were then labelled with anti-human IgG FITC (Jackson Laboratories), anti-CD98 Alexa647 (Abeam, #ab23495, labelled in-house with Alexa647, (ThermoFisher, #A20186)) and Texas Red Phalloidin (Life Technologies, #T7471). Cells were mounted between slide and slip-cover with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, #P36931). Slides were analyzed with a confocal microscope (AIR, Nikon Imaging Software NIS) with 60x/4 lens. Cells with and without capping were counted, and percentage capping was graphed for 100 cells per treatment.

Internalization of Antibodies

[0265] Isotype control IgGl (R&D), TNB-738 or daratumumab were labelled with pHrodo iFL-labeled Fab fragment (Zenon™ pHrodo™ iFL IgG Labeling Reagents, ThermoFisher) for 10 minutes. 0.5 M Daudi cells were incubated with Fc Block (BD Biosciences) for 30 min at 4°C. Cells were then washed and incubated at 37°C with isotype control IgGl (R&D), TNB-738, or daratumumab (10 pg/mL) for 60 minutes. Following incubation, cells were washed with cold PBS, stained with DAPI, and fluorescence was measured using a FACS Verse cytometer with FACSuite Software version 1.0.6; post acquisition analysis was performed using FlowJo software.

Assessment of hPBMC Activation

[0266] Healthy volunteers' blood was collected at the Etablissement Francais du Sang (Nantes, France) from healthy donors. Written informed consent was provided according to institutional guidelines. PBMCs were isolated by Ficoll-Paque density -gradient centrifugation (Eurobio, Courtaboeuf, France). Remaining red blood cells and platelets were eliminated with a hypotonic solution. The cells were then washed and counted. 5 x 10⁵ hPBMC were incubated overnight at 37°C without antibodies or with TNB-738 at 5, 1 or 0.1 pg/mL or with anti CD3 (wells were pre-coated with OKT3 anti-CD3 clone at 10 pg/mL) and anti-CD28 (10 pg/mL, in the supernatant). Following incubation, Brefeldin A was added for 4 hours, and the cells were subsequently stained with Fixable Viability Dye eFluor 506 (ThermoFisher), anti-hCD3 PeCy7 (BD Biosciences), anti-hCD4 PercpCy5.5 (BD Biosciences), anti- CD 16 (clone 3G8, labeled in house with Alexa Fluor 488 IgG labeling kit from ThermoFisher), and anti-CD25 APC Cy7 (BD Biosciences). Cells were fixed and permeabilized (FIX & PERM Cell Fixation & Cell Permeabilization Kit, ThermoFisher) and stained with anti-hFoxP3 PE (BD Biosciences) and anti-hlFNy V450 (BD Biosciences). Fluorescence was measured using a FACS Verse cytometer with FACSuite Software version 1.0.6; post-acquisition analysis was performed using FlowJo software. Complement Dependent Cytotoxicity

[0267] Antibodies were incubated with tumor cells in a 96-well plate for 10 minutes at room temperature. After incubation, a 1:10 dilution of rabbit complement serum was added to each well and incubated for an additional 30 minutes at 37°C and 8% C02. Cell Titer Glo 2.0 was then added to each well and luminescence was measured on a SpectraMax i3x plate reader. Percent viability was calculated as follows: 100 * (RLUTest Article / RLUUntreated).

Antibody Dependent Cellular Cytotoxicity

[0268] NK cells were isolated from healthy -donor PBMCs using a commercial NK cell isolation kit (Miltenyi, Cat. # 130-092-657). The NK cells were then incubated with tumor cells and antibodies in a 96-well plate for 4 hours at 37°C and 8% C02. Following incubation, the supernatant was harvested and combined with LDH assay substrate and incubated for 30 minutes at room temperature. After incubation, a stop solution was added to each well. Absorbance (490 nm) was then analyzed on a SpectraMax i3x plate reader. Percent killing was calculated using the following formula: (ODsample well-ODtarget only) / (ODtarget+lysis-ODtarget only-ODNK only) %.

Direct Apoptosis

[0269] Antibodies were incubated with tumor cells in a 96-well plate for 24 hours at 37°C and 8% C02. The cells were then centrifuged and resuspended in a mixture of Annexin V binding buffer, FITC- conjugated Annexin V, and 7-AAD for 10 minutes at room temperature. Following incubation, the cells were analyzed flow cytometry to assess cell viability. A quad gate was used to distinguish between early apoptotic (Annexin V+, 7-AAD-), late apoptotic (Annexin V+, 7-AAD+), and viable cells (Annexin V-, 7-AAD-).

Assessment of TNB-738 on Timor Growth

[0270] A549 cells were plated at 5,000 cells/well in non-tissue culture treated plates with anti adherence rinsing solution and grown for a week to form spheroids. The spheroids were then treated with TNB-738, isotype control, or EGF on days 0, 3, 6, and 8 post-growth week. Tumor area was measured using a microscope camera and ImageJ Shape Descriptor Plugin and was plotted on days 0, 3, 6, 8, and 10. On day 10, NAD+ concentration was measured using the Cell Biolab NAD+ assay kit according to manufacturer’s instructions.

Measurement of NAD+ Concentration

[0271] Cells were plated in a 96-well plate and incubated with antibody for 24 hours at 37°C and 8% C02. The cells were then washed once with PBS and resuspended in IX Extraction Buffer from the Cell Biolab NAD+ assay kit. Cell lysates were made by homogenizing cells followed by centrifugation at 4°C and 14000 rpm for 5 minutes. The supernatant was harvested and NAD+ concentration was measured using Cell Biolab NAD+ assay kit, according to manufacturer’s instructions. Measurement of SIRT Activity

[0272] Cells were plated in a 96-well plate and incubated with antibody for 24 hours at 37°C and 5% C02. The cells were then washed once with PBS and resuspended in M-PER Mammalian Protein Extraction Reagent. Cell lysates were made by homogenizing cells followed by centrifugation at 4°C and 14000 rpm for 5 minutes. The supernatant was harvested and incubated in a 1:30 dilution of anti- SIRT1 or anti-SIRT3 antibody for 4 hours on a rotator at 4°C. Protein A slurry was then added, and samples were incubated for an additional 2 hours at room temperature. Immunoprecipitation buffer was added, followed by centrifugation at 2500 g for 2 minutes. The wash steps with IP buffer were repeated. IgG elution buffer was added for 5 minutes, after which the eluate was centrifuged at 2500 g for 2 minutes. The supernatant was neutralized with 1 M Tris (pH 9) at 1:10. SIRT activity was measured using the SIRT Glo assay kit, according to manufacturer’s instructions.

Epitope Binning

[0273] Binning experiments were done with Octet QK386 (Forte Bio). All the reagents were diluted in Kinetic Buffer (KB). KB consists of PBS containing 0.1% BSA, 0.02% Tween 20, and 1% sodium azide. Ni-NTA (Fortebio, Cat: 18-5101) sensors were loaded with 5 pg/ml of his-tagged recombinant human CD38 (Sino Biological: 10818-H08H) for 120 seconds. A brief baseline was set for 60 seconds in kinetic buffer. The first antibody was then bound to the antigen on the sensor for 180 seconds. This was followed by another baseline for 60 seconds. Then the sensor was dipped into the second antibody for 180 seconds followed by dissociation for 240 seconds. All reagents were made in kinetic buffer. The antibodies were tested at 200 nM. The sensors were regenerated and recharged with nickel between runs.

[0274] Additional binning experiments were run on BioRad ProteOn SPR biosensor using a GLC sensor chip. Each antibody was amine coupled to the chip surface using a standard 5 -minute activation with sulfo-NHS/EDC followed by a 5-minute injection of each mAb at 15 pg/ml in 10 mM sodium acetate pH 5.0 and a 5-minute blocking step with 1M ethanolamine. Human CD38 was also coupled to a surface as a control. Running buffer contained DPBS pH 7.4 with 0.05% tween-20. All data were collected at 25°C. Next, a stacking study was run over each of the antibody and huCD38 surfaces. The first injection was the binding of huCD38 at 400 nM for 90 seconds at 100 pl/min, followed by a second injection that contained separately each antibody as well as recombinant huCD38 at 400 nM. Expression and Purification of CD38 and UniDab

[0275] CD38 (UNP: P28907), modified to mutate glycolsylation sites and to optimize expression while maintaining the overall structure of CD38, was cloned in Pichia pastoris based expression vector to yield the expression of 45-300 amino acid of CD38 in fusion with His-tag at its C-terminus. The fusion protein was expressed by transforming in Pichia Pastoris strain, BICC 9450. The protein was purified from the supernatant by affinity column chromatography. To obtain homogenous form of the protein, it was further purified by gel filtration chromatography using Superdex-75 column. The protein was eluted in 20 mM HEPES buffer, pH7.2 with 50 mM NaCl.

[0276] UniDabFl 1 A (antigen binding fragment of heavy chain only antibodies) expressing construct was generated by cloning the gene fragment in pCDNA3.1 vector. The expression plasmid allows expression of UniDabFl 1 A in fusion with C-terminal His tag in ExpiCHO cells. The transfection was carried using ExpiFectamine™ CHO Transfection Kit as per manufacturer’s protocol. The resultant clarified supernatant was subjected to affinity column chromatography using Ni-NTA Sepharose beads. The protein obtained after affinity-based purification was further purified using gel filtration chromatography. The protein was eluted in 20 mM HEPES pH7.2 with 50 mM NaCl.

Crystallization of the CD38/F11A Complex. Data Collection and Structure Determination

[0277] To obtain the binary complex, CD38 protein was mixed with UniDabFl 1 A at a molar ratio of 1.2:1. The mixture was incubated at 4°C for 16 hours. The mixture was then loaded onto the Superdex 75 16/60 column and the elute fractions were analyzed. Fractions having both CD38 and UniDabFl 1 A were pooled and concentrated using a 3 kDa cut-off centrifugal concentrators.

[0278] Crystallization of CD38-UniDabFllA complex was set up using commercially available screens. Crystallization was set up using sitting drop vapor diffusion method with 300 nl drop at 1:2 ratio of protein and reservoir solution. Initial crystals were obtained with 0.1 M Sodium Citrate pH 5.5, and 8% PEG 8000. Crystallization condition was further optimized by pH and precipitant grid screen using initial crystals as microseeds.

[0279] Crystals of CD38-UniDabFl 1A complex was cryo-protected in mother liquor containing 25% ethylene glycol and further flash-cooled in liquid nitrogen. The crystals were diffracted, and diffraction data was collected at MX-2 beamline at ANSTO synchrotron facility. The crystals belong to P 32 2 1 space group and have one molecule each of CD38 and UniDabFl 1 A in the crystallographic asymmetric unit. Diffraction data was processed and integrated using Mosflrn. Data was scaled and merged; the merged intensities were converted to structure factor using Truncate program from CCP4 Suite. The structure was solved using molecular replacement method. PDB 1YH3 chain A for CD38 and PDB 6PZW chain F for UniDabFl 1 A were used as template for molecular replacement search models.

[0280] The model was first refined using rigid-body refinement in Refmac5 of CCP4. It was further refined by iterative cycles of manual model building in COOT and positional refinement in Refmac5. The data collection and refinement statistics are summarized in the table. The atomic coordinates were deposited with the Protein Data Bank with accession code 7VKE.

Internalization Assay

[0281] Daudi cells were incubated with 10 pg/mL TNB-738 or daratumumab for 0, 30, 60, or 120 min at 37°C then fixed and permeabilized (FIX & PERM™ Cell Permeabilization Kit, ThermoFisher). Cells were then labelled with anti-human IgG FITC (Jackson laboratories), anti-CD98 Alexa647 (AbCam) and Texas Red Phalloidin (Life Technologies). Cells were mounted between slide and slip cover with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen). Slides were analyzed with a confocal microscope (AIR, Nikon Imaging Software NIS) with 60x/4 lens. Cells with and without capping were counted, and percentage capping was graphed for 100 cells per treatment.

Insilico epitope manning of F 12 A and confirmation

[0282] MapTope was used to determine the F12A epitope on CD38 (PMID: 30322966). Briefly, MAbTope is a docking-based method which generates 5.108 poses for the antibody-antigen complex and which, using several scoring functions, fdters these poses in order to obtain the 30 best solutions. The interfaces analysis of these 30 top-ranked solutions allowed identification of the residues which exhibit the highest probability of being implicated in the interaction (FIG. 27A). For in vitro validation, CD38 sequence disclosed in the Uniprot ID P28907 was used to construct genes. A flag tag was added to the N-terminal end of CD38, and no other modifications were made to the protein. The wildtype and mutated CD38 were designed in silico, and reverse translated in DNA using EMBL-EBI emboss - backtranseq tool, using a human codon usage table, and avoiding the Hind III and Xhol restriction sites used for the cloning in pcDNA3.1+. A stop codon and the Kozak sequence (GCCACC) were added to the DNA sequence. The genes were synthesized and cloned in pcDNA3.1+ by Twist Bioscience (South San Francisco, CA, USA).

[0283] One million HEK293 cells were transiently transfected with 1 ug of either one of the constructs or a mock vector using Metafectene (Biontex Laboratories, Munich, Germany) according to the manufacturer’s instructions. After 24 hrs, the cells were trypsinized, fixed and permeabilized according to the BD bioscience CytoFix/CytoPerm kit’s procedure and distributed in 96 wells plates. Fifty thousand cells were incubated with 2.5 ug of F12A or Human IgGl isotype (Biolegend, cat# BLE403502) in 100 ul of wash buffer - 20mM Citrate, lOOmM NaCl, 2mM EDTA, 1% FBS pH 6.2 for 1 hours at 4C. The cells were washed, and the cell pellet was re suspended in 20 ul of 20mM Citrate, lOOmM NaCl, 2mM EDTA, 1% FCS pH 6.2 with 0.1 ul of an APC-coupled Ms Anti-Human IgGl (Miltenyi, 130-119-857) and 0.03 ul of a PE-coupled human anti-Flag antibody (Miltenyi, cat#130-101- 576) and left in dark at 45 min at 4C. The cells were washed in the wash buffer and once again with 20mM Citrate, lOOmM NaCl, 2mM EDTA, pH 6.2. Finally, the fluorescence was measured with a MACSQ analyzer 10. The initial gatings for cell selection were performed on unstained cells. The experiment was repeated 4 times. Cytometry data was analyzed using FlowJo V10. Graphs and statistical analysis were performed with GraphPad Prism9.

Example 1 : Epitope Binning by Octet

[0284] Epitope bins of FI 1A (309157) and F12A (330304) on human CD38 were determined by Octet QK386. Ni-NTA sensors were loaded with 5 ug/ml of his-tagged recombinant huCD38. A brief baseline was set for 60 seconds in kinetic buffer. The first antibody was then bound to the antigen on the sensor for 180 seconds. This was followed by another baseline for 60 seconds. Then the sensor was dipped into the second antibody for 180 seconds followed by dissociation for 240 seconds. All reagents were made in kinetic buffer. The antibodies were tested at 200nM. The sensors were regenerated and recharged with nickel between rims.

[0285] The results are shown in FIGS. 7-9 and demonstrate that 309157 and 330304 bind to different sites of huCD38. 330304 blocks binding of Isatuximab and Daratumab blocks binding of 309157 to recombinant huCD38, respectively. In summary, 309157 and Daratumab compete for binding to huCD38 and recognize overlapping epitopes in huCD38, whereas 330304 and Isatuximab also recognize overlapping epitopes in huCD38, which is distinct from the 309157/Daratumab epitope.

Example 2: Sequence Alignment of FI 1 A andF12A family members

[0286] Sequence Alignment of FI 1A and F12A family members along with percent CD38 hydrolase inhibition was used to identify critical amino acid residues involved in enzyme inhibition. A family was defined as a set of UniAbs whose CDR3 amino acid sequence is greater than 90% similar. FIGS. 10 and 11 show the VH sequences of F11A and F12A family members with percent CD38 enzyme inhibition. The VHs were then aligned and the positions with high variability were determined using an entropy plot. FIGS. 12 and 13 show the entropy plot for family FI 1A and F12A members.

[0287] An F test was used to determine the significant amino acid residues with respect to enzyme inhibition. FIGS. 14 and 15 show the plot of neg_logl0_p_value for family FI 1A and F12A members. Positions whose p value was less than 0.05 were crucial for enzyme inhibition. FIGS. 16 and 17 summarize the position and the corresponding amino acids for F11A and F12A, respectively.

Example 3: Epitope Mappins by crystallography

[0288] The epitope of FI 1 A (301957) on human CD38 was determined by crystallography. VHs also known as UniDab for F11A was expressed using ExpiCHO system and purified using Ni Sepharose resin. Human CD38 ECD (residues Arg 45 to lie 299) with his tag on C-terminus was expressed using Pichia system and purified using Ni Sepharose resin. CD38-F11A UniDab complex was made with molar excess (1:1.2) of FI 1 A UniDab.

[0289] CD38 ECD with F11A crystals were obtained at condition containing 100-140 mM Sodium citrate pH 5.5, 15-22% PEG 6000. CD38-F11A complex crystal diffracted to 2.5 A. A full diffraction data set was collected from a rotating anode X-ray source and processed. FIG. 18 shows X-ray data collection and refinement summary. Structure was resolved using the data to a maximum resolution of 2.9 A. [0290] Images were integrated and scaled data was imported. Intensities were converted to structure factor amplitudes using CCP4 truncate program. Molecular replacement program Phaser provided a solution containing one chain each of CD38ECD and UniDab F11A in a unit cell. This model was refined further with rebuilding the chains between refinement cycles. FIG. 19 summarizes the surface interactions between FI 1A UniDab (Monomer 1) and human CD38 (Monomer 2).

Example 4: Comparison of Epitopes of FI 1 A, Daratumuab and Isatuximab on huCD38

[0291] Crystal structure and co-ordinate information for Daratumumab and Isatuximab were obtained from Protein Data Bank under the accession code 7DHA and 4CMH, respectively. On aligning the interfacing residues of F11A, Daratumuab and Isatuximab on huCD38, it stands out that F11A and Daratumumab compete. F11A and Daratumuab have overlapping epitopes, predominantly binding to C-terminus of human CD38 ECD. The sequence alignment also reveals that Isatuximab has a different epitope compared to Daratumumab and FI 1A. This data strengthens epitope binning results.

[0292] Daratumumab does not inhibit enzymatic activity of human CD38. F11A causes reduction in enzyme activity by 40 percent. Though the epitopes are overlapping, F11A UniAb causes enzyme inhibition due to a conformational change of huCD38 upon it’s binding to an allosteric site.

[0293] Isatuximab is known to inhibit 80% of CD38 hydrolase activity. F12A elicits 60% inhibition of humanCD38. F11A and F12A synergize to elicit greater than 90 percent inhibition while Isatuximab and Daratumumab do not synergize. Thus, combining the two UniAbs results in superior inhibition.

[0294] FIG. 20 shows interfacing residues of FI 1 A, Daratumumab and Isatuximab on human CD38 ECD.

Example 5: Discovery ofTNB-738

[0295] Anti-CD38 heavy chain only antibodies (UniAbs) were identified using a proprietary next- generation sequencing (NGS) approach on immunized UniRats (PMID: 30666250). In total, 797 UniAbs were selected for gene assembly, recombinant expression, and functional screening. High- throughput EFISA and cell binding screens identified 155 CD38-specific binders from 52 unique CDR3 clonotype families. Binders were further evaluated in a CD38 NADase inhibition assay, which identified only partial inhibitors of CD38 (FIG. 21A). No UniAbs inhibited CD38 hydrolase activity greater than 50%. Subsequently, an all by all enzyme inhibition experiment comprised of the partial inhibitors was performed to identify a synergistic pair that, in combination, strongly inhibits CD38 enzyme activity (data not shown). UniAbs FI 1A and F12A emerged as a synergistic combination that inhibited CD38 activity greater than 85% (FIG. 21B). TNB-738 was generated by pairing F11A and F12A using knob-into-hole technology (FIG. 21C). A light chain and CHI domain were added to the F11A arm of TNB-738 to optimize manufacturability with no loss in its ability to inhibit CD38. Pharmacokinetics of TNB-738 was evaluated in mice and cynomolgus monkeys (FIG. 31). Since TNB- 738 does not cross-react with CD38 in mice or cynomolgus monkey, the observed linear PK was consistent with non-specific clearance mechanisms dominating PK, demonstrating stability of TNB- 738 in serum.

Example 6: TNB-738 Binds to CD38 and Inhibits CD38 Enzymatic Activity

[0296] Binding of TNB-738 to CD38⁺and CD38 cell lines was evaluated by flow cytometry. On-target binding was assessed on Daudi, Ramos, and CHO cells stably transfected to express CD38 (CHO- HuCD38), while off-target binding was assessed on K562, HL-60, and HEK 293F cells. TNB-738 bound to Daudi, Ramos, and CHO-HuCD38 cells with EC50 values of 39.7, 50.3, and 70.2 nM, respectively (FIG. 22A). No binding was observed on CD38 cells (FIG. 22B), demonstrating target- specificity of TNB-738. Binding of TNB-738 was also evaluated on human PBMCs, with similar immune cell subsets bound by TNB-738 compared to daratumumab (FIG. 34).

[0297] Additionally, TNB-738 did not induce capping and internalization upon binding of CD38 on cells (FIG. 22C). Capping is the process that precedes internalization in which antibody-receptor complexes accumulate on one side of the cell. The percentage of cells with capping was assessed by visualizing the TNB-738-CD38 complexes via immunofluorescence staining. Representative immunofluorescence images are shown in FIG. 33A. Additionally, internalization was assessed by flow cytometry using pH-sensitive fluoroprobes; these pH-sensitive fluoroprobes are non-fluorescent outside of the cell but fluoresce in low pH environments such as the lysosome. Following incubation of Daudi cells with TNB-738, daratumumab, or isotype control coupled to pHrodo iFL (ThermoFisher), MFI was measured by flow cytometry to assess internalization. TNB-738 did not induce internalization, as evident by the lack of increase in MFI, compared to daratumumab (FIG. 33B).

[0298] Capping and internalization are facilitated by the cytoskeleton, and phosphorylation of several proteins is required. Upon internalization, antibody-receptor complexes are transported to lysosomal compartments where these complexes are degraded. Pierce SK, et al. Immunol Rev. 1988;106:149-180. Studies by Fonaro et al. have indicated that capping and internalization of CD38 by antibodies leads to transmembrane signaling and activation of B and T cells. Since TNB-738 neither induces capping nor is actively internalized by CD38+ cells, this suggests that TNB-738 does not activate CD38+ cells, including B and T cells. Funaro A, et al. Eur J Immunol. 1993;23(10):2407-2411. To further investigate the effects of TNB-738 on immune cell activation, PBMCs were incubated with TNB-738 and activation markers (CD25 and IFNy) were assessed by flow cytometry. Treatment of CD38+ cells did not result in increased expression of CD25 or IFNy, demonstrating that TNB-738 does not activate T cells (FIG. 35). [0299] TNB-738-mediated inhibition of CD38 hydrolase activity was evaluated in vitro. CD38- expressing Daudi, Ramos, and CHO-HuCD38 cells were incubated with sNAD+ in the presence of increasing concentrations of TNB-738. Hydrolysis of sNAD+, which leads to an increase in fluorescence at wavelength 310 nm, was measured over time using a microplate reader (PMID 30112426). TNB-738 dose-dependently inhibited cell surface CD38 hydrolase activity with an average maximum percent inhibition of 87 ± 2.3% across the three cell lines tested (FIG. 22D). TNB-738 also inhibited recombinant CD38 hydrolase activity with IC₅₀ and maximum percent inhibition of 6.4 nM and 68%, respectively (FIG. 22E).

Example 7: TNB-738-Mediated Inhibition of CD38 Ectoenzvme Activity Increases NAD+ Levels and SIRT1 Activity

[0300] To assess whether blocking CD38 enzyme activity with TNB-738 led to increased cellular NAD+ levels, CD38-expressing Jurkat and Ramos cells were incubated with TNB-738 for 48 h and NAD+ concentrations were quantified. As demonstrated in FIG. 23 A, TNB-738-treatment significantly increased intracellular NAD+ levels in Jurkat cells with an EC₅₀ of 29.7 nM and a maximum NAD+ concentration of 17.5 pmol/well. Similarly, an increase in NAD+ was observed in Ramos cells following treatment with TNB-738 (FIG. 23B). No increase in NAD+ levels was observed using the isotype control.

[0301] The downstream effects of increased NAD+ was assessed by measuring the activity of SIRTs, which are NAD-dependent deacetylases involved in cellular health and metabolic regulation. SIRT1 activity was measured using the SIRT-Glo assay (Promega) following treatment of either Jurkat (FIG. 23C) or Ramos (FIG. 23D) cells with TNB-738 or isotype control for 48 h. Only in the presence of TNB-738 was an increase in SIRT1 activity observed in the CD38-expressing cells. In addition to SIRT1, increased activity of other NAD-dependent enzymes, including SIRT3 (FIG. 32A) and PARP (FIG. 32B), was observed.

[0302] In all cases, combination of TNB-738 with NMN supplementation further boostedNAD+ levels and SIRT activity compared to TNB-738-treatment alone (FIG. 23). The increase in NAD+ levels and SIRT activity was dependent on CD38 inhibition; NMN treatment alone without TNB-738 had no effect. In all likelihood, NMN is rapidly degraded by CD38 thus preventing its uptake and conversion to NAD+ intracellularly . Altogether, the data indicate that CD38 inhibition is required in order to effectively boost NAD+ levels in CD38+ cells. Example 8: TNB-738 Neither Induces Lysis of CD38-Expressine Cells Nor Promotes the Growth of CD38-Expressins Tumors

[0303] Since the desired mechanism of action of TNB-738 is strictly enzyme inhibition rather than depletion of CD38⁺ cells, TNB-738 was engineered on a silenced IgG4 Fc. To confirm that TNB-738 does not deplete CD38⁺ cells, TNB-738 was evaluated in CDC, ADCC, and direct apoptosis assays. CDC is an Fc-mediated effector function in which proteins of the complement cascade are recruited to opsonized cells leading to formation of the membrane attack complex and lysis of the cell. Incubation of Daudi and Ramos cells with TNB-738 and 5% complement serum did not induce complement- mediated lysis of cells, as measured using Cell-Titer Glo 2.0, compared to the positive control, daratumumab (FIG. 24A). Likewise, incubation of Daudi and Ramos cells with TNB-738 andNK cells did not result in ADCC, an Fc-mediated effector function that induces NK cell-mediated killing of target cells (FIG. 24B). TNB-738 also did not cause direct apoptosis of cells, as demonstrated by the lack of 7-AAD uptake and Annexin V staining on TNB-738-treated Daudi and Ramos cells (FIG. 24C). Cells treated with isatuximab, in contrast, had much lower viability compared to the untreated control. Altogether, the data demonstrated that TNB-738-treatment does not induce lysis of CD38-expressing cells.

[0304] To assess the effect of increased NAD+ levels due to TNB-738-mediated CD38 inhibition on tumor growth, CD38-expressing A549 tumor spheroids were treated with TNB-738 and tumor size was monitored over time. Although substantially increased NAD+ levels were detected in the TNB-738- treated tumors (FIG. 24Diii), it did not affect tumor growth as TNB-738-treated tumors grew at the same rate as untreated and isotype control-treated tumors (FIG. 24Di and FIG. 24Dii). Representative spheroid images are shown in FIG. 24Dii.

Example 9: TNB-738 Binds to Two Distinct Epitopes on CD38

[0305] Epitope binning experiments were performed using biolayer interferometry (Fortebio’s Octet QK386). In-tandem format was used to bin F11A, F12A, isatuximab, and daratumumab. The epitope binning results demonstrated that FI 1A and F12A bind to different epitopes on CD38 (FIG. 7-9). FIG. 7 shows epitope binning curves with CD38 on sensor, Abl is F11A and Ab2 is F12A. FIG. 8 shows epitope binning curves with CD38 on sensor, Abl is Daratamumab and Ab2 is F11A. FIG. 9 shows epitope binning curves with CD38 on sensor, Abl is F12A and Ab2 is Isatuximab.

[0306] Epitope binning experiments were also performed using SPR (BioRad ProteOn). A sandwich format was used to bin F11A, F12A, isatuximab, and daratumumab. The epitope binning results demonstrated that F11A and F12A bind to different epitopes on CD38 (FIG. 36). F11A and daratumumab compete for binding to CD38 and recognize overlapping epitopes on CD38. Similarly, F12A and isatuximab also recognize overlapping epitopes on CD38, which was distinct from the FI 1 A/daratumumab epitope. Though SPR data suggests that isatuximab and daratumumab compete for binding, crystallography data showed that they bind to distinct epitopes on CD38 (PDB: 7DHA, 4CMH). The apparent competition between isatuximab and daratumumab is caused by the steric collision at the C terminus of CD38, as predicted by insilico super imposition. Lee HT, et al. Biochem Biophys Res Commun. 2021;536:26-31.

Example 10: Structure of CD38/F11A Complex

[0307] The crystal structure of the C38-F11 A complex was determined by X-ray crystallography. The CD38-F11A complex diffracted to a resolution of 1.9 A. Protein Interfaces, Surfaces, and Assemblies (PISA) program was used to analyze the interaction between F11A and CD38 (FIG. 25). F11A recognizes 9 residues on CD38 via hydrogen bonds and salt bridges. The data collection and refinement parameters are listed in FIG. 26. Most of the residues that interact with CD38 are present in the CDRs of F11A except for F11AY60 and F11A K65. Interacting residues on CD38 include CD38Q236, CD38E292, CD38E239, CD38P291, CD38E299, CD38Q272, CD38K276, CD38N277, CD38D252 at the C-terminus. Of the 9 residues that F11A interacts with on CD38 via hydrogen bonding and salt bridges, daratumumab shares 7. Daratumumab and F11A bind to synonymous residues in the b4, b5, b6 strands and the a9 helix at the C-terminus of extracellular CD38.

Example 11: F12A Epitope Prediction

[0308] The epitope of F12A was predicted by a docking-based insilico tool called MabTope. The residues of CD38 that likely belong to the F12A epitope are highlighted in FIG. 27. They are divided into four categories as a function of their raw probability to belong to the epitope, from violet for the highest probability to cyan for the lowest. These residues constitute the three peptides which were validated by expression of mutated CD38 and loss of F12A binding. A fourth peptide was suggested based on the binding site of a small molecule LX- 102 which competes with isatuximab (PMID: 31081167) The constructs were named: CD38_12ml, CD38_12m2, CD38_12m3, CD38_12m4. The numbering of the mutations corresponds to the sequence of the structural template PDB:203S. Mutations were selected among the residues whose lateral chains are exposed to solvent and thereby can interact with F12A. In order to not affect the 3D structure of CD38 proteins, prolines and other amino acids whose lateral chains were not exposed at the surface were not mutated.

[0309] In vitro validation was done by overexpressing these mutants on HEK cells. The antigen was flag tagged (DYKDDDDK) to monitor its expression using a PE-coupled Anti-FLAG antibody. Binding of F12A to mutants was monitored using F12A coupled with APC. The mutants CD38_12m2 and CD38_12m4 exhibited a decreased binding to F12A compared to the wildtype CD38. Hence, the mutated residues K25, E28, R151, E154 are part of the epitope. This suggests that F12A has a conformational epitope as the identified residues are discontinuous. Competition experiments using Octet confirmed that F12A and isatuximab have overlapping epitopes. Altogether, the results indicate that TNB-738 is an allosteric inhibitor of the enzyme functions of CD38.

[0310] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS:

1. A multispecific antibody that binds to two different epitopes on a CD38 protein, comprising: a first binding unit that binds to a first epitope on the CD38 protein; and a second binding unit that binds to a second epitope on the CD38 protein, wherein the first binding unit competes for binding to the first epitope with an anti-CD38 heavy chain-only antibody comprising a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 11, and a CDR3 sequence of SEQ ID NO: 22.

2. The multispecific antibody of claim 1, wherein the first binding unit comprises a heavy chain variable region paired with a light chain variable region.

3. The multispecific antibody of claim 2, wherein the light chain variable region is a fixed light chain variable region.

4. The multispecific antibody of any one of claims 1-3, wherein the first binding unit comprises a heavy chain-only variable region.

5. The multispecific antibody of claim 4, wherein the first binding unit lacks a light chain.

6. The multispecific antibody of claim 5, wherein the heavy chain-only variable region is in a monovalent or bivalent configuration.

7. The multispecific antibody of any one of claims 1-6, wherein the first binding unit comprises a CDR3 sequence having at least 88% identity to SEQ ID NO: 22.

8. The multispecific antibody of claim 7, wherein the first binding unit comprises a CDR3 sequence having at least 94% identity to SEQ ID NO: 22.

9. The multispecific antibody of claim 8, wherein the first binding unit comprises a CDR3 sequence having 100% identity to SEQ ID NO: 22.

10. The multispecific antibody of any one of claims 1-6, wherein the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 93% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22.

11. The multispecific antibody of claim 10, wherein the first binding unit comprises a full set of CDRs 1, 2, and 3 having at least 96% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1, 11 and 22.

12. The multispecific antibody of claim 11, wherein the first binding unit comprises a full set of CDRs 1, 2, and 3 having 100% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 1,

11 and 22.

13. The multispecific antibody of any one of claims 1-6, wherein the first binding unit comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 28-71.

14. The multispecific antibody of claim 13, wherein the first binding unit comprises a heavy chain variable region sequence comprising SEQ ID NO: 28.

15. The multispecific antibody of claim 14, wherein the first binding unit comprises a heavy chain variable region sequence having at least 98% identity to SEQ ID NO: 28.

16. The multispecific antibody of claim 15, wherein the first binding unit comprises a heavy chain variable region sequence having at least 99% identity to SEQ ID NO: 28.

17. The multispecific antibody of any one of claims 1-16, wherein the first binding unit comprises a heavy chain variable region sequence comprising one or more amino acid residues selected from the group consisting of: S30, S31, Y32, R45, W47, D53, K58, Y59, Y60, A61, D62, K65, D99, R100, G101, T102, M103, R104, V105, V106, V107, Y108, D109, T110, Llll, and W114.

18. The multispecific antibody claim 17, wherein the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues S30, S31 and Y32.

19. The multispecific antibody claim 17, wherein the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues K58, Y59, Y60, A61, and D62.

20. The multispecific antibody claim 17, wherein the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues D99, R100, G101, T102, M103, R104, V105, VI 06, VI 07, Y108, D109, T110, and Llll.

21. The multispecific antibody of any one of claims 1-16, wherein the first binding unit comprises a heavy chain variable region sequence comprising one or more amino acid residues selected from the group consisting of: S31, D53, Y60, K65, D99, T102, M103, Y108, D109, T110, and LI 11.

22. The multispecific antibody of claim 21, wherein the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues T102 and Ml 03.

23. The multispecific antibody of claim 21, wherein the first binding unit comprises a heavy chain variable region sequence comprising amino acid residues Y108, D109, T110, and LI 11.

24. The multispecific antibody of claim 1, wherein the second binding unit comprises a heavy chain variable region paired with a light chain variable region.

25. The multispecific antibody of claim 24, wherein the light chain variable region is a fixed light chain variable region.

26. The multispecific antibody of any one of claims 1, 24 and 25, wherein the second binding unit comprises a heavy chain-only variable region.

27. The multispecific antibody of claim 26, wherein the second binding unit lacks a light chain.

28. The multispecific antibody of claim 27, wherein the heavy chain-only variable region is in a monovalent or bivalent configuration.

29. The multispecific antibody of any one of claims 1 and 24-28, wherein the second binding unit competes for binding to the second epitope with an anti-CD38 heavy chain-only antibody comprising a heavy chain variable region comprising a CDR1 sequence of SEQ ID NO: 72, a CDR2 sequence of SEQ ID NO: 86, and a CDR3 sequence of SEQ ID NO: 94.

30. The multispecific antibody of claim 29, wherein the second binding unit comprises a CDR3 sequence having at least 90% identity to SEQ ID NO: 94.

31. The multispecific antibody of claim 29, wherein the second binding unit comprises a CDR3 sequence having 100% identity to SEQ ID NO: 94.

32. The multispecific antibody of claim 29, comprising a full set of CDRs 1, 2, and 3 having at least 96% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94.

33. The multispecific antibody of claim 32, comprising a full set of CDRs 1, 2, and 3 having 100% identity to a full set of CDRs 1, 2, and 3 defined by SEQ ID NOs: 72, 86 and 94.

34. The multispecific antibody of claim 29, wherein the second binding unit comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 96-135.

35. The multispecific antibody of claim 34, wherein the second binding unit comprises a heavy chain variable region sequence comprising SEQ ID NO: 96.

36. The multispecific antibody of claim 34, wherein the second binding unit comprises a heavy chain variable region sequence having at least 98% identity to SEQ ID NO: 96.

37. The multispecific antibody of claim 36, wherein the second binding unit comprises a heavy chain variable region sequence having at least 99% identity to SEQ ID NO: 96.

38. The multispecific antibody of any one of claims 1 and 24-37, wherein the second binding unit competes with Isatuximab for binding to the second epitope on the CD38 protein.

39. The multispecific antibody of any one of claims 1-38, wherein the CD38 protein is a human CD38 protein (SEQ ID NO: 136).

40. The multispecific antibody of any one of claims 1-39, wherein the first epitope is a conformational epitope comprising two or more amino acid residues selected from the group consisting of amino acid residues 120, 135, 139, 142, 202, 203, 205, 236, 237, 239, 241, 252, 254, 255, 272-279, 284, 287, 288, 291-294, 296, 297, 299 and 300 of SEQ ID NO: 136.

41. The multispecific antibody of claim 40, wherein the first epitope is a conformational epitope comprising amino acid residues 120, 135, 139, 142, 202, 203, 205, 236, 237, 239, 241, 252, 254, 255, 272-279, 284, 287, 288, 291-294, 296, 297, 299 and 300 of SEQ ID NO: 136.

42. The multispecific antibody of any one of claims 1-39, wherein the second epitope is a conformational epitope comprising two or more amino acid residues selected from the group consisting of amino acid residues 188-201, 262-264, and 275-284 of SEQ ID NO: 136.

43. The multispecific antibody of claim 42, wherein the second epitope is a conformational epitope comprising amino acid residues 188-201, 262-264, and 275-284 of SEQ ID NO: 136.

44. The multispecific antibody of any one of claims 1-43, further comprising at least one heavy chain constant region sequence in the absence of a CHI sequence.

45. The multispecific antibody of claim 41, comprising two heavy chain constant region sequences that each lack a CHI sequence.

46. The multispecific antibody of any one of claims 1-45, which is bispecific.

47. The multispecific antibody of any one of claims 1-46, which is a three chain antibody-like molecule (TCA) comprising a first binding unit comprising a heavy chain variable region and a light chain variable region, and a second binding unit comprising a heavy chain-only variable region, in a monovalent or bivalent configuration.

48. A pharmaceutical composition comprising a multispecific antibody of any one of claims 1-47.

49. A polynucleotide encoding a multispecific antibody of any one of claims 1-47.

50. A vector comprising the polynucleotide of claim 49.

51. A cell comprising the vector of claim 50.

52. A method of producing a multispecific antibody of any one of claims 1-47, the method comprising growing a cell according to claim 51 under conditions permissive for expression of the antibody, and isolating the antibody from the cell and/or a cell culture medium in which the cell is grown.

53. A method of making a multispecific antibody of any one of claims 1-47, the method comprising immunizing a UniRat animal with a CD38 protein and identifying CD38 protein-binding heavy chain sequences.

54. A method for the treatment of a disorder characterized by expression of CD38, comprising administering to a subject with said disorder a multispecific antibody of any one of claims 1-47, or a pharmaceutical composition of claim 48.

55. The method of claim 54, wherein the disease or disorder is characterized by a hydrolase enzymatic activity of CD38, a cyclase enzymatic activity of CD38, or a combination thereof.

56. The method of claim 54 or 55, wherein the disease or disorder is a telomere shortening disease.

57. The method of claim 56, wherein the telomere shortening disease is selected from the group consisting of: accelerated aging, aplastic anemia, dyskeratosis congenita, Franconi’s anemia, and idiopathic pulmonary fibrosis.

58. The method of claim 54 or 55, wherein the disease or disorder is an inflammatory disease.

59. The method of claim 58, wherein the inflammatory disease is selected from the group consisting of: ulcerative colitis, graft versus host disease (GvHD), acute GvHD, chronic GvHD, transplant-associated GvHD, and acute kidney injury.

60. The method of claim 54 or 55, wherein the disease or disorder is a fibrosis-associated disorder.

61. The method of claim 60, wherein the fibrosis-associated disorder is scleroderma.

62. The method of claim 54 or 55, wherein the disease or disorder is a metabolic syndrome.

63. The method of claim 62, wherein the metabolic syndrome is selected from the group consisting of: type II diabetes mellitus (T2DM), obesity, and systemic inflammation.

64. The method of claim 54 or 55, wherein the disease or disorder is doxorubicin-induced toxicity.

65. The method of claim 64, wherein the doxorubicin-induced toxicity comprises cardiotoxicity.

66. The method of claim 54 or 55, wherein the disease or disorder is an organ transplantation- associated disease or disorder.

67. The method of claim 66, wherein the organ transplantation-associated disease or disorder is a skin transplantation-associated disease or disorder or a kidney transplantation-associated disease or disorder.

68. The method of claim 54 or 55, wherein the disease or disorder is a cardiovascular disease or disorder.

69. The method of claim 68, wherein the cardiovascular disease or disorder comprises heart failure.

70. A method of treating a disease or disorder characterized by reduced sirtuin activity, the method comprising administering to a subject with the disease or disorder a multispecific antibody according to any one of claims 1-47, or a pharmaceutical composition of claim 48.

71. The method of claim 70, further comprising administering nicotinamide mononucleotide (NMN) to the subject.

72. The method of claim 70 or 71, wherein the disease or disorder is selected from the group consisting of: a metabolic disease or disorder, a cardiovascular disease or disorder, and a neurodegenerative disease or disorder.

73. The method of claim 70 or 71, wherein the disease or disorder is a cancer.

74. A method of increasing nicotinamide adenine dinucleotide (NAD+) concentration in a cell, the method comprising contacting the cell with a multispecific antibody according to any one of claims 1-47, or a pharmaceutical composition of claim 48.

75. The method of claim 74, further comprising contacting the cell with NMN.

76. A method of increasing sirtuin activity in a cell, the method comprising contacting the cell with a multispecific antibody according to any one of claims 1-47, or a pharmaceutical composition of claim 48.

77. The method of claim 76, further comprising contacting the cell with NMN.

78. A method of treating a disease or disorder characterized by expression of CD38, the method comprising administering to a subject with the disease or disorder a multispecific antibody according to any one of claims 1-47, or a pharmaceutical composition of claim 48.

79. The method of claim 78, wherein the disease or disorder is characterized by a hydrolase enzymatic activity of CD38, a cyclase enzymatic activity of CD38, or a combination thereof.

80. The method of any one of claims 78-79, wherein administering the multispecific antibody to the subject results in an inhibition of CD38 enzymatic activity.

81. A method of increasing nicotinamide adenine dinucleotide (NAD+) concentration in a cell without depleting or activating CD38 expressing cells, the method comprising contacting the cell with a multispecific antibody according to any one of claims 1-47, or a pharmaceutical composition of claim 48.

82. The multispecific antibody according to any one of claims 1-47, wherein the multispecific antibody inhibits CD38 enzymatic activity without directly or indirectly lysing CD38+ cells.