TNB-738, a biparatopic antibody, boosts intracellular NAD+ by inhibiting CD38 ecto-enzyme activity

ABSTRACT Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolizes nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose or cyclic ADP-ribose and nicotinamide. Other substrates of CD38 include nicotinamide adenine dinucleotide phosphate and nicotinamide mononucleotide, a critical NAD+ precursor in the salvage pathway. NAD+ is an important coenzyme involved in several metabolic pathways and is a required cofactor for the function of sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases. Declines in NAD+ levels are associated with metabolic and inflammatory diseases, aging, and neurodegenerative disorders. To inhibit CD38 enzyme activity and boost NAD+ levels, we developed TNB-738, an anti-CD38 biparatopic antibody that pairs two non-competing heavy chain-only antibodies in a bispecific format. By simultaneously binding two distinct epitopes on CD38, TNB-738 potently inhibited its enzymatic activity, which in turn boosted intracellular NAD+ levels and SIRT activities. Due to its silenced IgG4 Fc, TNB-738 did not deplete CD38-expressing cells, in contrast to the clinically available anti-CD38 antibodies, daratumumab, and isatuximab. TNB-738 offers numerous advantages compared to other NAD-boosting therapeutics, including small molecules, and supplements, due to its long half-life, specificity, safety profile, and activity. Overall, TNB-738 represents a novel treatment with broad therapeutic potential for metabolic and inflammatory diseases associated with NAD+ deficiencies. Abbreviations: 7-AAD: 7-aminoactinomycin D; ADCC: antibody dependent cell-mediated cytotoxicity; ADCP: antibody dependent cell-mediated phagocytosis; ADPR: adenosine diphosphate ribose; APC: allophycocyanin; cADPR: cyclic ADP-ribose; cDNA: complementary DNA; BSA: bovine serum albumin; CD38: cluster of differentiation 38; CDC: complement dependent cytotoxicity; CFA: Freund’s complete adjuvant; CHO: Chinese hamster ovary; CCP4: collaborative computational project, number 4; COOT: crystallographic object-oriented toolkit; DAPI: 4′,6-diamidino-2-phenylindole; DNA: deoxyribonucleic acid; DSC: differential scanning calorimetry; 3D: three dimensional; εNAD+: nicotinamide 1,N6-ethenoadenine dinucleotide; ECD: extracellular domain; EGF: epidermal growth factor; FACS: fluorescence activated cell sorting; FcγR: Fc gamma receptors; FITC: fluorescein isothiocyanate; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: immunoglobulin; IFA: incomplete Freund’s adjuvant; IFNγ: Interferon gamma; KB: kinetic buffer; kDa: kilodalton; KEGG: kyoto encyclopedia of genes and genomes; LDH: lactate dehydrogenase; M: molar; mM: millimolar; MFI: mean fluorescent intensity; NA: nicotinic acid; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NAM: nicotinamide; NGS: next-generation sequencing; NHS/EDC: N-Hydroxysuccinimide/ ethyl (dimethylamino propyl) carbodiimide; Ni-NTA: nickel-nitrilotriacetic acid; nL: nanoliter; NK: natural killer; NMN: nicotinamide mononucleotide; OD: optical density; PARP: poly (adenosine diphosphate-ribose) polymerase; PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell; PDB: protein data bank; PE: phycoerythrin; PISA: protein interfaces, surfaces, and assemblies: PK: pharmacokinetics; mol: picomolar; RNA: ribonucleic acid; RLU: relative luminescence units; rpm: rotations per minute; RU: resonance unit; SEC: size exclusion chromatography; SEM: standard error of the mean; SIRT: sirtuins; SPR: surface plasmon resonance; µg: microgram; µM: micromolar; µL: microliter

[1]  S. Deaglio,et al.  The Extracellular NADome Modulates Immune Responses , 2021, Frontiers in Immunology.

[2]  A. Lapucci,et al.  SIRT1-dependent restoration of NAD+ homeostasis after increased extracellular NAD+ exposure , 2021, The Journal of biological chemistry.

[3]  E. Verdin,et al.  NAD+ metabolism and its roles in cellular processes during ageing , 2020, Nature Reviews Molecular Cell Biology.

[4]  Y. Heo,et al.  Crystal structure of CD38 in complex with daratumumab, a first-in-class anti-CD38 antibody drug for treating multiple myeloma. , 2020, Biochemical and biophysical research communications.

[5]  P. Huber,et al.  NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential , 2020, Signal Transduction and Targeted Therapy.

[6]  J. Rabinowitz,et al.  CD38 ecto-enzyme in immune cells is induced during aging regulating NAD+ and NMN levels , 2020, Nature Metabolism.

[7]  D. Wiederschain,et al.  Isatuximab Acts Through Fc-Dependent, Independent, and Direct Pathways to Kill Multiple Myeloma Cells , 2020, Frontiers in Immunology.

[8]  S. Bhattacharyya,et al.  Targeting CD38-dependent NAD+ metabolism to mitigate multiple organ fibrosis , 2020, iScience.

[9]  J. Baur,et al.  Age-related NAD+ decline , 2020, Experimental Gerontology.

[10]  G. Sutphin,et al.  Kynurenine pathway, NAD+ synthesis, and mitochondrial function: Targeting tryptophan metabolism to promote longevity and healthspan , 2020, Experimental Gerontology.

[11]  D. Wiederschain,et al.  Therapeutic Opportunities with Pharmacological Inhibition of CD38 with Isatuximab , 2019, Cells.

[12]  L. Zhang,et al.  Rational Design and Identification of Small‐Molecule Allosteric Inhibitors of CD38 , 2019, Chembiochem : a European journal of chemical biology.

[13]  M. Mattson,et al.  NAD+ in Brain Aging and Neurodegenerative Disorders. , 2019, Cell metabolism.

[14]  E. Chini,et al.  The Multi-faceted Ecto-enzyme CD38: Roles in Immunomodulation, Cancer, Aging, and Metabolic Diseases , 2019, Front. Immunol..

[15]  E. Chini,et al.  The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD+ decline. , 2019, Biochemical and biophysical research communications.

[16]  J. Zweier,et al.  Inhibition of CD38 with the Thiazoloquin(az)olin(on)e 78c Protects the Heart against Postischemic Injury , 2019, The Journal of Pharmacology and Experimental Therapeutics.

[17]  Michael J. Osborn,et al.  Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies , 2019, Front. Immunol..

[18]  S. Bruzzone,et al.  Slc12a8 is a nicotinamide mononucleotide transporter , 2018, Nature Metabolism.

[19]  Amit Singhal,et al.  Host NAD+ metabolism and infections: therapeutic implications , 2018, International immunology.

[20]  A. Poupon,et al.  MAbTope: A Method for Improved Epitope Mapping , 2018, The Journal of Immunology.

[21]  A. Sboner,et al.  CD38 is methylated in prostate cancer and regulates extracellular NAD+ , 2018, Cancer & Metabolism.

[22]  E. Chini,et al.  Measuring CD38 Hydrolase and Cyclase Activities: 1,N6-Ethenonicotinamide Adenine Dinucleotide (ε-NAD) and Nicotinamide Guanine Dinucleotide (NGD) Fluorescence-based Methods. , 2018, Bio-protocol.

[23]  J. Zweier,et al.  A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline. , 2018, Cell metabolism.

[24]  E. Chini,et al.  The Pharmacology of CD38/NADase: An Emerging Target in Cancer and Diseases of Aging. , 2018, Trends in pharmacological sciences.

[25]  D. Sinclair,et al.  Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence. , 2018, Cell metabolism.

[26]  A. Boudreau,et al.  Multispecific antibodies targeting CD38 show potent tumor-specific cytotoxicity. , 2018 .

[27]  P. Richardson,et al.  CD38 antibodies in multiple myeloma: back to the future. , 2018, Blood.

[28]  J. Baur,et al.  NAD+ Intermediates: The Biology and Therapeutic Potential of NMN and NR. , 2017, Cell metabolism.

[29]  M. Mathieu,et al.  IgG Fc engineering to modulate antibody effector functions , 2017, Protein & Cell.

[30]  J. Reid,et al.  CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism. , 2016, Cell metabolism.

[31]  D. Seals,et al.  Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice , 2016, Aging cell.

[32]  J. Linden,et al.  Purinergic regulation of the immune system , 2016, Nature Reviews Immunology.

[33]  P. Parren,et al.  Monoclonal antibodies targeting CD38 in hematological malignancies and beyond , 2016, Immunological reviews.

[34]  Hao Jiang,et al.  SAR650984 directly induces multiple myeloma cell death via lysosomal-associated and apoptotic pathways, which is further enhanced by pomalidomide , 2016, Leukemia.

[35]  E. Verdin.  NAD+ in aging, metabolism, and neurodegeneration , 2015, Science.

[36]  A. Nikiforov,et al.  Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells* , 2015, The Journal of Biological Chemistry.

[37]  D. Rajpal,et al.  Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility , 2015, PloS one.

[38]  H. Mittrücker,et al.  CD38 Is Expressed on Inflammatory Cells of the Intestine and Promotes Intestinal Inflammation , 2015, PloS one.

[39]  J. D. Stuart,et al.  Discovery, Synthesis, and Biological Evaluation of Thiazoloquin(az)olin(on)es as Potent CD38 Inhibitors. , 2015, Journal of medicinal chemistry.

[40]  L. Guarente,et al.  NAD+ and sirtuins in aging and disease. , 2014, Trends in cell biology.

[41]  D. Sinclair,et al.  Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38: Implications for Cellular NAD+ Metabolism, Protein Acetylation, and Treatment of Metabolic Syndrome. Diabetes 2013;62:1084–1093 , 2014, Diabetes.

[42]  D. Sinclair,et al.  Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38 , 2013, Diabetes.

[43]  J. Yue,et al.  Design, synthesis and biological characterization of novel inhibitors of CD38. , 2011, Organic & biomolecular chemistry.

[44]  Owen Johnson,et al.  iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM , 2011, Acta crystallographica. Section D, Biological crystallography.

[45]  S. Ward,et al.  β-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and nonhuman primate colons. , 2011, Gastroenterology.

[46]  P. Canonico,et al.  Characterization of NAD Uptake in Mammalian Cells* , 2008, Journal of Biological Chemistry.

[47]  T. Seike,et al.  CD38 is critical for social behaviour by regulating oxytocin secretion , 2007, Nature.

[48]  Hon Cheung Lee.  Structure and Enzymatic Functions of Human CD38 , 2006, Molecular medicine.

[49]  Qun Liu,et al.  Crystal structure of human CD38 extracellular domain. , 2005, Structure.

[50]  D. F. Niven,et al.  Levels of nicotinamide adenine dinucleotide in extracellular body fluids of pigs may be growth-limiting for Actinobacillus pleuropneumoniae and Haemophilus parasuis. , 2003, Canadian journal of veterinary research = Revue canadienne de recherche veterinaire.

[51]  E. Zocchi,et al.  Connexin 43 hemichannels mediate Ca2+‐regulated transmembrane NAD+ fluxes in intact cells , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[52]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[53]  U. Dianzani,et al.  Human CD38 is associated to distinct molecules which mediate transmembrane signaling in different lineages , 1993, European journal of immunology.

[54]  S. Pierce,et al.  Antigen‐Presenting Function of B Lymphocytes , 1988, Immunological reviews.

[55]  T. Arndt.  Crystal , 2019, Springer Reference Medizin.

[56]  J. Dürig,et al.  CD38 expression is an important prognostic marker in chronic lymphocytic leukaemia , 2002, Leukemia.