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 metabolize 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,N 6 -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 pro...

Biparatopic antibodies: therapeutic applications and prospects

“… 43 Furthermore, bpAbs against CD38 and CD73, including the clinical bpAb TNB-738, were shown to act as enzyme inhibitors by locking the respective extracellular domains in catalytically inactive states ( Figure 2c ). 24 , 44 , 45 Whereas individual parent antibodies did not show any activity, anti-CD73 bpAbs had sub-nanomolar activity and achieved > 90% inhibition. 44 …”

“… 18–20 ISB 1442 21 and SAR441236 22 are examples of asymmetric bpAbs that expand the immunoglobulin G (IgG) architecture with additional Fab or scFv domains, respectively, to achieve higher valency and even triparatopic binding in the case of SAR441236. Smaller asymmetric formats such as the fragment-Fab-based zanidatamab 23 or the fragment–fragment-based TNB-738 24 can be exploited as well. Zanidatamab pairs an scFv domain with a full Fab arm while TNB-738 derives its binding arms from two sdAbs with an inert LC added for manufacturability.…”

“…Most clinical bpAbs function as naked antibodies to treat viral infections, inhibit or promote cell signaling via the respective receptor or, as in the case of TNB-738, 24 by inhibiting enzymatic activity. Increasingly, bpAbs are used for payload delivery as part of ADCs 11 , 23 , 56 due to their unique MoAs that promote internalization and lysosomal trafficking, as discussed above.…”

“…Ancora Biotech’s TNB-738 is an asymmetric anti-CD38 bpAb consisting of two sdAbs derived from Teneobio’s proprietary transgenic UniRat platform that bind to distinct CD38 epitopes with high affinity. 24 TNB-738 uses the knobs-into-holes mutations to achieve heavy-chain pairing, a fully silenced IgG4 Fc to eliminate effector functions, and an inert kappa LC and CH1 on one arm to improve manufacturability. Currently in Phase I dose escalation in healthy volunteers (NCT05215912), TNB-738 functions through enzyme inhibition.…”

Targeting CD38 in Neoplasms and Non-Cancer Diseases

“…Interestingly, by pairing two non-competing heavy chain antibodies in a bispecific format, TNB-738 antibody allows for the simultaneous binding of two epitopes on CD38. TNB-738 inhibits CD38’s enzymatic activity, elevating the intracellular NAD+ content and SIRT activity [ 64 ]. In January 2022, TNB-738 underwent a single- and multiple-dosing study for targeting CD38 in healthy volunteers (NCT05215912).…”