Synthesis of site-specific Fab-drug conjugates using ADP-ribosyl cyclases.

Targeted delivery of small-molecule drugs via covalent attachments to monoclonal antibodies has proved successful in clinic. For this purpose, full-length antibodies are mainly used as drug-carrying vehicles. Despite their flexible conjugation sites and versatile biological activities, intact immunoglobulins with conjugated drugs, which feature relatively large molecular weights, tend to have restricted tissue distribution and penetration and low fractions of payloads. Linking small-molecule therapeutics to other formats of antibody may lead to conjugates with optimal properties. Here, we designed and synthesized ADP-ribosyl cyclase-enabled fragment antigen-binding (Fab) drug conjugates (ARC-FDCs) by utilizing CD38 catalytic activity. Through rapidly forming a stable covalent bond with a nicotinamide adenine dinucleotide (NAD+ )-based drug linker at its active site, CD38 genetically fused with Fab mediates robust site-specific drug conjugations via enzymatic reactions. Generated ARC-FDCs with defined drug-to-Fab ratios display potent and antigen-dependent cytotoxicity against breast cancer cells. This work demonstrates a new strategy for developing site-specific FDCs. It may be applicable to different antibody scaffolds for therapeutic conjugations, leading to novel targeted agents.

CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells.

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.

Ex Vivo Efficacy of SAR442257 Anti-CD38 Trispecific T-cell Engager in Multiple Myeloma Relapsed After Daratumumab and BCMA-targeted Therapies.

T cell-engaging antibodies (TCEs) are showing promising efficacy in relapsed/refractory multiple myeloma, even in patients that relapsed after B-cell maturation antigen (BCMA)-targeted therapy. Patients with multiple myeloma may have compromised T-cell health unaccounted for by preclinical models. Here, we use Myeloma Drug Sensitivity Testing (My-DST) for ex vivo measurement of anti-multiple myeloma cytotoxicity for the trispecific CD38/CD28xCD3 TCE SAR442257 through activation of the patients' own endogenous T cells to inform clinical development of the compound in multiple myeloma. My-DST incubates primary mononuclear cells in humanized media for 48 hours followed by flow cytometry for multiple myeloma cell viability with or without drug treatment. SAR442257 was tested on 34 samples from patients with multiple myeloma across disease settings. Potential biomarkers, T-cell dependence, and degranulation were assessed. SAR442257 was effective at low dose in My-DST cultures. High ex vivo response rates were observed in primary aspirates taken from patients with multiple myeloma at diagnosis, with modestly reduced response in multiple myeloma recently treated with anti-CD38 mAbs. SAR442257 was highly effective in patients relapsing after BCMA therapy. The CD38/CD28xCD3 trispecific format was substantially more effective than a conventional bispecific CD38/CD3 antibody format and CD38 mAbs. Anti-multiple myeloma cell cytotoxicity was dependent on the presence of endogenous T cells. Surface CD38 expression was the strongest biomarker of TCE response. My-DST is capable of measuring T cell-dependent killing using the multiple myeloma patient's own bone marrow-derived T cells. SAR442257 shows promise for multiple myeloma and may be best suited for patients declared resistant to both CD38 mAbs and BCMA-targeted therapy. SIGNIFICANCE: This study introduces the use of My-DST to measure and characterize sensitivity to anti-CD38 T-cell engager SAR442257 in primary samples using matched endogenous T cells. Preclinical testing in samples from patients with diverse treatment history supports further testing in post-chimeric antigen receptor T-cell multiple myeloma.

CD38 promotes hematopoietic stem cell dormancy.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

Epigenetic regulation of CD38/CD48 by KDM6A mediates NK cell response in multiple myeloma.

Anti-CD38 monoclonal antibodies like Daratumumab (Dara) are effective in multiple myeloma (MM); however, drug resistance ultimately occurs and the mechanisms behind this are poorly understood. Here, we identify, via two in vitro genome-wide CRISPR screens probing Daratumumab resistance, KDM6A as an important regulator of sensitivity to Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Loss of KDM6A leads to increased levels of H3K27me3 on the promoter of CD38, resulting in a marked downregulation in CD38 expression, which may cause resistance to Daratumumab-mediated ADCC. Re-introducing CD38 does not reverse Daratumumab-mediated ADCC fully, which suggests that additional KDM6A targets, including CD48 which is also downregulated upon KDM6A loss, contribute to Daratumumab-mediated ADCC. Inhibition of H3K27me3 with an EZH2 inhibitor resulted in CD38 and CD48 upregulation and restored sensitivity to Daratumumab. These findings suggest KDM6A loss as a mechanism of Daratumumab resistance and lay down the proof of principle for the therapeutic application of EZH2 inhibitors, one of which is already FDA-approved, in improving MM responsiveness to Daratumumab.

Ecto-CD38-NADase inhibition modulates cardiac metabolism and protects mice against doxorubicin-induced cardiotoxicity.

AIMS: Doxorubicin (DXR) is a chemotherapeutic agent that causes dose-dependent cardiotoxicity. Recently, it has been proposed that the NADase CD38 may play a role in doxorubicin-induced cardiotoxicity (DIC). CD38 is the main NAD+-catabolizing enzyme in mammalian tissues. Interestingly, in the heart, CD38 is mostly expressed as an ecto-enzyme that can be targeted by specific inhibitory antibodies. The goal of the present study is to characterize the role of CD38 ecto-enzymatic activity in cardiac metabolism and the development of DIC. METHODS AND RESULTS: Using both a transgenic animal model and a non-cytotoxic enzymatic anti-CD38 antibody, we investigated the role of CD38 and its ecto-NADase activity in DIC in pre-clinical models. First, we observed that DIC was prevented in the CD38 catalytically inactive (CD38-CI) transgenic mice. Both left ventricular systolic function and exercise capacity were decreased in wild-type but not in CD38-CI mice treated with DXR. Second, blocking CD38-NADase activity with the specific antibody 68 (Ab68) likewise protected mice against DIC and decreased DXR-related mortality by 50%. A reduction of DXR-induced mitochondrial dysfunction, energy deficiency, and inflammation gene expression were identified as the main mechanisms mediating the protective effects. CONCLUSION: NAD+-preserving strategies by inactivation of CD38 via a genetic or a pharmacological-based approach improve cardiac energetics and reduce cardiac inflammation and dysfunction otherwise seen in an acute DXR cardiotoxicity model.

Targeting CD38/ ADP-ribosyl cyclase as a novel therapeutic strategy for identification of three potent agonists for leukopenia treatment.

Leukopenia is the most common side effect of chemotherapy and radiotherapy. It potentially deteriorates into a life-threatening complication in cancer patients. Despite several agents being approved for clinical administration, there are still high incidences of pathogen-related disease due to a lack of functional immune cells. ADP-ribosyl cyclase of CD38 displays a regulatory effect on leukopoiesis and the immune system. To explore whether the ADP-ribosyl cyclase was a potential therapeutic target of leukopenia. We established a drug screening model based on an ADP-ribosyl cyclase-based pharmacophore generation algorithm and discovered three novel ADP-ribosyl cyclase agonists: ziyuglycoside II (ZGSII), brevifolincarboxylic acid (BA), and 3,4-dihydroxy-5-methoxybenzoic acid (DMA). Then, in vitro experiments demonstrated that these three natural compounds significantly promoted myeloid differentiation and antibacterial activity in NB4 cells. In vivo, experiments confirmed that the compounds also stimulated the recovery of leukocytes in irradiation-induced mice and zebrafish. The mechanism was investigated by network pharmacology, and the top 12 biological processes and the top 20 signaling pathways were obtained by intersecting target genes among ZGSII, BA, DMA, and leukopenia. The potential signaling molecules involved were further explored through experiments. Finally, the ADP-ribosyl cyclase agonists (ZGSII, BA, and DMA) has been found to regenerate microbicidal myeloid cells to effectively ameliorate leukopenia-associated infection by activating CD38/ADP-ribosyl cyclase-Ca2+-NFAT. In summary, this study constructs a drug screening model to discover active compounds against leukopenia, reveals the critical roles of ADP-ribosyl cyclase in promoting myeloid differentiation and the immune response, and provides a promising strategy for the treatment of radiation-induced leukopenia.

Retention of stress susceptibility in the mdx mouse model of Duchenne muscular dystrophy after PGC-1α overexpression or ablation of IDO1 or CD38.

Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle wasting disease caused by the loss of the structural protein dystrophin with secondary pathological manifestations including metabolic dysfunction, mood and behavioral disorders. In the mildly affected mdx mouse model of DMD, brief scruff stress causes inactivity, while more severe subordination stress results in lethality. Here, we investigated the kynurenine pathway of tryptophan degradation and the nicotinamide adenine dinucleotide (NAD+) metabolic pathway in mdx mice and their involvement as possible mediators of mdx stress-related pathology. We identified downregulation of the kynurenic acid shunt, a neuroprotective branch of the kynurenine pathway, in mdx skeletal muscle associated with attenuated peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) transcriptional regulatory activity. Restoring the kynurenic acid shunt by skeletal muscle-specific PGC-1α overexpression in mdx mice did not prevent scruff -induced inactivity, nor did abrogating extrahepatic kynurenine pathway activity by genetic deletion of the pathway rate-limiting enzyme, indoleamine oxygenase 1. We further show that reduced NAD+ production in mdx skeletal muscle after subordination stress exposure corresponded with elevated levels of NAD+ catabolites produced by ectoenzyme cluster of differentiation 38 (CD38) that have been implicated in lethal mdx response to pharmacological ß-adrenergic receptor agonism. However, genetic CD38 ablation did not prevent mdx scruff-induced inactivity. Our data do not support a direct contribution by the kynurenine pathway or CD38 metabolic dysfunction to the exaggerated stress response of mdx mice.

Flow cytometric analysis of CD34+ CD38- cells; cell frequency and immunophenotype based on CD45RA expression pattern.

INTRODUCTION: The CD34+ CD38- population in bone marrow includes hematopoietic stem/progenitor cells. Recently, in acute myeloid leukemia, the focus has shifted to flow cytometry analysis targeting CD34+ CD38- leukemic cells due to their effectiveness in minimal/measurable residual disease detection and prognosis prediction. Nevertheless, the immunophenotype and cell frequency of these cells in the bone marrow, in the absence of leukemic cells, remains unknown. We aimed to evaluate detailed characteristics of CD34+ CD38- cells in both normal and leukemic cells by flow cytometry. METHODS: We compared the cell frequency and immunophenotype of the CD34+ CD38- fraction in the following groups: patients with idiopathic thrombocytopenic purpura and malignant lymphoma as controls (n = 17), post-treatment patients without abnormal blasts (n = 35), and patients with myeloid malignancies (n = 86). The comparison was based on the presence or absence of CD45RA expression, a marker commonly used to prospectively isolate lymphoid-primed cell populations within the CD34+ CD38- fraction. RESULTS: The CD34+ CD38- CD45RA+ cell population exhibited a significant expansion in bone marrow without leukemic cells 1 month after cord blood transplantation and in various type of myeloid malignancies, compared to the control group (p < 0.01). Continuous CD45RA expression and notable expansion of the CD34+ CD38- CD45RA- population were exclusively observed in myelodysplastic syndrome-related diseases. The CD34+ CD38- CD45RA+ population displayed frequent expression of various markers in both leukemic and non-leukemic cells, in contrast to the CD34+ CD38- CD45RA- population. CONCLUSIONS: The CD34+ CD38- fraction should be carefully evaluated considering the nature of normal hematopoietic precursor cells, their cell frequency and immunophenotype, including CD45RA expression pattern, for improving the accuracy of myeloid malignancy diagnosis.

CD38 x ICAM-1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma.

The cluster of differentiation 38 (CD38) is a well-validated target for treating multiple myeloma. Although anti-CD38 mAbs have demonstrated outstanding initial responses in patients with multiple myeloma, nearly all patients eventually develop resistance and relapse. In addition, currently approved CD38 targeting therapies have failed to show monotherapy efficacy in lymphomas, where CD38 expression is present but at lower levels. To effectively target CD38 on tumor cells, we generated an antibody-dependent cellular cytotoxicity (ADCC) enhanced bispecific CD38 x intercellular cell adhesion molecule 1 (ICAM-1) antibody, VP301. This bispecific antibody targets unique epitopes on CD38 and ICAM-1 on tumor cells with reduced red blood cell binding compared with the benchmark CD38 antibody daratumumab. VP301 demonstrated potent ADCC and antibody-dependent cellular phagocytosis activities on a selected set of myeloma and lymphoma cell lines even those with low CD38 expression. In an ex vivo drug sensitivity assay, we observed responses to VP301 in multiple myeloma primary samples from relapsed/refractory patients. Moreover, VP301 demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma models. Interestingly, combination of VP301 with the immunomodulatory drug, lenalidomide, led to synergistic antitumor growth activity in an in vivo efficacy study. In conclusion, the CD38 x ICAM-1 bispecific antibody VP301 demonstrated promising efficacy and specificity toward CD38+ and ICAM-1+ tumor cells and represents a novel approach for treating multiple myeloma and lymphoma.

The role of CD38 in inflammation-induced depression-like behavior and the antidepressant effect of (R)-ketamine.

CD38 is involved in immune responses, cell proliferation, and has been identified in the brain, where it is implicated in inflammation processes and psychiatric disorders. We hypothesized that dysfunctional CD38 activity in the brain may contribute to the pathogenesis of depression. To investigate the underlying mechanisms, we used a lipopolysaccharide (LPS)-induced depression-like model and conducted behavioral tests, molecular and morphological methods, along with optogenetic techniques. We microinjected adeno-associated virus into the hippocampal CA3 region with stereotaxic instrumentation. Our results showed a marked increase in CD38 expression in both the hippocampus and cortex of LPS-treated mice. Additionally, pharmacological inhibition and genetic knockout of CD38 effectively alleviated neuroinflammation, microglia activation, synaptic defects, and Sirt1/STAT3 signaling, subsequently improving depression-like behaviors. Moreover, optogenetic activation of glutamatergic neurons of hippocampal CA3 reduced the susceptibility of mice to depression-like behaviors, accompanied by reduced CD38 expression. We also found that (R)-ketamine, which displayed antidepressant effects, was linked to its anti-inflammatory properties by suppressing increased CD38 expression and reversing synaptic defects. In conclusion, hippocampal CD38 is closely linked to depression-like behaviors in an inflammation model, highlighting its potential as a therapeutic target for antidepressant development.

NADase CD38 is a key determinant of ovarian aging.

The ovary ages earlier than most other tissues, yet the underlying mechanisms remain elusive. Here a comprehensive analysis of transcriptomic landscapes in different organs in young and middle-aged mice revealed that the ovaries showed earlier expression of age-associated genes, identifying increased NADase CD38 expression and decreased NAD+ levels in the ovary of middle-aged mice. Bulk and single-cell RNA sequencing revealed that CD38 deletion mitigated ovarian aging, preserving fertility and follicle reserve in aged mice by countering age-related gene expression changes and intercellular communication alterations. Mechanistically, the earlier onset of inflammation induced higher expression levels of CD38 and decreased NAD+ levels in the ovary, thereby accelerating ovarian aging. Consistently, pharmacological inhibition of CD38 enhanced fertility in middle-aged mice. Our findings revealed the mechanisms underlying the earlier aging of the ovary relative to other organs, providing a potential therapeutic target for ameliorating age-related female infertility.

Heavy-chain antibody targeting of CD38 NAD+ hydrolase ectoenzyme to prevent fibrosis in multiple organs.

The functionally pleiotropic ectoenzyme CD38 is a glycohydrolase widely expressed on immune and non-hematopoietic cells. By converting NAD+ to ADP-ribose and nicotinamide, CD38 governs organismal NAD+ homeostasis and the activity of NAD+-dependent cellular enzymes. CD38 has emerged as a major driver of age-related NAD+ decline underlying adverse metabolic states, frailty and reduced health span. CD38 is upregulated in systemic sclerosis (SSc), a chronic disease characterized by fibrosis in multiple organs. We sought to test the hypothesis that inhibition of the CD38 ecto-enzymatic activity using a heavy-chain monoclonal antibody Ab68 will, via augmenting organismal NAD+, prevent fibrosis in a mouse model of SSc characterized by NAD+ depletion. Here we show that treatment of mice with a non-cytotoxic heavy-chain antibody that selectively inhibits CD38 ectoenzyme resulted in NAD+ boosting that was associated with significant protection from fibrosis in multiple organs. These findings suggest that targeted inhibition of CD38 ecto-enzymatic activity could be a potential pharmacological approach for SSc fibrosis treatment.

[Research Progress and Application of Daratumumab in Non-Multiple Myeloma--Review].

Daratumumab is the first CD38 monoclonal antibody drug approved for the treatment of patients with multiple myeloma. It can bind to CD38 expressed by tumor cells, inhibit tumor cell growth and induce myeloma cell apoptosis through a variety of immune-related mechanisms. Meanwhile, CD38 is also expressed in other cells, including regulatory T cells, regulatory B cells and myeloid-derived suppressor cells, which provides a theoretical basis for the treatment of hematological tumor diseases other than non-multiple myeloma diseases. This article reviews the research progress and application of this part.

Immunomodulatory properties of CD38 antibodies and their effect on anticancer efficacy in multiple myeloma.

BACKGROUND: CD38 has been established as an important therapeutic target for multiple myeloma (MM), for which two CD38 antibodies are currently approved-daratumumab and isatuximab. CD38 is an ectoenzyme that degrades NAD and its precursors and is involved in the production of adenosine and other metabolites. AIM: Among the various mechanisms by which CD38 antibodies can induce MM cell death is immunomodulation, including multiple pathways for CD38-mediated T-cell activation. Patients who respond to anti-CD38 targeting treatment experience more marked changes in T-cell expansion, activity, and clonality than nonresponders. IMPLICATIONS: Resistance mechanisms that undermine the immunomodulatory effects of CD38-targeting therapies can be tumor intrinsic, such as the downregulation of CD38 surface expression and expression of complement inhibitor proteins, and immune microenvironment-related, such as changes to the natural killer (NK) cell numbers and function in the bone marrow niche. There are numerous strategies to overcome this resistance, which include identifying and targeting other therapeutic targets involved in, for example, adenosine production, the activation of NK cells or monocytes through immunomodulatory drugs and their combination with elotuzumab, or with bispecific T-cell engagers.

The NADase CD38 may not dictate NAD levels in brain mitochondria of aged mice but regulates hydrogen peroxide generation.

Aging is a time-related functional decline that affects many species. One of the hallmarks of aging is mitochondrial dysfunction, which leads to metabolic decline. The NAD decline during aging, in several tissues, correlates with increase in NADase activity of CD38. Knock out or pharmacological inhibition of CD38 activity can rescue mitochondrial function in several tissues, however, the role of CD38 in controlling NAD levels and metabolic function in the aging brain is unknown. In this work, we investigated CD38 NADase activity controlling NAD levels and mitochondrial function in mice brain with aging. We demonstrate that NADase activity of CD38 does not dictate NAD total levels in brain of aging mice and does not control mitochondrial oxygen consumption nor other oxygen parameters markers of mitochondrial dysfunction. However, for the first time we show that CD38 regulates hydrogen peroxide (H2O2) generation, one of the reactive oxygen species (ROS) in aging brain, through regulation of pyruvate dehydrogenase and alfa-ketoglutarate dehydrogenase, as mitochondria H2O2 leakage sites. The effect may be related to mitochondrial calcium handling differences in CD38 absence. Our study highlights a novel role of CD38 in brain energy metabolism and aging.