Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule

Abstract

In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule's regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

Figures

Figure 1

Confocal microscopy analysis of CD38 internalization. DL06 myeloma cells were incubated with saturating amounts of FITC-labeled agonistic IB4 mAb for 30 min at 4°C. After rinsing in RPMI-1640 medium (Sigma, Milano, Italy) with 5% fetal calf serum (Euroclone, Milano, Italy), affinity-purified goat anti-mouse IgG2a (SouthernBiotech, Birmingham, AL, USA) was added for 10 min at 4°C. Cells were successively incubated at 37°C for selected times. DL06 were then fixed at 4°C for 20 min in PBS containing 4% formalin and rinsed in PBS. The samples were analyzed with an Olympus FV300 laser scanning confocal microscope equipped with a Blue Argon (488 nm) laser and FluoView 300 software (Olympus Biosystems, Hamburg, Germany).

Figure 2

Comparative microarray analysis of the DL06 cells after CD38 ligation (2 h versus 24 h). RNA was obtained from the DL06 cells incubated at 37°C with anti-CD38 (clone IB4) mAb for 2 h and 24 h. An irrelevant isotype-matched anti-TCRvβ3 mAb was used as control for the same interval times. Four independent experimental settings were analyzed using the two-color comparative hybridization procedure in three cases and the one color protocol in one. mRNA amplification and labeling was followed by hybridization on 8×60K Human Whole Genome Oligo Microarrays (Agilent Technologies, Santa Clara, CA, USA) with technical replication (details in [68]). Images were analyzed using Feature Extraction software v10.5 (Agilent Technologies) and raw data processed within R statistical environment, using the limma library (http://www.bioconductor.org/packages/2.11/bioc/html/limma.html). The raw intensity values were background-corrected with the normexp method and normalized with the quantile method, in the case of one-color hybridization. Two-color hybridization arrays were first subjected to Loess intraarray normalization and then to quantile interarray normalization. For each treatment time, technical replicates were combined and the empirical Bayes method was applied to retrieve any modulated probe in treated versus control comparisons (Benjamini-Hochberg corrected p value <0.01). Each point refers to 1 of the 14 transcripts with adjusted p value ≤0.01 in at least one of the experimental samples analyzed. Red represents upregulated expression and green downregulated expression.

Figure 3

Heat map representation of miRNAs modulated in human DL06 myeloma line by CD38-mediated signals. Total RNA was isolated from DL06 line treated with anti-CD38 (clone IB4) mAb for 0, 2, 6 and 24 h at 4°C. An irrelevant isotype-matched anti-TCRvβ3 mAb was used as control for the same interval times. Two biological replicates for 0, 24 h and control samples were hybridized. miRNA expression was investigated using the Agilent Human miRNA microarray (G4470B, Agilent Technologies), as described (69). By GeneSpring GX 12 software (Agilent Technologies), data transformation was applied to set all the negative raw values at 1.0, followed by a quantile normalization and a log2 transformation. miRNA expression was referred to time 0 h of each biological series. Filters on gene expression were used to keep only the miRNAs that were detected in at least one sample. miRNAs significantly modulated in the DL06 model after 24-h treatment with agonistic anti-CD38 mAb were identified using unpaired t test (corrected p value <0.05). Treatment was included in cluster analysis. The expression values of the genes represented on the heat map correspond to the values normalized on time 0 sample of the same biological series. Red represents upregulated expression and green downregulated expression.

Figure 4

Schematic representation of the mechanisms of action of ATRA and tamibarotene on CD38 gene.

Figure 5

CD38 expression on fresh myelomas (A) and human myeloma cell lines (B) after 24 h exposure to ATRA. CD38 expression on fresh myelomas (C) and human myeloma cell lines (D) after 24 h exposure to tamibarotene. Cells were cultured at 37°C with retinoid for 24 h. Cells were then incubated at 4°C for 20 min with the appropriate conjugated antibodies panel. Flow cytometric analyses were performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA), using a WinMDI 2.9 software.

Figure 6

CD38 expression in ATRA-treated CD19+/CD5+ CLL cells. PBMC isolated from CLL patients (CD38+ CLL, n = 6; CD38−CLL, n = 9) and cultured (24 h at the indicated ATRA concentrations) were stained with anti-CD19, CD5 and CD38 mAbs, all locally produced. CD38 expression was analyzed using FACSDiva software (Becton Dickinson). Representative CD38 expression in CD38+ (A) and CD38− (B) CLL cells from patients treated with ATRA, by evaluating rMFI.

Figure 7

Schematic representation of the CD38/PC-1/CD73 axis, which runs the production of ADO along with different products. NAM, nicotinamide; NAMPT, nicotinamide phosphoribosyltransferase; NMN, nicotinamide mononucleotide; NMNAT, NMN-adenylyltransferase; PRPPi, phosphoribosylpyrophosphate; TNAP, tissue nonspecific alkaline phosphatase.