Preclinical studies have revealed that both CDC and ADCC are more effective when the expression of CD38 is high, but immediately following the first infusion with DARA, there is a pronounced reduction of the expression of CD38 on the remaining MM cells [9, 23, 27]. In vitro studies showed that all-trans retinoic acid (ATRA) improves the DARA-mediated CDC and ADCC by upregulation of CD38 on MM cells including MM cells, which were resistant to DARA monotherapy [27]. This point towards a potential clinical advantage of a high expression of CD38 on myeloma cells. Indeed, a high expression of CD38 before initiation of DARA is a predictor of obtaining a partial response or better [23]. Based on these observations, a phase 1/2 study combining ATRA with DARA in DARA-refractory patients was initiated [28]. Despite of the promising in vitro experiences, the results of the clinical study were disappointing. The overall response rate was only 5% and the mean PFS 2.8 months. Panobinostat can also upregulate the expression of CD38 by myeloma cells and enhance the anti-myeloma effect of DARA in vitro but given the disappointing clinical results with ATRA it is unlikely that this avenue will be pursued further [29].

The fact that CD38 expression by MM cells is downregulated immediately after initiation of treatment with DARA but a response may be maintained over many months or even years suggests that other modes of action of DARA than CDC, ADCC and ADCP are important to obtain a long term response [23]. The long-term effects of DARA may be more dependent on its immunomodulatory effects (reduced production of immunosuppressive adenosine, elimination of CD38 positive regulatory cells) or the inhibition of formation of nanotubes and transfer of mitochondria from stromal cells to the MM cells. Thus, low CD38 expression by myeloma cells may in fact be beneficial to maintain control of the disease and one may indeed envision CD38 as a growth and survival factor for MM cells. A clinical aspect of this discussion is whether the treatment with DARA should continue with a change of partner drug despite progression on a DARA containing regimen, or whether treatment with DARA should be paused, so the MM cells regain the CD38 expression, which takes about 6 months [23]. Recently, Agoston and colleagues did a retrospective, nationwide evaluation of all MM patients in Denmark receiving DARA before 2019. The course of therapy for 474 patients were evaluated, and the OS from the time point where each patient progressed on DARA for the first time was determined. They found that the median OS for patients continuing to receive DARA but combined with another anti-myeloma therapy than before the relapse was 23.6 months, and the OS for patients not receiving DARA after progressing on DARA was 11.3 months. The difference was clinically significant in a multivariate analysis counting for age, cytogenetic risk, time since diagnoses among others [30]. This points towards a benefit of continuing treatment with DARA without interruption and suggests that the low CD38 expression by MM cells imposed by DARA is compatible with and may in fact support a continued clinical response.

In the clinical setting complement activation caused by DARA has only been measured during the first eight weeks of treatment and only by measuring native complement factors, among these C2 [23]. The results show that there is a decrease of the native complement factors after initiating DARA therapy, but the signal disappears after a few weeks of treatment, indicating that the complement system is mainly active during these first weeks or that an increased production of complement factors compensate for the consumption. In an attempt to make CDC even more potent, a new CD38 mAb is under development [31]. This CD38 mAb called GEN3014 has a hexamerization-enhancing mutation, which increases the binding of C1q and thereby enhance the activation of the complement cascade [32]. The data from the first in human trials of DARA and GEN3014 confirms this assumption. There was a much more profound decrease in C2 in patients treated with GEN3014 than in patients treated with DARA [23, 31]. To explore the impact of complement activation during long-term treatment with DARA, the complement split product C3d was measured in PB of patients who had received DARA for a median of minimum 300 days. The level of C3d in patients receiving DARA that had obtained a partial response or better was compared to patients progressing on DARA. There were no significant difference between the two groups. This supports the hypothesis that CDC is of less importance for the long-term response to DARA.

The MM bone marrow microenvironment seems to be in an immunosuppressive state and both B- and T-cells shows significant impairment of their functions, possibly due to an increased concentration of adenosine in the bone marrow [33]. Such impairment is reflected by higher expression of markers of T-cell exhaustion such programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte associated protein-4 (CTLA-4) [34]. Several checkpoint inhibitors that block PD-1, PD-L1 or CTLA-4 have been approved for solid cancers but, so far, checkpoint inhibitors have not shown convincing efficacy in MM [35]. T-cell immunoglobulin and ITIM domains (TIGIT) is an inhibitory checkpoint molecule highly expressed by CD8+ T-cell from MM patients and may play a more important role in the inhibition of the T-cell response against MM [36].

When initiating treatment with DARA, the number of CD4⁺CD25⁺CD127^dimTregs decline immediately. These Tregs are also CD38⁺and therefore sensitive to DARA. This decline is followed by an expansion of CD8⁺ T cells and an increase in the ratios of CD8⁺:CD4⁺ and CD8⁺: Tregs [11]. These immunomodulatory changes are more pronounced in patients with a good, clinical response to DARA. Whether exhaustion of T-cells plays a role in the development of resistance to DARA is not fully clarified, but adding durvalumab, a PD-L1 inhibitor, to DARA in DARA-refractory patients did not induce a clinical response [37]. Iversen et al. examined the expression of checkpoint molecules on CD8+ T-cells isolated from the bone marrow of newly diagnosed MM patients (NDMM) and patients relapsing on DARA [38]. They found no difference in the expression of PD-1 on CD8⁺ T-cells in DARA-refractory patients compared to NDMM patients. Furthermore, PD-1 was only expressed on around 20% of CD8⁺ T-cells from MM patients, whereas TIGIT was present on around 80% (Figure 3). Paiva et al. confirmed the observation on the percentage of PD-1 expressing CD8⁺T-cells [39]. Two studies confirm that TIGIT is more frequently expressed by CD8⁺ T-cells in myeloma patients than PD-1 [36, 40]. Altogether, it seems that PD-1 is not a key immune regulatory checkpoint in MM. Iversen et al. did not find a difference in the expression of TIGIT on CD8⁺ T-cells when comparing NDMM to DARA-refractory patients, which was supported by the findings by Guillerey [36]. Neri et al . found a higher expression of TIGIT on T-cells from DARA-resistant patients compared to responders [41]. Therefore, it seems that there is a difference in the expression of TIGIT on T-cells when comparing DARA-refractory patients to patients currently in a clinical response, but not when comparing DARA-refratory patients to NDMM. Anti-TIGIT mAbs are currently being tested in clinical trials of patients with relapsed MM.