**4. Monoclonal antibodies**

Binding of monoclonal antibody (mAb) to its target antigen on MM cells has been shown to induce cell death through several mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), induction of apoptosis through FcγR-mediated crosslinking of tumour-bound antibodies and modulation of target antigen enzymatic activity after antibody binding [111]. Three mAbs, Daratumumab, Elotuzumab and Pembrolizumab have advanced to phase III clinical trials with Daratumumab the most successful of these.

CD38 is variably expressed on haemopoietic and some non-haemopoietic cells with surface expression depending on the differentiation and activation status of the cell. High cell surface expression occurs on benign and malignant plasma cells [111] with the fully-humanised anti-CD38 mAb Daratumumab demonstrating impressive outcomes when combined with Bortezomib (CASTOR) or Lenalidomide (POLLUX) in the relapsed/refractory MM setting [112, 113]. Other CD38 mAbs, such as Isatuximab (chimeric) and MOR202 (fully human), with differing biological activities from Daratumumab are currently being evaluated in clinical trials (Isatuximab: NCT03275285, NCT03319667, NCT02990338; MOR202: NCT01421186). Elotuzumab binds to signalling lymphocytic activation molecule family member 7 (SLAM7) reducing MM cell binding to bone marrow stroma and activating ADCC [114]. Interestingly, whilst no responses to Elotuzumab as a single agent were observed, the addition of Elotuzumab to Lenalidomide and Dexamethasone in relapsed/refractory MM patients (ELOQUENT-2 trial) resulted in improvements in ORR and PFS, and Elotuzumab is currently the subject of ongoing clinical trials (NCT01891643, NCT02495922, NCT01335399) [115]. Pembrolizumab targets the programmed death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway, a critical initiator of immune activation, playing a role in mediating tolerance [116]. However, two phase III trials KEYNOTE-183 and KEYNOTE-185 have recently been suspended by the US Food and Drug Administration due to more deaths being observed in the Pembrolizumab arms and further information on the use of Pembrolizumab in MM is pending.

#### **4.1. Mechanisms of resistance to monoclonal antibodies**

#### *4.1.1. Pre-clinical/clinical findings*

*3.1.2. Clinical studies to circumvent resistance*

80 Update on Multiple Myeloma

**4. Monoclonal antibodies**

sustained response with dual BRAF and MEK inhibition [110].

to phase III clinical trials with Daratumumab the most successful of these.

arms and further information on the use of Pembrolizumab in MM is pending.

Whilst the CRBN pathway has been shown to be pivotal in IMiD responsiveness, no clinical studies have eventuated that make use of this important biology as a strategy to overcome resistance to IMiDs and many questions remain such as how much functional CRBN is actually required to maintain IMiD sensitivity. Despite the controversies surrounding CRBN, activating mutations in Ras pathway components, such as KRAS G12D and BRAF V600E, could potentially be targeted with existing compounds in MM patients harbouring these mutations [109]. Such studies have not yet been conducted, although two patients with BRAF V600E positive relapsed/refractory MM achieved significant reductions in tumour burden when treated with the BRAF inhibitor Vemurafenib whilst a patient with highly resistant and rapidly progressive MM also harbouring the BRAF V600E mutation achieved a rapid and

Binding of monoclonal antibody (mAb) to its target antigen on MM cells has been shown to induce cell death through several mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), induction of apoptosis through FcγR-mediated crosslinking of tumour-bound antibodies and modulation of target antigen enzymatic activity after antibody binding [111]. Three mAbs, Daratumumab, Elotuzumab and Pembrolizumab have advanced

CD38 is variably expressed on haemopoietic and some non-haemopoietic cells with surface expression depending on the differentiation and activation status of the cell. High cell surface expression occurs on benign and malignant plasma cells [111] with the fully-humanised anti-CD38 mAb Daratumumab demonstrating impressive outcomes when combined with Bortezomib (CASTOR) or Lenalidomide (POLLUX) in the relapsed/refractory MM setting [112, 113]. Other CD38 mAbs, such as Isatuximab (chimeric) and MOR202 (fully human), with differing biological activities from Daratumumab are currently being evaluated in clinical trials (Isatuximab: NCT03275285, NCT03319667, NCT02990338; MOR202: NCT01421186). Elotuzumab binds to signalling lymphocytic activation molecule family member 7 (SLAM7) reducing MM cell binding to bone marrow stroma and activating ADCC [114]. Interestingly, whilst no responses to Elotuzumab as a single agent were observed, the addition of Elotuzumab to Lenalidomide and Dexamethasone in relapsed/refractory MM patients (ELOQUENT-2 trial) resulted in improvements in ORR and PFS, and Elotuzumab is currently the subject of ongoing clinical trials (NCT01891643, NCT02495922, NCT01335399) [115]. Pembrolizumab targets the programmed death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway, a critical initiator of immune activation, playing a role in mediating tolerance [116]. However, two phase III trials KEYNOTE-183 and KEYNOTE-185 have recently been suspended by the US Food and Drug Administration due to more deaths being observed in the Pembrolizumab The relatively recent addition of mAbs to MM pharmacotherapy means there is a paucity of studies examining resistance mechanisms although these are now being explored with their increasing clinical use (**Table 1** and **Figure 1C**). Examination of CD38 expression on MM cells in 102 patients treated with Daratumumab monotherapy has been insightful [117]. With regard to the effect of Daratumumab on residual bone marrow plasma cells, two important points were clear from this analysis. Firstly, CD38 cell surface expression on plasma cells is highest before Daratumumab treatment and is significantly decreased during treatment. At the time of progressive disease, plasma cells isolated from the bone marrow of these patients exhibited low expression of CD38 suggesting Daratumumab therapy would be less effective, a finding corroborated previously [76]. Secondly, pre-treatment CD38 expression on the surface of MM cells was higher in patients who achieved at least a PR compared to those who did not. Recently, it was shown that Daratumumab-CD38 complexes and accompanying cell membrane are actively transferred from MM cells to monocytes and granulocytes in a process called trogocytosis that was also associated with reduced MM cell surface expression of CD49d, CD56 and CD138 [118]. However, Daratumumab-induced reductions in CD38 expression on MM cells occur in patients with deep and durable responses suggesting reductions in CD38 alone are not responsible for Daratumumab resistance [118]. Cell surface expression of the complement-inhibitory proteins, CD46, CD55 and CD59, was not associated with clinical response but significantly increased only at the time of disease progression. Furthermore, all*trans* retinoic acid increased CD38 expression whilst decreasing expression of CD55 and CD59 on MM cells from patients who developed Daratumumab resistance to approximately pretreatment levels, resulting in enhancement of Daratumumab-mediated CDC [117].

In addition to the cell surface expression of target antigens on MM cells, several other potential mechanisms of resistance to mAbs may be at play. Soluble forms of CD38 [119] and SLAM7 [120] may affect the efficacy of Daratumumab and Elotuzumab, respectively. Another potential mechanism of resistance is the development of neutralising antibodies to the therapeutic antibody. This phenomenon was noted in 39% of patients treated with single agent Elotuzumab resulting in more pronounced effects on serum Elotuzumab concentrations [121]. Furthermore, in the ELOQUENT-2 trial, 15% of patients developed anti-Elotuzumab antibodies on at least one occasion [115], however, antibodies directed against Daratumumab have to this day not been detected. Other factors that may contribute to the clinical efficacy of mAb therapy include the frequency and activity of effector immune cells [122], Fcγ receptor polymorphisms [123] and even KIR and HLA genotypes [124].

#### *4.1.2. Clinical studies to circumvent resistance*

Whilst the mechanisms of resistance to mAbs are being elucidated, clinical studies specifically designed to overcome these biological processes are largely lacking with the exception of an ongoing phase I/II trial of Daratumumab in combination with all-*trans* retinoic acid for patients with relapsed/refractory MM (NCT02751255).
