Resistance to daratumumab in patients with multiple myeloma

Research output: ThesisPh.D. thesis

Abstract

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage, characterized by uncontrolledgrowth of malignant plasma cells primarily in the bone marrow (BM) but sometimes also at extramedullarysites. The main clinical manifestations of MM are anaemia, renal failure, hypercalcemia, and osteolyticlesions. Remarkable progress has been made in the treatment of MM with the introduction ofimmunomodulatory drugs, proteasome inhibitors, and most recently monoclonal antibodies. However,despite this progress, MM remains an incurable disease.

Daratumumab (DARA) is a CD38 antibody approved for the treatment of MM as monotherapy or in combination with a number of standards of care anti-myeloma drugs. DARA may induce direct killing of myeloma cells, affect the tumour microenvironment and the immune system. By these modes of action, DARA has improved both overall response, the progression free survival and the overall survival for myeloma patients. However, despite of this most patients treated with DARA eventually progress. The mean overall survival after progression on DARA is only 12 months, so there is a need to examine the reasons for failure of DARA therapy in order to improve the outcome.

Complement dependent cytotoxicity (CDC) is thought to be one of most important modes of action of DARA. It has been demonstrated that two complement inhibitory proteins (CD55 and CD59) are upregulated on myeloma cells at the time of progression on DARA. This may inhibit DARA-mediated killing of myeloma cells by CDC. We estimated the complement activation in patients treated with DARA by measuring the level of serum-C3d, a complement fragment, which is produced when the complement
cascade is activated. We compared the concentration of serum-C3d in patients progressing on DARA to patients with a partial response or better. We found no statistically significant difference in the level of C3d between responders and non-responders, indicating that the there was no difference in the activation of the
complement system between the two groups when assessed by this biomarker. The validity of C3d as a biomarker of complement activation has been documented in the literature and was further supported in our study by a significant increase of C3d when MM patients started treatment in the clinical trial GCT3014 with a CD38 antibody developed to trigger complement activation in a more efficient manner through hexamerization (“Hexabody CD38”). This suggests that CDC may not be the most important mode of action of DARA for a maintained control of the MM. The ongoing study GCT3014 will clarify if enhanced CDC may result in a better outcome for MM patients.

Extracellular vesicles (EVs) are a heterogeneous population of particles, ranging in size from 50 to 5000 nm and enclosed by a lipid bilayer, that are released from cells and play an important role in cell-cell communication in physiological and pathophysiological situations. EVs are secreted in significantly higher amounts by cancer cells compared to normal cells. These tumour-derived EVs mediate intercellular communication between tumour cells and normal cells within the tumour microenvironment via the horizontal transfer of functional protein, DNA and RNA. Extracellular vesicles may also act as a decoy that captures and neutralizes therapeutic antibodies. EVs were isolated from PB and BM from MM patients treated with DARA and PB of healthy controls. EV size, number, and the expression of CD38, CD55, CD59 and PD-L1 as well as the EV markers CD9, CD63, CD81, and CD147 were determined by flow cytometry. We used mass spectrometry to determine whether DARA was present on the EVs. All EV samples from both PB and BM express the EV markers (CD9, CD63, CD81, CD147) and CD CD38, PD-L1, CD55 and CD59. The level of CD55 and CD59 are elevated on MM PB EVs compared with healthy controls, and the level of PD-L1 on MM PB EVs is higher in patients responding to treatment with DARA. Furthermore, mass
spectrometry data show that MM PB EVs bind DARA. An interesting observation that deserves further study was the high level of CD147 expressed by EVs of MM patients because CD147 is correlated with various important aspects of malignancy.

It has been shown that DARA can deplete CD38+ regulatory cells of T-cell (CD4+), B-cell and monocyte origin, which leads to the expansion and activation of cytotoxic T-cells (CD8+). The expansion of cytotoxic T-cells during treatment with DARA is correlated to the clinical response and a maintained activity of these T-cells may be important for a continued control of the disease. Expression of checkpoint inhibitor molecules may over time lead to treatment failure because the T-cells become exhausted. We isolated CD4+ and CD8+ T-cells from peripheral blood (PB) and bone marrow (BM) from newly diagnosed myeloma
patients (NDMM) and myeloma patients progressing on DARA (DRMM). We assessed the expression of immune regulatory receptors (CTLA-4, PD-L1, DNAM-1 and TIGIT) on T-cell subsets from PB and BM using multicolour flow cytometry. We observed that CD4+ T-cells from both PB and BM of patients relapsing on daratumumab have a higher expression of the costimulatory checkpoint receptor DNAM-1. The potential role of DNAM-1+CD4+ T-cells in the development of resistance to daratumumab needs further exploration. We also observed that the inhibitory checkpoint receptor TIGIT is more frequently expressed by BM CD8+ T-cells from MM patients than PD-1 and CTLA-4, which supports the hypothesis that TIGIT may play a central role in the immune escape of the malignant plasma cells. This is now being tested in a number of clinical trials.
Original languageEnglish
Awarding Institution
  • University of Southern Denmark
Supervisors/Advisors
  • Plesner, Torben, Principal supervisor
  • McGee, Margaret, Supervisor, External person
Date of defence14. Jun 2023
Publisher
DOIs
Publication statusPublished - 21. Apr 2023

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