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\begin{document}

\title{Resistance to daratumumab in patients with multiple myeloma}



\author[1]{Katrine Fladeland Iversen}%
\affil[1]{Syddansk Universitet}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
MM is an incurable cancer in the bone marrow. The treatment of MM has
developed significantly during the last 20 years, which has resulted in
increased survival. Daratumumab is the first CD38 antibody approved for
treatment of MM. It has improved the treatment of MM even further. This
is an evaluation of the modes of action of daratumumab and a description
of the development of resistance with focus on inhibitory checkpoint
receptors on CD8+ T-cells, complement activation and extracellular
vesicles.%
\end{abstract}%



\sloppy


\subsection*{Resistance to daratumumab in patients with multiple
myeloma}

{\label{resistance-to-daratumumab-in-patients-with-multiple-myeloma}}

Katrine Fladeland Iversen\textsuperscript{1}

\textsuperscript{1} Institute of Regional Health Science, University of
Southern Denmark, and Department of Internal Medicine, Section of
Hematology, Lillebaelt Hospital, University Hospital of Southern
Denmark, Beriderbakken 4, 7100 Vejle, Denmark;
katrine.fladeland.iversen@rsyd.dk

Abstract: 80 words

Body: 3304 words

References: 50

\subsection*{Abstract / Plain English
summary}

{\label{abstract-plain-english-summary}}

MM is an incurable cancer in the bone marrow. The treatment of MM has
developed significantly during the last 20 years, which has resulted in
increased survival. Daratumumab is the first CD38 antibody approved for
treatment of MM. It has improved the treatment of MM even further. This
is an evaluation of the modes of action of daratumumab and a description
of the development of resistance with focus on inhibitory checkpoint
receptors on CD8+ T-cells, complement activation and extracellular
vesicles.

\subsection*{Multiple myeloma}

{\label{multiple-myeloma}}

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage,
characterized by neoplastic, monoclonal expansion of plasma cells in the
bone marrow (BM), which may cause anaemia, osteolytic lesions of the
bones, hypercalcemia and renal failure also known as CRAB criteria
{[}1{]}. MM accounts for 10\% of all hematologic cancers, which makes it
the second most prevalent hematologic malignant disorder after
non-Hodgkin's lymphomas. The mean age at diagnosis is 70 years, and five
year overall survival is around 50\%.

\subsubsection*{Treatment of multiple
myeloma}

{\label{treatment-of-multiple-myeloma}}

Since MM is an incurable disease, the main purpose of treatment is to
give the patients deep and durable responses with as few side effects as
possible. The typical disease course consists of remissions and
relapses. Generally, with increasing lines of therapy, the more
superficial is the response, and the shorter is the duration of the
remission {[}2, 3{]}. The following types of drugs constitute the
backbone of MM therapy: immunomodulatory drugs (IMIDs): thalidomide,
lenalidomide and pomalidomide; proteasome inhibitors (PIs): bortezomib,
carfilzomib, and ixazomib; alkylating drugs: melphalan and
cyclophosphamide; corticosteroids: dexamethasone and prednisone; and the
newcomer monoclonal antibodies targeting CD38: daratumumab (DARA) and
isatuximab or SLAM-F7: elotuzumab. Very recently, approved therapies
encompass B-cell maturation antigen (BCMA)-targeting approaches such as
Belantamab Mafodotin, bispecific antibodies, chimeric antigen receptor
(CAR) T-cells, and a whole new range of active therapies are in the
pipeline.

The initial treatment is dependent on the age and co-morbidities of the
patient. For young ([?] 70 years) patients without severe co-morbidities,
an induction regimen followed by high dose treatment (HDT) and
autologous stem cell treatment (ASCT) is standard first line. A typical
induction regimen consists of four cycles of lenalidomide, bortezomib
and dexamethasone {[}4{]}. For older (\textgreater{} 70 years) patients
the initial treatment usually also consists of lenalidomide, bortezomib
and dexamethasone until a proper disease control has been obtained or
side effects to bortezomib emerge {[}5{]}. Most patients with MM will
eventually relapse and be in need for a second line of therapy {[}3{]}.
Treatment for relapse is less standardized and more tailored to the
individual patient taking into account the former treatment, response to
treatment, duration of response and co-morbidities. In Denmark, most
patients are treated with DARA in combination with dexamethasone and
lenalidomide or bortezomib in their second line of treatment {[}6{]}.

\subsection*{Daratumumab}

{\label{daratumumab}}

\subsubsection*{Modes of action}

{\label{modes-of-action}}

Daratumumab (DARA) is a human immunoglobulin G1 (IgG1) kappa monoclonal
antibody (mAb) that targets CD38. CD38 is a 45-kDa type II transmembrane
glycoprotein acting as both a receptor and an ectoenzyme. It is highly
expressed on plasma cells (PCs) -- and especially on malignant PCs (MM
cells), and to a lesser extent on red blood cells, platelets, natural
killer (NK) cells, a subset of B and T cells, and numerous
non-haematological tissues such as airway epithelium, smooth muscle
cells, striated-muscle cells including heart tissue, central and
peripheral nerve tissue, glial cells, osteoclasts and endocrine cells of
the pancreas {[}7{]}.

Three different effector mechanisms seem to be essential for the direct
killing of malignant PCs by DARA: antibody-dependent cellular
phagocytosis (ADCP), where the Fc region of DARA bound to CD38 (the
``tumour antigen'') on MM cells, reacts with the Fc receptor on effector
cells, e.g. macrophages (\textbf{Figure 1} ). This induces phagocytosis
of the MM cell. If the antigen-bound DARA reacts with the Fc receptor on
a NK-cell or T-/NK-cell, they will release perforin and GrB, which will
result in cytolysis of the MM cell termed antibody-dependent cellular
cytotoxicity (ADCC). In the preclinical studies, it seems that
especially complement dependent cytotoxicity (CDC) is important for MM
cell lysis {[}8{]}. During complement activation, the complement protein
C1q binds to the Fc region of DARA attached to CD38. Each Fc region has
a single binding site for C1q, and each C1q molecule must bind to at
least two DARA Fc regions to become activated. Since each DARA molecule
has only one Fc region, multiple DARA molecules must be brought together
to initiate the process of complement activation {[}8, 9{]}. An in vitro
comparison of DARA and three other CD38 mAbs indicates that DARA is the
most efficient activator of CDC {[}10{]}.

Beside these abilities to directly kill tumour cells, DARA affects the
immune system through several different modes of action. Studies of
patient material collected during clinical trials where DARA was given
as monotherapy showed that DARA may eliminate
CD38\textsuperscript{+}populations of regulatory T-cells, B-cells and
monocytes/macrophages that impose a break on the cytotoxic T-cells
{[}11{]}. Consequently, cytotoxic T-cells proliferate and become
activated following treatment with DARA. This immune response correlates
with the clinical response to the treatment. Furthermore, DARA inhibits
MM cell adhesion to the bone marrow stromal cells (BMSCs) via CD38
internalization through the endocytic machinery rendering the myeloma
cells more sensitive to concomitant therapy {[}12{]}. Recently, another
function of CD38 was identified. Marlein et al. showed that
CD38-dependent tumour-derived tunnelling nanotubes (TNT) could be
established between BMSCs and MM cells. Mitochondrial transfer via these
TNT is a method for the MM cell to provide energy for further tumour
growth. This transfer was dependent on CD38 and was significantly
decreased when using a CD38 blocking antibody or ``knock down'' of CD38
in vitro {[}13{]}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\par\null

\subsubsection*{Clinical development}

{\label{clinical-development}}

The first clinical trials with DARA monotherapy included patients who
had relapsed or refractory disease after two or more prior lines of
treatment {[}14, 15{]}. The overall response rate (ORR) was 31\% and for
responders the median overall survival (OS) was \textgreater{} 20 months
in this heavily pre-treated cohort of patients who at that time were
only expected to have an OS of nine months on standard treatment
{[}16{]}. These studies led to the approval of DARA as treatment for
relapsed refractory multiple myeloma (RRMM) {[}17{]}. Interestingly, a
clear benefit in OS (18 months, a doubling of the expected OS) was also
found in the group of patients who only achieved SD or MR according to
IMWG criteria. Thus, although the reduction of the tumour load in these
patients is minimal, they live significantly longer than the expected
nine months. This can only be understood if other mechanisms than tumour
cell killing are important. This point to a potential role of DARA for
the immune system's ability to control myeloma and/or to a possible
importance of DARA's ability to block the transfer of mitochondria
through nanotubes and thereby inhibit MM cell proliferation and survival
as described earlier.

Many clinical studies of DARA in combination with standard of care
anti-myeloma drugs have been conducted or are under way. So far, all
studies showed that the addition of DARA to standard of care
anti-myeloma drugs increases the ORR, the progression free survival
(PFS) and the number of patients where the disease is no longer
traceable even with the most sensitive techniques such as next
generation flow cytometry or next generation sequencing, so the patient
has become minimal residual disease (MRD) negative. With the most recent
developments of technologies, the detection level of residual tumour
cells has come down to one cell in one million BM cells {[}18{]}. The
high quality responses have also translated into very significantly
improved survival {[}19{]}. Recently an OS benefit was also found by
adding DARA to established anti-myeloma therapies of NDMM or RRMM {[}20,
21{]}. In addition, progression free survival on the subsequent line of
therapy (PFS2) may be improved by the addition of DARA {[}21{]}.

\subsubsection*{Mechanism of resistance to
daratumumab}

{\label{mechanism-of-resistance-to-daratumumab}}

DARA has improved the treatment of MM significantly, and improved both
PFS, ORR, MRD negativity rates and OS {[}20, 21{]}. Nevertheless, even
though the clinical benefits of DARA are well documented, a recent
evaluation of all Danish MM patients, who have received DARA before
2019, showed that the majority of patients eventually relapse on DARA
and that the median OS after progression is only approximately 12 months
{[}22{]}. These findings highlight a need to examine the reasons for
failure of DARA therapy in order to improve the outcome.

Immediately following the first infusion with DARA, there is a
pronounced reduction of the expression of CD38 on the remaining MM cells
{[}23{]}. Whether this downregulation of the CD38 expression is good or
bad for the response to DARA is not yet clarified. The reduction of CD38
results in impaired CDC, ADCC and ADCP activity by DARA. On the other
hand, formation of immunosuppressive adenosine, potentially protective
adhesion of myeloma cells to stromal cells and the formation of
nanotubes transferring mitochondria from stromal cells to myeloma cells
that boost myeloma cells is also reduced by DARA. A high expression of
CD38 before initiation of treatment with DARA is associated with a
better chance of response, but it does not result in a longer PFS
{[}23{]}.

\subsubsection*{The complement system}

{\label{the-complement-system}}

The complement system is a part of the humoral immunity. Activation of
the complement system results in formation of the membrane attack
complex (MAC) on the surface of the target cell (Figure 2). The MAC
forms pores in the plasma membrane, which results in osmotic swelling
and rupture of the target cell. The plasma level of the complement
degradation and activation products, e.g. C3a, C3d, C5a, C5b-9, reflects
the extent of complement activation {[}24-26{]}. Normal cells have
several complement inhibitory proteins (CIPs) on the cell surface to
protect them from the effects of complement. CD59 is an important CIP,
which blocks the final formation of the MAC, and thereby inhibits the
function of this element of the complement system. CD55 accelerates the
decay of C3- and C5-convertases (Figure 2). Analysis of samples
collected during monotherapy with DARA has shown an increased level of
the CIPs CD55 and CD59 on the myeloma cells at the time of loss of
response and disease progression {[}23{]}. Thus, inhibition of CDC may
be a cause of development of resistance to DARA.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

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.

\subsection*{T cells}

{\label{t-cells}}

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\textsuperscript{+}CD25\textsuperscript{+}CD127\textsuperscript{dim}Tregs
decline immediately. These Tregs are also CD38\textsuperscript{+}and
therefore sensitive to DARA. This decline is followed by an expansion of
CD8\textsuperscript{+} T cells and an increase in the ratios of
CD8\textsuperscript{+}:CD4\textsuperscript{+} and
CD8\textsuperscript{+}: 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\textsuperscript{+} T-cells in DARA-refractory patients compared to
NDMM patients. Furthermore, PD-1 was only expressed on around 20\% of
CD8\textsuperscript{+} 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\textsuperscript{+}T-cells {[}39{]}. Two studies confirm that TIGIT
is more frequently expressed by CD8\textsuperscript{+} 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\textsuperscript{+}
T-cells when comparing NDMM to DARA-refractory patients, which was
supported by the findings by Guillerey {[}36{]}. Neri \emph{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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 3.} The expression of inhibitory immune checkpoint
receptors on CD8\textsuperscript{+} T-cells {[}38{]}.

\subsection*{Extracellular vesicles}

{\label{extracellular-vesicles}}

Extracellular vesicles (EVs) are membrane-bound organelles that are
released from all cells to the extracellular space and biological
fluids, e.g., blood. They can contain molecular cargo (e.g., RNA,
protein and metabolites) and carry cell-specific markers on their
surface, which unveils their cell of origin. In recent years, the focus
on EVs and their role in oncogenesis, metastatic disease and resistance
to cancer therapy has expanded {[}42{]}. Caivano \emph{et al} . showed
that many patients with haematological malignancies, including MM, had a
significantly higher amount of EVs in peripheral blood (PB) compared to
healthy individuals. Most EVs from the haematological patients expressed
a cancer antigen specific for the individual disease on their surface.
EVs from MM patients expressed CD38 {[}43{]}. The MM bone marrow
microenvironment is a complex network of several different cell types,
and EVs may contribute to the cross talk between malignant and
non-malignant cells. Multiple studies in different types of cancer
suggest that EVs may play a role in drug-resistance by several different
mechanisms. For example by suppression of immune cells, by transferring
drug-efflux pumps from drug-resistant cells to drug-sensitive cells, or
by binding therapeutic mAbs in the circulation and thereby preventing
them from reaching their target {[}44{]}. In vivo studies have revealed
that lenalidomide-resistance can be transmitted from resistant to
non-resistant MM cells via EVs {[}45{]}. Breast carcinoma cells, which
express human epidermal growth factor receptor 2 (HER2), can be treated
with trastuzumab, a mAb like DARA. These carcinoma cells secrete EVs
expressing HER2, which then binds trastuzumab off target and inhibits
the interaction of trastuzumab with the tumour cells {[}46{]}. The same
process of capturing and neutralizing a therapeutic antibody has been
observed with rituximab in lymphoma {[}47{]}. In line with the findings
of HER2 expressing cells in breast cancer and CD20 expressing cells in
lymphomas, an in vitro study has revealed that DARA treated MM cell
lines secrete EVs expressing a CD38/daratumumab complex {[}48{]}.
Likewise, EVs isolated from MM patients receiving DARA express CD38
{[}49{]}. Brennan et al. performed mass spectrometry on EVs from PB of
10 DARA treated patients (5 responding to DARA and 5 progressing on
DARA) and 10 untreated healthy control EV samples. The majority of
peptides identified in both the DARA treated MM EVs and healthy control
EVs matched the DARA sequence. This is due to the fact that DARA is a
fully human IgG. But in 9 out of 10 patients treated with DARA, they
found EVs containing a peptide sequence that was not detected in the 10
control samples, with several MM patients having multiple DARA-specific
peptides. This finding supports the hypothesis that DARA is present on
EVs from patients treated with DARA and thus bound off target. Whether
the binding of DARA off target contributes to the development of
resistance is not known. In addition, the expression of the CIPs CD55
and CD59 was higher on EVs isolated from DARA treated MM patients
compared to healthy controls {[}49{]}. In combination with the high
amounts of CD55 and CD59 on the plasma membrane of the myeloma cells
this may inhibit CDC mediated by DARA {[}23{]}.

\subsection*{Conclusion}

{\label{conclusion}}

DARA has improved the treatment of myeloma patients significantly, but
patients relapsing on DARA is still a clinical challenge. It seems that
a high expression of CD38 on malignant plasma is essential for the
initial response, but might be a disadvantage for the long-term
response. The immune checkpoint receptor TIGIT is highly expressed on
CD8+ T-cells from myeloma patients, but has not proved its importance in
the clinical setting yet. Whether exhaustion of T-cells is important for
the development of resistance to DARA is still not known. Furthermore,
the formation of circulating EVs binding DARA off target, could
potentially contribute to this development. Further research is needed.

\subsection*{Conflicts of interest}

{\label{conflicts-of-interest}}

The author declares no conflicts of interest.

\subsection*{Acknowledgements}

{\label{acknowledgements}}

This focused review is based on the PhD thesis Resistance to daratumumab
in patients with multiple myeloma {[}50{]}.

\subsection*{References}

{\label{references}}

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