Abstract
Introduction
Chronic myelomonocytic
leukaemia (CMML) is characterised by the aberrant proliferation of monocytic
cells. The initial mutations occur within the bone marrow, and treatment is palliative
due to current drugs only being able to kill cells in the peripheral blood stream.
Slow proliferation of bone marrow cells also results in widely used
chemotherapy agents that target proliferation being ineffective. Herein we
report a novel CCL2 conjugated drug delivery system that can selectively target
cancerous cells involved in CMML, including in bone marrow by utilizing a
Nampti inhibitor as the cytotoxic payload. These compounds are metabolic
cytotoxins, allowing induction of apoptosis and cell death in non-proliferating
cells.
Methods and Materials
Click protein
conjugation was carried out using standard conditions and protein purified and
isolated by PD size exclusion chromatography. Samples were analysed by MALDI-
TOF and MCP-1 ELISA assay. Conjugate compounds were incubated with THP1 and
Jurkat monocytic cells for 48 hrs and analysed using a WST-1 cell proliferation
assay.
In Vivo mouse studies were undertaken by injecting CCL2- SCY5
into WT and CCR2 KO mice at 1 mg/kg (10 μM in PBS, 0.1% BSA). After three hours
samples were taken from bone marrow and blood, labelled for standard markers
and analysed by FACS.
Results
Nampti as cytotoxic payload
High (10% FBS)
and low (2.5% FBS) proliferative THP1 were treated with FK866 or the typical
cytotoxic ADC payload DM-1. DM-1 showed a large drop off in potency in the
slower proliferating THP1 cells in 2.5% FBS, whereas FK866 showed minimal
reduction in efficacy.
Conjugates in vitro
Following
earlier studies on selective uptake of CCL2 conjugates by CCR2+ cells. THP1 and
Jurkat cells were incubated for 48h with a novel CCL2-NAMPTi and WST-1 was used
as cell proliferation assay, showing that CCL2-NAMPTi is high selectivity for
CCR2-expressing cells.
Conjugates in vivo
In
in vivostudies WT mice showed high levels of Cy5 uptake in the CCR2 positive
population in bone marrow as well as in the blood, while no uptake is observed
in any cell type for the CCR2 -/- mice.
Conclusion
NAMPT inhibitors
are metabolic cytotoxins that can target non-proliferating cells, however as a
result they have no useful therapeutic window. We have developed CCL2
conjugates that are selectively internalized by CCR2+ cells, and are able to
target the bone marrow
in vivo.
Combining these
findings with the initial THP1 results gives a very promising basis that a
CCL2-Nampti inhibitor will be able to target and kill cancerous bone marrow
cells in CMML.
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