Antibody‐based therapy in acute myeloid leukemia (AML) has been marred by significant hematologic toxicity due to targeting of both hematopoietic stem and progenitor cells (HSPCs). Achieving greater success with therapeutic antibodies requires careful characterization of the potential target molecules on AML. One potential target is CD300f, which is an immunoregulatory molecule expressed predominantly on myeloid lineage cells. To confirm the value of CD300f as a leukemic target, we showed that CD300f antibodies bind to AML from 85% of patient samples. While one CD300f monoclonal antibody (mAb) reportedly did not bind healthy hematopoietic stem cells, transcriptomic analysis found that CD300f transcripts are expressed by healthy HSPC. Several CD300f protein isoforms exist as a result of alternative splicing. Importantly for antibody targeting, the extracellular region of CD300f can be present with or without the exon 4‐encoded sequence. This results in CD300f isoforms that are differentially bound by CD300f‐specific antibodies. Furthermore, binding of one mAb, DCR‐2, to CD300f exposes a structural epitope recognized by a second CD300f mAb, UP‐D2. Detailed analysis of publicly available transcriptomic data indicated that CD34+ HSPC expressed fewer CD300f transcripts that lacked exon 4 compared to AML with monocytic differentiation. Analysis of a small cohort of AML cells revealed that the UP‐D2 conformational binding site could be induced in cells from AML patients with monocytic differentiation but not those from other AML or HSPC. This provides the opportunity to develop an antibody‐based strategy to target AMLs with monocytic differentiation but not healthy CD34+ HSPCs. This would be a major step forward in developing effective anti‐AML therapeutic antibodies with reduced hematologic toxicity.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly reduces the rate of relapse in acute myeloid leukemia (AML) but comes at the cost of significant treatment-related mortality. Despite the reduction in relapse overall, it remains common, especially in high-risk groups. The outcomes for patients who relapse after transplant remains very poor. A large proportion of the morbidity that prevents most patients from accessing allo-HSCT is due to toxic nonspecific conditioning agents that are required to remove recipient hematopoietic stem and progenitor cells (HSPCs), allowing for successful donor engraftment. CD300f is expressed evenly across HSPC subtypes. CD300f has transcription and protein expression equivalent to CD33 on AML. We have developed an anti-CD300f antibody that efficiently internalizes into target cells. We have generated a highly potent anti-CD300f antibody-drug conjugate (ADC) with a pyrrolobenzodiazepine warhead that selectively depletes AML cell lines and colony forming units in vitro. The ADC synergizes with fludarabine, making it a natural combination to use in a minimal toxicity conditioning regimen. Our ADC prolongs the survival of mice engrafted with human cell lines and depletes primary human AML engrafted with a single injection. In a humanized mouse model, a single injection of the ADC depletes CD34+ HSPCs and CD34+CD38−CD90+ hematopoietic stem cells. This work establishes an anti-CD300f ADC as an attractive potential therapeutic that, if validated in transplant models using a larger cohort of primary AML samples, will reduce relapse rate and toxicity for patients with AML undergoing allo-HSCT.
Myeloid lineage cells present in human peripheral blood include dendritic cells (DC) and monocytes. The DC are identified phenotypically as HLA-DR + cells that lack major cell surface lineage markers for T cells (CD3), B cells (CD19, CD20), NK cells (CD56), red blood cells (CD235a), hematopoietic stem cells (CD34), and Mo that express CD14. Both DC and Mo can be phenotypically divided into subsets. DC are divided into plasmacytoid DC, which are CD11c − , CD304 + , CD85g + , and myeloid DC that are CD11c +. The CD11c + DC are readily classified as CD1c + DC and CD141 + DC. Monocytes are broadly divided into the CD14 + CD16 − (classical) and CD14 dim CD16 + subsets (nonclassical). A population of myeloid-derived cells that have DC characteristics, that is, HLA-DR + and lacking lineage markers including CD14, but express CD16 are generally clustered with CD14 dim CD16 + monocytes. We used high-dimensional clustering analyses of fluorescence and mass cytometry data, to delineate CD14 + monocytes, CD14 dim CD16 + monocytes (CD16 + Mo), and CD14 − CD16 + DC (CD16 + DC). We sought to identify the functional and kinetic relationship of CD16 + DC to CD16 + Mo. We demonstrate that differentiation of CD16 + DC and CD16 + Mo during activation with IFN in vitro and as a result of an allo-hematopoietic cell transplant (HCT) in vivo resulted in distinct populations. Recovery of blood CD16 + DC in both auto-and allo-(HCT) patients after myeloablative conditioning showed similar reconstitution and activation kinetics to CD16 + Mo. Finally, we show that expression of the cell surface markers CD300c, CCR5, and CLEC5a can distinguish the cell populations phenotypically paving the way for functional differentiation as new reagents become available.
Antibodies targeting the activation marker CD83 can achieve immune suppression by targeting antigen-presenting mature dendritic cells (DC). This study investigated the immunosuppressive mechanisms of anti-CD83 antibody treatment in mice and tested its efficacy in a model of autoimmune rheumatoid arthritis. A rat anti-mouse CD83 IgG2a monoclonal antibody, DCR-5, was developed and functionally tested in mixed leukocyte reactions, demonstrating depletion of CD83+ conventional (c)DC, induction of regulatory DC (DCreg), and suppression of allogeneic T cell proliferation. DCR-5 injection into mice caused partial splenic cDC depletion for 2–4 days (mostly CD8+ and CD83+ cDC affected) with a concomitant increase in DCreg and regulatory T cells (Treg). Mice with collagen induced arthritis (CIA) treated with 2 or 6 mg/kg DCR-5 at baseline and every three days thereafter until euthanasia at day 36 exhibited significantly reduced arthritic paw scores and joint pathology compared to isotype control or untreated mice. While both doses reduced anti-collagen antibodies, only 6 mg/kg achieved significance. Treatment with 10 mg/kg DCR-5 was ineffective. Immunohistological staining of spleens at the end of CIA model with CD11c, CD83, and FoxP3 showed greater DC depletion and Treg induction in 6 mg/kg compared to 10 mg/kg DCR-5 treated mice. In conclusion, DCR-5 conferred protection from arthritis by targeting CD83, resulting in selective depletion of mature cDC and subsequent increases in DCreg and Treg. This highlights the potential for anti-CD83 antibodies as a targeted therapy for autoimmune diseases.
Background: Aldosterone increases exocytosis from endothelial cells, which may contribute to the detrimental actions of aldosterone in the vascular system. We propose a role for exocytosis of exosomes; these small extracellular particles contain host cell genetic material and once released they can circulate in the blood and transfer their contents (e.g. miRNAs) to recipient cells. We aimed to examine the effect of aldosterone on exosome release from endothelial cells, the miRNA content of exosomes and finally, exosome trafficking of miRNA from endothelial cells to smooth muscle cells. Methods and Results: Exosomes were isolated via ultracentrifugation from the media of human coronary artery endothelial cells (HCAECs) following incubation with aldosterone (100nM) or vehicle (DMSO). Exosomes were analyzed using western blot analysis testing for exosome and cell markers, and by nanoparticle-tracking analysis (NanoSight); aldosterone increased exosome concentration by 2.65 fold (19.1x1010 particles/mL) compared to the vehicle control (7.2x1010 particles/mL). RNA was extracted from isolated exosomes and host HCAECs and levels of miR-24 measured by qRT-PCR. miR-24 levels were unaffected by aldosterone stimulation in HCAECs (0.58±0.19; p=0.07) but were significantly increased in exosomes isolated from the media (2.35±0.07 fold; p=0.03). To test exosome paracrine trafficking, HCAECs were transfected with a c.elegans miRNA (cel-miR-39) and incubated adjacent to human coronary artery smooth muscle cells (HCASMCs) separated by a 0.4 μm filter, with or without aldosterone. Levels of cel-miR-39 in HCASMCs increased significantly (2.42±0.52 fold; p=0.02) in aldosterone-stimulated cells, compared to control. Blocking transcription with Dactinomycin (2.07±0.61 fold; p>0.05) or blocking the mineralocorticoid receptor had no effect on aldosterone-mediated trafficking (3.43±1.49 fold; p>0.05). Conclusion: This study demonstrates that aldosterone: 1) increases exosome secretion from endothelial cells, 2) increases miR-24 content of exosomes and 3) contributes to an increased uptake of exosome-packaged microRNAs by smooth muscle cells. Together, this represents a novel mechanism by which aldosterone contributes to vascular disease.
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