Characterization of high-affinity peptides and their feasibility for use in nanotherapeutics targeting leukemia stem cells

doi:10.1016/j.nano.2011.12.004

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 8, Issue 7, October 2012, Pages 1116-1124

https://doi.org/10.1016/j.nano.2011.12.004 Get rights and content

Abstract

Peptides featuring the LR(S/T) motif were identified that could specifically bind to the C-type lectin-like molecule-1 (CLL1), a protein preferentially expressed on acute myeloid leukemia stem cells (LSCs). Micellar nanoparticles were covalently decorated with CLL1-targeting peptides for targeted drug delivery. The resulting peptide-coated nanoparticles were 13.5 nm in diameter and could be loaded with 5 mg of daunorubicin per 20 mg of telodendrimers. These “targeting nanomicelles” transported the drug load to the interior of cells expressing CLL1 and to LSCs isolated from clinical specimens in vitro, but did not bind to normal blood or normal hematopoietic stem cells. The presence of CLL1-targeting peptides on the surface of the nanomicelles enabled the improved binding and delivery of substantially more daunorubicin into the cells expressing CLL1 and CD34⁺ leukemic cells compared with unmodified nanomicelles. In conclusion, nanomicelles coated with CLL1-targeting peptides are potentially useful for eradicating LSCs and improving leukemia therapy.

From the Clinical Editor

Micellar nanoparticles covalently decorated with targeting peptides were used for targeted drug delivery of daunorubicin to address acute myeloid leukemia stem cells.

Graphical Abstract

A chemotherapeutic drug, drug combinations and/or radioisotopes are loaded inside nanomicelles. Targeting ligands against leukemia stem cell (LSC) surface molecules are decorated on the surface of nanomicelles. After intravenous administration, these targeting nanomicelles can specifically deliver high-concentration therapeutic agents to and eradicate LSC. In addition, the therapeutic agents can diffuse into blood circulation to kill leukemic cells throughout the body. Furthermore, formulation of chemotherapeutic drugs in nanomicelles can decrease therapy-related toxicity and improve treatment outcomes.

Section snippets

Identification of ligands targeting CLL1

The CLL1 complementary DNA expression vector was purchased from the American Type Culture Collection (ATTC; Manassas, Virginia) and was subcloned to a pcDNA3.1 expression vector. The C7C phage-display peptide library was purchased from New England Biolabs (Ipswich, Massachusetts). CLL1 was expressed in 5637 (bladder transitional-cell carcinoma), A549 (non–small cell lung cancer), and HTB38 (colon cancer) cells for sequential panning to identify peptides that bind to CLL1. At the time this

Development of ligands targeting CLL1

The PhD C7C phage peptide-display library (New England Biolabs) was subtracted with various confounding cells before each round of panning (Supplementary Information S1, Figure A). After panning, a total of 36 phage clones were sequenced. After alignment, two consensus sequences were identified: CXLR(S/T)AAVC and CXLRSSGPC (Table 1), in which “X” represents several amino acids that can be replaceable. Serine (S) and threonine (T) belong to the same hydrophilic class of amino acids with only one

Discussion

To our knowledge, this is the first report demonstrating the screening and synthesis of peptides that specifically bind to cells expressing CLL1 and with low nonspecific binding to other cell types. The CLL1 molecule is expressed on LSCs but not on normal hematopoietic stem cells, which makes it an attractive cell surface target for LSC-seeking nanotherapeutics. We also showed that nanomicelles decorated with CLL1-targeting peptides bind to cells expressing CLL1, but more importantly, can

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Daunorubicin-containing CLL1-targeting nanomicelles have anti-leukemia stem cell activity in acute myeloid leukemia
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As illustrated in Figure 1, A, two types of telodendrimers, CLL1-peptide-PEG5K-CA8 and PEG5K-Cys4-L8-CA8 were synthesized and mixed with a 1:1 ratio (Figure 1, A). After DNR loading, disulfide crosslinks were formed from cysteine residues under oxidative conditions (2–4). DC-CTM-DNR was spherically-shaped (Figure 1, B) with a size distribution of 17+/−6 nm (Figure 1, B and C).
Patients with acute myeloid leukemia have a very poor prognosis related to a high rate of relapse and drug-related toxicity. The ability of leukemia stem cells (LSCs) to survive chemotherapy is primarily responsible for relapse, and eliminating LSCs is ultimately essential for cure. We developed novel disulfide-crosslinked CLL1-targeting micelles (DC-CTM), which can deliver high concentrations of daunorubicin (DNR) into both bulk leukemia cells and LSCs. Compared to free DNR, DC-CTM-DNR had a longer half-life, increased DNR area under the curve concentration by 11-fold, and exhibited a superior toxicity profile. In patient-derived AML xenograft models, DC-CTM-DNR treatment led to significant decreases in AML engraftment and impairment of secondary transplantation compared to control groups. Collectively, we demonstrate superior anti-LSC/AML efficacy, and preferable pharmacokinetic and toxicity profiles of DC-CTM-DNR compared to free DNR. DC-CTM-DNR has the potential to significantly improve treatment outcomes and reduce therapy-related morbidity and mortality for patients with AML.
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Zhang and coworkers have developed targeting nanomicelles loaded with daunorubicin and able to target cells expressing the CLL-1 antigen because have been decorated with peptides able to bind specifically to cells expressing CLL-1 [22]. These nanomicelles were able to display cytotoxicity against leukemic cell lines and primary AML cells, but their activity against LSC, or in in vivo models was not assayed [22]. More recently, a bispecific antibody, αCLL-1-αCD3, was developed: this antibody recruits cytotoxic T cells to CLL-1 positive cells and displays potent and selective cytotoxicity in vitro against several human AML cell lines and primary AML blasts [23]; furthermore, administration of αCLL-1-αCD3 abrogates leukemia development in an U937 AML cell line xenograft model [23].
Studies of xenotransplantation of bone marrow and blood cells of AML patients have supported the existence of rare leukemic stem cells, able to initiate and maintain the leukemic process and bearing the typical leukemic abnormalities. LSCs possess self-renewal capacity and are responsible for the growth of the more differentiated leukemic progeny in the bone marrow and in the blood. These cells are more resistant than bulk leukemic cells to anti-leukemic drugs, thus survive to treatment and are, at a large extent, responsible for leukemia relapse. During the last two decades, considerable progresses have been made in the understanding of the peculiar cellular and molecular properties of LSCs. In this context, particularly relevant was the discovery of several membrane markers, selectively or preferentially expressed on LSCs. These membrane markers offer now unique opportunities to identify LSCs and to distinguish them from normal HSCs, to monitor the response of the various anti-leukemic treatments at the level of the LSC compartment, to identify relevant therapeutic targets. Concerning this last point, the most promising therapeutic targets are CD33 and CD123.
Progress in RNAi-mediated molecular therapy of acute and chronic myeloid leukemia
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Antibody (Ab)-mediated delivery has been used to target surface proteins overexpressed or differentially-expressed on leukemic cells (Table 3). Other ligands were derived from peptides/proteins, aptamers, saccharides, benzamides, and ODNs with targets including transferrin receptor,46,47,60,61,62,63,64,65,66,67,68 low-density lipoprotein,69,70,71 matrix metalloproteinase receptors (MMP-2/9),72 toll-like receptor,73 C-type lectin-like molecule-1 (CLL1 receptor),74,75 lectins,76 protein tyrosine kinase 7 (PTK7),59,77,78,79 vitamin receptors for biotin,80,81 folate/folic acid receptor,67,68,82,83,84,85,86,87 alendronate (bone),87 and sigma receptors.88 Some of the ligands target “endocytosing” receptors on cell surface, while others such as CPPs facilitate uptake without necessarily undergoing endocytosis.30,89
Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to impaired regulation of proliferation, differentiation, apoptosis, and survival of the transformed cells. With the advent of RNA interference (RNAi) and the short interfering RNA (siRNA) as its pharmacological mediator, it is becoming possible to modulate specific targets at will. This article summarizes current attempts to utilize RNAi reagents for therapy of leukemias, focusing on acute and chronic myeloid leukemia. We first present unique aspects of RNAi-mediated therapy, followed by a brief background on the delivery technology of RNAi reagents. The need for leukemia-specific delivery of siRNA is discussed by describing approaches that targeted agents to leukemic cells. Pharmacokinetics and biodistribution of RNAi agents are then presented, highlighting the critical issues pertinent to emerging siRNA therapy. Efforts to deliver specific RNAi therapies are then summarized in the context of expected clinical outcomes, focusing on limiting leukemic cell survival, sensitizing malignant cells to chemotherapy, mobilization of leukemic cells, and eradication of leukemic stem cells. We conclude with a perspective on the future of RNAi therapy, emphasizing the technological requirements and mechanistic challenges for clinical entry.
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Uncovering the relationships between peptide and protein sequences and binding properties is critical for successfully predicting, re-designing and inhibiting protein–protein interactions. Systematically collected data that link protein sequence to binding are valuable for elucidating determinants of protein interaction but are rare in the literature because such data are experimentally difficult to generate. Here we describe SORTCERY, a high-throughput method that we have used to rank hundreds of yeast-displayed peptides according to their affinities for a target interaction partner. The procedure involves fluorescence-activated cell sorting of a library, deep sequencing of sorted pools and downstream computational analysis. We have developed theoretical models and statistical tools that assist in planning these stages. We demonstrate SORTCERY's utility by ranking 1026 BH3 (Bcl-2 homology 3) peptides with respect to their affinities for the anti-apoptotic protein Bcl-x_L. Our results are in striking agreement with measured affinities for 19 individual peptides with dissociation constants ranging from 0.1 to 60 nM. High-resolution ranking can be used to improve our understanding of sequence–function relationships and to support the development of computational models for predicting and designing novel interactions.
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Although it has been reported that rapid renal filtration occurred in the case of particles with a hydrodynamic diameter of ≤ 5.5 nm (Choi et al., 2007), intravenous (i.v.) administration of amino-functionalised single-walled carbon nanotubes (SWCNTs, rod-like shape) with average lengths of 200–300 nm also underwent rapid renal excretion (Lacerda et al., 2008), indicating that particle shape may play an important role in systemic circulation (reviewed by Decuzzi and Ferrari, 2010; Moghimi et al., 2012). Unlike solid tumours that primarily reside at the extravascular space and are accessible by NPs through the enhanced permeability and retention (EPR) effect (Du et al., 2014; Yang et al., 2014), leukaemic cells and LSCs are mainly found at two sites: in the blood circulation and within the BM, both of which can be directly accessed by NPs through i.v. administration (Zhang et al., 2012). As mentioned, reduced renal filtration occurs with particles larger than 10 nm however, large particles (> 100 nm) may induce uptake by the reticuloendothelial system (RES), now known as mononuclear phagocyte system (MPS).
Leukaemia is a bone marrow cancer occurring in acute and chronic subtypes. Acute leukaemia is a rapidly fatal cancer potentially causing death within a few weeks, if untreated. Leukaemia arises as a result of disruption to haematopoietic precursors, caused either by acquired gene fusions, gene mutations or inappropriate expression of the relevant oncogenes. Current treatment options have made significant progress, but the 5 year survival for acute leukaemia remains under 10% in elderly patients, and less than 50% for some types of acute leukaemia in younger adults. For chronic leukaemias longer survival is generally expected and for chronic myeloid leukaemia patients on tyrosine kinase inhibitors the median survival is not yet reached and is expected to exceed 10 years. Chemotherapy and haematopoietic stem cell transplantation (HSCT) for acute leukaemia provide the mainstay of therapy for patients under 65 and both carry significant morbidity and mortality. Alternative and superior therapeutic strategies for acute leukaemias are urgently required. Recent molecular-based knowledge of recurring chromosome rearrangements, in particular translocations and inversions, has resulted in significant advances in understanding the molecular pathogenesis of leukaemia. Identification of a number of unique fusion genes has facilitated the development of highly specific small interfering RNAs (siRNA). Although delivery of siRNA using multifunctional nanoparticles has been investigated to treat solid cancers, the application of this approach to blood cancers is at an early stage. This review describes current treatments for leukaemia and highlights the potential of leukaemic fusion genes as therapeutic targets for RNA interference (RNAi). In addition, the design of biomimetic nanoparticles which are capable of responding to the physiological environment of leukaemia and their potential to advance RNAi therapeutics to the clinic will be critically evaluated.
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View all citing articles on Scopus

: This project is supported by the California Institute for Regenerative Medicine New Faculty Award (C-X.P.).

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Research ArticleCharacterization of high-affinity peptides and their feasibility for use in nanotherapeutics targeting leukemia stem cells

Abstract

From the Clinical Editor

Graphical Abstract

Section snippets

Identification of ligands targeting CLL1

Development of ligands targeting CLL1

Discussion

Blood

Blood

Blood

Biomaterials

Anal Biochem

Cell Stem Cell

Blood

Implications of cancer stem cells in the treatment of cancer

Future Oncol

Targeting the leukemic stem cell: the Holy Grail of leukemia therapy

Leukemia

C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia

Cancer Res

Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia

Haematologica

Well-defined, size-tunable, multifunctional micelles for efficient paclitaxel delivery for cancer treatment

Bioconjug Chem

Research Article
Characterization of high-affinity peptides and their feasibility for use in nanotherapeutics targeting leukemia stem cells