Patent Application
20210088505
This application generally relates to the field of progenitor cells and, more specifically, to neutrophil progenitor cells, methods of preparation and use thereof.
Neutrophils represent the most abundant cell population in the innate immune system and are indispensable antagonists of microbial infection and facilitators of wound healing. More recently, the role of neutrophils has also been extended to cover immune-related conditions such as cancer (1-3). Indeed, a number of studies have suggested that neutrophils may have both pro- and anti-tumorigenic roles, which apparently differs with cancer type and disease stage (Treffers et al., Immunol. Rev. 2016 September; 273(1):312-28). Additionally, studies have also suggested that tumors may manipulate neutrophils, sometimes early in their differentiation process, to create diverse phenotypic and functional polarization states able to alter tumor behavior (Coffelt et al., Nature Reviews Cancer 16, 431-446, 2016).
Studies have reported that substantially increased numbers of neutrophils are found in the blood of many patients with advanced cancer, and that this is often associated with poor prognosis as has been demonstrated in various types of cancer, including melanoma, renal cancer, and lung cancer. The neutrophil-to-lymphocyte ratio (NLR) was introduced more recently to represent probably, at least in most cases, the same phenomenon, and this appears to be an even better predictor for poor disease and treatment outcome.
Although a high NLR appears associated with an increase in markers of a systemic inflammatory response, including elevated circulating concentrations of G-CSF, IL-8, MIP1, and PDGF, the biological mechanisms leading to an elevated NLR in cancer patients are still largely unknown.
Neutrophils and monocytes arise from the same progenitor cells, the Granulocyte Monocyte Progenitor (GMP) in the bone marrow (BM). In mouse BM, it is known that Hematopoietic Stem and Progenitor Cells (HSPCs) commit to a series of checkpoints for lineage decision from the Long-Term and Short-Term Hematopoietic Stem Cells (LT/ST-HSCs) into the Common Myeloid Progenitor (CMP) for myeloid cell production. CMPs give rise to both megakaryocyte-erythrocyte progenitors (MEPs) and GMPs (5). GMPs are the oligopotent progenitors for granulocytes, monocytes, macrophages, and dendritic cells (DCs) (6) and are reprogrammed in cancer to produce tumor-associated monocytes and neutrophils (7, 8). Unipotent neutrophil progenitor cells, however, have not yet been identified, therefore, specific studies of neutrophil biology in health and disease have been impeded.
In light of at least the above, there is a need to better understand the biological mechanisms underlying neutrophil differentiation biology in health and disease.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key aspects or essential aspects of the claimed subject matter.
The present disclosure aims to at least identify, characterize and/or isolate unipotent neutrophil progenitor cells.
The present disclosure relates broadly to a method of treating a subject, wherein the method comprises i) processing a biological sample from the subject, the sample being suspected of including neutrophil cells to determine a concentration level thereof, ii) comparing the concentration level to a reference level, and iii) treating said subject at least based on said comparison, the treating step including stimulating or inhibiting differentiation of unipotent neutrophil progenitor cells into neutrophil cells so as to modulate the concentration of said neutrophil cells in said subject.
In certain aspects of the present invention, a method for evaluating a condition status in a subject is provided, the condition being associated with neutropenia. The method comprises providing a biological sample from said subject, the sample being suspected of including unipotent neutrophil progenitor cells in said sample. The method further includes processing the sample to determine a concentration or activation level of said unipotent neutrophil progenitor cells in said sample. Optionally, the method may further include comparing the concentration or activation level to a reference level, and evaluating the condition status based on at least the comparison, the condition being associated with neutropenia.
In certain alternative aspects of the present invention, a method for evaluating cancer in a subject is provided, the method comprising providing a biological sample from said subject, the sample being suspected of including unipotent neutrophil progenitor cells. The method further includes processing the sample to determine a concentration or activation level of said unipotent neutrophil progenitor cells in said sample. Optionally, the method may further include comparing the concentration or activation level to a reference level, and evaluating the subject as having or not having cancer based on at least the comparison.
In certain aspects of the present invention, a method for determining response or resistance to cancer treatment in a subject undergoing cancer treatment is provided. The method comprises providing a biological sample from said subject, the sample being suspected of including unipotent neutrophil progenitor cells. The method further includes processing the sample to determine a concentration or activation level of said unipotent neutrophil progenitor cells in said sample. Optionally, the method may further include comparing the concentration or activation level to a reference level, and evaluating the response or resistance to the cancer treatment based on at least the comparison.
In other aspects of the present invention, a method for determining response to a treatment for a condition associated with neutropenia in a subject undergoing the treatment is provided. The method comprises providing a biological sample from said subject, the sample being suspected of including unipotent neutrophil progenitor cells. The method further includes processing the sample to determine a concentration or activation level of said unipotent neutrophil progenitor cells in said sample. Optionally, the method may further include comparing the concentration or activation level to a reference level, and evaluating the response or resistance to the treatment based on at least the comparison.
In alternative aspects of the present invention, a method of reducing risk of cancer progression or cancer relapse in a subject is provided, the method comprising i) providing a biological sample form said subject, the sample being suspected of including unipotent neutrophil progenitor cells, ii) processing the sample to determine a concentration or activation level of said unipotent neutrophil progenitor cells in said sample, iii) comparing the concentration or activation level to a reference level, and iv) selectively administering a cancer therapeutic agent at least based on the comparison in step (iii) so as to reduce risk of cancer progression or cancer relapse in the subject.
In certain aspects of the present invention, a method of reducing risk of a condition associated with neutropenia in a subject is provided, the method comprising i) providing a biological sample from said subject, the sample being suspected of including unipotent neutrophil progenitor cells, ii) processing the sample to determine a concentration or activation level of said unipotent neutrophil progenitor cells in said sample, iii) comparing the concentration or activation level to a reference level, and iv) selectively administering a therapeutic agent at least based on the comparison in step (iii) so as to reduce risk of the condition associated with neutropenia in the subject.
In certain other aspects of the present invention, a method for screening a candidate molecule for an activity on cell differentiation of unipotent neutrophil progenitor cells into neutrophils is provided. The method comprises i) contacting said unipotent neutrophil progenitor cells with the candidate molecule, and ii) determining the activity of the candidate molecule on the cell differentiation of said unipotent cells into neutrophils.
In alternative aspects of the present invention, a method for screening a candidate molecule for an activity on neutrophil differentiation is provided, the method comprising i) providing the candidate molecule, ii) causing the candidate molecule to contact unipotent neutrophil progenitor cells to determine the activity of the candidate molecule on the cell differentiation of said unipotent cells into neutrophils, and iii) receiving information conveying the activity of the candidate molecule on the cell differentiation of said unipotent cells into neutrophils.
In certain aspects of the present invention, a method for treatment or prevention of neutropenia in a subject is provided, the method comprising administering to the subject an effective amount of a purified unipotent neutrophil progenitor cell population. In alternative embodiments, said progenitor cells are autologous cells to the subject.
In certain aspects of the present invention, use of an effective amount of a purified unipotent neutrophil progenitor cell population for treatment or prevention of neutropenia in a subject is provided. In alternative embodiments, said progenitor cells are autologous cells to the subject.
In certain aspects of the present invention, use of an effective amount of a purified unipotent neutrophil progenitor cell population in the manufacture of a medicament for treatment or prevention of neutropenia in a subject is provided. In alternative embodiments, said progenitor cells are autologous cells to the subject.
In certain other aspects of the present invention, a method of inhibiting or preventing tumor growth in a subject is provided, the method comprising inhibiting differentiation of unipotent neutrophil progenitor cells into neutrophil cells in said subject.
In certain other aspects of the present invention, use of an inhibitor for inhibiting or preventing tumor growth in a subject is provided, where the inhibitor inhibits differentiation of unipotent neutrophil progenitor cells into neutrophil cells in the subject.
In certain other aspects of the present invention, use of an inhibitor in the manufacture of a medicament for inhibiting or preventing tumor growth in a subject is provided, where the inhibitor inhibits differentiation of unipotent neutrophil progenitor cells into neutrophil cells in the subject.
In certain aspects of the present invention, a pharmaceutical composition comprising isolated unipotent neutrophil progenitor cells and a pharmaceutically acceptable carrier is provided, wherein said progenitor cells are modified so as to have modified gene expression, modified cell function, or to include a ribonucleic acid interference (RNAi) causing molecule, or a conjugated therapeutic agent. In some aspects, the cells are genetically modified by CRISPR-cas9, lentivirus transduction or RNAi.
In some aspects of the present invention, the biological sample described herein includes blood or a cell fraction thereof. In still other aspects, said biological sample includes blood, spleen, tumor tissue or bone marrow, or a cell fraction thereof.
In certain aspects of the present invention, said reference level described herein is derived from a cohort of at least 20 reference individuals without disease condition. In certain alternative aspects, said reference level is derived from a sample from the subject, the sample being provided prior to or after a treatment performed to treat the subject.
In alternative aspects of the present invention, said subject is afflicted with neutropenia. In other aspects, said neutropenia is caused by a cancer.
In certain aspects of the present invention, the progenitor cells have at least the phenotype CD45+, CD41−, CD127 (IL-7Rα)−, CD19−, CD3−, CD161 (NK1.1)−, CD169 (Siglec 1)−, CD11c−, Siglec 8−, FcεRIα− and CD115 (CSF-1R)−. In other aspects, the progenitor cells have at least the phenotype CD161−, CD34+, CD38+, CD115−, Siglec8−, FcεRIα− and CD114+.
In some aspects of the present invention, the progenitor cells have at least the phenotype CD45+, CD235ab−, CD41−, CD127 (IL-7Rα)−, CD19−, CD3−, CD4−, CD161 (NK1.1)−, CD56−, CD169 (Siglec 1)−, CD64−, CD11c−, HLA-DR−, CD86−, CD123−, CD7−, CD10−, CD366−, CD90−, Siglec 8−, FcεRIα−, CD115 (CSF-1R)−, CD34+, CD38+, CD45RA+, CD66b+, CD16b+, CD15+, CD114+, CD14int, CD162int, and CD62Lint.
In some aspects of the present invention, the progenitor cells have at least the phenotype hSiglec 8−, hFcεRIα−, hCD3−, hCD7−, hCD10−, hCD11c−, hCD19−, hCD41−, hCD56−, hCD90 (Thy1)−, hCD123 (IL-3Rα)−, hCD125 (IL-5Rα)−, hCD127 (IL-7Rα)−, hCD161−, hCD169−, hCD235a−, hCD66b+, hCD117 (c-Kit)+, hCD38+, and hCD34+ (e.g. Subset A as described herein).
In some aspects of the present invention, the progenitor cells have at least the phenotype hSiglec 8−, hFcεRIα−, hCD3−, hCD7−, hCD10−, hCD11c−, hCD19−, hCD41−, hCD56−, hCD90 (Thy1)−, hCD123 (IL-3Rα)−, hCD125 (IL-5Rα)−, hCD127 (IL-7Rα)−, hCD161−, hCD169−, hCD235a−, hCD34−, hCD66b+, hCD117 (c-Kit)+, and hCD38+ (e.g. Subset B as described herein).
In certain aspects of the present invention, said subject is human. In alternative aspects, said subject is a mouse.
In some aspects of the present invention, the progenitor cells have at least the phenotype CD161−, CD117(c-Kit)+, Ly6A/E−, CD16/32+, CD115−, SiglecF−, FcεRIα− and Ly6G−/lo. In other aspects, the progenitor cells have at least the phenotype CD45+, Ter119−, CD41−, CD127 (IL-7Rα)−, CD19- or B220−, CD3−, TCRβ−, CD161 (NK1.1)−, CD335 (NKp46)−, CD169 (Siglec 1)−, F4/80−, CD11c−, MHCII−, CD117 (c-kit)+/int, Ly6A/E (Sca1)−, Siglec F (Siglec 8)−, FcεRIα−, CD115 (CSF-1R)−, Ly6C−/int, CD16/32 (FcγRIII/II)+, and Ly6G−/lo.
In certain aspects of the present invention, the progenitor cells have at least the phenotype CD41−, CD127(IL-7Rα)−, CD3−, CD19−, CD161(NK1.1)−, CD169(Siglec 1)−, CD11c−, Siglec F, FcERIα−, CD115(CSF-1R)−, Ly6A/E(Sca1)−, Ly6G−, CD162(PSGL-1) lo, CD48 lo, Ly6C lo, and CD117(c-Kit)+, CD16/32(FcγRIII/II)+, Ly6B+ and CD11a(LFA1α)+ (e.g. Cluster #C1 as described herein).
In certain aspects of the present invention, the progenitor cells have at least the phenotype CD41−, CD127(IL-7Rα)−, CD3, CD19−, CD161(NK1.1)−, CD169(Siglec 1)−, CD11c−, Siglec F−, FcERIα−, CD115(CSF-1R)−, Ly6A/E(Sca1)−, CD117(c-Kit)+, CD16/32(FcγRIII/II)+, Ly6B, CD11a(LFA1α)+, and Ly6G+ (e.g. Cluster #C2 as described herein).
In certain aspects of the present invention, a kit for sorting unipotent neutrophil progenitor cells from a biological sample is provided, the kit comprising detecting agents for CD161, CD34, CD38, CD115, Siglec8, FcεRIα and CD114. In alternative aspects, the kit comprises detecting agents for CD45, CD41, CD127 (IL-7Rα), CD19, CD3, CD161 (NK1.1), CD169 (Siglec 1), CD11c, Siglec 8, FcεRIα and CD115 (CSF-1R). In other aspects, the kit comprises detecting agents for CD45, CD235ab, CD41, CD127 (IL-7Rα), CD19, CD3, CD4, CD161 (NK1.1), CD56, CD169 (Siglec 1), CD64, CD11c, HLA-DR, CD86, CD123, CD7, CD10, CD366, CD90, Siglec 8, FcεRIα, CD115 (CSF-1R), CD34, CD38, CD45RA, CD66b, CD16b, CD15, CD114, CD14, CD162, and CD62L. In other aspects, the kit comprises detecting agents for hSiglec 8, hFcεRIα, hCD3, hCD7, hCD10, hCD11c, hCD19, hCD41, hCD56, hCD90 (Thy1), hCD123 (IL-3Rα), hCD125 (IL-5Rα), hCD127 (IL-7Rα), hCD161, hCD169, hCD235a, hCD66b, hCD117 (c-Kit), hCD38, and hCD34.
In certain aspects of the present invention, the use of a kit for sorting unipotent neutrophil progenitor cells from a biological sample is provided, where the kit is as defined herein.
All features of embodiments which are described in this disclosure and are not mutually exclusive can be combined with one another. Elements of one embodiment can be utilized in the other embodiments without further mention. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying Figures.
A detailed description of specific embodiments is provided herein below with reference to the accompanying drawings in which:
In the drawings, embodiments are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustrating certain embodiments and are an aid for understanding. They are not intended to be a definition of the limits of the invention.
Illustrative embodiments of the disclosure will now be more particularly described. While the making and using of various embodiments of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
While modern techniques such as multi-color flow cytometry techniques have enabled the discovery of several unipotent or oligopotent progenitors in hematopoiesis, including the Eosinophil Progenitor (EoP) (10, 11), Basophil/Mast Cell Progenitors (B/MCP) (12, 13), and multiple Monocyte Progenitors (here termed MonPs), which consist of the Monocyte/Dendritic Cell (DC) Progenitor (MDP), the common Monocyte Progenitor (cMoP), and the recently discovered Segregated-nucleus-containing atypical Monocyte Progenitor (SatMP) (14-17), however, it is unknown if unipotent neutrophil progenitor cells exist.
Although the existence of bonafide neutrophil precursors has been suggested in several studies (18-20), the unipotency of these precursors to strictly produce only neutrophils has not been shown (21).
Recently, high-dimensional mass cytometry (also known as cytometry by time-of-flight, CyTOF), which combines the advantages of both flow cytometry and mass spectrometry by utilizing antibodies conjugated to metal isotopes, has become a powerful tool to investigate the hematopoietic system (22-24). Using mass cytometry, high heterogeneity of hematopoietic progenitors within the BM has been demonstrated (22, 23). While, mass cytometry analysis of the mouse BM with a myeloid-selective panel of surface markers revealed a cell subset with morphology highly related to reported neutrophil precursors (22), however, the developmental potential of this subset was not evaluated.
In the present specification, the inventors describe an enriched or purified preparation of novel neutrophil progenitor population, methods of making a preparation of such neutrophil progenitor population, and methods of using same.
In the present specification, the inventors describe the discovery of a new, very early-stage, committed unipotent neutrophil progenitor (NeP) that is present in mouse and human bone marrow. The inventors have found that both the mouse and human NeP promoted primary tumor growth in vivo in established cancer models. Further, the presence of the human NeP (hNeP) in the blood of patients with recently diagnosed melanoma was identified, showing that this hNeP is released from the bone marrow in patients with cancer, and can be readily identified in human blood.
Importantly, a tumor-promoting role for this new early-stage neutrophil progenitor was discovered in both mice and humans. In tumor-bearing mice, frequencies of this NeP are increased in bone marrow, showing aberrant myelopoiesis in response to tumor growth (
The earliest committed neutrophil progenitor has remained elusive for decades. Most studies have focused on murine hematopoiesis. In this regard, the classic model of hematopoiesis shows that LSK+ (Lin−CD117+Ly6A/E+CD127) HSPCs give rise to CLP (Lin− CD117lo Ly6A/E+CD1271 for lymphopoiesis and to the Lin−CD117+Ly6A/E−CD127− HSPCs for myelopoiesis (Weissman et al., 2001). However, further examination of the Lin−CD117+Ly6A/E− HSPC fraction by mass cytometry showed 5 committed myeloid progenitors (
In sum, using mass cytometry the inventors have identified a novel, new, early-stage committed unipotent neutrophil progenitor that is present in both mouse and human bone marrow. This discovery provides new therapeutic and pharmaceutical targets for neutrophil-related diseases or treatment outcomes that are associated with chronic inflammation. For example, neutropenia leads to high susceptibility to infections and is often associated as a by-product of cancer treatments (Lyman et al., 2014). Without being bound to a particular theory, targeting hNeP may rescue patients from undesirable neutropenia. In addition, the inventors' observation of increased hNeP in blood of melanoma patients provides avenues for early detection for cancer diagnosis as a biomarker. As this hNeP also displays tumor-promoting effects, without being bound to a particular theory, this hNeP itself could be an immune-oncology target.
The progenitor population of the present disclosure is also referred to hereinafter as Neutrophil Progenitors (NePs). The progenitor population of the present disclosure includes progenitor cells that give rise, upon differentiation, to only neutrophils. Such ability can be tested in vitro and/or in vivo with the herein described methods or with methods that are readily available to the person of skill in the art. Accordingly, the NePs of the present disclosure are hereinafter also referred to as unipotent neutrophil progenitor cells.
In one embodiment, the progenitor population of the present disclosure includes a cell population having at least the phenotype CD115−, Siglec8− and FcERIα−.
In one embodiment, the progenitor population of the present disclosure includes a cell population having at least the phenotype CD45+, CD41−, CD127 (IL-7Rα)−, CD19−, CD3−, CD161 (NK1.1)−, CD169 (Siglec 1)−, CD11c−, Siglec 8−, FcERIα− and CD115 (CSF-1R)−.
In one embodiment, the progenitor population of the present disclosure includes a mouse cell population having at least the phenotype CD117(c-Kit)+, CD16/32+, CD115−, SiglecF−, FcERIα−.
In one embodiment, the progenitor population of the present disclosure includes a mouse cell population having at least the phenotype CD161−, CD117(c-Kit)+, Ly6A/E−, CD16/32+, CD115−, SiglecF−, FcERIα− and Ly6G−/lo.
In one embodiment, the progenitor population of the present disclosure includes a mouse cell population having at least the phenotype CD45+, Ter119−, CD41−, CD127 (IL-7Rα)−, CD19- or B220−, CD3−, TCRβ−, CD161 (NK1.1)−, CD335 (NKp46)−, CD169 (Siglec 1)−, F4/80−, CD11c−, MHCII−, CD117 (c-kit)+/int, Ly6A/E (Sca1)−, Siglec F (Siglec 8)−, FcERIα−, CD115 (CSF-1R)−, Ly6C−/int, CD16/32 (FcγRIII/II)+, and Ly6G−/lo.
In one embodiment, the progenitor population of the present disclosure includes a mouse cell population having at least the phenotype CD41−, CD127(IL-7Rα)−, CD3−, CD19−, CD161(NK1.1)−, CD169(Siglec 1)−, CD11c−, Siglec F, FcERIα−, CD115(CSF-1R)−, Ly6A/E(Sca1)−, Ly6G−, CD162(PSGL-1) lo, CD48 lo, Ly6C lo, and CD117(c-Kit)+, CD16/32(FcγRIII/II)+, Ly6B+ and CD11a(LFA1α)+.
In one embodiment, the progenitor population of the present disclosure includes a mouse cell population having at least the phenotype CD41−, CD127(IL-7Rα)−, CD3, CD19−, CD161(NK1.1)−, CD169(Siglec 1)−, CD11c−, Siglec F−, FcERIα−, CD115(CSF-1R)−, Ly6A/E(Sca1)−, CD117(c-Kit)+, CD16/32(FcγRIII/II)+, Ly6B, CD11a(LFA1α)+, and Ly6G+.
In one embodiment, the progenitor population of the present disclosure includes a human cell population having at least the phenotype CD34+, CD38+, CD115−, Siglec8− and FcERIα−.
In one embodiment, the progenitor population of the present disclosure includes a human cell population having at least the phenotype CD161−, CD34+, CD38+, CD115−, Siglec8−, FcERIα− and CD114+.
In one embodiment, the progenitor population of the present disclosure includes a human cell population having at least the phenotype CD45+, CD235ab−, CD41−, CD127 (IL-7Rα)−, CD19-, CD3−, CD4−, CD161 (NK1.1)−, CD56−, CD169 (Siglec 1)−, CD64−, CD11c−, HLA-DR−, CD86−, CD123−, CD7−, CD10−, CD366−, CD90−, Siglec 8−, FcERIα−, CD115 (CSF-1R)−, CD34+, CD38+, CD45RA+, CD66b+, CD16b+, CD15+, CD114+, CD14int, CD162int, and CD62Lint.
In one embodiment, the progenitor population of the present disclosure includes a human cell population having at least the phenotype hSiglec 8−, hFcεRIα−, hCD3−, hCD7−, hCD10−, hCD11c−, hCD19−, hCD41−, hCD56−, hCD90 (Thy1)−, hCD123 (IL-3Rα)−, hCD125 (IL-5Rα)−, hCD127 (IL-7Rα)−, hCD161−, hCD169−, hCD235a−, hCD66b+, hCD117 (c-Kit)+, hCD38+, and hCD34+.
In one embodiment, the progenitor population of the present disclosure includes a human cell population having at least the phenotype hSiglec 8−, hFcεRIα−, hCD3−, hCD7−, hCD10−, hCD11c−, hCD19−, hCD41−, hCD56−, hCD90 (Thy1)−, hCD123 (IL-3Rα)−, hCD125 (IL-5Rα)−, hCD127 (IL-7Rα)−, hCD161−, hCD169−, hCD235a−, hCD34−, hCD66b+, hCD117 (c-Kit)+, and hCD38+.
In the present disclosure, “−” refers to negative expression, “+” refers to positive expression, the term “lo” refers to negative or low expression levels, “int” refers to intermediate expression levels and “hi” refers to high expression levels.
In one embodiment, the progenitor population of the present disclosure includes a cell population that further expresses Lymphocyte antigen 6 complex locus G6D (hereinafter, a cell of further phenotype Ly6G+). In another embodiment, the progenitor population of the present disclosure includes a cell population that does not express Lymphocyte antigen 6 complex locus G6D (hereinafter, a cell of further phenotype Ly6G). In yet another embodiment, the progenitor population of the present disclosure includes a first cell population of further phenotype Ly6G+ and a second cell population of further phenotype Ly6G−.
In one embodiment, the progenitor population of the present disclosure includes a first cell population of further phenotype Ly6G+ and a second cell population of further phenotype Ly6G− in a ratio Ly6G+/Ly6G− which is selected based on a desired neutrophil differentiation kinetics when the progenitor population is introduced in a subject. The person of skill can, thus, prepare a composition comprising the progenitor population of the present disclosure where the composition includes a first cell population of further phenotype Ly6G+ and a second cell population of further phenotype Ly6G− in a ratio Ly6G+/Ly6G− which is selected based on a desired neutrophil differentiation kinetics when the progenitor population is introduced in a subject. Such composition, thus, does not exist in nature and is functionally different from a comparison composition which is extracted (e.g., cell sorted) from a natural biological sample since this composition will have different neutrophil differentiation kinetics when the progenitor population is introduced in a subject, where such kinetics are purposively selected by the person of skill by specifically designing the composition to have a given ratio Ly6G+/Ly6G−.
In one embodiment, the progenitor population of the present disclosure may include a cell population with cells that have been modified, for example but without being limited thereto, so as to have modified gene expression, modified cell function or to include a ribonucleic acid interference (RNAi)-causing molecule, or to have a conjugated therapeutic agent.
In one embodiment, the progenitor population of the present disclosure may include a cell population with cells that have been genetically modified by CRISPR-cas system (such as CRISPR/Cas9), Cre-lox recombination system, gene knock-down, gene knock-out, lentivirus transduction or RNAi-causing molecule.
For example, the progenitor population of the present disclosure may include a cell population with cells that have been further modified so as to include an RNAi-causing molecule such as a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro RNA (miRNA), or a plasmid DNA for expressing the shRNA, siRNA or miRNA. RNAi-causing molecules are well known in the art. For example, the person of skill will readily understand that miRNA are small (e.g., 18-25 nucleotides in length), noncoding RNAs that influence gene regulatory networks by post-transcriptional regulation of specific messenger RNA (mRNA) targets via specific base-pairing interactions. This ability of microRNAs to inhibit the production of their target proteins results in the regulation of many types of cellular activities, such as cell-fate determination, apoptosis, differentiation, and oncogenesis.
The person of skill will readily recognized that the progenitor population of the present disclosure may be modified in vitro and/or in vivo, with techniques that are readily available to the person of skill, so as to obtain cells having the desired characteristic.
In one embodiment, the progenitor population of the present disclosure may be extracted from a biological sample using a cell sorting technique. For example, the cell sorting technique may include flow-cytometry-based cell sorting, magnetic cell sorting, and/or antibody panning.
In one embodiment, the cell sorting technique may be carried out in a device adapted to separate or quantify cells on the basis of detecting agent(s) binding to specific cell markers in the progenitor population of the present disclosure. The detecting agent(s) may further include cell sorting agent(s), such as a chromophore or a metal. When the detecting agent includes a chromophore, the device may be, for example, a fluorescence-activated cell sorting (FACS) device. The specific markers of the progenitor population of the present disclosure may be intracellular markers and/or cell surface markers. For example, the detecting agent may include antibodies, which may further include a cell sorting agent as described above.
The cell measurements may be carried out, for example, by immunoassays including, but not limited to, western blots, immunohistochemistry, immunocytochemistry, in situ hybridization, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immune-radiometric assays, fluorescent immunoassays, immunofluorescence, or flow cytometry.
In one embodiment, the progenitor population of the present disclosure may be extracted from a biological sample using at least one of the gating strategies which are provided in Example 1.
As used herein, the progenitor population of the present disclosure may be extracted from a sample which includes bone marrow, tumor tissue, blood or spleen, or a cell fraction thereof.
In one embodiment, the present disclosure relates to a kit for cell sorting the progenitor cells of the present disclosure. For example, such kit may include a combination of detecting agents for any combination of the previously described markers.
For example, the kit may include a combination of detecting agents for markers such as at least CD115, Siglec8 and FcERIα; or at least CD45, CD41, CD127 (IL-7Rα), CD19, CD3, CD161 (NK1.1), CD169 (Siglec 1), CD11c, Siglec 8, FcERIα and CD115 (CSF-1R); or at least CD117(c-Kit), CD16/32, CD115, Siglec F, FcERIα; or at least CD34, CD38, CD115, Siglec 8 and FcERIα.
In another example, such kit may include a combination of detecting agents for markers such as at least CD161, CD117(c-Kit), Ly6A/E, CD16/32, CD115, SiglecF, FcERIα and Ly6G; or at least CD45, Ter119, CD41, CD127 (IL-7Rα), CD19 or B220, CD3, TCRβ, CD161 (NK1.1), CD335 (NKp46), CD169 (Siglec 1), F4/80, CD11c, MHCII, CD117 (c-kit), Ly6A/E (Sca1), Siglec F (Siglec 8), FcERIα, CD115 (CSF-1R), Ly6C, CD16/32 (FcγRIII/II), and Ly6G.
In another example, such kit may include a combination of detecting agents for markers such as at least CD41, CD127(IL-7Rα), CD3, CD19, CD161(NK1.1), CD169(Siglec 1), CD11c, Siglec F, FcERIα, CD115(CSF-1R), Ly6A/E(Sca1), Ly6G, CD162(PSGL-1), CD48, Ly6C, and CD117(c-Kit), CD16/32(FcγRIII/II), Ly6B and CD11a(LFA1α).
In another example, such kit may include a combination of detecting agents for markers such as at least CD41, CD127(IL-7Rα), CD3, CD19, CD161(NK1.1), CD169(Siglec 1), CD11c, Siglec F, FcERIα, CD115(CSF-1R), Ly6A/E(Sca1), CD117(c-Kit), CD16/32(FcγRIII/II), Ly6B, CD11a(LFA1α), and Ly6G.
In another example, such kit may include a combination of detecting agents for markers such as at least CD161, CD34, CD38, CD115, Siglec8, FcERIα and CD114; or at least CD45, CD235ab, CD41, CD127 (IL-7Rα), CD19, CD3, CD4, CD161 (NK1.1), CD56, CD169 (Siglec 1), CD64, CD11c, HLA-DR, CD86, CD123, CD7, CD10, CD366, CD90, Siglec 8, FcERIα, CD115 (CSF-1R), CD34, CD38, CD45RA, CD66b, CD16b, CD15, CD114, CD14, CD162, and CD62L.
In another example, such kit may include a combination of detecting agents for markers such as at least hSiglec 8, hFcεRIα, hCD3, hCD7, hCD10, hCD11c, hCD19, hCD41, hCD56, hCD90 (Thy1), hCD123 (IL-3Rα), hCD125 (IL-5Rα), hCD127 (IL-7Rα), hCD161, hCD169, hCD235a, hCD66b, hCD117 (c-Kit), hCD38, and hCD34.
The person of skill will readily recognize that various permutation of detecting agent may be included in such kits so as to obtain the desired result.
The present disclosure further describes methods which make use of the progenitor population of the present disclosure to obtain a desired result, which may be for example, but without being limited thereto, therapeutic and/or prophylactic, or which may further provide information on neutrophil biology in health and/or disease, or which may assist in evaluating the effectiveness of a given treatment, and the like.
In one embodiment, the present disclosure describes a method for treatment of a subject. The method may include activation or inhibition of the progenitor population of the present disclosure to differentiate into neutrophils. In other words, the person of skill may implement steps to target the progenitor population of the present disclosure. Such method may have therapeutic and/or prophylactic desired results. The activation or inhibition may occur in vitro, in which case, the resulting activated or inhibited progenitor population of the present disclosure can then be administered to the subject in order to obtain the desired result. Alternatively or additionally, the activation or inhibition may occur in vivo with the administration of a suitable activation or inhibition compound to the subject.
For example, activation of the progenitor population of the present disclosure may include contacting the progenitor population with a suitable compound targeting transcription factors such as Gfi1, Snai1, or KLF5. If the progenitor population of the present disclosure includes a human cell population, then the suitable activating compound may target CD114 (G-CSFR). In certain embodiments, activation of the progenitor population of the present disclosure may include administering a drug suitable for treatment of neutropenia (e.g., G-CSF, Docetaxel). Inhibition of the progenitor population of the present disclosure may include contacting the progenitor population with a suitable compound targeting transcription factors such as Gata1, IRF8, or KLF4. In certain embodiment, inhibition of the progenitor population of the present disclosure may include administering a drug suitable for treatment of neutrophilia (e.g., Imatinib).
In one embodiment, the suitable compound may be a ribonucleic acid interference inducing (RNAi) molecule, a small molecule, an antibody, a protein, a peptide, a ligand mimetic, and the like. The person of skill will readily understand what compound may be suitable to obtain the desired effect.
This method of treatment can be used conjunctly with an assessment step as described below.
In a first variant, the above method of treatment may further include an assessment step whereby one determines the levels of the progenitor population of the present disclosure which are present in the subject pre- and/or post-treatment. In order to do so, the person of skill may implement additional steps whereby the levels of the progenitor population of the present disclosure are determined in a biological sample of the subject. In certain non-limiting embodiments, the biological sample here includes blood, spleen, tumor tissue, or bone marrow, or a cell fraction thereof. Such additional steps may comprise processing the biological sample being suspected of including the progenitor population of the present disclosure to determine the concentration or activation level thereof. In one embodiment, such additional steps may make use of the cell sorting techniques described earlier to extract the progenitor population of the present disclosure from the biological sample.
In a second variant, the above method of treatment may further include an assessment step whereby one determines the levels of the neutrophil cells which are present in the subject pre- and/or post-treatment. In order to do, the person of skill may implement additional steps whereby the levels of the neutrophil cells are determined in another biological sample of the subject. In one non-limiting embodiment, the biological sample here includes blood or a cell fraction thereof. Such additional steps may comprise processing the biological sample being suspected of including the neutrophil cells to determine the level thereof. In one non-limiting embodiment, such additional steps may make use of cell sorting techniques, as described elsewhere in the present document or that are readily available to the person of skill in the art. In another variant, the person of skill may make use of readily available detecting agents that selectively recognize markers present on the neutrophil cells and which can be detected/quantified so as to indirectly determine the concentration level of neutrophils.
In a third variant, the above method of treatment may include a combination of the first and second variant.
As discussed elsewhere in the present document, the level which is determined from the biological sample can be compared to a reference level. In certain embodiments, the reference level can be derived from a sample of at least 20 reference individuals without condition (in other words that are not afflicted by the condition of the tested subject), or at least 30, or at least 40, or at least 50, or at least 60, or at least 100 reference individuals without condition. Alternatively or additionally, the reference level can be derived from levels determined in the subject pre and/or post treatment.
In one practical implementation, such variants can, thus, serve to determine the effectiveness of a given treatment by providing clinical information pertaining to a subject's neutrophil levels and/or NePs levels in pre and/or post treatment phase. For example, the person of skill can monitor the effectiveness of a method for treatment or prevention of cancer, neutropenia or related conditions. Such monitoring can be performed by implementing at least one of the herein described variants.
In one embodiment, neutropenia can be caused by a cancer. For example, a cancer selected from colon carcinomas, pancreatic cancer, breast cancer, lung carcinoma, prostate cancer, metastatic renal cell carcinoma (RCC), mammary carcinoma, lung cancer, thymoma, fibrosarcoma, and myeloid sarcoma.
In another embodiment, neutropenia can be caused by chemotherapy, severe microbial infection (such as Hepatitis, HIV/AIDS, malaria or Salmonella), sepsis (overwhelming blood infection that depletes neutrophils faster than they can be produced), Kostmann's syndrome, myelokathexis or other congenital disorders, leukemia, myelodysplastic syndromes, autoimmune disorders such as Rheumatoid arthritis, neonates with growth disorders or those born to mothers with preeclampsia or hypertension, or transplant.
The present disclosure also describes a method for evaluating a cancer in a subject. Generally speaking, this method includes determining a concentration or activation level of the neutrophil progenitor population of the present disclosure in a biological sample of the subject, which is suspected of including the neutrophil progenitor population of the present disclosure. The biological sample here may include blood, spleen, tumor tissue, bone marrow or a cell fraction thereof. In one embodiment, the biological sample may include blood or a cell fraction thereof. The method further includes comparing the concentration or activation level to a reference level. At least based on such comparison, the person of skill can then determine the likelihood that the subject has or does not have cancer. Indeed, the data presented in the present document provide factual basis for the person of skill to reasonably expect that the concentration or activation level of the neutrophil progenitor population of the present disclosure is indicative of the presence of cancer in a subject.
In a variant of such method for evaluating a cancer in a subject, the person of skill can also determine the response or resistance to cancer treatment in a subject undergoing cancer treatment. Indeed, following treatment, the person of skill can determine the concentration or activation level of the neutrophil progenitor population of the present disclosure which will be indicative of the progression of the cancer and accordingly, will provide information as to the response or resistance to cancer treatment in the subject undergoing cancer treatment. In other words, when comparing the concentration or activation level to a reference level, the person of skill can evaluate the response or resistance to the treatment based on at least the comparison.
In another variant, of such method for evaluating a cancer in a subject, the cancer may cause neutropenia. In such variant, the person of skill can also determine the response or resistance to a treatment for a condition associated with neutropenia in the subject undergoing the treatment. Indeed, following treatment, the person of skill can determine the concentration or activation level of the neutrophil progenitor population of the present disclosure which will be indicative of the neutrophil differentiation capability of the subject. In other words, when comparing the concentration or activation level to a reference level, the person of skill can evaluate the response or resistance to the treatment based on at least the comparison.
The present disclosure also describes a method for reducing risk of cancer progression or cancer relapse in a subject. The method includes determining a concentration or activation level of the neutrophil progenitor population of the present disclosure in a biological sample of the subject, which is suspected of including the neutrophil progenitor population of the present disclosure. The biological sample here may include blood, spleen, tumor tissue, bone marrow or a cell fraction thereof. In one embodiment, the biological sample may include blood or a cell fraction thereof. The method further includes comparing the concentration or activation level to a reference level. At least based on such comparison, the person of skill can then selectively administer a cancer therapeutic agent so as to reduce risk of cancer progression or cancer relapse in the subject.
In one embodiment, the present disclosure also describes a method for reducing risk of a condition associated with neutropenia in the subject. The method includes determining a concentration or activation level of the neutrophil progenitor population of the present disclosure in a biological sample of the subject, which is suspected of including the neutrophil progenitor population of the present disclosure. The biological sample here may include blood, spleen, tumor tissue, bone marrow or a cell fraction thereof. In one embodiment, the biological sample may include blood or a cell fraction thereof. The method further includes comparing the concentration or activation level to a reference level. At least based on such comparison, the person of skill can then selectively administer a therapeutic agent so as to reduce risk of the condition neutropenia in the subject.
The comparison step includes using a reference level. The reference level can be derived from a sample of at least 20 reference individuals without condition (in other words that are not afflicted by the condition of the tested subject), or at least 30, or at least 40, or at least 50, or at least 60, or at least 100 reference individuals without condition. Alternatively or additionally, the reference level can be derived from levels determined in the subject pre and/or post treatment.
In one embodiment, the present disclosure also describes a method for screening a candidate molecule for an activity on cell differentiation of the neutrophil progenitor population of the present disclosure into neutrophils. The method includes contacting the neutrophil progenitor population of the present disclosure with the candidate molecule and determining the activity of the candidate molecule on the cell differentiation of the neutrophil progenitor population of the present disclosure into neutrophils.
In one embodiment, the present disclosure also describes a method for treatment or prevention of neutropenia in a subject. The method includes administering to the subject an effective amount of a purified preparation of the neutrophil progenitor population of the present disclosure. Such administration can be used in conjunction with the assessment steps described earlier in this document, for example, to monitor the effectiveness of the treatment.
In one embodiment, the neutrophil progenitor population of the present disclosure which is administered to the subject includes progenitor cells that are autologous (cells from the subject being administered), allogeneic (cells from another individual), or syngenic (genetically identical, or sufficiently identical and immunologically compatible as to allow for transplantation), to the subject.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention pertains. As used herein, and unless stated otherwise or required otherwise by context, each of the following terms shall have the definition set forth below.
In one embodiment, the methods described herein make use of the measured levels of the progenitor population of the present disclosure to detect surges or declines in cell numbers as predictive measures. As used herein, a “surge” indicates a statistically significant increase in the level of relevant cells, typically from one measurement to one or more later measurements. In other instances, an increase in the level of relevant cells can be determined from one measure in a subject of interest relative to control (e.g., a value or a range of values for normal, i.e., healthy, individuals). Surges may be a two-fold increase in cell levels (i.e., a doubling of cell counts), a three-fold increase in cell levels (i.e., a tripling of cell numbers), a four-fold increase in cell levels (i.e., an increase by four times the number of cells in a previous measurement), or a five-fold or greater increase. In addition to the marked increase described as a surge, lesser increases in the levels of relevant cells may also have relevance to the methods of the present disclosure. Increases in cell levels may be described in terms of percentages. Surges may also be described in terms of percentages. For example, a surge or increase may be an increase in cell levels of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. A “decline” indicates a decrease from one measurement to one or more later measurements. A decline may be a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% or greater decrease in cell levels from one measurement to one or more later measurements. In other instances, a decrease in the level of relevant cells can be determined from one measure in a subject of interest relative to control (e.g., a value or a range of values for normal, i.e., healthy, individuals).
In one embodiment, the surges or declines in cell numbers can be determined based on a comparison with a reference level derived from samples of at least 20 reference individuals without condition, a non-patient population. The surges or declines in cell numbers in a sample can also refer to a level that is elevated in comparison to the level of the cell numbers reached upon treatment, for example with an anti-cancer compound.
In one embodiment, the term “cancer” refers to a class of diseases in which a group of cells display uncontrolled growth, invasion, and metastasis. The term is meant to include, but not limited to, a cancer of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, and parathyroid. The cancer may be a solid tumor, a non-solid tumor, or a distant metastasis of a tumor. Some specific examples of cancers include, but are not limited to, leukemia; lymphomas; multiple myelomas; bone and connective tissue sarcomas; brain tumors; breast cancer; adrenal cancer; thyroid cancer; pancreatic cancer; pituitary cancers; eye cancers; vaginal cancers; cervical cancers; uterine cancers; ovarian cancers; esophageal cancers; stomach cancers; colon cancers; rectal cancers; gastric cancers; liver cancers; bladder cancers; gallbladder cancers; cholangiocarcinoma; lung cancers; testicular cancers; prostate cancers; penile cancers; oral cancers; basal cancers; salivary gland cancers; pharynx cancers; skin cancers; kidney cancers; and Wilms' tumor. Examples of solid tumors include solid tumors of the breast, prostate, colon, pancreas, lung, gastric system, bladder, and bone/connective tissue. When making reference to neutropenia in particular, the cancer can be selected from colon carcinomas, pancreatic cancer, breast cancer, lung carcinoma, prostate cancer, metastatic RCC, mammary carcinoma, lung cancer, thymoma, fibrosarcoma, and myeloid sarcoma.
As used herein, “relapse” or “recurrence” may include the appearance of at least one new tumor lesions in a subject who previously had cancer but has had no overt evidence of cancer as a result of surgery and/or therapy until relapse. Such recurrence of cancer cells can be local, occurring in the same area as one or more previous tumor lesions, or distant, occurring in a previously lesion-free area, such as lymph nodes or other areas of the body.
As used herein, “response to treatment” may include complete response and partial response to treatment. A “complete response” (CR), in certain embodiments relating to e.g. cancer, is typically understood to include the disappearance of all target lesions and non-target lesions and normalization of tumor marker levels, whereas in other embodiments relating to e.g. neutropenia, is typically understood as the complete normalization of neutrophil levels in the subject. A “partial response” (PR), in certain embodiments relating to cancer, is typically understood to include an at least 30% decrease in the sum of the diameters of target lesions, whereas in other embodiments relating to neutropenia, is typically understood as a relative increase of neutrophil levels in a subject suffering from neutropenia of at least 30%. Generally speaking, in the context of embodiments relating to e.g. cancer, “response to treatment” may include an at least 30%-100% decrease in the sum of the diameters of target lesions, or disappearance of all target lesions and non-target lesions and normalization of tumor marker levels. Generally speaking, in the context of embodiments relating to e.g. neutropenia, “response to treatment” may include an at least 30%-100% increase in neutrophil levels. “Progression” or “progressive disease” (PD), in certain embodiments relating to e.g. cancer, is typically understood to include an at least 20% increase in the sum of the diameters of target lesions, progression (increase in size) of any existing non-target lesions, and is also typically determined upon appearance of at least one new lesion. Non-CR/non-PD, in certain embodiments relating to e.g. cancer, is typically understood to include the persistence of one or more non-target lesions and/or maintenance of above-normal tumor marker levels. “Stable disease” (SD) is typically understood to include an insufficient increase to qualify for PD, but an insufficient decrease to qualify for PR. While the concepts of CR, PR, PD, and SD have been discussed in the context of cancer and neutropenia, the person of skill will readily understand that these concepts may also apply to other disease/conditions, which are associated with aberrant neutrophil levels.
As used herein, the terms “treatment”, “treating”, and the like, may include amelioration or elimination of a developed disease or condition once it has been established or alleviation of the characteristic symptoms of such disease or condition. As used herein, these terms may also encompass, depending on the condition of the subject, preventing the onset of a disease or condition or of symptoms associated with the disease or condition, including for example reducing the severity of the disease or condition or symptoms associated therewith prior to affliction with the disease or condition. Such prevention or reduction prior to affliction may refer, in the context of cancer, to administration of at least one cancer therapeutic compound to a subject that is not at the time of administration afflicted with the disease or condition. “Preventing” may also encompass preventing the recurrence or relapse of a previously existing disease or condition or of symptoms associated therewith, for instance after a period of improvement.
The subject or patient can be any mammal, including a human. In particular, in the context of cancer, the subject can be a mammal who previously had cancer but appears to have recovered as a result of surgery and/or therapy, or who presently has cancer and is undergoing cancer therapy, or has completed a cancer therapeutic regime, or has received no cancer therapy.
As used herein, the terms “therapeutically effective amount” and “effective amount” are used interchangeably to refer to an amount of a composition of the disclosure that is sufficient to result in the prevention of the development, recurrence, or onset of a disease or condition. For example, in certain embodiments e.g. cancer, these terms refer to an amount of a composition of the invention that is sufficient to result in the prevention of the development, recurrence, or onset of cancer stem cells or cancer and one or more symptoms thereof, to enhance or improve the prophylactic effect(s) of another therapy, reduce the severity and duration of cancer, ameliorate one or more symptoms of cancer, prevent the advancement of cancer, cause regression of cancer, and/or enhance or improve the therapeutic effect(s) of additional anticancer treatment(s). For example, in certain embodiments e.g. neutropenia, these terms refer to an amount of a composition of the disclosure that is sufficient to result in the prevention of the development, recurrence, or onset of neutropenia or one or more symptoms thereof, to enhance or improve the prophylactic effect(s) of another therapy, reduce the severity and duration of neutropenia, ameliorate one or more symptoms of neutropenia, prevent the advancement of neutropenia (further decrease of neutrophil levels), and/or enhance or improve the therapeutic effect(s) of additional anti-neutropenia treatment(s).
A therapeutically effective amount can be administered to a patient in one or more doses sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease, or reduce the symptoms of the disease. The amelioration or reduction need not be permanent, but may be for a period of time ranging from at least one hour, at least one day, or at least one week or more. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the patient, the condition being treated, the severity of the condition, as well as the route of administration, dosage form and regimen and the desired result.
In one non-limiting embodiment, the biological sample from the subject which is suspected of including neutrophil cells includes blood or a cell fraction thereof.
In one non-limiting embodiment, the biological sample from the subject which is suspected of including the progenitor population of the present disclosure includes blood, spleen, tumor tissue, bone marrow or a cell fraction thereof.
As used herein, a “cell fraction” of a biological sample may be obtained using routine clinical cell fractionation techniques, such as gentle centrifugation, e.g., centrifugation at about 300-800×g for about five to about ten minutes, or fractionated by other standard methods.
In one non-limiting embodiment, the herein described sample can be obtained by any known technique, for example by drawing, by non-invasive techniques, or from sample collections or banks, etc.
In one non-limiting embodiment, the present disclosure provides a kit which includes reagents that may be useful for implementing at least some of the herein described methods. The herein described kit may include at least one detecting agent which is “packaged”. As used herein, the term “packaged” can refer to the use of a solid matrix or material such as glass, plastic, paper, fiber, foil and the like, capable of holding within fixed limits the at least one detection reagent. Thus, in one non-limiting embodiment, the kit may include the at least one detecting agent “packaged” in a glass vial used to contain microgram or milligram quantities of the at least one detecting agent. In another non-limiting embodiment, the kit may include the at least one detecting agent “packaged” in a microtiter plate well to which microgram quantities of the at least one detecting agent has been operatively affixed. In another non-limiting embodiment, the kit may include the at least one detecting agent coated on microparticles entrapped within a porous membrane or embedded in a test strip or dipstick, etc. In another non-limiting embodiment, the kit may include the at least one detecting agent directly coated onto a membrane, test strip or dipstick, etc. which contacts the sample fluid. Many other possibilities exist and will be readily recognized by those skilled in this art without departing from the invention. For example, the kit may include a combination of detecting agent which can be useful for cell sorting the progenitor cells of the present disclosure, as discussed elsewhere in the present document.
The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
As used herein, the term “transcription factor” refers to a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. In turn, this helps to regulate the expression of genes near that sequence.
By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
As used herein, a “purified cell population” refers to a cell population which has been processed so as to separate the cell population from other cell populations with which it is normally associated in its naturally occurring state. The purified cell population can, thus, represent an enriched cell population in that the relative concentration of the cell population in a sample can be increased following such processing in comparison to its natural state. In one embodiment, the purified cell population can refer to a cell population which is enriched in a composition in a relative amount of at least 80%, or at least 90%, or at least 95% or 100% in comparison to its natural state. Such purified cell population may, thus, represent a cell preparation which can be further processed so as to obtain commercially viable preparations. For example, in one embodiment, the cell preparation can be prepared for transportation or storage in a serum-based solution containing necessary additives (e.g., DMSO), which can then be stored or transported in a frozen form. In doing so, the person of skill will readily understand that the cell preparation is in a composition that includes a suitable carrier, which composition is significantly different from the natural occurring separate elements. For example, the serum-based preparation may comprise human serum or fetal bovine serum, which is a structural form that is markedly different from the form of the naturally occurring elements of the preparation. The resulting preparation includes cells that are in dormant state, for example, that may have slowed-down or stopped intracellular metabolic reactions and/or that may have structural modifications to its cellular membranes. The resulting preparation includes cells that can, thus, be packaged or shipped while minimizing cell loss which would otherwise occur with the naturally occurring cells. A person skilled in the art would be able to determine a suitable preparation without departing from the present disclosure.
The composition described herein may include one or more pharmaceutically acceptable carrier. As used herein, the term “carrier” refers to any carrier, diluent or excipient that is compatible with the herein described NePs and can be given to a subject without adverse effects. Suitable acceptable carriers known in the art include, but are not limited to, water, saline, glucose, dextrose, buffered solutions, and the like. Such a carrier is advantageously non-toxic to the NePs and not harmful to the subject. It may also be biodegradable. The carrier may be a solid or liquid acceptable carrier. A suitable solid acceptable carrier is a non-toxic carrier. For instance, this solid acceptable carrier may be a common solid micronized injectable such as the component of a typical injectable composition for example, but without being limited to, kaolin, talc, calcium carbonate, chitosan, starch, lactose, and the like. A suitable liquid acceptable carrier may be, for example, water, saline, DMSO, culture medium such as DMEM, and the like. The person skilled in the art will be able to determine a suitable acceptable carrier for a specific application without departing from the present disclosure.
As used herein, the term “about” for example with respect to a value relating to a particular parameter (e.g. concentration, such as “about 100 mM”) relates to the variation, deviation or error (e.g. determined via statistical analysis) associated with a device or method used to measure the parameter. For example, in the case where the value of a parameter is based on a device or method which is capable of measuring the parameter with an error of ±10%, “about” would encompass the range from less than 10% of the value to more than 10% of the value.
The invention is further illustrated by the following non-limiting examples.
The following examples describe some exemplary modes of making and practicing embodiments of the invention. It should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the compositions and methods described herein.
The following materials and methods were used to perform the practical examples described subsequently.
C57BL/6J, B6 CD45.1 congenic mice, and NSG-SGM3 mice were purchased from The Jackson Laboratory. Mice were fed a standard rodent chow diet and were housed in microisolator cages in a pathogen-free facility. Mice were euthanized by CO2 inhalation followed by cervical dislocation. All experiments followed approved guidelines of the La Jolla Institute for Allergy and Immunology Animal Care and Use Committee, and approval for use of rodents was obtained from the La Jolla Institute for Allergy and Immunology according to criteria outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health.
Animals were randomly assigned to groups from available mice bred in our facility or ordered from distributor. Experiments in this study used male animals 6-10 weeks of age in good health. If animals were observed with non-experiment related health conditions (i.e. malocclusion, injuries from fighting, etc.), animals were removed from study groups.
Fresh BM samples of anonymous healthy adult donors were obtained from AllCells, Inc. (Alameda, Calif.). The cells were stained for either flow cytometry or FACS-sorting following protocols described in the Flow Cytometry and Cell Sorting section.
For tumor studies, B16F10 melanoma cells and 143B human osteosarcoma cells were obtained from ATCC. Cell lines were tested for being pathogen free. Cell lines were maintained in DMEM medium containing 10% heat-inactivated FBS, 2 mmol/L 1-glutamine, 1 mmol/L sodium pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin.
Melanoma patient (no previous radiation, no prior chemo treatment) blood collected in EDTA-tubes were provided by the Biospecimen Repository Core Facility (BRCF) from University of Kansas Cancer Center. Cells were stained for flow cytometry followed by the protocol described in the Flow Cytometry and Cell Sorting section.
Heparinized blood from healthy volunteers was obtained after written informed consent under the guidelines of the Institutional Review Board of the La Jolla Institute for Allergy and Immunology and in accordance with US Dept. of Health and Human Services Policy for protection of Human Research Subjects (VD-057-0217). Cells were stained for flow cytometry followed by the protocol described in the Flow Cytometry and Cell Sorting section.
Bone marrow (BM) cells were harvested from femurs, and tibias of 6-10 week old mice. Bones were centrifuged for the collection of marrow. For the adoptive transfer experiments, donor BM cells were collected and stained under sterile conditions. Peripheral blood was obtained by cardiac puncture with an ethylenediaminetetraacetic acid (EDTA)-coated syringe. For
Metal-conjugated antibodies were purchased directly from Fluidigm™ for available targets. For all other targets, purified antibodies were purchased from the companies listed as provided in
For viability staining, cells were washed in PBS and stained with Cisplatin (Fluidigm) to a final concentration of 5 μM. Prior to surface staining, anti-CD16/32 (151Eu) antibody was added to cell suspension in ice-cold staining buffer (PBS+2 mM EDTA+0.1% BSA+0.05% NaN3) to stain and block the Fc receptors for 15 min. The surface antibody cocktail listed in
Antibodies for flow cytometry were purchased from commercial sources as follows: anti-CD3E (145-2C11; BD Biosciences); anti-CD19 (1D3; BD Biosciences); anti-CD161 (PK136; eBiosciences); anti-F4/80 (T45-2342; BD Biosciences); anti-CD11c (HL3; BD Biosciences); anti-CD45 (30-F11; BioLegend); anti-CD45.1 (A20; BioLegend); anti-CD45.2 (104; BioLegend); anti-CD117 (c-kit) (2B8; BioLegend); anti-Ly6A/E (Sca-1) (D7; BioLegend); anti-CD16/32 (FcγRIII/II (93; BioLegend); anti-CD11b (M1/70; BioLegend); anti-CD115 (M-CSFR) (AFS98; BioLegend); anti-Ly6G (1A8; BioLegend); anti-Ly6C (HK1.4; BioLegend); anti-Siglec F (E50-2446; BD Biosciences); anti-FcERIα (MAR-1; BioLegend); anti-Ki67 (SolA15; eBiosciences); anti-hCD45 (2D1; BioLegend); anti-hCD3E (HIT3a; BD Biosciences); anti-hCD7 (MT701; BD Biosciences); anti-hCD161 (HP-3G10; BioLegend); anti-hCD56 (B159; BD Biosciences); anti-hCD19 (HIB19; BD Biosciences); anti-hCD127 (A019D5; BioLegend); anti-hSiglec 8 (7C9; MACS Miltenyi Biotec); anti-hFcεRIα (AER-37; BioLegend); anti-hCD235a (GA-R2; BD Biosciences); anti-hCD41 (HIPS; BD Biosciences); anti-hCD169 (7-239; BD Biosciences); anti-hCD69 (10.1; BioLegend); anti-hCD11c (B-ly6; BD Biosciences); anti-hCD90 (5E10; BioLegend); anti-hCD86 (IT2.2; BioLegend); anti-hCD66B (G10F5; BioLegend); anti-hCD34 (581; BD Biosciences); anti-hCD117 (YB5.B8; BD Biosciences); anti-hCD38 (HB-7; BioLegend). Cell viability was determined with LIVE/DEAD™ Fixable Yellow (or Blue) Dead Cell Stain Kit (ThermoFisher).
All mouse FACS staining was performed in FACS buffer (DPBS+1% BSA+0.1% sodium azide+2 mM EDTA) on ice. All human FACS staining was performed in FACS buffer (DPBS+1% human serum+0.1% sodium azide+2 mM EDTA) on ice. Cells were filtered through sterile 70 μm cell strainers to obtain a single cell suspension (30,000 cells per μl for flow cytometry analysis, 0.5-2×107 per ml for sorting). Prior to surface staining, anti-CD16/32 (FITC) antibody (for mouse) or human Fc receptors blocking reagent (MACS' Miltenyi Biotec) was added for 15 min to stain and block the Fc receptors. Surface staining was performed for 30 minutes in a final volume of 500 μl for FACS sorts and 100 ul for regular flow cytometry. Cells were washed twice in at least 200 μl FACS buffer before acquisition. Cells were sorted using a FACS Aria™ II and Aria-Fusion (BD biosciences) and conventional flow cytometry using an LSRII or a LSR Fortessa™ (BD Biosciences). All flow cytometry was performed on live cells. Calculations of percentages of CD45+ immune cells were based on live cells as determined by forward and side scatter and viability analysis. All analyses and sorts were repeated at least 3 times, and purity of sorted fractions was checked visually and by FACS reanalysis of the surface markers. Data were analysed using Cytobank (the reader is referred to the Cytobank Internet website) and FlowJo™ (version 10.1r5).
Cells were FACS-sorted and resuspended in PBS. Following fixation in 4% methanol-free formaldehyde in PBS for 10 min at room temperature, cells were washed with PBS and resuspended in 5% normal donkey serum, 0.3% Triton™ X-100 in PBS for one hour. Cells were then incubated with a rabbit anti-Ki67 monoclonal antibody (clone SP6, Abcam, 1:150) or negative control (normal rabbit IgG) in 1% bovine serum albumin and 0.3% Triton X-100 in PBS overnight at 4° C. Cells were washed twice with PBS and incubated with anti-rabbit IgG (H+L) F(ab′)2 fragment conjugated to Alexa™ Fluor 647 (Cell Signalling, #4414, 1:500) and Hoechst (1:1000 of 10 mg/ml solution) for one hour at room temperature. After washing, cells were adhered to poly-L-lysine coated #1.5H coverslips and embedded in Prolong™ Gold (Thermo Fisher). Samples were imaged with a Zeiss LSM780 and Leica SP8 confocal microscopes using a 63×/1.40 NA oil-immersion objective. Images were processed with ZEN or Leica HyVolution™ software and 3D reconstructions of DNA were created in Imaris™ software. The mean and integrated fluorescence intensity (select the one you will show) of Ki67 within the nuclear regions were calculated in Image-Pro™ Premier. To reduce Z-stretching confocal images were deconvolved with Huygens Essential. Analysis of the surface area, volume and sphericity was performed in Imaris software.
Cytospins from sorted populations were fixed on slides with methanol, stained with solutions of May-Grünwald (eosin methylene blue) and Giemsa (eosin methylene blue; Merck) and analyzed on a Nikon Eclipse 80i microscope (Nikon).
Recipient mice were housed in a barrier facility under pathogen-free conditions before and after adoptive transfer. NSG-SGM3 recipient mice were maintained in sterile conditions at all times. CD45.1 recipient mice were fed with autoclaved acidified water with antibiotics (trimethoprimsulfamethoxazole) for 3 days before the adoptive transfer. Sub-lethally irradiated recipient mice received 600 Rads. Donor BM cells were collected and FACS sorted as described in the flow cytometry section. Mouse and human progenitor cells were sorted directly into sterile FBS and kept chilled during sorting. Cells then were washed and resuspended in ice-cold DPBS for injection. 5×104 donor progenitors in 200 μl DPBS were delivered into each recipient mouse for
Sorted progenitor cells (3×104) were seeded into 6-well plates and cultured for 10 days with Methocult™ GF M3434 media (Stem Cell Technologies) according to the manufacturer's protocol. The numbers of wells containing proliferated colonies were counted for colony-forming assays.
For tumor injection, the hair around the tumor injection area of the 6-10 week old mice or adoptive transfer recipients was removed before injection. For
Single cell RNA-Sequencing was performed using Chromium™ Single Cell 3′ v2 Reagent Kits (10× Genomics) following the manufacturer's protocol (Zheng et al., 2017). Briefly, after sort collection, cells were resuspended in PBS at concentration ranging between 400 to 600 cells per μ1. Between 5,000 to 10,000 cells were loaded for gel bead-in-emulsion generation and barcoding. To increase barcode diversity, samples were split in 2 technical replicates for all downstream steps: Reverse transcription, cDNA amplification, fragmentation and library preparation. Final libraries with size ranging between 200 to 1000 bp were size-selected using AMPure™ XP beads (Beckman Coulter). Quality and quantity of samples was controlled at multiple steps during the procedure by running small fraction (<5%) of sample on BioAnalyzer™ (high sensitivity DNA chip, Agilent). Libraries were sequenced on HiSeq2500 platform to obtain 26 (read1)×100 (read2) paired-end reads.
Using Cell Ranger v1.3.0 (10× genomics), reads were aligned on the mm10 reference genome for mouse and hg19 reference genome for human and unique molecular identifier gene expression profiles were generated for every single cell reaching standard sequencing quality threshold (default parameters). On average we obtained data for 2868 cells for mouse samples and 518 cells for human samples, and on average 46,477 reads per cell for mouse and 274,080 reads per cell for human. Only confidently mapped, non-PCR duplicates with valid barcodes and UMIs were used to generate a gene-barcode matrix for further analysis. Counts were normalized to get counts per million (CPM). Unbiased clustering of single cells was performed using Seurat (version 1.4) (R Development Core Team, 2016; Satija et al., 2015). Principal Component Analysis (PCA) was performed using a set of top variable genes (ranging between 647 to 2142 genes) and then dimensionality reduction was performed using t-SNE algorithm with top 10 to 18 PCAs. For FIG. 2A, tSNE 2D plots were obtained applying Seurat scRNA-Seq analysis R Package (using 12 first PCA, and 810 most variable genes with resolution parameter set at 0.03).
RNA purity and quantity was measured with a Nanodrop™ spectrophotometer (Thermo Scientific). Approximately 100 ng RNA was used for synthesis of cDNA with an Iscript™ cDNA Synthesis Kit (Bio-Rad). Total cDNA was diluted 1:20 in H2O, and a volume of 9 μl was used for each real-time condition with a MyIQ™ Single-Color Real-Time PCR Detection System (Bio-Rad) and TaqMan® Gene Expression Mastermix and TaqMan primers (Life Technologies). Data were analyzed and presented on the basis of the relative expression method. β-actin was used as ‘housekeeping’ gene for data normalization.
Data for all experiments were analyzed with Prism™ software (GraphPad). Unpaired t-tests and two-way analysis of variance were used for comparison of experimental groups. P values of *P<0.05, **P<0.01 were considered to indicate statistical significance. The data appeared to be normally distributed with similar standard deviation and error observed between and within experimental groups. No statistical methods were used to predetermine sample size. No animal or sample was excluded from the analysis.
In this example, the inventors demonstrate that the neutrophil progenitor cell population of the present disclosure can be extracted from a biological sample, in particular a mouse bone marrow (BM) sample.
Using mass cytometry, the inventors developed an antibody panel shown in
To identify neutrophil progenitors, the inventors focused efforts on further analysis of Cluster #C. Using Phenograph, a second unbiased clustering algorithm (Chen et al., 2016; Levine et al., 2015), it was found that Cluster #C consists of two major populations that display a continuum of Ly6G, Ly6C, and Ly6B expression (
In this example, the inventors used scRNA Seq analysis of Cluster #C to reveal two major subpopulations (#C1 and #C2).
The inventors further investigated Cluster #C by sorting Cluster #C from mouse BM for scRNA-Seq analysis using the gating strategy in
Next, a flow cytometry panel shown in
In this example, the inventors show that Cluster #C1 cells are unipotent neutrophil progenitors in vitro.
Comparison of #C1 and #C2 showed a gradient of Ly6G expression from negative in #C1 to intermediate in #C2 to high in mature BM Neuts (
The selective neutrophil potency of #C1 cells was first tested by examining in vitro methylcellulose colony-forming unit formation (
In this example, the inventors describe a functional analysis of the progenitor cell population of the present disclosure, showing the Cluster #C1 is the early-stage committed unipotent neutrophil progenitor (NeP) in vivo.
The function of #C1 in generating neutrophils in vivo was analyzed using adoptive transfer approaches. The experimental scheme is shown in
Donor-derived neutrophils appeared in recipient blood at Day 5 and Day 7 post-adoptive transfer in the groups reconstituted with #C1 and #C2, showing neutrophil potency in both populations and slower kinetics of the #C1 cells in producing neutrophils (
Neutrophil production peaks at day 14 in #C2 recipients but at day 28, neutrophils vanished from the #C2 recipients, showing limited developmental potency of #C2 (
Taken together, by using high dimensional mass cytometry and scRNA-Seq the inventors have discovered an early-stage committed neutrophil progenitor (#C1, termed NeP) in mouse bone marrow. This progenitor can be identified as Lin− CD117+Ly6A/E− Siglec F− FcERIα− CD16/32+ Ly6B+CD11a+ CD162lo CD48lo Ly6C10 CD115−Ly6G−.
In this example, the inventors further describe a functional analysis of the progenitor cell population of the present disclosure in the context of tumor growth.
Granulopoiesis is often associated with cancer (3). The inventors examined whether #C1 NeP progenitor cells were increased in the bone marrow and periphery of mice using a melanoma tumor model. B16F10 tumor cells SubQ were injected into the rear flank of wild-type C57BL/6J mice (Tumor). Age-matched, gender-matched wild-type mice received D-PBS to serve as healthy controls (Healthy). At 14 days post-injection, tissues were harvested for flow cytometry analysis. The inventors found an expansion of #C1 NeP progenitor cells, but not #E or #B (CD115+) cells, in the bone marrow of tumor-bearing mice (
To test whether NePs can contribute to tumor growth, #C1 NeP cells, #B (CD115+) cells, and #E cells were sorted from CD45.2 wild-type donor mice and adoptively transferred into irradiated CD45.1 recipient healthy mice. At day 1 after donor cell transfer, recipient mice were injected SubQ with B16F10 tumor cells into the rear flank. Tumor size was measured at day 12 after injection (
In further studies to confirm that NePs can be recruited directly to the tumor, NePs were sorted from CD45.2 wild-type donor mice and adoptively transferred into irradiated CD45.1/2 recipient healthy mice. At day 1 after donor NeP transfer, recipient mice were injected SubQ with B16F10 tumor cells into the rear flank. At day 8, the blood and early tumor were harvested for analysis (
To directly investigate the role of NePs in driving tumor progression, the inventors performed a series of extended adoptive transfers in a cancer model in vivo (
In this example, the inventors show the discovery of a heterogeneous hCD66b+hCD117+hCD38+hCD34+/− progenitor-like cell fraction in human bone marrow.
The inventors next analyzed healthy human bone marrow. Human CMP and GMP express hCD34, hCD38, and hCD117 and mirror the murine CMP/GMP paradigm in myeloid cell production (Doulatov et al., 2010; Edvardsson et al., 2006; Manz et al., 2002). CD66b is considered a marker of mature myeloid cells and, as such, is often excluded from flow cytometry panels geared towards hematopoietic progenitors. However, as this is an important marker for neutrophil identification, this marker was retained in the search for the early neutrophil progenitor in human bone marrow. Indeed, the inventors discovered that human bone marrow contains a heterogenous hCD66b+ population that expresses either CD34+ or CD117+ (
In this example, the inventors show both hCD66b+hCD117+hCD38+ subsets produce only neutrophils in NSG-SGM3 (NSG-M3) mice.
The inventors examined the neutrophil potency of these human neutrophil progenitor candidates (hCD34+ Subset A and hCD34− Subset B) in vivo by performing adoptive transfers of each subset into NSG-SGM3 (NSG-M3) mice. The triple transgenic NSG-M3 mice are immunodeficient NOD scid gamma (NSG™) mice that express the human cytokines Interleukin 3 (IL-3), granulocyte/macrophage-stimulating factor (GM-CSF) and SCF, also known as KITLG. This mouse model supports stable engraftment of human myeloid lineages. The two subsets were isolated from fresh human bone marrow by FACS using the sorting panel in
In this example, the inventors show hNeP increase in melanoma patient blood and promote early osteosarcoma tumor growth in humanized NSG-M3 mice.
The inventors examined whether hNeP played a role in tumorigenesis by examining osteosarcoma growth in NSG-M3 mice. Osteosarcoma is the most common type of cancer and is an important solid tumor target for immunotherapy (Anderson, 2017). Shown in
Finally, blood from human subjects with melanoma was analyzed for the presence of hNeP. Blood specimens collected from patients prior to treatment who were diagnosed with melanoma were used. Flow cytometry analysis of healthy donor blood as well as melanoma patient blood using the panel in
Other examples of implementations will become apparent to the reader in view of the teachings of the present description and as such, will not be further described here.
Note that titles or subtitles may be used throughout the present disclosure for convenience of a reader, but in no way should these limit the scope of the invention. Moreover, certain theories may be proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the present disclosure without regard for any particular theory or scheme of action.
All references cited throughout the specification are hereby incorporated by reference in their entirety for all purposes.
It will be understood by those of skill in the art that throughout the present specification, the term “a” used before a term encompasses embodiments containing one or more to what the term refers. It will also be understood by those of skill in the art that throughout the present specification, the term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, un-recited elements or method steps.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used in the present disclosure, the terms “around”, “about” or “approximately” shall generally mean within the error margin generally accepted in the art. Hence, numerical quantities given herein generally include such error margin such that the terms “around”, “about” or “approximately” can be inferred if not expressly stated.
With respect to ranges of values, the invention encompasses the upper and lower limits and each intervening value between the upper and lower limits of the range to at least a tenth of the upper and lower limit's unit, unless the context clearly indicates otherwise. Further, the invention encompasses any other stated intervening values.
Although various embodiments of the disclosure have been described and illustrated, it will be apparent to those skilled in the art in light of the present description that numerous modifications and variations can be made. The scope of the invention is defined more particularly in the appended claims.
The present application claims the benefit of U.S. provisional patent application Ser. No. 62/483,305 filed on Apr. 7, 2017 by Catherine Hedrick. The contents of the above-referenced document are incorporated herein by reference in their entirety.
This invention was made with government support under grant numbers R01HL134236, P01HL136275, R01CA202987, 1S100D018499-01 and ADA7-12-MN-31(04) awarded by the National Institute of Health. The U.S. Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/026613 | 4/6/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62483305 | Apr 2017 | US |
