Patent Application
20240375114
The present invention provides a biological sample holder and handler system for cell-based liquid biopsies. The system is useful for performing diagnostic assays, such as those based on a simple blood sample. For example, the system is useful for delivering precision cancer diagnoses that improve patient outcomes by optimizing treatment options, monitoring therapy efficacy, characterizing metastasis and assessing treatment toxicity.
Traditional solid tissue biopsies are the gold standard for cancer diagnosis. However, these assays are static and require fixation and staining of the samples for histologic analysis by conventional microscopic techniques. This limits sample size and thickness and does not present the opportunity to more readily sample and monitor tumors under dynamic conditions. For ongoing monitoring of disease progression and patient management, liquid biopsies offer a unique advantage because they involve non-invasive, blood-based testing of cell-free DNA (cfDNA) or circulating tumor cells (CTCs). Molecular analysis of cfDNA (including circulating tumor DNA, ctDNA) has shown high sensitivity and specificity for detecting driver mutations, structural rearrangements, copy number aberrations and changes in DNA methylation, therefore providing valuable information for disease monitoring.
However, a driver gene does not kill a patient. Rather, aggressive tumor cells do. CTC analysis permits the study of whole cells, and offers DNA, RNA and protein-based molecular profiling, as well as the opportunity for functional studies that can guide precision therapies.
CTCs have been detected in the blood of cancer patients in frequencies of 1:108 to 1:106 or higher. CTCs are released in the circulation from the primary tumor following a series of biological events also involving epithelial to mesenchymal transition (EMT). Single tumor cells or tumor cell clusters leave the primary tumor site, invade the blood vessels, and travel throughout the body until they leave the blood stream. The cells settle in different tissues, thereby generating the bud of metastasis formation. Detection of CTCs in the blood is challenging, both because of their scarcity and because they can express different phenotypes. These phenotypes include epithelial, mesenchymal and stemness-like CTCs, but with features that can change over time, converting from one state to another, and vice versa. See, Joosse et al (2015). Biology, detection, and clinical implications of circulating tumor cells. EMBO Mol Med 7: 1-11.
One of the early commercial CTC analysis systems was CellSearch® (Silicon Biosystems/Menarini) developed in the mid-2000's. This system is based on epithelial cell adhesion molecule (EpCAM) for CTC enrichment, followed by immunofluorescent staining for cytokeratin (positive CTC characterization), CD45 (which is also known as lymphocyte common antigen) to exclude leukocytes and diamidino-2-phenylindole (DAPI) for nuclear counterstaining. CellSearch validation studies showed the prognostic value of CTCs and led to FDA clearance for monitoring patients with metastatic breast, prostate and colon cancer. See, Hayes D F, et al. Circulating Tumor Cells at Each Follow-up Time Point during Therapy of Metastatic Breast Cancer Patients Predict Progression-Free and Overall Survival Clin Cancer Res 2006; 12(14): 4218-4224; de Bono, J S, et al. Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer. Clin Cancer Res 2008; 14:6302-6309; and Cohen S J, Punt C J A, Lannotti N, et al. J Clin Oncol. 2008; 26(19):3213-3221.
Since the development of early commercial systems, multiple studies and trials with different endpoints have analyzed the clinical utility of CellSearch CTC enumeration. It was shown that increased CTC numbers (5 per 7.5 mL of whole blood) are associated with poor prognosis in metastatic breast cancer (MBC). A pooled study of trials conducted between 2003 and 2012 and involving 2000 MBC patients, confirmed the independent prognostic effect of CTC-count on progression-free survival (PFS) and overall survival (OS). It also confirmed that CTC-count improves the prognostication of MBC when added to full clinicopathological predictive models, which cannot be done with serum tumor markers. See, Bidard, FC (2014). Clinical validity of circulating tumor cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol. 15:406-14.
Beyond enumeration, several studies addressed the question of genotypic and phenotypic characterization of CTCs. In metastatic breast cancer (MBC), because there are several drugs that target human epidermal growth factor receptor 2 (HER2) and are beneficial for patient management, testing for HER2 at the DNA, m.RNA and protein levels has been extensively used. In metastatic renal cancer, dynamic changes of live versus. apoptotic CTC subpopulations were shown to be a predictive marker of response to chemotherapy. See, Pestrin et al. (2012) Final results of a multicenter phase II clinical trial evaluating the activity of single-agent lapatinib in patients with HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells. A proof-of-concept study. Breast Cancer Res Treat 134: 283-289. In early breast cancer, several studies including more than 2800 patients, have shown that the detection of CTCs is independently associated with poor prognosis. See, Franken et al (2012). Circulating tumor cells, disease recurrence and survival in newly diagnosed breast cancer Breast Cancer Res, 14:R133.
Liquid biopsies offer non-invasive, precision blood-based testing of circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs). CTC liquid biopsies were shown to have prognostic value in different metastatic cancers [1,2,3], early disease [12] and to aid in management of metastatic cancer [10,13]. Current systems utilize CTC enrichment by antibody capture [4], filtration [5], dielectrophoresis [6] or other methods. Different CTC enrichment approaches impede direct comparison of results between studies while variable CTC phenotypes have different metastatic potential [7]. Quantitative detection and characterization of viable CTCs, may be valuable for the biology of cancer metastasis [8]. Single CTC isolation and whole genome analysis (WGA) is promising for targeted therapy selection and monitoring disease progression [9,11].
Almost every CTC diagnostic system utilized today requires enrichment to increase CTC abundance from less than or equal to about 1:106 in the patient blood to about 1:1000 nucleated cells which makes microscopic examination feasible. Without an enrichment step, it is very difficult to otherwise find and qualitatively or quantitatively assess the CTCs. After enrichment, the specimen is stained for CTC-specific biomarkers by immunostaining or in situ hybridization. Enrichment methods either utilize a cell surface marker (such as EpCAM) for antibody-based CTC capture or some physical separation method such as filtration. Either approach is making selective assumptions that lead to incomplete detection of all CTCs. Since CTCs are known to contribute to metastasis formation, it is important to identify and characterize them as accurately as possible.
CTC detection, including live characterization and characterization without enrichment, and using biomarkers for multiple phenotypes, will open the way for a standardized CTC definition and benefit precision cancer diagnosis.
For the present invention we have developed an automated microscope that offers deep quantitation of all cells in a blood or other body fluid specimen using fluorescence Selective Plane Illumination Microscopy (SPIM) [14]. SPIM microscopy uses laser-sheet illumination which can offer resolution comparable to confocal microscopy with much less phototoxicity. After staining for multiple biomarkers, cells are mixed in a hydrogel solution (e.g. agarose), aspirated and allowed to immobilize in a transparent tube (e.g., a FEP tube). By means of a fixture, they are placed in the aqueous-solution filled chamber which is part of the SPIM optical path. Three-dimensional cell images are acquired in multiple fluorescent channels and every cell in the specimen, e.g. white blood cells (WBCs), are analyzed for the detection of target cells (e.g. CTCs). The system can observe cells while perfused with substances contained in the surrounding aqueous solution. In analytical validation experiments, the system is able to detect cancer cells at a frequency of <1:500.000 blood cells.
Therefore, it is seen that the state of the art is limited either by conventional solid tissue biopsy methods or by CTC analysis systems that require a cumbersome enrichment step which can add error to the methodology. The present invention, which utilizes a new application of selective plane illumination microscopy (SPIM) provides a rapid analysis system for characterizing and quantifying CTCs under more realistic biological conditions without the need for an enrichment step, and therefore provides a useful alternative to conventional solid tissue biopsy methods. The present invention is centered on a cell sample holder and handler system for use with SPIM.
The present invention provides a biological sample holder and handler system for cell-based liquid biopsies. The system is useful for performing diagnostic assays, based on a simple blood sample. For example, the system is useful for delivering precision cancer diagnoses that to improve patient outcomes by optimizing treatment options, monitoring therapy efficacy, characterizing metastasis and assessing treatment toxicity.
The present invention relates to an apparatus for characterizing or quantitating particles in a biological sample comprising:
In further embodiments the present invention relates to an apparatus wherein the capillary tube comprises a plurality of pores or holes.
In further embodiments the present invention relates an apparatus wherein the capillary tube has an inner bore diameter of from about 0.5 mm to about 10 mm.
In further embodiments the present invention relates to an apparatus wherein the capillary tube has an inner bore diameter of about 1 mm.
In further embodiments the present invention relates to an apparatus wherein the plurality of pores or holes of the capillary tube each have a diameter from about 0.002 mm to about 0.05 mm.
In further embodiments the present invention relates to an apparatus wherein the illumination or excitation source is a light sheet source.
In further embodiments the present invention relates to an apparatus wherein the light sheet source is a laser light sheet source.
In further embodiments the present invention relates to an apparatus wherein the observation means is a microscope.
In further embodiments the present invention relates to an apparatus wherein the microscope is a microscope for performing selective plane illumination microscopy (SPIIM).
In further embodiments the present invention relates to an apparatus wherein the observation means is a digital camera, a UV/visible spectrophotometer, or a raman spectrophotometer.
In further embodiments the present invention relates to an apparatus for characterizing or quantitating particles in and further manipulating and isolating particles from a biological sample comprising:
In further embodiments the present invention relates to an apparatus wherein the (e) means for moving and positioning the sample is a syringe.
In further embodiments the present invention relates to an apparatus wherein the syringe of (e) is capable of being operated, moved, and rotated by a motorized device.
In further embodiments the present invention relates to an apparatus wherein the (f) means for removing the particles is a micropipette.
In further embodiments the present invention relates to an apparatus wherein the micropipette of (f) is capable of being operated, moved, and rotated by a motorized device.
In further embodiments the present invention relates to an apparatus wherein the biological sample is selected from bodily fluids such as for example, blood, urine, semen, saliva, amniotic fluid, spinal fluid, and seminal fluid.
In further embodiments the present invention relates to an apparatus wherein the biological sample is blood.
In further embodiments the present invention relates to an apparatus wherein the particles are selected from circulating tumor cells (CTCs), blood cells, rare immune cells (such as for the diagnosis of autoimmune disease), and pathogenic cells.
In further embodiments the present invention relates to an apparatus wherein the particles are circulating tumor cells (CTCs).
In further embodiments the present invention relates to an apparatus for characterizing or quantitating particles in a biological sample comprising the following components as illustrated in any of
glass syringe, glass capillary tube, a prepared sample in agarose gel, fluid output port, sample chamber and O-ring, illumination and detection lenses, lens holder, fluid manifold, micropipette, manipulator for the micropipette, and fluid connectors (input and output), wherein the capillary tube (for example a glass capillary tube) comprises one or more holes (for example micro laser drilled holes) to enable the introduction of reagents and washing steps to and from the capillary tube.
In further embodiments the present invention relates to a method for characterizing and quantitating target cells in a blood sample utilizing the apparatus of the present invention comprising the steps of:
In further embodiments the present invention relates to a method comprising the further step (g) of collecting one or more target cells.
In further embodiments the present invention relates to a method for determining or diagnosing a disease state in a subject utilizing the apparatus of the present invention comprising steps of:
In further embodiments the present invention relates to a method for determining or diagnosing a disease state comprising the further step (g) of collecting one or more target cells.
In further embodiments the present invention relates to a method for determining or diagnosing, and further treating a disease state in a subject utilizing the apparatus of the present invention comprising the steps of:
In further embodiments the present invention relates to a method for determining or diagnosing, and further treating a disease state comprising the further step (h) of collecting one or more target cells.
In further embodiments the present invention relates to such methods wherein the subject is a human subject.
In further embodiments the present invention relates to such methods wherein the subject is an animal subject, including mammals such as mice, rats, dogs, and other mammals used in cancer research.
In further embodiments the present invention relates to a method wherein the disease state is cancer.
In further embodiments the present invention relates to a method wherein the selected target cells are circulating tumor cells (CTCs).
In further embodiments the present invention relates to a method wherein the CTCs are characterized.
In further embodiments the present invention relates to a method wherein the CTCs are quantitated.
In further embodiments the present invention relates to use of the apparatus of the present invention in the manufacture of a medicament for characterizing and quantitating selected target cells in a biological sample.
In further embodiments the present invention relates to a method which does not require enrichment or concentration of the sample for the target cells.
In further embodiments the present invention relates to a method wherein the sample comprises about 1 or less target cells per about 1×106 total cells (i.e., total nucleated cells) in the sample.
In further embodiments the present invention relates to 1 method wherein the sample comprises about 1 or less target cells per about 1×105 total cells (i.e, total nucleated cells) in the sample.
In further embodiments the present invention relates to a method wherein the sample comprises about 1 or less target cells per about 1×104 total cells (i.e., total nucleated cells) in the sample.
In further embodiments the present invention relates to a method wherein the sample comprises about 1 or less target cells per about 1×103 total cells (i.e., total nucleated cells) in the sample.
These and other embodiments of the present invention will become apparent from the disclosure herein.
The skilled artisan will understand that the drawings primarily are for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily to scale; in some instances, various aspects of the inventive subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of different features. In the drawings, like reference characters generally refer to like features (e.g., functionally similar and/or structurally similar elements).
The biological sample holder and handler system of the present invention comprises several components and utilizes advanced characterization and quantitation techniques to provide high resolution, accuracy, and sensitivity.
Fluorescence light sheet microscopy (FLSM) is a fluorescence microscopy technique in which a sample is illuminated by a laser light sheet (i.e. a laser beam which is focused in only one direction) perpendicularly (i.e. orthogonally or 90 degrees to the direction of observation. The light sheet can be created using e.g. cylindrical lens or by a circular beam scanned in one direction to create the light sheet. As has been reported, only the actually observed section of a sample is illuminated. Therefore, this method is reported to reduce the photodamage and stress induced on a living sample. Also, it has been reported that the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Furthermore, selective plane illumination microscopy (SPIM) and fluorescence microscopy techniques in which a focused sheet of light serves to illuminate the sample have become increasingly popular in developmental studies. Fluorescence light-sheet microscopy bridges the gap in image quality between epifluorescence microscopy and high-resolution imaging of fixed tissue sections. In addition, high depth penetration, low bleaching and high acquisition speeds make light-sheet microscopy ideally suited for extended time-lapse experiments. See, Huisken et al (2009). Selective plane illumination microscopy techniques in developmental biology. Development 136, 1963-1975 doi:10.1242/dev.022426. FLSM systems can be purchased from various companies such as Zeiss, Leica, or Olympus. These systems can also be built for research purposes following designs offered through open source groups such as OpenSPIM or SPIM-fluid.
See, http://openspim.org/Welcome_to_the_OpenSPIM_Wiki; and https://doi.org/10.1364/BOE.6.004447.
The system and method of the present invention can accurately detect epithelial, mesenchymal, or stemness-like CTCs, including intermediate phenotypes, because it is designed to quantitatively detect multiple CTC biomarkers. It provides fully automated CTC detection in patient blood samples for clinical diagnosis, academic research and drug development. The present system does not require enrichment because its high-resolution and high-speed microscope can scan and analyze every nucleated cell from the patient sample and deliver very sensitive detection of CTCs or other blood cell subpopulations such as T-cells.
The system has the unique ability to observe live cell preparations in addition to detecting and characterizing CTCs without enrichment. The system enables (i) spatial and temporal characterization of disease progression, and (ii) real-time observation of live CTC phenotypes by ex vivo imaging.
The system of the present invention can include an aqueous solution filled cell observation chamber. This enables observation of either fixed or live cell preparation. The chamber can be equipped with a media recirculation system which enables perfusion of the cells with solutions that can contain: (i) biomarkers such as antibodies or fluorescence in situ hybridization (FISH) probes appropriately labeled for enumeration and quantitation, (ii) substances for staining DNA or other molecules, (iii) agents including therapeutic substances, viral suspensions etc. that can affect the physiology of targeted live cells, (iv) de-staining solutions, and (v) cleaning and de-contamination solutions.
The system's “lossless” CTC detection is applicable to different cancer types. By scanning every nucleated cell from the blood sample and utilizing multiple markers associated with different CTC phenotypes. The system enables detection of epithelial, mesenchymal and stemness-like CTCs. Quantitative imaging of biomarker levels also allows detection of CTCs transitioning between different CTC phenotypes.
Unlike other microscopic methods, the system's high-resolution microscopy has very low phototoxicity (i.e. the light-induced degradation of photosensitive components or in general adverse light-induced effects), which permits multiple imaging sessions of a given specimen, in successive time points.
The system of the present invention includes a cell aspiration device that allows removal of target cells from the specimen (including while live), for further molecular, single-cell testing. CTCs isolated by the system can be used as a tissue source for drug sensitivity testing by utilizing subsequent ex vivo cultures and for the detection of specific mutations in CTC-derived cell lines. In CTC-derived cell lines, cells can be studied for their resistance to specific chemotherapy or targeted therapies or combinations of the above. Drug sensitivity testing can be carried out also in mouse xenograft models. The clinical utility of the CTC models can depend on (i) the percentage of patients in which CTC will be detected and (ii) whether the CTC models can reliably capture response to different drugs. The system and methods of the present invention can aid in combining CTC genomic and transcriptomic analyses together with drug sensitivity testing in CTC-derived cell lines and mouse models; this can provide new insights for driving personalized cancer treatment.
The holder and handler of the present invention is a system that allows ex vivo observation of cells that have been stained with vital stains for CTC-specific biomarkers and maintained alive for periods of time supported by a 3-dimensional culture subsystem. A specially designed cell chamber will be fitted for input and output of culture media, gas regulation and control of environmental variables (temperature, pH etc). This will allow ex vivo observation of cells while perfused with culture media which may contain various substances. The chamber will be fitted with a micromanipulator (handler) used to isolate target cells under direct observation. Both the chamber and the micromanipulator can be operated automatically by a system computer and software system.
The ex vivo liquid biopsy will offer longitudinal observation of target cells, e.g. CTCs and WBCs and assessment of desired and undesired toxicity of therapeutic drug cocktails before used for patient treatment. This will drive precision medicine for improved outcomes and reduced adverse effects to the patient. Cell isolation will enable CTC genomic and transcriptomic analysis that may reveal improved therapeutic options, tuned to the patient's current disease status.
The sample holder and handler of the present invention, combined with deep quantitation of every cell the specimen, have the potential to become an important precision medicine tool. Deep CTC characterization and single-cell, genomic/transcriptomic analysis will enable the oncologist to select a treatment that is synchronized with the current disease stage. Ex vivo assessment of how a selected drug or drug combination affects CTCs and WBCs in the patient's blood will have to be studied against patient outcomes. However, it has the potential to revolutionize therapy selection and longitudinally, help in turning cancer from a devastating to a chronic disease.
A central computer system (not shown) operates a software package that (a) acquires and processes images of the biological specimen's features for identification and quantitation, (b) actuates the motorized components, pumps, sensors of the system, (c) operates a robotic arm that loads and unloads samples, and (d) handles digital information managed in local or wide area networks. The central computer system may utilize local or distributed processing protocols.
The system also includes or is coupled to a tunable laser source or multiple single wavelength laser sources, complete with light management optical path(s). An optical system modulating the light sheet can combine bilateral illumination to produce the sheet illumination for SPIM.
Imaging is done by illuminating the specimen with narrow spectrum excitation light provided by monochromatic and/or tunable laser sources. Images of the resulting emission are acquired by high sensitivity monochrome cameras on a field by field basis. These images are combined in 3-dimensional stacks, which are then analyzed for quantitative measurement of biomarker levels in the individual cells.
In operation, a biological specimen that can include live cells is stained with a variety of markers against proteins, nucleic acids or other cellular components and encased in an appropriately shaped cylindrical sheath to be fitted on a biological sample holder. The preparation is made by mixing the cell suspension with agarose or other hydrogels compatible with preserving the subcellular structure of the embedded cells, at a temperature where the solution is still liquid. In addition to the cells, fluorescent beads that serve the role of fiducial reference for the identified cells are added to the solution. The liquid cell/bead/gel suspension is aspirated in tubing that is chosen to be transparent to the fluorescence light regime utilized. After being allowed to solidify, the specimen can be visualized in the light path. The biological specimen is mounted on a specimen holder loaded onto the microscope stage.
All combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are part of the inventive subject matter disclosed herein. The terminology used herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.
A micropipette 9 for extracting cells or particles of interest and an access port for the micropipette 9A on the fluid chamber 5 is shown. It is important that the micropipette can access the sample at the intersection of the optical axes of the light paths from the illumination and detection lenses.
In the foregoing embodiments, it should be noted that the illumination lens 9A and the detection lens 9B, as well as the illumination and detection means, can be swapped around, so long as their orientation is orthogonal. Also, the openings 11A and 11B of the lens holder and the openings 14A and 14B of the sample chamber would also be concurrently swapped in such a situation.
A cell suspension can be observed in SPIM instrument mounted in fixture and embedded in hydrogels that allow cell perfusion with fluorescently labeled antibodies, fluorescence in situ hybridization FISH probes, and other stains as well as media that can sustain ex vivo cell observation.
The following steps are performed: Compare performance of embedding gels including agarose, collagen, polyacrylamide and tubing such as micro-perforated, fluorinated polyethylene (FPE) and glass both for fixed and live cells. Optimize fixation/permeabilization protocols. Assess need of antifading for fluorescence bleaching. Adapt SPIM image acquisition to materials chosen. Quantitative analysis of cell staining and morphology changes via 3d image analysis with QCDx imaging software.
Explore prototype chamber design fitted with computer-controlled, microfluidic media circulation, gas exchange mechanism and environmental sensors. Evaluate vital fluorescence stains for immunostaining and nuclear counterstaining and viability stains. Quantitative morphological changes in target cells to establish acceptable longitudinal ex vivo imaging periods.
The holder and handler has design requirements for the chamber, specimen fixture, tube and embedding gel that will enable longitudinal imaging of cell suspensions, fixed or ex vivo.
The present invention can comprise instruments and kits for the detection and characterization of CTCs and other target cell populations
The following references have been cited above corresponding to the following numbering.
While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
Also, various inventive concepts may be embodied as one or more methods, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity’ such as “either”’ “one of” “only one of” or “exactly one of” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of and “consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
In the specification, the singular forms also include the plural forms, unless the context clearly dictates otherwise. Unless defined otherwise, 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 belongs. In the case of conflict, the present specification will control.
The entire disclosure of each of the patent documents, including certificates of correction, patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls.
The present application is a continuation of U.S. application Ser. No. 17/132,046, filed Dec. 23, 2020, which is a continuation of International Application No. PCT/US19/39244, filed Jun. 26, 2019, which claims the benefit of U.S. Provisional Application No. 62/690,402, filed Jun. 27, 2018, each of which is entirely incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62690402 | Jun 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17132046 | Dec 2020 | US |
| Child | 18395977 | US | |
| Parent | PCT/US19/39244 | Jun 2019 | WO |
| Child | 17132046 | US |
