TWI589877B - Method for selecting a pool of molecules - Google Patents

Description

Method of screening a molecular group

The present invention relates to a method of screening a population of molecules. More specifically, the present invention relates to a method of screening a population of molecules having high selectivity for certain markers of interest.

All biological reactions require intermolecular interactions. The interaction between the antibody and the antigen and between the ligand and the receptor is critical to initiate a series of pathways in response to external stimuli. Identifying the specific molecules involved in the interaction is important for research and pharmaceutical development.

For example, most human expression genes have been identified after the completion of the human genome program. However, in the emerging proteomics era, proteins play an important role in these revealed genes. In order to effectively solve the mystery of these proteins, many efforts have been made to produce at least one antibody against each human expression gene in the human genome. Because of its inherent sensitivity and specificity, antibodies are the most functional tool for studying the proteins of their one-to-one relationship. However, one of the intricacies is that when placed in different cells (such as cancer cells), these proteins do not always behave in the same way. Many proteins are known to have translocational abilities in different cellular environments and, therefore, their corresponding antibodies recognize unique locations in a given cell. Furthermore, in many cases, protein translocations are associated with activation of cancer cells. In the future, detecting these activities may help diagnose the type and stage of cancer. Direct screening of high-throughput antigens on cell membranes Methods may reveal potential therapeutic targets as well as new disease markers.

However, even though many useful antibodies have been generated, there are two main limitations, namely, (1) how to directly detect specific antigens (such as membranes or surface markers) in a given cell, and (2) how quickly Identify useful antibodies that recognize these antigens.

In the case of membrane-bound proteins, many membrane-bound proteins are associated with specific disease states, such as lung cancer, and are often an attractive therapeutic target. Systematic and quantitative analysis of membrane-bound proteins enhances our understanding of the role of biological processes in different disease states. In the most frequently sequenced genomes, it is estimated that about 20 to 30% of the open reading frame encodes a complete membrane-bound protein (Blonder, J., et al., Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography). -tandem mass spectrometry.Journal of Proteome Research,2002.1(4):p.351-360;Han,DK,et al.,Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nature Biotechnology, 2001.19(10): p.946-951). However, membrane-bound protein bodies have not been mapped and are still experimentally challenging due to the small amount of membrane-bound proteins. In addition, using high-throughput proteomics and gene-based wafer-based screening studies, the novel targets identified often lack antibodies to establish their location and function, further preventing researchers from studying potential membrane-bound proteins. Direct performance analysis methods are necessary to identify antibodies and recognize receptors (surface markers or membrane-bound proteins).

Random screening is time consuming and often not feasible due to differences in physiological conditions. It is also not possible to use a single antibody to screen for a new receptor, as it must have more than 25,000 antibodies to apply to the entire genome. In addition, cancer cells often have multiple over-expressed receptors and the same target protein may have different positions in different cell types. Therefore, it is necessary to develop a fast and efficient method to solve these problems at the same time.

The present invention provides a small population performance screening to screen for specifics of interest Biomarkers are one of a group of molecules that are highly selective.

The present invention provides a method of screening a population of molecules comprising detecting whether the population of molecules has binding specificity for a medium.

The invention further provides a method of screening a biomarker population in a cell or on a cell, comprising the method described above, wherein the population of molecules is the biomarker population.

The invention further provides a composition comprising a population of molecules selected by the above methods.

The invention further provides a method of delivering a therapeutic agent comprising administering a composition of the above to a cell or a body.

The invention further provides a method of diagnosing a condition in a body, comprising: providing a biological sample; contacting the composition with the biological sample; and identifying whether the composition is associated with the biological sample, wherein the binding exists This means that the individual has this condition.

The invention will be described in detail in the following paragraphs, and other features, objects, and advantages of the invention will be apparent from the following description and claims.

Figure 1: Schematic representation of the adsorption of antibody-targeted cancer cells by LPPC (liposome/PEI/PEG complex). The antibody was mixed with DiO-labeled LPPC and then blocked with PEG 1500. The PEG-blocking LPPC/antibody complex is incubated with cells (such as A549) and then analyzed by FACScan flow cytometry to determine its potency for binding to the cell surface. The fluorescent mean of each sample was normalized to a positive control group (100% DiO-labeled LPPC on cells) and a negative control group (unbound antibody). The fluorescent mean of each sample was first divided by the fluorescent mean of the positive control, which was then divided by the fluorescent mean of the negative control.

Figure 2: Schematic representation of antibody-based liposomes for drug screening. A total of 450 antibodies were divided into 50 populations for use in the formation of liposome/antibody (LPPC/antibody) complexes. To screen for the potency of antibody binding to the cell surface, 50 LPPC/antibody complexes were incubated with different cancer cell lines at 4 °C. LPPC/antibody with high binding efficiency to cell surface The complex was preferentially analyzed by a FACScan flow cytometer. Individual antibodies from selected populations (eg, Group 3A) were then incubated with LPPCs and subsequently analyzed by FACScan to determine their potency for binding to the cell surface. Immunofluorescence is used as a secondary screening to determine the subcellular location of the selected target.

Figure 3: Screening of liposome-regulated antibody populations to identify specific receptors in different cancer cell lines. Fifty individual antibody populations were mixed with DiO-labeled LPPCs, then incubated with three different cell lines (A549, HT29 and MCF7) and analyzed by FACScan flow cytometry. Five populations (2G, 2I, 3A, 3B, and Y1C populations) exhibited better binding potency than other antibody populations. These populations were again divided into individual antibodies to test their binding potency to LPPC, as shown in Figures 4 to 7. The "cell" means a single cell that is not stained by DiO, and "NC Ab" means an unbound antibody on the LPPC.

Figure 4: Confirmation of subcellular localization of preferential proteins in the 3B population. (A) Individual antibodies in the 3B population were tested with three different cancer cells using the LPPC/antibody complex system. Many of these selected antibodies were further confirmed for their subcellular localization by immunofluorescence. (B) 3B-4 (MAPK8, red) is located on the cell membrane of HT29 cells (colon cancer cells), but not in A549 cells (lung cancer cells).

Figure 5: Confirmation of subcellular localization of preferential proteins in the 2G population. (A) Individual antibodies in the 2G population were tested with three different cancer cells using the LPPC/antibody complex system. Many of these selected antibodies were further confirmed for their subcellular localization by immunofluorescence. (B) 2G-4 (NU98, red) is located on the cell membrane of A549 cells, and 2G-7 (MAG1A9, red) is located on the cell membrane of A549 and HT29 cells.

Figure 6: Confirmation of subcellular localization of preferential proteins in the 2I population. (A) Individual antibodies in the 2I population were tested with three different cancer cells using the LPPC/antibody complex system. Two of these selected antibodies (2I-3 and 2I-6) were further confirmed for their subcellular localization by immunofluorescence. (B) 2I-3 (PIK3R4, red) and (C) 2I-6 (NPR2, red) are located on the cell membrane of MCF7 cells.

Figure 7: Confirmation of subcellular localization of preferential proteins in the Y1C population. (A) Individual antibodies in the Y1C test population were tested in six different cancer cells using the LPPC/antibody complex system. Many of these selected antibodies were further confirmed for their subcellular localization by immunofluorescence. (B) Y1C-4 (SPAG5, red) is located on the cell membrane of Mahlavu (hepatocellular carcinoma cells) and MCF7 (breast cancer cell line), but in A549 cells (lung cancer cell lines), hepatocytes and Huh7 (hepatocyte cancer cells) Not in the middle. (C) Y1C-5 (POLR2A, red) is located on the cell membrane of A549 cells.

The present invention provides a method of screening a population of molecules comprising detecting whether the population of molecules has binding specificity for a medium.

The term "molecular" as used herein refers to a small molecule or macromolecule involved in a biochemical reaction. Preferably, the molecule is a large molecule such as a protein, peptide, nucleic acid, oligonucleotide or polynucleotide. The molecule can be natural or artificial. In another aspect, the molecule can be purified or mixed with other ingredients. In a preferred embodiment of the invention, the molecule has a different manifestation in a normal state and an abnormal state (e.g., a disease). In another preferred embodiment of the invention, the molecule has different expression patterns in different cell types. In still another preferred embodiment of the invention, the molecule is an antibody, antigen, enzyme, matrix, ligand, receptor, membrane-bound protein or cell surface marker. In a further preferred embodiment of the invention, the molecule is an antibody.

The term "one molecule group" in the present invention means a group of molecules in which the molecules may be the same or different.

The term "medium" in the context of the present invention means a small molecule or macromolecule involved in a biochemical reaction. Preferably, the medium is a large molecule such as a protein, peptide, nucleic acid, oligonucleotide or polynucleotide. The medium can be natural or artificial. In another aspect, the medium can be purified or mixed with other ingredients. Preferably, the medium is located within the cell or on the cell. In a preferred embodiment of the invention, the molecule is in a normal state and an abnormal state There are different manifestations under the state (such as a disease). In another preferred embodiment of the invention, the molecule has different expression patterns in different cell types. In still another preferred embodiment of the invention, the molecule is an antibody, antigen, enzyme, matrix, ligand, receptor, membrane-bound protein or cell surface marker. In a further preferred embodiment of the invention, the molecule is an antigen.

The term "binding specificity" in the present invention means a property that a molecule having only a specific shape complementary to the active region can be bound to the active region of the medium even if a different molecule is present.

For ease of handling, the population of molecules is preferably located on a carrier. The manner in which the molecule is localized can occur naturally or artificially bind the population of molecules to the carrier. Depending on the type of the carrier and the molecule, one of ordinary skill in the art to which the invention pertains may choose a suitable manner to achieve the combination. For example, U.S. Patent No. 7,880,882 discloses a cationic immunoliposome or polymer complex for the preparation of an antibody or an antibody fragment. This disclosure is hereby incorporated by reference. In one embodiment of the invention, the carrier is a liposome, a micelle, a timed release capsule, a vesicle, a microsphere, a nanoparticle, a polyplex or a cell; preferably a liposome.

Most currently available lipid systems are prepared by covalently conjugating a target molecule to a lipid component, such as a cholesterol or polymer modified lipid side chain. The coupling reaction may violently impair the activity of certain target molecules (Nobs, L., et al., Current methods for attaching targeting ligands to liposomes and nanoparticles. Journal of Pharmaceutical Sciences, 2004. 93 (8): p. 1980-1992; Kocbek, P., et al., Targeting cancer cells using PLGA nanoparticles surface modified with monoclonal antibody. Journal of Controlled Release, 2007. 120 (1-2): p. 18-26). One way to avoid this problem is to use non-covalently adhering the target molecule to the cationic liposome. However, another problem with the dissociation of the target molecule from the liposome is the activity of the liposome (Nobs, L., et al.). This is mainly due to the weak interaction between the target molecule and the liposome. At present, a swollen liposome vector, LPPC (Liposome/PEI/PEG complex), has been developed, which not only can conveniently load anti-tumor drugs, but also strongly adsorbs tumor-specific antibodies on its surface, so that the particles can be For this cancer cell (Liu, YK, et al., A unique and potent protein binding nature of liposomes containing polyethylenimine and polyethylene glycol: a nondisplaceable property. Biotechnology and Bioengineering, 2011. 108 (6): p. 1318-1327). In addition, the LPPC can also be isolated by centrifugation, allowing the LPPC/antibody complex to be readily purified from unbound antibodies. Empty LPPC can be readily combined with fluorescent dyes to form fluorescent nanoparticles, which provide the potential to develop specific probes when adsorbed to specific antibodies.

As used herein, the cell is preferably a normal cell, a cancer cell, a stem cell or a cancer stem cell. The specific binding between the molecule and the medium preferably occurs in cells under physiological conditions or in vivo.

For ease of operation, the carrier preferably includes a detectable label. Depending on the type of carrier or the detectable label, one of ordinary skill in the art may select a suitable means to mark the carrier to detectable indicia. In a preferred embodiment of the invention, the detectable label is a fluorescent or radioisotope.

To apply the method, in accordance with other aspects of the invention, such as drug delivery, the vector preferably further comprises at least one drug, cytotoxic drug, growth factor, cytokine, vaccine, and oligonucleotide.

Preferably, the method of the invention further comprises identifying the molecule. Those of ordinary skill in the art to which the invention pertains may apply appropriate chemical, physical or biological analysis to identify the molecule.

In a preferred embodiment of the present invention, the method comprises the steps of: (a) providing a candidate molecular group; (b) dividing the candidate molecular group into a plurality of subgroups; (c) loading each subgroup on a carrier to form a complex; and (d) contacting the complex of step (c) with a medium to detect whether the subgroup comprises binding specificity to the medium The molecule is used to screen out the molecular group.

The term "candidate molecule" as used herein refers to a group of molecules suspected to be the molecule sought by the present invention. The candidate population can be a natural extraction or an artificial combination, such as a fusion tumor cell population that can express one of the antibodies.

The contact state of the step (d) is preferably equivalent to the physiological state. Those of ordinary skill in the art to which the present invention pertains may select a suitable state.

In a preferred embodiment of the invention, the method further comprises, after step (b), separating the subgroup into further subgroups. This step and steps (b) and (c) and (d) can be repeated to reduce the number of molecules in the population.

Preferably, step (d) further comprises contacting the complex of step (c) with a cell to detect whether the complex binds to the cell, wherein the medium is located within the cell or on the cell. As used herein, the cell is preferably a normal cell, a cancer cell, a stem cell or a cancer stem cell. The specific binding between the molecule and the medium preferably occurs in cells under physiological conditions or in vivo.

Preferably, the step (d) further comprises contacting the complex of the step (c) with a cell to detect whether the complex poisons the cell, wherein the medium is located in the cell or on the cell. As used herein, the cell is preferably a cancer cell.

In accordance with the present invention, this small group concept is provided to identify "mixed-pool antibodies" to reveal new receptors that are overexpressed in a given cell, such as a cancer cell or stem cell. The underlying concept is to subdivide all antibody repertoires into smaller populations to increase the likelihood of detecting potential receptors (surface markers or membrane-bound proteins) in a given cancer cell type. When a candidate population antibody is identified, this method allows for easier and faster separation of a single antibody; in addition, unlike other proteomics and microarray-based screening studies, those identified are often lacking to demonstrate their Position on the cell membrane The necessary antibodies, the methods of the invention recognize novel antibody recognition receptors and are immediately applicable to a variety of applications.

The invention also provides a method of screening a biomarker population in a cell or on a cell, comprising the method of any of the above, wherein the biomarker is a tumor-specific antigen, a membrane-bound protein or a cell surface marker.

The invention also provides a composition comprising a population of molecules, wherein the population of molecules is screened by a method as described above.

The invention also provides a method of delivering a therapeutic agent comprising administering a composition as described above to a cell or a body.

The invention also provides a method of diagnosing a condition in a body, comprising: providing a biological sample; contacting the composition with the biological sample; and identifying whether the composition is associated with the biological sample, wherein the binding Existence means that the individual has this condition.

Preferably, the biological sample is a cancer cell, a cancer stem cell, a tumor slice or a cultured tissue.

Preferably, the system is a human.

The method of the invention has many potential applications. In one embodiment of the invention, the screening is to identify differences in the expression of disease-associated receptors by comparing normal cells with diseased cells, conjugated to one or more antibodies via the liposome, the tumor cells can receive and normal tissue Another therapeutic strategy is provided compared to one of the relatively high doses of the drug. The identified receptor can be used for drug delivery, for example, through liposome delivery.

In another embodiment of the invention, the identification of specific cell surface antigens can be performed under different conditions. For example, this method can be used to identify surface markers in stem cell studies such as mesenchymal stem cells (MSCs), neural stem cells (NSCs), pluripotent stem cells, and cancer stem cells, and to identify cell type-specific differentiation markers. The single or multiple identified surface markers can be used to identify and isolate cells of interest (or subgroups) from a different population of cells. In other words, the identified cell surface marker can be used Cells are analyzed and sorted by means of flow cytometry.

In yet another embodiment of the invention, the method rapidly recognizes potential receptors (surface markers or membrane-bound proteins) from a large collection of single or multiple antibodies produced by a peptide or bacteriophage. In general, there is a lack of a systematic analytical method to identify potential receptors from these antibody stocks. Using this basic small antibody population screening, thousands of antibodies can be screened in a short period of time.

The invention is illustrated by the following examples, which are not intended to limit the invention.

Example method Incubate cells with LPPC/antibody complex

In this example, LPPC (lipid/PEI/PEG complex) was labeled with green fluorescent dye DiO for 30 minutes. Next, 20 μg of Dio-labeled LPPC and 1 μg of the antibody group containing 9 antibodies were incubated for 30 minutes. The LPPC/antibody complex was then blocked with PEG 1500 for 30 minutes and centrifuged at 5,900 xg for 5 minutes to remove excess PEG. Finally, blocking of PEG- LPPC / antibody complex with 3 × 10 ⁵ cells were incubated for 30 minutes to an antigen on the cell surface binding. Binding potency was measured using a FACScan flow cytometer. The mean fluorescence of each sample was normalized to a positive control group (100% DiO-labeled LPPC on cells) and a negative control group (non-binding antibody). The fluorescence average of each sample was first divided by the fluorescent mean of the positive control group, and then the normalized value was divided by the fluorescent mean of the negative control group; the threshold value of 3 times was classified as a positive result.

Immunofluorescence

To characterize the localization of the selected protein, cells are stained by immunofluorescence using antibodies. The cells were washed with phosphate buffered saline (PBS) and fixed with 3.7% in formaldehyde in PBS at 25 ° C for 5 minutes, followed by washing 3 times with PBS for 10 minutes. Including cells 0.5% Triton X-100 PBS was permeated for 5 minutes and blocked with 5% normal goat serum for 30 minutes. The fixed cells were mixed overnight in a PBS containing primary antibody (eg SPAG5) and 1% normal goat serum at 4 °C. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, 2 μg/mL). Immunofluorescence cell imaging was continued with an Olympus LSM Fluoview FV1000 confocal laser scanning microscope (Olympus).

result Establishment of LPPC/antibody complexes for screening novel receptors and membrane-bound proteins

The LPPC/antibody complex can be readily purified from unbound antibodies. In addition, empty LPPCs can readily load fluorescent dyes to form fluorescent nanoparticles, and the fluorescent LPPCs, upon adsorption of specific antibodies, offer the potential to develop specific probes. The antibody/fluorescent LPPC complex does not require chemical conjugation as an excellent tool for detecting surface antigens. The binding potency was measured by FACScan flow cytometry (Figure 1). These procedures can be performed simply and in a small tube and the results indicate that the method of the invention is useful for identifying new receptors and/or membrane-bound proteins.

Screening of small antibody populations for identifying cancer-specific receptors and/or membrane-bound proteins

The problem to be solved by the present invention is whether or not the receptor (surface marker or membrane-bound protein) is discriminated by antibody screening. To prove the feasibility of this idea, the first is to collect 450 antibodies. Next, before loading the LPPC, randomly select 9 antibodies and mix them together into a group, a total of 50 LPPC-antibody groups, and 3 different cell lines, lung cancer (A549), colon cancer (HT29) and breast cancer. (MCF7) cells were subjected to preliminary culture. The antibody-LPPC complex was then subjected to FACScan analysis (Fig. 2). Based on the results of the preliminary screening, many populations, including the 3A, 3B, Y1C, 2I, and 2G populations, showed higher membrane binding capacity in cancer cell assay analysis compared to the other populations (Fig. 3). Use GO (cell component) and PubMed search, Careful examination of the subcellular location of each of the five selected populations revealed that these five selected populations each contained a known membrane-bound protein and, of course, had better membrane binding potency. However, the 2I population showed different binding potency in cell line assays (eg, higher binding potency in AHT and MCF7 cells in HT29 cells), with different antibodies that may be recognized in different cells in the test protein. The possibility of different positioning. Therefore, we hypothesized that we could identify new receptors and/or membrane-bound proteins using individual antibodies from each of the five selected groups for further study (Figures 4-7). Therefore, after the first screening, 9 individual antibodies from a positive population (eg 3A) were tested by reloading them onto fluorescent LPPC to determine which antibodies are potential receptors for cancer cells (or membranes) Binding protein) (Figure 2). Several antibodies from these five groups, such as 3A-9 (or PDGFRB) and Y1C-4 (or SPAG5) (Table 1), not only rediscovered as known receptors, but also revealed potential in cancer cells. The possibility of membrane-bound proteins.

Screening for subcellular localization of specific preferential proteins by small populations

Several known receptors were re-discovered in this screening, such as 3A-9 (or PDGFRB, see Table 1) and 3B-7 (or KRAS2, see Table 1). In addition, many antibodies exhibit different affinities for different cell lines. For example, Y1C-5 has a higher affinity in A549 and HT29 cells than in MCF7 cells, whereas Y1C-4 has a higher affinity in MCF7 cells than A549 and HT29 cells. To further validate these observations, we established a secondary immunofluorescence screen to confirm the membrane localization of the preferential proteins identified in the primary LPPC screen. In order to expand the application of this method, we not only specified the subcellular localization of selected targets from the original lung cancer (A549), colon cancer (HT29) and breast cancer cells (MCF7), but also extended to other cancer cell lines, including hepatocellular carcinoma. Cells (Mahlavu). The results show that 3B-4 (Fig. 4), 2G-4 and 2G-7 (Fig. 5), 2I-3 and 2I-6 (Fig. 6), and Y1C-4 and Y1C-5 (Fig. 7) are located. The cell membrane is cell type specific. For example, Y1C-4 (or SPAG5, see Table 1) is located in the cell membrane of hepatocyte cancer cells (Mahlavu) rather than lung cancer cells (A549, Figure 7). SPAG5, mitotic spindle-related egg White, known to regulate the correct alignment of chromosomes in the mid-term, is associated with breast cancer and non-small cell lung cancer. In addition, the absence of SPAG5 results in the cohesiveness of sister staining. In general, SPAG5 performs very low during the interim period and is adjusted upwards in the medium term and is located on the centromere and spindle. Further research is needed to analyze how SPAG5 mediates the signaling pathway on the cell membrane of liver cancer. A summary of the results of immunofluorescence is shown in Table 1.

The above-described embodiments are merely illustrative of the principles and effects of the invention, and are not intended to limit the invention. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.

Claims (11)

A method of screening a molecular group, the method comprising the steps of: (a) providing a candidate molecular group; (b) dividing the candidate molecular group into a plurality of subgroups; (c) non-covalent loading of each subgroup Forming a complex on a carrier; and (d) contacting the complex of the step (c) with a medium to detect whether the subgroup contains a molecule having binding specificity to the medium, thereby screening the group of molecules Wherein the molecule is an antibody or an antigen; the vector is a liposome; and the medium is an antibody or an antigen. The method of claim 1, wherein the anti-system is a monoclonal antibody or a plurality of antibodies. The method of claim 1, wherein the medium is located within the cell or on the cell. The method of claim 3, wherein the cell line is a normal cell, a cancer cell, a stem cell or a cancer stem cell. The method of claim 1, wherein the carrier comprises a detectable marker. The method of claim 5, wherein the detectable label is a fluorescent or radioisotope. The method of claim 1, wherein the vector further comprises at least one drug, cytotoxic drug, growth factor, cytokine, vaccine or oligonucleotide. The method of claim 1, further comprising identifying the molecule. The method of claim 1, wherein the step (d) further comprises contacting the complex of the step (c) with a cell to detect whether the complex binds to the cell, wherein the medium is located in the cell or on the cell. . The method of claim 1, wherein the step (d) further comprises contacting the complex of the step (c) with a cell to detect whether the complex poisons the cell, wherein the medium is located in or on the cell. A method of screening a biomarker population in a cell or a cell, comprising the method of any one of claims 1 to 10, wherein the biomarker is a tumor-specific antigen, a membrane-bound protein or a cell surface marker.