Method for spatial identification and isolation of immune cells using variable region targets of immune cells

Abstract

The present invention relates to recovery of molecular information through spatial targeting and isolation of immune cell variable regions. More specifically, unlike conventional methods in which spatial information is lost and data having all molecular information mixed therein is obtained or single-cell information is obtained but with high cost required, the present invention can identify a precise spatial position of targeted immune cells and obtain additional molecular information at the corresponding position through isolation. The invention pertains to a technology for targeting immune cells in present spatial contexts, isolating same, and recovering desired molecular information.

Description

A method for identifying and separating the spatial location of immune cells using variable region targets of immune cells.

[0001] The present invention relates to a method for identifying and separating the spatial location of an immune cell using a variable region target of an immune cell, and more specifically, unlike existing methods that lose spatial information and obtain data by mixing all molecular information, or obtain information on a single cell but require high costs, the present invention relates to a method for identifying and separating the spatial location of an immune cell using a variable region target of an immune cell, which can identify the precise spatial location of a targeted immune cell and obtain additional molecular information at that location through separation.

[0002] This invention was conducted with the support of Seoul National University (Industry-Academic Cooperation Foundation) (Project No. 0534-20230045).

[0003] Immune cells are primarily composed of T cells and B cells, each of which plays a crucial role in the immune system. T cells regulate cell-mediated immune responses and are involved in recognizing and destroying infected cells. B cells, on the other hand, contribute to the immune response by producing antibodies to respond to foreign antigens.

[0004] Hypervariable regions exist in immune cell receptors. Hypervariable regions are regions expressed in cell receptors and enable diverse responses to antigens. However, these hypervariable regions increase the heterogeneity of immune cells, resulting in countless different cell subpopulations within a specific cell population (10 15 (more than one) exist, which makes it difficult to analyze immune cells (see Figure 1).

[0005] T cells possess T cell receptors (TCRs) that contain various chains: alpha (α), beta (β), gamma (γ), and delta (δ). The combination of these various chains allows T cells to respond to various antigens. T cell receptors with alpha and beta chains are primarily expressed on CD4+ or CD8+ T cells and primarily recognize antigens by binding to MHC class I or class II molecules. Gamma-delta T cells are primarily involved in tissue-specific immunity and play a crucial role in detecting specific antigens within infected tissues.

[0006] B cells possess antibody receptors composed of heavy and light chains. The heavy chain of an antibody has various hypervariable and constant regions, which play a crucial role in determining the antibody's function. The light chain, along with the heavy chain, plays a crucial role in determining the antibody's variability. B cells produce each antibody receptor, which reacts specifically to foreign antigens. These diverse antibodies enable the immune system to respond specifically to a variety of antigens.

[0007] The present invention represents a significant advance in research targeting the hypervariable regions of immune cells, particularly T and B cells. This approach is not limited to a single experimental technique, but rather encompasses a universal approach that allows for the effective recovery of secondary DNA / RNA / protein information from target cells by spatially isolating them through a variety of experimental methodologies.

[0008] Existing methods have had several limitations in obtaining genetic information as well as spatial information about cells. In particular, considering the diversity and complexity of immune cells, precise genetic profiling at the individual cell level has been a significant challenge.

[0009] Among existing technologies, scRNA seq can recover intact heavy and light chains from B cells (or alpha and beta chains from T cells). However, these technologies require high costs of approximately 10 million won per analysis and are known to have very low throughput, recovering information on only less than 10,000 cells at a time. Therefore, they have limitations in covering the human immune system, which contains billions of diverse cells (see Figure 2).

[0010] Combinatorial methods utilize the frequency of probabilistic combinations to obtain combinations of heavy and light chains in B cells (or alpha and beta chains in T cells) from a large number of immune cells. However, because this process relies on probability to obtain a pair of sequence combinations, a large number of cells must be extracted to obtain reliable results. Furthermore, if a specific immune cell's frequency within the sample (frequency within the repertoire) is excessively high or low, the sequence combination cannot be identified. Furthermore, similar to scRNA-seq, the cost of a single experimental kit exceeds 10 million won, making it difficult to utilize in laboratories. Furthermore, this method also has limitations such as a high rate of data loss during the experimental process (see Figure 3).

[0011] The present invention provides a technological advancement to address these issues. The process of precisely isolating target cells and recovering additional genetic information enables in-depth analysis and new discoveries in areas such as disease diagnosis, understanding immune responses, and developing therapeutics.

[0012] In order to solve the above-mentioned problem, the present invention provides a method for identifying and separating the spatial location of an immune cell using a variable region target of an immune cell, which can identify the precise spatial location of a targeted immune cell and obtain additional molecular information at that location through separation, unlike existing methods that lose spatial information and obtain data by mixing all molecular information or that obtain information on a single cell at a high cost.

[0013] In order to solve the above-described problem, the present invention provides a method for identifying and separating the spatial location of an immune cell using a variable region target of an immune cell, the method comprising the steps of: generating a cell receptor data group formed based on genetic information of an antibody; selecting a specific antibody from the data group; producing or selecting a probe capable of binding to a variable region of the selected antibody; identifying a desired immune cell by mixing the probe with a sample containing immune cells; and separating the identified immune cell.

[0014] In one embodiment, the genetic information may be heavy chain genetic information.

[0015] In one embodiment, the cell receptor may be a B cell receptor or a T cell receptor.

[0016] In one embodiment, the specific antibody may include an antibody comprising a sequence commonly found among patients sharing the same symptom; an antibody that persists in the body of a specific patient for a predetermined period of time; an antibody in which somatic hypermutation (SHM) has been introduced in the variable region (V gene region) a predetermined number of times or more; an antibody having a frequency of 0.1% or more in the cell receptor data group; an antibody in which class switching recombination has occurred; or an antibody in which the antibody sequence exhibits structurally or sequence-specific characteristics in the antigen binding region (CDR).

[0017] In one embodiment, the identification step can be performed by analyzing a fluorescent signal, a fluorescent marker location, a genetic information-based barcode, or a hybridization location of the probe.

[0018] In one embodiment, the identification step can be performed using a device including a fluorescence-activated cell analyzer (FACS), a flow cytometer, a fluorescence microscope, a confocal microscope, super-resolution imaging, an image-based analysis device, a spatial transcriptomics device, or a combination thereof.

[0019] In one embodiment, the probe may be capable of complementarily binding to a CDR3 region or an adjacent region of the CDR3 region among the variable regions.

[0020] In one embodiment, the probe may comprise a fluorescent detection domain, a unique molecular identifier (UMI), a barcode for sample identification, or a signal amplification sequence.

[0021] In one embodiment, the probe may comprise a label that generates an optical or spectroscopic signal.

[0022] In one embodiment, the label that generates the optical or spectroscopic signal may be a fluorophore or a fluorescent quencher.

[0023] In one embodiment, the step of identifying the immune cell may be a step of identifying the immune cell based on the fluorescent signal of the probe.

[0024] In one embodiment, the step of identifying the immune cells can be performed using an optical microscope, a confocal microscope, a super-resolution imaging device, a spectral imaging device, or a combination thereof.

[0025] In one embodiment, after the step of isolating the identified immune cells, the method may further include a step of generating a light chain capable of binding to the heavy chain of the antibody based on genetic information within the separated immune cells; and a step of isolating and confirming the generated light chain.

[0026] The method for retrieving molecular information through spatial targeting and separation of immune cell variable regions according to the present invention has the advantage of targeting desired immune cells at low cost and identifying their characteristics. Specifically, when limited to B cells, this study is expected to enable the selection of highly functional antibody candidates in a shorter period of time and reduce inefficient analysis costs. Since even a single antibody therapeutic can have a significant impact, this research has the potential to accelerate therapeutic development and lead to personalized treatments for B cell-specific diseases. Furthermore, it can provide information not only on antibody receptors but also on the expression of genes expressed. Based on this information, various information on targeted cells can be collected and used to identify diagnostic or pharmaceutical targets.

[0027] Figure 1 is a diagram showing the diversity of antibodies in the human body.

[0028] Figure 2 shows the scRNA seq and TCR / BCR-seq used previously.

[0029] Figure 3 shows the combinatorial method used in the past.

[0030] Figure 4 briefly illustrates the composition of an antibody according to one embodiment of the present invention and a method for securing an antibody sequence using a conventional method.

[0031] Figure 5 briefly illustrates an antibody separation method according to one embodiment of the present invention.

[0032] Figure 6 shows BCR repertoire data generated from the blood of a patient infected with coronavirus (COVID19) according to one embodiment of the present invention.

[0033] Figure 7 shows BCR repertoire data and its ratio generated from the blood of 17 patients infected with coronavirus (COVID19) according to one embodiment of the present invention.

[0034] Figure 8 shows target location and sequence information during the BCR repertoire data production process according to one embodiment of the present invention.

[0035] Figure 9 illustrates a primer list used to create an NGS library according to one embodiment of the present invention.

[0036] Figure 10 shows the position targeted by the primer used when targeting the J gene instead of the C gene according to one embodiment of the present invention.

[0037] Figure 11 shows a primer sequence used when targeting the J gene instead of the C gene according to one embodiment of the present invention.

[0038] Figure 12 shows the sequence information obtained by NGS of the cdr3 aa heavy chain sequence selected as a target according to one embodiment of the present invention and the heavy chain recovered by separation after in situ sequencing.

[0039] Figure 13 is a result of confirming spatial information of B cells having a heavy chain according to one embodiment of the present invention.

[0040] Figure 14 briefly illustrates a method for obtaining a light chain according to one embodiment of the present invention.

[0041] Figure 15 is a graph showing the results of confirming through qPCR whether the light chain of the second PCR portion is well amplified in cells where ISS has been completed according to one embodiment of the present invention.

[0042] Figure 16 shows the gel electrophoresis results for the recovery of the heavy chain (upper row), the light chain (lower row), and the kappa chain for verification in isolated cells according to one embodiment of the present invention.

[0043] Figure 17 shows the gel electrophoresis results for the recovery of Lambda chains among Light chains for verification in isolated cells according to one embodiment of the present invention.

[0044] Figure 18 shows the index primer sequence for library preparation for the final NGS run according to one embodiment of the present invention.

[0045] Figure 19 shows sequence information recovered based on a cell line according to one embodiment of the present invention and sequence information recovered through NGS.

[0046] Hereinafter, preferred embodiments of the present invention will be described in detail. In describing the present invention, if a detailed description of related known technology is judged to obscure the gist of the present invention, the detailed description thereof will be omitted. Throughout the specification, singular expressions should be understood to include plural expressions unless the context clearly indicates otherwise, and terms such as “comprise” or “have” should be understood to indicate the presence of described features, numbers, steps, operations, components, parts, or combinations thereof, but not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. In addition, in performing a method or a manufacturing method, each step constituting the method may occur in a different order from the stated order, unless the context clearly indicates a specific order. That is, each step may occur in the same order as the stated order, may be performed substantially simultaneously, or may be performed in the reverse order.

[0047] The technology disclosed in this specification is not limited to the implementation examples described herein and may be embodied in other forms. However, the implementation examples introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the technical spirit of the present technology can be sufficiently conveyed to those skilled in the art. In the drawings, the dimensions of each device component, such as width and thickness, are somewhat enlarged to clearly represent the components. The drawings are described from the perspective of an observer, and when an element is mentioned as being positioned above another element, this includes the meaning that the element is positioned directly above the other element or that additional elements may be interposed between them. Furthermore, those skilled in the art will be able to implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in multiple drawings indicate substantially the same elements.

[0048] As used herein, the term "and / or" includes a combination of multiple listed items or any one of multiple listed items. As used herein, "A or B" can include "A," "B," or "both A and B."

[0049] Antibodies are substances that respond to foreign substances entering the body. Antibodies bind to antigens through their heavy and light chains, and these two elements are essential for antigen binding. Developing antibody therapeutics, which are currently attracting attention, requires securing both the heavy and light chains simultaneously, which are known to play a crucial role in antibody performance.

[0050] However, existing antibody discovery technologies fail to capture both heavy and light chains from the diversity of antibodies in the body. Droplet-based single-cell analysis technologies capture both chains, but their throughput cannot keep pace with the diversity of antibodies in the body. Furthermore, methods that randomly combine light chains from target heavy chains have limited performance.

[0051] Recently, technologies for analyzing the heavy or light chains of antibodies in the body using next-generation sequencing are being developed. These analytical techniques enable the identification of heavy or light chains corresponding to each antigen from a variety of disease groups. However, while these next-generation sequencing technologies can cover the diversity of antibodies in the body, they only provide information on a single chain, making it essential to secure the corresponding chain.

[0052] Antibodies expressed in B cells are known to bind to specific antigens through the combination of heavy and light chains. These two elements are essential for antigen binding and play a crucial role in determining antibody efficacy and specificity. However, conventional antibody discovery methods separate the heavy and light chains during cell destruction, making it difficult to simultaneously obtain them.

[0053] Traditionally, antibody discovery techniques target the C gene (constant region) of the heavy chain to generate bulk BCR repertoire data to obtain sequence information within the immune system of an individual (or a patient with a disease) as diverse as possible for antibody discovery. Tens of thousands to thousands of light chains are then randomly linked to the target heavy chain using a vector to express them, allowing antibodies that bind well to a specific antigen to be selected. This process is being experimented with under the name of light chain shuffling. However, as mentioned above, light chain shuffling has the disadvantage of randomly introducing light chains, which can lead to random light chains binding to heavy chains, thus reducing antibody selectivity (see Figure 4).

[0054] Accordingly, the present invention proposes a technology that can connect actual Light chains existing within the body, rather than random Light chains supplied from outside, based on a group of produced cell receptor data.

[0055] Accordingly, the present invention relates to a method for identifying and separating spatial locations of immune cells using a variable region target of immune cells, comprising the steps of: generating a cell receptor data group formed based on genetic information of an antibody; selecting a specific antibody from the data group; producing or selecting a probe capable of binding to a variable region of the selected antibody; identifying a desired immune cell by mixing the probe with a sample containing immune cells; and separating the identified immune cell (see FIG. 5).

[0056] The genetic information for the antibody used in the present invention may be present in the heavy chain, light chain, or variable region. The antigen-binding site of an antibody is primarily determined by the heavy chain, and the heavy chain has a greater influence on the specificity and function of the antibody than the light chain. Therefore, the present invention preferentially utilizes a method of identifying and isolating target B cells based on heavy chain genetic information.

[0057]

[0058] The above cell receptor may be a B cell receptor or a T cell receptor. Various cells and antibodies may be used in an immune response, but in the case of the present invention, since the location identification and separation of immune cells are performed using the antibody-related cells, the cells may be B cells or T cells, and accordingly, the cell receptor may also be a B cell receptor or a T cell receptor.

[0059] The above cell receptor data group refers to a collection of cell receptor data generated as described above. At this time, the cell receptor data group may be a data group of all immune cells collected from one patient, but data groups of multiple patients, animal experiment data groups, cell experiment data groups, etc. may be used. In addition, in addition to the data group of naturally formed immune cells as described above, data groups that do not exist in nature, such as data groups of recombinant immune cells, recombinant genetic information, genetic information data groups through simulation, genetic information data groups generated by AI, etc., may also be used.

[0060] A specific antibody can be selected from the above data group. The specific antibody refers to an antibody predicted to have an effect on an antigen, and may include an antibody containing a sequence commonly found among patients sharing the same symptoms; an antibody that persists in the body of a specific patient for a predetermined period of time; an antibody in which somatic hypermutation (SHM) has been introduced in the variable region (V gene region) a predetermined number of times or more; an antibody having a frequency of 0.1% or more in the above cell receptor data group; an antibody in which class switching recombination has occurred; or an antibody in which the antibody sequence exhibits structurally or sequence-specific characteristics in the antigen binding region (CDR).

[0061] At this time, in the case of an antibody in which somatic hypermutation (SHM) has been applied to the variable region (V gene region) more than a certain number of times, if SHM (somatic hypermutation) of the V gene region is excessively applied to the heavy chain sequence of a B cell, especially if it has been applied more than 10 times, it can be used as a specific antibody.

[0062] Additionally, an antibody having a frequency of 0.1% or more, preferably a frequency of 0.5% or more, and more preferably a frequency of 1% or more in the above cell receptor data group can be used.

[0063] As an example, Figure 6 shows a portion of BCR repertoire data cultured from the blood of a patient infected with coronavirus (COVID19).

[0064] As shown in Figure 6, when hundreds of thousands of BCR heavy chain sequences are obtained from one patient (or one animal model), it is possible to select targets based on indicators related to the B cell maturation process (Frequency, SHM, CSR, etc.) from the entire BCR repertoire data.

[0065] At this time, in the case of an antibody in which somatic hypermutation (SHM) has been applied to the variable region (V gene region) more than a certain number of times, if SHM (somatic hypermutation) of the V gene region is excessively applied to the heavy chain sequence of a B cell, especially if it has been applied more than 10 times, it can be used as a specific target antibody.

[0066] The best candidates are those with high frequency, a high degree of SHM authorization, and class switch recombination (CSR).

[0067] Figure 7 shows the sequences shared between each patient and the ratio of those sequences based on the Bulk BCR repertoire data formed from the blood of 17 patients infected with COVID-19.

[0068] The bar in the upper right corner of Figure 7 represents the number and proportion of sequences shared within the BCR repertoire data of other autoimmune diseases, dementia, and healthy individuals, in addition to COVID-19. Sequences shared between patients, shared with other disease groups, and sequences found over a long period of time within patients can be selected as target sequences based on indicators separate from Frequency, SHM, and CSR. Furthermore, sequences that exhibit similar characteristics to sequences known to bind to publicly available protein databases can also be selected as target sequences.

[0069] After the selection of a specific antibody of interest has been completed as described above, a probe capable of binding to the variable region of the selected antibody can be selected. At this time, it is more preferable to select a probe capable of complementarily binding to the CDR3 region of the variable region or a region adjacent to the CDR3 region. At this time, the probe can be designed by performing 5' phosphorylation modification (5' phosphorylation).

[0070] In addition, the probe selected as above can be amplified through various methods, but it is preferably amplified using RCA (rolling circle amplification).

[0071] In addition, the probe may include a fluorescent detection domain, a unique molecular identifier (UMI), a barcode for sample identification, or a signal amplification sequence. In the case of the probe, it may perform a role of identifying immune cells, such as B cells or T cells, by binding to the antibody. In this case, the probe may be labeled through various known methods, but in the case of the present invention, the probe includes a fluorescent detection domain, a unique molecular identifier (UMI), a barcode for sample identification, or a signal amplification sequence, so that the desired probe and the immune cells bound thereto can be identified.

[0072] In addition, it is more preferable that the probe include a label that generates an optical or spectroscopic signal, and most preferably, it is preferable that the probe include a fluorophore or fluorescent quencher to distinguish the desired immune cells. In the case of the fluorophore or fluorescent quencher, any fluorophore or fluorescent quencher that is currently used in probes may be used without limitation.

[0073] After the probe is selected as described above, the desired immune cells can be identified by mixing the probe with a sample containing immune cells. Typically, a sample contains a variety of immune cells mixed in a two-dimensional or three-dimensional space. Conventional methods make it nearly impossible to isolate the desired immune cells from the sample, so hemolysis, separation, and amplification are performed in the mixed state described above. Therefore, the conventional method has the disadvantage of very low selectivity for antibodies. However, the present invention utilizes a probe that binds to a specific antibody as described above to label the desired immune cells and simultaneously spatially identify them, thereby enabling the isolation of the desired antibody with high selectivity.

[0074] The desired immune cell may be a cell containing a specific antibody sequence.

[0075] The above identification step can be performed using a device including a fluorescence-activated cell analyzer (FACS), a flow cytometer, a fluorescence microscope, a confocal microscope, a super-resolution imaging device, an image-based analysis device, a spatial transcriptomics device, or a combination thereof.

[0076] Additionally, the identification step can be performed by analyzing the intensity of the fluorescent signal, the location of the fluorescent marker, the genetic information-based barcode, or the hybridization location of the probe.

[0077] This analytical method enables the individual identification of multiple immune cells within the same sample, and simultaneous detection of various molecular targets, including heavy chains, light chains, other antibody sequences, transcripts, microRNAs, intestinal RNAs, specific gene sequences, or protein-coding / non-coding RNAs.

[0078] Therefore, the present invention is not limited to a single target, but can identify and isolate desired immune cells with high selectivity based on multiple targets.

[0079] At this time, it is preferable that the immune cells be identified optically using the fluorescent material included in the probe as described above. That is, the step of spatially identifying the immune cells may be a step of identifying them based on the fluorescent signal of the probe. In addition, the step of identifying the immune cells is preferably performed using an optical microscope, a confocal microscope, super-resolution imaging, a spectral imaging device, or a combination thereof.

[0080] Immune cells identified as described above can be isolated from a sample. Conventional methods can be used to isolate the immune cells, but LDM or SLACS can also be used.

[0081] Laser Dissection Microscopy (LDM) (LCM: Laser Capture Microdissection) is a technique that uses a laser to selectively isolate specific cells or cell populations from a tissue sample, while retaining spatial location information of the isolated cells. This technique is used when analysis requires consideration of the original location of the cells and their relationship to surrounding cells, and is useful for understanding intercellular heterogeneity and the roles of specific cells within tissues. This includes microdissection methods utilizing UV and catapulting methods utilizing IR, such as thermoplastics. It also includes laser ablation methods utilizing IR.

[0082] The above-mentioned SLACS (Spatially Resolved Laser Activated Cell Sorter) is a spatially resolved laser-activated cell sorter. It uses a high-precision laser to precisely isolate and sort single cells within tissue samples. This method maintains the spatial location information of single cells while separating them, making it extremely useful for understanding interactions between cells and between cells and their surrounding environment within tissues. SLACS is particularly suitable for biological studies that consider the spatial arrangement of cells and for analyzing the heterogeneity of complex cell populations.

[0083] After separating the labeled immune cells as described above, the method may further include a step of generating a light chain capable of binding to the heavy chain of the antibody based on the genetic information within the separated immune cells; and a step of separating and confirming the generated light chain. As described above, in the case of the present invention, since only immune cells having the desired antibody are isolated, when a light chain is generated based on the genetic information within the immune cells, it is possible to generate a light chain that precisely corresponds to the heavy chain of the desired antibody described above. At this time, the separated immune cells are lysed, and then a primer targeting the light chain within the immune cells is used as a target for the total RNA, thereby obtaining the desired light chain.

[0084] In other words, since the separated immune cells have a fluorescent marker attached to the probe, the mRNA is amplified before hemolysis, and then light chain target PCR targeting the light chain is performed to selectively amplify the desired light chain.

[0085] In the case of the present invention, the following usefulness can be achieved through this separation method.

[0086] The technology developed through this invention can serve as a valuable tool in research for the development of antibody therapeutics. By simultaneously securing both heavy and light chains, more specific and effective antibodies can be discovered, contributing to the development of treatments for a variety of diseases.

[0087] In Situ Sequencing (ISS) allows for the recovery of additional secondary DNA / RNA information from experimental cells (tissues) based on spatial information. While existing ISS technologies can quantify the location and expression of genes within tissue samples, the recovery of additional genetic information after a series of experiments is limited. The present invention overcomes these limitations and possesses technological features that enable the acquisition of more in-depth genetic information from the experimental cells.

[0088] By providing detailed genetic profiles at the cellular level, along with the spatial distribution of cells within a tissue sample, the present invention offers a new opportunity to more clearly understand cell function and status, as well as their relationship to disease states. In particular, this technology can significantly contribute to understanding the heterogeneity and complexity of immune cells, particularly hypervariable regions of T and B cells.

[0089] The present invention can be applied to the discovery of antibodies against various disease groups based on specific chains. In particular, based on bulk T-cell receptor (Bulk TCR) repertoire data, specific sequences thought to be problematic for patients with autoimmune diseases or other diseases can be selected, and selection can be performed using probes in tissue or blood smear samples based on the hypermutant region of the sequence. In this case, the selection can be performed using SLACS equipment to isolate single cells, and RNA-seq, DNA-seq, or methylation analysis can be performed on the isolated target sample.

[0090] This technological approach could have the following expected effects:

[0091] Improving Diagnosis through Accurate Sequence Analysis: By selecting disease-specific sequences based on existing large-scale TCR sequence data, the diagnostic accuracy of a given disease can be improved. In particular, selecting tissue and blood smear samples based on hypervariable regions (HVRs) is expected to provide greater diagnostic reliability, as it allows for the accurate detection of specific antibody responses in disease-related cells or tissues.

[0092] Understanding immunological mechanisms: Selected specific sequences can provide a detailed understanding of antibody production and response mechanisms in patients with autoimmune diseases or other conditions. This can contribute to the development of treatments by elucidating the mechanisms of disease onset and progression.

[0093] Developing personalized treatments: Analysis of antibodies based on specific sequences can enhance understanding of a patient's individual immune response. This will enable the development of personalized treatments and the development of effective treatment and prevention strategies.

[0094] Furthermore, utilizing the isolation method of the present invention, target B cells or T cells can be precisely isolated, and DNA, RNA, epigenome, proteome, and metabolome information can be obtained from them. This multi-omics data enables not only the identification of complete antibody sequences but also the following advanced analyses.

[0095] Analysis of interactions between cell subpopulations: Data obtained from isolated cells can be used to analyze interactions between B cells and T cells, or between various subpopulations within cells. For example, one can investigate how a T cell subpopulation with a specific TCR mutation interacts with B cells with a specific BCR mutation. This can elucidate intercellular networks and signaling pathways within the immune system and understand how these networks change in disease states.

[0096] Identification and Analysis of Disease-Specific Antibody Responses: Multi-omics data obtained from target cells can be used to identify and analyze disease-specific antibody responses. By analyzing the patterns of antibody sequences expressed in a specific disease state, specific immune responses to that disease can be identified. This information can be used to identify disease diagnostic markers and therapeutic targets.

[0097] Multidimensional Analysis of Cell Function and State Changes: By integrating and analyzing multi-omics data obtained from isolated cells, we can gain a multidimensional understanding of cellular function and state changes. For example, simultaneous analysis of a cell's response to a specific drug or treatment at the DNA, RNA, protein, and metabolite levels allows for a comprehensive assessment of the drug's effects on the cell. This can be particularly useful for studying drug response and adverse effect mechanisms.

[0098] Development and Optimization of Immunotherapies: Multi-omics data obtained from target cells can be used to develop and optimize immunotherapies. For example, analyzing B and T cell responses to immune checkpoint inhibitors using multi-omics data can lead to the development of effective therapeutic combinations and therapies.

[0099]

[0100] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily implement them. Furthermore, when describing the present invention, detailed descriptions of related, known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention. Furthermore, certain features presented in the drawings may be enlarged, reduced, or simplified for ease of explanation, and the drawings and their components are not necessarily drawn to scale. However, those skilled in the art will readily understand these details.

[0101] The following examples were conducted on human-derived B cell lines (see Figures 6 and 7).

[0102] Example

[0103] NGS sample prep (cell receptor data group - BCR repertoire data - generation)

[0104] The gene encoding the V gene and a portion of the C gene or J gene domain were amplified using specific primers. Complementary DNA (cDNA) was synthesized using the SuperScript IV First-Strand Synthesis System (Invitrogen) using total RNA as a template and specific primers targeting specific domains (see Fig. 8).

[0105] The Primer List used to create the NGS Library at this time is as shown in Figure 9. Figure 9 shows primers targeting the V gene and the C gene, and the two types of primers at the bottom represent primers targeting two types of germlines of the Light chain, respectively.

[0106] Also, Figures 10 and 11 show primers used when targeting the J gene instead of the C gene.

[0107] After cDNA synthesis, cDNA was purified using SPRI beads (Beckman Coulter, AMPure XP) 1.8X and dissolved in 35 μl of water. The purified cDNA (15 μl) was used for second-strand synthesis in a 25 μl reaction volume using IGHV gene-specific primers and the KAPA Biosystems kit (Roche, KAPA HiFi HotStart).

[0108] PCR conditions were as follows: 95°C for 3 min, 98°C for 30 s, 60°C for 45 s, and 72°C for 6 min. After second-strand synthesis, dsDNA was purified using SPRI Bead 1X as described above.

[0109] Purified dsDNA (15 μl) was amplified in a 25 μl reaction volume using primers containing the index sequence and a KAPA Biosystems kit.

[0110] PCR conditions were as follows: 95°C for 3 min; 25 cycles of 98°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and 72°C for 5 min. PCR products were electrophoresed on a 1.5% agarose gel and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc.) according to the manufacturer's instructions. Gel-purified PCR products were repurified using 1x SPRI beads and dissolved in 20 μl of water. SPRI-purified sequencing libraries were quantified using the D1000 ScreenTape assay on a 4200 TapeStation System (Agilent Technologies) and used for NGS on the Illumina platform.

[0111]

[0112] Selecting a specific heavy chain from the BCR repertoire

[0113] Among the BCR repertoire data generated as above, those in which SHM (somatic hypermutation) in the V gene region was approved 10 or more times were selected and used as specific target antibodies.

[0114] As shown in Figure 12, the upper red box is the cdr3 aa heavy chain sequence selected as the target, and the sequence in the lower red box is the sequence information obtained by NGS of the heavy chain recovered after in situ sequencing.

[0115] The above results are the result of identifying the actual targeted heavy chain sequence for validation purposes, since separation is based on spatial information when targeting the heavy chain sequence.

[0116] Among the generated NGS libraries, 99% were obtained with identical heavy chains.

[0117]

[0118] Designing padlock probes based on target sequence

[0119] A region (position on the sequence) containing the CDR3 adjacent unique sequence of the target heavy chain is defined, left and right binding arm candidates of 18 to 25 nucleotides in length are generated upstream and downstream of the window, and then, for each candidate, a melting temperature of 58 to 64°C, GC 40 to 60%, and avoidance of repeating the same base 4 or more times are applied.

[0120] To prevent hairpin / dimer formation, scoring is performed to satisfy avoidance conditions below the free energy threshold, and off-targets are removed by proximity matching analysis against human transcriptome and immunoglobulin references.

[0121] After rearranging the binding sites to avoid mutation-prone areas, the left and right arm combinations with ΔTm less than 2°C and the lowest off-target risk are selected.

[0122] A padlock probe with 5' phosphorylation was constructed by placing a backbone containing a docking sequence for signal detection and, if necessary, a UMI and a sample barcode between the selected left and right arms, and then cDNA was formed with a reverse transcription primer in the tissue, and the padlock was hybridized, ligated, and RCA was performed to obtain a fluorescent signal, and cells labeled with SLACS were automatically separated using the signal coordinates, and the light chain sequence was recovered from the separated cells.

[0123]

[0124] cell culture

[0125] Depending on the cell status, culture in petri dishes / 6, 8, 12 well.

[0126] Increase the temperature of the media 30 minutes before (RPMI + p / s + FBS).

[0127] Transfer all cells from the existing dish to a 15ml tube.

[0128]

[0129] cell smear

[0130] Prepare general slides and Cosmo slides

[0131] Centrifuge under the same conditions as when subculturing, and add less than 1 ml of media. (If you add more than that, if there aren't many cells, they will all fly away when smearing.)

[0132] Gently pipette and dispense only 10ul onto the right side of the Cosmo slide.

[0133] Hold the general slide horizontally and smear it at an appropriate speed while holding both ends.

[0134] Transfer to a petri dish and dry thoroughly. (Refrigerate for 10 minutes recommended)

[0135] Fixed at 4% FA for 10 minutes.

[0136]

[0137] cell cytospin

[0138] Use the luna machine to make a buffer in a 1.5ml tube with a concentration of 1*10e5=1ml.

[0139] Take out the tools for fitting the slides from the drawer underneath the machine.

[0140] After fixing it firmly and adjusting the balance, turn it according to the conditions (ex: 600 / 10min).

[0141] Take it out, organize it, and fix it on ice with 4% FA for 10 minutes.

[0142]

[0143] In situ sequencing

[0144] In the cell fixation step, the sample is fixed with FA for 10 minutes and then washed twice with PBST.

[0145] Attach a chamber suitable for the sample, dispense HCl into the sample, and wait for 1 minute. Use HCl stored at room temperature.

[0146] After fixing with FA for 10 minutes, wash three times with PBST.

[0147] In the reverse transcription step, prepare a mixture of H2O, BSA, specific primer, DNTP, RNase inhibitor, RT buffer, and RT reagent. Dispense the prepared mixture into the sample and incubate at 45°C to 50°C for 45 minutes.

[0148] After that, in the post fixation step, fix FA for 10 minutes and wash 3 times with PBST.

[0149] For padlock probe hybridization, prepare a mixture of H2O, BSA, KCl, Formamide, padlock probe 1, padlock probe 2, Ampligase, and Rnase H reagents.

[0150] Dispense the prepared mixture into the sample and incubate at 37°C for 2 hours, followed by 45°C for 4 hours.

[0151] After that, wash 3 times with PBST.

[0152] In the RCA step, a mixture is prepared using H2O, BSA, glycerol, DNTP, phi 29 buffer, and phi 29 polymerase reagent. The prepared mixture is dispensed into the sample and incubated overnight at 37°C.

[0153] After overnight, wash the sample three times with PBST.

[0154] In the anchor primer hybridization step, prepare a mixture using H2O, SSC buffer, formamide, DAPI (or Hoechst), and anchor probe reagent. Dispense the prepared mixture into the sample and incubate at 37°C for 1 hour.

[0155] After that, wash twice with PBST.

[0156] In the sequencing probe ligation step, a mixture is prepared using H2O, T4 ligation buffer, BSA, sequencing probe, and T4 ligase reagent. The prepared mixture is dispensed onto the sample and incubated at room temperature for 16 hours.

[0157] Once incubation is complete, wash three times with PBST.

[0158] After that, dispense 70% ethanol into the sample and wait for 2 minutes.

[0159] Remove the attached chamber and incubate in sequence with 70% ethanol for 1 minute, 85% ethanol for 3 minutes, and 100% ethanol for 3 minutes.

[0160] Figure 13 shows the results of targeting a human B cell line grown on a slide as a smear and in situ sequencing. It was confirmed that B cells containing heavy chains could be detected while maintaining spatial information. Based on this, SLACS was performed.

[0161]

[0162] Separation based on SLACS equipment

[0163] Samples are prepared in various forms, such as blood or tissue sections. The prepared samples are pretreated with plasma and mounted on slides. A full-face image of the mounted slide is acquired. The acquired images are loaded into the user interface software via a server. The user navigates the image and selects target cells. Once selected, the software generates a location information file and an overlay image. The two files are loaded into the SLACS instrument control software. The control software controls the slit, changes the objective lens, and moves the stage. The instrument moves to the designated location, dissects, and isolates the cells. The isolated cells are collected in a collection device. The recovered cells are immediately used for subsequent analysis.

[0164]

[0165] Light chain prep (see Fig. 14)

[0166] Prepare eight plasma-treated PCR tubes and lids.

[0167] Prepare lysis buffer (NFW, proteinase K (PK))

[0168] Pour Lysis buffer into the PCR tube lid.

[0169] Using SLACS, separate the target cells into the desired tube caps. Close the caps and allow the cells to settle for 1 minute.

[0170] Cells were incubated at 50°C for 1 hour, then maintained at 72°C for 10 minutes.

[0171] After the lysis step, samples must be rapidly reverse-transcribed. Add 6 μl of reverse transcription reagent to each sample and incubate at 42°C for 90 minutes, 50°C for 2 minutes, 42°C for 2 minutes, and 70°C for 15 minutes. Steps 2 and 3 should be repeated 20 times.

[0172] In step 1 of PCR, DNA must be amplified. Add 15 μl of PCR1 reagent to each sample and perform PCR. PCR should be performed at 98°C for 2 minutes, followed by 20 cycles at 98°C, 15 seconds at 67°C, and 6 minutes at 72°C. Steps 2-4 should be repeated 25 times.

[0173] Before bead purification, the beads must be activated at room temperature for approximately 30 minutes. The amount of beads is 0.8 times the amount of each sample. 0.8 times the amount of each sample (20 μl) is added to a 1.5 ml tube. Add the samples to each tube and mix gently. Incubate for 8 minutes, place the tubes on a magnet, and wait 3 minutes for the samples to bind to the magnet. Then, aspirate the supernatant using a pipette and discard. After washing with 80% EtOH, aspirate the EtOH using 190 μl of fresh EtOH, and then aspirate the remaining EtOH using a 10 μl pipette and discard. Disaggregate the aggregated beads using 17.5 μl of NFW. Incubate for 8 minutes, place the tubes on a magnet, and wait 3 minutes for the samples to bind to the magnet. This time, since the supernatant is the sample, not the beads, 15 μl of the sample is removed and placed in eight new PCR tubes.

[0174] Figure 15 shows qPCR data that quantifies and tracks the process of amplifying only the light chain through target PCR in isolated specific cells. The top three lines represent the heavy chain, and the central six lines represent the light chain. Figure 15 demonstrates that both the heavy and light chains are sufficiently amplified with each repetition of the cycle.

[0175] Figure 16 shows the gel electrophoresis results for the recovery of the heavy chain (top row), the light chain (bottom row), and the kappa chain (bottom row) for verification in isolated cells, as shown in bands. The target length is distributed between approximately 400 and 550 bp, demonstrating that each chain was amplified to the desired length.

[0176] Figure 17 shows the gel electrophoresis results for the recovery of Lambda chains from Light chains for verification in isolated cells, as shown in bands. The target length is distributed between approximately 400 and 550 bp, demonstrating that each chain was amplified to the desired length.

[0177]

[0178] NGS sample prep (light chain target PCR and heavy chain PCR for verification)

[0179] Re-amplification was performed using specific primers targeting the V gene encoding the VH and VL genes and a portion of the C gene or J gene domain. Complementary DNA (cDNA) was amplified using total RNA as a template.

[0180] PCR conditions were as follows: 95°C for 3 min, 98°C for 30 s, 60°C for 45 s, and 72°C for 6 min. After second-strand synthesis, dsDNA was purified using SPRI Bead 1X as described above.

[0181] Purified dsDNA (15 μl) was amplified in a 25 μl reaction volume using primers containing the index sequence (Fig. 18) and a KAPA Biosystems kit.

[0182] PCR conditions were as follows: 95°C for 3 min; 25 cycles of 98°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and 72°C for 5 min. PCR products were electrophoresed on a 1.5% agarose gel and purified using the QIAquick Gel Extraction Kit (QIAGEN Inc.) according to the manufacturer's instructions. Gel-purified PCR products were repurified using 1x SPRI beads and dissolved in 20 μl of water. SPRI-purified sequencing libraries were quantified using the D1000 ScreenTape assay on a 4200 TapeStation System (Agilent Technologies) and used for NGS on the Illumina platform.

[0183]

[0184] NGS data processing process

[0185] B cell antibody sequences obtained from PBMCs or tissues are analyzed through an NGS data analysis pipeline. This pipeline includes adapter trimming, quality filtering, unique molecular identifier (UMI) processing, V(D)J gene annotation, clustering, quality control, and diversity analysis.

[0186] Forward reads (R1) and reverse reads (R2) are merged using the paired-end read merger (PEAR) v0.9.10 with default settings, and the merged reads are filtered under the q20p95 condition to ensure that 95% of the reads have a Phred score ≥20. Primer positions are identified in the quality-filtered reads, and the primer region is trimmed to remove the influence of synthesis errors, allowing for one substitution or deletion.

[0187] Based on the primer recognition results, UMI sequences are extracted, and reads are clustered according to the UMI sequences. To eliminate indexing errors, clustered reads are subclustered based on read similarity (with five mismatches allowed in each subcluster), ensuring that multiple subclusters match the UMI. Subclustered reads are multi-sequence aligned using Clustal Omega v1.2.4, and consensus calling is performed by selecting the dominant frequency base at all positions in the aligned sequences. The number of reads in a consensus sequence is redefined as the number of UMI subclusters belonging to the consensus sequence.

[0188] Sequence annotation includes isotype annotation and V(D)J annotation, and the consensus sequence is divided into the V(D)J region and the constant region. The isotype is annotated by aligning the extracted constant region with the constant gene of the International Immunogenomics Information System (IMGT), and the V(D)J region of the sequence is annotated using an updated version of IgBLAST (v1.17.1). The number of IGHV genes, IGHJ genes, HCDR3 sequences, and SHMs are extracted from the annotation results and can be used for further analysis.

[0189] The CDR3 sequence information in the upper box of Figure 19 is sequence information recovered based on the cell line. The box below is sequence information when the light chain sequence was separated and data was recovered through NGS, and it can be confirmed that a 100% identical Lambda chain sequence was obtained within the library.

[0190]

[0191] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A step of generating a group of cell receptor data formed based on the genetic information of an antibody; A step of selecting a specific antibody from the above data group; A step of producing or selecting a probe capable of binding to the variable region of the above-mentioned selected antibody; A step of identifying desired immune cells by mixing the probe into a sample containing immune cells; and A step of isolating the identified immune cells; A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells including .

2. In paragraph 1, A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells, characterized in that the above genetic information is genetic information of a heavy chain.

3. In paragraph 1, A method for identifying and separating the spatial location of an immune cell using a variable region target of an immune cell, characterized in that the above cell receptor is a B cell receptor or a T cell receptor.

4. In paragraph 1, The above specific antibody is, Antibodies containing sequences commonly found among patients who share the same symptoms; Antibodies that persist in a specific patient's body for a certain period of time; An antibody in which somatic hypermutation (SHM) has been introduced in the variable region (V gene region) a predetermined number of times; An antibody having a frequency of 0.1% or more in the above cell receptor data group; Antibodies in which class switching recombination has occurred; or An antibody wherein the antibody sequence exhibits structurally or sequence-specific characteristics in the antigen binding region (CDR); A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells, characterized in that it includes.

5. In paragraph 1, A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells, characterized in that the above identification step is performed by analyzing a fluorescent signal, a fluorescent marker location, a genetic information-based barcode, or a hybridization location of the probe.

6. In paragraph 5, A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells, characterized in that the above identification step is performed using a device including a fluorescence-activated cell analyzer (FACS), a flow cytometer, a fluorescence microscope, a confocal microscope, super-resolution imaging, an image-based analysis device, a spatial transcriptomics device, or a combination thereof.

7. In paragraph 1, A method for identifying and separating the spatial location of an immune cell using a variable region target of an immune cell, characterized in that the probe can complementarily bind to a CDR3 region or an adjacent region of the CDR3 region among the variable regions.

8. In paragraph 7, A method for spatially identifying and separating immune cells using a variable region target of immune cells, characterized in that the probe comprises a fluorescent detection domain, a unique molecular identifier (UMI), a barcode for sample identification, or a signal amplification sequence.

9. In paragraph 1, A method for spatial location identification and separation of immune cells using a variable region target of immune cells, characterized in that the probe includes a label that generates an optical or spectroscopic signal.

10. In paragraph 9, A method for spatial location identification and separation of immune cells using a variable region target of immune cells, characterized in that the label generating the optical or spectroscopic signal is a fluorescent substance or a fluorescent quencher.

11. In paragraph 9, A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells, characterized in that the step of identifying the immune cells is a step of identifying the immune cells based on the fluorescent signal of the probe.

12. In paragraph 11, A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells, characterized in that the step of identifying the immune cells is performed using an optical microscope, a confocal microscope, a super-resolution imaging device, a spectral imaging device, or a combination thereof.

13. In paragraph 1, After the step of isolating the identified immune cells, A step of generating a light chain capable of binding to the heavy chain of the antibody based on the genetic information within the separated immune cells; and A step of separating and confirming the light chain generated above; A method for identifying and separating the spatial location of immune cells using a variable region target of immune cells, characterized in that it further includes.

Images