Detection of coexisting receptor encoding nucleic acid segments

Abstract

A method for identifying the co-existence of nucleic acid segments in a nucleic acid sample from a specimen, comprising obtaining a nucleic acid sample from the specimen, determining the sequences of a first nucleic acid segment and a second nucleic acid segment in nucleic acid fragments of the sample to generate a first set of sequences and a second set of sequences, generating a first set of probes and a second set of probes from the first set of sequences and the second set of sequences, exposing a test sample to members of the first set of probes and members of the second set of probes, performing a hybridization analysis to determine whether members of the first set of probes and the second set of probes hybridize with the test sample, and determining whether the first nucleic acid segment and the second nucleic acid segment co-exist in a common cell in the specimen.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 747,785, filed on October 19, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to detecting the co-presence of nucleic acid segments, such as coding segments for cell surface receptors. Background of the Invention

[0005] Neoantigen-specific T cells direct antitumor immune responses in tissues, and in turn, neoantigen specificity is mediated through surface-expressed T cell receptors. T cell receptors are heterodimers composed of an α and β subunit pair. Technologies exist for sequencing nucleic acid segments encoding both subunit pairs. However, identifying coexisting coding segment pairs from a population of fragments extracted from tissue samples remains a challenging problem. SUMMARY OF THE INVENTION

[0007] The present disclosure features a method for identifying coexisting nucleic acid segments in a sample. The segment sequences can be used to engineer T cells that express new antigen-specific T cell receptors, which are then infused into patients for therapeutic applications. The method can be used for a variety of samples, including fresh and fresh frozen tissue sections, formalin-fixed paraffin-embedded (FFPE) tissue sections, and substrate-mounted smears (e.g., blood-derived leukocytes, such as peripheral blood mononuclear cells). A first portion of a probe derived from a first sample or sample is hybridized with a second portion of a second sample or sample, and a specific hybridization event associated with the probe (which is associated with a specific segment sequence) is detected to determine coexisting segment pairs.

[0008] In one aspect, the disclosure features a method for identifying the co-occurrence of nucleic acid segments in a nucleic acid sample from a specimen, the method comprising: obtaining a nucleic acid sample from the specimen, wherein the nucleic acid sample is characterized by a plurality of nucleic acid fragments associated with expression of an antigen receptor molecule in the specimen; determining the sequence of a first nucleic acid segment and a second nucleic acid segment in the nucleic acid fragments of the sample to generate a first set of sequences corresponding to the first nucleic acid segment in the sample and a second set of sequences corresponding to the second nucleic acid; generating a first set of probes from the first set of sequences, wherein each member of the first set of probes is characterized by an oligonucleotide linked to a detection portion, the oligonucleotide corresponding to a different sequence of the first set of sequences; generating a second set of probes from the second set of sequences, wherein each member of the second set of probes is characterized by an oligonucleotide linked to a detection portion, the oligonucleotide corresponding to a different sequence of the second set of sequences; exposing a test sample obtained from the specimen to members of the first set of probes and members of the second set of probes; performing a hybridization analysis to determine whether members of the first set of probes hybridize to the test sample, and to determine whether members of the second set of probes hybridize to the test sample; and determining whether the first nucleic acid segment and the second nucleic acid segment co-occur in a common cell in the specimen.

[0009] Embodiments of the method may include any one or more of the following features.

[0010] The antigen receptor molecule may be a T cell antigen receptor molecule. The first nucleic acid segment may be associated with the alpha chain of the T cell antigen receptor molecule, and the second nucleic acid segment may be associated with the beta chain of the T cell antigen receptor molecule.

[0011] The antigen receptor molecule may be a B cell antigen receptor molecule. The first nucleic acid segment may be associated with a heavy chain of a B cell antigen receptor molecule, and the second nucleic acid segment may be associated with a light chain of a B cell antigen receptor molecule.

[0012] The first nucleic acid segment and the second nucleic acid segment may be located in a complementarity determining region 3 (CDR3) portion of the nucleic acid segment.

[0013] The nucleic acid sample may include genomic DNA. The nucleic acid sample may include total RNA. The nucleic acid sample may include nucleic acid molecules from tumor infiltrating lymphocytes.

[0014] The specimen may include a fresh or frozen tumor tissue specimen. Obtaining a nucleic acid sample from the specimen may include: fixing the specimen and embedding it in paraffin, resecting a portion of the specimen, and extracting the nucleic acid sample from the resected portion of the specimen. The resected portion of the specimen may include target immune cells. The target immune cells may include tumor infiltrating lymphocytes.

[0015] The sequences of the first nucleic acid segment and the second nucleic acid segment can each include 50 to 200 base pairs (e.g., 75 to 150 base pairs). The first set of sequences and the second set of sequences can each include at least 20 different sequences (e.g., at least 50 different sequences).

[0016] The first set of sequences may include N different sequences, and generating the first set of probes may include: for each of the M different sequences expressed with the highest abundance in the nucleic acid sample, generating a population of oligonucleotides corresponding to the sequence, and connecting each member of the population of oligonucleotides to a detection portion. M may be equal to N or less than N. M may be 48 or less (e.g., 24 or less).

[0017] The oligonucleotides of the population may include a DNA sequence. The DNA sequence may be complementary to the M different sequences. The oligonucleotides of the population may include an RNA sequence. The RNA sequence may be an antisense RNA sequence. The antisense RNA sequence may be complementary to an RNA transcript sequence corresponding to the M different sequences.

[0018] The oligonucleotides of the population may include modified nucleic acids. The modified nucleic acids may include peptide nucleic acids.

[0019] The detection moiety may include biotin or a derivative thereof. The detection moiety may include a hapten. The detection moiety may include a fluorescent moiety. The detection moiety may include at least one chelated metal ion. The detection moiety may include a reactive moiety that reacts with a reagent to produce chemiluminescence. The detection moiety may include a molecular barcode. The molecular barcode may include an oligonucleotide. Each member of the population may be connected to a common detection moiety that includes the same molecular barcode.

[0020] In a population of nucleotides corresponding to M different sequences, the common detection moiety is unique to one oligonucleotide population corresponding to only one of the M different sequences. The oligonucleotide may comprise a DNA sequence, and wherein the DNA sequence may be attached to the 3' end of a member of the oligonucleotide population.

[0021] The test portion may include a tissue section. The tissue section may include a formalin-fixed paraffin-embedded tissue section. The test sample may include a portion of the specimen extracted to obtain the remaining portion of the specimen after the nucleic acid sample is obtained. The test sample may include a leukocyte smear. The leukocytes may include peripheral blood mononuclear cells (PBMC).

[0022] Exposing the test sample to members of the first set of probes and the second set of probes may include: contacting the test sample with a composition characterized by all members of the first set of probes and the second set of probes; and removing members of the first set of probes and the second set of probes that do not hybridize to the test sample from contact with the test sample.

[0023] The first group of probes may include J different types of probes, each of the J different types of probes corresponding to one of the first group of sequences, the second group of probes may include K different types of probes, each of the K different types of probes corresponding to one of the second group of sequences, each member of the first group of probes and the second group of probes may correspond to only one of the J different types of probes, or correspond to only one of the K different types of probes, each member of the first group of probes and the second group of probes includes a detection portion characterized by a molecular barcode that is unique to only one probe type among the J different types of probes and the K different types of probes; and the molecular barcode may include an oligonucleotide.

[0024] Performing a hybridization analysis may include: (a) exposing a test sample to a detection probe set, each member of the detection probe set being characterized by an oligonucleotide sequence that hybridizes to a single type of molecular barcode and a fluorescent portion attached to the oligonucleotide sequence; (b) detecting fluorescence emission from members of the detection probe set that hybridize to the molecular barcode in the test sample; (c) removing detection probes that hybridize to the molecular barcode in the sample from the test sample; and (d) repeating steps (a)-(c) using other detection probe sets.

[0025] The first set of probes may include J different types of probes, each of the J different types of probes corresponding to one of the first set of sequences, the second set of probes may include K different types of probes, each of the K different types of probes corresponding to one of the second set of sequences, and exposing the test sample to members of the first and second sets of probes may include: contacting the test sample with a composition characterized by one of the J different types of probes and one of the K different types of probes, and removing any probes of the composition that do not hybridize with the test sample from contact with the test sample. Performing a hybridization analysis may include: detecting a detection moiety attached to one of the J different types of probes, and detecting a detection moiety attached to one of the K different types of probes.

[0026] The detection moiety attached to one of the J different types of probes may include biotin or a derivative thereof, and detecting the detection moiety may include: combining the detection moiety with a detection probe characterized by a moiety that binds to biotin or a derivative thereof and a fluorescent moiety, and detecting fluorescence emission from the fluorescent moiety after the detection moiety binds to the detection probe. The moiety that binds to biotin or a derivative thereof may include at least one of avidin and streptavidin.

[0027] The detection moiety attached to one of the J different types of probes may include a hapten, and detecting the detection moiety may include: combining the detection moiety with a detection probe characterized by a moiety that binds to the hapten and a fluorescent moiety, and detecting fluorescence emission from the fluorescent moiety after the detection moiety binds to the detection probe. The moiety that binds to the hapten may include at least one member selected from the group consisting of a protein, a polypeptide, a polysaccharide, or a liposome.

[0028] The detection moiety attached to one of the J different types of probes may include a fluorescent moiety, and detecting the detection moiety may include detecting fluorescent emission from the fluorescent moiety.

[0029] The detection moiety attached to one of the J different types of probes may include at least one chelated metal ion, and detecting the detection moiety may include: releasing the chelated metal ion from the detection moiety; and detecting the released metal ion by mass spectrometry.

[0030] The detection moiety attached to one of the J different types of probes may include a reactive group, and detecting the detection moiety may include: exposing the reactive group to a reagent that reacts with the reactive group to produce chemiluminescent emission, and detecting the chemiluminescent emission after the reaction.

[0031] Embodiments of the method may also include any other features described herein, and may include any combination of features, including combinations of those described in relation to different embodiments, unless explicitly stated otherwise.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this article, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification including definitions shall prevail. In addition, materials, methods and embodiments are illustrative only and are not intended to be restrictive.

[0033] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a flow chart illustrating a series of example steps for identifying the co-occurrence of nucleic acid segments in a sample.

[0036] Figure 2 is a table showing example CDR3 sequences derived from specific nucleic acid segments of nucleic acid fragments extracted from a sample.

[0037] Figure 3 is a schematic diagram of a nucleic acid segment probe.

[0038] Figure 4 is a schematic diagram of the detection probe.

[0039] Figure 5 is a schematic diagram of a system for obtaining fluorescent and chemiluminescent images indicative of probe hybridization of nucleic acid segments in a sample.

[0040] Fig. 6A is a schematic diagram of a fluorescence image showing fluorescence emission corresponding to nucleic acid segments coexisting in two sample cells.

[0041] Figure 6B is a schematic diagram of a set of two fluorescence images showing the co-existence of fluorescence emissions corresponding to different nucleic acid segments in a sample.

[0042] Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE INVENTION

[0044] Chimeric antigen receptor (CAR) T cell immunotherapy can be used to treat a variety of immune diseases and is readily applicable to cancers where circulating T cells are extracted from the patient's blood. The extracted T cells can be reengineered for surface expression of the CAR, replicated in large numbers for administration, and then infused into the patient to specifically target tumor cells. Commercial therapies exist, particularly for lymphomas, and target surface antigens common in heme malignancies, such as CD19.

[0045] Developing therapies targeting solid tumors is more challenging, in part because the cells of such tumors typically express fewer surface markers. However, neoantigens are generated in such tumors through somatic mutations and can be expressed on the surface of solid tumor cells. Neoantigen-specific T cells direct the patient's immune anti-tumor response. Therefore, a promising approach for immunotherapy of solid tumors involves reengineering T cells to express surface antigen receptors that interact with the patient's neoantigens. Cloning and infusing reengineered T cells into patients can produce more effective therapeutic outcomes than other more conventional treatments.

[0046] Neoantigen specificity is mediated by the T cell receptor, which is a heterodimer composed of an alpha (α) and beta (β) subunit pair. There are commercial methods to determine the nucleic acid sequences that specifically encode the α and β chain fragments in the T cell receptor. However, for samples extracted from tumor tissue and containing a large number of α and β chain encoding nucleic acid fragments, sequencing results in the distribution of the coding sequences of the two chain fragments without indicating which pair of coding sequences (i.e., α and β coding sequence pairs) produces a functional T cell receptor.

[0047] Identifying T cell receptors with anti-tumor function is a prerequisite for engineered T cell therapy. By sequencing nucleic acids extracted from such samples and identifying the co-existing nucleic acid encoding segments of α and β receptor chain fragments in the α and β coding segment population, specific functional T cell receptors can be identified. The co-existence information of specific α and β coding segments can then be used to engineer a population of T cells with functional neoantigen receptors, thereby having anti-tumor function.

[0048] Next generation sequencing methods can be readily used to obtain T cell receptor α and β coding sequences. However, identifying which of the tens or hundreds of α coding segments obtained from a typical sample is functionally paired with which of the tens or hundreds of β coding segments obtained from the same sample remains a challenging problem. In addition, certain existing analytical methods are not applicable to solid tumor tissues, especially formalin-fixed paraffin-embedded samples, which is the preferred clinical form of most tumor specimens.

[0049] The present disclosure features a method for identifying the coexistence of nucleic acid segments in a biological sample. The method can be used for a variety of different samples, including tissue sections and blood smears. Sample-specific probes are generated from nucleic acids extracted from the sample, and the probes are hybridized to the sample in series or in parallel. Detecting hybridization events associated with a particular single probe provides information about which nucleic acid segments are present in the sample and, therefore, which polypeptide chain fragments are co-expressed in the sample. In the case where the polypeptide chain fragments are paired chain fragments corresponding to functional receptors, the co-expression information directly leads to the identification of functional receptors in the sample.

[0050] As described above, an important application of the above method is to provide engineered T cell therapy targeting solid tumors. For such applications, the methods described herein can be applied to identify co-existing nucleic acid segments encoding co-expressed α and β chain fragments of functional T cell neoantigen receptors in a sample. The following discussion describes such applications in detail to provide illustrative examples of the methods.

[0051] However, the methods described herein are not limited to the co-existence analysis of nucleic acid segments encoding α and β chain fragments of T cells. Instead, the method can be used for the co-existence analysis of nucleic acid segments encoding various polypeptides (or fragments thereof) in many different types of cells and samples. In some embodiments, for example, the method can be used to determine the co-existence of nucleic acid segments encoding other polypeptides (including other chain fragments) in T cells.

[0052] In certain embodiments, the method can be used to determine the coexistence of nucleic acid segments encoding polypeptides or fragments thereof in other types of cells. For example, B cells can be engineered to provide immunoglobulin therapy, as long as functional B cells can be identified. B cells contain paired heavy and light chain fragments, and identifying which B cells are functional for engineering and cloning B cells for effective therapeutic purposes involves determining the coexistence of nucleic acid segments encoding heavy and light chain fragments in a sample. Using this information, B cells containing coexisting nucleic acid segments can be engineered and will show therapeutic efficacy.

[0053] Similarly, B cells expressing a particular antigen-specific antibody protein can be used to produce sufficient quantities of the antibody so that the antibody can be delivered as a therapeutic agent, or it can be linked to a separate therapeutic moiety, which can be a small molecule or other therapeutic immune cell used to target the moiety to a tumor or other cell type of interest.

[0054] Figure 1 1 is a flow chart 100 showing an example set of steps (e.g., a workflow) for identifying co-occurrence of nucleic acid segments. In a first step 102, a nucleic acid sample is obtained from a specimen. The specimen is typically a tissue sample removed from an organism, such as by biopsy. Suitable specimens can be obtained from a variety of organisms, for example, human specimens, or specimens removed from any other mammalian or non-mammalian organism, including mice, rats, avian organisms, and simian organisms.

[0055] Specimens can generally be of various types. In some embodiments, for example, specimens are fresh tissue samples. In certain embodiments, specimens are fresh frozen tissue samples. Specimens can also be fixed in formalin (or other fixatives), embedded in a structural medium, and then sliced ​​(e.g., in a microtome) to form tissue sections (e.g., formalin-fixed paraffin embedded (FFPE)).

[0056] The specimen can be derived from any of a variety of different types of tissue. In some embodiments, for example, the specimen is extracted from tumor tissue in a patient. The tumor can be a solid tumor, removed by biopsy or dissection during or after surgery.

[0057] In certain embodiments, the specimen corresponds to one or more cells circulating in a body fluid. For example, the specimen may include one or more leukocytes derived from a patient's blood sample. Suitable leukocytes include, but are not limited to, for example, peripheral blood mononuclear cells (PBMCs).

[0058] After obtaining a suitable specimen, a nucleic acid sample is extracted from the specimen. In some embodiments, for example, where the specimen is a tissue section, the specimen can be stained with one or more chromogenic dyes and imaged to identify different parts of the specimen. For specimens including tumor tissue, chromogenic staining can be used to reveal tumor margins and identify cells suitable for nucleic acid extraction. More generally, any of a variety of techniques can be used to identify cells in a specimen suitable for nucleic acid extraction.

[0059] Once the appropriate one or more cells are identified, the one or more cells are isolated from the specimen and nucleic acid is extracted from the one or more cells. Various techniques can be used to isolate one or more cells from the specimen. For example, in some embodiments, laser capture microdissection is used to isolate one or more cells from the specimen. Methods for performing laser capture microdissection are disclosed in, for example, Emmert-Buck et al., Science 274:998-1001 (1996) and Espina et al., Expert Rev. Mol. Diagn. 7(5):647-657 (2007), each of which is incorporated herein by reference in its entirety.

[0060] Various types of cells (eg, target immune cells) can be isolated from the specimen for nucleic acid extraction. In certain embodiments, for example, the isolated cells are tumor infiltrating lymphocytes, which are functional lymphocytes responsible for the immune response to tumor cells in the specimen.

[0061] Next, a nucleic acid sample is extracted from one of the isolated cells. Depending on the nature of the nucleic acid, various methods can be used for nucleic acid extraction. In order to extract DNA, techniques such as cesium chloride gradient centrifugation and solid phase extraction can be used. Suitable examples of such methods include, but are not limited to, those described in Ali et al., Biomed. Res. Int. 2017: 9306564 (2017), the entire contents of which are incorporated herein by reference. In order to extract RNA, techniques such as guanidine thiocyanate-phenol-chloroform extraction can be used. Examples of RNA extraction and separation methods are described in, for example, Doleshal et al., J. Mol. Diagn. 10 (3): 203–211 (2008), Peirson et al., Methods Mol. Biol. 362: 315–327 (2007) and Chomcyznski et al., Nat. Protocols 1 (2): 581–585 (2006), the entire contents of each of which are incorporated herein by reference.

[0062] As can be seen from the previous discussion, the nucleic acid sample extracted from the specimen can be a DNA sample or an RNA sample. In some embodiments, the nucleic acid sample corresponds to genomic DNA extracted from the nucleus of one or more cells such as tumor infiltrating lymphocytes. In certain embodiments, the nucleic acid sample corresponds to total RNA isolated from one or more cells (e.g., tumor infiltrating lymphocytes) isolated from the specimen.

[0063] Although in some cases, RNA isolation is more difficult due to the presence of resistant RNases in the cells that degrade the isolated RNA chains, in some embodiments, RNA as a nucleic acid sample provides certain advantages. First, the relative abundance of RNA in isolated cells can more accurately reflect the underlying biology of T cells, where the most activated tumor-specific T cells can express higher amounts of new antigen-specific receptor transcripts. In order to clone therapeutically effective T cell populations based on co-existence information, this intrinsic mapping of relative T cell activation and RNA abundance may generate co-existence information that can be used to engineer T cell populations and improve efficacy.

[0064] Secondly, in some embodiments, it may be easier to obtain sequence information from RNA samples than from corresponding DNA samples, depending on the portion of the nucleic acid sample that is sequenced. For the segments encoding the complementarity determining region 3 (CDR3) region of the T cell receptor alpha and beta chains, there is a 5 kb intron between the hypervariable diversity / junction and constant genes spliced ​​out in the RNA, which increases the chances of capturing the complete receptor coding sequence, including the CDR3 region, by sequencing the RNA rather than the genomic DNA. As will be explained in more detail below, sequencing the CDR3 region may be particularly relevant for identifying the coexistence of nucleic acid segments encoding functional T cell receptors.

[0065] exist Figure 1 The nucleic acid sample obtained from the specimen in step 102 includes a plurality of nucleic acid fragments associated with the expression of a protein or polypeptide of interest in the specimen. As discussed above, in some embodiments, the expressed polypeptide is a functional T cell neoantigen receptor, particularly the α and β chains of the neoantigen receptor. Therefore, the nucleic acid fragments correspond to partial or complete coding fragments of the α and β chains of the neoantigen receptor.

[0066] To initially determine whether two nucleic acid segments co-exist in a nucleic acid sample, the sequences of the two nucleic acid segments in each sample fragment are determined in step 104. Generally, any two nucleic acid segments in each sample nucleic acid fragment can be tested for co-occurrence. However, certain segments may be more correlated than other segments in some circumstances.

[0067] The nucleic acid sequences encoding the α and β chain portions of the new antigen receptor may be particularly present in the complementary determining regions 1, 2 and 3 (CDR1, CDR2, CDR3). Among CDR1, CDR2 and CDR3, the CDR3 region is particularly highly variable due to natural VDJ recombination in T cells and B cells. In these cells, variable (V), diversity (D) and joining (J) gene segments are randomly assembled to produce unique antigen (or new antigen) receptors that can recognize different antigens and new antigens. When a T cell or B cell successfully recognizes an antigen or a new antigen, signal transduction occurs, and the gene segment encoding the receptor enters immune memory.

[0068] Due to the high variable diversity in the CDR3 region, the nucleic acid sequences in this region are almost absolutely unique. In other words, each cell (e.g., T cell or B cell) will have a different nucleic acid sequence in this region, which acts as a type of molecular barcode for the cell. Therefore, by comparing nucleic acid segments within the CDR3 region of nucleic acid fragments, the coexistence of two specific segments can be easily determined, and the chance of error due to the non-uniqueness of the segment sequence is relatively small.

[0069] To determine the sequences of the two nucleic acid segments of interest in step 104, various public and commercially available next generation sequencing methods can be used. Suitable sequencing methods include, but are not limited to, massively parallel signature sequencing, polymerase clone (polony) sequencing, 454 sequencing, Illumina sequencing, ion torrent sequencing, SOLiD DNA sequencing technology, and DNA nanoball sequencing. Aspects of suitable sequencing methods are described in, for example, Rajesh et al., Current Developments in Biotechnology and Bioengineering, pp. 143-158 (2017), the entire contents of which are incorporated herein by reference.

[0070] Sequencing nucleic acid segments of nucleic acid fragments derived from a plurality of cells produces a distribution of segment sequences, each segment sequence being present at a different frequency in the nucleic acid sample. Figure 2 is a table showing a portion of the distribution of nucleic acid segment sequences obtained from different nucleic acid fragments in a sample. Figure 2 Each sequence in the table corresponds to the same portion of different nucleic acid fragments in the sample (e.g., a portion of the CDR3 region in multiple fragments). The left column of the table shows each segment sequence, and the right column shows the frequency of each sequence in the nucleic acid sample.

[0071] Sequencing the two nucleic acid segments in each nucleic acid fragment of the sample generates two sets of sequences: a first set of sequences corresponding to the first nucleic acid segment of each fragment, and a second set of sequences corresponding to the second nucleic acid segment of each fragment. In each set of sequences, different sequences exist at different frequencies, and the most common sequences correspond to the most common cells in the specimen.

[0072] The number of base pairs of the first nucleic acid segment, the second nucleic acid segment, or both the first nucleic acid segment and the second nucleic acid segment can generally be selected as desired. In some embodiments, for example, the number of base pairs is 50 to 200 (e.g., 50 to 190, 60 to 190, 60 to 180, 70 to 180, 70 to 170, 70 to 160, 75 to 150, 80 to 140, 80 to 130, 80 to 120).

[0073] The first set of sequences, the second set of sequences, or both the first set of sequences and the second set of sequences can generally include any number of different sequences, depending on the number of specimen cells from which the nucleic acid sample is derived. In some embodiments, for example, the number of different sequences is 10 or more (e.g., 12 or more, 15 or more, 20 or more, 30 or more, 40 or more, 45 or more, 48 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, or even more).

[0074] Back to Figure 1 , in the next step 106, a first set of probes is generated from the first set of sequences determined in step 104. Optionally, step 104 may also include sorting the first set of sequences in frequency order, and the most frequent sequences appear earlier. Further, in certain embodiments, sequences in the first set that occur less frequently relative to other sequences may be eliminated from the first group so that the first group includes only N different most frequently occurring sequences. N may be, for example, 10 or more (e.g., 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 48 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, or even more).

[0075] The first set of sequences (optionally limited to the N most frequent sequences, as described above) is then used to generate a first set of probes for the specimen. To generate the first set of probes, M different sequences from the first set of different (or N different) sequences can be used (e.g., the M most abundant sequences in the first set). M can be equal to or less than N, and in some embodiments, can be 48 or less (e.g., 40 or less, 30 or less, 24 or less, 20 or less, 10 or less).

[0076] To generate the first set of probes, a population of oligonucleotides is synthesized for each of the M different sequences. Each oligonucleotide in a given population corresponds to only one of the M different sequences and is linked to a detection moiety to form a probe. Figure 3 is a schematic diagram showing a probe comprising an oligonucleotide 302 attached to a detection moiety 304. Just as the oligonucleotide 302 corresponds to only one of the M different sequences, the detection moiety 304 also corresponds to only one of the M different sequences. Thus, after cycling through each of the M different sequences, M different populations of probes are obtained, such that each population of probes corresponds to only one of the M different sequences, and the probes of the population include an oligonucleotide 302 and a detection moiety 304 that are unique to only one of the M different sequences.

[0077] Typically, the oligonucleotides synthesized for probe generation may correspond to different types of nucleic acids. In some embodiments, for example, the oligonucleotide may include a DNA sequence (e.g., a DNA sequence complementary to each of the above-mentioned M different sequences). In certain embodiments, the oligonucleotide may include an RNA sequence (e.g., an RNA antisense sequence hybridized to M different RNA transcript sequences). In some embodiments, the oligonucleotide may be a synthetic and / or modified nucleic acid-containing substance, such as a peptide nucleic acid (PNA) and / or a heterologous nucleic acid (XNA).

[0078] Can use a variety of different methods to synthesize oligonucleotides from 24 different sequences.The example of this type of method includes but is not limited to the method based on phosphoramidite, the method based on H-phosphonate and the phosphotriester method.The method for oligonucleotide and peptide nucleic acid synthesis is described in, for example Herdewijn, P. (ed.), Oligonucleotide Synthesis, Springer (2005), and Braasch et al., "Synthesis and purification of peptide nucleic acids," Current Protocols in Nucleic Acid Chemistry, Chapter 4 (2002), the entire contents of which are incorporated herein by reference.

[0079] After the oligonucleotide colony is synthesized, each oligonucleotide 302 is connected to a detection portion 304 to form a probe. The detection portion 304 promotes the hybridization detection of each probe. Typically, the detection portion can correspond to a molecular fragment that produces a detectable signal, or alternatively, corresponds to a molecular fragment that is bonded to another fragment that produces a detectable signal. A variety of different detection portions can be used to form the above-mentioned probe.

[0080] In some embodiments, the detection portion 304 includes biotin or a derivative thereof. Methods for attaching biotin and its derivatives to the oligonucleotide 302 include, for example, by reacting with N-biotin-6-aminohexanoic acid N-hydroxysuccinimide ester, binding the biotin substituent to the allylamino residue of the functionalized oligonucleotide, extending RNA with poly(A) polymerase, and reacting the N-hydroxysulfosuccinimide ester attached to biotin with the primary oligonucleotide amine. Other methods for attaching biotin and its derivatives to the oligonucleotide 302 are described in, for example, Cook et al., Nucleic Acids Res. 16 (9): 4077-4095 (1988), in Moritz et al., RNA, 20 (3): 421-427 (2014), and Soukup et al., Bioconj. Chem. 6: 135-138 (1995), the entire contents of each of which are incorporated herein by reference.

[0081] In certain embodiments, the detection moiety 304 comprises a hapten. Suitable methods for conjugating a hapten to the oligonucleotide 302 (sometimes referred to as a hapten tag) include phosphoramidite-based methods described, for example, in Luehrsen et al., J. Histochem. Cytochem. 48(1):133-145 (2000), the entire contents of which are incorporated herein by reference.

[0082] In some embodiments, the detection moiety 304 includes a fluorescent moiety. A variety of different fluorescent moieties can be used, including, but not limited to: xanthene-based fluorophores, such as fluorescein, rhodamine, Oregon green, eosin, and Texas red; cyanine-based fluorophores, such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine; squaryl-based fluorophores, including squaryl rotaxane derivatives; naphthalene-based fluorophores, coumarin-based fluorophores, oxadiazole-based fluorophores, such as pyridine oxazole, nitrobenzoxadiazole, and benzoxadiazole; anthracene-based fluorophores, such as anthraquinone, pyrene-based fluorophores; oxazine-based fluorophores, such as Nile red, Nile blue, cresyl violet, and malachite green; and tetrapyrrole-based fluorophores, such as porphyrin, phthalocyanine, and bilirubin. Methods for attaching fluorophores to oligonucleotide 302 are described, for example, in Proudnikov et al., Nucl. Acids Res. 24(22):4535-4542 (1996), the entire contents of which are incorporated herein by reference.

[0083] In certain embodiments, the detection portion 304 includes at least one chelated metal ion. The probe with the chelated metal ion can be detected by releasing the metal ion from the probe and detecting the released ion using mass spectrometry. Suitable types of metal ions include but are not limited to lanthanide metal ions. The chelating portion of such ions can be realized as one or more metal chelating groups combined with the polymer backbone. Examples of such chelating portions and methods for preparing oligonucleotide-metal chelates are described in Majoris et al., Anal. Chem. 82 (21): 8961-8969 (2010), and Kwiatskowski et al., Nucl. Acids. Res. 22 (13): 2604-2611 (1994), the entire contents of which are incorporated herein by reference.

[0084] In some embodiments, detection portion 304 includes a molecular barcode. Typically, a molecular barcode is an oligonucleotide with a specific sequence that is unique to a specific probe population. Each probe in a specific population (corresponding to a single sequence in M ​​sequences) includes the same molecular barcode. However, the molecular barcode is not connected to the oligonucleotide in any other population. In this way, the probes of each population are contained in a common molecular barcode that is unique in different probe populations.

[0085] The oligonucleotide used as a molecular barcode can generally be a DNA sequence, an RNA sequence or a modified nucleic acid sequence, such as a peptide nucleic acid sequence. The oligonucleotide can include any number of bases or base pairs (e.g., 5 to 200, 10 to 200, 15 to 200, 20 to 200, 20 to 180, 20 to 150, 30 to 150, 20 to 150, 15 to 200, 15 to 200, 20 to 150, 20 to 150, 40 to 150, 50 to 150, 50 to 100, 10 to 50, 10 to 40, 10 to 30, 20 to 50, 30 to 50).

[0086] In certain embodiments, each oligonucleotide sequence corresponding to a unique The barcode is attached to the oligonucleotide 302 (eg, at the 3' end of the oligonucleotide 302). Barcodes are available from Akoya Biosciences (Menlo Park, CA). Methods for attaching these barcodes to oligonucleotides are described, for example, in Goltsev et al., Cell 174(4):968-981 (2018) and U.S. Pat. No. 9,909,167, the entire contents of each of which are incorporated herein by reference.

[0087] In some embodiments, the detection part 403 includes a reaction part that produces chemiluminescence. Specifically, the reaction part reacts with the second substance introduced during the detection step to produce luminescence that can be detected to identify the presence of the probe. Various reaction parts can be connected to the oligonucleotide 302 to produce a chemiluminescent probe. Examples of such reaction parts include but are not limited to luminol and its derivatives. Horseradish peroxidase (HRP) catalyzes luminol to be converted into 3-aminophthalate. When carried out in the presence of an enhancer, chemiluminescence is easily observed and allows detection of extremely low probe concentrations. The method for connecting the reaction part for chemiluminescent detection to the oligonucleotide is described in, for example, Khan et al., Appl.Biochem.Biotechnol.173 (2): 333-355 (2014), the entire contents of which are incorporated herein by reference.

[0088] The M probe populations generated as described above form a first set of probes corresponding to the first nucleic acid segment. After the first set of probes is generated, a second set of probes corresponding to the second nucleic acid fragment is generated in step 108. The method for generating the second set of probes in step 108 can generally correspond to any of the methods for forming the first set of probes in step 106 described above, and thus are not repeated. After completing step 108, a first set of probes corresponding to the first nucleic acid segment (e.g., a segment encoding a portion of a T cell receptor alpha chain) and a second set of probes corresponding to the second nucleic acid segment (e.g., a segment encoding a portion of a T cell receptor beta chain) have been generated.

[0089] Next, the test sample is exposed to one or more members of the first and second sets of probes in step 110. Exposure of the probe sets can be performed in different ways, depending on the nature of the subsequent hybridization analysis to determine which probes hybridize to the test sample.

[0090] In some embodiments, the test sample is exposed to the probe in a "paired" manner. The test sample is exposed to a probe population corresponding to one of the M sequences of the first nucleic acid segment and a probe population corresponding to one of the M sequences of the second nucleic acid segment. Therefore, the sample is exposed to only two different types of probes (for example, one corresponding to the α chain encoding fragment and one corresponding to the β chain encoding fragment) at the same time. After hybridization analysis (discussed further below), the two different types of probes can be removed (e.g., by dehybridization) or quenched from the test sample, and then the test sample is reused by exposing the test sample to another pair of different probe populations corresponding to the first nucleic acid segment and the second nucleic acid segment, respectively, or a new test sample is obtained and exposed to the next pair of probe populations. Repeat the paired exposure cycle until all paired combinations of probe populations corresponding to the first nucleic acid segment and the second nucleic acid segment have been hybridized and analyzed.

[0091] In certain embodiments, the test sample is exposed to the probe in a "continuous" manner. That is, the test sample is exposed to only one type of probe population, which corresponds to one of the M sequences of the first nucleic acid segment or the second nucleic acid segment. Therefore, the sample is exposed to only one type of probe at the same time. After the hybridization analysis, the type of probe is removed from the test sample (e.g., by dehybridization) or quenched, and then the test sample is reused by exposing the test sample to another single probe population corresponding to one of the first nucleic acid segment and the second nucleic acid segment, or a new test sample is obtained and exposed to the next probe population. Repeat the single population exposure cycle until all probe populations corresponding to the first nucleic acid segment and the second nucleic acid segment have been hybridized and analyzed separately.

[0092] In some embodiments, the test sample is exposed to the probes in a "pooled" manner. In other words, the test sample is exposed to more than one type of probe population corresponding to more than one of the M sequences of the first nucleic acid segment, and / or is exposed to more than one type of probe population corresponding to more than one of the M sequences of the second nucleic acid segment. After the hybridization analysis, the probe is removed from the test sample (e.g., by dehybridization) or quenched, and the test sample is exposed using a new combination of different types of probes. Alternatively, a new test sample can be obtained and exposed to a new combination of different types of probes. The exposure cycle is repeated until one or more test samples have been exposed to all combinations of different probe types corresponding to the first nucleic acid segment and the second nucleic acid segment, so that each pair of different sequences corresponding to the first nucleic acid segment and the second nucleic acid segment can be identified.

[0093] In some embodiments, the test sample is exposed to the first set of probes and the second set of probes in a fully combined manner. In other words, each different type of probe population corresponding to the first nucleic acid segment and the second nucleic acid segment is combined and hybridized to the test sample at the same time. By using a fully combined exposure strategy, the coexistence of all pairs of nucleic acid segment sequences can be evaluated using only a single hybridization cycle, thereby greatly simplifying the workflow and correspondingly reducing the assay time.

[0094] The test sample is typically obtained from the same specimen as the nucleic acid sample described in step 102. In some embodiments, the test sample corresponds to the remainder of a sample from which the nucleic acid sample has been excised using a technique such as laser capture microdissection (e.g., a FFPE tissue section). In certain embodiments, the test sample corresponds to a separate tissue section obtained from the specimen, and can be a fresh section, a fresh frozen section, or a FFPE tissue section.

[0095] Alternatively, in some embodiments, the test sample is a smear of cells obtained from a body fluid. For example, the test sample may correspond to a blood smear mounted on a substrate and comprising leukocytes (eg, peripheral blood mononuclear cells).

[0096] Back to Figure 1 , interleaved with the exposure step in step 110, a hybridization analysis is performed in step 112 to determine which of the different types of probes hybridize to the test sample. Typically, the first step of the hybridization analysis involves washing the unhybridized probes from the test sample so that such probes do not generate a measurement signal. After this washing step, the nature of the hybridization analysis depends on the nature of the detection moiety present in the probe.

[0097] Hybridization analysis determines which of the probes exposed by the test sample hybridize with the test sample. Therefore, hybridization analysis includes detecting the detection probe of hybridization, and more specifically, detecting the detection part of the detection probe of hybridization. The method of detecting the detection part depends on the nature of the detection part.

[0098] As described above, in some embodiments, the detection portion 304 is directly detectable. For example, in the case where the detection portion 304 includes a fluorescent portion, the probe hybridized with the detection sample can be directly detected by measuring the fluorescence emission from the detection sample, as will be discussed in more detail below. In the case where the detection portion 304 includes a chelated metal ion, the probe hybridized with the detection sample can be directly detected by releasing the metal ion and using mass spectrometry to detect the metal ion. The mass spectrometry-based detection method of metal ion labeled samples is described in, for example, Keren et al., "MIBI-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure," Sci. Adv. 5 (10): eaax5851 (2019), and Angelo et al., Nat. Medicine 20: 436-442 (2014), the entire contents of each of which are incorporated herein by reference.

[0099] In contrast, in certain embodiments, the detection moiety 304 is not directly detectable, and a detection probe is conjugated to the detection moiety 304 to allow for hybridization of the detection probe. Figure 4 400. The probe 400 includes a binding group 402 and a label 404. Typically, the label 404 includes a fluorescent portion that produces fluorescent emission, which is detected to identify hybridization of the probe with the test sample. Any fluorescent portion and its derivatives discussed above can generally be used in the label 404.

[0100] The nature of the binding group 402 depends on the nature of the probe detection portion 304. In embodiments where the detection portion 304 includes biotin or a derivative thereof, the binding group may include a moiety that binds to biotin or a derivative thereof. Examples of such moieties include, but are not limited to, avidin or streptavidin-based binding groups conjugated to biotin to attach the detection probe 400 to the detection portion 304.

[0101] In embodiments where the detection moiety 304 comprises a hapten, the binding group 402 may comprise a moiety that binds to the hapten. Examples of suitable moieties include, but are not limited to, proteins, polypeptides, polysaccharides, and liposomes.

[0102] In embodiments where the detection portion 304 includes a reactive portion that produces chemiluminescence, the probe detection portion 304 is typically not conjugated to the detection probe 400. Instead, the detection portion 304 is exposed to a reagent that reacts with the reactive portion to produce detectable chemiluminescence in the test sample. A variety of different reagents can be used, depending on the nature of the reactive portion. For example, where the reactive portion is based on luminol, the reagent may include an enzyme that reacts with luminol to produce chemiluminescence, such as horseradish peroxidase.

[0103] In embodiments where the detection moiety 304 is an oligonucleotide such as a molecular barcode, the binding group 402 can be an oligonucleotide that is at least partially complementary to the detection moiety 304 and hybridizes to the detection moiety 304. The degree of complementarity between the detection moiety 304 and the binding group 402 can be 70% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or even 100%).

[0104] In the detection section 304 is In the embodiment of barcode (obtained from Akoya Biosciences), the detection probe 400 may correspond to a complementary sequence consisting of a dye-labeled reporter sequence. Report material. Hybridization of the reporter to the detection moiety 304 produces a conjugated probe structure from which fluorescent emission can be detected to identify hybridization of a specific probe to the test sample.

[0105] remove In addition to the reporter molecule, the detection probe 400 may more broadly correspond to a variety of fluorescent in situ hybridization (FISH) probes. Such probes generally include an oligonucleotide binding group 402 and a conjugated fluorophore. Measuring the fluorescence emission from the fluorophore shows the hybridization of the probe conjugated to the corresponding FISH probe. In certain embodiments, the FISH detection probe may include a detection probe 400, which includes an oligonucleotide binding group 402 and a plurality of coupling groups. Suitable coupling groups include, for example, antigens. After the FISH detection probe is hybridized with the detection portion 304, a plurality of amplification probes each consisting of a fluorophore conjugated to a complementary coupling group are introduced and bound to the FISH detection probe. Suitable complementary coupling groups include, for example, antibodies. In this way, the amplified FISH detection probe can be hybridized with the nucleic acid segment probe of the sample hybridization, thereby increasing the intensity of the fluorescence emission indicating the hybridization of the nucleic acid segment probe.

[0106] The nucleic acid characteristics of the binding group 402 depend on the nucleic acid characteristics of the detection portion 304. For example, when the detection group 304 is an oligonucleotide having a double-stranded DNA sequence, the binding group 402 corresponds to a complementary single-stranded DNA sequence. The detection portion 304 can be denatured before the binding group 402 hybridizes with one of the double strands of the detection portion 304. When the detection portion 304 is a single-stranded DNA sequence, the hybridization of the binding group 402 can be performed directly. When the detection portion 304 is an oligonucleotide having an RNA sequence, the binding group 402 corresponds to an antisense RNA sequence that is complementary to at least a portion of the RNA sequence.

[0107] As described above, when the detection moiety 304 reacts to produce chemiluminescence, or when the detection moiety 304 includes a fluorescent moiety (or is attached to a detection probe 400 that includes a fluorescent moiety), light emission from the detection sample is measured to determine which probes are hybridized to the detection sample. Figure 5 A schematic diagram of a system 500 for detecting light emission (e.g., chemiluminescence, fluorescence emission) from a test sample is shown. The system 500 includes a light source 502, an optical assembly 504, a stage 506 that supports a test sample 508, a wavelength-dependent reflective element 510, a filter assembly 512, and a detector 514. To detect chemiluminescence from the test sample 508, the light source 502, the reflective element 510, and the filter assembly 512 can optionally be omitted from the system 500.

[0108] Detector 514 is an imaging detector such as a CCD detector, and obtains one or more images of light emitted from the test sample 514. Chemiluminescence is directly generated by the probes in the test sample and imaged by the detector 514. To measure fluorescence from the probes in the test sample, the light source 502 (e.g., a multi-diode array) generates excitation light in a selected wavelength band, which passes through the reflective element 510 and is directed to the test sample 508 by the optical assembly 504. The excitation light excites fluorescence emission from the hybridized probes in the test sample 508, and the emitted fluorescence is reflected by the reflective element 510, passes through the filter assembly 512 and is detected by the detector 514. Note that in the following discussion, fluorescence measurement is discussed by way of example, but it should be understood that the described methods are equally applicable to detecting probe hybridization in the test sample by measuring chemiluminescence.

[0109] The nature of the image acquired by the detector 514 depends on the nature of the hybridization analysis in step 112. In general, images of fluorescence emission in a single band or in multiple bands can be acquired by the system 500. In some embodiments, in the case of using a continuous probe exposure strategy as described above, and optionally, in the case of using a paired or merged probe exposure strategy, an image of the detection sample showing fluorescence emission in a single band is acquired. Thus, for example, in a paired exposure strategy in which a different fluorescent marker is attached to each of two different types of probes hybridized to the sample, two fluorescent images of the sample can be obtained, each image corresponding to a different band of fluorescence emission associated with one of the fluorescent markers. For a merged probe exposure strategy, in which Q different fluorescent markers are respectively attached to one of Q different and corresponding hybridization sample probe types, Q fluorescent images of the detection sample are obtained, each Q image corresponding to emission from a different probe type from the Q probe types.

[0110] In certain embodiments, when paired or combined exposure strategies are used so that the test sample includes multiple different types of hybridization probes, a multi-band fluorescence image can be obtained that displays fluorescence emission in multiple spectral bands, each corresponding to the emission from a different fluorescent label attached to a different type of probe.

[0111] In some embodiments, as described above, the probes in each population may include probes corresponding to molecular barcodes such as The barcode detection portion 304 can be combined to form a composition including probes corresponding to all sequences corresponding to the first nucleic acid segment and the second nucleic acid segment. The fully combined probes can then be hybridized to the test sample simultaneously in a single hybridization step. Barcoding facilitates probe hybridization detection, Reporters can be used in a multiplexed fashion to perform multiple detection cycles, with each cycle producing a sample fluorescence image in which fluorescence from multiple Report emission.

[0112] For example, in the first detection cycle, a plurality (e.g., 3, 4, 5 or even more; usually k) of The reporter is hybridized to k nucleic acid segment probes, which are hybridized to the test sample. A fluorescence image of the test sample is obtained that shows fluorescence emission in k different spectral bands, wherein each spectral band corresponds to a different k One of the reporters thus corresponds to one of the different hybridized nucleic acid segment probes. The reporter is washed out of the test sample and a new analysis cycle begins, in which a new k different The reporter set is hybridized to a different k nucleic acid segment probe set, which is hybridized to the test sample. Another fluorescent image showing emission light in k different spectral bands is obtained, each spectral band corresponding to one of the different new k nucleic acid segment probe sets, and then the new k nucleic acid segment probe sets are washed out from the test sample. Reporting substance group. The hybridization-imaging-washing cycle continues until hybridization of each different probe type in the test sample has been analyzed.

[0113] For each different type of probe, the extent of hybridization of the probe to the test sample is determined by the presence of fluorescence emission corresponding to the probe in the sample image. If the probe does not show fluorescence emission, the probe has not hybridized to the test sample (presumably having been washed out of the sample before imaging). If the probe shows fluorescence emission in the sample image, the probe is hybridized to the sample. Since each type of probe is associated with one of the sequences corresponding to the first nucleic acid segment or the second nucleic acid segment, hybridization of a particular type of probe to the test sample indicates the presence of a nucleic acid segment in the test sample corresponding to the sequence associated with the hybridized probe. Therefore, by imaging the fluorescence emission from each different type of probe, the presence of each type of nucleic acid segment sequence in the test sample can be assessed.

[0114] It should also be noted that while the foregoing and subsequent discussions involve fluorescence measurements to identify the presence of nucleic acid segments in a sample, other methods described herein (e.g., detection of released metal ions, chemiluminescence) may also be used as a similar analysis workflow as non-fluorescent images.

[0115] Back to Figure 1Once hybridization of different probes to the test sample is determined and the presence of a specific nucleic acid segment sequence has been identified, the co-localization of the specific nucleic acid segments in a single cell can be determined. Typically, for a specific sample cell, if the fluorescence emission corresponding to the two nucleic acid segments is co-localized in the sample fluorescence image, then the two specific nucleic acid segments co-exist in the sample cell. If the fluorescence image shows emission corresponding to the two nucleic acid segments in the same image, co-localization is easy to identify because the fluorescence emission comes from a common spatial location in the image.

[0116] For example, Fig. 6A 602 and 604 in the image, fluorescence emission corresponding to each nucleic acid segment probe is observed. Therefore, the nucleic acid segments coexist in the cells at positions 602 and 604 in the sample, but not in other sample cells.

[0117] In the case where fluorescence emission from two nucleic acid segments is shown in different sample images, the spatial position of the fluorescence emission in the different sample images is measured. If the spatial position in the different images corresponds to within the error threshold of the assay, the two nucleic acid segments are considered to coexist in the cell. If the difference in spatial position is too large, the two nucleic acid segments do not coexist in the cell.

[0118] For example, Figure 6B Two schematic fluorescence images are shown, each of which shows fluorescence emission corresponding to a different nucleic acid segment probe hybridized to the sample. At most positions in the two images, no fluorescence emission is observed, or fluorescence emission corresponding to only one nucleic acid segment probe is observed. However, at position 606 in the left figure, fluorescence emission corresponding to the first nucleic acid segment probe is observed, and at position 606 in the right figure, fluorescence emission corresponding to the second nucleic acid segment probe is observed. Therefore, the two nucleic acid segments coexist in the cells at position 606 in the sample, but not in other sample cells.

[0119] Once a sample fluorescence image is obtained, the aforementioned analysis can be quickly applied to determine which pair of nucleic acid segments coexist in each of a plurality of sample cells. For specimens in which the first nucleic acid segment and the second nucleic acid segment correspond to the α chain and β chain coding sequences of functional T cells, the coexistence information shows which combinations of α chain and β chain coding sequences can produce functional T cells. This information can then be used to engineer specific T cell clonal populations for targeted immunotherapy.

[0120] application

[0121] Information about the coexistence of nucleic acid segments can be used for a variety of different therapeutic applications, especially for the synthesis of therapeutic agents for delivery to patients. As described above, due in part to the lack of reliable biomarkers, the development of therapies for the treatment of solid tumor malignancies has not been as rapid as the development of therapies for hematological malignancies. However, solid tumor cells produce new antigens that are recognized by surface neoantigen receptors of functional T cells. Therefore, the coexistence information of nucleic acid segments corresponding to the alpha chain and beta chain of the neoantigen receptor of functional T cells can be used to engineer a population of functional T cells, which can then be therapeutically administered to patients.

[0122] A variety of different methods for T cell engineering using coexistence information obtained as described herein can be used to prepare therapeutic functional T cell populations. Examples of such methods are described in, for example, Li et al., Signal Transduction and Targeted Therapy 4:35 (2019), the entire contents of which are incorporated herein by reference.

[0123] Information about the coexistence of nucleic acid segments can also be used as part of a treatment program for targeting B cell engineering. In particular, the coexistence information of nucleic acid segments encoding the heavy chain and light chain fragments of the B cell receptor can be used to identify functional B cells. The therapeutic B cell population can then be engineered and delivered to the patient. Suitable methods for B cell engineering of therapeutic B cell lines are described in, for example, Moffett et al., Science Immunology 4 (35): eaax0644 (2019), Wu et al., J. Immunol. Methods 457: 33-40 (2018) and Johnson et al., Sci. Reports 8: 12144 (2018), the entire contents of each of which are incorporated herein by reference.

[0124] Other Implementations

[0125] Although the present disclosure describes specific embodiments, these should not be interpreted as limitations on the scope of the present disclosure, but should be interpreted as feature descriptions in certain embodiments. Features described in the context of separate embodiments may also be implemented in combination in a single embodiment. On the contrary, the various features described in the context of a single embodiment may also be implemented in multiple embodiments or in any suitable sub-combination, respectively. Moreover, although the above features may be described as existing in certain combinations, and even initially claimed as such, one or more features in the claimed combination may be removed from the combination, and the claimed combination may involve a sub-combination or a variation of a sub-combination.

[0126] In addition to the embodiments explicitly disclosed herein, it will be understood that various modifications may be made to the described embodiments without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A method for identifying the co-existence of nucleic acid segments in a nucleic acid sample from a specimen, the method comprising: Obtaining a nucleic acid sample from a specimen, wherein the nucleic acid sample comprises a plurality of nucleic acid fragments corresponding to a portion or all of the coding fragments of an antigen receptor molecule expressed in the specimen; Determining the sequences of the first and second nucleic acid segments in the nucleic acid fragments of the sample to generate a first set of sequences corresponding to the first nucleic acid segment in the sample and a second set of sequences corresponding to the second nucleic acid segment; generating a first set of probes from said first set of sequences, wherein each member of said first set of probes comprises an oligonucleotide linked to a detection moiety, said oligonucleotide corresponding to a different sequence of said first set of sequences; generating a second set of probes from the second set of sequences, wherein each member of the second set of probes comprises an oligonucleotide linked to a detection moiety, the oligonucleotide corresponding to a different sequence of the second set of sequences; exposing a test sample obtained from the specimen to members of the first set of probes and members of the second set of probes; performing a hybridization analysis to determine whether members of the first set of probes hybridize to the test sample, and to determine whether members of the second set of probes hybridize to the test sample; as well as It is determined whether the first and second nucleic acid segments co-exist in a common cell in the specimen.

2. The method of claim 1, wherein the antigen receptor molecule is a T cell antigen receptor molecule.

3. The method of claim 2, wherein the first nucleic acid segment corresponds to a coding fragment of part or all of the α chain of the T cell antigen receptor molecule, and wherein the second nucleic acid segment corresponds to a coding fragment of part or all of the β chain of the T cell antigen receptor molecule.

4. The method of claim 1, wherein the antigen receptor molecule is a B cell antigen receptor molecule.

5. The method of claim 4, wherein the first nucleic acid segment corresponds to a portion or all of the coding fragment of the heavy chain of the B-cell antigen receptor molecule, and wherein the second nucleic acid segment corresponds to a portion or all of the coding fragment of the light chain of the B-cell antigen receptor molecule.

6. The method of claim 3, wherein the first and second nucleic acid segments are located in the complementarity determining region 3 (CDR3) portion of the nucleic acid fragment.

7. The method of claim 1, wherein the nucleic acid sample comprises genomic DNA.

8. The method of claim 1, wherein the nucleic acid sample comprises total RNA.

9. The method of claim 1, wherein the nucleic acid sample comprises nucleic acid molecules from tumor infiltrating lymphocytes.

10. The method of claim 1, wherein the specimen comprises a fresh or frozen tumor tissue specimen.

11. The method of claim 10, wherein obtaining the nucleic acid sample from the specimen comprises: fixing and embedding the specimens in paraffin; resecting a portion of the specimen; and The nucleic acid sample is extracted from the excised portion of the specimen.

12. The method of claim 11, wherein the resected portion of the specimen comprises target immune cells.

13. The method of claim 12, wherein the target immune cells comprise tumor infiltrating lymphocytes.

14. The method of claim 1, wherein the sequences of the first and second nucleic acid segments each comprise 50 to 200 base pairs.

15. The method of claim 1, wherein the sequences of the first and second nucleic acid segments each comprise 75 to 150 base pairs.

16. The method of claim 1, wherein the first and second sets of sequences each comprise at least 20 different sequences.

17. The method of claim 16, wherein the first and second sets of sequences each comprise at least 50 different sequences.

18. The method of claim 1, wherein the first set of sequences comprises N different sequences, and wherein generating the first set of probes comprises: For each of the M different sequences that are expressed with the highest abundance in the nucleic acid sample, generating a population of oligonucleotides corresponding to the sequence; and Each member of the oligonucleotide population is linked to a detection moiety.

19. The method of claim 18, wherein M equals N.

20. The method of claim 18, wherein M < N.

21. The method of claim 18, wherein M is 48 or less.

22. The method of claim 18, wherein M is 24 or less.

23. The method of claim 18, wherein the population of oligonucleotides comprises DNA sequences.

24. The method of claim 23, wherein the DNA sequence is complementary to the M different sequences.

25. The method of claim 18, wherein the population of oligonucleotides comprises RNA sequences.

26. The method of claim 25, wherein the RNA sequence is an antisense RNA sequence.

27. The method of claim 26, wherein the antisense RNA sequence is complementary to an RNA transcript sequence corresponding to M different sequences.

28. The method of claim 18, wherein the population of oligonucleotides comprises modified nucleic acids.

29. The method of claim 28, wherein the modified nucleic acid comprises a peptide nucleic acid.

30. The method of claim 18, wherein the detection moiety comprises biotin or a derivative thereof.

31. The method of claim 18, wherein the detection moiety comprises a hapten.

32. The method of claim 18, wherein the detection moiety comprises a fluorescent moiety.

33. The method of claim 18, wherein the detection moiety comprises at least one chelated metal ion.

34. The method of claim 18, wherein the detection moiety comprises a molecular barcode.

35. The method of claim 34, wherein the molecular barcode comprises an oligonucleotide.

36. The method of claim 34, wherein each member of the population is linked to a common detection moiety comprising the same molecular barcode.

37. The method of claim 36, wherein among the population of nucleotides corresponding to the M different sequences, the common detection portion is unique to a population of oligonucleotides corresponding to only one of the M different sequences.

38. The method of claim 36, wherein the oligonucleotide comprises a DNA sequence, and wherein the DNA sequence is linked to the 3' end of a member of the population of oligonucleotides.

39. The method of claim 18, wherein the detection moiety comprises a reactive moiety that reacts with a reagent to produce chemiluminescence.

40. The method of claim 1, wherein the test sample comprises a tissue section.

41. The method of claim 40, wherein the tissue sections comprise formalin-fixed, paraffin-embedded tissue sections.

42. The method of claim 40, wherein the detecting the sample comprises a remaining portion of the specimen after extracting a portion of the specimen to obtain the nucleic acid sample.

43. The method of claim 1, wherein the test sample comprises a leukocyte smear.

44. The method of claim 43, wherein the leukocytes comprise peripheral blood mononuclear cells (PBMCs).

45. The method of claim 1, wherein exposing the test sample to members of the first and second sets of probes comprises: contacting the test sample with a composition comprising all members of the first and second sets of probes; and Members of the first and second sets of probes that do not hybridize to the test sample are removed from contact with the test sample.

46. ​​The method of claim 45, wherein: The first group of probes includes J different types of probes, each of the J different types of probes corresponds to one of the first group of sequences; The second group of probes includes K different types of probes, each of the K different types of probes corresponds to one of the second group of sequences; Each member of the first and second sets of probes corresponds to only one of the J different types of probes, or corresponds to only one of the K different types of probes; Each member of the first and second sets of probes comprises a detection moiety comprising a molecular barcode that is unique to only one of the J and K different types of probes; and The molecular barcode comprises an oligonucleotide.

47. The method of claim 46, wherein performing the hybridization analysis comprises: (a) exposing the test sample to a detection probe set, each member of the detection probe set comprising an oligonucleotide sequence hybridized to a single type of molecular barcode and a fluorescent moiety attached to the oligonucleotide sequence; (b) detecting fluorescence emission from members of the detection probe set hybridized to the molecular barcode in the detection sample; (c) removing the detection probe hybridized to the molecular barcode in the sample from the detection sample; and (d) Repeat steps (a)-(c) using other detection probe sets.

48. The method of claim 1, wherein: The first group of probes includes J different types of probes, each of the J different types of probes corresponds to one of the first group of sequences; The second group of probes includes K different types of probes, each of the K different types of probes corresponds to one of the second group of sequences; and Exposing the test sample to members of the first and second sets of probes comprises contacting the test sample with a composition comprising one of the J different types of probes and one of the K different types of probes, and removing any probes of the composition that do not hybridize with the test sample from contact with the test sample.

49. The method of claim 48, wherein performing the hybridization analysis comprises: The detection moiety attached to one of the J different types of probes is detected, and the detection moiety attached to one of the K different types of probes is detected.

50. The method of claim 49, wherein the detection moiety attached to one of the J different types of probes comprises biotin or a derivative thereof, and wherein detecting the detection moiety comprises: combining the detection moiety with a detection probe comprising a moiety that binds to biotin or a derivative thereof and a fluorescent moiety; and Fluorescent emission from the fluorescent moiety is detected after binding of the detection moiety to the detection probe.

51. The method of claim 50, wherein the moiety that binds to biotin or a derivative thereof comprises at least one of avidin and streptavidin.

52. The method of claim 49, wherein the detection moiety attached to one of the J different types of probes comprises a hapten, and wherein detecting the detection moiety comprises: combining the detection moiety with a detection probe comprising a moiety that binds to a hapten and a fluorescent moiety; and Fluorescent emission from the fluorescent moiety is detected after binding of the detection moiety to the detection probe.

53. The method of claim 52, wherein the moiety that binds to the hapten comprises at least one member selected from the group consisting of a protein, a polypeptide, a polysaccharide, or a liposome.

54. The method of claim 49, wherein the detection moiety attached to one of the J different types of probes comprises a fluorescent moiety, and wherein detecting the detection moiety comprises: Fluorescent emission from the fluorescent moiety is detected.

55. The method of claim 49, wherein the detection moiety attached to one of the J different types of probes comprises at least one chelated metal ion, and wherein detecting the detection moiety comprises: releasing the chelated metal ions from the detection moiety; and The released metal ions were detected by mass spectrometry.

56. The method of claim 49, wherein the detection moiety attached to one of the J different types of probes comprises a reactive group, and wherein detecting the detection moiety comprises: exposing the reactive group to a reagent that reacts with the reactive group to produce chemiluminescent emission; and The reaction is followed by detection of the chemiluminescent emission.

Images