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  • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

• This present disclosure relates to systems, methods, and compositions useful for profiling T cell receptor (TCR) and B cell receptor (BCR) repertoire using next-generation sequencing (NGS) methods.
• TCR T cell receptor
• BCR B cell receptor
• NGS next-generation sequencing
• the present disclosure also relates to systems and methods for diagnosing, treating, or predicting infection, disease, conditions, or therapeutic outcome, or efficacy based on the TCR/BCR profile data from a subject in need thereof.
• the methods comprise detecting SARS-CoV-2 exposure.
• the vertebrate immune system is comprised of two main arms: the innate arm and the adaptive arm.
• the innate arm of the immune system has evolved to quickly and effectively respond to foreign antigens or danger signals. However, in many cases an innate immune response is not sufficient to provide sterilizing immunity.
• the adaptive arm of the immune system has no capacity for “memory,” meaning that a more effective response to a pathogen cannot be made upon subsequent challenges by the same pathogen or a similar pathogen. Therefore, the innate arm of the immune system (and/or non-immune cells, for example, infected cells) presents antigens to the adaptive immune system, which can then begin the process of selection of antigen-specific immune cells, T lymphocytes (T cells) and B lymphocytes (B cells). This process is facilitated by the presence of an enormous diversity of antigen-specific cells to be available to respond to any antigenic challenge.
• the method comprises a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes and enriching for a targeted whole transcriptome panel using a set of transcriptome hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; and e) analyzing the sequencing data to determine the TCR/BCR profile of the patient.
• the set of TCR/BCR hybrid-capture probes comprises a first pool comprising BCR constant region probes, a second pool comprising BCR non-constant region probes, a third pool comprising TCR constant region probes, a fourth pool comprising TCR non-constant region probes, and a fifth pool comprising transcriptome hybrid-capture probes.
• the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100. In some embodiments, the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1:2.5:100:100:10. In some embodiments, 2% or less of the reads in the sequencing data map to TCR/BCR genes. In some embodiments, the sample is a blood sample.
• step (d) comprises identifying a plurality of TCR/BCR clones in the sample, and/or comprises identifying the most abundant TCR/BCR clones in the sample, and/or comprises identifying the most abundant non-constant region sequences in the sample.
• step (c) comprises whole transcriptome sequencing or shortread sequencing.
• the patient's BCR/TCR profile is compared with a control TCR/BCR profile and the patient is identified as having a disease or medical condition based on the comparison.
• the disease or condition is an infectious disease, a cancer, an autoimmune disease, or an allergy.
• the cancer or infectious disease is one or more provided in the list in embodiment 114.
• the infectious disease comprises exposure to SARS-CoV-2.
• the subject is suspected of having or has been diagnosed with COVID-19.
• the disease is cancer.
• analyzing comprises determining the presence or extent of tumor lymphocyte infiltration.
• the methods comprises treating the patient with a therapy.
• the therapy comprises an immunotherapeutic agent.
• the immunotherapeutic agent is a vaccine.
• the immunotherapeutic agent is a chimeric antigen receptor (CAR) T cell.
• step (d) comprises identifying the most abundant TCR non-constant region sequences in the sample, and wherein the treatment administered in step (e) comprises administering a CAR-T cell therapy, wherein the CAR-T cell comprises at least one of the most abundant TCR non-constant region sequences.
• methods for characterizing the effect of a therapy on the TCR/BCR profile of a patient comprise a) at a first time point; i) isolating RNA from a sample from the patient; ii) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes and enriching for a targeted whole transcriptome panel using a set of transcriptome hybrid-capture probes; iii) determining the sequence of the RNA of (b) to generate sequencing data; and iv) analyzing the sequencing data to determine the TCR/BCR profile of the patient; and b) at a second time point: i) isolating RNA from a sample from the patient; ii) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes and enriching for a targeted whole transcriptome panel using a set of transcriptome hybrid-capture
• the first time point is before a therapy has been administered and the second time point is a time after the therapy has been administered.
• the first time point comprises a first time during a first course of therapy, and the second time point is at a second time during treatment with a first therapy, or is after the course of treatment with the first therapy has finished.
• a third, fourth, fifth or Nth time point is analyzed.
• One or more of the Nth time points may be time points used in longitudinal testing, e.g., during the course of a therapy, during the course of a clinical trial, or before, during or after multiple therapies.
• the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100. In some embodiments, the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1:2.5:100:100:10. In some embodiments, 2% or less of the reads in the sequencing data map to TCR/BCR genes. In some embodiments, the sample is a blood sample.
• the sample comprises a blood sample or a solid tumor sample.
• step (c) comprises whole-transcriptome sequencing or short-read sequencing.
• a method of determining the TCR/BCR profile of a patient who has COVID-19 or another disease comprises a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes and enriching for a targeted whole transcriptome panel using a set of whole transcriptome hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; and d) analyzing the sequencing data to determine the TCR/BCR profile of the patient, wherein the set of TCR/BCR hybrid-capture probes comprises a first pool comprising TCR constant region probes, a second pool comprising TCR non-constant region probes, a third pool comprising BCR constant region probes, a fourth pool comprising BCR non-constant region probes, and a fifth pool comprising transcriptome hybrid-capture probes.
• the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100. In some embodiments, the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1:2.5:100:100:10. In some embodiments, 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• the sample is a blood sample. In some embodiments, the patient's TCR/BCR profile is compared to a SARS- CoV-2 TCR/BCR positive control profile, and in some embodiments, a determination of whether the patient has been exposed to SARS CoV-2 is made. In some embodiments, the subject is treated if the determining indicates exposure to SARS-CoV-2.
• a method of determining SARS CoV-2 exposure in a patient comprises a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid- capture probes and enriching for a targeted whole transcriptome panel using a set of transcriptome hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; and d) analyzing the sequencing data to determine the TCR/BCR profile of the patient; and e) comparing the TCR/BCR profile of the patient to a positive control to determine SARS-CoV-2 exposure; wherein the set of TCR/BCR hybrid-capture probes comprises a first pool comprising TCR constant region probes, a second pool comprising TCR non-constant region probes, a third pool comprising BCR constant region probes, and a fourth pool comprising BCR non-constant region
• the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100. In some embodiments, the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1:2.5:100:100:10. In some embodiments, 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• the sample is a blood sample. In some embodiments, the patient has been exposed to or is suspected to have been exposed to SARS-CoV-2. In some embodiments, the patient is experiencing flu-like symptoms or symptoms associated with a respiratory disease. In some embodiments, the method comprises treating the patient for SARS-CoV-2 exposure, if the patient is determined to have been exposed to SARS-CoV-2.
• step (c) comprises whole- transcriptome sequencing or short-read sequencing.
• FIG. 1A-B presents an exemplary TCR/BCR immune repertoire display (report) illustrating additional or alternative fields for review by a physician.
• A presents an exemplary TCR/BCR immune repertoire display (report) illustrating additional or alternative fields for review by a physician.
• B An example TCR/BCR immune repertoire display (report), showing patient clonality after analysis with novel hybrid- capture approach, in this case related to BCR clonality.
• Fig. 11. (Left) is a scatter-plot showing the number of TCR Beta productive clonotypes vs. the normalized Shannon entropy within each immune profile from 501 human cancer samples that were sequenced using the novel hybrid-capture approach disclosed herein. A higher normalized Shannon entropy is correlated with increased diversity in clonotypes within the sample.
• transcriptome refers to the full range of messenger RNA molecules expressed by an organism, a particular tissue, or a particular cell.
• a transcriptome can be defined at a particular point in time, for example, at a particular developmental stage, in a particular disease stage, etc.
• “Whole transcriptome” refers to the coding and non-coding RNA expressed in cells, tissues, organs and/or an entire body.
• “Whole transcriptome sequencing” or “whole transcriptome profile” refers to the measurement of the complete complement of transcripts in a sample at a given time. Whole transcriptome sequencing captures both coding (mRNA) and non-coding transcripts (such as miRNA, tRNA, rRNA, if rRNA is of interest), and provides a "snapshot" of expression levels, exons, introns, and variants. In some embodiments, whole transcriptome sequencing starts with the removal of rRNA from the sample (rRNA typically takes up a majority of the sequencing reads).
• whole exome sequencing refers to sequencing the protein-coding regions of the genome, typically using NGS sequencing methods.
• the human exome represents less than 2% of the genome, but contains -85% of known disease-related variants, making this method a cost-effective alternative to whole-genome sequencing.
• whole exome sequencing is performed comprising an exome enrichment step, using an exome targeting panel to enrich for exome sequences (and to omit non-coding sequences, for example).
• exome targeting panel to enrich for exome sequences (and to omit non-coding sequences, for example).
• Such panels are commercially available, and typically include probes to enrich 5,000, 10,000, 20,000 genes or more.
• a non-limiting exome panel is Integrated DNA Technologies xGen Exome Research Panel v2.
• targeted panel and “targeted gene sequencing panel” or “targeting panel” are used interchangeably herein to refer to a probe set directed to a select set of genes or gene regions of interest. Targeted panels are useful tools for detecting a set of specific sequences in a given sample.
• a targeted panel produces a smaller, more manageable data set (e.g., TCR/BCR profile) as compared to broader approaches such as whole-genome sequencing.
• a targeted panel comprises a whole exome panel, or a whole transcriptome panel, and encompasses 5,000, 10,000, 20,000, or more targets.
• a targeted panel comprises hybrid capture probes.
• Hybridization-capture probes or “hybrid-capture probes,” as used herein refer to biotinylated oligonucleotides that contain a region of complementary to nucleic acid sequences of interest sufficient to bind (hybridize to) the nucleic acid sequences of interest and provide a means for their enrichment through the use of streptavidin linked capture moieties linked to a solid support structure, e.g. beads. In various embodiments, other capture moieties may be used instead of streptavidin and biotinylation. Examples of binding moieties include but are not limited to biotin: streptavidin, biotin: avidin, biotin:haba:streptavidin, antibody: antigen, antibody: antibody, covalent chemical linkage (ex. click chemistry).
• probe pools directed to cancer-specific sequences may be included with a BCR/TCR panel, instead of a whole transcriptome panel, a whole exome panel, or in addition to a whole transcriptome panel or a whole exome panel.
• nucleic acid refers to a covalently linked sequence of nucleotides (i.e., ribonucleotides for RNA and deoxyribonucleotides for DNA) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next.
• Sequenced nucleotides may be of any form of nucleic acid, including, but not limited to RNA, DNA and cfDNA molecules.
• RNA DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase.
• mRNA messenger RNA
• miRNA microRNA
• polynucleotide includes, without limitation, single- and double-stranded polynucleotide.
• the term “gene” refers to a nucleic acid sequence that encodes a gene product, either a polypeptide or functional RNA molecule.
• the term “gene” is to be interpreted broadly herein, encompassing both the genomic DNA form of a gene (i.e., a particular portion of a particular chromosome), and mRNA and cDNA forms of the gene produced therefrom.
• genomic DNA is transcribed into RNA, which can be immediately functional or can be translated into a polypeptide that performs a function.
• a gene comprises "noncoding regions".
• Genes may also comprise "insulator" elements that protect promoters from inappropriate regulation. Insulators may function by either blocking interaction with an enhancer or silencer or by acting as a barrier that prevents the spreading of condensed chromatin. While enhancers and silencers are generally not considered to be part of a gene per se (given that a single enhancer or silencer may regulate the expression of multiple genes), as used herein, the term gene encompasses those distal elements that influence its expression.
• sequences are used herein to refer to the series of nucleotides present in a DNA, RNA or cDNA molecule. In the context of the present invention, sequences are determined by sequencing nucleic acids present in a biological specimen.
• read refers to a DNA sequence of sufficient length (e.g., at least about 30 bp) that can be used to identify a larger sequence or region, e.g., by aligning it with a chromosome, genomic region, or gene.
• a read may be a paired-end or single-end read.
• reference genome refers to any particular known genome sequence, whether partial or complete, of any organism or virus which may be used to reference identified sequences from a subject. Many reference genomes are provided by the National Center for Biotechnology Information at www.ncbi.nlm.nih.gov.
• a “genome” refers to the complete genetic information of an organism or virus, expressed in nucleic acid sequences.
• alignment refers to a process used to identify regions of similarity.
• alignment refers to matching sequences with positions in a reference genome based on the order of their nucleotides in these sequences.
• Alignment can be performed manually or by a computer algorithm, for example, using the Efficient Local Alignment of Nucleotide Data (ELAND) computer program distributed as part of the Illumina Genomics Analysis pipeline. Alignment can refer to a either a 100% sequence match or a match that is less than 100% (non-perfect match). In various examples, alignment includes pseudo-alignment.
• library and “sequencing library” are used herein to refer to a pool of DNA fragments with adapters attached.
• Adapters are commonly designed to interact with a specific sequencing platform, e.g., the surface of a flow-cell (Illumina) or beads (Ion Torrent), to facilitate a sequencing reaction.
• RNA read count is used herein to refer to the number of sequencing reads generated from a genetic analyzer.
• RNA read count is often used to refer to the number of reads overlapping a given feature (e.g., a gene or chromosome).
• RNA or protein molecule may or may not be normalized using standard methods (e.g., counts per million, finding the base 10 logarithm of the raw read count) generated by a gene or other genetic regulatory region (e.g. long non-coding RNAs, enhancers), which may be defined by a chromosomal location or other genetic mapping indicator.
• standard methods e.g., counts per million, finding the base 10 logarithm of the raw read count
• a gene or other genetic regulatory region e.g. long non-coding RNAs, enhancers
• enriched or enrichment refers to the process of enhancing the amount of one or more nucleic acid species in a sample.
• exemplary enrichment methods may include chemical and/or mechanical means, and may also include amplifying nucleic acids contained in a sample.
• enrichment may include the use of hybrid-capture probes, and the polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• Enrichment can be sequence specific (for example using hybrid-capture probes or target-specific PCR primers) or nonspecific (i.e., involving any of the nucleic acids present in a sample).
• Enriched refers to an increased level or amount of the one or more biomolecules, such as nucleic acid or protein, as compared to a control level, or as compared to the other biomolecules in the sample (as a relative amount).
• enrichment refers to statistical enrichment.
• cancer refers to any one or more of a wide range of benign or malignant tumors, including those that are capable of invasive growth and metastases through a human or animal body or a part thereof, such as, for example, via the lymphatic system and/or the blood stream.
• tumor includes both benign and malignant tumors and solid growths.
• Typical cancers include but are not limited to carcinomas, lymphomas, or sarcomas, such as, for example, ovarian cancer, colon cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, urinary tract cancer, uterine cancer, acute lymphatic leukemia, Hodgkin's disease, small cell carcinoma of the lung, melanoma, neuroblastoma, glioma, and soft tissue sarcoma of humans.
• the therapeutic agent may be administered before, during or after the onset of disease or injury.
• the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest.
• the subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
• the term “effective amount” refers to an amount of an active agent that is sufficient to exhibit a detectable therapeutic effect without excessive adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of the present disclosure.
• the effective amount for a patient will depend upon the type of patient, the patient's size and health, the nature and severity of the condition to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on knowledge in the art and the information provided herein. The optimum dosing regimen can be determined by one skilled in the art without undue experimentation.
• lymphocyte receptors T cell receptors, "TCRs” and B cell receptors “BCRs”
• TCRs T cell receptors
• BCRs B cell receptors
• NGS Next Generation Sequencing
• the field of immune profiling leverages technology known as Next Generation Sequencing (NGS) to accurately sequence the immune receptor repertoire in an individual subject.
• NGS is capable of producing millions of sequencing “reads” which are then aligned to a reference genome or transcriptome to give a relatively complete picture of an individual's genome or a sample’s transcriptome.
• the technical and computational challenges, however, in assembling and analyzing reads to detect and assess T and B cell receptors are significant.
• High quality sequencing data depends on two factors known as breadth and depth of sequencing.
• the breadth of sequencing refers to the number of genome bases that are covered by the sequencing, or the percentage of total, while the depth of sequencing refers to roughly how many times a particular base or region is covered by the sequencing run.
• T and B cell receptors are made up of discrete genes that are rearranged to form the large repertoire present in an individual.
• any strategy to selectively enrich T and B cell receptor transcripts must be tailored to combat the low abundance of transcripts encoding lymphocyte receptors, and the variety of different genes that are assembled to encode recombined antigen receptors.
• sequencing reads mapping to TCRs and BCRs may not be balanced, causing a bias toward the detection of either TCR or BCR clones in a sample.
• the most critical information for immune profiling lies in the hypervariable (non constant) regions, and not in the constant regions. Thus, sequences for hypervariable regions may require enrichment.
• sample volume and/or quality can be limiting because it is derived from biopsies or other precious samples. Therefore, there is a need in the art for a method that can extract both high-quality RNA sequencing and also provide deep and accurate immune profiling at scale, from a single sample or sequencing run.
• the methods and systems of the present disclosure address this need in the art.
• T cells and B cells can be a powerful tool for mapping the immune system ("immunome") in cancer and other conditions, such as auto-immune disease, infectious disease, and transplantation.
• immunoome immune system
• Each non-clonal T cell and B cell is unique at the DNA level, and in particular, they differ at the T cell receptor (TCR) gene or B cell receptor (BCR) gene that determines the pathogen or antigen the cell will respond to.
• TCR T cell receptor
• BCR B cell receptor
• the immune system can be more accurately mapped, and a new class of immune-specific features can be generated to, for example, predict immune responses; diagnose or confirm diseases, conditions, or pathogen exposure; determine disease severity; measure or confirm therapeutic effect and efficacy; determine minimal residual disease (MRD); and provide the information necessary to produce specific therapies such as chimeric antigen receptor (CAR) T cells (CAR-T cells), NK cells (CAR- NK cells), macrophages (CAR-M cells), or another cell type engineered to express a CAR, Immune mobilizing monoclonal T-cell receptors against Cancer (ImmTAC), another adoptive cell therapy, and vaccines.
• CAR chimeric antigen receptor
• the set of hybrid-capture probes comprises a first pool comprising BCR constant region probes, a second pool comprising BCR non-constant region probes, a third pool comprising TCR constant region probes, and a fourth pool comprising TCR non-constant region probes.
• a TCR/BCR probe set is used that is obtained according to the methods of example 1.
• TCR/BCR profiling may be performed as a standalone assay.
• the TCR/BCR profiling methods and systems as disclosed herein may be configured for use within the context of a broader RNAseq whole transcriptome or whole exome RNA panel, thereby providing a novel and valuable method to conserve precious patient samples, speed time to diagnosis or therapy recommendation, and obtain, in addition to gene expression data and related genetic data (such as but not limited to alternative splicing events, fusions, and genetic variants), specific information about the subject's immune profile.
• TCR/BCR profiling may have expanded utility and an even more unique and valuable resource for insight generation.
• TCR/BCR profiling may be used to profile and track multiple disease states and related immune responses, including cancer, infectious disease, transplantation, allergic diseases (triggered by airway, food, or other allergens), and autoimmunity.
• Allergic diseases may include contact dermatitis, asthma, anaphylaxis, non-IgE-mediated food allergies related to atopic dermatitis, etc.
• Autoimmune diseases may include type 1 diabetes, rheumatoid arthritis, lupus, celiac disease, Sjogren’s syndrome, multiple sclerosis, polymyalgia rheumatica, ankylosing spondylitis, alopecia areata, vasculitis, temporal arteritis, etc.
• TCR/BCR profiling may include allele typing and may be used for biomarker discovery, for predicting immune response, health outcomes, and/or disease severity.
• TCR/BCR profiling may be included within the context of a broader RNAseq whole transcriptome panel. In an oncology or a more general profiling environment, TCR/BCR profiling may be added to other analysis on the rest of the transcriptome, such as cytokine expression, immune cell composition, potential viral/bacterial signals, and inflammatory signatures. Whole transcriptome analysis allows for the capture of that data while also providing a TCR/BCR snapshot.
• transcriptome analysis the study of the complete set of RNA transcripts that are produced by a cell (i.e., the transcriptome), and exome analysis, the study of RNAs that encode a protein product, offers a promising means to identify genetic variants that are correlated with disease state and disease progression.
• transcriptome and/or exome analysis may be performed on a sample collected from a patient that contains cancer cells. Suitable patient samples include tissue samples, tumors (e.g., a solid tumor), biopsies, lymph nodes, and bodily fluids (e.g., blood, serum, plasma, lymph, sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears, saliva).
• the first step in extracting RNA from a sample is often to lyse the cells present in that sample.
• Several physical disruption methods are commonly used to lyse cells, including, for example, mechanical disruption (e.g., using a blender or tissue homogenizer), liquid homogenization (e.g., using a dounce or French press), high frequency sound waves (e.g., using a sonicator), freeze/thaw cycles, heating, manual grinding (e.g., using a mortar and pestle), and bead beating (e.g., using a Mini-beadbeater-96 from BioSpec).
• mechanical disruption e.g., using a blender or tissue homogenizer
• liquid homogenization e.g., using a dounce or French press
• high frequency sound waves e.g., using a sonicator
• freeze/thaw cycles e.g., heating, manual grinding (e.g., using a mortar and pestle)
• bead beating
• RNA from degradation may also be lysed using reagents that contain a detergent, many of which are commercially available (e.g., QIAzol Lysis Reagent from QIAGEN, FastBreakTM Cell Lysis Reagent from Promega).
• a detergent many of which are commercially available (e.g., QIAzol Lysis Reagent from QIAGEN, FastBreakTM Cell Lysis Reagent from Promega).
• lysis buffers such as detergents or proteases (e.g., proteinase K) that increase the efficiency of lysis.
• Homogenization buffers may also include anti-foaming agents and/or RNase inhibitors to protect RNA from degradation.
• lysis techniques may be required to obtain the best possible yield from different tissues. Techniques that minimize the degradation of the released RNA and that avoid the release of nuclear chromatin are preferred.
• RNA can be separated from other cellular components.
• Total RNA is commonly isolated using guanidinium thiocyanate-phenol-chloroform extraction (e.g., using TRIzol) or by performing trichloroacetic acid/acetone precipitation followed by phenol extraction.
• guanidinium thiocyanate-phenol-chloroform extraction e.g., using TRIzol
• trichloroacetic acid/acetone precipitation followed by phenol extraction.
• column-based systems for extracting RNA e.g., PureLink RNA Mini Kit by Invitrogen and Direct-zol Miniprep kit by Zymo Research.
• the isolated RNA will contain very little DNA and enzymatic contamination.
• the isolation method may utilize agents that eliminate DNA (e.g., TURBO DNase-I), and/or remove enzymatic proteins from the sample (e.g., Agencourt® RNAClean® XP beads from Beckman Coulter).
• RNAs microRNAs
• siRNAs small interfering RNAs
• kits that have been designed to efficiently recover small RNAs (e.g., mirVanaTM miRNA Isolation Kit from Invitrogen).
• RNA is converted into a form that is suitable for next-generation sequencing (NGS).
• NGS next-generation sequencing
• the RNA is converted into a collection of DNA fragments known as a "sequencing library.”
• the resulting sequencing "reads" are aligned to a reference genome or transcriptome to determine the expression profile of the analyzed cells.
• library preparation is automated to enable higher sample throughput, minimize errors, and reduce hands-on time.
• Fully automated library preparation can be performed, for example, using a liquid handling robot (e.g., SciClone® NGSx from PerkinElmer).
• library preparation is automated to enable higher sample throughput, minimize errors, and reduce hands-on time.
• Fully automated library preparation can be performed, for example, using a liquid handling robot (e.g., SciClone® NGSx from PerkinElmer).
• RNA is converted to more stable, double-stranded complementary DNA (cDNA) using reverse transcription (RT).
• RT reverse transcription
• reverse transcription is performed directly on a sample lysate, prior to RNA isolation. In other cases, reverse transcription is performed on isolated RNA.
• Reverse transcription is catalyzed by reverse transcriptase, an enzyme that uses an RNA template and a short primer complementary to the 3' end of the RNA to synthesize a complementary strand of cDNA.
• This first strand of cDNA is then made double-stranded, either by subjecting it to PCR or using a combination of DNA Polymerase I and DNA Ligase.
• an RNase e.g., RNase H
• RNase H is commonly used to digest the RNA strand, allowing the first cDNA strand to serve as a template for synthesis of the second cDNA strand.
• an exonuclease e.g., Exonuclease I
• Exonuclease I may be added to the samples to degrade any primers that remain from the reaction, preventing them from interfering in subsequent amplification steps.
• Targeted sequencing may be used to study a select set of genes or specific genomic elements. Libraries that are enriched for target sequences are commonly prepared using hybridization based methods (i.e., hybridization capture-based target enrichment). Hybridization may be performed either on a solid surface (microarray) or in solution. In the solution based method, a pool of biotinylated oligonucleotide probes that specifically hybridize with the genes or genomic elements of interest is added to the library. The probes are then captured and purified using streptavidin-coated magnetic beads, and the sequences that hybridized to these probes are subsequently amplified and sequenced.
• probe panels for library enrichment are commercially available, including those from IDT (e.g., xGen Exome Research Panel vl.O and v2.0 probes) and Roche (e.g., SeqCap® probes). Many available probe panels can be customized, allowing investigators to design sets of capture probes that are precisely tailored to a particular application.
• kits e.g., SeqCap EZ MedExome Target Enrichment Kit from Roche
• hybridization mixes e.g., xGen Lockdown from IDT
• libraries are commonly treated with oligonucleotides that bind to adapter sequences (e.g., xGen Blocking Oligos) or to repetitive sequences (e.g., human Cot DNA) to reduce non-specific binding to the capture probes.
• adapter sequences e.g., xGen Blocking Oligos
• repetitive sequences e.g., human Cot DNA
• library preparation typically includes at least one amplification step to enrich for sequencing-competent DNA fragments (i.e.., fragments with adapter ligated ends) and to generate a sufficient amount of library material for downstream processing.
• Amplification may be performed using a standard polymerase chain reaction (PCR) technique.
• PCR polymerase chain reaction
• high-fidelity DNA polymerases e.g., KAPA HiFi DNA Polymerase from Roche
• PCR master mix e.g., NEBNext® High-Fidelity 2X PCR Master Mix from New England BioLabs
• kit e.g., KAPA HiFi Library Amplification kit by Roche.
• PCR conditions must be fine-tuned for each sequencing experiment, even when a highly-optimized PCR protocol is used. For example, depending on the initial concentration of DNA in the library and on the input requirement of the sequencer to be used, it may be desirable to subject the library to anywhere from 4-14 cycles of PCR.
• library preparation protocols include multiple rounds of library amplification. For example, in some cases, an additional round of amplification followed by PCR clean-up is performed after the libraries have been pooled.
• sequencing data may be normalized to accurately identify changes across experimental conditions. Normalization may be useful, for example, to address global changes in transcription between different experimental conditions.
• a "spike-in control" may be added to the sequencing libraries for normalization.
• the spike-in control constitutes DNA sequences that are added at a known ratio to, for instance, the specimens.
• the control DNA can be any DNA that is readily distinguished from the experimental cDNA during data analysis.
• control libraries commonly comprise synthetic DNA or DNA from an organism other than the organism of interest (e.g., a PhiX spike-in control may be added to a human-derived library).
• DNA is commonly fragmented into uniform pieces prior to sequencing.
• the optimal fragment length depends on both the sample type and the sequencing platform to be used. For example, whole genome sequencing typically works best with fragments of DNA that are -350 bp long, while targeted sequencing using hybridization capture (see Section 2G) works best with fragments of DNA that are -200 bp long.
• fragmentation is performed after reverse transcription (i.e., on cDNA).
• Suitable methods for fragmenting DNA include physical methods (e.g., using sonication, acoustics, nebulization, centrifugal force, needles, or hydrodynamics), enzymatic methods (e.g., using NEBNext dsDNA Fragmentase from New England BioLabs), and tagmentation (e.g., using the NexteraTM system from Illumina).
• a size selection step may subsequently be performed to enrich the library for fragments of an optimal length or range of lengths.
• size selection was accomplished by separating differentially sized fragments using agarose gel electrophoresis, cutting out the fragments of the desired sizes, and performing a gel extraction (e.g., using a MinElute Gel Extraction KitTM from Qiagen).
• size selection is now commonly accomplished using magnetic bead-based systems (e.g., AMPure XPTM from Beckman Coulter, ProNex® Size- Selective Purification System from Promega).
• Sequencing adapters are short DNA oligonucleotides that contain (1) sequences needed to amplify the cDNA fragment during the sequencing reaction, and (2) sequences that interact with the NGS platform (e.g., the surface of the Illumina flow-cell or Ion Torrent beads). Accordingly, adapters must be selected based on the sequencing platform that is to be used.
• Adapters may also include unique molecular identifiers (UMIs), short sequences, often with degenerate bases, that incorporate a unique barcode onto each molecule within a given sample library.
• UMIs unique molecular identifiers
• Many index sequences and adapter sets are commercially available including, for example, SeqCap Dual End Adapters from Roche, xGen Dual Index UMI Adapters from IDT, and TruSeq UD Indexes from Illumina.
• the amplified DNA is typically purified to remove enzymes, nucleotides, primers, and buffer components that remain from the reaction. Purification is commonly accomplished using phenol-chloroform extraction followed by ethanol precipitation or using a spin column that contains a silica matrix to which DNA selectively binds in the presence of chaotropic salts. Many column-based PCR cleanup kits are commercially available including, for example, those from Qiagen (e.g., MinElute PCR Purification Kit), Zymo ResearchTM (DNA Clean & ConcentratorTM-5), and Invitrogen (e.g., PureLinkTM PCR Purification Kit). Alternatively, purification may be accomplished using paramagnetic beads (e.g., AxygenTM AxyPrep MagTM PCR Clean-up Kit).
• Qiagen e.g., MinElute PCR Purification Kit
• Zymo ResearchTM DNA Clean & ConcentratorTM-5
• Invitrogen e.g., PureLinkTM PCR Purification Kit
• sequencing adapter ligation To keep sequencing cost-effective, clinical laboratory technicians or researchers often pool together multiple libraries, each with a unique barcode (see “sequencing adapter ligation," above), to be sequenced in a single run.
• the sequencer to be used and the desired sequencing depth should dictate the number of samples that are pooled. For example, for some applications it is advantageous to pool fewer than 12 libraries to achieve greater sequencing depth, whereas for other applications it may be advisable to pool more than 100 libraries.
• care should be taken to ensure that the sequencing coverage is roughly equal for each library. To this end, an equal amount of each library (based on molarity) should be pooled.
• the total molarity of the pooled libraries must be compatible with the sequencer. Thus, it is important to accurately quantify the DNA in the libraries (e.g., using the methods discussed in "Quality Control,” below) and to perform the necessary calculations before pooling the libraries. In some cases, to achieve a suitable total molarity, it may be necessary to concentrate the pooled libraries, e.g., using a vacufuge.
• pooling is performed twice.
• sequencer adapter ligation and pooling (for example, pooling approximately 5-10 samples) are performed before enrichment/library amplification and a second pooling step is performed after library clean-up.
• DNA quantification Prior to sequencing, libraries may be evaluated to ensure that they comprise DNA of sufficient quantity and quality to generate useful sequencing results. To verify that the concentration of the library is sufficient for loading on the sequencer, the DNA may be quantified. Commonly used methods of DNA quantification include gel electrophoresis, UV spectrophotometry (e.g., NanoDrop®), fluorometry (e.g., QubitTM, PicofluorTM), real-time PCR (also known as quantitative PCR), or droplet digital emulsion PCR (ddPCR). DNA quantification is often aided by the use of dyes and stains, of which an extensive assortment is commercially available (e.g., ethidium bromide, SYBR Green, RiboGreen®). Notably, given that the recommended input range is very narrow for NGS, it is preferable that a highly precise method of quantitation is used to verify that the concentration of the final library is suitable.
• UV spectrophotometry e.g., NanoDrop®
• amplification To sequence a library, the library is applied to a device, typically a flow cell (Illumina) or chip (Ion Torrent), in which the sequencing chemistry occurs. These devices are decorated with short oligonucleotides that are complementary to the adapter sequences, allowing the cDNAs in the library to attach to the device.
• clonal amplification e.g., by cluster generation (Illumina) or by microemulsion PCR (Ion Torrent)
• clonal amplification is performed using a commercially available kit (e.g., Paired-end Cluster Kit from Illumina).
• the library is ready for sequencing.
• the probe set includes probes targeting one or more loci not encoding a protein, for example, regulatory loci, miRNA loci, and other non-coding loci, e.g., that have been found, for example, to be associated with one or more particular disease or medical conditions (for example cancer).
• the plurality of loci include at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 750, 1000, 2500, 5000, or more human genomic loci.
• probes for enrichment of nucleic acids include DNA, RNA, or a modified nucleic acid structure with a base sequence that is complementary to a locus of interest.
• a probe designed to hybridize to a locus in a cDNA molecule can contain a sequence that is complementary to either strand, because the cDNA molecules may be double stranded.
• each probe in the plurality of probes includes a nucleic acid sequence that is identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive bases of a locus of interest.
• each probe in the plurality of probes includes a nucleic acid sequence that is identical or complementary to at least 20, 25, 30, 40, 50, 75, 100, 150, 200, or more consecutive bases of a locus of interest.
• probe sequences may be selected in accordance with the methods set forth in FastPCR Software for PCR Primer and Probe Design and Repeat Search (Kalendar et al., 2009 Genes, Genomes, and Genomics, 3 (Special Issue 1), pp. 1- 14) which is incorporated by reference herein.
• Targeted-panels provide several benefits for nucleic acid sequencing.
• panels targeting genes with high variability among individual subjects, humans, or even cells within subjects or humans may facilitate bioinformatics processing to determine the sequences of those genes. For example, if a “whole exome” or targeted sequencing panel is not generating a sufficient number of sequencing reads mapping to the high- variable genes, probes targeting the high-variable genes may be added to the whole exome or targeted sequence panel probes to increase the number of reads mapping to high-variable genes.
• the gene panel is a whole-exome panel that analyzes the exomes of a biological sample. In some embodiments, the gene panel is a whole-genome panel that analyzes the genome of a specimen. In some embodiments, the gene panel is a whole- transcriptome panel that analyzes the transcriptome of a specimen. In some embodiments, the gene panel is a targeted whole-transcriptome panel that analyzes the transcriptome of a specimen. In some embodiments, the gene panel is used in conjunction with a TCR/BCR gene panel (for example, to provide clinical decision support related to immunological profiles or immunomes).
• each read may be aligned to a predetermined immunological receptor gene sequence.
• a plurality of anchor windows may be identified, each window being associated with a plurality of reads that exceed a threshold value.
• Reads that align to a region of an anchor window called “anchor reads,” may be used to generate an anchor sequence from the anchor reads.
• the anchor windows, the anchor sequences, and the un-aligned reads may be provided to an assembly process to generate a contig sequence.
• Each contig sequence may be annotated or otherwise associated with at least one immunological gene region class selected from one of V, D , J, and C.
• portions of each contig sequence located outside of CDR3 region may be deleted. The number of contig sequences annotated and/or associated with each class may be quantified.
• each row represents a SARS-CoV-2 peptide and corresponding SARS- CoV-1 peptide that could be recognized by a T cell receptor.
• the table includes information about the T cell type (CD4 or CD8) of the TCR that recognizes the peptide and the protein/amino acid position of the peptide origin within the viral protein. See, Le Bert, N., Tan, A.T., Kunasegaran, K. et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 584, 457-462 (2020). https://doi.org/10.1038/s41586-020-2550-z, the contents of which are incorporated herein by reference in their entirety for all purposes.
• Table 2 includes human coronavirus peptides and corresponding amino acid positions and the source viral protein for each peptide. Peptides in the same row are homologous peptides from distinct coronaviruses. See, Mateus et al, DOI: 10.1126/science. abd3871, the contents of which are incorporated herein by reference in their entirety for all purposes.
• column 1 "VP” is viral protein
• column 2 "1st AA" is the position of first amino acid.
• Table 3 includes SARS-CoV-2 peptides, corresponding amino acid positions and the source viral protein for each peptide, and a compatible HLA for each peptide. See, Sekine et al, Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19, Cell (2020), doi: https://doi.Org/10.1016/j.cell.2020.08.017, the contents of which are incorporated herein by reference in their entirety for all purposes.
• Embodiments may include a single microservice for executing and delivering TCR/BCR profiling information or may include a plurality of microservices each having a particular role which together implement one or more of the embodiments above.
• a first microservice may execute TCR/BCR profiling in order to deliver profile results to a second microservice for reporting.
• the genetic analyzer system may include targeted panels and/or sequencing probes.
• a targeted panel is disclosed, for example, in U.S. Patent Application Nos. 16/789,288 and 15/930,234, filed February 12, 2020 and May 12, 2020, respectively which are incorporated herein by reference and in its entirety for all purposes.
• targeted panels may enable the delivery of next generation sequencing results for genes having a high degree of sequence variability among individuals and/or cells within an individual, including immunological genes (for example, TCR and BCR genes) according to an embodiment, above.
• An example of the design of next-generation sequencing probes is disclosed, for example, in U.S. Patent Application No. 17/706,704, titled “Systems and Methods for Next Generation Sequencing Uniform Probe Design”, and filed 10/21/20, which is incorporated herein by reference and in its entirety for all purposes.
• the methods and systems described above may be utilized after completion or substantial completion of the systems and methods utilized in the bioinformatics pipeline.
• the bioinformatics pipeline may receive next-generation genetic sequencing results and return a set of binary files, such as one or more BAM files, reflecting DNA and/or RNA read counts aligned to a reference genome.
• the methods and systems described above may be utilized, for example, to ingest the DNA and/or RNA read counts and produce TCR/BCR sequence profiling as a result.
• any RNA read counts may be normalized before processing embodiments as described above.
• An example of an RNA data normalizer is disclosed, for example, in U.S. Patent Application No. 16/581,706, titled “Methods of Normalizing and Correcting RNA Expression Data”, and filed 9/24/19, which is incorporated herein by reference and in its entirety for all purposes.
• the digital and laboratory health care platform further includes application of one or more of the above in combination with or as part of a medical device or a laboratory developed test that is generally targeted to medical care and research, such laboratory developed test or medical device results may be enhanced and personalized through the use of artificial intelligence.
• An example of laboratory developed tests, especially those that may be enhanced by artificial intelligence, is disclosed, for example, in U.S. Provisional Patent Application No. 62/924,515, titled “Artificial Intelligence Assisted Precision Medicine Enhancements to Standardized Laboratory Diagnostic Testing”, and filed 10/22/19, which is incorporated herein by reference and in its entirety for all purposes.
• the present disclosure provides methods to analyze the number of clones and distribution of clones of T cell receptors (TCRs) andB cells receptors (BCRs). Sequences encoding TCRs and BCRs contain a variety of information that is useful for medical and research applications. For example, by performing immune profiling, the clonality of the T and B cell repertoire can be determined.
• T and B cells that are specific to the pathogen SARS-CoV-2 are activated and expand following infection that is accompanied by no obvious symptoms. Immune profiling of an individual in such a case would reveal the expansion of SARS-CoV-2 specific lymphocytes. Furthermore, humoral immune responses to SARS-CoV-2 have been shown to wane over time, leaving fewer SARS-CoV-2 specific antibodies in the circulation (Self WH et al. MMWR Morb Mortal Wkly Rep 2020;69:1762-1766). This feature of SARS-CoV-2 infection (COVID-19) reduces the potential effectiveness of tests for SARS-CoV-2 exposure based on virus- specific antibody titer.
• Immune profiling is also important, for example, in detecting T and B cell lymphomas, as these cancers generally have dominant clones that arise and expand as the cancer progresses. The presence of dominant T or B cell clones could be assessed in an individual that would aid in determining the extent or severity of disease.
• the technology of the current application combines complete next generation sequencing of DNA or RNA based samples with immune profiling.
• the sample material would have to be split into two separate assays and the data combined after sequencing.
• the method of the present application allows for the analysis of both genomic/transcriptomic data and immune profiling in one assay without compromising the quality of the data derived from either component. Therefore, the method of the present application has superior efficiency that could be translated to provide precision medicine at a scale that would make it viable for routine use by medical practitioners for a variety of potential applications.
• the method of the present disclosure leverages hybrid capture probes to enrich sequences most vital to understanding the T and B cell repertoire in an individual subject.
• Novel probes are designed to tile constant and non-constant regions of TCR and BCR sequences.
• the probe sets are designed so that sequencing is deep in critical areas of the TCR and BCR sequences so that a complete immune profile can be developed with fewer reads than traditional assays.
• the probe sets are formulated to provide productive sequences that cover both TCRs and BCRs.
• the formulation of probes may be further tuned to each individual application to provide maximum coverage of the immune repertoire.
• TCR and BCR sequences can be used to predict, for example, past infections and potentially which T cells are killing tumor cells. While standard RNAseq allows us to infer the proportion of T cells in a tumor (infiltration), TCR sequencing can tell us whether the majority of the T cells in the tumor are specific for a single neoantigen or arise from a diverse pool. By tracking TCRs and BCRs over the entirety of a patient cohort, it is possible to identify specific receptors that recur in patients with the same alterations, generating information that may be directed to TCR-based/CAR cell therapies.
• TCR and BCR sequencing results may be useful for characterizing an infection and/or an immune response to infection.
• TCR/BCR sequencing is performed as part of a whole-exome RNAseq assay, RNA sequences and expression levels of various immune genes (for example, cytokines, checkpoint molecules, innate immune genes) may also contribute to that characterization.
• immune genes for example, cytokines, checkpoint molecules, innate immune genes
• TCR/BCR profiling results may be used to: determine whether an individual has been exposed to one or more infectious pathogens; detect whether an individual has TCR or BCR sequences associated with sterilizing immunity and/or neutralizing antibodies for a group of infectious pathogens or a specific infectious pathogen; identify an adaptive immune response to a particular pathogen or antigen; analyze and improve treatment protocols for the infectious disease for the general patient population or a patient subpopulation; identify associations between severity of disease and immune profile; categorize or predict the severity of an individual’s disease (for example, see SchultheiB et al, 2020, Immunity, https://doi.Org/10.1016/j.immuni.2020.06.024, which is incorporated by reference herein in its entirety), assist a physician in selecting treatment protocols, tailor a treatment protocol to an individual’s immune response, develop and/or assess the efficacy of therapeutics or preventative treatments (for example, vaccines), design clinical trials or better define patient cohorts, and/or
• T cell response In some cases, infections that do not elicit a strong B cell response may still be controlled and cleared by an individual, and one of the hypothesized mechanisms for this control in the absence of a B cell response is a T cell response (see, Gallais et al, 2020, MedRxiv https://doi.org/10.1101/2020.06.21.20132449)
• a number of assays may be used to analyze an individual’s T cell response and/or memory B cells that are specific to a particular pathogen and/or antigen.
• these assays often require cell culturing techniques and/or an incubation period that limits the number of tests that can be performed each day.
• TCR/BCR sequencing may be more amenable to high volume testing allowing many samples to be processed each day.
• the aforementioned T cell, B cell, and antibody assays detect TCR and BCR that react to the antigens included in the assay and may not detect TCR or BCR that react to antigens generated during an infection, cancer, or other disease state that are not included in the assay. Furthermore, these assays do not automatically provide the genetic sequence (and thus, the protein structure) of the BCR or TCR molecule, which is another advantage of methods of TCR/BCR sequencing disclosed herein.
• patient samples are collected including blood or tumor samples, and the severity of disease is evaluated.
• the severity of disease for hematological malignancies including T and B cell lymphoma may be evaluated by performing TCR/BCR hybrid-capture and sequencing to develop an immune profile for the patient.
• the immune profile provides information regarding the clonality of normal and of the malignant cells. This information can be used by a healthcare practitioner to develop an understanding of the tumor burden in the patient, and to help guide treatment decisions.
• a therapy is recommended or matched based on a TCR/BCR profile.
• the TCR/BCR profile provides information regarding the major clones that make up the malignancy. Therefore, the TCR/BCR profile may help inform treatment decisions made by a healthcare practitioner.
• therapies recommended subsequent to TCR/BCR profiling may include adoptive cell therapy/ ACT, CAR-T cell therapy, chimeric antigen receptor macrophage (CAR-M) therapy, or other classes of cells engineered to express a chimeric antigen receptor (CAR).
• Additional therapies include, but are not limited to, cancer vaccine, immuno-oncology drugs, immunotherapy, checkpoint blockade, immune checkpoint inhibitors, chemotherapy, a cancer specific treatment, vaccine, antivirals, antibiotics, antiparasitics, antifungals, one or more antibodies (could be monoclonal, polyclonal, etc., could be isolated from another patient after recovery from infection), anti-histamines, nasal sprays, antileukotriene, leukotriene modifier, leukotriene receptor antagonist, allergy shots or another method to induce isotype switching from an allergenic IgE to a more tolerable IgG, anti inflammatory treatment, steroids, oral corticosteroid, prednisone, anti -rheumatic drugs (DMARDS), biologies that target common anti-inflammatory pathways, TNF pathway antagonists (including Remicade), B cell depletion (including Rituxan), immunosuppressant, insulin, bone marrow transplant, anti-inflammatory dietary restrictions, physical therapy, surgery, topical medication,
• the present technology is used to perform only one or several of the following functions simultaneously: evaluate the presence and extent of lymphocyte infiltration in a solid tumor sample, to measure/confirm disease severity, or detect infiltration biomarker.
• TCR/BCR profiles of patient samples derived from a solid tumor provide information about the frequency and clonality of tumor infiltrating lymphocytes (TILs).
• TILs tumor infiltrating lymphocytes
• a therapy is recommended based on the analysis of TILs made using TCR/BCR profiling.
• treatments that may be recommended following TCR/BCR profiling include: ACT, CAR-T, and/or other immune oncological (10) modalities.
• TCR/BCR data is combined with other infiltration predictors (engines), and/or as a feature to refine those prediction models.
• An example of an immune infiltration engine is disclosed, for example, in U.S. Patent Application No. 16/533,676, titled “A Multi-Modal Approach to Predicting Immune Infiltration Based on Integrated RNA Expression and Imaging Features”, and filed 8/6/19, which is incorporated herein by reference and in its entirety for all purposes.
• An additional example of an immune infiltration engine is disclosed, for example, in U.S. Patent Application No. 62/804,509, titled “Comprehensive Evaluation of RNA Immune System for the Identification of Patients with an Immunologically Active Tumor Microenvironment”, and filed 2/12/19, which is incorporated herein by reference and in its entirety for all purposes.
• a TCR/BCR profile may be generated for a patient having non-small cell lung cancer (NSCLC) and an EGFR mutation.
• NSCLC non-small cell lung cancer
• This TCR/BCR profile may be analyzed with or without output from infiltration predictors used to analyze the patient’s data. These results may be used to match a therapy (for example, immunotherapy, checkpoint blockade, etc.) with the patient.
• a therapy for example, immunotherapy, checkpoint blockade, etc.
• the present technology is used to identify whether a therapeutic immune cell has infiltrated a target tumor.
• cells that are detected using TCR/BCR profiling include CAR-T cells, or cells delivered through adoptive cell transfer (ACT) therapy.
• ACT adoptive cell transfer
• additional probes may be added to specifically target a sequence unique to the particular therapeutic modality.
• the present technology can be used to perform longitudinal testing, for example, before and after administration of immune-affecting therapy, such as chemotherapy.
• Chemotherapeutic drugs often adversely affect a patient’s immune system.
• chemotherapeutic drugs may include Anthracyclines, such as doxorubicin and epirubicin, taxanes such as paclitaxel and docetaxel, 5-fluorouracil, cyclophosphamide, or carboplatin, or other drugs used in the treatment of cell proliferative diseases.
• the present technology can be used to monitor the extent and nature of immune repertoire adverse events or off target effects.
• the TCR/BCR profile is used to understand whether the subject is more susceptible to infection or other disease associated with being immunocompromised.
• the TCR/BCR profile allows a physician to provide additional directed therapy in response to TCR/BCR profile analysis.
• TCR/BCR profile may lead to administration of cytokines known to positively affect the immune system, and in some embodiments, longitudinal TCR/BCR analysis provides data to determine the extent and nature of immune repertoire improvement, and to provide data to modify the treatment as necessary.
• the present technology is used to determine a TCR/BCR profile for the patient or patient sample taken from, for example, malignant tissue.
• the methods are useful to determine the presence and extent of lymphocyte infiltration in a tumor sample and identify the most abundant clones in a tumor sample.
• such profiles can allow selection of highly represented clones to be expanded for patient-specific Adoptive Cell Transfer, and/or used to identify receptor non-constant regions that are highly expressed to generate patient-specific chimeric receptors. This information provides a basis for developing a personalized medicine treatment approach using, for example, CAR-T cell therapy.
• the present technology is used to determine the TCR/BCR profile of a patient suffering from or suspected of having a pathogenic infection.
• the most abundant clones in a patient sample e.g., blood sample
• the receptor non constant regions for this patient sample are identified to generate patient-specific chimeric receptors for use in CAR cell therapy.
• the TCR/BCR data is used in combination with other pathogen detection or prediction methods and/or can be used as a feature to revise those prediction models.
• An example of a pathogen detection or prediction method is disclosed, for example, in U.S. Patent Application No. 16/802,126, filed February 26, 2020, which is incorporated herein by reference and in its entirety for all purposes.
• Another example of a pathogen detection or prediction method is disclosed, for example, in PCT/US21/18619, filed February 18, 2021, which is incorporated herein by reference and in its entirety for all purposes.
• data could be used to predict whether a patient is protected from future infection.
• this information would be valuable for patients following vaccination or natural infection with a virus.
• analysis could be performed longitudinally to characterize the immune response to a pathogenic organism.
• immune profiling may be used to predict whether the patient has/had an infection and can guide treatment decisions made by a healthcare practitioner.
• a list of receptor sequences associated with a pathogen (see the application of the technology above) is provided.
• a large dataset with positive controls and negative controls to determine which TCR/BCR sequences are associated with, for example, a given disease, pathogen, or antigen is provided (see e.g., Example 2).
• an example of utilizing the present TCR/BCR profiling methods for a diagnostic or confirmatory diagnostic application is provided below.
• Each row in Table 4 represents a BCR sequence, which includes a V, D, J family classification for the heavy chain and an amino acid sequence for the heavy chain CDR3, and a V and J family classification for the light chain and an amino acid sequence for the light chain CDR3.
• the amino acid sequence may represent a consensus sequence of the amino acids that are present in multiple CDR3 sequences when aligned and compared, and a tilde ( ⁇ ) may indicate a location in the sequence that does not have the same amino acid in the aligned CDR3 sequences.
• two BCR sequences may be paired.
• the third and fourth rows may represent two alleles that are expressed by the same cell to create a heterodimer protein BCR structure.
• the fifth and sixth rows may be paired sequences
• the seventh and eighth rows may be paired
• the ninth and tenth rows may be paired, etc.
• GAGGTGCAGCT EVQLVESGGGLIQP
• GAAATTGTGTTG EIVLTQSPGTLSL
• CTTTATTGTAGT AAAC A AGGGAC C AC G GTCACCGTCTCC TCA
• Embodiment 1 in a first embodiment, a method of determining a TCR/BCR profile of a patient is provided.
• the method comprises (a) isolating RNA from a sample from the patient; (b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; (c) determining the sequence of the RNA of (b) to generate sequencing data; and (d) analyzing the sequencing data to determine the TCR/BCR profile of the patient.
• Embodiment 9 The method of embodiment 1 wherein step (d) comprises identifying the most abundant non-constant region sequences in the sample.
• Embodiment 12 The method of embodiment 11, wherein the disease or condition comprises one or more of cancer, an infection, an autoimmune condition, allergy, or graft versus host disease.
• Embodiment 13 The method of embodiment 12, wherein the cancer or infection (infectious disease) is one or more provided in the list in embodiment 114.
• Embodiment 15 In some methods of any of the previous embodiments, a control TCR/BCR panel for a disease (such as cancer or an infection), or medical condition is provided.
• a disease such as cancer or an infection
• a method of diagnosing a patient with a disease or condition based on the patient's TCR/BCR profile comprises: a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; d) analyzing the sequencing data to determine the TCR/BCR profile of the patient; and e) comparing the TCR/BCR profile of the patient to a set of standards to diagnose the patient with a disease or condition; wherein the set of TCR/BCR hybrid- capture probes comprises a first pool of TCR constant region probes, a second pool of TCR non constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non constant region probes.
• Embodiment 17 The method of embodiment 16, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 18 The method of embodiment 16, wherein step (b) further comprises enriching for a targeted whole-transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 21 The method of embodiment 16, wherein step (b) further comprises enriching for a targeted whole-exome panel using a fifth pool of hybrid-capture probes.
• Embodiment 25 In some embodiments, a control TCR/BCR panel for a disease (such as cancer or an infection), or medical condition is provided. [0341] Embodiment 26. In some embodiments, a method of evaluating the severity or progression of a disease or condition based on the TCR/BCR profile of a patient is provided.
• Embodiment 27 The method of embodiment 26, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 29 The method of embodiment 28, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 31 The method of embodiment 26, wherein step (b) further comprises enriching for a targeted whole exome panel using a fifth pool of hybrid-capture probes.
• Embodiment 32 The method of embodiment 26, wherein the disease is an infectious disease, a cancer, an autoimmune disease, or an allergy.
• Embodiment 33 The method of embodiment 29, wherein the sample is a solid tumor sample.
• Embodiment 34 The method of embodiment 30, wherein step (e) comprises determining the presence or extent of tumor lymphocyte infiltration.
• Embodiment 35 The method of embodiment 32, wherein the cancer or infection (infectious disease) is one or more provided in the list in embodiment 114.
• Embodiment 36 The method of embodiment 35, wherein the diagnosing comprises comparing the subject's TCR/BCR profile to a control, wherein if the subject's BCR/TCR profile is similar to the control (for example, the abundance, identity, and/or clonality of one or more BCR/TCR receptors is similar to or identical to the control) the subject is diagnosed as having the disease or condition.
• a method for treating a disease or condition of a patient based on the patient's TCR/BCR profile comprises: a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; d) analyzing the sequencing data to determine the TCR/BCR profile of the patient; and e) administering a treatment based on the TCR/BCR profile of the patient; wherein the set of TCR/BCR hybrid-capture probes comprises a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non-constant region probes.
• Embodiment 39 The method of embodiment 38, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 40 The method of embodiment 38, wherein step (b) further comprises enriching for a targeted whole transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 41 The method of embodiment 40, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 42 The method of embodiment 41, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 43 The method of embodiment 38, wherein step (b) further comprises enriching for a targeted whole exome panel using a fifth pool of hybrid-capture probes.
• Embodiment 44 The method of embodiment 38, wherein step (d) comprises identifying the most abundant TCR/BCR clone in the sample, and wherein the treatment administered in step (e) comprises expanding the most abundant clones in vitro and re-administering the expanded cells to the patient.
• Embodiment 45 The method of embodiment 38, wherein step (d) comprises identifying the most abundant TCR non-constant region sequences in the sample, and wherein the treatment administered in step (e) comprises administering a CAR-T cell therapy comprising at least one of the most abundant TCR non-constant region sequences.
• Embodiment 46 The method of embodiment 38, wherein the disease or condition is an infectious disease, a cancer, an autoimmune disease, or an allergy.
• a method for characterizing the effect of a therapy on the TCR/BCR profile of a patient comprises: (a) at a first time point before the therapy is administered: (i) isolating RNA from a sample from the patient; (ii) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid- capture probes; (iii) determining the sequence of the RNA of (ii) to generate sequencing data; and (iv) analyzing the sequencing data to determine the TCR/BCR profile of the patient; and (b) at a second time point after the therapy has been administered: (i) isolating RNA from a sample from the patient; (ii) enriching the isolated RNA for TCR/BCR genes using a set of hybrid-capture probes; (iii) determining the sequence of the RNA of (ii) to generate sequencing data; and (iv) analyzing the
• Embodiment 49 The method of embodiment 48, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 50 The method of embodiment 48, wherein step (b) further comprises enriching for a targeted whole transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 51 The method of embodiment 50, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 52 The method of embodiment 51, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 53 The method of embodiment 48, wherein step (b) further comprises enriching for a targeted whole exome panel using a fifth pool of hybrid-capture probes.
• Embodiment 54 The method of embodiment 48, wherein the therapy is an immunotherapeutic agent.
• Embodiment 55 The method of embodiment 54, wherein the immunotherapeutic agent is a vaccine.
• Embodiment 56 The method of embodiment 54, wherein the immunotherapeutic agent is a chimeric antigen receptor (CAR) T cell.
• the immunotherapeutic agent is a chimeric antigen receptor (CAR) T cell.
• Embodiment 57 The method of any one of embodiments 48-57 further comprising modifying the treatment prescribed to the patient based on the observed effect.
• Embodiment 58 a method of identifying TCR/BCR non-constant region sequences that are enriched in a cohort of patients that have a specific disease or condition is provided.
• the method comprises: a) isolating RNA from a sample from each patient in the cohort; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; wherein the set of hybrid-capture probes comprises a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non-constant region probes; c) determining the sequence of the RNA of (b) to generate sequencing data; d) analyzing the sequencing data to determine the TCR/BCR profile of the patients in the cohort; and e) identifying TCR/BCR non constant region sequences that are enriched in the cohort as compared to
• Embodiment 59 The method of embodiment 58, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 60 The method of embodiment 58, wherein the set of hybrid-capture probes further comprises a fifth pool of probes comprising a targeted whole transcriptome panel.
• Embodiment 61 The method of embodiment 60, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 62 The method of embodiment 61, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 63 The method of embodiment 58, wherein the set of hybrid-capture probes further comprises a fifth pool of probes comprising a targeted whole exome panel.
• Embodiment 64 The method of embodiment 58, wherein the disease or condition is an infection, an autoimmune disease, an allergy, or cancer.
• Embodiment 65 The method of embodiment 58 further comprising using the enriched TCR/BCR non-constant region sequences to identify disease-specific antigens.
• Embodiment 66 The method of embodiment 65 further comprising producing a vaccine comprising the disease-specific antigens.
• Embodiment 67 The method of embodiments 65 or 66, wherein the disease-specific antigens are tumor antigens.
• Embodiment 68 The method of embodiment 64, wherein the cancer or infection (infectious disease) is one or more provided in the list in embodiment 114.
• kits for determining the TCR/BCR profile of a patient comprises a set of TCR/BCR hybrid-capture probes.
• a method of determining the TCR/BCR profile of a patient comprises: a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes and enriching for a targeted whole transcriptome panel using a set of hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; and d) analyzing the sequencing data to determine the TCR/BCR profile of the patient, wherein the set of TCR/BCR hybrid-capture probes comprises a first pool of BCR constant region probes, a second pool of BCR non-constant region probes, a third pool of TCR constant region probes, and a fourth pool of TCR non-constant region probes, wherein the ratio of the whole transcriptome-targeting panel, first pool, second pool, third pool, and fourth
• a method of determining the TCR/BCR profile of a patient comprises: a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; and d) analyzing the sequencing data to determine the TCR/BCR profile of the patient, wherein the patient has been exposed to or is suspected to have been exposed to SARS- CoV-2, wherein the set of TCR/BCR hybrid-capture probes comprises a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non-constant region probes.
• Embodiment 72 The method of embodiment 71, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 73 The method of embodiment 71, wherein step (b) further comprises enriching for a targeted whole-transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 74 The method of embodiment 73, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 75 The method of embodiment 74, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 76 The method of embodiment 71, wherein step (b) further comprises enriching for a whole exome targeting panel using a fifth pool of hybrid-capture probes.
• Embodiment 77 The method of embodiment 71 further comprising identifying a plurality of TCR/BCR clones in the sample.
• Embodiment 78 The method of embodiment 71 further comprising identifying the most abundant TCR/BCR clone in the sample.
• Embodiment 79 The method of embodiment 71 further comprising identifying the most abundant non-constant region sequences in the sample.
• Embodiment 80 The method of embodiment 71, wherein the sample is a blood sample or a solid tumor sample.
• a method of evaluating the severity or progression of COVID-19 based on the TCR/BCR profile of a patient comprises: a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; d) analyzing the sequencing data to determine the TCR/BCR profile of the patient; and e) comparing the TCR/BCR profile of the patient to a set of standards to characterize the severity or progression of the disease; wherein the set of TCR/BCR hybrid-capture probes comprises a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non-constant region probes.
• Embodiment 83 The method of claim 81, wherein step (b) further comprises enriching for a targeted transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 84 The method of claim 83, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 85 The method of claim 84, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 86 The method of claim 81, wherein step (b) further comprises enriching for a targeted whole exome panel using a fifth pool of hybrid-capture probes.
• a method for treating COVID-19 based on the patient's TCR/BCR profile comprises: a) isolating RNA from a sample from the patient; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; d) analyzing the sequencing data to determine the TCR/BCR profile of the patient; and e) administering a treatment based on the TCR/BCR profile of the patient; wherein the set of TCR/BCR hybrid-capture probes comprises a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non-constant region probes.
• Embodiment 88 The method of embodiment 87, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 89 The method of embodiment 87, wherein step (b) further comprises enriching for a targeted whole transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 90 The method of embodiment 89, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 91 The method of embodiment 90, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 92 The method of embodiment 87, wherein step (b) further comprises enriching for a targeted whole transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 93 The method of embodiment 87, wherein step (d) comprises identifying the most abundant TCR/BCR clone in the sample, and wherein the treatment administered in step (e) comprises expanding the most abundant clones in vitro and re-administering the expanded cells to the patient.
• Embodiment 94 The method of claim 1, wherein step (d) comprises identifying the most abundant TCR non-constant region sequences in the sample, and wherein the treatment administered in step (e) comprises administering a CAR-T cell therapy comprising at least one of the most abundant TCR non-constant region sequences.
• Embodiment 95 a method for characterizing the effect of a COVID- 19 therapy on the TCR/BCR profile of a patient is provided.
• the method comprises a) at a first time point before the therapy is administered: i. isolating RNA from a sample from the patient; ii. enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; iii. determining the sequence of the RNA of (aii) to generate sequencing data; and iv.
• Embodiment 97 The method of embodiment 95, wherein step (aii) and (bii) further comprises enriching for a targeted whole transcriptome panel using a fifth pool of hybrid-capture probes.
• Embodiment 98 The method of embodiment 97, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 99 The method of embodiment 98, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 100 The method of embodiment 95, wherein step (aii) and (bii) further comprises enriching for a targeted whole exome panel using a fifth pool of hybrid-capture probes.
• Embodiment 101 The method of embodiment 95, wherein the therapy is an immunotherapeutic agent.
• Embodiment 102 The method of embodiment 101, wherein the immunotherapeutic agent is a vaccine.
• Embodiment 103 The method of embodiment 101, wherein the immunotherapeutic agent is a chimeric antigen receptor (CAR) T cell.
• the immunotherapeutic agent is a chimeric antigen receptor (CAR) T cell.
• Embodiment 104 The method of any one of embodiments 95-104 further comprising modifying the treatment prescribed to the patient based on the observed effect.
• a method of identifying TCR/BCR non-constant region sequences that are enriched in a cohort of patients with SARS-CoV-2 comprises: a) isolating RNA from a sample from each patient in the cohort; b) enriching the isolated RNA for TCR/BCR genes using a set of TCR/BCR hybrid-capture probes; c) determining the sequence of the RNA of (b) to generate sequencing data; d) analyzing the sequencing data to determine the TCR/BCR profile of the patients in the cohort; and e) identifying TCR/BCR non-constant region sequences that are enriched in the cohort as compared to a control group without the disease or condition, wherein the set of hybrid-capture probes comprises a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool
• Embodiment 106 The method of embodiment 105, wherein the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1 :2.5: 100: 100.
• Embodiment 107 The method of embodiment 105, wherein the set of hybrid-capture probes further comprises a fifth pool of probes comprising a whole transcriptome targeting panel.
• Embodiment 108 The method of embodiment 107, wherein the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• Embodiment 109 The method of embodiment 108, wherein 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 110 The method of embodiment 105, wherein the set of hybrid-capture probes further comprises a fifth pool of probes comprising a whole exome targeting panel.
• Embodiment 111 The method of embodiment 105 further comprising using the enriched TCR/BCR non-constant region sequences to identify SARS-CoV-2-specific antigens.
• Embodiment 112. The method of embodiment 108 further comprising producing a vaccine comprising the SARS-CoV-2-specific antigens.
• kits for determining the TCR/BCR profile of a patient with COVID-19 comprises a set TCR/BCR hybrid capture probes.
• the set of probes is provided as four separate pools, comprising a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non-constant region probes.
• the ratio of the first pool, second pool, third pool, and fourth pool within the set is 1:2.5:100:100.
• the probe set is used in combination with one of (1) a whole transcriptome targeting panel, (2) a whole exome targeting panel; or (3) a targeted panel directed to 10-20,000 targets of interest, as a fifth pool of probes, and the ratio of the first pool, second pool, third pool, fourth pool, and fifth pool within the set is 1 :2.5: 100: 100: 10.
• the TCR/BCR panels are configured such that 2% or less of the reads in the sequencing data map to TCR/BCR genes.
• Embodiment 114 In some of the above embodiments, (a) a subject or a cohort is diagnosed with, suspected of having, or is suffering from a disease or medical condition, such as a cancer or infection (infectious disease); or (b) a method for diagnosing a disease or medical condition, such as a cancer or infection (infectious disease) is provided.
• a disease or medical condition such as a cancer or infection (infectious disease)
• a method for diagnosing a disease or medical condition such as a cancer or infection (infectious disease) is provided.
• the cancer may be one or more of chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone/osteosarcoma, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, myxosarcoma, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioblastoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male
• sarcomas of primary cutaneous origin e.g. dermatofibrosarcoma protuberans
• lymphomas of primary cutaneous origin e.g. dermatofibrosarcoma protuberans
• bronchial adenomas/carcinoids small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, Kaposi sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, or liposarcoma.
• EHE epithelioid hemangioendothelioma
• the infection may be one or more of: Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), Amoebiasis, Anaplasmosis, Angiostrongyliasis, Anisakiasis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial meningitis, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Bartonellosis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystosis, Blastomycosis, Venezuelan hemorrhagic fever, Botulism (and Infant botulism),
• Embodiment 115 A method of sequencing at least one TCR or BCR region of a specimen using a plurality of probes, wherein the probes comprise: a first pool of TCR constant region probes, a second pool of TCR non-constant region probes, a third pool of BCR constant region probes, and a fourth pool of BCR non-constant region probes, wherein the first pool has a first concentration level, the second pool has a second concentration level, the third pool has a third concentration level, and the fourth pool has a fourth concentration level.
• Embodiment 116 The method of embodiment 115, wherein the first concentration level, the second concentration level, the third concentration level, and the fourth concentration level are different from each other.
• Embodiment 117 The method of embodiment 115 or 116, wherein the concentration level of probes in the first pool is less than the concentration level of probes in the second pool, and wherein the concentration level of probes in the third pool is less than the concentration level of probes in the fourth pool. In some embodiments, the concentration level of probes in the first pool is about the same as the concentration level of probes in the second pool, and wherein the concentration level of probes in the third pool is less than the concentration level of probes in the fourth pool.
• Embodiment 118 The method of any one of embodiments 115-117, wherein the concentration level of probes in the first and third pool are independently at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold less the concentration level of probes in the second and fourth pools.
• the concentration level of probes in the third and fourth pool are independently at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold less than the concentration level of probes in the first and second pools.
• Embodiment 119 A method of sequencing at least one TCR or BCR region of a specimen, comprising, selecting more than one probe from a set of probes to form a pool, wherein the more than one probes in the pool are selected to omit at least a portion of a constant region of the at least one TCR or BCR region.
• Embodiment 120 The method of embodiment 119, wherein the pool comprises probes for sequencing at least a portion of the constant region of the TCR or BCR.
• Embodiment 121 The method of any of embodiments 119-120, wherein the sequencing is whole-transcriptome sequencing.
• Embodiment 122 The method of any of embodiments 117-121, wherein the sequencing is short-read sequencing.
• Embodiment 123 The method of embodiment 115, wherein the sequencing is performed on a specimen collected from a patient and the results are used to predict the disease susceptibility of the patient.
• Embodiment 124 The method of embodiment 115, wherein the TCR or BCR region is associated with a viral infection, and the specimen is collected prior to the administration to the patient of a vaccine designed to protect against the viral infection.
• Embodiment 125 The method of embodiment 115, wherein the sequencing is performed on a specimen collected from a patient, wherein the patient was exposed to an infectious pathogen prior to specimen collection.
• Embodiment 126 The method of embodiment 125, wherein the patient generated antibodies against the infectious pathogen.
• Embodiment 127 The method of embodiment 125, wherein the patient did not generate a substantial amount of antibodies against the infectious pathogen.
• Embodiment 128 The method of embodiment 125, wherein the infectious pathogen did not cause seroconversion.
• Embodiment 129 The method of embodiment 125, wherein high concentrations of the infectious pathogen were not detectable in the patient’s blood.
• Embodiment 130 The method of embodiment 125, wherein the infectious pathogen is SARS-CoV-2.
• Embodiment 131 The method of embodiment 115, wherein the sequencing is performed on a specimen collected from a patient, wherein the patient is experiencing symptoms associated with respiratory disease.
• Embodiment 134 The method of embodiment 115, wherein the specimen is a tumor specimen.
• Embodiment 137 The method of claim 115, wherein the specimen is a mucus specimen.
• Embodiment 139 The method of claim 115, wherein the sequencing is conducted in whole- transcriptome sequencing.
• Embodiment 144 The method of embodiment 143, further comprising identifying the proportion of at least one BCR clone in the plurality of BCR clones in the specimen.
• Embodiment 145 The method of any of embodiments 115-141, wherein the set comprises at least one oligonucleotide from a TCR constant region pool, a TCR non-constant region pool, a BCR constant region pool, and a BCR non-constant region pool.
• Embodiment 146 The method of any of the previous embodiments, wherein the TCR/BCR probe set is obtained as described in Example 1.
• KAPA RNA HyperPrep Kit for Illumina P/N KK8544
• UMI IDT unique dual indexed
• IDT UDI-UMI adapters were ligated to cDNA and the adapter-ligated libraries were cleaned using a magnetic bead-based method (Roche Diagnostics, P/N KK8002).
• the libraries were amplified with high fidelity, low-bias PCR using primers complementary to adapter sequences. Amplified libraries were subjected to magnetic bead based clean-up (Axygen, P/N MAG-PCR-CL-250) to eliminate unused primers, and quantity was assessed.
• 1,074 of those detected clonotypes were determined to be productive sequences (for example, they did not include a stop codon, were not out of frame, were not partial sequences, etc.).
• V_allele - (e.g. IGLV3-25*03 -> IGLV3-25) o. V_allele - (e.g. IGLV3-25*03 -> 03) p. D gene family q. D gene r. D allele s. J gene family t. J gene u. J allele v. IGH isotype - null if not called/applicable, otherwise: ⁇ 'Ar,'A2VD','E','GlVG2','G3VG4VM' ⁇ w. has_CDR3nt_twin - “True” entered if there are duplicates of this clonotype's nt sequence in the repertoire x. has_CDR3aa_twin - “True” entered if there are duplicates of this clonotype's aa sequence in the repertoire
• the clonotype frequency and/or gene clonotype assignments may be determined by a TCR or BCR sequence assembly algorithm included in the systems and methods described herein.
• a reference dataset may be generated or an existing reference dataset may be selected.
• the data may be de-identified data.
• the data may have protected health information (PHI) removed.
• the reference dataset can include TCR/BCR sequencing data associated with annotated clinical documentation as well as additional NGS-based outputs (including but not limited to, patient HLA type or matched NGS DNA/RNA sequencing, viral/pathogen sequencing, whole exome or targeted panel sequencing of patient specimen(s)).
• HLA data may be used to further annotate or contextualize the TCR sequence data.
• certain combinations of TCR sequences and HLA types may be incompatible, implying that certain TCR sequences may be expected in the context of certain HLA types.
• the absence of that sequence may be expected if the patient does not have HLA types that are compatible with that TCR sequence.
• This reference dataset may be mined to identify TCR or BCR sequences that are enriched in patients that are responding to or have recently recovered from disease caused by a specific pathogen or combination of pathogens. For example, see Emerson, R., DeWitt, W., Vignali, M. et al. Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire. Nat Genet 49, 659-665 (2017). https://doi.org/10.1038/ng.3822, the contents of which are incorporated by reference herein in their entirety for all purposes.
• the mining may include the use of machine learning clustering techniques on the TCR/BCR sequence database.
• Example methods include detecting a pathogen-associated TCR or BCR sequence in data from patients having a particular cancer type. These sequences could be used as a biomarker, an indication for prescribing checkpoint inhibitors or predicting response to IO.
• Cross-reactivity means the sequence could be present in a higher percentage of patients, especially if the infection by the pathogen is common, so these are more likely to be the first sequences discovered that are common to many patients.
• the patients have non-small cell lung cancer (NSCLC) and a viral- associated TCR sequence.
• NSCLC non-small cell lung cancer
• TCRbeta chains were grouped into affinity groups (based on similar amino acid structure).
• Certain viral associated TCRs cross react with cancer antigens (for patients having the same HLA).
• TCR and BCR sequences can then be analyzed by a predictive model trained on the reference dataset or a subset of the reference dataset (for example, only records having data deemed relevant to the prediction, including data associated with a known negative or positive status or numeric score related to the prediction target or category of prediction) to calculate a likelihood of the patient having an infection status, exposure history, and/or potential protection or resistance to infection associated with any of the TCR and/or BCR sequences, where the associations may be based on associations or trends captured in the reference dataset.
• the reference dataset may also be analyzed to find associations between the severity of disease and various genetic, immunological, or clinical factors or characteristics.
• factors may include alleles or variants associated with the ABO blood type gene, the gene located on chromosome 9q34.2, immunological genes, genes located on chromosomes 3 or 6, HLA genes, etc., immunological characteristics, clinical data/status (age, history of cardiac disease, diabetic, blood sugar levels, hypertension, obesity, asthma, COPD, etc.), and/or the presence of specific TCR and/or BCR sequences.

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