WO2024035951A2 - Méthodes d'évaluation de lymphocytes t thérapeutiques pour un herpèsvirus humain 6 latent et réactivé - Google Patents

Méthodes d'évaluation de lymphocytes t thérapeutiques pour un herpèsvirus humain 6 latent et réactivé Download PDF

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WO2024035951A2
WO2024035951A2 PCT/US2023/030111 US2023030111W WO2024035951A2 WO 2024035951 A2 WO2024035951 A2 WO 2024035951A2 US 2023030111 W US2023030111 W US 2023030111W WO 2024035951 A2 WO2024035951 A2 WO 2024035951A2
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hhv
cells
cell
sequence reads
rna sequence
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WO2024035951A3 (fr
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Caleb Lareau
Ansuman Satpathy
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • viruses that can infect humans including viruses from the Herpesviridae, Polyomaviridae, Adenoviridae, and Parvoviridae families, can become ‘quiescent,’ resulting in a latent phase of the virus that is stably maintained in healthy individuals.
  • acute stress conditions such as fever, hematopoietic stem cell transplant (HSCT), or trauma, latent viruses can become reactivated, leading to a variety of complex clinical manifestations.
  • HSCT hematopoietic stem cell transplant
  • Cell therapies have yielded durable clinical benefits for patients with cancer but have been accompanied by unexpected side effects of treatment. There is a current lack of understanding of the mechanisms of toxicity observed in patients receiving cell therapies, including encephalitis caused by human herpesvirus 6 (HHV-6).
  • HHV-6 human herpesvirus 6
  • the methods comprise obtaining from the subject a biological sample comprising the therapeutic T cells, and assessing the therapeutic T cells for HHV-6 reactivation. Also provided are methods comprising assessing an in vitro culture comprising candidate therapeutic human T cells for HHV- 6 reactivation, wherein the in vitro culture is assessed for a level of one or more HHV-6 analytes by quantitative nucleic acid sequencing.
  • the present disclosure further provides non-transitory computer-readable media and computer devices that find use in practicing the methods of the present disclosure.
  • FIG. 1A-1 G Petabase-scale analysis of viral nucleic acids reveals that HHV-6 is reactivated in human T cells.
  • 1A Schematic of viral life cycles of endogenous human viruses. Viruses can enter a latent phase after primary infection and become reactivated based on environmental cues.
  • 1 B Enumeration of BioSamples with human viruses capable of reactivation confidently expressed in human SRA samples.
  • 1 C Summary of BioSamples with viral expression in human T cells. Viruses shown in (b) but not (c) had no evidence of reactivation in T cells.
  • 1 D Reanalysis of Shytaj et al. 1 ⁇ RNA-seq data.
  • CD4+ T cells from 3 separate donors were treated with either an HIV or mock infection and cultured for ⁇ 2 weeks. Shown is the % of RNA molecules aligning to the HHV-6B reference Serratus reference. 1 E: Reanalysis of the LaMere et al.— data. Naive and memory CD4+ T cells were separated and cultured for two weeks. Shown is the % of RNA molecules aligning to the HHV-6B reference Serratus reference. 1 F: Quantification of HHV- 6B episomal DNA from a reanalysis of ChlP-seq from LaMere et al.— data.
  • FIG. 2A-2I scRNA-seq identifies rare HHV-6 expressing cells in CAR T cell culture.
  • 2A Longitudinal sampling of the HHV-6B U31 transcript CAR T cell culture from two donors.
  • 2B Summary of HHV-6B qPCR expression at Day 19 for four donors normalized to the total cell count. Bars are sorted in increasing order of expression.
  • 2C Schematic of single-cell sequencing workflow to detect HHV-6+ cells from the CAR T culture. Two models are presented that would explain HHV-6 reactivation: Model 1 (top) where cells all express HHV-6 transcripts or Model 2 (bottom) where only a subset of cells express HHV-6B.
  • Both the host and HHV-6B viral RNA can be directly quantified using our 10x Genomics RNA-seq workflow.
  • 2D Summary of HHV-6B expression from an individual donor (D98). The top 0.2% of cells contain 99% of the HHV-6B transcript UMIs from this experiment.
  • 2E Tabulated summary of scRNA-seq profiling for four CAR T donors, including number of cells profiled, % expressing HHV-6, U31 transcript qPCR value, and number of shared TCR clones between the HHV-6B + cells.
  • 2F Extended longitudinal sampling of the HHV-6B U31 transcript CAR T cell culture for two donors.
  • 2G Schematic and summary of HHV-6B expression in the D34 donor after 19 and 25 days, showing evidence of HHV-6B spreading in the culture as depicted in the schematic.
  • 2H Correlation analyses of host factor gene expression with HHV-6B expression in individual cells. The per-gene correlation statistics are shown in black against a permutation of the HHV-6B expression in gray. Noteworthy genes are indicated.
  • 2I Pathway enrichment analysis of GSEA/Msigdb Hallmark gene sets. A positive Normalized Enrichment Score (NES) corresponds to genes that are overexpressed in cells with high amounts of HHV-6B transcript.
  • NES Normalized Enrichment Score
  • FIG. 3A-3E Clinically validated and FDA-approved CAR T cells harbor HHV-6 in vivo.
  • 3A Schematic and summary of patient cohorts, noting different cell therapy products (axi-cel and tisa-cel) and two different timepoints for sampling (preinfusion product and 7 days post transfusion). The number of total cells analyzed and HHV-6+ cells detected are noted underneath.
  • 3B Summary heatmap of 7 HHV-6 positive cells from cohort 2 ex vivo follow up. Total HHV-6 expression is noted in blue boxes; non-zero host expression noted in green boxes (see Methods).
  • 3C Heatmap of HHV-6B transcripts (columns) by same individual cells (rows, as in (3B)) grouped by viral gene class (immediate early; early; late expressing genes as previously described).
  • 3E Summary of SJCAR19-09 patients, including PCR of HHV-6 and T cell scRNA-seq abundance. Treatment regimen with Foscarnet is noted with initial dose administered at day +24.
  • FIG. 4A-4B Characterization of 0X40 expression in bulk sequencing experiments.
  • 4A Expression of TNFRSF4 (0X40), the canonical receptor of HHV-6B in unstimulated and stimulated immune cell populations.— 0X40 is not expressed in unstimulated immune cells but highly expressed in CD4 and CD8 T cells after activation/stimulation of CD3/CD28 and IL-2.
  • 4B Broad expression of 0X40 across healthy tissues from the GTEx bulk atlas.
  • FIG. 5A-5B Characterization of 0X40 expression in resting and stimulated endothelial cells.
  • 5A Refinement of HHV-6B expression using the single-cell GTEx atlas—.
  • 5B Pseudobulk expression of 0X40 across the human fetal expression atlas—. Induction of 0X40 expression on endothelial cell lines in the presence of TNF-a; RNA-seq dataset from Richards et al.—
  • FIG. 6A-6D Supporting analyses for HHV-6 reactivation using Serratus.
  • 6A Heatmap of HHV-6B transcripts across the four highest RNA-seq libraries from Serratus. Shown are the first 40 genes (based on genomic coordinate order) from the HHV-6B transcriptome and the number of reads that pseudoalign to each transcript.
  • 6B Summary of naive CD4+ culture in the LaMere et al. dataset; compare to FIG. 1 E.
  • 6C Summary of HHV-6B expression in the Qu et al 2017 ATAC-seq atlas, showing sorted T cells from Patient 59, an individual with CTCL, had detectable levels of HHV-6B DNA within cells.
  • 6D Smoothed coverage (rollmean of 500 base pairs) over the four libraries from Patient 59, indicating coverage across the HHV-6B reference genome.
  • FIG. 7A-7F Supporting analyses for HHV-6 expression during in vitro CAR T cell culture.
  • 7A Summary of observed (red) and permuted (gray) HHV-6B expression for four donors at day 19 in culture. Dotted line is 10 UMIs, the threshold for a super-expressor.
  • 7B Heatmap of HHV- 6B expression for selected cells across 3 donors with detectable super expressors. Columns are grouped based on HHV-6B gene programs (immediate early; early; late).
  • 7C Uniform manifold approximation and projection for 3 samples, noting marker genes and HHV-6B UMI expression (log transformed). The WPRE feature indicates the presence of the CAR transgene.
  • 7D Summary of HHV-6B +/- cells from differential testing for host factors. Arrows indicate lymphotoxin a (LTa/LTA) and downregulation of lymphotoxin p (LTp/LTB).
  • 7E Summary of HHV- 6B expression in re-cultured samples for donors D61 and D34.
  • 7F Correlation statistics of HHV- 6B transcript signatures with 0X40 expression across 3 re-cultured samples, including p-values from Pearson's product moment correlation coefficient. The consistently positive, significant correlation statistic represents an association uniquely between 0X40 expression and the immediate early HHV-6B gene signature. From left to right in each of the four groups: D34 Day 25, D34 Day 27 and D61 Day 27.
  • FIG. 8 A flow diagram of a computer-implemented single cell RNA sequencing-based method for identifying individual human cells expressing HHV-6B RNA, according to embodiments of the present disclosure.
  • FIG. 9 A flow diagram of a second computer-implemented single cell RNA sequencingbased method for identifying individual human cells expressing HHV-6B RNA, according to embodiments of the present disclosure.
  • FIG. 10 A flow diagram of a computer-implemented method for identifying a particular cell type susceptible to reactivation by a particular virus, according to embodiments of the present disclosure.
  • FIG. 11 Schematic of a re-culture experiment where CAR T cells were recovered from the infusion product and then re-cultured with TransAct and IL7/15.
  • CPM RNA counts per million
  • FIG. 12A-12F Mitigation of HHV-6 reactivation and spreading via foscarnet treatment in vitro.
  • 12A Schematic of CAR T product re-culture experiment. Donor D97, which at day 19 showed a low but detectable level of HHV-6, was selected for re-culture for five days.
  • 12B Summary of RT-qPCR at the control and two treatment levels of Foscarnet. Each dot represents a technical replicate.
  • 12C Schematic of D34 re-culture +/- foscarnet at 1 mM.
  • 12D Difference between untreated and treated in the abundance of HHV-6+ cells.
  • aspects of the present disclosure include methods of assessing in vitro candidate therapeutic human T cells for latent and reactivated Human Herpesvirus 6 (HHV-6), e.g., HHV- 6B.
  • HHV-6 Human Herpesvirus 6
  • the methods are based in part on the surprising findings, demonstrated herein, that HHV-6 can become reactivated in human T cells in standard in vitro cultures, as well as the identification of a rare population of HHV-6 “super-expressor” cells that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture that may spread rapidly to infect other cells in vitro.
  • CAR chimeric antigen receptor
  • the methods find use in a variety of contexts, including the context of screening candidate therapeutic human T cells for latent and/or reactivated HHV-6 to determine whether such cells are suitable for administration to a subject in a cell based therapy. Details regarding the in vitro methods will now be described.
  • HHV-6 analytes e.g., one or more HHV-6B analytes.
  • candidate therapeutic human T cells are human T cells to be used in a cell based therapy, subject to the result of the assessment for the one or more HHV-6 analytes and/or HHV-6 reactivation.
  • Candidate therapeutic human T cells determined to be negative for the one or more HHV-6 analytes and/or HHV-6 reactivation may then be identified as therapeutic human T cells, which in turn may be administered to a subject in need thereof as a cell based therapy.
  • a “cell based therapy” or “cell therapy” refers to the transfer of autologous or allogeneic cellular material into a subject for medical purposes.
  • Non-limiting examples of cellbased therapies include CAR T cell therapy, engineered T cell therapy (e.g., T cells that express a recombinant T cell receptor (TCR)), a therapy comprising administering T cells which do not express a recombinant receptor, and the like.
  • CAR T cell therapy e.g., engineered T cell therapy (e.g., T cells that express a recombinant T cell receptor (TCR)), a therapy comprising administering T cells which do not express a recombinant receptor, and the like.
  • TCR recombinant T cell receptor
  • the one or more HHV-6 analytes comprise an HHV-6B nucleic acid.
  • nucleic acid and “polynucleotide” are used interchangeably herein to describe a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.
  • the one or more HHV-6 analytes comprise an HHV-6 deoxyribonucleic acid (DNA).
  • the one or more HHV-6 analytes comprise an HHV-6B ribonucleic acid (RNA).
  • HHV-6B DNAs and/or RNAs A variety of approaches are available for assessing the in vitro culture for one or more HHV-6B DNAs and/or RNAs.
  • the HHV-6B genome and transcriptome have been sequenced.
  • the HHV-6B reference genome and transcriptome are available at Genbank (Genbank AF157706).
  • Genbank AF157706 Genbank AF157706
  • the presence or absence of one or more selected HHV-6B DNAs and/or RNAs may be determined by hybridization (e.g., Southern analysis, Northern analysis, microarray analysis, or the like), nucleic acid sequencing, and/or the like.
  • Sequencing HHV-6B DNA and/or RNA may be performed using any of a variety of available high throughput nucleic acid sequencing machines and systems.
  • Illustrative sequencing systems include the Illumina iSeq 100, Miniseq, MiSeq series, NextSeq series (e.g., NextSeq 500 series, NextSeq 1000, NextSeq 2000), and NovaSeq sequencing systems (Illumina, Inc., San Diego, Calif.), the Pacific Biosciences Sequel (e.g., Sequel II) sequencing system (Pacific Biosciences, Menlo Park, Calif.), the Oxford Nanopore Technologies MinlONTM, GridlONx5 TM , PromethlONTM, or SmidglONTM nanopore-based sequencing systems (Oxford Nanopore Technologies, Oxford, UK), and other systems having similar capabilities.
  • sequencing is achieved using a set of sequencing platform-specific oligonucleotides that hybridize to a defined region within amplified HHV-6B DNA and/or RNA
  • the raw sequence data is preprocessed to remove errors in the primary sequence of each read and to compress the data.
  • a nearest neighbor algorithm can be used to collapse the data into unique sequences by merging closely related sequences, to remove both PCR and sequencing errors. See, e.g., US2012/0058902; US2010/033057; WO201 1/106738; US2015/0299785; or WO2012/027503, which is each incorporated by reference in its entirety.
  • the one or more HHV-6 analytes comprise an HHV-6B protein.
  • protein polypeptide
  • peptide are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • HHV-6B proteins A variety of approaches are available for assessing the in vitro culture for one or more HHV-6B proteins. For example, based on the available amino acid sequence information for proteins encoded by the HHV-6B transcriptome, the presence or absence of one or more selected HHV-6B proteins may be assessed, e.g., by a variety of immunoassays using available antibodies specific for one or more HHV-6B proteins.
  • an “immunoassay” is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody.
  • the immunoassay is an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, a fluorescent immunoassay, a chemiluminescent immunoassay, a lateral flow immunoassay, or an immunoblot assay.
  • ELISA enzyme-linked immunosorbent assay
  • radioimmunoassay a radioimmunoassay
  • fluorescent immunoassay a fluorescent immunoassay
  • chemiluminescent immunoassay a chemiluminescent immunoassay
  • lateral flow immunoassay a lateral flow immunoassay
  • the presence or amount of one or more HHV-6B proteins can be determined using antibodies and detecting specific binding to the one or more HHV-6B proteins.
  • Any immunoassay may be utilized.
  • the immunoassay may be an enzyme-linked immunoassay (ELISA), a competitive inhibition assay, such as forward or reverse competitive inhibition assays, or a competitive binding assay, for example.
  • ELISA enzyme-linked immunoassay
  • a competitive inhibition assay such as forward or reverse competitive inhibition assays
  • a competitive binding assay for example.
  • one tag is attached to the capture antibody and the detection antibody.
  • a microparticle or nanoparticle employed for capture also can function for detection, e.g., where it is attached or associated by some means to a cleavable linker.
  • a heterogeneous format may be used. For example, after a test sample from the in vitro culture comprising the candidate human therapeutic T cells is obtained, a first mixture is prepared. The mixture contains the test sample being assessed for a HHV-6B protein and a first specific binding partner, wherein the first specific binding partner and HHV-6B protein form a first specific binding partner-HHV-6B protein complex.
  • the first specific binding partner may be an anti-HHV-6B protein antibody or a fragment thereof.
  • the order in which the test sample and the first specific binding partner are added to form the mixture is not critical.
  • the first specific binding partner may be immobilized on a solid phase.
  • the solid phase used in the immunoassay can be any solid phase known in the art, such as, but not limited to, a magnetic particle, a bead a nanobead, a microbead, a nanoparticle, a microparticle, a membrane, a scaffolding molecule, a film, a filter paper, a disc, or a chip (e.g., a microfluidic chip).
  • any unbound analyte may be removed from the complex using any technique known in the art.
  • the unbound analyte can be removed by washing.
  • the first specific binding partner is present in excess of any HHV-6B protein present in the test sample, such that all HHV-6B protein that is present in the test sample is bound by the first specific binding partner.
  • a second specific binding partner is added to the mixture to form a first specific binding partner- HHV-6B protein-second specific binding partner complex.
  • the second specific binding partner is preferably an anti-HHV-6B protein antibody that binds to an epitope on the HHV-6B protein that differs from the epitope on the HHV-6B protein bound by the first specific binding partner.
  • the second specific binding partner may be labeled with or contains a detectable label, e.g., tag attached by a cleavable linker. The use of immobilized antibodies or fragments thereof may be incorporated into the immunoassay.
  • the antibodies may be immobilized onto a variety of supports, such as magnetic or chromatographic matrix particles, latex particles or modified surface latex particles, polymer or polymer film, plastic or plastic film, planar substrate, a microfluidic surface, pieces of a solid substrate material, and the like.
  • supports such as magnetic or chromatographic matrix particles, latex particles or modified surface latex particles, polymer or polymer film, plastic or plastic film, planar substrate, a microfluidic surface, pieces of a solid substrate material, and the like.
  • the in vitro culture comprises candidate therapeutic human T cells.
  • the type of human T cells may vary. Examples of T cells include naive T cells (TN), cytotoxic T cells (TCTL), memory T cells (TMEM), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), tissue resident memory T cells (T RM ), effector T cells (TEFF), regulatory T cells (T EGS), helper T cells (T H , T H 1 , T H 2, T H 17), CD4+ T cells, CD8+ T cells, virusspecific T cells, alpha beta T cells (T a p), and gamma delta T cells (T v o).
  • the candidate therapeutic human T cells comprise candidate therapeutic CD4+ human T cells.
  • the candidate therapeutic human T cells are genetically modified.
  • the genetic modification comprises engineering the candidate therapeutic human T cells to express a receptor (e.g., a recombinant receptor) on the surface thereof.
  • a receptor e.g., a recombinant receptor
  • a variety of suitable approaches for genetically modifying cells are available.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes.
  • vector construct e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes
  • vector construct e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes
  • vector construct e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes.
  • a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector, e.g., using recombinant DNA techniques known in the art.
  • viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • expression vectors include, but are not limited to pCIneo vectors (Promega) for expression in mammalian cells; pLenti4/V 5-DESTTM, pLenti6/V 5- DESTTM, murine stem cell virus (MSCV), MSGV, moloney murine leukemia virus (MMLV), and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • a nucleic acid sequence encoding a polypeptide to be expressed in the cells may be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
  • Expression control sequences, control elements, or regulatory sequences present in an expression vector are those non-translated regions of the vector - origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence), introns, a polyadenylation sequence, 5' and 3' untranslated regions, and/or the like - which interact with host cellular proteins to carry out transcription and translation.
  • Such elements may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including ubiquitous promoters and inducible promoters may be used.
  • Components of the expression vector are operably linked such that they are in a relationship permitting them to function in their intended manner.
  • the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g. , a nucleic acid encoding the polypeptide, where the expression control sequence directs transcription of the nucleic acid encoding the polypeptide.
  • the expression vector is an episomal vector or a vector that is maintained extrachromosomally.
  • the term “episomal” refers to a vector that is able to replicate without integration into the host cell’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
  • Such a vector may be engineered to harbor the sequence coding for the origin of DNA replication or "ori" from an alpha, beta, or gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, a yeast, or the like.
  • the host cell may include a viral replication transactivator protein that activates the replication.
  • Alpha herpes viruses have a relatively short reproductive cycle, variable host range, efficiently destroy infected cells and establish latent infections primarily in sensory ganglia.
  • alpha herpes viruses include HSV 1 , HSV 2, and VZV.
  • Beta herpesviruses have long reproductive cycles and a restricted host range. Infected cells often enlarge.
  • Non-limiting examples of beta herpes viruses include CMV, HHV-6 and HHV-7.
  • Gamma-herpesviruses are specific for either T or B lymphocytes, and latency is often demonstrated in lymphoid tissue.
  • Illustrative examples of gamma herpes viruses include EBV and HHV-8.
  • the candidate therapeutic human T cells are engineered to express a chimeric antigen receptor (CAR), a T cell receptor (TCR) such as a recombinant TCR, a chimeric cytokine receptor (CCR), a chimeric chemokine receptor, a synthetic notch receptor (synNotch), a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a generalized extracellular molecule sensor (GEMS) receptor, a growth factor receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, a hormone receptor, a receptor tyrosine kinase, an immune receptor such as CD28, CD80,
  • CAR T
  • such a receptor is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.
  • a T cell receptor a B cell receptor
  • a natural killer (NK) cell receptor a macrophage receptor
  • monocyte receptor a neutrophil receptor
  • a dendritic cell receptor a mast cell receptor
  • basophil receptor eosinophil receptor
  • the candidate therapeutic human T cells are engineered to express a chimeric antigen receptor (CAR).
  • the candidate therapeutic human T cells are engineered to express a recombinant TCR.
  • the candidate therapeutic human T cells may be engineered to express a CAR.
  • the extracellular binding domain of the CAR may comprise a single chain antibody.
  • the single-chain antibody may be a monoclonal single-chain antibody, a chimeric single-chain antibody, a humanized single-chain antibody, a fully human single-chain antibody, and/or the like.
  • the single chain antibody is a single chain variable fragment (scFv).
  • the extracellular binding domain of the CAR is a single-chain version (e.g. , an scFv version) of an antibody approved by the United States Food and Drug Administration and/or the European Medicines Agency (EMA) for use as a therapeutic antibody.
  • EMA European Medicines Agency
  • Non-limiting examples of single-chain antibodies which may be employed when the protein of interest is a CAR include single-chain versions (e.g., scFv versions) of Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab,
  • Emibetuzumab Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab,
  • Lumretuzumab Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab,
  • Polatuzumab Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab,
  • Tucotuzumab Vandortuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or an antigen-binding variant thereof.
  • the receptor may include one or more linker sequences between the various domains.
  • a “variable region linking sequence” is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that includes the same light and heavy chain variable regions.
  • a non-limiting example of a variable region linking sequence is a glycine-serine linker, such as a (G4S)s linker.
  • a linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, co-stimulatory domains, and/or primary signaling domains.
  • the receptor e.g., CAR
  • the receptor includes one, two, three, four, or five or more linkers.
  • the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids.
  • the linker is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more amino acids in length.
  • the antigen binding domain of the receptor e.g., CAR
  • the spacer domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3.
  • the spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • the spacer domain includes the CH2 and/or CH3 of lgG1 , lgG4, or IgD.
  • Illustrative spacer domains suitable for use in the receptors (e.g., CARs) described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD4, which may be wild-type hinge regions from these molecules or variants thereof.
  • the hinge domain includes a CD8a hinge region.
  • the hinge is a PD-1 hinge or CD152 hinge.
  • the hinge is an lgG4 hinge.
  • the “transmembrane domain” is the portion of the receptor (e.g., CAR) that fuses the extracellular binding portion and intracellular signaling domain and anchors the receptor to the plasma membrane of the cell (e.g., T-cell, such as a Treg).
  • the Tm domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • the Tm domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell receptor, CD35, CD3 , CD3y, CD30, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.
  • a receptor (e.g., CAR) includes a Tm domain derived from CD28.
  • a receptor includes a Tm domain derived from CD28 and a short oligo- or polypeptide linker, e.g., between 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the Tm domain and the intracellular signaling domain of the receptor.
  • a glycine-serine linker may be employed as such a linker, for example.
  • intracellular signaling domain of a receptor refers to the part of the receptor that participates in transducing the signal from binding to a target molecule/antigen into the interior of the cell to elicit cell function.
  • intracellular signaling domain refers to the portion of a protein which transduces the signal and that directs the cell to perform a specialized function. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of a full-length intracellular signaling domain as long as it transduces the signal.
  • intracellular signaling domain is meant to include any truncated portion of an intracellular signaling domain sufficient for transducing signal. Signals generated through the T cell receptor (TOR) alone are insufficient for full activation of the T cell, and a secondary or costimulatory signal is also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TOR (e.g., a TCR/CD3 complex) and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal.
  • primary signaling domains that initiate antigen-dependent primary activation through the TOR
  • costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal.
  • a receptor expressed by a genetically modified cell may include an intracellular signaling domain that includes one or more (e.g., 1 , 2, or more) “costimulatory signaling domains” and a “primary signaling domain.”
  • Primary signaling domains regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner.
  • Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Non-limiting examples of ITAM-containing primary signaling domains suitable for use in a receptor of the present disclosure include those derived from FcRy, FcRp, CD3y, CD35, CD3s, CD3 ⁇ , CD22, CD79a, CD79p, and CD665.
  • a receptor includes a CD3 ⁇ primary signaling domain and one or more costimulatory signaling domains.
  • the intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.
  • the receptor e.g., CAR
  • the receptor includes one or more costimulatory signaling domains to enhance the efficacy and expansion of immune effector cells (e.g., T cells) expressing the receptor.
  • costimulatory signaling domain or “costimulatory domain” refers to an intracellular signaling domain of a costimulatory molecule or an active fragment thereof.
  • Example costimulatory molecules suitable for use in receptors contemplated in particular embodiments include TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD1 1 , CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1 BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70.
  • the receptor e.g., CAR
  • the receptor includes one or more costimulatory signaling domains selected from the group consisting of 4-1 BB (CD137), CD28, and CD134, and a CD3 ⁇ primary signaling domain.
  • a receptor expressed by a cell genetically modified according to the methods of the present disclosure may include any variety of suitable domains including but not limited to a leader sequence; hinge, spacer and/or linker domain(s); transmembrane domain(s); costimulatory domain(s); signaling domain(s) (e.g., CD3 domain(s)); ribosomal skip element(s); restriction enzyme sequence(s); reporter protein domains; and/or the like.
  • suitable domains including but not limited to a leader sequence; hinge, spacer and/or linker domain(s); transmembrane domain(s); costimulatory domain(s); signaling domain(s) (e.g., CD3 domain(s)); ribosomal skip element(s); restriction enzyme sequence(s); reporter protein domains; and/or the like.
  • the extracellular binding domain of the receptor specifically binds a tumor antigen expressed on the surface of a cancer cell.
  • Non-limiting examples of tumor antigens to which the extracellular binding domain of the receptor may specifically bind include 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase/phosphodieste
  • the candidate therapeutic human T cells are genetically modified to express an antibody.
  • antibody also used interchangeably with “immunoglobulin” encompasses antibodies of any isotype (e.g., IgG (e.g., lgG1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the antigen, including, but not limited to single chain Fv (scFv), Fab, (Fab’) 2 , (SCFV’)2, and diabodies; chimeric antibodies; monoclonal antibodies, humanized antibodies, human antibodies; and fusion proteins comprising an antigen-
  • isotype e.
  • Immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, lgG 2 , IgGa, lgG4), delta, epsilon and mu heavy chains or equivalents in other species.
  • Full-length immunoglobulin “light chains” (usually of about 25 kDa or about 214 amino acids) comprise a variable region of about 1 10 amino acids at the NH 2 -terminus and a kappa or lambda constant region at the COOH-terminus.
  • Full-length immunoglobulin “heavy chains” (of about 150 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
  • An immunoglobulin light or heavy chain variable region (VL and VH, respectively) is composed of a “framework” region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, E. Kabat et al., Sequences of proteins of immunological interest, 4th ed. U.S. Dept.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs.
  • the CDRs are primarily responsible for binding to an epitope of an antigen. All CDRs and framework provided by the present disclosure are defined according to Kabat, supra, unless otherwise indicated.
  • an “antibody” thus encompasses a protein having one or more polypeptides that can be genetically encodable, e.g., by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • an antibody of the present disclosure is an IgG antibody, e.g., an lgG1 antibody, such as a human lgG1 antibody.
  • the cell expresses an antibody that comprises a human Fc domain.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • the candidate therapeutic human T cells are not genetically modified to express an engineered receptor.
  • the candidate therapeutic human T cells may be expanding during a therapeutic T cell manufacturing process.
  • expanding or “expanded” is meant the cells are cultured under conditions in which the cells proliferate. Suitable conditions may vary depending upon, e.g., the type of T cells being expanded.
  • Such conditions may include culturing the T cells in a suitable container (e.g., a cell culture plate or well thereof, a cassette, tube, bottle or bag suitable for use in an automated therapeutic cell manufacturing system, e.g., a closed automated therapeutic cell manufacturing system such as the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, or the like), in suitable medium (e.g., cell culture medium, such as RPMI, DMEM, IMDM, MEM, DMEM/F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO 2 , e.g., 3% to 10%, such as 5%.
  • T cells e.g., therapeutic T cells and the like
  • methods for activating and expanding cells for therapy are known in the art and are described, e.g., in U.S. Patent Nos. 6,905,874; 6,867,041 ; and 6,797,514; and PCT Publication No. WO 2012/079000, the contents of which are hereby incorporated by reference in their entirety.
  • T cells such methods may include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2.
  • a stimulatory agent and costimulatory agent such as anti-CD3 and anti-CD28 antibodies
  • Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC).
  • APC antigen presenting cell
  • One example is the Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells.
  • the T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012/129514, the contents of which are hereby incorporated by reference in their entirety.
  • the candidate therapeutic human T cells are expanded using an automated system designed for the manufacture of therapeutic cells.
  • automated system designed for the manufacture of therapeutic cells.
  • Non-limiting examples of such systems include the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, etc.
  • Detailed guidance and protocols for manufacturing therapeutic cells on such systems are available from the providers of such systems.
  • the methods comprise assessing the in vitro culture for the one or more HHV-6B analytes two or more times during expansion of the candidate therapeutic human T cells.
  • the in vitro culture may be assessed 2 or more, 3 or more, 4 or more, or 5 or more times during expansion of the candidate therapeutic human T cells.
  • the methods comprise assessing the in vitro culture for the one or more HHV-6B analytes one or more times at from 0 to 15 days post-activation, e.g., at from 0 to 20 days post-activation.
  • the methods may comprise harvesting the cells, i.e., removing the cells from the container(s)/bioreactor(s) in which the cells were expanding at the time of harvest.
  • the cells Prior to or subsequent to harvesting the cells, the cells may be concentrated if desired, e.g., by centrifugation, a suitable cell separation technique (e.g., magnetic beads), and/or the like.
  • the harvested cells are cryopreserved.
  • cryopreserved refers to cells that have been preserved or maintained by cooling to low subzero temperatures, such as 77 K or -196 deg. C. (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped.
  • Useful methods of cryopreservation and thawing cryopreserved cells, as well as processes and reagents related thereto include but are not limited to e.g., those described in U.S. Patent Nos.
  • freshness may refer to cells that have not been cryopreserved and, e.g., may have been directly obtained and/or used (e.g., transplanted, cultured, etc.) following collection from a subject or organ thereof.
  • a cell suspension is aliquoted into one or more vessels and pelleted by centrifugation.
  • Cell pellets may then be resuspended in cryopreservation media under cold conditions to reach a desired final concentration, such as e.g., 10 million live cells per mL, and the resuspended cells kept at 4-8 deg. C.
  • Cells prepared for cryopreservation may then be aliquoted into freezing containers and frozen using a controlled rate freezer. After controlled rate freezing is complete, cryopreserved may then be transferred to vapor phase liquid nitrogen for storage.
  • the methods of the present disclosure find use in screening the candidate therapeutic human T cells for the one or more HHV-6 analytes and/or HHV-6 reactivation to determine whether the candidate therapeutic human T cells are suitable for use in a cell based therapy.
  • the methods may further comprise identifying the candidate therapeutic human T cells as therapeutic T cells.
  • Such methods may further comprise administering to a subject in need thereof the therapeutic human T cells or progeny thereof.
  • the methods may further comprise administering to a subject in need thereof therapeutic human T cells other than the candidate therapeutic T cells, wherein the administered therapeutic human T cells are known to be HHV- 6B negative at the time of administration.
  • HHV-6B negative is meant it has been determined that the administered therapeutic human T cells do not comprise detectable latent or reactivated HHV-6B.
  • aspects of the present disclosure further include methods comprising assessing an in vitro culture comprising candidate therapeutic human T cells for HHV-6B reactivation.
  • HHV- 6B reactivation is meant a process by which latent HHV-6B switches to a lytic phase of replication.
  • assessing for HHV-6B reactivation comprises assessing the in vitro culture for a level of one or more HHV-6B analytes.
  • the one or more HHV-6B analytes may comprise any of the HHV-6B analytes described elsewhere herein.
  • the assessing comprises quantitative nucleic acid sequencing.
  • the quantitative nucleic acid sequencing comprises single cell nucleic acid sequencing.
  • single cell RNA sequencing scRNA-Seq
  • scRNA-Seq is a genomic approach for the detection and quantitative analysis of messenger RNA molecules in a biological sample.
  • scRNA-seq permits comparison of the transcriptomes of individual cells.
  • C1 SMARTer
  • Smart-seq2 e.g., see Picelli et al. (2013) Nat Methods 10:1096-8
  • MATQ-seq e.g., see Sheng et al.
  • MARS-seq e.g., see Jaitin et al. (2014) Science 343:776-9
  • CEL-seq e.g., see Hashimshony et al. (2012) Cell Rep. 2:666-73
  • Drop-seq e.g., see Macosko et al. (2015) Cell 161 :1202-14
  • InDrop e.g., see Klein et al. (2015) Cell 161 :1 187-201
  • Chromium e.g., see Zheng et al. (2017) Nat Commun. 8:14049
  • SEQ-well e.g., see Gierahn et al.
  • performing scRNA-seq comprising labeling the cells according to the Biolegend TotalSeqTM-A protocol (www.biolegend.com/en-us/protocols/totalseq-a- antibodies-and-cell-hashing-with-10x-single-cell-3-reagent-kit-v3-3-1 -protocol), performing the 10x 3’ Chromium Single-Cell RNA-Sequencing Protocol (support.10xgenomics.com/single-cell- gene-expression/library-prep/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide- v31 -chemistry), and sequencing at about 300-400 M reads per 10X library and about 25 M reads per Biolegend TotalSeqTM Library. Data from the single cell RNA sequencing may be deconvoluted into single cell transcriptomes, e.g., using barcode sequence information.
  • the assessing comprises aligning RNA sequence reads of single cells of the candidate therapeutic human T cells to a reference comprising human genomic DNA sequences and HHV- 6B RNA sequences, and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference.
  • reactivation may be determined to be present in a single cell when a threshold number of RNA sequence reads from the single cell map to the HHV-6B RNA sequences of the reference.
  • the threshold number of RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 1 1 , or about 10 RNA sequence reads.
  • Such methods may be computer-implemented as described in further detail below.
  • the assessing comprises performing scRNA-Seq
  • the assessing comprises pseudoaligning RNA sequence reads of single cells of the candidate therapeutic human T cells to an HHV-6B reference index, removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome, and counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index.
  • HHV-6B reactivation is determined to be present in a cell of the candidate therapeutic human T cells when a threshold number of remaining RNA sequence reads are pseudoaligned to the HHV-6B RNA reference index.
  • the threshold number is the threshold number of RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 1 1 , or about 10 RNA sequence reads.
  • Nucleic acids may be obtained from a given sample (e.g., a candidate therapeutic T cell culture) using extraction methods known to those skilled in the art. Such methods may initially include lysis, inactivation of nucleases, and separation of nucleic acids from cell debris. Methods for isolating nucleic acids from extracts employ combinations of extraction/precipitation, chromatography, centrifugation, electrophoresis and affinity separation. Additional methods for isolating nucleic acids will be recognized by those skilled in the art. In some cases, separation of the total nucleic acid from other components of the sample may not be performed.
  • extraction/precipitation methods may include solvent extraction performed to eliminate contaminants from nucleic acids (e.g., phenol-chloroform extraction), selective precipitation of nucleic acids using high concentrations of salt or changes in pH to precipitate proteins, and nucleic acid precipitation using isopropanol or ethanol.
  • solvent extraction performed to eliminate contaminants from nucleic acids (e.g., phenol-chloroform extraction)
  • selective precipitation of nucleic acids using high concentrations of salt or changes in pH to precipitate proteins e.g., phenol-chloroform extraction
  • selective precipitation of nucleic acids using high concentrations of salt or changes in pH to precipitate proteins
  • nucleic acid precipitation using isopropanol or ethanol e.g., phenol-chloroform extraction
  • nucleic acids can be isolated using methods that combine affinity immobilization with magnetic separation.
  • poly(A) mRNA may be bound to streptavidin-coated magnetic particles by biotin-labeled oligo(dT) and the particle complex removed from unbound contaminants using a magnet.
  • Such methods can replace several centrifugation, organic extraction and phase separation steps with a rapid magnetic separation step.
  • chromatography methods to isolate nucleic acids may utilize gel filtration, ion exchange, selective adsorption or affinity binding.
  • nucleic acids may be isolated from extracts by adsorption chromatography which relies on the nucleic acid-binding properties of silica or glass particles in the presence of chaotropic agents (see, e.g., U.S Patent Nos. 5,234,809 and 7,517,969, herein incorporated by reference).
  • chromatography and affinity separation is used in combination to isolate nucleic acids from any given sample. For example, silica or glass coated magnetic particles may be added to a sample containing nucleic acids.
  • a chaotropic agent Upon addition of a chaotropic agent, nucleic acids in the sample will bind to the silica or glass coating. Nucleic acids are then separated from unbound contaminants using a magnet.
  • Suitable chaotropic agents are substances that disrupt the structure of, and denatures, macromolecules such as proteins and nucleic acids, and includes, e.g., butanol, ethanol, guanidinium chloride, guanidinium isothiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, urea, and the like.
  • total nucleic acid may be isolated from a given sample by first lysing the sample so that nucleic acids are released into solution.
  • Silica or glass coated magnetic particles are added to the lysed sample together with an effective amount of a chaotropic agent (e.g., 8 M guanidinium chloride), to allow nucleic acids to adsorb onto the surfaces of the magnetic particles.
  • a chaotropic agent e.g. 8 M guanidinium chloride
  • a release agent is added to release the nucleic acids from the magnetic particles.
  • Nucleic acids are then eluted into a buffer of choice before proceeding to downstream processing (e.g., nucleic acid amplification and detection).
  • total nucleic acid may be isolated from a given sample by using a method described in Jangam, et al., known as filtration isolation of nucleic acids (FINA) (Jangam, et al., J. Clin. Microbiol., 47(8), 2363-2368 (2009)).
  • a method for isolation of HIV proviral DNA from leukocyte DNA from whole blood includes the use of a cell separation membrane disk placed in direct contact with an absorbent pad, which drives fluid flow by capillary pressure. Upon transfer of a sample of whole blood onto the disk, leukocytes and erythrocytes are trapped in the cell separation membrane, while plasma flows through into the absorbent pad.
  • Membrane-entrapped cells are lysed, and cell debris etc., are wicked into the absorbent pad.
  • the released nucleic acids are trapped within the membrane for further elution and processing.
  • total nucleic acid may be isolated from a given sample by using commercially available nucleic acid isolation kits that result in isolated nucleic acids ready for downstream processing (e.g., sequencing).
  • nucleic acids may be extracted and purification of nucleic acids as described in Sur et al. J. Mol. Diagn., 2010, 12 (5): 620-628.
  • a single pass of paramagnetic particles (PMPs), on which nucleic acids are adsorbed, through an immiscible hydrophobic liquid yields pure nucleic acid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification.
  • the PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a cartridge. Transporting PMPs through the immiscible phase yields DNA and RNA with equivalent purity as methods that utilize extensive wash steps.
  • nucleic acids may be isolated by exposing a sample comprising cells containing nucleic acids to an aqueous mixture comprising a lytic reagent and one or more beads capable of binding the nucleic acid released from the cells to form a nucleic acid-bead complex; and passing the nucleic acid-bead complex through an immiscible liquid layer to separate the nucleic acid from the aqueous mixture, where the one or more beads are magnetic, and the nucleic acid-bead complex is passed through and separated from the immiscible liquid layer with an applied magnetic field.
  • the immiscible liquid layer may be an organic liquid or a wax layer.
  • aspects of the present disclosure include methods of assessing in vivo therapeutic T cells for HHV-6B reactivation.
  • methods of monitoring a human subject receiving a T cell based therapy for HHV-6B reactivation in therapeutic T cells present in the subject comprise obtaining from the subject a biological sample comprising the therapeutic T cells, and assessing the therapeutic T cells for HHV-6B reactivation.
  • the manner of the assessment will vary depending upon the type of HHV-6B analyte(s) used to assess reactivation.
  • an immunoassay may be performed on the sample when an HHV-6B protein is used to assess for reactivation. Any of the immunoassays described elsewhere herein may be employed.
  • quantitative nucleic acid sequencing e.g., quantitative single cell nucleic acid sequencing
  • assessing the therapeutic T cells for HHV-6B reactivation comprises performing scRNA-Seq on the therapeutic T cells.
  • the assessing comprises pseudoaligning RNA sequence reads of single cells of the therapeutic T cells to an HHV-6B reference index, and removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome.
  • Such methods further comprise counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index.
  • HHV-6B reactivation is determined to be present in a cell of the therapeutic T cells when a threshold number of remaining RNA sequence reads are pseudoaligned to the HHV-6B RNA reference index.
  • the highly homologous regions of the HHV-6B reference index and human T cell transcriptome comprise a homologous region between HHV- 6B DR1 and human KDM2A.
  • the threshold number of remaining RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • the assessing comprises aligning RNA sequence reads of single cells of the therapeutic T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences, and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference, where HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • the methods further comprise ceasing the T cell based therapy. Such methods may further comprise administering to the subject a new T cell based therapy with cells determined to be HHV-6B negative.
  • the method further comprises administering an antiviral therapy to the subject.
  • Suitable antiviral therapies include, but are not limited to, administering to the subject an agent approved for treating HHV-6B infection in humans.
  • the antiviral therapy comprises administering ganciclovir, cidofovir, foscarnet, or any combination thereof to the subject.
  • the antiviral therapy comprises administering foscarnet to the subject.
  • the methods comprise continuing the T cell based therapy, e.g., by further administering therapeutic T cells from the same source as one or more previous administrations.
  • Biological samples of interest include those that comprise T cells, including but not limited to, whole blood samples (e.g., a peripheral blood sample), a fraction of whole blood comprising peripheral blood mononuclear cells (e.g., blood plasma), serum, a peripheral blood mononuclear cell (PBMC) sample, a gut tissue sample, urine, buffy coat, synovial fluid, bone marrow, cerebrospinal fluid, saliva, lymph fluid, seminal fluid, vaginal secretions, urethral secretions, exudate, transdermal exudates, pharyngeal exudates, nasal secretions, sputum, sweat, bronchoalveolar lavage, tracheal aspirations, fluid from joints, or vitreous fluid.
  • whole blood samples e.g., a peripheral blood sample
  • a fraction of whole blood comprising peripheral blood mononuclear cells e.g., blood plasma
  • serum e.g., a peripheral blood mononuclear cell (PBMC)
  • T cells can also be obtained from biological samples which may be derived from, for example, solid tissue samples.
  • T cells may be helper T cells (effector T cells or Th cells), cytotoxic T cells (CTLs), memory T cells, and regulatory T cells.
  • CTLs cytotoxic T cells
  • memory T cells e.g., adenosar cells
  • regulatory T cells e.g., regulatory T cells.
  • PBMC peripheral blood mononuclear cells isolated by techniques known to those of skill in the art, e.g., by Ficoll-Hypaque® density gradient separation.
  • Nucleic acid such as, genomic DNA or RNA may be extracted from lymphoid cells by methods known to those of skill in the art. Examples include using the QIAamp® DNA blood Mini Kit or a Qiagen DNeasy Blood extraction kit (both commercially available from Qiagen, Gaithersburg, Md., USA) to extract genomic DNA. Alternatively, total nucleic acid can be isolated from cells, including both genomic DNA and mRNA. In other embodiments, cDNA is transcribed from mRNA and then used as templates for amplification. The RNA molecules can be transcribed to cDNA using known reverse-transcription kits, such as the SMARTerTM Ultra Low RNA kit for Illumina sequencing (Clontech, Mountain View, Calif.) essentially according to the supplier's instructions.
  • known reverse-transcription kits such as the SMARTerTM Ultra Low RNA kit for Illumina sequencing (Clontech, Mountain View, Calif.) essentially according to the supplier's instructions.
  • aspects of the present disclosure further include methods of monitoring a therapeutic T cell product for HHV-6B reactivation.
  • the methods comprise administering a first population of therapeutic T cells from the therapeutic T cell product to a subject in need thereof, and culturing a second population of therapeutic T cells from the therapeutic T cell product, wherein the culturing comprises culturing the therapeutic T cells during a period subsequent to administration of the first population of therapeutic T cells to the subject.
  • Such methods further comprise, during the period subsequent to administration of the first population of therapeutic T cells to the subject, monitoring the T cell culture for HHV-6B reactivation.
  • the culturing commences prior to administration of the first population of therapeutic T cells to the subject (e.g., 7 or fewer days prior to administration), at the time of administration of the first population of therapeutic T cells to the subject, or subsequent to administration of the first population of therapeutic T cells to the subject, e.g., commencing within one, two, three, four or five days of administration of the first population of therapeutic T cells to the subject.
  • the duration of the culturing is from three days to two weeks, e.g., from three to 14 days, from three to 13 days, from three to 12 days, from three to 1 1 days, from three to 10 days, from three to 9 days, from three to 8 days, from three to 7 days, from three to 6 days, or from three to 5 days.
  • the culturing comprises continuation of the culture of the therapeutic T cells once a phase of manufacturing the therapeutic T cell product has concluded. For example, an aliquot (first population) of the expanded therapeutic T cells may be administered to the subject, and the remaining population (second population) may be cultured in parallel with administration of the first population to the subject.
  • Suitable culture conditions may include culturing the second population of therapeutic T cells from the therapeutic T cell product in a suitable container (e.g., a cell culture plate or well thereof, a cassette, tube, bottle, or the like) in suitable medium (e.g., cell culture medium, such as RPMI, DMEM, IMDM, MEM, DMEM/F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO2, e.g., 3% to 10%, such as 5%.
  • suitable medium e.g., cell culture medium, such as RPMI, DMEM, IMDM, MEM, DMEM/F-12, or the like
  • suitable temperature e.g., 32°C - 42°C, such as 37°C
  • pH e.g., pH 7.0 - 7.7, such as
  • Monitoring the T cell culture for HHV- 6B reactivation may include one or more (e.g., two or more, or three or more) assessments for HHV-6B reactivation. Any convenient manner of assessment may be employed. In some embodiments, the one or more assessments are performed using any of the methods of the present disclosure described herein for assessing HHV-6B reactivation, e.g., any of the quantitative nucleic acid sequencing approaches described herein, which are not reiterated here for purposes of brevity.
  • aspects of the present disclosure further include methods of preventing or mitigating viral reactivation in therapeutic human cells during a therapeutic human cell manufacturing process.
  • such methods comprise expanding the therapeutic human cells in the presence of an anti-viral agent at a concentration effective to prevent or mitigate viral reactivation in the expanding therapeutic human cells.
  • the therapeutic human cells are therapeutic human T cells.
  • HHV-6A, HHV-6B, and/or HHV-7 viral reactivation is prevented or mitigated.
  • the therapeutic human cells are therapeutic human induced pluripotent stem cells (iPSCs).
  • Non-limiting examples of anti-viral agents that may be employed include ganciclovir, cidofovir, foscarnet, or any combination thereof.
  • the effective concentration may vary depending upon the particular anti-viral agent employed and can be readily ascertained by one of skill in the art with the benefit of the present disclosure.
  • the therapeutic human cells are genetically modified to express an engineered receptor. Such cells are described elsewhere herein and not reiterated for purposes of brevity.
  • the therapeutic human cell manufacturing process may comprise expanding the human therapeutic cells.
  • Suitable manufacturing processes may be carried out, e.g., in an automated therapeutic cell manufacturing system, e.g., a closed automated therapeutic cell manufacturing system such as the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, or the like), in suitable medium (e.g., cell culture medium, such as RPMI, DMEM, IMDM, MEM, DMEM/F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO 2 , e.g., 3% to 10%, such as 5%.
  • suitable medium e.g., cell culture medium, such as RPMI, DMEM, IMDM,
  • Also provided by the present disclosure are computer-implemented methods, computer- readable media and computer devices.
  • the computer-readable media and computer devices find use, e.g., in practicing the computer-implemented methods of the present disclosure.
  • computer-implemented is meant at least one step of the method is implemented using one or more processors and one or more non-transitory computer-readable media.
  • the computer-implemented methods of the present disclosure may further comprise one or more steps that are not computer-implemented, e.g., obtaining a sample (e.g., a blood sample, or the like) from a subject, manufacturing T cells, preparing a sample for assessment (e.g., nucleic acid sequencing, immunoassay, and/or the like), administering a therapy to a subject based on the assessment, and/or the like.
  • a sample e.g., a blood sample, or the like
  • HHV-6B reactivation comprises aligning RNA sequence reads of single cells of the candidate human T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences, and counting the number of RNA sequence reads of the single cells mapping to the HHV-6B RNA sequences of the reference.
  • HHV-6B reactivation is determined to be present in a single cell when a threshold number of RNA sequence reads from the single cell map to the HHV-6B RNA sequences of the reference.
  • FIG. 8 is a flow diagram illustrating a non-limiting embodiment of such methods. According to this embodiment, the steps of the method may be performed as follows: Step 1 : Create a joint HHV-6B and host reference genome/ transcriptome
  • RNA-seq library that are from HHV-6
  • a software with the capacity to identify them is provided.
  • a combined reference of both human and HHV-6 nucleic acids is created. This requires both a combined genome (.fa file) and transcriptome (.gtf).
  • Step 2 Align single-cell datasets to combined reference
  • the single-cell sequencing library e.g. sample_with_hhv6
  • read mapping is performed to look for molecules that align to the HHV-6B genome/transcriptome.
  • Step 3 Count per single-cell reads mapping to the HHV-6B transcriptome
  • identifying potential super expressors or other HHV-6B+ cells requires summarizing the number of HHV-6B-associated reads by identifying single-cell barcodes with at least 10 HHV- 6B reads.
  • Computer-readable media and computer devices related to the above-described methods are also provided.
  • one or more non-transitory computer readable media having stored thereon instructions that cause a computer device to: align RNA sequence reads of single cells of a population of candidate human T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and count the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference.
  • a computer device comprising one or more processors and one or more non-transitory computer readable media.
  • the one or more non-transitory computer readable media have stored thereon instructions that cause the computer device to align RNA sequence reads of single cells of a population of human T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences, and count the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference.
  • HHV-6B reactivation in therapeutic T cells present in a subject receiving a T cell based therapy.
  • Such methods comprise pseudoaligning RNA sequence reads of single cells of the therapeutic T cells to an HHV-6B reference index, removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome, and counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index.
  • HHV-6B reactivation may be determined to be present in a cell of the therapeutic T cells when a threshold number of remaining RNA sequence reads are pseudoaligned to the HHV-6B RNA reference index.
  • the highly homologous regions of the HHV-6B reference index and human T cell transcriptome comprise a homologous region between HHV-6B DR1 and human KDM2A.
  • the threshold number of remaining RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 1 1 , or about 10 RNA sequence reads. Shown in FIG. 9 is a flow diagram illustrating a non-limiting embodiment of such methods. According to this embodiment, the steps of the method may be performed as follows:
  • Step 1 Create HHV-6B reference index for pseudoalignment
  • the specific example here utilizes the kallisto
  • Other tools such as salmon and older tools like grep can also be used.
  • an index must be built with a defined k-mer size (size 31 is used here, which is default): kallisto index -k 31 - 1 HHV6b_transcript ome . idx AF 157706 . fa
  • Step 2 Pseudoalign scRNA-seq datasets to only the HHV-6B reference index
  • Step 2b Optional Mapping of scRNA-seq data to host transcriptome
  • HHV-6+ cells While not required to identify HHV-6+ cells, this is a necessary step to derive host gene correlations with viral expression and/or reactivation. This can be performed using a standard command such as cellranger count:
  • Step 3 Remove reads mapping to highly homologous regions of HHV-6B and the human transcriptome
  • Step 4 Count number of HHV-6B aligned reads per single-cell barcode
  • This step similarly groups the HHV-6 expression by single-cell barcode to identify super expressor cells of HHV-6B transcripts. This works within the unix environment but can be similarly executed in various software environments.
  • Computer-readable media and computer devices related to the above-described methods are also provided.
  • one or more non-transitory computer readable media having stored thereon instructions that cause a computer device to: pseudoalign RNA sequence reads of single cells of therapeutic T cells to an HHV-6B reference index; remove RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and count the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index.
  • a computer device comprising one or more processors and one or more non-transitory computer readable media.
  • the one or more non-transitory computer readable media have stored thereon instructions that cause the computer device to pseudoalign RNA sequence reads of single cells of candidate therapeutic T cells to an HHV-6B reference index; remove RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and count the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index.
  • identifying a human therapeutic cell type susceptible to reactivation by a virus comprise (i) obtaining from a sequencing database of nucleic acid sequencing samples having at least one annotated read from a virus known to infect humans; (ii) performing step (i) for one or more viruses known to infect humans; (iii) obtaining metadata for the annotated biosample that was linked to specific sequencing reads, wherein the metadata comprises sample name, descriptor, biosample ID, derived organism, or any combination thereof; and (iv) identifying relevant cell types for a specific virus and identifying virus-specific reactivation, or identifying viral reactivation for a specific cell type and identifying cell type-specific reactivation. Shown in FIG. 10 is a flow diagram illustrating a non-limiting embodiment of such methods. According to this embodiment, the steps of the method may be performed as follows:
  • Step 1 Perform a Serratus API call for a specific virus using a valid nuccore ID
  • NC_001401 .1 the human adeno-associated virus. This command plugs into the Serratus API to stream all RNA-seq samples that have at least one annotated read being derived from the virus with the specific nuccore id.
  • Step 1 requires executing the API call for multiple viruses, such as all of those that have been known to infect humans (see attached table).
  • Step 2 Annotate relevant meta data onto the Serratus hits
  • RNA- seq libraries where one or more reads may be viral derived. Many of these will not be relevant for human samples. Explicitly, there are nearly 5.7 million RNA-seq libraries in Serratus, and it is not feasible to investigate each of them without specific methodologies. Performed here is a secondary API call to the NCBI Gene Expression Omnibus to include meta data (i.e. sample names, descriptors, biosample IDs, derived organism, etc.). Second, it was determined that more stringent filtering of the Serratus values per RNA-seq sample most reliably yields high- confidence viral reactivation pairs.
  • Explicitly required is a minimum of 100 reads in the overall library to be viral derived and with a minimum Serratus identity score of 50. These thresholds were found to be both sufficiently inclusive so as not to miss potential reactivation but that reduce the search space from 5.7M samples to -6,000 for viruses from the Herpesviridae, Polyomaviridae, Adenoviridae, and Parvoviridae families. library(data.table) library(dplyr) library(stringr)
  • Step 3 Specific filtering of API output
  • Step 3a Identification of virus-specific reactivation
  • a specific virus e.g. Human cytomegalovirus
  • a specific virus e.g. Human cytomegalovirus
  • RNA-seq samples that have reads mapping to dozens of viruses in low complexity repeat regions that we can exclude only when considering multiple viruses within the same analytical environment.
  • Step 3b Identification of cell type-specific reactivation
  • T cells e.g. T cells, IPSCs, etc.
  • a specific celltype e.g. T cells, IPSCs, etc.
  • regular expression filtering for a specific cell type. Examples of this for T cells are shown below:
  • Viruses that may be screened according to the above-described methods include, but are not limited, to those in the table below:
  • processor-based systems may be employed to implement the embodiments of the present disclosure.
  • Such systems may include system architecture wherein the components of the system are in electrical communication with each other using a bus.
  • System architecture can include a processing unit (CPU or processor), as well as a cache, that are variously coupled to the system bus.
  • the bus couples various system components including system memory, (e.g., read only memory (ROM) and random access memory (RAM), to the processor.
  • system memory e.g., read only memory (ROM) and random access memory (RAM)
  • System architecture can include a cache of high-speed memory connected directly with, in close proximity to, or integrated as part of the processor.
  • System architecture can copy data from the memory and/or the storage device to the cache for quick access by the processor. In this way, the cache can provide a performance boost that avoids processor delays while waiting for data.
  • These and other modules can control or be configured to control the processor to perform various actions.
  • Other system memory may be available for use as well.
  • Memory can include multiple different types of memory with different performance characteristics.
  • Processor can include any general purpose processor and a hardware module or software module, such as first, second and third modules stored in the storage device, configured to control the processor as well as a special-purpose processor where software instructions are incorporated into the actual processor design.
  • the processor may essentially be a completely self-contained computing system, containing multiple cores or processors, a bus, memory controller, cache, etc.
  • a multi-core processor may be symmetric or asymmetric.
  • an input device can represent any number of input mechanisms, such as a microphone for speech, a touch-sensitive screen for gesture or graphical input, keyboard, mouse, motion input, speech and so forth.
  • An output device can also be one or more of a number of output mechanisms.
  • multimodal systems can enable a user to provide multiple types of input to communicate with the computing system architecture.
  • a communications interface can generally govern and manage the user input and system output.
  • the storage device is typically a non-volatile memory and can be a hard disk or other types of computer-readable media which can store data that are accessible by a computer, such as magnetic cassettes, flash memory cards, solid state memory devices, digital versatile disks, cartridges, random access memories (RAMs), read only memory (ROM), and hybrids thereof.
  • a computer such as magnetic cassettes, flash memory cards, solid state memory devices, digital versatile disks, cartridges, random access memories (RAMs), read only memory (ROM), and hybrids thereof.
  • the storage device can include software modules for controlling the processor. Other hardware or software modules are contemplated.
  • the storage device can be connected to the system bus.
  • a hardware module that performs a particular function can include the software component stored in a computer-readable medium in connection with the necessary hardware components, such as the processor, bus, output device, and so forth, to carry out various functions of the disclosed technology.
  • Embodiments within the scope of the present disclosure may also include tangible and/or non-transitory computer-readable storage media or devices for carrying or having computerexecutable instructions or data structures stored thereon.
  • Such tangible computer-readable storage devices can be any available device that can be accessed by a general purpose or special purpose computer, including the functional design of any special purpose processor as described above.
  • such tangible computer-readable devices can include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other device which can be used to carry or store desired program code in the form of computer-executable instructions, data structures, or processor chip design.
  • a network or another communications connection either hardwired, wireless, or combination thereof
  • the computer properly views the connection as a computer-readable medium.
  • any such connection is properly termed a computer-readable medium.
  • Computer-executable instructions include, for example, instructions and data which cause a general purpose computer, special purpose computer, or special purpose processing device to perform a certain function or group of functions.
  • Computer-executable instructions also include program modules that are executed by computers in stand-alone or network environments.
  • program modules include routines, programs, components, data structures, objects, and the functions inherent in the design of special-purpose processors, etc. that perform tasks or implement abstract data types.
  • Computer-executable instructions, associated data structures, and program modules represent examples of the program code means for executing steps of the methods disclosed herein. The particular sequence of such executable instructions or associated data structures represents examples of corresponding acts for implementing the functions described in such steps.
  • Embodiments of the disclosure may be practiced in network computing environments with many types of computer system configurations, including personal computers, hand-held devices, multi-processor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, and the like. Embodiments may also be practiced in distributed computing environments where tasks are performed by local and remote processing devices that are linked (either by hardwired links, wireless links, or by a combination thereof) through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.
  • a method of monitoring a human subject receiving a T cell based therapy for HHV-6B reactivation in therapeutic T cells present in the subject comprising: obtaining from the subject a biological sample comprising the therapeutic T cells; and assessing the therapeutic T cells for HHV-6B reactivation.
  • the assessing comprises: aligning RNA sequence reads of single cells of the therapeutic T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • a computer-implemented method of assessing for HHV-6B reactivation in therapeutic T cells present in a subject receiving a T cell based therapy comprising: aligning RNA sequence reads of single cells of the therapeutic T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • RNA sequence reads 9. The method according to embodiment 7 or 8, wherein the threshold number of RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • assessing comprises: pseudoaligning RNA sequence reads of single cells of the therapeutic T cells to an
  • HHV-6B reference index removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index, wherein HHV-6B reactivation is determined to be present in a cell of the therapeutic T cells when a threshold number of remaining RNA sequence reads are pseudoaligned to the HHV-6B RNA reference index.
  • a computer-implemented method of assessing for HHV-6B reactivation in therapeutic T cells present in a subject receiving a T cell based therapy comprising: pseudoaligning RNA sequence reads of single cells of the therapeutic T cells to an HHV-6B reference index; removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index, wherein HHV-6B reactivation is determined to be present when a threshold number of remaining RNA sequence reads of a cell of the therapeutic T cells are pseudoaligned to the HHV-6B RNA reference index.
  • RNA sequence reads are from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • the method further comprises ceasing the T cell based therapy.
  • the method further comprises administering an antiviral therapy to the subject.
  • antiviral therapy comprises administering to the subject an agent approved for treating HHV-6B infection in humans.
  • antiviral therapy comprises administering ganciclovir, cidofovir, foscarnet, or any combination thereof to the subject.
  • the method comprises continuing the T cell based therapy.
  • a method comprising assessing an in vitro culture comprising candidate therapeutic human T cells for HHV-6B reactivation, wherein the in vitro culture is assessed for a level of one or more HHV-6B analytes by quantitative nucleic acid sequencing.
  • the assessing comprises: aligning RNA sequence reads of single cells of the candidate therapeutic human T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • a computer-implemented method of assessing an in vitro culture comprising candidate therapeutic human T cells for HHV-6B reactivation comprising: aligning RNA sequence reads of single cells of the candidate therapeutic human T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • 25 The method according to embodiment 23 or 24, wherein the threshold number of RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • HHV-6B reactivation is determined to be present in a cell of the candidate therapeutic human T cells when a threshold number of remaining RNA sequence reads are pseudoaligned to the HHV-6B RNA reference index.
  • a computer-implemented method of assessing an in vitro culture comprising candidate therapeutic human T cells for HHV-6B reactivation comprising: pseudoaligning RNA sequence reads of single cells of the candidate therapeutic human T cells to an HHV-6B reference index; removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index, wherein HHV-6B reactivation is determined to be present when a threshold number of remaining RNA sequence reads of a cell of the candidate therapeutic human T cells are pseudoaligned to the HHV-6B RNA reference index.
  • RNA sequence reads are from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • the engineered receptor is a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric cytokine receptor (OCR), a chimeric chemokine receptor, a synthetic notch receptor (synNotch), a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • OCR a chimeric cytokine receptor
  • SNK chimeric chemokine receptor
  • synNotch synthetic notch receptor
  • MEA Modular Extracellular Sensor Architecture
  • Tango receptor a Tango receptor
  • ChaCha receptor a generalized extracellular molecule sensor
  • a computer-implemented method of identifying a human therapeutic cell type susceptible to reactivation by a virus comprising:
  • step (ii) performing step (i) for one or more viruses known to infect humans;
  • a method of monitoring a therapeutic T cell product for HHV-6B reactivation comprising: administering a first population of therapeutic T cells from the therapeutic T cell product to a subject in need thereof; culturing a second population of therapeutic T cells from the therapeutic T cell product, wherein the culturing comprises culturing the therapeutic T cells during a period subsequent to administration of the first population of therapeutic T cells to the subject; and during the period subsequent to administration of the first population of therapeutic T cells to the subject, monitoring the T cell culture for HHV-6B reactivation.
  • the monitoring comprises: aligning RNA sequence reads of single cells of the therapeutic T cell product to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • a computer-implemented method of monitoring a therapeutic T cell product for HHV-6B reactivation comprising: aligning RNA sequence reads of single cells of the therapeutic T cell product to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and counting the number of RNA sequence reads of single cells mapping to the HHV-6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • the threshold number of RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • the monitoring comprises: pseudoaligning RNA sequence reads of single cells of the therapeutic T cell product to an HHV-6B reference index; removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index, wherein HHV-6B reactivation is determined to be present in a cell of the therapeutic T cell product when a threshold number of remaining RNA sequence reads are pseudoaligned to the HHV-6B RNA reference index.
  • a computer-implemented method of monitoring a therapeutic T cell product for HHV-6B reactivation comprising: pseudoaligning RNA sequence reads of single cells of the therapeutic T cell product to an HHV-6B reference index; removing RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and counting the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index, wherein HHV-6B reactivation is determined to be present when a threshold number of remaining RNA sequence reads of a cell of the therapeutic T cell product are pseudoaligned to the HHV-6B RNA reference index.
  • RNA sequence reads The method according to any one of embodiments 51 to 53, wherein the threshold number of remaining RNA sequence reads is from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • a method of preventing or mitigating viral reactivation in therapeutic human cells during a therapeutic human cell manufacturing process comprising expanding the therapeutic human cells in the presence of an anti-viral agent at a concentration effective to prevent or mitigate viral reactivation in the expanding therapeutic human cells.
  • anti-viral agent is selected from ganciclovir, cidofovir, foscarnet, or any combination thereof.
  • therapeutic human cells are therapeutic human induced pluripotent stem cells (IPSCs).
  • ISCs therapeutic human induced pluripotent stem cells
  • the engineered receptor is a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric cytokine receptor (CCR), a chimeric chemokine receptor, a synthetic notch receptor (synNotch), a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • CCR a chimeric cytokine receptor
  • GEMS generalized extracellular molecule sensor
  • One or more non-transitory computer readable media having stored thereon instructions that cause a computer device to: align RNA sequence reads of single T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and count the number of RNA sequence reads of single T cells mapping to the HHV-6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • a computer device comprising one or more processors; and one or more non-transitory computer readable media having stored thereon instructions that cause the computer device to: align RNA sequence reads of single T cells to a reference comprising human genomic DNA sequences and HHV-6B RNA sequences; and count the number of RNA sequence reads of single T cells mapping to the HHV- 6B RNA sequences of the reference, wherein HHV-6B reactivation is determined to be present when a threshold number of RNA sequence reads from a single cell map to the HHV-6B RNA sequences of the reference.
  • RNA sequence reads are from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • One or more non-transitory computer readable media having stored thereon instructions that cause a computer device to: pseudoalign RNA sequence reads of single cells of therapeutic T cells to an HHV-6B reference index; remove RNA sequence reads pseudoaligned to highly homologous regions of the HHV- 6B reference index and human T cell transcriptome; and count the number of remaining RNA sequence reads pseudoaligned to the HHV-6B reference index.
  • RNA sequence reads are from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • a computer device comprising one or more processors; and one or more non-transitory computer readable media having stored thereon instructions that cause the computer device to: pseudoalign RNA sequence reads of single cells of candidate therapeutic T cells to an HHV-6B reference index; remove RNA sequence reads pseudoaligned to highly homologous regions of the HHV-6B reference index and human T cell transcriptome; and count the number of remaining RNA sequence reads pseudoaligned to the HHV- 6B reference index.
  • RNA sequence reads are from 5 to 15, 6 to 14, 7 to 13, 8 to 12, 9 to 11 , or about 10 RNA sequence reads.
  • One or more non-transitory computer readable media having stored thereon instructions that cause a computer device to:
  • step (ii) perform step (i) for one or more viruses known to infect humans
  • a computer device comprising: one or more processors; and one or more non-transitory computer readable media having stored thereon instructions that cause the computer device to:
  • step (ii) perform step (i) for one or more viruses known to infect humans; (iii) obtain metadata for the annotated biosample that was linked to specific sequencing reads, wherein the metadata comprises sample name, descriptor, biosample ID, derived organism, or any combination thereof; and
  • Example 1 - HHV-6 is reactivated in human T cells
  • acute stress conditions such as fever, hematopoietic stem cell transplant (HSCT)— , or trauma 2 , latent viruses can become reactivated, leading to a variety of complex clinical manifestations— (FIG. 1 A).
  • HHV-6A infections are largely confined to sub-Saharan Africa—, the HHV-6B virus accounts for 97-100% of HHV-6 infections reported in the United States, United Kingdom, and Japan.
  • the canonical receptor for HHV-6B, TNFRSF4 (CD134 or 0X40) is upregulated during both CD4 and CD8 T cell activation (among other healthy tissues; FIG. 4, 2), supporting the notion that this viral strain could spread during in vitro T cell culture and expansion.
  • HHV-6 encephalitis In light of eight cases of HHV-6 encephalitis from patients receiving CAR T cell therapy, including in three clinical trials, jt was hypothesized that the lytic HHV-6 may be derived from cell therapy products.
  • Using research grade CAR T cell culture conditions cells were cultured over a 19 day period and screened by qPCR to detect the U31 transcript from the HHV-6B virus (Methods). Over the course of screening, four exemplar donors that by day 19 expressed a range of HHV-6B expression from 0.0015-0.90 copies of U31 per cell were identified (FIG. 2a).
  • Example 3 Clinically validated and FDA-approved CAR T cells harbor HHV-6 in vivo
  • this patient was clinically diagnosed with immune effector associated neurotoxicity syndrome (ICANS) following axi-cel treatment and subsequently developed altered mental status with transient hypotension beginning on day +9, which peaked on day +12 (grade 3a by CTCAE criteria, grade 2 by ASTCT) before returning to baseline by day +14 (FIG. 3d).
  • ICANS immune effector associated neurotoxicity syndrome
  • a spike in HHV-6+ cells at day +7 was observed that was resolved by day +14.
  • HHV6+ super-expressors at days +14 were detected that persisted in vivo until day +21 before these cells were undetectable at day 27.
  • the products used in cohorts 2 and 3 were cultured for 7-10 days before infusion, one can infer that HHV-6 may be reactivated in some cells when the therapy is administered but the virus was not yet transcriptionally active or abundant enough to be detected via single-cell sequencing.
  • these rare HHV6+ super expressors were detectable in peripheral blood draws.
  • the SJCAR19-09 case vignette suggests that Foscarnet may be an efficacious agent to mitigate the effects of viral infection during the course of therapy if HHV-6 expressing cells are identified during the course of treatment with CAR T cells.
  • the present work implicates the cell therapy as the source of a lytic virus, HHV-6. From the inferences using the Serratus database, latent viral reactivation may be limited but is likely to extend beyond HHV-6 and T cells to other viruses or forms of cell therapy. For example, reactivation of HSV-1 from reprogrammed induced pluripotent stem cells (iPSCs) in 18 RNA-seq libraries, as well as EBV expression in T cells (FIG. 1 c), was observed. The EBV receptor, CD21 , is expressed in gamma-delta T cells at levels exceeding B cell subsets in resting and stimulated conditions (FIG.
  • iPSCs reprogrammed induced pluripotent stem cells
  • Example 4 - HHV-6 is reactivated in clinical autologous CAR T cell products
  • CAR protein from flow-enriched cells
  • CAR transcript from flow-enriched cells
  • TCR a/p transcripts TCR a/p transcripts
  • CD3 expression the cellular identity of 28 total HHV-6B+ cells were verified as engineered CAR T cells that expressed a diversity of HHV-6B transcripts. Having confirmed the presence of HHV- 6B+, CAR+ T cells in vivo, the course of two patients with super-expressors in the post-infusion blood draws was more closely examined.
  • this patient was clinically diagnosed with immune effector-associated neurotoxicity syndrome (ICANS) following axi-cel treatment and subsequently developed altered mental status with tremulousness and word-finding difficulties beginning on day +9, which peaked on day +12 (grade 3a by CTCAE criteria, grade 2 by ASTCT) before returning to baseline by day +14.
  • ICANS immune effector-associated neurotoxicity syndrome
  • a spike in HHV-6+ cells was observed at day +7 that was resolved by day +14, and the dynamics of viral reactivation and clearance could be observed within 1 week of each other.
  • the symptoms correlated with the presence of HHV-6+ CAR T cells in the blood, it could not be concluded that the detection of HHV-6 in peripheral blood is causal for the patient’s neurotoxicity.
  • delirium and neurocognitive decline are common symptoms in patients with HHV-6 viremia receiving HSCT, whereas ICANS is not, HHV-6 is unlikely to be the pathogenic agent for ICANS in most patients receiving CAR T cell therapy
  • HHV-6 super-expressing cells were detected from patient SJCAR19-09 in cohort 3.
  • SJCAR19-09 tested positive, aligning with our single-cell-resolved detection of HHV-6+ CAR T cells in this individual.
  • SJCAR19-09 continued to test positive via qPCR at day +21 post-CAR T cell infusion and was started on foscarnet at day +24. After another positive qPCR test on day +27, the patient no longer had detectable levels of HHV-6 at day +34.
  • HHV-6+ super-expressors were detected at day +14 that persisted in vivo until day +21 before these cells were undetectable at day +27.
  • CAR T cells used in cohorts 2 and 3 were only cultured for 7-10 days before infusion, it was reasoned that HHV-6 may reactivate in vivo only after the therapy is administered. It was hypothesized that an extension of the culture, as is performed in many allogeneic manufacturing contexts to achieve higher cell numbers for multiple patients, could lead to viral reactivation in vitro. To test this, the culture of the SJCAR19-09 infusion was extended for an additional two weeks and examined at five time points during that period using scRNA-seq.
  • foscarnet may be a productive agent to mitigate the effects of viral infection during therapy. It was hypothesized that the addition of foscarnet to the allogeneic CAR T cell cultures could mitigate viral reactivation and spreading. Based on prior reports in other contexts of cell cultures being treated with 1 , 2.5, and 5mM concentrations of foscarnet (Stenberg et al. (1985) Antimicrob. Agents Chemother. 28:802-806), additional day 19 vials from the allogeneic CAR T cell donors were thawed and the culture was extended for another 5 days to assess viral levels.
  • the Serratus— database for 129 human viruses curated from ViralZone— was queried using the NG genome identification provided on the ViralZone webpage as input in the Serratus API.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • Lentivirus containing proprietary CAR sequences were administered early in culture. These T cell cultures were allowed to expand for up to 19 days and then frozen using a controlled rate freezer prior to storage in liquid nitrogen. For selected donors (FIG. 2f), the 19 day cryovial was thawed and re-cultured and expanded up to 7 additional days.
  • the master mix used was TaqMan Fast Virus 1 -Step Master Mix (ThermoFisher). Manufacturer’s protocol was followed to add the appropriate volumes of master mix, water, DNA and primers. All samples were prepared in triplicate and run on a QuantStudio 6/7 Flex Real-Time PCR System (ThermoFisher). Primer and probe sequences for designated HHV-6 marker (U31 ) were employed. Measurements were taken in triplicates and the mean value is shown for qPCR plots.
  • Single-cell RNA-seq scRNA-seq libraries were generated using the 10x Chromium Controller and the Chromium Single Cell 5' Library Construction Kit and human B cell and T cell V(D)J enrichment kit according to the manufacturer’s instructions. Briefly, the suspended cells were loaded on a Chromium controller Single-Cell Instrument to generate single-cell Gel Bead-In-Emulsions (GEMs) followed by reverse transcription and sample indexing using a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (BioRad). After breaking the GEMs, the barcoded cDNA was purified and amplified, followed by fragmenting, A-tailing and ligation with adaptors.
  • GEMs Gel Bead-In-Emulsions
  • BioRad 96-Deep Well Reaction Module
  • PCR amplification was performed to enable sample indexing and enrichment of scRNA- Seq libraries.
  • target enrichment from cDNA was conducted according to the manufacturer’s instructions.
  • the final libraries were quantified using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent).
  • 10x scRNA-seq libraries were sequenced as recommended by the manufacturer (-20,000 reads I cell) via a Nova-seq 6000 using an S4 flow cell.
  • Raw sequencing data was demultiplexed using CellRanger mkfastq and aligned to the host reference genome using CellRanger v6.0 and TCR sequences were processed using the CellRanger vdj pipeline with default settings.
  • a kallistolbustools 3 ⁇ workflow was developed to rapidly quantify reads from either single-cell or bulk transcriptomes.
  • GenBank AF157706 reference transcriptome was downloaded and a kallisto index using the default -k 31 (kmer) parameter was created.
  • raw sequencing reads were processed using the kallisto 'bus' command with appropriate hyperparameters for each version of the single-cell chemistry (either 14 or 16 bp sequence barcode and 10 or 12 bases of UMI sequence). After barcode and UMI correction, a plain text sparse matrix was emitted, corresponding to unique HHV-6B reads mapping to individual cells in the single-cell sequencing library.
  • the same index could be utilized with the standard kallisto 'quant' execution.
  • HHV-6B UMIs per cell based on empirical expression (e.g., FIG. 2d), and the ease of use/interpretation of the number 10, were used.
  • One cell in the in vivo dataset expressed 8 UMIs (FIG. 3b), which likely is a super expressor but under sequenced based on the number of overall UMIs detected for this cell.
  • FIG. 7a For visualization across the Day 19 samples (FIG. 7a), shown are both the rank-ordered expression per cell based on total HHV-6B UMIs as well as a null model where HHV-6B reads are allocated proportional to the number of total UMIs detected for that particular cell barcode.
  • D34 and D38 which had the highest number of HHV-6+ cells, were considered. This was achieved using the 'FindMarkers' function from Seurat after segregating HHV-6+ cells as those that have a minimum of 2 UMIs (to increase power) and HHV-6" as those with identically 0 HHV-6 UMIs. From the FindMarkers output, a statistic was computed, - log10(p.val)*log2FC, that preserves the direction of over/under expression while partially scaling the gene expression difference by the magnitude of the statistical association, resulting in the plot (FIG. 7d).
  • lymphotoxin genes LTA and LTB were distinguished as the consistent associations.
  • the homotrimer LTa 3 is secreted but the heterotrimer (LTaip 2 ) is membrane-bound 33 , this suggests that changes in the LT expression upon HHV-6 reactivation lead to a secreted signal via LTa 3 , but due to the pleiotropic effects of this cytokine trimer 33 the impact of its possible increased secretion is not evident.
  • targets of the E2F transcription factor were upregulated as the HHV-6B infection spread throughout culture, consistent with prior reports of E2F1 -induced HHV-6A expression in T cells—. Furthermore, observed was an increase in genes associated with oxidative phosphorylation, a pathway previously reported to be co-opted during active viral infection 32 . Overall, the results largely recapitulate known pro- and anti-viral responses, indicating that the host transcriptome at the single-cell level permits or resists HHV- 6B infection from spreading within the T cell culture.
  • Table 1 represents a summation of four large datasets reanalyzed for HHV-6B expression.
  • the number of cells and raw sequencing reads analyzed are reported, alongside the number of high-confidence HHV-6B UMIs from the quantification pipeline.
  • the number of HHV-6+ cells are barcodes annotated as cells before quality-control filtering in the original publication with at least one HHV-6 UMI.
  • Many super-expressors contain high levels of mitochondrial and ribosomal transcripts, potentially due to these cells nearing apoptosis.
  • HHV-6+ cells form the in vivo fusion product were from CAR T cells
  • the presence of the CAR transgene was assessed via RNA (kallisto pseudoalignment to the axi-cell transcript) and host gene expression values were noted (FIG. 3).
  • the CAR ‘FACS’ cell was from the 10x channel that enriched for CAR+ cells via flow cytometry but due to the mechanisms of 10x Genomics library preparation protocol, the specific protein abundance for this individual cell is not discernable.
  • the ‘cytotoxic’ signature was the sum of the UMIs for GZMK, GNLY, KLRG1 , ZEB2, and NKG7 genes.
  • the TCR a/p transcript number was the total number of UMIs aligning per barcode in the TCR vdj libraries.
  • HHV-6 reactivation and expression To evaluate other possible sources of HHV-6 reactivation and expression, the landscape of cell types that express TNFRSF4 (0X40), the canonical receptor of HHV-6B, and CR2 (CD21 ), the canonical receptor of EBV, was considered. Processed count data and/or summary plots were downloaded from the GTEx portal—, fetal development—, sorted resting and stimulated immune cells—, and endothelial cell lines—.
  • 0X40 While the overall expression of 0X40 appears to be 1 -2 orders of magnitude higher in activated T cells, the application of the present atlas-level analyses of the HHV-6B viral receptor suggests that endothelial cells may be targets of active HHV-6B infection, either from the primary infection or from cell therapy-mediated reactivation. Moreover, brain endothelial cell expression of 0X40 might underly the encephalitis observed in severe infections/reactivation.
  • Tang, H. etal. CD134 is a cellular receptor specific for human herpesvirus-6B entry. Proc. Natl. Acad. Sci. U. S. A. 110, 9096-9099 (2013).

Abstract

L'invention concerne des méthodes de surveillance d'un sujet humain recevant une thérapie basée sur des lymphocytes T pour une réactivation de HHV-6 dans des lymphocytes T thérapeutiques présentes chez le sujet. Dans certains modes de réalisation, les méthodes consistent à obtenir, à partir du sujet, un échantillon biologique comprenant les lymphocytes T thérapeutiques, et à évaluer les lymphocytes T thérapeutiques pour une réactivation de HHV-6. L'invention concerne également des méthodes comprenant l'évaluation d'une culture in vitro comprenant des lymphocytes T humains thérapeutiques candidats pour une réactivation HHV-6, la culture in vitro étant évaluée pour un niveau d'un ou de plusieurs analytes HHV-6 par séquençage d'acide nucléique quantitatif. La présente divulgation concerne en outre des supports lisibles par ordinateur non transitoires et des dispositifs informatiques qui trouvent une utilisation dans la mise en œuvre des méthodes de la présente divulgation.
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