WO2017165869A1 - Evaluaton of transgenes of genetically modified cells in bodily fluids - Google Patents

Evaluaton of transgenes of genetically modified cells in bodily fluids

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Publication number
WO2017165869A1
WO2017165869A1 PCT/US2017/024189 US2017024189W WO2017165869A1 WO 2017165869 A1 WO2017165869 A1 WO 2017165869A1 US 2017024189 W US2017024189 W US 2017024189W WO 2017165869 A1 WO2017165869 A1 WO 2017165869A1
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Prior art keywords
cells
transgene
subject
method
sample
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PCT/US2017/024189
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French (fr)
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Karsten Schmidt
Nicholas Nelson
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Trovagene, Inc.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/17Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48Reproductive organs
    • A61K35/54Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/7051T-cell receptor (TcR)-CD3 complex
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA

Abstract

Provided is a method of detecting the presence of genetically modified cells in a subject. The method comprises detecting a first transgene from the cells in a cell-free fraction of a first sample from the subject. In this method, the sample is a bodily fluid, the transgene is not part of the genome of the subject, and the transgene is not part of the natural genome of the cells.

Description

EVALUATION OF TRANSGENES OF GENETICALLY MODIFIED CELLS IN

BODILY FLUIDS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/313,461, filed March 25, 2016, and incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present application generally relates to the detection of transgenes in bodily fluids. More specifically, the application is directed to the detection of transgenes in cells introduced into a subject to characterize the state of the cells in the subject.

(2) Description of the related art

Numerous therapies under development utilize transgenic cells that are introduced into the body. These therapies include treatment of metabolic diseases (Aathira and Jain, 2014), blood disorders (Nienhuis, 2013), infectious disease (Lam and Bollard, 2013), immune disease (Chinen and Buckley, 2010), diseases involving enzyme deficiencies (Doerfler et al., 2016), and, especially, cancer therapeutics (Kim et al., 2016 and references cited therein). Cells utilized for these therapies include hematopoietic stem cells derived from umbilical cord blood, peripheral blood, or bone marrow; and immune cells including dendritic cells, T cells, and natural killer (NK) cells.

Approaches for the cancer therapies include (i) therapy with cells that give rise to a new immune system which may be better able to recognize and kill tumor cells through the infusion of hematopoietic stem cells, (ii) therapy with immune cells such as dendritic cells which are designed to activate the patient's own resident immune cells (e.g. T cells) to kill tumor cells, and (iii) direct infusion of immune cells such as T cells and NK cells which are prepared to find, recognize, and kill cancer cells directly. In all three approaches, therapeutic cells are harvested, generally from the patient, transformed with a vector comprising transgenes and proliferated in the laboratory, then infused into the patient.

The most popular of these transgenic cell therapies utilize chimeric antigen receptor- modified T (CAR-T) cells. These cells are transduced with genes encoding fusion proteins of antigen -recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. Second- and third- generations of these cells further comprise transgenic intracellular signaling domains to enhance the effectiveness of the cells (Kim et al., 2016). CAR-T cells can also comprise a suicide gene system, such as a system utilizing inducible Caspase-9 to limit off-tumor toxicity of the CAR-T cells (Gargett and Brown, 2014).

Another example of a transgenic cell therapy under active development is genetically modified NK cells. These therapies seek to enhance the tumor cell-killing abilities of NK cells. Various modifications of NK cells being developed or contemplated include addition of transgenes encoding cytokines (e.g., IL-2 or IL-15), antigen receptors (e.g., a chimeric antigen receptor as described above for CAR-T cells), activating receptors, and silencers of inhibitory receptors. See, e.g., Dahlberg (2015) and references cited therein.

Still another example of a transgenic cell therapy under development is induced pluripotent stem cells (iPSCs), which are transformed with transcription factors. Because iPSCs have a propensity for tumor formation, the iPSCs under development for cancer therapy generally use nonintegrative reprogramming strategies and/or suicide mechanisms such as inducible Caspase-9 (as discussed above) and/or other mechanisms to increase their safety (Kim et al., 2015).

Since the fate of these cells after infusion can vary greatly with construct, vector, therapeutic target, cell type and individual patient, methods to monitor the progress of these therapies, both while under development and as a clinical tool, would be useful. The present invention addresses that need.

BRIEF SUMMARY OF THE INVENTION

Provided is a method of detecting the presence of genetically modified cells in a subject. The method comprises detecting a first transgene from the cells in a cell-free fraction of a first sample from the subject. In this method, the sample is a bodily fluid; the transgene is not part of the genome of the subject; and the transgene is not part of the natural genome of the cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, the use of "or" is intended to include "and/or", unless the context clearly indicates otherwise. As used herein, a "transgene" is genetic material that has been transferred by genetic engineering techniques to a cell.

Provided is a method of detecting the presence of genetically modified cells in a subject. The method comprises detecting a first transgene from the cells in a cell-free fraction of a first sample from the subject. In this method, the sample is a bodily fluid; the transgene is not part of the genome of the subject; and the transgene is not part of the natural genome of the cells.

As used herein, a transgene is not part of the natural genome of a cell if the genetic material of the transgene would not be present in the cell without the genetic material being genetically engineered into the cell or its ancestor cells.

As used herein, a transgene is not part of the genome of a subject if that transgene would not be in the subject without the genetic material of the transgene being genetically engineered into a cell, or its ancestor cells, in the subject.

In these methods, the subject can be any vertebrate, for example a bird or mammal. In some embodiments the subject is a mammal, for example a human, or a domesticated animal such as a companion animal or a farm animal. In some of those embodiments, the subject is a human.

The present invention also encompasses transgenes that are part of the genome of the subject, but are expressed in greater or lesser amount as a result of the transgene being introduced into the cells.

The sample can be any bodily fluid that would be expected to have cell-free nucleic acids. Non-limiting examples of bodily fluids include, but are not limited to, peripheral blood, serum, plasma, urine, lymph fluid, amniotic fluid, and cerebrospinal fluid. In some embodiments, the bodily fluid is blood, plasma, serum or urine.

Urine is known to comprise nucleic acids that reflect somatic cell, including cancer cell, death. See, e.g., US Patents RE39920E1; 6,287,820; 6,492,144; and 8,486,626; US Patent Application Publications 2008/0139801; 2010/0068711; 2015/0139946; and 2016/0002740; and PCT Publication WO 2015/073162.

The cell free transgene detected in the bodily fluid can be DNA or mRNA. In various embodiments, the cell free transgene is DNA.

The transgene in the bodily fluid can be detected by any method known in the art. Nonlimiting examples include MALDI-TOF, HR-melting, di-deoxy-sequencing, single-molecule sequencing, use of probes, pyrosequencing, second generation high-throughput sequencing, SSCP, RFLP, dHPLC, CCM, or methods utilizing the polymerase chain reaction (PCR), e.g., digital PCR, quantitative-PCR, or allele- specific PCR (where the primer or probe is complementary to the variable gene sequence). Where the transgene sequence is similar to a naturally occurring sequence, the transgene can be detected by PCR using blocking sequences that block amplification of the native sequences, e.g., as taught in WO 2015/073162 and references cited therein.

To detect the transgene, only a small segment of the nucleic acid need be detected, if that small segment distinguishes the transgene from any sequence in the non-genetically engineered genome of the subject.

In various embodiments, the first transgene is quantified, using any appropriate method.

The cells of the invention methods can be introduced into the subject for any purpose, including to introduce a trait to the subject, or enhance a trait of the subject, where the subject is otherwise healthy. In some embodiments, the cells are introduced into the subject as a treatment for a disease or disorder, e.g., a genetic disease, a metabolic disease, a blood disorder, an infectious disease, an immune disease, or cancer.

In some embodiments, the cells are derived from cells of an individual other than the subject. Nonlimiting examples of such cells include nonautologous umbilical cord cells, nonautologous stem cells and transplanted cells, such as a tissue transplant, blood transfusion, or stem cell transplantation. In other embodiments, the cells are derived from the subject's cells, e.g., cells removed from the subject, genetically engineered with the transgene, proliferated in vitro, then infused into the subject.

In various embodiments, the cells are stem cells, e.g., hematopoietic stem cells or cancer stem cells. In other embodiments, the cells are immune cells, e.g., macrophages, B-cells, hematopoietic stem cells, dendritic cells, T cells or natural killer (NK) cells.

In some embodiments, the cells are chimeric antigen receptor T-cells (CAR-T cells). In these embodiments, the transgene can be any component of the DNA that has been genetically engineered into the cells. In various embodiments, the transgene encodes a single-chain variable fragment (scFv) of an antibody. In some of those embodiments, the scFv specifically binds to a disease-associated antigen, for example a tumor-associated antigen. Nonlimiting examples of disease-associated antigens are TRAIL-receptorl, CD19, CD20, HER2, HA-1, H/HLA-A2, ERBB2, EGFRvIII, CD138, GD2, ERB2, dectinl, CEA, CSPG4, PSMA, CS 1, EphA2, NY- ESO-1, MOG, B7, ERBB receptor, mesothelin, IL-l lRa, FITC, IL13Ra2, IL13R, a-folate receptor, ROR-1, CD22, CD30, CD33, CD123, LewisY, kappa, NKG2D, CD171, PSMA, folate receptor, IL-13 zetakin, erbB T4+, and FAP.

In additional embodiments where the cells are CAR-T cells, the transgene is a portion of a suicide safety switch gene cassette, e.g., caspase 9 or a drug-binding domain. See, e.g., Gargett and Brown, 2014. Alternatively, the transgene can encode an intracellular signaling domain from a costimulatory protein receptor, a portion of the vector, or a noncoding sequence.

In other embodiments, the cells are genetically modified NK cells. The invention methods are not narrowly limited to detecting any particular transgene in genetically modified NK cells. In some embodiments, the first transgene encodes a cytokine, an antigen receptor, an activating receptor, or a silencer of an inhibitory receptor. In various embodiments, the first transgene encodes a chimeric antigen receptor.

In additional embodiments, the cells are induced pluripotent stem cells (iPSTs). Any transgene in those cells may be used as the first transgene in these methods. In some embodiments, the first transgene encodes a transcription factor used to induce the iPSTs or a gene involved in an inducible safety switch gene cassette.

The detection of the transgene using these methods can be used to determine the presence or persistence of the cells, as well as the characterization, measurement, or prediction of a specific side effect or symptom of the cells in a subject. The quantitation of the transgene in the sample provides further refinement of those determinations. For example, the excess proliferation of the cells may predict cytokine release syndrome (described in Kim et al., 2016).

In some embodiments of these methods, the first transgene is detected in a second sample of a bodily fluid from the subject. In these embodiments, the second sample was taken from the subject at a different time from the first sample. A third, fourth, etc. sample can also be taken. In this way, the presence, absence, or quantity of the transgene and the transgenic cells can be determined over time. When the transgene is quantified in the sample, a dynamic picture of the pharmacokinetics, distribution, and/or retention of the genetically modified cells in the subject can be established. For example, a specific side effect or symptom resulting from the treatment can be characterized, measured, or predicted.

The quantities of the transgene in the first sample and second sample can also be correlated with a parameter of a disease in the subject, for example tumor burden or therapeutic window, i.e., the quantity of the cell in the patient that causes a therapeutic effect. In various embodiments, a second gene is quantified in the first and second sample. In some of these embodiments, the second gene is a gene from the genome of the subject. Nonlimiting examples include a gene that characterizes a negative effect of the treatment or an aspect of the immune status of the subject, e.g., a gene characteristic of a particular immune cell or an mRNA characteristic of a particular activated immune cell, to determine whether the immune system is over-activated or to determine the effectiveness of the genetically modified cells to stimulate production or activation of those immune cells.

Thus, these embodiments are useful for characterizing, measuring or predicting a specific side effect or symptom resulting from the treatment.

In other embodiments, the second gene is a gene having a mutation associated with a cancer in the subject. This allows for the characterization, measurement, or prediction of a specific side effect or symptom of the treatment. For example, cytokine release syndrome is known to be correlated with tumor burden at injection time of CAR-T cells (Kim et al., 2016).

Nonlimiting examples include mutations associated with a cancer in ABL1, BRAF, CHEK1, FANCC, GAT A3, JAK2, MITF, PDCD1LG2, RBM10, STAT4, ABL2, BRCA1, CHEK2, FANCD2, GATA4, JAK3, MLH1, PDGFRA, RET, STK11, ACVR1B, BRCA2, CIC, FANCE, GATA6, JUN, MPL, PDGFRB, RICTOR, SUFU, AKT1, BRD4, CREBBP, FANCF, GID4(C17orf39), KAT6A (MYST3), MRE11A, PDK1, RNF43, SYK, AKT2, BRIP1, CRKL, FANCG, GLI1, KDM5A, MSH2, PIK3C2B, ROS 1, TAF1, AKT3, BTG1, CRLF2, FANCL, GNA11, KDM5C, MSH6, PIK3CA, RPTOR, TBX3, ALK, BTK, CSF1R, FAS, GNA13, KDM6A, MTOR, PIK3CB, RUNX1, TERC, AMER1 (FAM123B), Cl lorf30 (EMSY), CTCF, FAT1, GNAQ, KDR, MUTYH, PIK3CG, RUNX1T1, TERT promoter, APC, CARD11, CTNNA1, FBXW7, GNAS, KEAP1, MYC, PIK3R1, SDHA, TET2, AR, CBFB, CTNNB 1, FGF10, GPR124, KEL, MYCL (MYCL1), PIK3R2, SDHB, TGFBR2, ARAF, CBL, CUL3, FGF14, GRIN2A, KIT, MYCN, PLCG2, SDHC, TNFAIP3, ARFRP1, CCND1, CYLD, FGF19, GRM,3 KLHL6, MYD88, PMS2, SDHD, TNFRSF14, ARID 1 A, CCND2, DAXX, FGF23, GSK3B, KMT2A (MLL), NF1, POLD1, SETD2, TOPI, ARID IB, CCND3, DDR2, FGF3, H3F3A, KMT2C (MLL3), NF2, POLE, SF3B 1, TOP2A, ARID2, CCNE1, DICERl, FGF4, HGF, KMT2D (MLL2), NFE2L2, PPP2R1A, SLIT2, TP53, ASXL1, CD274, DNMT3A, FGF6, HNF1A, KRAS, NFKBIA, PRDM1, SMAD2, TSC1, ATM, CD79A, DOT1L, FGFR1, HRAS, LMOl, NKX2-1, PREX2, SMAD3, TSC2, ATR, CD79B, EGFR, FGFR2, HSD3B 1, LRP1B, NOTCH1, PRKAR1A, SMAD4, TSHR, ATRX, CDC73, EP300, FGFR3, HSP90AA1, LYN, NOTCH2, PRKCI, SMARCA4, U2AF1, AURKA, CDH1, EPHA3, FGFR4, IDH1, LZTR1, NOTCH3, PRKDC, SMARCB 1, VEGFA, AURKB, CDK12, EPHA5, FH, IDH2, MAGI2, NPM1, PRSS8, SMO, VHL, AXIN1, CDK4, EPHA7, FLCN, IGF1R, MAP2K1, NRAS, PTCH1, SNCAIP, WISP3, AXL, CDK6, EPHB 1, FLT1, IGF2, MAP2K2, NSD1, PTEN, SOCS 1, WT1, BAP1, CDK8, ERBB2, FLT3, IKBKE, MAP2K4, NTRK1, PTPN11, SOX10, XPOl, BARD1, CDKN1A, ERBB3, FLT4, IKZF1, MAP3K1, NTRK2, QKI, SOX2, ZBTB2, BCL2, CDKNIB, ERBB4, FOXL2, IL7R, MCLl, NTRK3, RACl, SOX9, ZNF217, BCL2L1, CDKN2A, ERG, FOXP1, INHBA, MDM2, NUP93, RAD50, SPEN, ZNF703, BCL2L2, CDKN2B, ERRFI1, FRS2, INPP4B, MDM4, PAK3, RAD51, SPOP, BCL6, CDKN2C, ESR1, FUBP1, IRF2, MED 12, PALB2, RAF1, SPTA1, BCOR, CEBPA, EZH2, GABRA6, IRF4, MEF2B, PARK2, RANBP2, SRC, BCORL1, CHD2, FAM46C, GATA1, IRS2, MEN1, PAX5, RARA, STAG2, BLM, CHD4, FANCA, GATA2, JAK1, MET, PBRM1, RB I, and STAT3.

In additional embodiments, the second gene is a second transgene from the cells. In these embodiments, the quantities of the first transgene and the second transgene in the first sample and the second sample may be compared to each other to determine genetic integrity of the cell population or persistence of vector sequences in the cell population. In some of these embodiments where the cells are CAR-T cells, the first CAR-T transgene encodes a tumor- associated antigen and the second CAR-T transgene is a vector sequence or encodes caspase 9, a drug-binding domain, or an intracellular signaling domain from a costimulatory protein receptor.

Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification be considered exemplary only, with the scope and spirit of the invention being indicated by the claims.

References

Aathira and Jain, 2014, World J. Diabetes 15:689-696.

Chinen and Buckley, 2010, J. Allergy Clin. Immunol. 125:S324-325.

Dahlberg et al., 2015, Frontiers in Immunol. Article 605.

Doerfler et al., 2016, Mol. Ther. Methods Clin. Dev. 3: 15053.

Kim, JJ, 2015, Biomarker Insights 10(S 1): 125- 131.

Kim et al., 2016, Arch. Pharm. Res. sl22272-016-0719-7. Gargett and Brown, 2014, Frontiers in Pharmacol. Article 235.

Lam and Bollard, 2013, Immunotherapy 5:407-414.

Neinhius, 2013, Blood 122: 1556-1564.

In view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

Claims

What is claimed is:
1. A method of detecting the presence of genetically modified cells in a subject, the method comprising detecting a first transgene from the cells in a cell-free fraction of a first sample from the subject, wherein
the sample is a bodily fluid;
the transgene is not part of the genome of the subject; and
the transgene is not part of the natural genome of the cells.
2. The method of claim 1, wherein the cells are not genetically engineered cells from the subject.
3. The method of claim 1, wherein the cells are derived from cells removed from the subject, genetically engineered with the transgene, proliferated in vitro, then infused into the subject.
4. The method claim 1, wherein the cells are stem cells.
5. The method of claim 4, wherein the stem cells are hematopoietic stem cells or cancer stem cells.
6. The method of claim 1, wherein the cells are immune cells.
7. The method of claim 6, wherein the immune cells are macrophages, B-cells, hematopoietic stem cells, dendritic cells, T cells or natural killer cells.
8. The method of claim 3, wherein the cells are chimeric antigen receptor T-cells (CAR- T cells).
9. The method of claim 8, wherein the first CAR-T transgene encodes a single-chain variable fragment (scFv).
10. The method of claim 9, wherein the scFv specifically binds to a disease-associated antigen.
11. The method of claim 10, wherein the disease-associated antigen is a tumor- associated antigen.
12. The method of claim 10, wherein the disease-associated antigen is TRAIL-receptorl, CD19, CD20, HER2, HA-1, H/HLA-A2, ERBB2, EGFRvIII, CD138, GD2, ERB2, dectinl, CEA, CSPG4, PSMA, CS 1, EphA2, NY-ESO-1, MOG, B7, ERBB receptor, mesothelin, IL- l lRa, FITC, IL13Ra2, IL13R, a-folate receptor, ROR-1, CD22, CD30, CD33, CD123, LewisY, kappa, NKG2D, CD171, PSMA, folate receptor, IL-13 zetakin, erbB T4+, or FAP.
13. The method of claim 8, wherein the first CAR-T transgene is a portion of a suicide safety switch gene cassette.
14. The method of claim 13, wherein the portion of the suicide safety switch gene cassette is caspase 9 or a drug-binding domain.
15. The method of claim 8, wherein the first CAR-T transgene encodes an intracellular signaling domain from a costimulatory protein receptor.
16. The method of claim 1, wherein the first transgene encodes a portion of a viral vector or is a noncoding sequence.
17. The method of claim 3, wherein the cells are genetically modified NK cells.
18. The method of claim 17, wherein the first transgene encodes a cytokine, an antigen receptor, an activating receptor, a silencer of an inhibitory receptor, a portion of a viral vector, or a noncoding sequence.
19. The method of claim 17, wherein the first transgene encodes a chimeric antigen receptor.
20. The method of claim 3, wherein the cells are induced pluripotent stem cells (iPSTs).
21. The method of claim 30, wherein the first transgene encodes a transcription factor used to induce the iPSTs or a gene involved in an inducible safety switch gene cassette.
22. The method of claim 1, wherein the first transgene in the sample is quantified.
23. The method of claim 1, wherein the bodily fluid is blood, plasma, serum or urine.
24. The method of claim 1, wherein the bodily fluid is urine.
25. The method of claim 1, further comprising detecting the first transgene in a second sample of a bodily fluid from the subject, wherein the second sample was taken from the subject at a different time from the first sample.
26. The method of claim 25, wherein the first transgene in the first sample and second sample are quantified.
27. The method of claim 26, wherein the quantities of the first transgene in the first sample and second sample are compared to each other to determine pharmacokinetics, distribution, and/or retention of the genetically modified cells in the subject.
28. The method of claim 26, wherein the quantities of the first transgene in the first sample and second sample are compared to each other and correlated with a parameter of a disease in the subject.
29. The method of claim 27, wherein the parameter of a disease is tumor burden or therapeutic window.
30. The method of claim 26, wherein a second gene is quantified in the first sample and second sample.
31. The method of claim 30, wherein the second gene is a gene from the genome of the subject.
32. The method of claim 31, wherein the second gene is a gene that characterizes an aspect of the immune status of the subject or a negative effect of the cells.
33. The method of claim 32, wherein the second gene is quantified to characterize, measure or predict a specific side effect or symptom associated with the presence of the cells in the subject.
34. The method of claim 30, wherein the second gene is a gene having a mutation associated with a cancer in the subject.
35. The method of claim 30, wherein the second gene is a second transgene from the cells.
36. The method of claim 35, wherein a the quantities of the first transgene and the second transgene in the first sample and the second sample are compared to each other to determine genetic integrity of the cell population or persistence of vector sequences in the cell population.
37. The method of claim 36, wherein the cells are CAR-T cells, the first transgene encodes a tumor- associated antigen, and the second transgene is a vector sequence or encodes caspase 9, a drug-binding domain, or an intracellular signaling domain from a costimulatory protein receptor.
PCT/US2017/024189 2016-03-25 2017-03-25 Evaluaton of transgenes of genetically modified cells in bodily fluids WO2017165869A1 (en)

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