# US 12,178,831 B2 · Techniques for generating cell-based therapeutics using recombinant T cell receptor genes

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- Number: US 12,178,831 B2
- Document ID: US12178831B2
- Status: Granted
- Kind: B2
- Application number: 16/976,983
- Filed: 2019-02-27
- Published: 2021-01-07
- Granted: 2024-12-31
- Earliest claimed: 2018-03-01
- Assignees: University of Kansas
- Applicants: University of Kansas
- Inventors: Brandon Dekosky; Cheng-Yu Chung
- Web page: https://patentgrep.com/patent/US12178831B2

## CPC

- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/22: Immunosuppressive or immunotolerising
- A61K 40/32: T-cell receptors [TCR]
- A61K 40/418: {Antigens related to induction of tolerance to non-self}
- A61K 40/42: Cancer antigens
- A61K 40/46: Viral antigens
- A61P 35/00: Antineoplastic agents
- C12N 5/0636: {T lymphocytes}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2239/31: characterized by the route of administration
- A61K 2239/50: Colon
- A61K 2239/56: Kidney
- A61K 2239/59: Reproductive system, e.g. uterus, ovaries, cervix or testes
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present technology relates generally to compositions and methods for creating recombinant T cell receptor (TCR) libraries and methods of their therapeutic use. The compositions and methods of the present technology are useful for rapid isolation of antigen-specific TCR repertoires as personalized, targeted therapies for cancer and viral infection.

## Claims

### Claim 1

A method for preparing a recombinant TCR library, the method comprising
(a) lysing a single T cell in a compartment having a volume of 5 nL or less,
(b) isolating mRNA of the lysed single T cell in the compartment using an mRNA capture reagent;
(c) reverse transcribing the mRNA into cDNA in the compartment by overlap extension reverse transcription PCR (OE-RT-PCR) to generate linked cDNA products, wherein the linked cDNA products comprise
(i) a first polynucleotide encoding a T cell receptor (TCR) a polypeptide and a second polynucleotide encoding a TCRβ polypeptide; or
(ii) a first polynucleotide encoding a TCRγ polypeptide and a second polynucleotide encoding a TCRδ polypeptide,
wherein the first and second polynucleotides are a cognate pair and are operably linked via a linker polynucleotide;
(d) amplifying the linked cDNA products using nested PCR to generate TCR amplicons;
(e) generating a recombinant TCR library by introducing the TCR amplicons into an expression vector or a population of cells.

### Claim 2 (depends on 1)

The method of claim 1, further comprising screening the library for specific binding to a target cell, optionally wherein
the target cell is a cancer cell or a cell infected with a virus, or
the cell was isolated from a subject.

### Claim 3 (depends on 1)

The method of claim 1, further comprising screening the library for specific binding to an antigen: MHC complex, optionally wherein
the antigen of the antigen: MHC complex is a viral antigen derived from a virus selected from the group consisting of adenovirus, CMV, coronavirus, coxsackievirus, Dengue virus, Epstein-Barr virus (EBV), enterovirus 71 (EV71), Ebola virus, hepatitis A (HAV), hepatitis B (HBV), cytomegalovirus (CMV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human T-lymphotropic virus (HTLV), influenza A virus, influenza B virus, Japanese encephalitis, leukemia virus, measles virus, molluscum contagiosum, orf virus, parvovirus, rabies virus, respiratory syncytial virus, rift valley fever virus, rubella virus, rotavirus, tick-borne encephalitis (TBEV), simian immunodeficiency virus, tobacco etch virus (TEV), varicella zoster virus, variola, West Nile virus, Zika virus, and Chikungunya virus, or
the antigen of the antigen: MHC complex is a tumor antigen selected from the group consisting of CD45, glypican-3, IGF2B3, Kallikrein 4, KIF20A, Lengsin, Meloe, mucin 5AC (MUC5AC), survivin, cyclin-A1, MAGE-A1, MAGE-C1, MAGE-C2, SSX2, XAGE1b/GAGED2A, CD19, CD20, CD22, CD52, EGFR, HER2, TRAILR1, RANKL, IGF1R, EpCAM, and CEA.

### Claim 4 (depends on 1)

The method of claim 1, further comprising
screening the library for T cell phenotypic markers; or
screening the library for activity in a co-culture system, wherein the co-culture system comprises at least one of the following:
(a) a cancer cell line;
(b) a plurality of cells infected with a known virus;
(c) a plurality of tumor cells isolated from a cancer patient;
(d) an immortalized cell line; or
(e) a plurality of cells derived from a patient tissue biopsy; or
performing in vitro activation of the transformed population of cells, optionally wherein in vitro activation is performed using one or more of the following stimulants: anti-CD3 antibody, anti-CD8 antibody, anti-CD27 antibody, IL-2, IL-4, IL-21, anti-PD1 antibody, anti-CTLA4 antibody, tumor cell lysate, cellular co-culture with virus-infected cells, and tumor cell lines; or
transforming the population of cells with a polynucleotide encoding a transcription factor, optionally wherein the transcription factor is selected from the group consisting of FOXP3, BLIMP-1, Ikaros, Helios and TGF-beta; or
selecting individual vectors for inclusion in the recombinant TCR library on the basis of one or more of the following characteristics: TCR clonal prevalence, TCR enrichment characteristics from in vitro assays, TCR binding specificity, TCR V segment sequence, TCR D segment sequence, TCR J segment sequence, TCR gene motifs, and/or CDR3 gene motifs, optionally wherein selection comprises mixing individual vectors at a defined ratio to generate a synthetically-derived TCR library.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first polynucleotide and the second polynucleotide have been altered to incorporate at least one target restriction endonuclease site selected from the group consisting of: Age I, SpeI, XhoI, NheI, BsiWI, BstZI7I, BstBI, and SphI or wherein the target restriction endonuclease site comprises a silent mutation; or
wherein step (e) is performed by cleaving at a target restriction endonuclease site, optionally wherein the target restriction endonuclease site is natively found in TCR genes at low frequency; or
wherein the population of cells are hematopoietic stem cells, hematopoietic progenitor cells, T cells, or NK cells.