# US 2024/0201171 A1 · Assay to measure the potency of receptor-ligand interactions in nanomedicines

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- Number: US 2024/0201171 A1
- Document ID: US20240201171A1
- Status: Published application
- Kind: A1
- Application number: 18/459,960
- Filed: 2023-09-01
- Published: 2024-06-20
- Earliest claimed: 2017-04-07
- Applicants: UTI Limited Partnership
- Inventors: Pedro SANTAMARIA
- Web page: https://patentgrep.com/patent/US20240201171A1

## CPC

- G01N 33/505: {involving T-cells}
- A61K 39/0008: {Antigens related to auto-immune diseases; Preparations to induce self-tolerance}
- A61K 47/6929: {the form being a nanoparticle, e.g. an immuno-nanoparticle}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C12N 5/0636: {T lymphocytes}
- G01N 33/502: {for testing non-proliferative effects}
- G01N 33/56977: {HLA or MHC typing}
- A61K 2039/55516: {Proteins; Peptides}
- A61K 2039/605: {MHC molecules or ligands thereof}
- G01N 2333/90241: acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Described herein, is an isolated cell comprising a recombinant T cell receptor (TCR) and a TCR-pathway-dependent reporter, wherein the recombinant T cell receptor is specific for a disease-relevant antigen bound to an MHC molecule. Also described are methods of use for the isolated cell as an assay to determine the function or potency of a peptide-major histocompatibility complex (pMHC) coupled to a nanoparticle (pMHC-NP) that can be used as a medicine for treating an autoimmune disease or cancer.

## Claims

### Claim at position 1

1-48. (canceled)

### Claim 49

An in vitro method for assaying agonistic activity of a nanomedicine, wherein the nanomedicine comprises a nanoparticle coupled to a construct comprising a disease-relevant antigen bound to a major histocompatibility (MHC) molecule, the method comprising:
a) contacting the nanomedicine with a cell comprising:
a recombinant T cell receptor (TCR) comprising a TCR alpha chain and a TCR beta chain; and
a T cell receptor-pathway-dependent reporter, wherein the recombinant T cell receptor is specific for the disease-relevant antigen bound to the MHC molecule coupled to the nanoparticle; and
b) detecting a signal produced by the T cell receptor-pathway-dependent reporter.

### Claim 50 (depends on 49)

The method of claim 49, wherein the nanomedicine comprises a plurality of nanoparticles coupled to a construct comprising a disease-relevant antigen bound to the MHC molecules.

### Claim 51 (depends on 50)

The method of claim 50, wherein each nanoparticle of the plurality of nanoparticles comprises a plurality of disease-relevant antigens bound to the MHC molecule coupled to the nanoparticle.

### Claim 52 (depends on 51)

The method of claim 51, wherein the disease-relevant antigen is an autoimmune or inflammatory disease-relevant antigen.

### Claim 53 (depends on 52)

The method of claim 52, wherein the autoimmune or inflammatory disease-relevant antigen is chosen from a diabetes mellitus Type I antigen, an asthma or allergic asthma antigen, a multiple sclerosis antigen, a peripheral neuropathy antigen, a primary biliary cirrhosis antigen, a neuromyelitis optica spectrum disorder antigen, a stiff-person syndrome antigen, an autoimmune encephalitis antigen, a pemphigus vulgaris antigen, a pemphigus foliaceus antigen, a psoriasis antigen, a Sjogren's disease/syndrome antigen, an inflammatory bowel disease antigen, an arthritis or rheumatoid arthritis antigen, a systemic lupus erythematosus antigen, a scleroderma antigen, an ANCA-associated vasculitis antigen, a Goodpasture syndrome antigen, a Kawasaki's disease antigen, a celiac disease, an autoimmune cardiomyopathy antigen, a myasthenia gravis antigen, an autoimmune uveitis antigen, a Grave's disease antigen, an anti-phospholipid syndrome antigen, an autoimmune hepatitis antigen, a sclerosing cholangitis antigen, a primary sclerosing cholangitis antigen, chronic obstructive pulmonary disease antigen, or a uveitis relevant antigen, and combinations thereof.

### Claim 54 (depends on 50)

The method of claim 50, wherein each nanoparticle of the plurality of nanoparticles comprises a diameter of from 1 nanometer to about 100 nanometers.

### Claim 55 (depends on 49)

The method of claim 49, further comprising quantifying the T cell receptor-pathway-dependent reporter signal.

### Claim 56 (depends on 55)

The method of claim 55, wherein the quantifying step comprises determining a concentration of the nanomedicine that initiates a response that is about 50% of a maximal response, wherein the maximal response is the response initiated at the highest concentration of nanomedicine contacted with the cell or a population of cells when a plurality of concentrations of the nanomedicine are contacted with the cell or population of cells.

### Claim 57 (depends on 56)

The method of claim 56, wherein the plurality of the concentrations of the nanomedicine are contacted with the cell or the population of cells in the same assay.

### Claim 58 (depends on 55)

The method of claim 55, wherein the quantifying step comprises determining a concentration of the nanomedicine that initiates a response that is at least about 200%, of a negative control, wherein the negative control comprises a nanomedicine that does not specifically interact with the recombinant T cell receptor (TCR) of the cell or a population of cells.

### Claim 59 (depends on 49)

The method of claim 49, wherein the signal is produced by an enzyme.

### Claim 60 (depends on 59)

The method of claim 59, wherein the enzyme is luciferase or peroxidase.

### Claim 61 (depends on 49)

The method of claim 49, wherein the signal is a fluorescent signal.

### Claim 62

(canceled)

### Claim 63 (depends on 49)

The method of claim 49, used as a quality control step in a manufacturing process.

### Claim 64 (depends on 49)

The method of claim 49, wherein the T cell receptor-pathway-dependent reporter is actively transcribed.

### Claim 65 (depends on 49)

The method of claim 49, wherein the T cell receptor-pathway-dependent reporter activates transcription of a gene chosen from a luciferase gene, a beta lactamase gene, a chloramphenicol acetyltransferase (CAT) gene, a secreted embryonic alkaline phosphatase (SEAP) gene, a fluorescent protein gene, and combinations thereof.

### Claim 66 (depends on 49)

The method of claim 49, wherein the T cell receptor-pathway-dependent reporter comprises a polynucleotide sequence chosen from a nuclear factor of activated T cells (NFAT) transcription factor-binding DNA sequence or promoter, an NF-κB transcription factor-binding DNA sequence or promoter, an AP1 transcription factor-binding DNA sequence or promoter, an IL-2 transcription factor-binding DNA sequence or promoter, and combinations thereof.

### Claim 67 (depends on 49)

The method of claim 49, wherein the cell is selected from JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4.

### Claim 68 (depends on 49)

The method of claim 49, wherein the disease-relevant antigen is a polypeptide consisting of any one of SEQ ID NOs: 1 to 352 and combinations thereof.

### Claim 69 (depends on 49)

The method of claim 49, wherein the disease-relevant antigen is a polypeptide consisting of any one of SEQ ID NOs: 353 to 455 and combinations thereof.

### Claim 70 (depends on 49)

The method of claim 49, wherein the TCR alpha chain and TCR beta chain are translated as a single polypeptide.

### Claim 71 (depends on 70)

The method of claim 70, wherein the TCR alpha chain and TCR beta chain of the single polypeptide are separated by a ribosome skipping sequence.

### Claim 72 (depends on 71)

The method of claim 71, wherein the ribosome skipping sequence is set forth in any one of SEQ ID NOs: 456 to 523.

### Claim 73 (depends on 70)

The method of claim 70, wherein the single polypeptide comprises an amino acid sequence at least 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 527, 533, or 538.

### Claim 74 (depends on 49)

The method of claim 49, wherein the TCR alpha chain and TCR beta chain are translated as separate polypeptides.

### Claim 75 (depends on 49)

The method of claim 49, wherein the TCR alpha chain comprises an amino acid sequence at least 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 528, 530, 534, 536 539, 541, and the TCR beta chain comprises an amino acid sequence at least 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOs: 529, 531, 535, 537, 540, or 542.

### Claim 76 (depends on 49)

The method of claim 49, wherein the TCR alpha chain and TCR beta chain are expressed at the surface of the cell.

### Claim 77 (depends on 49)

The method of claim 49, wherein the cell comprises at least one exogenous polynucleotide encoding the TCR alpha chain and the TCR beta chain.

### Claim 78 (depends on 77)

The method of claim 77, wherein the at least one exogenous polynucleotide comprises an internal ribosome entry site (IRES) nucleic acid sequence.

### Claim 79 (depends on 78)

The method of claim 78, wherein the IRES nucleic acid sequence is set forth in any one of SEQ ID NOs: 524 to 526.

### Claim 80 (depends on 79)

The method of claim 79, wherein the at least one exogenous polynucleotide comprises a nucleic acid sequence at least 80%, 90%, 95%, or 100% homologous to that set forth in any one of SEQ ID NOs: 532 or 557.

### Claim 81 (depends on 49)

The method of claim 49, wherein the nanomedicine is for use in a human individual.