# US 12,007,398 B2 · Cell-based assay for detecting anti-CD3 homodimers

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- Number: US 12,007,398 B2
- Document ID: US12007398B2
- Status: Granted
- Kind: B2
- Application number: 16/874,550
- Filed: 2020-05-14
- Published: 2020-12-03
- Granted: 2024-06-11
- Earliest claimed: 2015-05-28
- Assignees: Genentech, Inc.
- Applicants: Genentech, Inc.
- Inventors: Kendall Carey
- Web page: https://patentgrep.com/patent/US12007398B2

## CPC

- G01N 33/6854: {Immunoglobulins}
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/2887: {against CD20}
- C12N 5/0636: {T lymphocytes}
- G01N 33/505: {involving T-cells}
- C07K 2317/31: multispecific
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C12N 2510/00: Genetically modified cells
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- 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)
- 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

The present invention provides a cell-based assay for identifying and/or quantifying anti-CD3 homodimers in a composition comprising a T cell-dependent Bispecific antibody (TDB). In some aspects, the invention T cells comprising a T cell activation responsive reporter are contacted with the TDB to detect the presence of anti-CD3 homodimers. Compositions of reporter T cells and kits are also contemplated.

## Claims

### Claim 1

A method for quantitating an amount of anti-CD3 homodimer antibody in a composition comprising a T cell dependent bispecific antibody (TDB), the method comprising:
(a) contacting a population of T cells with the composition at one or more concentrations of the TDB, wherein:
(i) the population of T cells comprise a nucleic acid encoding a reporter operably linked to a response element that is responsive to T cell activation, and wherein expression of the reporter indicates the presence of anti-CD3 homodimer antibodies,
(ii) the TDB comprises a target antigen binding fragment and a CD3 binding fragment, and
(iii) the population of T cells does not comprise the target antigen; and
(b) quantitating the amount of anti-CD3 homodimer antibody in the composition by correlating the expression of the reporter as a function of anti-CD3 homodimer antibody concentration in the composition using a standard curve, wherein the standard curve is generated by contacting T cells comprising the nucleic acid with different concentrations of a purified anti-CD3 homodimer antibody.

### Claim 2 (depends on 1)

The method of claim 1, wherein the reporter is a luciferase, a fluorescent protein, an alkaline phosphatase, beta lactamase, or a beta galactosidase.

### Claim 3 (depends on 2)

The method of claim 2, wherein the luciferase is a firefly luciferase, a Renilla luciferase, or a nanoluciferase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the response element that is responsive to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter.

### Claim 5 (depends on 4)

The method of claim 4, wherein the response element that is responsive to T cell activation comprises T cell activation responsive elements from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF.

### Claim 6 (depends on 1)

The method of claim 1, wherein the population of T cells is a population of CD4+ T cells or CD8+ T cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the population of T cells is a population of Jurkat T cells or CTLL-2 T cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein the one or more concentrations of the TDB range from about 0.01 ng/mL to about 50 ng/mL and the purified anti-CD3 homodimer antibody concentrations range from about 0.01 ng/mL to about 50 ng/mL.

### Claim 9 (depends on 1)

The method of claim 1, wherein the purified anti-CD3 homodimer antibody concentrations range from about 0.01 ng/mL to about 50 ng/mL.

### Claim 10 (depends on 1)

The method of claim 1, wherein the reporter is detected after any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20 or 24 hours after contacting the population of T cells with the composition.