# US 2023/0055005 A1 · New binding agent and assay for pivka

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- Number: US 2023/0055005 A1
- Document ID: US20230055005A1
- Status: Published application
- Kind: A1
- Application number: 17/821,937
- Filed: 2022-08-24
- Published: 2023-02-23
- Earliest claimed: 2017-07-13
- Assignees: Roche Diagnostics Operations, Inc.
- Applicants: Roche Diagnostics Operations, Inc.
- Inventors: Bernhard Eckert; Michael Gerg; Johann Karl; Martin Kaufmann; Julia Riedlinger; Magdelena Swiatek-de-Lange; Lars Hillringhaus; Klaus Hirzel; Marcus-Rene Lisy
- Web page: https://patentgrep.com/patent/US20230055005A1

## CPC

- C07K 16/36: against blood coagulation factors
- C07K 16/3076: {against structure-related tumour-associated moieties}
- C07K 16/40: against enzymes
- G01N 33/57525: of the liver or pancreas
- G01N 33/86: involving blood coagulating time {or factors, or their receptors}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- G01N 2333/745: Assays involving non-enzymic blood coagulation factors
- 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)
- 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)

## Abstract

The present disclosure relates to specific binding agents binding to different PIVKA-II forms as compared to antibodies known so far in the art. The present disclosure also relates to methods of using the specific binding agents to detect the presence of PIVKA-II.

## Claims

### Claim 1

A method for the production of an antibody that specifically binds to PIVKA-II characterized in that it binds to a synthetic peptide of SEQ ID NO:1, has an at least 10-fold binding preference for the peptide of SEQ ID NO:1 as compared to a synthetic peptide of SEQ ID NO:2, and binds to a synthetic peptide of SEQ ID NO:3 at least as well as compared to the peptide of SEQ ID NO:1, the method comprising culturing a host cell comprising one or more vectors, the one or more vectors comprising one or more nucleic acid molecules encoding the antibody, and isolating the antibody produced.

### Claim 2 (depends on 1)

The method of claim 1, wherein the antibody contains CDRs that comprise the following amino acid sequences or variants thereof, wherein the variants differ in at most one amino acid substitution per CDR:
(i) a CDR1 comprising the amino acid sequence of SEQ ID NO:10, a CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 comprising the amino acid sequence of SEQ ID NO:12 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:13, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain;
(ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR3 comprising the amino acid sequence of SEQ ID NO:20 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:21, a CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain;
(iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 comprising the amino acid sequence of SEQ ID NO:20 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:21, a CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain; or
(iv) a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 comprising the amino acid sequence of SEQ ID NO:28 in the light chain variable domain, and a CDR1 comprising the amino acid sequence of SEQ ID NO:29, a CDR2 comprising the amino acid sequence of SEQ ID NO:25, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15 in the heavy chain variable domain.

### Claim 3 (depends on 2)

The method of claim 2, wherein the amino acid substitutions are conservative amino acid substitutions.

### Claim 4 (depends on 1)

The method of claim 1, wherein the antibody comprises as light chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:16 and as heavy chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 17.

### Claim 5 (depends on 1)

The method of claim 1, wherein the antibody comprises as light chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:23 and as heavy chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:24.

### Claim 6 (depends on 1)

The method of claim 1, wherein the antibody comprises as light chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:26 and as heavy chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:27.

### Claim 7 (depends on 1)

The method of claim 1, wherein the antibody comprises as light chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:30 and as heavy chain variable domain an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:31.

### Claim 8 (depends on 1)

The method of claim 1, wherein the one or more vectors independently comprise an expression vector.

### Claim 9 (depends on 1)

The method of claim 1, wherein the host cell is selected from the group consisting of a HeLa, HEK293, H9, Per.C6, Jurkat cells, mouse NIH3T3, NS/0, SP2/0, C127, COS 1, COS 7, CV1, quail QC1-3, mouse L, mouse sarcoma, Bowes melanoma, and Chinese hamster ovary (CHO) cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the host cell is a bacterium selected from the group consisting of E. coli, S. typhimurium, Serratia marcescens, Corynebacterium (glutamicum), Pseudomonas (fluorescens), Lactobacillus, Streptomyces, Salmonella and Bacillus subtilis.

### Claim 11 (depends on 1)

The method of claim 1, wherein the isolated antibody is further purified to
(i) greater than 95% by weight;
(ii) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or
(iii) to homogeneity by SDS-PAGE under reducing or nonreducing conditions.

### Claim 12 (depends on 1)

The method of claim 1, wherein the method further comprises an initial step of selecting the antibody to be produced, wherein the antibody is selected by:
(i) incubating a candidate antibody with a synthetic peptide of SEQ ID NO:1 and measuring their reactivity;
(ii) incubating the candidate antibody with a synthetic peptide of SEQ ID NO:2 and measuring their reactivity;
(iii) incubating the candidate antibody with a synthetic peptide of SEQ ID NO:3 and measuring their reactivity;
(iv) comparing the reactivity of the candidate antibody with the synthetic peptide of SEQ ID NO:1 and the reactivity of the candidate antibody with the synthetic peptide of SEQ ID NO:2;
(iv) comparing the reactivity of the candidate antibody with the synthetic peptide of SEQ ID NO:1 and the reactivity of the candidate antibody with the synthetic peptide of SEQ ID NO:3; and
(iv) selecting the candidate antibody as the antibody of the method if the candidate antibody binds to a synthetic peptide of SEQ ID NO:1, has at least 10-fold binding preference for the peptide of SEQ ID NO:1 as compared to a synthetic peptide of SEQ ID NO:2, and binds to a synthetic peptide of SEQ ID NO:3 at least as well as compared to the peptide of SEQ ID NO:1.

### Claim 13 (depends on 12)

An antibody prepared according to the method of claim 12.

### Claim 14 (depends on 13)

A method of detecting PIVKA-II in a sample comprising the steps of:
a) contacting the sample with the antibody of claim 13 for a time and under conditions sufficient for the formation of an antibody-PIVKA-II complex; and
b) detecting the presence of the antibody-PIVKA-II complex formed in (a), wherein the presence of the antibody-PIVKA-II complex indicates the presence of PIVKA-II in the sample.

### Claim 15 (depends on 14)

The method of claim 14 wherein
step a) further comprises contacting the sample with a second specific binding agent to PIVKA-II, wherein the second specific binding agent is detectably labeled, for a time and under conditions sufficient to form an antibody-PIVKA-II-second specific binding agent complex, wherein
step b) comprises measuring the complex formed in a), thereby detecting PIVKA-II in the sample.

### Claim 16 (depends on 15)

The method according to claim 15, wherein the second specific binding agent is a polyclonal or a monoclonal antibody.

### Claim 17 (depends on 15)

The method according to claim 15, wherein the epitope on PIVKA-II bound by the second specific binding agent is comprised within F1/F2.

### Claim 18 (depends on 15)

The method according to claim 15, wherein the epitope on PIVKA-II bound by the second specific binding agent is comprised within F1.

### Claim 19 (depends on 15)

The method according to claim 15, wherein the epitope on PIVKA-II bound by the second specific binding agent is comprised within the Gla-domain of PIVKA-II.

### Claim 20 (depends on 13)

A method of diagnosing hepatocellular carcinoma (HCC) in a patient suspected of having HCC, comprising the steps of:
a) obtaining a sample from the patient;
b) contacting the sample with the antibody of claim 13 and a second specific binding agent to PIVKA-II, wherein the second specific binding agent is detectably labeled, for a time and under conditions sufficient to form an antibody-PIVKA-II-second specific binding agent complex; and
c) measuring the complex formed in (b), thereby measuring the amount of PIVKA-II present in the sample, wherein an amount of PIVKA-II greater than a reference level is indicative of the presence of HCC in the patient.