# US 2022/0033484 A1 · Antibody against human cardiac troponin I and use thereof

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- Number: US 2022/0033484 A1
- Document ID: US20220033484A1
- Status: Published application
- Kind: A1
- Application number: 17/278,345
- Filed: 2019-09-27
- Published: 2022-02-03
- Earliest claimed: 2018-10-10
- Assignees: Guagndong Feipeng Biological Co., Ltd.
- Applicants: Guagndong Feipeng Biological Co., Ltd.
- Inventors: Peng CUI; Zhiqiang HE; Yuan MENG; Dongmei ZHONG
- Web page: https://patentgrep.com/patent/US20220033484A1

## CPC

- C07K 16/18: against material from animals or humans
- G01N 33/577: involving monoclonal antibodies {binding reaction mechanisms characterised by the use of monoclonal antibodies (G01N33/5302 - G01N33/576 take precedence)}
- G01N 33/6887: {from muscle, cartilage or connective tissue}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- G01N 2333/4712: Muscle proteins, e.g. myosin, actin, protein
- G01N 2470/04: Sandwich assay format
- G01N 2800/12: Pulmonary diseases
- G01N 2800/324: Coronary artery diseases, e.g. angina pectoris, myocardial infarction
- 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

Disclosed is an isolated binding protein including a cardiac troponin I (cTnI) antigen binding domain, and preparation and a use and the like of the binding protein. The antigen binding domain includes at least one complementarity determining region selected from an amino acid sequence defined in this article, or has at least 80% sequence identity with the complementarity determining region of the amino acid sequence and has KD≤9.96×10−8 mon affinity with cTnI. The binding protein may be used in the detection field of the cTnI protein.

## Claims

### Claim 1

An isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequences, or has at least 80% sequence identity with the complementarity determining region of the following amino acid sequences and has KD≤9.96×10−8 mol/L affinity with a cTnI;
a complementarity determining region CDR-VH1 is G-Y-X1-F-T-X2-Y-V-X3-H, wherein,
the X1 is S or T, X2 is I or L, and the X3 is V, L or I;
a complementarity determining region CDR-VH2 is Y-I-X1-P-Y-X2-D-G-T-X3-Y-N-E-K, wherein,
the X1 is Q, N or Y, the X2 is I or L, and the X3 is R or K;
a complementarity determining region CDR-VH3 is R-X1-G-Y-G-X2-Y-G-L-A, wherein
the X1 is S or T, and the X2 is Q, N or G;
a complementarity determining region CDR-VL1 is S-X1-G-A-X2-T-T-S-X3-Y-A-N, wherein,
the X1 is S or T, the X2 is A or V, and the X3 is Q or N;
a complementarity determining region CDR-VL2 is G-S-X1-N-R-X2-P, wherein,
the X1 is N or Q, and the X2 is A or V;
a complementarity determining region CDR-VL3 is A-X1-V-Y-S-N-X2-W, wherein,
the X1 is I or L, and the X2 is Q, H or N.

### Claim 2 (depends on 1)

The binding protein as claimed in claim 1, wherein in the complementarity determining region CDR-VH1, the X1 is T;
in the complementarity determining region CDR-VH2, the X3 is K;
in the complementarity determining region CDR-VH3, the X1 is S;
in the complementarity determining region CDR-VL1, the X1 is T;
in the complementarity determining region CDR-VL2, the X2 is A; and
in the complementarity determining region CDR-VL3, the X1 is L.
further, in the complementarity determining region CDR-VH1 the X2 is I, and the X3 is V;
further, in the complementarity determining region CDR-VH1, the X2 is I, and the X3 is L;
further, in the complementarity determining region CDR-VH1, the X2 is I, and the X3 is I;
further, in the complementarity determining region CDR-VH1, the X2 is L, and the X3 is V;
further, in the complementarity determining region CDR-VH1, the X2 is L, and the X3 is L;
further, in the complementarity determining region CDR-VH1, the X2 is L, and the X3 is I;
further, in the complementarity determining region CDR-VH2, the X1 is Q, and the X2 is I;
further, in the complementarity determining region CDR-VH2, the X1 is Q, and the X2 is L;
further, in the complementarity determining region CDR-VH2, the X1 is N, and the X2 is I;
further, in the complementarity determining region CDR-VH2, the X1 is N, and the X2 is L;
further, in the complementarity determining region CDR-VH2, the X1 is Y, and the X2 is I;
further, in the complementarity determining region CDR-VH2, the X1 is Y, and the X2 is L;
further, in the complementarity determining region CDR-VH3, the X2 is Q;
further, in the complementarity determining region CDR-VH3, the X2 is N;
further, in the complementarity determining region CDR-VH3, the X2 is G;
further, in the complementarity determining region CDR-VL1, the X2 is A, and the X3 is Q;
further, in the complementarity determining region CDR-VL1, the X2 is A, and the X3 is N;
further, in the complementarity determining region CDR-VL1, the X2 is V, and the X3 is Q;
further, in the complementarity determining region CDR-VL1, the X2 is V, and the X3 is N;
further, in the complementarity determining region CDR-VL2, the X1 is N;
further, in the complementarity determining region CDR-VL2, the X1 is Q;
further, in the complementarity determining region CDR-VL3, the X2 is Q;
further, in the complementarity determining region CDR-VL3, the X2 is H;
further, in the complementarity determining region CDR-VL3, the X2 is N.

### Claim 3 (depends on 1)

The binding protein as claimed in claim 1, wherein the binding protein comprises at least 3 CDRs; or the binding protein comprises at least 6 CDRs.

### Claim 4 (depends on 1)

The binding protein as claimed in claim 1, wherein the binding protein further comprises an antibody constant region sequence;
further, the constant region is derived from the mouse;
a light-chain constant region sequence is as shown in SEQ ID NO: 9; and
a heavy-chain constant region sequence is as shown in SEQ ID NO: 10.

### Claim 5 (depends on 1)

An isolated nucleic acid, wherein the binding protein as claimed in claim 1 is encoded by the nucleic acid.

### Claim 6 (depends on 5)

A vector, wherein the vector comprises the nucleic acid as claimed in claim 5.

### Claim 7 (depends on 5)

A host cell, wherein the host cell comprises the nucleic acid as claimed in claim 5.

### Claim 8 (depends on 1)

A method for producing the binding protein as claimed in any one of claim 1, wherein the method comprises the following steps:
culturing a host cells comprising a nucleic acid encoding the binding protein as claimed in claim 1 in a culture medium, and recovering the generated binding protein from the culture medium or from the cultured host cells.

### Claim 9

(canceled)

### Claim 10 (depends on 1)

A kit, wherein the kit comprises one or more of the binding protein as claimed in claim 1;
preferably, the kit further comprises a label for labeling the binding protein.

### Claim 11 (depends on 1)

A method for detecting a troponin I antigen in a test sample, comprising:
a) in a condition sufficient for an antibody/antigen binding reaction, enabling the troponin I antigen in the test sample to contact with the binding protein as claimed in claim 1 to form an immune complex; and
b) detecting the presence of the immune complex, the presence of the immune complex indicates the presence of the troponin I antigen in the test sample;
preferably, the immune complex further comprises a second antibody, and the second antibody is bound with the binding protein;
preferably, in the step a), the immune complex further comprises a second antibody, and the second antibody is bound with the troponin I antigen; and
preferably, the troponin I antigen is a cardiac troponin I antigen.

### Claim 12

(canceled)

### Claim 13

(canceled)

### Claim 14 (depends on 11)

The method as claimed in claim 11, wherein the method is based on a fluorescence immunoassay, a chemiluminescence technology, a colloidal gold immunotechnology, a radioimmunoassay and/or an enzyme-linked immunoassay.

### Claim 15 (depends on 11)

The method as claimed in claim 11, wherein the test sample is selected from at least one of whole blood, peripheral blood, serum, plasma or a myocardial tissue.

### Claim 16 (depends on 11)

The method as claimed in claim 11, wherein the subject which the test sample is derived from is a mammal, preferably a primate, and more preferably a human.

### Claim 17

(canceled)

### Claim 18

(canceled)

### Claim 19 (depends on 1)

The binding protein as claimed in claim 1, wherein preferably, a mutation site of each complementarity determining region is selected from any one of the following mutation combinations:
CDR-
CDR-
CDR-
CDR-
CDR-
CDR-
VH1
VH2
VH3
VL1
VL2
VL3
Site
X2/X3
X1/X2
X2
X2/X3
X1
X2
Mutation
L/V
Q/L
G
A/Q
N
H
combination 1
Mutation
I/L
Q/I
N
A/N
Q
Q
combination 2
Mutation
I/I
Q/I
G
V/Q
N
N
combination 3
Mutation
L/V
Q/I
Q
V/N
Q
Q
combination 4
Mutation
L/L
Q/I
N
A/Q
N
N
combination 5
Mutation
L/I
Q/I
G
A/N
Q
H
combination 6
Mutation
I/V
Q/L
Q
V/Q
N
N
combination 7
Mutation
I/L
Q/L
N
V/N
Q
H
combination 8
Mutation
I/I
Q/L
G
A/Q
N
Q
combination 9
Mutation
L/V
Q/L
Q
A/N
Q
H
combination 10
Mutation
L/L
Q/L
N
V/Q
N
Q
combination 11
Mutation
L/I
N/I
G
V/N
Q
N
combination 12
Mutation
I/V
N/I
Q
A/Q
N
Q
combination 13
Mutation
I/L
N/I
N
A/N
Q
N
combination 14
Mutation
I/I
N/I
G
V/Q
N
H
combination 15
Mutation
L/V
N/I
Q
V/N
Q
N
combination 16
Mutation
L/L
N/L
N
A/Q
N
H
combination 17
Mutation
L/I
N/L
G
A/N
Q
Q
combination 18
Mutation
I/V
N/L
Q
V/Q
N
H
combination 19
Mutation
IL
N/L
N
V/N
Q
Q
combination 20
Mutation
I/I
Y/I
G
A/Q
N
N
combination 21
Mutation
L/V
Y/I
Q
A/N
Q
Q
combination 22
Mutation
L/L
Y/I
N
V/Q
N
N
combination 23
Mutation
L/I
Y/I
G
V/N
Q
H
combination 24
Mutation
I/V
Y/L
Q
A/Q
N
N
combination 25
Mutation
I/L
Y/L
N
A/N
Q
H
combination 26
Mutation
I/I
Y/L
G
V/Q
N
Q
combination 27
Mutation
L/V
Y/L
Q
V/N
Q
H
combination 28
Mutation
L/L
Y/L
N
A/Q
N
Q
combination 29
Mutation
L/I
Q/I
G
A/Q
Q
N
combination 30
Mutation
I/V
Q/L
Q
A/Q
N
Q
combination 31
Mutation
I/L
N/I
N
A/Q
Q
N
combination 32
Mutation
I/I
N/L
G
A/N
N
H
combination 33
Mutation
L/V
Y/I
G
A/Q
Q
N
combination 34
Mutation
L/L
Q/I
N
A/N
N
H
combination 35
Mutation
L/I
Q/L
G
A/N
Q
Q
combination 36
Mutation
I/V
N/I
Q
V/Q
N
H
combination 37
Mutation
I/L
N/L
N
V/Q
Q
Q
combination 38
Mutation
I/I
Y/I
G
V/Q
N
N
combination 39
Mutation
L/V
Q/L
Q
V/N
Q
Q
combination 40
Mutation
I/L
Q/L
N
V/N
N
N
combination 41
Mutation
L/I
N/I
N
V/N
Q
Q
combination 42
Mutation
L/V
N/L
Q
V/N
N
H
combination 43
Mutation
I/L
Y/I
N
A/Q
Q
Q
combination 44
Mutation
I/I
Q/I
G
A/N
N
N
combination 45
Mutation
L/V
Q/L
Q
V/Q
Q
Q
combination 46
Mutation
I/L
N/I
N
V/N
N
N
combination 47
Mutation
L/I
N/L
G
A/Q
Q
H
combination 48
Mutation
I/V
Y/I
Q
A/N
N
Q
combination 49
Mutation
L/L
N/L
N
V/Q
Q
N
combination 50
Mutation
I/I
Y/I
G
V/N
Q
H
combination 51
Mutation
I/V
Q/I
Q
A/Q
N
H
combination 52
Mutation
I/V
N/L
N
A/Q
N
N
combination 53
Mutation
I/V
Y/I
G
V/N
Q
Q
combination 54
Mutation
I/V
N/L
Q
A/N
N
Q
combination 55
Mutation
I/V
Y/I
G
V/Q
Q
N
combination 56

### Claim 20 (depends on 1)

The binding protein as claimed in claim 1, wherein the binding protein is one of a nano-antibody, F(ab′)2, Fab′, Fab, Fv, scFv, a bispecific antibody and an antibody minimum recognition unit.

### Claim 21 (depends on 1)

The binding protein as claimed in claim 1, wherein the binding protein comprises light-chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 of which sequences are successively shown in SEQ ID NO: 1-4, and/or heavy-chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 of which sequences are successively shown in SEQ ID NO: 5-8.

### Claim 22 (depends on 1)

The binding protein as claimed in claim 1 wherein the constant region sequence is selected from a sequence of any one constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

### Claim 23 (depends on 1)

The binding protein as claimed in claim 1, wherein a species source of the constant region is cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, gamecock or human.

### Claim 24 (depends on 1)

The binding protein as claimed in claim 1, wherein the binding protein is labeled by an indicator which shows signal intensity.

### Claim 25 (depends on 11)

The method as claimed in claim 11, wherein the presence of the immune complex indicates the presence of the disease related to the cardiac troponin I.