# US 12,180,301 B2 · Serine protease inhibitor kazal (SPIK) compositions and methods

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- Number: US 12,180,301 B2
- Document ID: US12180301B2
- Status: Granted
- Kind: B2
- Application number: 16/978,634
- Filed: 2019-03-06
- Published: 2021-08-19
- Granted: 2024-12-31
- Earliest claimed: 2018-03-06
- Assignees: ImCare Biotech, LLC
- Applicants: Imcare Biotech, LLC
- Inventors: Xuanyong Lu; Felix Lu
- Web page: https://patentgrep.com/patent/US12180301B2

## CPC

- C07K 16/30: from tumour cells
- C07K 16/38: against protease inhibitors of peptide structure
- A61P 35/00: Antineoplastic agents
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/57525: of the liver or pancreas
- G01N 33/6893: {related to diseases not provided for elsewhere}
- C07K 2317/31: multispecific
- C07K 2317/32: specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- 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 2317/567: Framework region [FR]
- C07K 2317/90: characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- G01N 2800/085: Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
- G01N 2800/50: Determining the risk of developing a disease
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

Anti-AS-SPIK antibodies are disclosed, along with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to diagnose disorders characterized by the expression of AS-SPIK (e.g., liver cancer). Diagnostic methods and kits comprising the anti-AS-SPIK antibodies are also disclosed.

## Claims

### Claim 1

An isolated anti-Abnormal Secreted Serine Protease Inhibitor Kazal (anti-AS-SPIK) antibody, or an antigen-binding fragment thereof, that specifically binds to Abnormal Secreted Serine Protease Inhibitor Kazal (AS-SPIK), and does not bind to Normal Secreted Serine Protease Inhibitor Kazal (NS-SPIK), comprising:
(a) a CDRH1 sequence comprising SEQ ID NO: 15, a CDRH2 sequence comprising SEQ ID NO: 19, a CDRH3 sequence comprising SEQ ID NO: 23, a CDRL1 sequence comprising SEQ ID NO: 27, a CDRL2 sequence comprising SEQ ID NO: 31, and a CDRL3 sequence comprising SEQ ID NO: 35; or
(b) a CDRH1 sequence comprising SEQ ID NO: 16, a CDRH2 sequence comprising SEQ ID NO: 20, a CDRH3 sequence comprising SEQ ID NO: 24, a CDRL1 sequence comprising SEQ ID NO: 28, a CDRL2 sequence comprising SEQ ID NO: 32, and a CDRL3 sequence comprising SEQ ID NO: 36; or
(c) a CDRH1 sequence comprising SEQ ID NO: 17, a CDRH2 sequence comprising SEQ ID NO: 21, a CDRH3 sequence comprising SEQ ID NO: 25, a CDRL1 sequence comprising SEQ ID NO: 29, a CDRL2 sequence comprising SEQ ID NO: 33, and a CDRL3 sequence comprising SEQ ID NO: 37; or
(d) a CDRH1 sequence comprising SEQ ID NO: 18, a CDRH2 sequence comprising SEQ ID NO: 22, a CDRH3 sequence comprising SEQ ID NO: 26, a CDRL1 sequence comprising SEQ ID NO: 30, a CDRL2 sequence comprising SEQ ID NO: 34, and a CDRL3 sequence comprising SEQ ID NO: 38.

### Claim 2 (depends on 1)

The antibody or antigen-binding fragment of claim 1, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences are present within a framework sequence.

### Claim 3 (depends on 2)

The antibody or antigen-binding fragment of claim 2, wherein at least a portion of the framework sequence comprises a human consensus framework sequence.

### Claim 4 (depends on 3)

The antibody or antigen-binding fragment of claim 3, comprising:
(a) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 7 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 11; or
(b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 8 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 12; or
(c) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 9 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 13; or
(d) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 10 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 14.

### Claim 5 (depends on 4)

The antibody or antigen-binding fragment of claim 4, comprising:
(a) a heavy chain variable region sequence comprising SEQ ID NO: 7 and a light chain variable region sequence comprising SEQ ID NO: 11; or
(b) a heavy chain variable region sequence comprising SEQ ID NO: 8 and a light chain variable region sequence comprising SEQ ID NO: 12; or
(c) a heavy chain variable region sequence comprising SEQ ID NO: 9 and a light chain variable region sequence comprising SEQ ID NO: 13; or
(d) a heavy chain variable region sequence comprising SEQ ID NO: 10 and a light chain variable region sequence comprising SEQ ID NO: 14.

### Claim 6 (depends on 1)

A multispecific antibody comprising the anti-AS-SPIK antibody or antigen binding fragment thereof of claim 1.

### Claim 7 (depends on 1)

A bispecific antibody comprising the anti-AS-SPIK antibody or antigen binding fragment thereof of claim 1.

### Claim 8 (depends on 1)

The antibody or antigen-binding fragment of claim 1, which is monoclonal.

### Claim 9

A diagnostic method for determining whether a subject has hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), or cirrhosis of the liver, the method comprising:
(a) contacting a biological test sample from the subject with an anti-Abnormal Secreted Serine Protease Inhibitor Kazal (anti-AS-SPIK) antibody, or an antigen-binding fragment thereof, that specifically binds to AS-SPIK, and does not bind to Normal Secreted Serine Protease Inhibitor Kazal (NS-SPIK), to generate an AS-SPIK-antibody complex, wherein the anti-AS-SPIK antibody or antigen-binding fragment thereof comprises:
(i) a CDRH1 sequence comprising SEQ ID NO: 15, a CDRH2 sequence comprising SEQ ID NO: 19, a CDRH3 sequence comprising SEQ ID NO: 23, a CDRL1 sequence comprising SEQ ID NO: 27, a CDRL2 sequence comprising SEQ ID NO: 31, and a CDRL3 sequence comprising SEQ ID NO: 35; or
(ii) a CDRH1 sequence comprising SEQ ID NO: 16, a CDRH2 sequence comprising SEQ ID NO: 20, a CDRH3 sequence comprising SEQ ID NO: 24, a CDRL1 sequence comprising SEQ ID NO: 28, a CDRL2 sequence comprising SEQ ID NO: 32, and a CDRL3 sequence comprising SEQ ID NO: 36; or
(iii) a CDRH1 sequence comprising SEQ ID NO: 17, a CDRH2 sequence comprising SEQ ID NO: 21, a CDRH3 sequence comprising SEQ ID NO: 25, a CDRL1 sequence comprising SEQ ID NO: 29, a CDRL2 sequence comprising SEQ ID NO: 33, and a CDRL3 sequence comprising SEQ ID NO: 37; or
(iv) a CDRH1 sequence comprising SEQ ID NO: 18, a CDRH2 sequence comprising SEQ ID NO: 22, a CDRH3 sequence comprising SEQ ID NO: 26, a CDRL1 sequence comprising SEQ ID NO: 30, a CDRL2 sequence comprising SEQ ID NO: 34, and a CDRL3 sequence comprising SEQ ID NO: 38;
(b) detecting a concentration of the AS-SPIK-antibody complex in the biological test sample; and
(c) comparing the concentration of the AS-SPIK-antibody complex to a reference value to determine whether the subject has hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), or cirrhosis of the liver.

### Claim 10 (depends on 9)

The diagnostic method of claim 9, wherein the anti-AS-SPIK antibody or antigen-binding fragment thereof comprises a detectable label.

### Claim 11

A kit comprising an anti-Abnormal Secreted Serine Protease Inhibitor Kazal (anti-AS-SPIK) antibody, or an antigen-binding fragment thereof, that specifically binds to Abnormal Secreted Serine Protease Inhibitor Kazal (AS-SPIK), and does not bind to Normal Secreted Serine Protease Inhibitor Kazal (NS-SPIK), comprising:
(a) a CDRH1 sequence comprising SEQ ID NO: 15, a CDRH2 sequence comprising SEQ ID NO: 19, a CDRH3 sequence comprising SEQ ID NO: 23, a CDRL1 sequence comprising SEQ ID NO: 27, a CDRL2 sequence comprising SEQ ID NO: 31, and a CDRL3 sequence comprising SEQ ID NO: 35; or
(b) a CDRH1 sequence comprising SEQ ID NO: 16, a CDRH2 sequence comprising SEQ ID NO: 20, a CDRH3 sequence comprising SEQ ID NO: 24, a CDRL1 sequence comprising SEQ ID NO: 28, a CDRL2 sequence comprising SEQ ID NO: 32, and a CDRL3 sequence comprising SEQ ID NO: 36; or
(c) a CDRH1 sequence comprising SEQ ID NO: 17, a CDRH2 sequence comprising SEQ ID NO: 21, a CDRH3 sequence comprising SEQ ID NO: 25, a CDRL1 sequence comprising SEQ ID NO: 29, a CDRL2 sequence comprising SEQ ID NO: 33, and a CDRL3 sequence comprising SEQ ID NO: 37; or
(d) a CDRH1 sequence comprising SEQ ID NO: 18, a CDRH2 sequence comprising SEQ ID NO: 22, a CDRH3 sequence comprising SEQ ID NO: 26, a CDRL1 sequence comprising SEQ ID NO: 30, a CDRL2 sequence comprising SEQ ID NO: 34, and a CDRL3 sequence comprising SEQ ID NO: 38.

### Claim 12 (depends on 11)

The kit of claim 11, further comprising an antibody or antigen-binding fragment thereof that specifically binds to SPIK.

### Claim 13 (depends on 11)

The kit of claim 11, further comprising an antibody or antigen-binding fragment thereof that specifically binds to both AS-SPIK and NS-SPIK.

### Claim 14 (depends on 11)

The kit of claim 11, wherein the antibody or antigen-binding fragment thereof that specifically binds to AS-SPIK, and does not bind to NS-SPIK, is pre-bound to an assay device.