# US 2025/0155441 A1 · HIGH AFFINITY ANTIBODIES SPECIFICALLY BINDING TO a-1,6-CORE-FUCOSYLATED ALPHA-FETOPROTEIN

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- Number: US 2025/0155441 A1
- Document ID: US20250155441A1
- Status: Published application
- Kind: A1
- Application number: 18/951,180
- Filed: 2024-11-18
- Published: 2025-05-15
- Earliest claimed: 2022-05-16
- Assignees: Roche Diagnostics Operations, Inc.
- Applicants: Roche Diagnostics Operations, Inc.
- Inventors: Michael Gerg; Lars Hillringhaus; Ute Jucknischke; Anna Christine Muth; Tobias Oelschlaegel; Boris Pinchuk; Michael Schraeml; Joanna Siebenhaar; Laurence Thirault
- Web page: https://patentgrep.com/patent/US20250155441A1

## CPC

- G01N 33/57525: of the liver or pancreas
- C07K 16/18: against material from animals or humans
- G01N 33/57585: {involving compounds identifiable in body fluids}
- G01N 33/689: {related to pregnancy or the gonads}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/30: characterized by aspects of specificity or valency
- 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 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- G01N 2333/471: Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
- G01N 2400/00: Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N 2440/38: addition of carbohydrates, e.g. glycosylation, glycation
- 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)

## Abstract

The present invention relates to monoclonal antibodies and antigen binding fragments that specifically bind to α-1,6-core-fucosylated alpha-fetoprotein (AFP), which is the core component of AFP-L3. Thus, the antibodies and antigen binding fragments provided herein may also be referred to as AFP-L3 antibodies. Also provided are polynucleotides encoding the antibodies or antigen binding fragments of the invention, host cells expressing the antibodies and antigen binding fragments of the invention, methods for producing the antibodies and antigen binding fragments of the invention, and uses of the antibodies and antigen binding fragments of the invention. Also provided herein is a pretreatment agent facilitating the binding of the antibodies and antigen binding fragments of the invention to α-1,6-core-fucosylated AFP. The present invention further relates to kits comprising the antibodies and antigen binding fragments of the invention and optionally the pretreatment agent of the invention.

## Claims

### Claim 1

A monoclonal antibody or antigen binding fragment thereof specifically binding to α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP comprising said α-1,6-core-fucosylation, wherein the monoclonal antibody or antigen binding fragment comprises
(i) a heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6; and
(ii) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10.

### Claim 2 (depends on 1)

The monoclonal antibody or antigen binding fragment according to claim 1, wherein the partial sequence of AFP comprising said α-1,6-core-fucosylation comprises or consists of a glycopeptide of Formula I,

### Claim 3 (depends on 2)

The monoclonal antibody or antigen binding fragment according to claim 2, wherein the monoclonal antibody or antigen binding fragment specifically bind the glycopeptide of Formula I and the glycopeptide of Formula II,

### Claim 4 (depends on 3)

The monoclonal antibody or antigen binding fragment according to claim 3, wherein the ratio between the KD for the binding to glycopeptide of Formula II and the glycopeptide of Formula I is at least 2.

### Claim 5 (depends on 1)

The monoclonal antibody or antigen binding fragment according to claim 1, wherein
(i) the heavy chain variable domain (VH) has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:11 or 12; and
(ii) the light chain variable domain (VL) has an amino acid sequence with at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 13 or 14.

### Claim 6 (depends on 1)

The monoclonal antibody or antigen binding fragment according claim 1, wherein
(i) the heavy chain variable domain (VH) has the amino acid sequence SEQ ID NO: 11 or 12; and
(ii) the light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 13 or 14.

### Claim 7 (depends on 1)

The monoclonal antibody or antigen binding fragment according to claim 1, which binds to the glycopeptide of Formula I,
with a KD of 100 nM or less, 20 nM or less, 10 nM or less or 3.1 nM or less, optionally wherein the KD is measured at 37° C.

### Claim 8 (depends on 1)

The monoclonal antibody or antigen binding fragment according to claim 1, which binds to the glycopeptide of Formula I,
with a KD that is less than 10, 8, 6, 4, or 2 times the KD of a rabbit IgG antibody comprising the heavy chain variable domain of SEQ ID NO: 11 or 12 and a light chain variable domain of SEQ ID NO: 13 or 14 towards the glycopeptide of Formula I, wherein the KD values are measured under the same conditions and using the same method.

### Claim 9 (depends on 1)

A polynucleotide or a set of polynucleotides encoding
(i) the heavy chain or heavy chain variable domain of the monoclonal antibody or antigen binding fragment according to claim 1; and/or
(ii) the light chain or light chain variable domain of the monoclonal antibody or antigen binding fragment according to claim 1.

### Claim 10 (depends on 9)

A vector comprising the polynucleotide or the set of polynucleotides according to claim 9.

### Claim 11 (depends on 1)

A host cell comprising a polynucleotide or a set of polynucleotides encoding (i) the heavy chain or heavy chain variable domain of the monoclonal antibody or antigen binding fragment according to claim 1; and/or (ii) the light chain or light chain variable domain of the monoclonal antibody or antigen binding fragment according to claim 1, and expressing the antibody or antigen binding fragment of claim 1.

### Claim 12 (depends on 1)

A method of producing the monoclonal antibody or antigen binding fragment according to claim 1 comprising culturing a host cell comprising a polynucleotide or a set of polynucleotides encoding (i) the heavy chain or heavy chain variable domain of the monoclonal antibody or antigen binding fragment according to claim 1; and/or (ii) the light chain or light chain variable domain of the monoclonal antibody or antigen binding fragment according to claim 1, and isolating said antibody or antigen binding fragment.

### Claim 13 (depends on 1)

A composition comprising the antibody or antigen binding fragment according to claim 1.

### Claim 14

(canceled)

### Claim 15 (depends on 1)

An in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation in a sample using the antibody or antigen binding fragment as defined in claim 1.

### Claim 16

A pretreatment reagent for treating a sample comprising α-1,6-core-fucosylated AFP, said pretreatment reagent comprising a reducing agent.

### Claim 17 (depends on 1)

A kit comprising the antibody or antigen binding fragment as defined in claim 1.

### Claim 18 (depends on 5)

The monoclonal antibody or antigen binding fragment according to claim 5, wherein
(i) the heavy chain variable domain (VH) has an amino acid sequence with at least 97.5% sequence identity to SEQ ID NO: 11 or 12; and
(ii) the light chain variable domain (VL) has an amino acid sequence with at least 97.3% sequence identity to SEQ ID NO: 13 or 14.

### Claim 19 (depends on 15)

The in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence of claim 15, wherein the sample is pretreated with a pretreatment reagent comprising a reducing agent.

### Claim 20 (depends on 19)

The in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence of claim 19, wherein the reducing agent is a protein reducing agent.

### Claim 21 (depends on 16)

The pretreatment reagent for treating a sample comprising α-1,6-core-fucosylated AFP according to claim 16, wherein the reducing agent is a protein reducing agent.