WO2024022478A1

WO2024022478A1 - Antibody binding to cannabinoid receptor cb1 and use thereof

Info

Publication number: WO2024022478A1
Application number: PCT/CN2023/109811
Authority: WO
Inventors: 马彦彬; 金磊; 王思勤; 陈达锴; 胡少平; 陈兰兰; 金后聪; 申红霞; 孙杲; 康立山
Original assignee: 长春金赛药业有限责任公司
Priority date: 2022-07-28
Filing date: 2023-07-28
Publication date: 2024-02-01

Abstract

An antibody specifically binding to CB1 or an antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof and a host cell comprising the nucleic acid molecule, and therapeutic and diagnostic use of these antibodies or the antigen-binding fragments thereof.

Description

Antibodies that bind cannabinoid receptor CB1 and their uses

Technical field

The present invention relates to antibodies that specifically bind CB1 or antigen-binding fragments thereof, pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, host cells containing the same, and related use. Furthermore, the invention relates to the therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.

Background technique

Cannabinoid receptors CB1 and CB2 are key components of the endocannabinoid system and are the primary targets of D9-tetrahydrocannabinol (D9-THC), a compound extracted from cannabis with a wide range of therapeutic uses. of psychoactive chemicals (Lemberger, 1980). Cannabinoid receptor 1 (CB1) is activated by the lipid tetrahydrocannabinol (THC) and is associated with psychoactive, neuromodulatory, and analgesic effects. CB1 and CB2 play important roles in a variety of physiological processes, including appetite, pain perception, memory, and immune regulation (Guzma'n, 2003). The two cannabinoid receptors, CB1 and CB2, share 44% total sequence homology and 68% sequence similarity in the transmembrane region (Munro et al., 1993). However, they have different tissue distribution and play different functions in the endocannabinoid system, with CB1 and CB2 being mainly expressed in the central nervous system and immune system, respectively. The development of therapeutic applications is hampered by the difficulty of specifically modulating individual cannabinoid receptors due to their high sequence similarity. CB1 receptors are expressed in the central nervous system, lungs, liver, adipose tissue and kidneys. Studies have found that CB1 receptors are involved in the occurrence and development of many diseases, such as obesity, diabetic nephropathy, liver fibrosis, and pulmonary fibrosis. The expression of CB1 receptors and their ligands is significantly up-regulated under pathological conditions.

Rimonabant developed by the French company Sanofi-Synthelabo is the world's first type 1 cannabinoid receptor (CB1) inhibitor weight loss drug, which has a good weight loss effect. It also has the effect of sensitizing insulin and improving lipid metabolism disorders, and has a certain effect in assisting smoking cessation. It was approved for marketing in the EU in 2006 as a new weight loss drug. However, clinical studies and post-marketing studies found that Rimonabant can increase serious mental adverse reactions, namely depression and anxiety, and also increase the risk of suicide in users. Therefore, Rimonabant was withdrawn from the European market.

An article published in J Clin Invest in 2017 demonstrated that CB1 deletion in adipocytes can prevent obesity and metabolic disorders through peripheral knockout of CB1 receptors, indicating that peripheral CB1 blockade has reasonable biological significance in reducing body weight and liver fat in mice. Compared with small molecules that can pass through the blood-brain barrier (BBB), the development of peripherally restricted macromolecule drugs retains their efficacy and reduces central nervous system side effects, and is expected to become the best drug in its class. Currently, there are companies in the world that are developing CB1 receptor antibodies. For example, the antibody GFB-024 (see patent: WO2019211665) developed by Takeda is currently in Phase 1 clinical trials. However, currently existing antibodies targeting CB1 receptors do not have cross-activity between humans and mice. Therefore, it is still necessary to develop new and more effective antibodies targeting CB1 receptors to provide more clinical application options.

Contents of the invention

Through extensive research, the inventors of the present application have obtained a mouse antibody that specifically binds to human CB1 and has significant inhibitory effect on CB1 activity. In certain embodiments, the antibodies provided herein have cross-binding activity against human CB1 and murine CB1. In addition, the inventor of the present application further obtained a humanized antibody with CB1 binding activity and CB1 activity inhibitory effect based on the mouse-derived antibody.

Based on this, the present application also provides pharmaceutical compositions containing the antibody or antigen-binding fragment thereof, nucleic acid molecules encoding the antibody or antigen-binding fragment thereof, host cells containing the same, and related uses.

Antibodies or antigen-binding fragments thereof

mAb010 and its humanized antibodies

Therefore, in a first aspect, the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding CB1, the antibody or an antigen-binding fragment thereof comprising:

VH CDR1 or a variant thereof, a VH CDR2 or a variant thereof, and a VH CDR3 or a variant thereof contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 22 or 40; and/or, as in SEQ ID NO. VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in any one of NO: 27 and 41-43;

Wherein, the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions, eg conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 22 or 40; and/or, as shown in any of SEQ ID NO: 27, 41-43, the light chain variable region 3 CDRs contained in the area (VL).

In certain embodiments, the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system. In certain embodiments, the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat numbering system.

The heavy chain variable region (VH) contains the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:23, VH CDR2 with the sequence SEQ ID NO:24, and VH CDR2 with the sequence SEQ ID NO:25 The VH CDR3 of , VL CDR3 whose sequence is SEQ ID NO:30;

Wherein, the CDR is defined by kabat numbering system.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises framework sequence derived from a murine immunoglobulin.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a murine heavy chain germline sequence, and/or a light chain framework region of a murine light chain germline sequence.

Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 22 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 27 or its variations;

Wherein, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:22 and a VL comprising the sequence set forth in SEQ ID NO:27.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises framework sequence derived from a human immunoglobulin.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence of a human heavy chain germline sequence, and/or a light chain framework region of a human light chain germline sequence.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:40 or a variant thereof; and/or, comprising a sequence set forth in SEQ ID NO:40 or a variant thereof; The light chain variable region (VL) of the sequence shown in any one of NO: 41-43 or a variant thereof;

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:40 and a VL comprising the sequence set forth in SEQ ID NO:41.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:40 and a VL comprising the sequence set forth in SEQ ID NO:42.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:40 and a VL comprising the sequence set forth in SEQ ID NO:43.

In certain embodiments, the antibody or antigen-binding fragment thereof has cross-binding activity against human CB1 and murine CB1.

mAb003 and its humanized antibodies

Therefore, in a second aspect, the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding CB1, the antibody or an antigen-binding fragment thereof comprising:

VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO: 2, 31-32; and/ Or, VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in any one of SEQ ID NO: 7 and 33-35;

3 CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO:2, 31-32; and/or, as shown in any one of SEQ ID NO:7, 33-35 The light chain variable region (VL) contains 3 CDRs.

Heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): VH CDR1 with sequence SEQ ID NO:3, VH CDR2 with sequence SEQ ID NO:4, and VH CDR2 with sequence SEQ ID NO:5 The VH CDR3 of , VL CDR3 whose sequence is SEQ ID NO:10;

Wherein, the CDR is defined by kabat numbering system.

Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 2 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 7 or its variations;

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:2 and a VL comprising the sequence set forth in SEQ ID NO:7.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 31 or 32 or a variant thereof; and/or comprising as The light chain variable region (VL) of the sequence shown in any one of SEQ ID NO: 33-35 or a variant thereof;

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO: 31 and a VL comprising the sequence set forth in SEQ ID NO: 34.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:31 and a VL comprising the sequence set forth in SEQ ID NO:35.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:32 and a VL comprising the sequence set forth in SEQ ID NO:33.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:32 and a VL comprising the sequence set forth in SEQ ID NO:34.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:32 and a VL comprising the sequence set forth in SEQ ID NO:35.

mAb006 and its humanized antibodies

Therefore, in a third aspect, the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding CB1, the antibody or an antigen-binding fragment thereof comprising:

VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO: 12, 36-37; and/ Or, VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in any one of SEQ ID NO: 17 and 38-39;

3 CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO:12, 36-37; and/or, as shown in any one of SEQ ID NO:17, 38-39 The light chain variable region (VL) contains 3 CDRs.

The heavy chain variable region (VH) contains the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:13, VH CDR2 with the sequence SEQ ID NO:14, and VH CDR2 with the sequence SEQ ID NO:15 The VH CDR3 of , VL CDR3 whose sequence is SEQ ID NO:20;

Wherein, the CDR is defined by kabat numbering system.

Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 12 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 17 or its variations;

Wherein, the variant has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO: 12 and a VL comprising the sequence set forth in SEQ ID NO: 17.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 36 or 37 or a variant thereof; and/or comprising as The light chain variable region (VL) of the sequence shown in SEQ ID NO: 38 or 39 or a variant thereof;

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:36 and a VL comprising the sequence set forth in SEQ ID NO:38.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:37 and a VL comprising the sequence set forth in SEQ ID NO:38.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:37 and a VL comprising the sequence set forth in SEQ ID NO:39.

mAb016 and its humanized antibodies

Therefore, in a fourth aspect, the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding CB1, the antibody or an antigen-binding fragment thereof comprising:

VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 46; and/or, as in SEQ ID NO: VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in 50;

3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46; and/or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:50 CDR.

The heavy chain variable region (VH) contains the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:47, VH CDR2 with the sequence SEQ ID NO:48, and VH CDR2 with the sequence SEQ ID NO:49 The VH CDR3 of , VL CDR3 whose sequence is SEQ ID NO:53;

Wherein, the CDR is defined by kabat numbering system.

Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 46 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 50 or its variations;

Wherein, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, to Less than 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity sexual sequence.

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:46 and a VL comprising the sequence set forth in SEQ ID NO:50.

mAb030 and its humanized antibodies

Therefore, in a fifth aspect, the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding CB1, the antibody or an antigen-binding fragment thereof comprising:

VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 54; and/or, as in SEQ ID NO: VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in 58;

3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:54; and/or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:58 CDR.

A heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:55, VH CDR2 with the sequence SEQ ID NO:56, and SEQ ID NO:57 VH CDR3; and/or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 whose sequence is SEQ ID NO:59, VL CDR2 whose sequence is SEQ ID NO:60, and whose sequence is SEQ ID NO:60 VL CDR3 of ID NO:61;

Wherein, the CDR is defined by kabat numbering system.

Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 54 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 58 or its variations;

In certain embodiments, the substitutions are conservative substitutions.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:54 and a VL comprising the sequence set forth in SEQ ID NO:58.

In certain embodiments, in the first, second, third, fourth or fifth aspect, the antibody or antigen-binding fragment thereof further comprises an immune The constant region of a globulin.

In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and/or the antibody or antigen-binding fragment thereof The light chain of the antigen-binding fragment comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).

In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant of a human immunoglobulin. region (CH) or a variant thereof having one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 10) compared to the sequence from which it is derived. Substitution, deletion or addition of 5 amino acids; e.g. substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); and/or,

The light chain of the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions compared to the sequence from which it is derived, Deletion or addition (e.g., substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, substitution, deletion of 1, 2, 3, 4, or 5 amino acids or add). The substitutions may be conservative substitutions or non-conservative substitutions.

In some embodiments, the variant of the heavy chain constant region (CH) may have one or more conservative substitutions of amino acids compared to the sequence from which it is derived. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same properties as the wild-type sequence from which it is derived.

In other embodiments, the variants of the heavy chain constant region (CH) may comprise one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the invention: Fc receptor binding , antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc. Effector function can be altered by replacing at least one amino acid residue in the constant region of the antibody with a different residue or by chemical modification to produce a functional change, for example, changing the affinity of the antibody for an effector ligand such as FcR or complement C1q. (e.g. lower or boost). The Fc region of antibodies mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

In certain embodiments, the antibodies of the invention, or antigen-binding fragments thereof, comprise variants of the human immunoglobulin heavy chain constant region (CH) that may have reduced or Eliminated effector functions.

In certain embodiments, the antibodies of the invention, or antigen-binding fragments thereof, comprise variants of the human immunoglobulin heavy chain constant region (CH) that may have enhanced potency compared to the wild-type sequence from which they are derived. Effector functions.

In certain embodiments, the antibodies of the invention, or antigen-binding fragments thereof, comprise a human immunoglobulin heavy chain constant region (CH) or a variant thereof, which variant may have the same sequence as the wild-type sequence from which it is derived. or essentially the same effector function.

In certain embodiments, the heavy chain constant region (CH) is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.

In certain embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

In certain exemplary embodiments, in the first, second, third, fourth or fifth aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises as set forth in SEQ ID NO:44 The heavy chain constant region (CH) shown in SEQ ID NO: 45, and/or the light chain of the antibody or antigen-binding fragment thereof includes the light chain constant region (CL) shown in SEQ ID NO: 45.

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) A heavy chain comprising VH of the sequence shown in SEQ ID NO:40 and CH shown in SEQ ID NO:44, and, a light chain comprising a VL of the sequence set forth in SEQ ID NO:41 and a CL set forth in SEQ ID NO:45;

(2) A heavy chain comprising VH of the sequence shown in SEQ ID NO:40 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:42 and SEQ ID NO: The light chain of CL shown in 45;

(3) A heavy chain comprising VH of the sequence shown in SEQ ID NO:40 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:43 and SEQ ID NO: The light chain of CL shown in 45;

(4) A heavy chain comprising VH of the sequence shown in SEQ ID NO:31 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:34 and SEQ ID NO: The light chain of CL shown in 45;

(5) A heavy chain comprising VH of the sequence shown in SEQ ID NO:31 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:35 and SEQ ID NO: The light chain of CL shown in 45;

(6) A heavy chain comprising VH of the sequence shown in SEQ ID NO:32 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:33 and SEQ ID NO: The light chain of CL shown in 45;

(7) A heavy chain comprising VH of the sequence shown in SEQ ID NO:32 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:34 and SEQ ID NO: The light chain of CL shown in 45;

(8) A heavy chain comprising VH of the sequence shown in SEQ ID NO:32 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:35 and SEQ ID NO: The light chain of CL shown in 45;

(9) A heavy chain comprising VH of the sequence shown in SEQ ID NO:36 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:38 and SEQ ID NO: The light chain of CL shown in 45;

(10) A heavy chain comprising VH of the sequence shown in SEQ ID NO:37 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:38 and SEQ ID NO: The light chain of CL shown in 45; or,

(11) A heavy chain comprising VH of the sequence shown in SEQ ID NO:37 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:39 and SEQ ID NO: The light chain of CL is shown in 45.

In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a murine immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4), the antibody or antigen-binding fragment thereof The light chain includes a light chain constant region derived from a murine immunoglobulin (eg, kappa or lambda).

In certain exemplary embodiments, in a first aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 21, and/or, the antibody or antigen-binding fragment thereof The light chain includes the sequence shown in SEQ ID NO:26.

In certain exemplary embodiments, in the second aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 1, and/or, the antibody or antigen-binding fragment thereof The light chain contains the sequence shown in SEQ ID NO:6.

In certain exemplary embodiments, in the third aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises, e.g. The sequence shown in SEQ ID NO: 11, and/or the light chain of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 16.

In certain embodiments, in the first, second, third, fourth or fifth aspect, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') ₂ , Fd, Fv , disulfide bond-linked Fv, scFv, di-scFv, (scFv) ₂ , diabody and single domain antibody (sdAb); and/or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody hybrid, bispecific, or multispecific antibodies.

In a sixth aspect, the application also provides an isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, or a heavy chain thereof, as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect. Variable domain and/or light chain variable domain, or heavy chain and/or light chain thereof.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a first nucleotide sequence encoding said antibody or antigen-binding fragment thereof A second nucleotide sequence of a light chain or light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different separate nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said second nucleotide sequence.

In a seventh aspect, the present application also provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a light chain encoding said antibody or antigen-binding fragment thereof or a second nucleotide sequence of the light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence exist on different vectors, the vector of the present invention includes a first vector containing the first nucleotide sequence and a vector containing the A second vector for a second nucleotide sequence.

In an eighth aspect, the present application also provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.

The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) . Alternatively, these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )). For example, Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F(ab′) ₂ fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). In addition, Fv, Fab or F(ab') ₂ fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.

In a ninth aspect, the present application also provides a method for preparing an antibody or an antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, which includes, allowing the antibody to The host cells as described above are cultured under conditions for expression of the antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.

therapeutic use

In a tenth aspect, the present application also provides a pharmaceutical composition, which includes the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect, or the antibody or antigen-binding fragment thereof as described in the sixth aspect. The isolated nucleic acid molecule described above, the vector as described in the seventh aspect or the host cell as described in the eighth aspect, and pharmaceutically acceptable carriers and/or excipients.

In certain embodiments, the pharmaceutical composition further includes an additional pharmaceutically active agent.

In certain exemplary embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% Glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

In the eleventh aspect, the application also provides the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect, and the isolated nucleic acid as described in the sixth aspect. The use of molecules, vectors as described in the seventh aspect, host cells as described in the eighth aspect or pharmaceutical compositions as described in the tenth aspect for the preparation of medicaments for preventing and/or treating subjects of CB1-related diseases.

In certain embodiments, the CB1-related diseases will benefit from antagonism of CB1 signaling.

In certain embodiments, the disease associated with CB1 is: obesity-related disease (e.g., Prader-Willi syndrome), diabetes (e.g., type 1 diabetes, type 2 diabetes), kidney disease (e.g., diabetes kidney disease), hepatic fibrosis, pulmonary fibrosis, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver disease (e.g., non-alcoholic fatty liver disease), or combinations thereof.

In certain embodiments, the subject is a mammal, such as a human or a mouse.

In certain embodiments, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent, such as administered simultaneously or sequentially.

In a twelfth aspect, the present application also provides a method for preventing and/or treating CB1-related diseases in a subject, which includes: administering to a subject in need an effective amount of, e.g. The antibody or antigen-binding fragment thereof described in the first, second, third, fourth or fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the vector described in the seventh aspect, The host cell as described in the eighth aspect or the pharmaceutical composition as described in the tenth aspect.

In certain embodiments, the subject is a mammal, such as a human or a mouse.

In certain embodiments, the antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, the isolated nucleic acid molecule as described in the sixth aspect, such as The vector according to the seventh aspect, the host cell according to the eighth aspect or the pharmaceutical composition according to the tenth aspect may be administered in combination with another pharmaceutically active agent, for example simultaneously or sequentially.

The antibody or antigen-binding fragment thereof or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules , elixirs, tablets, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention should be sterile and stable under the conditions of production and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions may be prepared by incorporating in an appropriate solvent the requisite dose of an antibody of the invention, or antigen-binding fragment thereof, and, optionally, other desired ingredients including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, preservatives, diluents, or any combination thereof) followed by filtered sterilization. In addition, it can Sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (for example, 0.9% NaCl), glucose solution (for example, 5 % glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present application may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, eyeball, topical, parenteral, rectal, intrathecal , intracytoplasmic reticulum, groin, bladder, topically (eg, powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will understand that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.

Testing purposes

In a thirteenth aspect, the present application also provides a conjugate comprising an antibody or an antigen-binding fragment thereof as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect, and with said A detectable label attached to an antibody or antigen-binding fragment thereof.

In certain embodiments, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.

In the fourteenth aspect, the application also provides a kit, which includes the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect or the thirteenth aspect. the above conjugates.

In certain embodiments, the kit includes a detection buffer.

In certain embodiments, the kit includes the conjugate of the thirteenth aspect.

In certain embodiments, the kit comprises an antibody or an antigen-binding fragment thereof as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect, and specifically recognizes the antibody or a second antibody to an antigen-binding fragment thereof; optionally, the second antibody further includes a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.

In a fifteenth aspect, the present application also provides a method for detecting the presence or level of CB1 in a sample, which includes This includes using the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect or the conjugate as described in the thirteenth aspect.

In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.

In certain embodiments, the method is an immunological assay, such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

In certain embodiments, the method includes using the conjugate of the thirteenth aspect.

In certain embodiments, the method includes using an antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, and the method further includes using a carrier Detectable labels (such as enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes ( The antibody or antigen-binding fragment thereof is detected by a secondary antibody such as fluorescein or fluorescent protein), radionuclide or biotin).

In certain embodiments, the method includes: (1) contacting the sample with an antibody of the invention, or an antigen-binding fragment or conjugate thereof; (2) detecting the formation of an antigen-antibody immune complex or detecting the resulting The amount of immune complexes. The formation of the immune complex indicates the presence of CB1 or cells expressing CB1.

The present application also provides a method of diagnosing a disease related to CB1, which includes detecting the presence of CB1 or the level thereof in a sample from a subject using the method described in the fifteenth aspect. In certain embodiments, when CB 1 is present or the level of CB 1 is increased compared to a reference level (eg, compared to healthy controls), the subject has a disease associated with CB 1 .

In certain embodiments, the subject is a mammal, such as a human or a mouse.

In the sixteenth aspect, the application also provides the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect, the third aspect, the fourth aspect or the fifth aspect or the conjugate as described in the thirteenth aspect Use in the preparation of detection reagents for detecting the presence or level of CB1 in a sample and/or diagnosing CB1-related diseases.

In certain embodiments, the detection reagent detects the presence or level of CB1 in the sample by the method described in the fifteenth aspect.

In certain embodiments, the detection reagent detects the presence of CB1 or its level in a sample through the method described in the fifteenth aspect to diagnose a disease related to CB1. In certain embodiments, when CB1 is present or compared to a reference When the levels of CB1 are increased compared to levels (e.g., compared to healthy controls), it indicates that the subject suffers from a CB1-related disease.

In certain embodiments, the sample is a tissue sample (eg, central nervous tissue, lung tissue, liver tissue, adipose tissue, or kidney tissue) from a subject (eg, a mammal, preferably human or mouse).

Definition of Terms

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the virology, biochemistry, and immunology laboratory procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

When the terms "such as," "such as," "such as," "including," "including," or variations thereof are used herein, these terms will not be considered limiting terms and will instead be interpreted to mean "without limitation ” or “without limitation.”

Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" as well as "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover Singular and plural.

The term "antibody" as used herein refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. The target antigen. When referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments capable of specifically binding a target antigen, unless the context clearly indicates otherwise. An "intact antibody" typically consists of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q). The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site. The assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof, are preferably determined by the Kabat, Chothia, or IMGT numbering systems.

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

The term "antibody" is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. It can be obtained by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of the antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, disulfide-linked Fv, scFv , di-scFv, diabodies, single domain antibodies, and polypeptides containing at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al. , 2005; Nat Biotechnol, 23:1126-1136.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab') ₂ fragment "F(ab') fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fab'fragment" means the disulfide bond connecting the two heavy chain fragments in the F(ab') ₂ fragment by reduction The fragment obtained is composed of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.

As used herein, the term "Fc" means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain. Antibody fragments. The Fc fragment of an antibody has many different functions but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In certain embodiments of the invention, scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody. In certain embodiments of the invention, scFv can form (scFv) ₂ , which refers to two or more individual scFvs joining in parallel to form an antibody.

As used herein, the term "diabody" means one whose VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of the other chain and creates two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds. Single domain antibodies are also called nanobodies.

As used herein, the term "bispecific antibody" refers to an antibody that has binding specificities for two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity for at least two (eg, three or four) different antigens (or epitopes). Bispecific antibodies or multispecific antibodies contain multiple antigen-binding domains with binding specificities for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and/or target molecules. Each antigen-binding domain comprised by a bispecific antibody or a multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or an antigen-binding fragment thereof (e.g., Fv fragment, Fab fragment, F(ab')2 fragment or scFv). In some cases, the individual antigen binding domains are linked by a peptide linker.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.

Antigen-binding fragments of an antibody (e.g., the above-described antibody fragments) can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.

As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology to that of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source. Immunoglobulins (receptor antibodies). In certain embodiments, the CDR regions of the humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and/or constant region) are derived from a human source. Immunoglobulins (receptor antibodies). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In this application, the donor antibody may be a murine antibody with desired properties (eg, antigen specificity, affinity, reactivity, etc.). To prepare humanized antibodies, the CDR regions of the donor antibody can be inserted into human framework sequences using methods known in the art. In some cases, the human framework sequences may contain amino acid mutations that are replaced by corresponding non-human residues. In addition, humanized antibodies can also contain residues that are not found in either the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or human framework sequences to further improve or optimize Performance of this humanized antibody.

As used herein, the term "chimeric antibody" refers to an antibody in which part of the light chain and/or heavy chain is derived from an antibody (which may be derived from a specific species or belonging to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of How, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy chain variable region and light chain variable region of the antibody are derived from the first antibody, and the heavy chain constant region and light chain constant region of the antibody are from secondary antibodies.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, both sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877. Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide). For example, and without limitation, polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K _D ) of the interaction. In the present invention, the term " _KD " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate. "Association rate constant" (ka or kon) and "Dissociation rate" Both "rate constants" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio kdis/kon is equal to dissociation Constant K _D (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The K _D , kon and kdis values can be measured by any effective method. In certain embodiments, surface plasmon resonance can be used (SPR) in Biacore to measure dissociation constants. Alternatively, bioluminescence interferometry or Kinexa can be used to measure dissociation constants.

As used herein, a detectable label of the invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., ^3H , ^125I , ^35S , ^14C , or ^32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , fluorescent proteins (such as phycoerythrin (PE)), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa750)), luminescent substances (such as chemiluminescent reagents, such as acridinium esters) compounds, luminol and its derivatives, ruthenium derivatives such as ruthenium terpyridine), magnetic beads (for example, ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and avidin modified to bind the above labels (e.g., streptavidin ) of biotin. In certain embodiments, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin. In certain embodiments, detectable labels as described above can be linked to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or fibroblast cells, CHO cells, COS cells, NSO cells, Animal cells such as HeLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999) ; and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).

The twenty conventional amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives, stabilizers. For example, pH adjusting agents include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers generally understood by those skilled in the art Meaning, it is capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose) , amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% Glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to expected survival if not receiving treatment.

As used herein, the term "subject" refers to a mammal, such as a human or a mouse. In certain embodiments, the subject (e.g., human, murine) has a CB1-related disease (e.g., obesity-related disease (e.g., Prader-Willi syndrome), diabetes (e.g., type 1 Diabetes mellitus, type 2 diabetes), renal disease (eg, diabetic nephropathy), liver fibrosis, pulmonary fibrosis, dyslipidemia (eg, hyperlipidemia, hypercholesterolemia), fatty liver disease (eg, nonalcoholic fatty liver disease) ), or, are at risk for the above conditions.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, at least in part, the desired effect. For example, prevention of disease (e.g., obesity-related disease (e.g., Prader-Willi syndrome), diabetes (e.g., type 1 diabetes, type 2 diabetes), kidney disease (e.g., diabetic nephropathy), liver fibrosis, pulmonary fibrosis (e.g., hyperlipidemia, hypercholesterolemia), fatty liver disease (e.g., non-alcoholic fatty liver disease)), the effective amount refers to the amount sufficient to prevent, prevent, or delay the occurrence of the diseases; A therapeutically effective amount is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.

Beneficial effects of the invention

The antibody provided in this application has strong binding activity against human CB1, and it has significant inhibitory effect on CB1 activity. In addition, the antibody of the present application is naturally distributed in the periphery, and compared with other antagonists that can pass through the BBB, it can have It can effectively reduce the side effects on the central nervous system and has good clinical application prospects. In certain embodiments, the antibodies provided herein have cross-binding activity against human CB1 and murine CB1.

In addition, in certain embodiments, the antibodies of the present application also possess inhibitory activity superior to GFB-024.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.

Description of drawings

Figure 1 shows the inhibition of CB1 activity by murine antibody mAb003.

Figure 2 shows the inhibition of CB1 activity by murine antibody mAb006.

Figure 3 shows the inhibition of CB1 activity by murine antibody mAb010.

Figure 4 shows the inhibition of CB1 activity by murine antibody mAb016.

Figure 5 shows the inhibition of CB1 activity by murine antibody mAb030.

Figure 6 shows the fibrosis area percentage of each group of animals in Example 6; Two sample t-tests; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

sequence information

A description of the sequences covered by this application is provided in the table below.

Table 1: Sequence information

Detailed ways

The invention will now be described with reference to the following examples which are intended to illustrate but not to limit the invention.

Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out according to the method described in F.M. Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the product manufacturer. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: Antibody preparation

1. Preparation of hybridoma antibodies:

1.1 After immunizing mice with CB1 receptor antigen, select mice with higher titers for cell fusion and hybridoma preparation.

1.2 Prepare myeloma cells. Before fusion, use a counter to count and detect viability. Collect SP2/0 cells, centrifuge at 400g at room temperature for 5 minutes, discard the culture medium, and resuspend the cell pellet in 20 ml of fusion culture medium.

1.3 Collect and culture spleen cells, select mice with strong response to the immunogen for preparation of hybridoma cells, use carbon dioxide to euthanize mice according to the method approved by IACUC, collect pre-fusion plasma, separate and collect serum (FB) as Positive control for hybridoma supernatant screening.

1.4 Soak the mouse in 70% alcohol for disinfection, and then immediately transfer it to a biological safety cabinet for operation. Collect the spleen, place it in a sterile petri dish, place the spleen on the filter, and squeeze it repeatedly with the plug of a sterile syringe. Remove the spleen until there is no obvious clump, then rinse the filter with fusion culture medium to obtain the spleen cell suspension, and transfer all of it to a 50 ml centrifuge tube.

1.5 Centrifuge at 400g for 5 minutes, discard the supernatant, use electrofusion method to prepare fused cells, resuspend the spleen cell pellet in 5ml of red blood cell lysis buffer, then place it at 4 degrees for 5 minutes, and use 50ml of 10% FBS added. DEME culture medium terminated the reaction. Collect the spleen cell pellet by centrifugation, then add fusion culture medium to resuspend the cells, invert the centrifuge tube 3-5 times, and then count. Centrifuge at 400 g for 5 minutes, discard the supernatant, keep the cell pellet, and then resuspend the cells in 40 ml of fusion culture medium. Let the splenocytes stand at room temperature for 2-3 minutes, then transfer to a new 50 ml centrifuge tube. According to the ratio of splenocytes to SP2/0 cells of 4:1, add SP2/0 cells to splenocytes, and adjust the volume to 50 ml with fusion culture medium. Centrifuge the cell mixture, discard the supernatant, and loosen the cell pellet by repeatedly tapping the bottom of the tube. Fusion, for electrofusion, place the cell mixture in the electroporation tank and perform electroporation fusion according to the optimized procedure.

1.6 After electrofusion, the fused cells need to rest in the electroporation tank for an additional 10 minutes. Resuspend in DMEM culture medium supplemented with 20% FBS and HT, and inoculate into a 96-well microplate at a volume of 100 μl per well. After 24 hours, DMEM culture medium supplemented with 20% FBS and HAT was added to the well plate, 100 μl per well. Transfer all confluent plates back to the CO2 incubator, 37°C, 5% _CO2 . Monitor cell growth rate and presence of microbial contamination daily. When hybridoma clones When the hybridoma grows to a size of 1-2 mm in diameter (generally 10-14 days after fusion), use Acumen to screen the hybridoma supernatant.

1.7 Subcloning and expansion culture. The positive clones were transferred to a 24-well plate and cultured with amplification medium. The hybridoma culture supernatant was collected for FACS detection. By limiting dilution method, the positive mother clone was subcloned to obtain a single clone. Place the subclone 96-well plate in a carbon dioxide incubator and culture it for 7 days, and then detect and screen the activity of the supernatant. To ensure a single clone, 1-2 rounds of subcloning are required. Based on the screening results, select the four most active single clones from each subclone plate and transfer them to a 24-well plate for amplification and culture.

1.8 Cell cryopreservation. The amplified subcloned cell lines were cryopreserved, stored in DMEM culture medium supplemented with 20% FBS and 10% DMSO, and placed in a liquid nitrogen tank for long-term storage.

The mouse monoclonal antibodies mAb003, mAb006, mAb010, mAb016 and mAb030 were prepared by the above method, and their CDR and variable region sequences are shown in Table 2.

Table 2 Mouse antibody variable region and CDR sequence information

2. Humanization of mouse antibodies:

Use CDR transplantation to design humanized antibodies. BLAST the sequence closest to the mouse sequence in the human antibody as the variable region skeleton, replace the mouse framework region, and transplant the CDRs of the parent antibody into the human recipient to obtain each parent antibody. of humanized light chain and humanized heavy chain. The humanized light and heavy chain variable regions of each parent antibody were paired with each other for affinity ranking experiments. The heavy chain variable region and the light chain variable region are then connected to the human IgG4 heavy chain constant region (SEQ ID NO:44) and the human kappa light chain constant region (SEQ ID NO:45) respectively to obtain the humanized antibody heavy chain. and full-length light chain sequence.

The DNA sequences encoding the humanized antibody heavy chain and light chain were synthesized and inserted into the pcDNA3.4 vector to construct a full-length expression plasmid. Humanized antibodies were expressed in Expi293F cell cultures, and the supernatant was purified using a protein A affinity column. Buffer exchange the purified antibody into PBS using a PD-10 desalting column. The concentration and purity of the purified protein were determined by OD280 and SDS-PAGE, respectively.

Humanized antibodies of mouse monoclonal antibodies mAb003, mAb006 and mAb010 were obtained by the above method. The amino acid sequences of the light and heavy chain variable regions of each humanized antibody are shown in SEQ ID NO: 31-43.

Example 2: Murine Antibody Cell Binding Experiment

Experimental steps:

The cells used were CHOK1 stably transduced cell lines expressing human CB1 (P21554, Uniprot)/mouse CB1 (P47746, Uniprot) constructed through lentivirus based on CHOK1 cells, hereinafter referred to as CHOK1-hCB1, CHOK1-mCB1.

1 Digest cells with TrypLE (gibco; 12604-021).

2. Stop the digestion with complete culture medium and count with a cell counter (Countstar; IC1000).

3. Centrifuge at 1000rpm for 5 minutes and discard the supernatant to collect the cell pellet.

4. Use cold FACS buffer (500ml DMEM (CORNING; 10-013-CVRC) + 50ml FBS (BI; 04-002-1A)) to resuspend the cells and adjust the cell density to 5 × 10 ⁶ cells per ml FACS buffer: 500ml.

5. Let the cell suspension stand at room temperature for 10 to 20 minutes.

6. Centrifuge the cell plate at 1000 rpm for 5 minutes, then discard the supernatant.

7. Dilute the antibody to be tested with FACS buffer, add 100 μl to each well, and incubate at 4°C for 1 hour.

8. Wash the cells three times with FACS buffer, add 250 μl to each well, and centrifuge at 300 g (4 degrees Celsius) for 5 minutes.

9. Prepare the secondary antibody anti-mouse IgG-Alexa488 antibody (invitrogen; A21202) (dilution of 1:1k) in FACS buffer.

10. Resuspend the cell pellet in secondary antibody diluent, add 100 μl to each well, and incubate in the dark at 4 degrees for 1 hour.

11. Wash the cells three times with FACS buffer, add 250 μl to each well, and centrifuge at 300 g (4 degrees Celsius) for 5 minutes.

12. Resuspend the cell pellet in 4°C FACS buffer, add 100 μl to each well, and place the cells in a dark place for FACS (BECKMAN COULTER; B53013) analysis.

Data processing (data processing methods) and experimental data:

Use graphpad to perform nonlinear fitting of the binding signal value corresponding to each concentration of the antibody. Calculate the EC50 based on the maximum binding signal of the antibody and curve fitting, as shown in Table 3-4.

Table 3 max MFI value and EC50 of each group

Table 4 max MFI value and EC50 of each group

Among them, mouse IgG1 (mIgG1) is a negative control; Tab06 is a positive control antibody, whose sequence is derived from the antibody with clone number 10D10 in Amgen's CB1 antibody patent (WO2014210205A1). Specifically, SEQ ID NO:4 in Amgen's patent WO2014210205A1 is 10D10 The heavy chain sequence, SEQ ID NO:5 is the light chain sequence of 10D10. In addition, since 10D10 in the patent is a humanized antibody, in order to serve as a control for mouse anti-screening, this application replaced its hIgG constant region with the mIgG1 constant region to prepare the control antibody Tab06.

Experimental results:

On CHOK1-hCB1 cells overexpressing human CB1, mAb006 and mAb010 have higher maximum binding signals than the positive control Tab06, and the EC50 of mAb006 and mAb010 are both smaller than the positive control; on cells overexpressing mouse CB1 CHOK1-mCB1 , according to the max MFI value, mAb003, mAb006 and mAb010 all show certain binding, while the positive control antibody Tab06 has no binding.

In addition, mAb016 and mAb030 also showed good binding activity to CHOK1-hCB1, and the binding EC50 of mAb016 to CHOK1-mCB1 was 1.51 μg/ml, showing good human-mouse cross-binding activity.

Example 3: Murine-derived antibody cell cAMP experiment

Experimental steps:

1. Use Trypsin-EDTA (Invitrogen, 25200072) to digest 293-hCB1 cells (the cells are stable transducers expressing human CB1 constructed based on 293 cells using liposome transfection) (37 degrees Celsius for 3 minutes).

2. Stop digestion with complete culture medium and count using a cell counter (Invitrogen, Countess II).

3. Centrifuge at 1200rpm for 3 minutes and discard the supernatant to collect the cell pellet.

4. Use assay buffer 1×DPBS (Hyclone, SH30028.02) + 500 μM IBMX (Sigma, I5879) + 0.1% BSA (Sigma, A7030)) to resuspend the cells and adjust the cell density to 5×10 ⁵ cells per ml.

5. Add 1μl of the antibody to be tested (10×, final concentration 200nM) in the 384-well plate (784075Greiner).

6. Use MultiDrop Combi (set to low speed) (Thermo) to add 4 μl of cell suspension to each well so that the number of cells in each well is 2000.

7. Incubate the cell plate in a 37°C incubator for 10 minutes.

8. Use MultiDrop Combi (set to high speed) to add assay buffer containing 15μM Forskolin (Sigma, F6886) and 0.5nM CP55940 (Sigma, C1112) to each well.

9. Incubate the plate in a 37°C incubator for 30 minutes.

10. Use cAMP kit (62AM4PEC, Cisbio) to detect cAMP content: add 5 μl cAMP-D2 antibody solution to each well, and then add 5 μl Eu-Anti-cAMP solution.

11. Seal the plate, incubate at room temperature in the dark for 60 minutes, and use Envision (PerkinElmer) to read the plate. The signals at 665nM and 615nM wavelengths, the signal ratio at wavelength 665nM/615nM is inversely proportional to the relative content of cAMP, indicating the relative activity of CB1.

Data processing (data processing methods) and experimental data:

The ratio of the signals at wavelengths 665nM and 615nM is calculated. The ratio is inversely proportional to the relative content of cAMP, indicating the relative activity of CB1. Make a nonlinear regression curve between the antibody concentration and the corresponding 665nM/615nM ratio to calculate IC50.

The results are shown in Figure 1-5.

Experimental results:

mAb003, mAb006, mAb010, mAb016 and mAb030 all showed significant inhibition of CB1 activity.

Example 4: Humanized antibody cell binding experiment

Experimental steps:

1 Use TrypLE (gibco; 12604-021) to digest CHOK1-hCB1 and CHOK1-mCB1 cells (the cells are stably transduced strains expressing human CB1/mouse CB1 constructed based on CHOK1 cells using lentivirus).

4. Use cold FACS buffer (500ml DMEM (CORNING; 10-013-CVRC) + 50ml FBS (BI; 04-002-1A) to resuspend the cells and adjust the cell density to 5 × 10 ⁶ cells per ml FACS buffer: 500ml.

5. Let the cell suspension stand at room temperature for 10 to 20 minutes.

7. Dilute the antibody to be tested with FACS buffer, add 100 μl to each well, and incubate at 4 degrees Celsius for 1 hour.

9. Prepare the secondary antibody anti-human IgG-Alexa488 antibody (invitrogen; A-11013) (dilution of 1:1k) in FACS buffer.

Data processing (data processing methods) and experimental data:

Use Graphpad to perform nonlinear fitting of the binding signal value corresponding to each concentration of the antibody. Calculate the EC50 based on the maximum binding signal of the antibody and curve fitting, as shown in Table 5-7.

Table 5 Maximum binding signal and EC50 of humanized antibody of mAb003

Table 6 Maximum binding signal and EC50 of humanized antibody of mAb010

Table 7 Maximum binding signal and EC50 of humanized antibody of mAb006

Among them, human IgG4 (hIgG4) is a negative control; Tab04-hIgG4 (S228P) is a positive control, and its sequence is derived from the CB1 antibody named GFB-024 by Takeda Company, which corresponds to the antibody named M7-H5 in Takeda patent WO2019211665A1. The heavy chain is SEQ ID NO: 161 in WO2019211665A1; the light chain is SEQ ID NO: 167 in WO2019211665A1.

Experimental results:

Different humanized antibodies from 3 antibodies all maintained binding signals and activities to CHOK1-hCB1 cells expressing human CB1. Except for cAb06-VH3+VL1, which had equivalent activity to Tab04-hIgG4 (S228P), the others were stronger than Positive control Tab04-hIgG4(S228P); In addition, each humanized antibody showed cross-activity against CHOK1-hCB1 cells expressing human CB1 and CHOK1-mCB1 cells expressing mouse CB1, while the positive control Tab04-hIgG4(S228P) CHOK1-mCB1 cells expressing mouse CB1 had no binding activity.

Example 5: Humanized antibody cell cAMP experiment

Experimental steps:

1. Use Trypsin-EDTA (Invitrogen, 25200072) to digest 293-hCB1 cells (the cells are 293 cell stably transduced strains expressing human CB1 constructed based on 293 cells using liposome transfection) (37 degrees Celsius for 3 minutes).

7. Incubate the cell plate in a 37°C incubator for 10 minutes.

9. Incubate the plate in a 37°C incubator for 30 minutes.

11. Seal the plate and incubate in the dark at room temperature for 60 minutes. Use Envision (PerkinElmer) to read the plate and detect the signals at 665nM and 615nM wavelengths. The signal ratio of 665nM/615nM wavelength is inversely proportional to the relative content of cAMP, indicating the relative activity of CB1. .

Data processing (data processing methods) and experimental data:

The ratio of the signals at wavelengths 665nM and 615nM is calculated. The ratio is inversely proportional to the relative content of cAMP, indicating the relative activity of CB1. Make a nonlinear regression curve between the antibody concentration and the corresponding 665nM/615nM ratio to calculate the IC50. The results are shown in Table 8.

Table 8 IC50 of humanized antibodies to mAb010

Experimental results:

Based on the cell cAMP activity data, the inhibitory effects of cAb10-VH4+VL3 and cAb10-VH4+VL4 are stronger than the control Positive antibody Tab04-hlgG4(S228P).

Example 6: mAb010 improves carbon tetrachloride-induced liver fibrosis in mice

Animals: 30 female BALB/c mice, SPF grade, 8 weeks old, weighing about 20 g, were purchased from Beijing Vitong Lever Experimental Animal Technology Co., Ltd. All animals maintained a 12-hour alternating circadian rhythm and had free access to food and drink.

Instruments: automatic biochemical analyzer, animal balance, tissue dehydration and embedding machine, Leica automatic microtome, automatic staining machine.

Reagents: mouse antibody mAb010; carbon tetrachloride (purchased from Sinopharm Chemical Reagent Co., Ltd.), olive oil (purchased from Acros Organics).

experiment procedure:

1. Animal grouping and modeling

After one week of adaptive feeding, the mice were randomly divided into 5 groups according to their body weight: control group (Oil), model group (CCl ₄ ), and drug administration group (mAb010). The mice were fed a normal diet and water, and the model was established for 8 weeks. The model group and the administration group were injected with 25% CCl ₄ oil solution twice a week at a dose of 4 mL/kg, and the control group was injected with the same volume of oil solvent.

2. Administration

Administration was started at the same time as modeling; the administration group was intravenously injected with mAb010 (60 mg/kg) once a week; the control group and model group were intragastrically administered the same volume of control solvent every day. After 8 weeks of administration, the mice were allowed to eat and drink normally. Mice in each group were weighed every day, and their weight, hair, feces and activity were carefully observed and recorded.

3. Get materials

Forty-eight hours after the 16th injection of CCl ₄ , the specimens were dissected and harvested. Blood was collected from the eye sockets, and the liver was collected at euthanasia. The left lobe, middle right lobe and caudal lobe tissue of the liver were fixed in 10% formalin for at least 48 hours and used for HE and Sirius red staining sections. Part of the remaining liver tissue was quickly frozen in liquid nitrogen for subsequent testing of other indicators.

4. Analysis of liver tissue Sirius red stained sections

Liver fibrosis positive area analysis uses BIOTOPIXTM analysis system (visopharm), fibrosis area (%) = fibrosis area/liver area*100%

5.Experimental results

Through pathological research methods, the anti-liver fibrosis effect of mAb010 was observed. Sirius red staining results showed that the percentage of fibrosis area in the administration group was reduced by 44.57% compared with the model group. mAb010 could significantly reduce the deposition of collagen in the mouse liver (Figure 6).

In summary, mAb010 has a protective effect on liver fibrosis mouse models, suggesting that mAb010 has a therapeutic effect on liver fibrosis-related diseases and can be used to prepare preventive and therapeutic drugs against liver fibrosis-related diseases and liver cirrhosis.

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

An antibody or an antigen-binding fragment thereof capable of specifically binding CB1, the antibody or an antigen-binding fragment thereof comprising:

(a) VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 22 or 40; and/or, VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in any one of SEQ ID NO: 27 and 41-43;

(b) VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO: 2 and 31-32 ; and/or, VL CDR1 or its variant, VL CDR2 or its variant and VL CDR3 or its variant contained in the light chain variable region (VL) as shown in any one of SEQ ID NO:7, 33-35 Variants;

(c) VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO: 12, 36-37 ; and/or, VL CDR1 or its variant, VL CDR2 or its variant and VL CDR3 or its variant contained in the light chain variable region (VL) as shown in any one of SEQ ID NO: 17, 38-39 Variants;

(d) VH CDR1 or a variant thereof, a VH CDR2 or a variant thereof, and a VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as set forth in SEQ ID NO: 46; and/or, as in SEQ VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in ID NO:50; or,

(e) VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as set forth in SEQ ID NO: 54; and/or, as in SEQ VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) shown in ID NO:58;

Wherein, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2 or 3 amino acid substitutions, deletions or additions, such as conservative substitutions); preferably Land, the replacement is a conservative replacement;

Preferably, the antibody or antigen-binding fragment thereof comprises:

(a) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 22 or 40; and/or, as shown in any of SEQ ID NO: 27, 41-43, light 3 CDRs contained in the chain variable region (VL);

(b) 3 CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO: 2 and 31-32; and/or, as shown in any one of SEQ ID NO: 7 and 33-35 3 CDRs contained in the light chain variable region (VL) shown in the item;

(c) 3 CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO: 12, 36-37; and/or, as shown in any one of SEQ ID NO: 17, 38-39 3 CDRs contained in the light chain variable region (VL) shown in the item;

(d) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46; and/or, as contained in the light chain variable region (VL) as shown in SEQ ID NO:50 3 CDRs; or,

(e) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:54; and/or, as contained in the light chain variable region (VL) as shown in SEQ ID NO:58 3 CDRs;

Preferably, the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.
The antibody or antigen-binding fragment thereof according to claim 1, comprising:

(a) A heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:23, VH CDR2 with the sequence SEQ ID NO:24, and VH CDR2 with the sequence SEQ ID NO:24 VH CDR3 of NO:25; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO:28, VL CDR1 of SEQ ID NO:29 VL CDR2, VL CDR3 with sequence SEQ ID NO:30;

(b) A heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:3, VH CDR2 with the sequence SEQ ID NO:4, and VH CDR2 with the sequence SEQ ID NO:4 VH CDR3 of NO:5; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO:8, VL CDR1 of SEQ ID NO:9 VL CDR2, VL CDR3 with sequence SEQ ID NO:10;

(c) A heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:13, VH CDR2 with the sequence SEQ ID NO:14, and VH CDR2 with the sequence SEQ ID NO:14 VH CDR3 of NO:15; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 whose sequence is SEQ ID NO:18, sequence SEQ ID NO:19 VL CDR2, VL CDR3 with sequence SEQ ID NO:20;

(d) A heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:47, VH CDR2 with the sequence SEQ ID NO:48, and VH CDR2 with the sequence SEQ ID NO:48 VH CDR3 of NO:49; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 whose sequence is SEQ ID NO:51, and whose sequence is SEQ ID NO:52 VL CDR2, VL CDR3 with sequence SEQ ID NO:53; or,

(e) A heavy chain variable region (VH) containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:55, VH CDR2 with the sequence SEQ ID NO:56, and VH CDR2 with the sequence SEQ ID NO:56 VH CDR3 of NO:57; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 whose sequence is SEQ ID NO:59, and whose sequence is SEQ ID NO:60 VL CDR2, VL CDR3 with sequence SEQ ID NO:61;

Wherein, the CDR is defined by the Kabat numbering system.
The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

(a) Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 22 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 27 The sequence or its variant;

(b) Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 2 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 7 The sequence or its variant;

(c) Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 12 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 17 The sequence or its variant;

(d) Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 46 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 50 sequence or a variant thereof; or,

(e) Heavy chain variable region (VH), which includes the sequence shown in SEQ ID NO: 54 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 58 The sequence or its variant;

Wherein, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity; preferably, the substitution is a conservative substitution;

Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 22 and a VL comprising the sequence shown in SEQ ID NO: 27;

Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:2 and a VL comprising the sequence shown in SEQ ID NO:7;

Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 12 and a VL comprising the sequence shown in SEQ ID NO: 17;

Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 46 and a VL comprising the sequence shown in SEQ ID NO: 50;

Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 54 and a VL comprising the sequence shown in SEQ ID NO: 58.
The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from human immunoglobulin;

Preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a human heavy chain germline sequence, and/or a light chain framework region of a human light chain germline sequence.
The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:40 or a variant thereof; and/or, comprising a sequence shown in any one of SEQ ID NO:41-43 or a variant thereof; Variant light chain variable region (VL);

(b) A heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 31 or 32 or a variant thereof; and/or, comprising the sequence set forth in any one of SEQ ID NO: 33-35 or the light chain variable region (VL) of a variant thereof;

or,

(c) A heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 36 or 37 or a variant thereof; and/or, a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 38 or 39 or a variant thereof; The light chain variable region (VL) of the body;

Wherein, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity;

Preferably, the substitution is a conservative substitution;

Preferably, the antibody or antigen-binding fragment thereof comprises:

(1) VH containing the sequence shown in SEQ ID NO:40 and VL containing the sequence shown in SEQ ID NO:41;

(2) VH containing the sequence shown in SEQ ID NO:40 and VL containing the sequence shown in SEQ ID NO:42;

(3) VH containing the sequence shown in SEQ ID NO:40 and VL containing the sequence shown in SEQ ID NO:43;

(4) VH containing the sequence shown in SEQ ID NO:31 and VL containing the sequence shown in SEQ ID NO:34;

(5) VH containing the sequence shown in SEQ ID NO:31 and VL containing the sequence shown in SEQ ID NO:35;

(6) VH containing the sequence shown in SEQ ID NO:32 and VL containing the sequence shown in SEQ ID NO:33;

(7) VH containing the sequence shown in SEQ ID NO:32 and VL containing the sequence shown in SEQ ID NO:34;

(8) VH containing the sequence shown in SEQ ID NO:32 and VL containing the sequence shown in SEQ ID NO:35;

(9) VH containing the sequence shown in SEQ ID NO:36 and VL containing the sequence shown in SEQ ID NO:38;

(10) VH comprising the sequence shown in SEQ ID NO:37 and VH comprising the sequence shown in SEQ ID NO:38 VL; or,

(11) VH comprising the sequence shown in SEQ ID NO:37 and VL comprising the sequence shown in SEQ ID NO:39.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (such as human or mouse) immunoglobulin;

Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3 or IgG4), and/or the antibody or antigen-binding fragment thereof The light chain includes a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda);

Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO:44, and/or the light chain of the antibody or antigen-binding fragment thereof comprises as SEQ ID NO: The light chain constant region shown in 45;

Preferably, the antibody or antigen-binding fragment thereof comprises:

(1) A heavy chain comprising VH of the sequence shown in SEQ ID NO:40 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:41 and SEQ ID NO: The light chain of CL shown in 45;

(2) A heavy chain comprising VH of the sequence shown in SEQ ID NO:40 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:42 and SEQ ID NO: The light chain of CL shown in 45;

(3) A heavy chain comprising VH of the sequence shown in SEQ ID NO:40 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:43 and SEQ ID NO: The light chain of CL shown in 45;

(4) A heavy chain comprising VH of the sequence shown in SEQ ID NO:31 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:34 and SEQ ID NO: The light chain of CL shown in 45;

(5) A heavy chain comprising VH of the sequence shown in SEQ ID NO:31 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:35 and SEQ ID NO: The light chain of CL shown in 45;

(6) A heavy chain comprising VH of the sequence shown in SEQ ID NO:32 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:33 and SEQ ID NO: The light chain of CL shown in 45;

(7) A heavy chain comprising VH of the sequence shown in SEQ ID NO:32 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:34 and SEQ ID NO: The light chain of CL shown in 45;

(8) A heavy chain comprising VH of the sequence shown in SEQ ID NO:32 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:35 and SEQ ID NO: The light chain of CL shown in 45;

(9) A heavy chain comprising VH of the sequence shown in SEQ ID NO:36 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:38 and SEQ ID NO: The light chain of CL shown in 45;

(10) A heavy chain comprising VH of the sequence shown in SEQ ID NO:37 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:38 and SEQ ID NO: The light chain of CL shown in 45; or,

(11) A heavy chain comprising VH of the sequence shown in SEQ ID NO:37 and CH shown in SEQ ID NO:44, and, VL comprising the sequence shown in SEQ ID NO:39 and SEQ ID NO: The light chain of CL shown in 45;

Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a murine immunoglobulin (eg, IgG1, IgG2, IgG3 or IgG4), and/or the antibody or antigen-binding fragment thereof The light chain includes a light chain constant region derived from a murine immunoglobulin (eg, kappa or lambda).
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fd, Fv, disulfide-linked Fv, scFv , di-scFv, (scFv) 2 , diabody and single domain antibody (sdAb); and/or, the antibody is a mouse antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a polypeptide specific antibodies.
Isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-7, or its heavy chain variable region and/or light chain variable region, or its heavy chain and/or light chain .
A vector comprising the isolated nucleic acid molecule of claim 8; preferably, the vector is a cloning vector or an expression vector.
A host cell comprising the isolated nucleic acid molecule of claim 8 or the vector of claim 9.
A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising culturing the host cell according to claim 10 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the isolated nucleic acid molecule according to claim 8, the vector according to claim 9 or the host according to claim 10 cells, and pharmaceutically acceptable carriers and/or excipients;

Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the isolated nucleic acid molecule according to claim 8, the vector according to claim 9, the host cell according to claim 10 or the host cell according to claim 12. The use of the above-mentioned pharmaceutical composition for preparing a medicament for preventing and/or treating CB1-related diseases in a subject;

Preferably, the CB1-related diseases will benefit from antagonism of CB1 signaling;

Preferably, the diseases related to CB1 are: obesity-related diseases (such as Prader-Willi syndrome), diabetes (such as type 1 diabetes, type 2 diabetes), kidney disease (such as diabetic nephropathy), liver disease Fibrosis, pulmonary fibrosis, dyslipidemia (eg, hyperlipidemia, hypercholesterolemia), fatty liver disease (eg, nonalcoholic fatty liver disease), or combinations thereof;

Preferably, the subject is a mammal, such as a human or a mouse;

Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent.
A method for preventing and/or treating CB1-related diseases in a subject, comprising: administering an effective amount of any one of claims 1-7 to a subject in need thereof. Antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10 or the pharmaceutical composition of claim 12;

Preferably, the CB1-related diseases will benefit from antagonism of CB1 signaling;

Preferably, the diseases related to CB1 are: obesity-related diseases (such as Prader-Willi syndrome), diabetes (such as type 1 diabetes, type 2 diabetes), kidney disease (such as diabetic nephropathy), liver disease Fibrosis, pulmonary fibrosis, dyslipidemia (eg, hyperlipidemia, hypercholesterolemia), fatty liver disease (eg, nonalcoholic fatty liver disease), or combinations thereof;

Preferably, the subject is a mammal, such as a human or a mouse;

Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent.
A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7, and a detectable label connected to the antibody or antigen-binding fragment thereof;

Preferably, the detectable label is selected from enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives) ), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7 or the conjugate according to claim 15.
The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the conjugate according to claim 15 in preparing a detection reagent for detecting the presence of CB1 in a sample or its levels and/or diagnosis of CB1-related diseases;

Preferably, the CB1-related diseases are: obesity-related diseases (such as Prader-Willi syndrome), sugar Diabetes (eg, type 1 diabetes, type 2 diabetes), kidney disease (eg, diabetic nephropathy), liver fibrosis, pulmonary fibrosis, dyslipidemia (eg, hyperlipidemia, hypercholesterolemia), fatty liver (eg, , non-alcoholic fatty liver disease) or combinations thereof;

Preferably, the sample is a tissue sample (eg central nervous tissue, lung tissue, liver tissue, adipose tissue or kidney tissue) from a subject (eg mammal, preferably human or mouse).
A method for detecting the presence or level of CB1 in a sample, comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1-7 or the conjugate according to claim 15;

Preferably, the method is an immunological detection, such as Western blotting, enzyme immunoassay (such as ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay;

Preferably, the method comprises using the conjugate of claim 15;

Preferably, the method includes using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and the method further includes using an antibody carrying a detectable label (such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin) a second antibody to detect said antibody or antigen-binding fragment thereof;

Preferably, the method includes: (1) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1-7 or the conjugate according to claim 15; (2) detecting the antigen. - The formation of antibody immune complexes or the detection of the amount of said immune complexes, the formation of which indicates the presence of CB1 or cells expressing CB1.
A method for diagnosing a disease associated with CB1, comprising detecting the presence or level of CB1 in a sample from a subject using the method of claim 18;

Preferably, the subject suffers from a disease associated with CB1 when CB1 is present or the level of CB1 is increased compared to a reference level (eg compared to healthy controls).