Antibodies that bind to cannabinoid receptor CB1 and uses thereof

Abstract

An antibody specifically binding to CB1 or an antigen-binding fragment thereof, a pharmaceutical composition containing 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 same, and therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.

Description

Antibodies that bind to cannabinoid receptor CB1 and their uses Technical Field

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to CB1, pharmaceutical compositions containing such antibodies or antigen-binding fragments, nucleic acid molecules encoding such antibodies or antigen-binding fragments, host cells containing such antibodies or antigen-binding fragments, and related uses. Furthermore, the invention relates to therapeutic and diagnostic uses of such antibodies or antigen-binding fragments. Background Art

[0002] Cannabinoid receptors CB1 and CB2 are key components of the endocannabinoid system and the primary targets of D9-tetrahydrocannabinol (D9-THC), a psychoactive chemical extracted from cannabis with widespread therapeutic use (Lemberger, 1980). Cannabinoid receptor 1 (CB1) is activated by the lipids of 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 (Guzman, 2003). The two cannabinoid receptors, CB1 and CB2, share 44% overall sequence homology and 68% sequence similarity in their transmembrane regions (Munro et al., 1993). However, they have distinct tissue distributions and play distinct roles in the endocannabinoid system, with CB1 and CB2 being primarily expressed in the central nervous system and immune system, respectively. Due to their high sequence similarity, the development of therapeutic applications has been hampered by the difficulty of specifically modulating individual cannabinoid receptors. CB1 receptors are expressed in the central nervous system, lungs, liver, adipose tissue, and kidneys. Studies have shown that CB1 receptors are involved in the development and progression of many diseases, such as obesity, diabetic nephropathy, liver fibrosis, and pulmonary fibrosis. The expression of CB1 receptors and their ligands is significantly upregulated under pathological conditions.

[0003] Rimonabant, developed by Sanofi-Synthelabo of France, is the world's first cannabinoid receptor type 1 (CB1) inhibitor weight loss drug. It boasts excellent weight loss effects, insulin sensitization, improved lipid metabolism, and some smoking cessation support. It was approved for marketing in the European Union in 2006 as a new weight loss drug. However, clinical studies and post-marketing findings revealed that rimonabant can increase serious psychiatric adverse reactions, including depression and anxiety, and also increase the risk of suicide in users. Consequently, rimonabant was withdrawn from the European market.

[0004] A 2017 article published in J Clin Invest demonstrated that adipocyte CB1 deficiency prevents obesity and metabolic disorders by peripherally knocking out the CB1 receptor, indicating that peripheral CB1 blockade has reasonable biological significance in reducing mouse body weight and liver fat. Compared with small molecules that can cross the blood-brain barrier (BBB), the development of peripherally restricted large molecule drugs that retain efficacy and reduce central nervous system side effects has the potential to become best-in-class drugs. Currently, international companies are developing CB1 receptor antibodies. For example, Takeda's antibody GFB-024 (see patent: WO2019211665) is in Phase 1 clinical trials. However, existing antibodies targeting the CB1 receptor do not have human-mouse cross-reactivity. Therefore, there is still a need to develop new and more effective antibodies targeting the CB1 receptor to provide more clinical application options.

[0005] Summary of the Invention

[0006] Through extensive research, the inventors of this application have obtained murine antibodies that specifically bind to human CB1 and exhibit significant CB1 activity inhibition. In certain embodiments, the antibodies provided herein exhibit cross-binding activity against both human and murine CB1. Furthermore, based on these murine antibodies, the inventors of this application have further developed humanized antibodies that exhibit both CB1 binding activity and CB1 activity inhibition.

[0007] Based on this, the present application also provides a pharmaceutical composition containing the antibody or its antigen-binding fragment, a nucleic acid molecule encoding the antibody or its antigen-binding fragment and a host cell containing the same, as well as related uses.

[0008] Antibodies or antigen-binding fragments thereof

[0009] mAb010 and its humanized antibodies

[0010] Therefore, in a first aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to CB1, wherein the antibody or the antigen-binding fragment thereof comprises:

[0011] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 22 or 40; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 27, 41-43;

[0012] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0014] The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 22 or 40; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NO: 27, 41-43.

[0015] 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 systems. 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.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0017] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 23, VH CDR2 of SEQ ID NO: 24, and VH CDR3 of SEQ ID NO: 25; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 28, VL CDR2 of SEQ ID NO: 29, and VL CDR3 of SEQ ID NO: 30;

[0018] Wherein, the CDRs are defined by the Kabat numbering system.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0020] 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.

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0022] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 27 or a variant thereof;

[0023] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0024] In certain embodiments, the substitutions are conservative substitutions.

[0025] In certain embodiments, 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.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an 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 sequence of a human light chain germline sequence.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 40 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in any one of SEQ ID NOs: 41-43 or a variant thereof;

[0029] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0030] In certain embodiments, the substitutions are conservative substitutions.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:40 and a VL comprising the sequence shown in SEQ ID NO:41.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:40 and a VL comprising the sequence shown in SEQ ID NO:42.

[0033] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:40 and a VL comprising the sequence shown in SEQ ID NO:43.

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof has cross-binding activity against human CB1 and murine CB1.

[0035] mAb003 and its humanized antibodies

[0036] Therefore, in a second aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to CB1, wherein the antibody or the antigen-binding fragment thereof comprises:

[0037] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 2 and 31-32; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 7 and 33-35;

[0038] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0040] The three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 2 and 31-32; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 7 and 33-35.

[0041] In certain embodiments, the 3 CDRs contained in the VH and / or the 3 CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering systems. In certain embodiments, the 3 CDRs contained in the VH and / or the 3 CDRs contained in the VL are defined by the Kabat numbering system.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0043] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 3, VH CDR2 of SEQ ID NO: 4, and VH CDR3 of SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 8, VL CDR2 of SEQ ID NO: 9, and VL CDR3 of SEQ ID NO: 10;

[0044] Wherein, the CDRs are defined by the Kabat numbering system.

[0045] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0046] 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.

[0047] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0048] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 7 or a variant thereof;

[0049] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0050] In certain embodiments, the substitutions are conservative substitutions.

[0051] In certain embodiments, 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.

[0052] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0053] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an 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 sequence of a human light chain germline sequence.

[0054] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 31 or 32 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in any one of SEQ ID NO: 33-35 or a variant thereof;

[0055] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0056] In certain embodiments, the substitutions are conservative substitutions.

[0057] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:31 and a VL comprising the sequence shown in SEQ ID NO:34.

[0058] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:31 and a VL comprising the sequence shown in SEQ ID NO:35.

[0059] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:32 and a VL comprising the sequence shown in SEQ ID NO:33.

[0060] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:32 and a VL comprising the sequence shown in SEQ ID NO:34.

[0061] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:32 and a VL comprising the sequence shown in SEQ ID NO:35.

[0062] In certain embodiments, the antibody or antigen-binding fragment thereof has cross-binding activity against human CB1 and murine CB1.

[0063] mAb006 and its humanized antibodies

[0064] Therefore, in a third aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to CB1, wherein the antibody or the antigen-binding fragment thereof comprises:

[0065] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 12, 36-37; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 17, 38-39;

[0066] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0067] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0068] The three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 12, 36-37; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 17, 38-39.

[0069] 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 systems. 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.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0071] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 13, VH CDR2 of SEQ ID NO: 14, and VH CDR3 of SEQ ID NO: 15; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 18, VL CDR2 of SEQ ID NO: 19, and VL CDR3 of SEQ ID NO: 20;

[0072] Wherein, the CDRs are defined by the Kabat numbering system.

[0073] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0074] 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.

[0075] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0076] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 17 or a variant thereof;

[0077] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0078] In certain embodiments, the substitutions are conservative substitutions.

[0079] In certain embodiments, 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.

[0080] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0081] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an 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 sequence of a human light chain germline sequence.

[0082] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 36 or 37 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 38 or 39 or a variant thereof;

[0083] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0084] In certain embodiments, the substitutions are conservative substitutions.

[0085] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:36 and a VL comprising the sequence shown in SEQ ID NO:38.

[0086] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:37 and a VL comprising the sequence shown in SEQ ID NO:38.

[0087] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:37 and a VL comprising the sequence shown in SEQ ID NO:39.

[0088] In certain embodiments, the antibody or antigen-binding fragment thereof has cross-binding activity against human CB1 and murine CB1.

[0089] mAb016 and its humanized antibodies

[0090] Therefore, in a fourth aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to CB1, wherein the antibody or the antigen-binding fragment thereof comprises:

[0091] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as set forth in SEQ ID NO: 46; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as set forth in SEQ ID NO: 50;

[0092] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0093] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0094] The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 46; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 50.

[0095] 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 systems. 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.

[0096] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0097] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 47, VH CDR2 of SEQ ID NO: 48, and VH CDR3 of SEQ ID NO: 49; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 51, VL CDR2 of SEQ ID NO: 52, and VL CDR3 of SEQ ID NO: 53;

[0098] Wherein, the CDRs are defined by the Kabat numbering system.

[0099] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0100] 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.

[0101] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0102] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 46 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 50 or a variant thereof;

[0103] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0104] In certain embodiments, the substitutions are conservative substitutions.

[0105] In certain embodiments, 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.

[0106] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0107] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an 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 sequence of a human light chain germline sequence.

[0108] In certain embodiments, the antibody or antigen-binding fragment thereof has cross-binding activity against human CB1 and murine CB1.

[0109] mAb030 and its humanized antibodies

[0110] Therefore, in a fifth aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to CB1, wherein the antibody or the antigen-binding fragment thereof comprises:

[0111] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as set forth in SEQ ID NO: 54; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as set forth in SEQ ID NO: 58;

[0112] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0113] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0114] The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 54; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 58.

[0115] 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 systems. 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.

[0116] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0117] a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 55, VH CDR2 of SEQ ID NO: 56, and VH CDR3 of SEQ ID NO: 57; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 59, VL CDR2 of SEQ ID NO: 60, and VL CDR3 of SEQ ID NO: 61;

[0118] Wherein, the CDRs are defined by the Kabat numbering system.

[0119] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0120] 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.

[0121] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0122] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 54 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 58 or a variant thereof;

[0123] wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity.

[0124] In certain embodiments, the substitutions are conservative substitutions.

[0125] In certain embodiments, 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.

[0126] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0127] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region comprised in an 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 sequence of a human light chain germline sequence.

[0128] In certain embodiments, in the first, second, third, fourth or fifth aspects, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (eg, human or murine) immunoglobulin.

[0129] 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 light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0130] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin 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 5 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; and / or,

[0131] The light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. The substitutions can be conservative or non-conservative.

[0132] 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 compared to the wild-type sequence from which it is derived.

[0133] In other embodiments, the variant 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, the number of cysteine ​​residues, effector cell function, or complement function, etc. Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue or chemically modifying it, for example, by altering the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering effector function (e.g., reducing or enhancing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0134] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise variants of the human immunoglobulin heavy chain constant region (CH), which variants may have reduced or abolished effector function compared to the wild-type sequence from which they are derived.

[0135] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise variants of the human immunoglobulin heavy chain constant region (CH), which variants may have enhanced effector function compared to the wild-type sequence from which they are derived.

[0136] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a human immunoglobulin heavy chain constant region (CH) or a variant thereof, which may have the same or substantially the same effector function as the wild-type sequence from which it is derived.

[0137] In certain embodiments, the heavy chain constant region (CH) is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0138] In certain embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0139] In certain exemplary embodiments, in the first, second, third, fourth or fifth aspects, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:44, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO:45.

[0140] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises:

[0141] (1) a heavy chain comprising the VH sequence shown in SEQ ID NO: 40 and the CH sequence shown in SEQ ID NO: 44, and a light chain comprising the VL sequence shown in SEQ ID NO: 41 and the CL sequence shown in SEQ ID NO: 45;

[0142] (2) a heavy chain comprising the VH of SEQ ID NO: 40 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 42 and the CL of SEQ ID NO: 45;

[0143] (3) a heavy chain comprising the VH of SEQ ID NO: 40 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 43 and the CL of SEQ ID NO: 45;

[0144] (4) a heavy chain comprising the VH of SEQ ID NO: 31 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 34 and the CL of SEQ ID NO: 45;

[0145] (5) a heavy chain comprising the VH of SEQ ID NO: 31 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 35 and the CL of SEQ ID NO: 45;

[0146] (6) a heavy chain comprising the VH of SEQ ID NO: 32 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 33 and the CL of SEQ ID NO: 45;

[0147] (7) a heavy chain comprising the VH of SEQ ID NO: 32 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 34 and the CL of SEQ ID NO: 45;

[0148] (8) a heavy chain comprising the VH of SEQ ID NO: 32 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 35 and the CL of SEQ ID NO: 45;

[0149] (9) a heavy chain comprising the VH of SEQ ID NO: 36 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 38 and the CL of SEQ ID NO: 45;

[0150] (10) a heavy chain comprising the VH of SEQ ID NO: 37 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 38 and the CL of SEQ ID NO: 45; or

[0151] (11) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 37 and the CH of the sequence shown in SEQ ID NO: 44, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 39 and the CL of the sequence shown in SEQ ID NO: 45.

[0152] 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., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a murine immunoglobulin (e.g., κ or λ).

[0153] In certain exemplary embodiments, in the first aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 21, and / or the light chain of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 26.

[0154] In certain exemplary embodiments, in the second aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 1, and / or the light chain of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 6.

[0155] In certain exemplary embodiments, in the third aspect, the heavy chain of the antibody or antigen-binding fragment thereof comprises 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.

[0156] In certain embodiments, in the first, second, third, fourth or fifth aspects, the antigen-binding fragment is selected from 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 murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody.

[0157] In a sixth aspect, the present application also provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, or its heavy chain variable region and / or light chain variable region, or its heavy chain and / or light chain.

[0158] 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 present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated 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 comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0159] In a seventh aspect, the present application further 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.

[0160] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0161] In an eighth aspect, the present application also provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0162] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0163] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0164] 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 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 recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0165] In a ninth aspect, the present application also provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0166] Therapeutic uses

[0167] In the tenth aspect, the present application also provides a pharmaceutical composition comprising 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, the isolated nucleic acid molecule as described in the sixth aspect, the vector as described in the seventh aspect or the host cell as described in the eighth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0168] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0169] In certain exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is 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), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0170] In the eleventh aspect, the present application also provides the use of 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, the vector as described in the seventh aspect, the host cell as described in the eighth aspect or the pharmaceutical composition as described in the tenth aspect for preparing a drug for preventing and / or treating a CB1-related disease in a subject.

[0171] In certain embodiments, the CB1-related disease would benefit from antagonism of CB1 signaling.

[0172] In certain embodiments, the CB1-related disease is: 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, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease), or a combination thereof.

[0173] In certain embodiments, the subject is a mammal, such as a human or mouse.

[0174] 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, eg, administered simultaneously or sequentially.

[0175] In a twelfth aspect, the present application also provides a method for preventing and / or treating a CB1-related disease in a subject, comprising: administering to a subject in need thereof an effective amount of 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, the vector as described in the seventh aspect, the host cell as described in the eighth aspect, or the pharmaceutical composition as described in the tenth aspect.

[0176] In certain embodiments, the CB1-related disease would benefit from antagonism of CB1 signaling.

[0177] In certain embodiments, the CB1-related disease is: 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, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease), or a combination thereof.

[0178] In certain embodiments, the subject is a mammal, such as a human or mouse.

[0179] In certain embodiments, the antibody or antigen-binding fragment thereof of the first, second, third, fourth or fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the vector of the seventh aspect, the host cell of the eighth aspect or the pharmaceutical composition of the tenth aspect can be administered in combination with another pharmaceutically active agent, for example, simultaneously or sequentially.

[0180] The antibodies or antigen-binding fragments thereof or pharmaceutical compositions 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, lozenges, 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 present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof of the present invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy 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 (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0181] The antibody of the present application or its Fab or pharmaceutical composition can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In certain embodiments, antibody of the present invention or its Fab or pharmaceutical composition are given by intravenous injection or push injection.

[0182] Detection purpose

[0183] In a thirteenth aspect, the present application also provides a conjugate comprising the antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, and a detectable label linked to the antibody or antigen-binding fragment thereof.

[0184] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0185] In a fourteenth aspect, the present application further provides a kit comprising the antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, or the conjugate as described in the thirteenth aspect.

[0186] In certain embodiments, the kit comprises a detection buffer.

[0187] In certain embodiments, the kit comprises the conjugate of the thirteenth aspect.

[0188] In certain embodiments, the kit comprises the antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises 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), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin.

[0189] In a fifteenth aspect, the present application also provides a method for detecting the presence or level of CB1 in a sample, which comprises using the antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect or the conjugate as described in the thirteenth aspect.

[0190] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0191] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0192] In certain embodiments, the method comprises using the conjugate of the thirteenth aspect.

[0193] In certain embodiments, the method comprises using the antibody or antigen-binding fragment thereof of the first, second, third, fourth or fifth aspect, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin).

[0194] In certain embodiments, the method comprises: (1) contacting the sample with an antibody, or antigen-binding fragment thereof, or conjugate thereof; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of CB1 or cells expressing CB1.

[0195] The present application also provides 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 described in Aspect 15. In certain embodiments, the presence of CB1 or an increase in the level of CB1 compared to a reference level (e.g., compared to a healthy control) indicates that the subject suffers from a disease associated with CB1.

[0196] In certain embodiments, the CB1-related disease would benefit from antagonism of CB1 signaling.

[0197] In certain embodiments, the CB1-related disease is: 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, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease), or a combination thereof.

[0198] In certain embodiments, the subject is a mammal, such as a human or mouse.

[0199] In the sixteenth aspect, the present application also provides the use of the antibody or antigen-binding fragment thereof as described in the first, second, third, fourth or fifth aspect, or the conjugate as described in the thirteenth aspect, in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of CB1 in a sample and / or diagnose a disease related to CB1.

[0200] In certain embodiments, the detection reagent detects the presence or level of CB1 in a sample by the method of aspect 15.

[0201] In certain embodiments, the detection reagent detects the presence or level of CB1 in a sample by the method described in aspect 15 to diagnose a disease associated with CB1. In certain embodiments, the presence of CB1 or an increase in the level of CB1 compared to a reference level (e.g., compared to a healthy control) indicates that the subject suffers from a disease associated with CB1.

[0202] In certain embodiments, the sample is a tissue sample (eg, central nervous system tissue, lung tissue, liver tissue, adipose tissue, or kidney tissue) from a subject (eg, a mammal, preferably a human or mouse).

[0203] Definition of terms

[0204] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0205] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0206] The terms "a" and "an" and "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 both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0207] The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing 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 is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0208] 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 and light chains each contain three CDRs, designated 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), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0209] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments 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 or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.

[0210] 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.

[0211] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0212] 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 bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." 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. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. 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 that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0213] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0214] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0215] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0216] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are 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 that can be used in the present invention are described 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, 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 be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0217] As used herein, the term "diabodies" means antibodies whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create 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)).

[0218] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody. Single-domain antibodies are also called nanobodies.

[0219] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity to at least two or more (e.g., three or four) different antigens (or epitopes). Bispecific antibodies or multispecific antibodies comprise multiple antigen-binding domains that have binding specificity to 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 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, each antigen-binding domain is connected by a peptide linker.

[0220] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0221] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0222] 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 improve the homology with the sequence 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 (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). In certain embodiments, the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a mouse antibody with expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human framework sequence using methods known in the art. In some cases, the humanized framework sequence may include amino acid mutations that are replaced by corresponding non-human residues. In addition, the humanized antibody may also include residues that are not found in the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or human framework sequence to further improve or optimize the performance of the humanized antibody.

[0223] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or 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 in any case, 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 a first antibody, and the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0224] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order 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., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. 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 molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0225] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0226] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, 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, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0227] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0228] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (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 of kdis / kon is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0229] As used herein, the detectable label of the present invention can be any substance that can be detected 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, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), fluorescent proteins (e.g., phycoerythrin (PE)), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa750)), luminescent substances (e.g., chemiluminescent agents, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin. In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0230] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0231] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0232] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change 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 substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the 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 amino acids 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, 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 amino acid substitutions 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 are incorporated herein by reference).

[0233] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, 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. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0234] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, and stabilizers. For example, pH regulators include, but are not limited to, phosphate buffers. 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, sugars, 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). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are 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 dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier 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), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0235] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0236] As used herein, the term "subject" refers to a mammal, such as a human or mouse. In certain embodiments, the subject (e.g., a human or mouse) suffers from a disease associated with CB1 (e.g., obesity-related diseases (e.g., Prader-Willi syndrome), diabetes (e.g., type 1 diabetes, type 2 diabetes), kidney disease (e.g., diabetic nephropathy), liver fibrosis, pulmonary fibrosis, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease)), or is at risk of suffering from the above-mentioned disease.

[0237] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., obesity-related diseases (e.g., Prader-Willi syndrome), diabetes (e.g., type 1 diabetes, type 2 diabetes), kidney disease (e.g., diabetic nephropathy), liver fibrosis, pulmonary fibrosis, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease)) refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0238] Advantageous Effects of the Invention

[0239] The antibodies provided herein have strong binding activity against human CB1 and exhibit significant inhibitory effects on CB1 activity. Furthermore, these antibodies are naturally distributed in the periphery, effectively reducing central nervous system side effects compared to other antagonists that can cross the BBB, thus offering promising clinical application prospects. In certain embodiments, the antibodies provided herein exhibit cross-binding activity against both human and mouse CB1.

[0240] In addition, in certain embodiments, the antibodies of the present application also have inhibitory activity superior to that of GFB-024.

[0241] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0242] FIG1 shows the inhibition of CB1 activity by the murine antibody mAb003.

[0243] FIG2 shows the inhibition of CB1 activity by the murine antibody mAb006.

[0244] FIG3 shows the inhibition of CB1 activity by the murine antibody mAb010.

[0245] FIG4 shows the inhibition of CB1 activity by the murine antibody mAb016.

[0246] FIG5 shows the inhibition of CB1 activity by the murine antibody mAb030.

[0247] FIG6 shows the percentage of fibrosis area in each group of animals in Example 6; Two sample t-tests; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0248] Sequence information

[0249] A description of the sequences involved in this application is provided in the table below.

[0250] Table 1: Sequence information DETAILED DESCRIPTION

[0251] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0252] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0253] Example 1: Antibody Preparation

[0254] 1. Preparation of hybridoma antibodies:

[0255] 1.1 After immunizing mice with CB1 receptor antigen, mice with higher titers were selected for cell fusion and hybridoma preparation.

[0256] 1.2 Prepare myeloma cells. Before fusion, count and test the viability of the cells using a cell counter. Collect SP2 / 0 cells and centrifuge at 400g for 5 minutes at room temperature. Discard the culture medium and resuspend the cell pellet in 20 ml of fusion culture medium.

[0257] 1.3 Collect and culture spleen cells, select mice with strong immunogenic responses for hybridoma cell preparation, and euthanize mice with carbon dioxide according to IACUC-approved methods. Collect pre-fusion plasma, separate and collect serum (FB) as a positive control for hybridoma supernatant screening.

[0258] 1.4 Soak the mouse in 70% alcohol for disinfection, then immediately transfer it to a biosafety cabinet for operation. Collect the spleen and place it in a sterile culture dish. Place the spleen on a filter and repeatedly squeeze the spleen with the plunger of a sterile syringe until there are no obvious clumps. Then rinse the filter with fusion culture medium to obtain a spleen cell suspension and transfer it all to a 50 ml centrifuge tube.

[0259] 1.5 Centrifuge at 400g for 5 minutes and discard the supernatant. Prepare fused cells using electrofusion. Resuspend the spleen cell pellet in 5 ml of red blood cell lysis buffer, then incubate at 4°C for 5 minutes. Terminate the reaction with 50 ml of DEME medium supplemented with 10% FBS. Collect the spleen cell pellet by centrifugation and resuspend the cells in fusion medium. Invert the tube 3-5 times and count the cells. Centrifuge at 400g for 5 minutes, discard the supernatant, retain the cell pellet, and resuspend the cells in 40 ml of fusion medium. Let the spleen cells stand at room temperature for 2-3 minutes, then transfer them to a new 50 ml centrifuge tube. Add SP2 / 0 cells to the spleen cells at a ratio of 4:1 spleen cells to SP2 / 0 cells and make up to 50 ml with fusion medium. Centrifuge the cell mixture, discard the supernatant, and repeatedly tap the bottom of the tube to loosen the cell pellet. For electrofusion, place the cell mixture in an electroporation chamber and perform electroporation according to the optimized protocol.

[0260] After electrofusion, allow the fused cells to rest in the electroporation chamber for an additional 10 minutes. Resuspend the cells in DMEM supplemented with 20% FBS and HAT and plate into 96-well microplates at a volume of 100 μl per well. After 24 hours, add DMEM supplemented with 20% FBS and HAT to the wells of the plate at a volume of 100 μl per well. Transfer all fusion plates back to the CO2 incubator at 37°C, 5% CO2. Monitor cell growth and microbial contamination daily. When hybridoma clones reach a diameter of 1-2 mm (typically 10-14 days after fusion), screen the hybridoma supernatants using Acumen.

[0261] 1.7 Subcloning and expansion culture. Transfer the positive clones to a 24-well plate and culture with expansion culture medium. Collect the hybridoma culture supernatant for FACS detection. Subclone the positive mother clone to obtain a single clone by limiting dilution. Place the subclone 96-well plate in a carbon dioxide incubator, culture for 7 days, and then detect the activity of the screening supernatant. To ensure monoclonality, 1-2 rounds of subcloning are required. Based on the screening results, select the 4 most active monoclones from each subclone plate and transfer them to a 24-well plate for expansion culture.

[0262] 1.8 Cell cryopreservation. Cryopreserve the expanded subcloned cell lines in DMEM supplemented with 20% FBS and 10% DMSO and store them in a liquid nitrogen tank for long-term storage.

[0263] Murine 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.

[0264] Table 2. Mouse antibody variable region and CDR sequence information

[0265] 2. Humanization of mouse antibodies:

[0266] Humanized antibodies were designed using CDR grafting. The sequence closest to the mouse sequence in the BLAST human antibody was used as the variable region skeleton, replacing the mouse framework region. The CDRs of the parent antibody were transplanted into the human receptor to obtain the humanized light chain and humanized heavy chain of each parent antibody. 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 light chain variable region were 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 full-length sequences of the humanized antibody heavy and light chains.

[0267] DNA sequences encoding the heavy and light chains of the humanized antibody were synthesized and inserted into the pcDNA3.4 vector to construct a full-length expression plasmid. The humanized antibody was expressed in Expi293F cell culture, and the supernatant was purified using a Protein A affinity column. The purified antibody buffer was exchanged into PBS using a PD-10 desalting column. The concentration and purity of the purified protein were determined by OD280 and SDS-PAGE, respectively.

[0268] Humanized antibodies of murine 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 NOs: 31-43.

[0269] Example 2: Mouse Antibody Cell Binding Experiment

[0270] Experimental steps:

[0271] The cells used were CHOK1 stably transfected cell lines expressing human CB1 (P21554, Uniprot) / mouse CB1 (P47746, Uniprot) constructed by lentivirus based on CHOK1 cells, hereinafter referred to as CHOK1-hCB1 and CHOK1-mCB1.

[0272] 1. Digest the cells with TrypLE (gibco; 12604-021).

[0273] 2. Terminate the digestion with complete culture medium and count the cells using a cell counter (Countstar; IC1000).

[0274] 3. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant to collect the cell pellet.

[0275] 4. Resuspend the cells in cold FACS buffer (500 ml DMEM (CORNING; 10-013-CVRC) + 50 ml FBS (BI; 04-002-1A)) and adjust the cell density to 5 × 10 6 cells per ml FACS buffer: 500 ml.

[0276] 5. Allow the cell suspension to stand at room temperature for 10 to 20 minutes.

[0277] 6. Centrifuge the cell plate at 1000 rpm for 5 minutes and discard the supernatant.

[0278] 7. Dilute the antibody to be tested with FACS buffer, add 100 μl to each well, and incubate at 4°C for 1 hour.

[0279] 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.

[0280] 9. Prepare the secondary antibody anti-mouse IgG-Alexa488 antibody (Invitrogen; A21202) in FACS buffer (1:1k dilution).

[0281] 10. Resuspend the cell pellet with secondary antibody diluent, add 100 μl to each well, and incubate at 4°C in the dark for 1 hour.

[0282] 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.

[0283] 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.

[0284] Data processing (data processing methods) and experimental data:

[0285] The binding signal values ​​corresponding to each concentration of the antibody were nonlinearly fitted using GraphPad, and the EC50 was calculated based on the maximum binding signal of the antibody and curve fitting, as shown in Table 3-4.

[0286] Table 3 Max MFI values ​​and EC50 of each group

[0287] Table 4 Max MFI values ​​and EC50 of each group

[0288] Mouse IgG1 (mIgG1) served as a negative control, while Tab06 served as a positive control antibody. Its sequence was derived from the antibody clone 10D10 in Amgen's CB1 antibody patent (WO2014210205A1). Specifically, SEQ ID NO: 4 in Amgen's patent WO2014210205A1 represents the heavy chain sequence of 10D10, and SEQ ID NO: 5 represents the light chain sequence of 10D10. Furthermore, since 10D10 is a humanized antibody in the patent, its hIgG constant region was replaced with an mIgG1 constant region to serve as a control for mouse antibody screening in this application to prepare the control antibody Tab06.

[0289] Experimental conclusion:

[0290] In CHOK1-hCB1 cells overexpressing human CB1, mAb006 and mAb010 had higher maximum binding signals than the positive control Tab06, and the EC50 of mAb006 and mAb010 were both lower than that of the positive control. In CHOK1-mCB1 cells overexpressing mouse CB1, mAb003, mAb006, and mAb010 all exhibited some binding based on the max MFI values, while the positive control antibody Tab06 showed no binding.

[0291] In addition, mAb016 and mAb030 also showed good binding activity with CHOK1-hCB1, and the binding EC50 of mAb016 to CHOK1-mCB1 was 1.51 μg / ml, showing good human-mouse cross-binding activity.

[0292] Example 3: Mouse Antibody Cell cAMP Experiment

[0293] Experimental steps:

[0294] 1. Digest 293-hCB1 cells (a stable strain expressing human CB1 constructed by lipofectamine transfection based on 293 cells) with Trypsin-EDTA (Invitrogen, 25200072) (37 degrees Celsius for 3 minutes).

[0295] 2. Terminate the digestion with complete culture medium and count the cells using a cell counter (Invitrogen, Countess II).

[0296] 3. Centrifuge at 1200 rpm for 3 minutes and discard the supernatant to collect the cell pellet.

[0297] 4. Resuspend the cells in assay buffer 1×DPBS (Hyclone, SH30028.02) + 500 μM IBMX (Sigma, I5879) + 0.1% BSA (Sigma, A7030) and adjust the cell density to 5×10 5 cells per milliliter.

[0298] 5. Add 1 μl of the antibody to be tested (10×, final concentration 200 nM) to a 384-well plate (784075 Greiner).

[0299] 6. Add 4 μl of cell suspension to each well using a MultiDrop Combi (set to low speed) (Thermo) to ensure that the number of cells in each well is 2000.

[0300] 7. Incubate the cell plate in a 37°C incubator for 10 minutes.

[0301] 8. Using a MultiDrop Combi (set to high speed), add assay buffer containing 15 μM Forskolin (Sigma, F6886) and 0.5 nM CP55940 (Sigma, C1112) to each well.

[0302] 9. Incubate the plate in a 37°C incubator for 30 minutes.

[0303] 10. Detect cAMP content using cAMP kit (62AM4PEC, Cisbio): Add 5 μl of cAMP-D2 antibody solution to each well, followed by 5 μl of Eu-Anti-cAMP solution.

[0304] 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 665 nM and 615 nM wavelengths. The signal ratio at 665 nM / 615 nM is inversely proportional to the relative level of cAMP, indicating the relative activity of CB1.

[0305] Data processing (data processing methods) and experimental data:

[0306] The ratio of the signals at wavelengths of 665nM and 615nM is calculated. This ratio is inversely proportional to the relative level of cAMP and represents the relative activity of CB1. A nonlinear regression curve is constructed between the antibody concentration and the corresponding 665nM / 615nM ratio to calculate the IC50.

[0307] The results are shown in Figures 1-5.

[0308] Experimental conclusion:

[0309] mAb003, mAb006, mAb010, mAb016 and mAb030 all showed significant inhibition of CB1 activity.

[0310] Example 4: Humanized Antibody Cell Binding Experiment

[0311] Experimental steps:

[0312] 1. CHOK1-hCB1 and CHOK1-mCB1 cells (the cells are stable transgenic lines expressing human CB1 / mouse CB1 constructed using lentivirus based on CHOK1 cells) were digested with TrypLE (gibco; 12604-021).

[0313] 2. Terminate the digestion with complete culture medium and count the cells using a cell counter (Countstar; IC1000).

[0314] 3. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant to collect the cell pellet.

[0315] 4. Resuspend the cells in cold FACS buffer (500 ml DMEM (CORNING; 10-013-CVRC) + 50 ml FBS (BI; 04-002-1A) and adjust the cell density to 5 × 10 6 cells per ml FACS buffer: 500 ml.

[0316] 5. Allow the cell suspension to stand at room temperature for 10 to 20 minutes.

[0317] 6. Centrifuge the cell plate at 1000 rpm for 5 minutes and discard the supernatant.

[0318] 7. Dilute the antibody to be tested in FACS buffer, add 100 μl to each well, and incubate at 4°C for 1 hour.

[0319] 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.

[0320] 9. Prepare the secondary antibody anti-human IgG-Alexa 488 antibody (Invitrogen; A-11013) in FACS buffer (1:1k dilution).

[0321] 10. Resuspend the cell pellet with secondary antibody diluent, add 100 μl to each well, and incubate at 4°C in the dark for 1 hour.

[0322] 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.

[0323] 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.

[0324] Data processing (data processing methods) and experimental data:

[0325] The binding signal values ​​corresponding to each antibody concentration were subjected to nonlinear fitting using Graphpad, and the EC50 was calculated based on the maximum binding signal of the antibody and curve fitting, as shown in Tables 5-7.

[0326] Table 5 Maximum binding signal and EC50 of humanized antibody of mAb003

[0327] Table 6 Maximum binding signal and EC50 of humanized antibody of mAb010

[0328] Table 7 Maximum binding signal and EC50 of humanized antibody of mAb006

[0329] Among them, human IgG4 (hIgG4) is a negative control; Tab04-hIgG4 (S228P) is a positive control, whose sequence is derived from the CB1 antibody named GFB-024 by Takeda, corresponding to the antibody named M7-H5 in Takeda patent WO2019211665A1, and its heavy chain is SEQ ID NO: 161 in WO2019211665A1; the light chain is SEQ ID NO: 167 in WO2019211665A1.

[0330] Experimental conclusion:

[0331] The different humanized antibodies from the three antibodies all maintained binding signals and activity against CHOK1-hCB1 cells expressing human CB1. With the exception of cAb06-VH3+VL1, which had comparable activity to Tab04-hIgG4(S228P), the others were all stronger than the positive control Tab04-hIgG4(S228P). Furthermore, each humanized antibody showed cross-reactivity against CHOK1-hCB1 cells expressing human CB1 and CHOK1-mCB1 cells expressing mouse CB1, while the positive control Tab04-hIgG4(S228P) had no binding activity against CHOK1-mCB1 cells expressing mouse CB1.

[0332] Example 5: Humanized Antibody Cellular cAMP Experiment

[0333] Experimental steps:

[0334] 1. Digest 293-hCB1 cells (a stable 293 cell line expressing human CB1 constructed by lipofectamine transfection based on 293 cells) with Trypsin-EDTA (Invitrogen, 25200072) (37 degrees Celsius for 3 minutes).

[0335] 2. Terminate the digestion with complete culture medium and count the cells using a cell counter (Invitrogen, Countess II).

[0336] 3. Centrifuge at 1200 rpm for 3 minutes and discard the supernatant to collect the cell pellet.

[0337] 4. Resuspend the cells in assay buffer 1×DPBS (Hyclone, SH30028.02) + 500 μM IBMX (Sigma, I5879) + 0.1% BSA (Sigma, A7030) and adjust the cell density to 5×10 5 cells per milliliter.

[0338] 5. Add 1 μl of the antibody to be tested (10×, final concentration 200 nM) to a 384-well plate (784075 Greiner).

[0339] 6. Add 4 μl of cell suspension to each well using a MultiDrop Combi (set to low speed) (Thermo) to ensure that the number of cells in each well is 2000.

[0340] 7. Incubate the cell plate in a 37°C incubator for 10 minutes.

[0341] 8. Using a MultiDrop Combi (set to high speed), add assay buffer containing 15 μM Forskolin (Sigma, F6886) and 0.5 nM CP55940 (Sigma, C1112) to each well.

[0342] 9. Incubate the plate in a 37°C incubator for 30 minutes.

[0343] 10. Detect cAMP content using cAMP kit (62AM4PEC, Cisbio): Add 5 μl of cAMP-D2 antibody solution to each well, followed by 5 μl of Eu-Anti-cAMP solution.

[0344] 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 665 nM and 615 nM wavelengths. The signal ratio at 665 nM / 615 nM is inversely proportional to the relative level of cAMP, indicating the relative activity of CB1.

[0345] Data processing (data processing methods) and experimental data:

[0346] The ratio of the signals at wavelengths of 665 nM and 615 nM was calculated. This ratio is inversely proportional to the relative level of cAMP and represents the relative activity of CB1. A nonlinear regression curve was constructed between the antibody concentration and the corresponding 665 nM / 615 nM ratio to calculate the IC50. The results are shown in Table 8.

[0347] Table 8 IC50 of humanized antibodies of mAb010

[0348] Experimental conclusion:

[0349] Based on the cell cAMP activity data, the inhibitory effects of cAb10-VH4+VL3 and cAb10-VH4+VL4 were stronger than those of the control positive antibody Tab04-hIgG4(S228P).

[0350] Example 6: mAb010 improves liver fibrosis in mice induced by carbon tetrachloride

[0351] Animals: Thirty female BALB / c mice, SPF grade, 8 weeks old, weighing approximately 20 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All animals were maintained on a 12-h alternating circadian rhythm and had free access to food.

[0352] Instruments: automatic biochemical analyzer, animal balance, tissue dehydration and tissue embedding machine, Leica automatic slicer, automatic staining machine.

[0353] Reagents: mouse antibody mAb010; carbon tetrachloride (purchased from Sinopharm Chemical Reagent Co., Ltd.), olive oil (purchased from Acros Organics).

[0354] Experimental process:

[0355] 1. Animal Grouping and Modeling

[0356] After one week of adaptive feeding, the mice were randomly divided into five groups based on body weight: a control group (oil), a model group (CCl4), and a treatment group (mAb010). All mice received a normal diet and water, and the model was maintained for 8 weeks. The model and treatment groups were injected twice weekly with a 25% CCl4 oil solution at a dose of 4 mL / kg, while the control group received an equal volume of oil solvent.

[0357] 2. Medication

[0358] Drug administration began simultaneously with model establishment; the treatment group received mAb010 (60 mg / kg) intravenously once weekly; the control and model groups received an equal volume of control solvent via gavage daily. For 8 weeks, the mice maintained a normal diet and water intake. Each group of mice was weighed daily, and their body weight, hair, feces, and activity were carefully observed and recorded.

[0359] 3. Get materials

[0360] Forty-eight hours after the 16th CCl₄ injection, the animals were dissected and sampled. Orbital blood was collected, and the livers were harvested after euthanasia. The left, middle, and caudal lobes of the liver were fixed in 10% formalin for at least 48 hours and sectioned for H&E and Sirius red staining. The remaining liver tissue was snap-frozen in liquid nitrogen for subsequent analysis of other parameters.

[0361] 4. Analysis of Liver Tissue Sirius Red Staining Sections

[0362] The positive area of ​​liver fibrosis was analyzed using the BIOTOPIX™ analysis system (visopharm). Fibrosis area (%) = fibrosis area / liver area*100%

[0363] 5. Experimental Results

[0364] Pathological studies examined the anti-hepatic fibrosis effects of mAb010. Sirius red staining revealed a 44.57% decrease in the percentage of fibrosis area in the treatment group compared to the model group, demonstrating that mAb010 significantly reduced collagen deposition in the mouse liver (Figure 6).

[0365] In summary, mAb010 has a protective effect on the liver fibrosis mouse model, suggesting that mAb010 has a therapeutic effect on liver fibrosis-related diseases and can be used to prepare preventive and therapeutic drugs for liver fibrosis-related diseases and cirrhosis.

[0366] 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 the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CB1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH CDR1 or variant thereof, a VH CDR2 or variant thereof, and a VH CDR3 or variant thereof contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 22 or 40; and / or a VL CDR1 or variant thereof, a VL CDR2 or variant thereof, and a VL CDR3 or variant thereof contained in a light chain variable region (VL) as shown in any one of SEQ ID NOs: 27, 41-43; (b) a VH CDR1 or variant thereof, a VH CDR2 or variant thereof, and a VH CDR3 or variant thereof contained in a heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 2 and 31-32; and / or a VL CDR1 or variant thereof, a VL CDR2 or variant thereof, and a VL CDR3 or variant thereof contained in a light chain variable region (VL) as shown in any one of SEQ ID NOs: 7 and 33-35; (c) a VH CDR1 or variant thereof, a VH CDR2 or variant thereof, and a VH CDR3 or variant thereof contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 12, 36-37; and / or a VL CDR1 or variant thereof, a VL CDR2 or variant thereof, and a VL CDR3 or variant thereof contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 17, 38-39; (d) VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 46; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 50; or (e) VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 54; and / or VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 58; in, The variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) the three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 22 or 40; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NO: 27, 41-43; (b) the three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 2 and 31-32; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 7 and 33-35; (c) the three CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 12, 36-37; and / or the three CDRs contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 17, 38-39; (d) the three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 46; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 50; or (e) the three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 54; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 58; 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 systems.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 23, VH CDR2 of SEQ ID NO: 24, and VH CDR3 of SEQ ID NO: 25; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 28, VL CDR2 of SEQ ID NO: 29, and VL CDR3 of SEQ ID NO: 30; (b) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 3, VH CDR2 of SEQ ID NO: 4, and VH CDR3 of SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 8, VL CDR2 of SEQ ID NO: 9, and VL CDR3 of SEQ ID NO: 10; (c) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 13, VH CDR2 of SEQ ID NO: 14, and VH CDR3 of SEQ ID NO: 15; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 18, VL CDR2 of SEQ ID NO: 19, and VL CDR3 of SEQ ID NO: 20; (d) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 47, VH CDR2 of SEQ ID NO: 48, and VH CDR3 of SEQ ID NO: 49; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 51, VL CDR2 of SEQ ID NO: 52, and VL CDR3 of SEQ ID NO: 53; or, (e) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 55, VH CDR2 of SEQ ID NO: 56, and VH CDR3 of SEQ ID NO: 57; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 59, VL CDR2 of SEQ ID NO: 60, and VL CDR3 of SEQ ID NO: 61; in, The CDRs are defined by the Kabat numbering system.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: (a) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 27 or a variant thereof; (b) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 7 or a variant thereof; (c) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 17 or a variant thereof; (d) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 46 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 50 or a variant thereof; or (e) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 54 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 58 or a variant thereof; in, The variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity; preferably, the substitutions are conservative substitutions; 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.

4. 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 a 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.

5. 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 a light chain variable region (VL) comprising the sequence shown in any one of SEQ ID NOs: 41-43 or a variant thereof; (b) a heavy chain variable region (VH) comprising a sequence as shown in SEQ ID NO: 31 or 32 or a variant thereof; and / or a light chain variable region (VL) comprising a sequence as shown in any one of SEQ ID NO: 33-35 or a variant thereof; or, (c) a heavy chain variable region (VH) comprising a sequence as shown in SEQ ID NO: 36 or 37 or a variant thereof; and / or a light chain variable region (VL) comprising a sequence as shown in SEQ ID NO: 38 or 39 or a variant thereof; wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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 100% sequence identity; Preferably, the substitution is a conservative substitution; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a VH comprising the sequence shown in SEQ ID NO: 40 and a VL comprising the sequence shown in SEQ ID NO: 41; (2) a VH comprising the sequence shown in SEQ ID NO: 40 and a VL comprising the sequence shown in SEQ ID NO: 42; (3) a VH comprising the sequence shown in SEQ ID NO: 40 and a VL comprising the sequence shown in SEQ ID NO: 43; (4) a VH comprising the sequence shown in SEQ ID NO: 31 and a VL comprising the sequence shown in SEQ ID NO: 34; (5) a VH comprising the sequence shown in SEQ ID NO: 31 and a VL comprising the sequence shown in SEQ ID NO: 35; (6) a VH comprising the sequence shown in SEQ ID NO: 32 and a VL comprising the sequence shown in SEQ ID NO: 33; (7) a VH comprising the sequence shown in SEQ ID NO: 32 and a VL comprising the sequence shown in SEQ ID NO: 34; (8) a VH comprising the sequence shown in SEQ ID NO: 32 and a VL comprising the sequence shown in SEQ ID NO: 35; (9) a VH comprising the sequence shown in SEQ ID NO: 36 and a VL comprising 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.

6. 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 (e.g., 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 (e.g., IgG1, IgG2, IgG3 or IgG4), and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ); 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 a light chain constant region as shown in SEQ ID NO: 45; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising the VH sequence shown in SEQ ID NO: 40 and the CH sequence shown in SEQ ID NO: 44, and a light chain comprising the VL sequence shown in SEQ ID NO: 41 and the CL sequence shown in SEQ ID NO: 45; (2) a heavy chain comprising the VH of SEQ ID NO: 40 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 42 and the CL of SEQ ID NO: 45; (3) a heavy chain comprising the VH of SEQ ID NO: 40 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 43 and the CL of SEQ ID NO: 45; (4) a heavy chain comprising the VH of SEQ ID NO: 31 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 34 and the CL of SEQ ID NO: 45; (5) a heavy chain comprising the VH of SEQ ID NO: 31 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 35 and the CL of SEQ ID NO: 45; (6) a heavy chain comprising the VH of SEQ ID NO: 32 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 33 and the CL of SEQ ID NO: 45; (7) a heavy chain comprising the VH of SEQ ID NO: 32 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 34 and the CL of SEQ ID NO: 45; (8) a heavy chain comprising the VH of SEQ ID NO: 32 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 35 and the CL of SEQ ID NO: 45; (9) a heavy chain comprising the VH of SEQ ID NO: 36 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 38 and the CL of SEQ ID NO: 45; (10) a heavy chain comprising the VH of SEQ ID NO: 37 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 38 and the CL of SEQ ID NO: 45; or (11) a heavy chain comprising the VH of SEQ ID NO: 37 and the CH of SEQ ID NO: 44, and a light chain comprising the VL of SEQ ID NO: 39 and the CL of SEQ ID NO: 45; Preferably, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from a murine immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4), and / or the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from a murine immunoglobulin (e.g., κ or λ).

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antigen-binding fragment is selected from 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 murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody.

8. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the heavy chain variable region and / or light chain variable region thereof, or the heavy chain and / or light chain thereof.

9. A vector comprising the isolated nucleic acid molecule according to claim 8; preferably, the vector is a cloning vector or an expression vector.

10. A host cell comprising the isolated nucleic acid molecule of claim 8 or the vector of claim 9.

11. 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 recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

12. 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 cell according to claim 10, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

13. Use of 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 pharmaceutical composition according to claim 12 for preparing a medicament for preventing and / or treating a disease associated with CB1 in a subject; Preferably, the CB1-related disease will benefit from antagonism of CB1 signaling; Preferably, the CB1-related disease is: 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, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease), or a combination 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.

14. A method for preventing and / or treating a CB1-related disease in a subject, comprising: administering to a subject in need thereof an effective amount of 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 pharmaceutical composition according to claim 12; Preferably, the CB1-related disease will benefit from antagonism of CB1 signaling; Preferably, the CB1-related disease is: 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, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease), or a combination 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.

15. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and a detectable label linked to the antibody or antigen-binding fragment thereof; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

16. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the conjugate according to claim 15.

17. 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 the preparation of a detection reagent for detecting the presence or level of CB1 in a sample and / or diagnosing a disease associated with CB1; Preferably, the diseases associated with CB1 are: obesity-related diseases (such as Prader-Willi syndrome), diabetes Diabetes (e.g., type 1 diabetes, type 2 diabetes), kidney disease (e.g., diabetic nephropathy), liver fibrosis, pulmonary fibrosis, dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia), fatty liver (e.g., non-alcoholic fatty liver disease), or a combination thereof; Preferably, the sample is a tissue sample (eg, central nervous system tissue, lung tissue, liver tissue, adipose tissue or kidney tissue) from a subject (eg, a mammal, preferably a human or mouse).

18. 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 to 7 or the conjugate according to claim 15; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g. ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises using the conjugate of claim 15; Preferably, the method comprises using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin); Preferably, the method comprises: (1) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the conjugate according to claim 15; (2) Detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of CB1 or cells expressing CB1.

19. 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 presence of CB1 or an increase in the level of CB1 compared to a reference level (eg, compared to a healthy control) indicates that the subject suffers from a disease associated with CB1.

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