WO2024141063A1

WO2024141063A1 - Anti-rsv antibody, composition and preparation, and use thereof

Info

Publication number: WO2024141063A1
Application number: PCT/CN2023/143459
Authority: WO
Inventors: 焦娇; 李若薇; 王茜; 王娇; 车黎明; 张新月
Original assignee: 甘李药业股份有限公司
Priority date: 2022-12-30
Filing date: 2023-12-29
Publication date: 2024-07-04

Abstract

Provided are an antibody or an antigen-binding fragment, composition and preparation thereof having an effective neutralizing activity against RSV. The anti-RSV antibody or the antigen-binding fragment, composition and preparation thereof provided by the present invention can be used as a drug for treating or preventing RSV infections or RSV infection-associated symptoms.

Description

Anti-RSV virus antibodies, compositions, preparations and applications thereof

Technical Field

The present invention relates to anti-RSV virus antibodies, compositions, preparations, methods for preparing the antibodies, pharmaceutical compositions comprising the antibodies, and uses of the antibodies, compositions, preparations in preparing drugs for treating and/or preventing respiratory syncytial virus (RSV) infection or RSV-related symptoms.

Background technique

Respiratory syncytial virus (RSV) is a negative sense, single stranded RNA virus. It is one of the most common viral pathogens that cause respiratory diseases in infants, the elderly and immunocompromised adults. RSV is the leading cause of lower respiratory tract infections in infants and young children, and the leading cause of hospitalization for respiratory diseases in young children. Almost all children have experienced one or more infections before the age of 4 years, and the peak age of infection is 2 to 8 months. Many studies have shown that severe infection in infants is a high-risk factor for the development of asthma in the future, and its severity far exceeds that of other microbial pathogens. RSV can also infect the adult population. In this population, RSV mainly causes upper respiratory tract diseases. Elderly patients and immunocompromised adults, especially bone marrow transplant patients, may be at greater risk of severe infection and pneumonia.

The immunity produced by natural RSV infection is insufficient and cannot produce lasting immunity. Therefore, a notable feature of RSV infection is that the antibodies produced in the body by the previous infection cannot provide permanent protection. In the same epidemic season, different subtypes of RSV can cause reinfection. Even multiple natural RSV infections cannot induce lifelong immune protection of the upper respiratory tract against viral infections. Therefore, repeated infections are very common.

Currently, several approaches to prevent and treat RSV infection have been studied, including vaccine development, antiviral compounds (ribavirin), antisense drugs, RNA interference technology, and antibody products. Ribavirin is a nucleoside antimetabolite that is severely toxic and teratogenic. Although vaccines may be useful, several candidate vaccines have been abandoned and some are under development. To date, no vaccine products against RSV have been approved for marketing. Two glycoproteins on the surface of RSV, F and G, have been shown to be As the target of neutralizing antibodies, an antibody specific to the antigenic determinant region of the RSV-F protein, Palivizumab, has been approved for use in pediatric patients at a monthly dose of 15 mg/kg during the entire RSV epidemic season (November to April in the northern hemisphere) to prevent severe lower respiratory tract diseases caused by RSV, however, Palivizumab is an expensive humanized monoclonal antibody and can only be used for prophylactic treatment of pediatric patients. Therefore, there is an urgent need to develop new anti-RSV drugs, especially drugs that can treat and/or prevent RSV infection in a wider population.

Summary of the invention

In order to solve the problems existing in the prior art, the purpose of the present invention is to provide an antibody against respiratory syncytial virus and its application.

The present invention successfully obtains RSV-specific antibodies with comparable or better properties than previous RSV-specific antibodies, including attacks against RSV subtype A and/or RSV subtype B, affinity for RSV, IC50 value and animal efficacy results, etc.

In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof, comprising:

The three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment with the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15; and/or

The three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment with the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16.

The present invention also provides an antibody or an antigen-binding fragment thereof, comprising:

1) HCDR3, wherein the HCDR3 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 19, 25, 31, 37, 43, 49, 55, and 61; and

2) LCDR3, wherein LCDR3 comprises any amino acid sequence in the group consisting of the following amino acid sequences or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 22, 28, 34, 40, 46, 52, 58, 64, and 70.

1) HCDR1, wherein the HCDR1 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 17, 23, 29, 35, 41, 47, 53, and 59; and

2) LCDR1, wherein the LCDR1 comprises any one of the following amino acid sequences: An amino acid sequence or any one of the amino acid sequences in the group consisting of SEQ ID NO: 20, 26, 32, 38, 44, 50, 56 and 62;

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

3) HCDR2, wherein the HCDR2 comprises or consists of any one of the group consisting of the following amino acid sequences: SEQ ID NO: 18, 24, 30, 36, 42, 48, 54 and 60; and

4) LCDR2, wherein LCDR2 comprises any amino acid sequence in the group consisting of the following amino acid sequences or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 21, 27, 33, 39, 45, 51, 57 and 63.

Optionally, the antibody or antigen-binding fragment thereof provided by the present invention comprises:

1) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 1, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:2; or

2) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 3, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:4; or

3) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO:5, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:6; or

4) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO:7, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:8; or

5) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO:9, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO: 10; or

6) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 11, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:12; or

7) The amino acid sequence of the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 13, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:14; or

8) The amino acid sequence of the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 15, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown as SEQ ID NO:16.

1) HCDR1 comprising or consisting of SEQ ID NO: 17,

HCDR2 comprising or consisting of SEQ ID NO:18,

HCDR3 comprising or consisting of SEQ ID NO:19,

LCDR1 comprising or consisting of SEQ ID NO:20,

LCDR2 comprising or consisting of SEQ ID NO:21, and

LCDR3 comprising or consisting of SEQ ID NO:22; or

2) HCDR1 comprising or consisting of SEQ ID NO: 23,

HCDR2 comprising or consisting of SEQ ID NO:24,

HCDR3 comprising or consisting of SEQ ID NO:25,

LCDR1 comprising or consisting of SEQ ID NO:26,

LCDR2 comprising or consisting of SEQ ID NO:27, and

LCDR3 comprising or consisting of SEQ ID NO:28; or

3) comprising HCDR1 represented by or consisting of SEQ ID NO: 29,

HCDR2 comprising or consisting of SEQ ID NO:30,

HCDR3 comprising or consisting of SEQ ID NO:31,

LCDR1 comprising or consisting of SEQ ID NO:32,

LCDR2 comprising or consisting of SEQ ID NO: 33, and

LCDR3 comprising or consisting of SEQ ID NO:34; or

4) HCDR1 comprising or consisting of SEQ ID NO:35,

HCDR2 comprising or consisting of SEQ ID NO:36,

HCDR3 comprising or consisting of SEQ ID NO:37,

LCDR1 comprising or consisting of SEQ ID NO:38,

LCDR2 comprising or consisting of SEQ ID NO:39, and

LCDR3 comprising or consisting of SEQ ID NO:40; or

5) HCDR1 comprising or consisting of SEQ ID NO:41,

HCDR2 comprising or consisting of SEQ ID NO:42,

HCDR3 comprising or consisting of SEQ ID NO:43,

LCDR1 comprising or consisting of SEQ ID NO:44,

LCDR2 comprising or consisting of SEQ ID NO:45, and

LCDR3 comprising or consisting of SEQ ID NO:46; or

6) HCDR1 comprising or consisting of SEQ ID NO:47,

HCDR2 comprising or consisting of SEQ ID NO:48,

HCDR3 comprising or consisting of SEQ ID NO:49,

LCDR1 comprising or consisting of SEQ ID NO:50,

LCDR2 comprising or consisting of SEQ ID NO:51, and

LCDR3 comprising or consisting of SEQ ID NO:52; or

7) HCDR1 comprising or consisting of SEQ ID NO:53,

HCDR2 comprising or consisting of SEQ ID NO:54,

HCDR3 comprising or consisting of SEQ ID NO:55,

LCDR1 comprising or consisting of SEQ ID NO:56,

LCDR2 comprising or consisting of SEQ ID NO:57, and

LCDR3 comprising or consisting of SEQ ID NO:58; or

8) HCDR1 comprising or consisting of SEQ ID NO:59,

HCDR2 comprising or consisting of SEQ ID NO:60,

HCDR3 comprising or consisting of SEQ ID NO:61,

LCDR1 comprising or consisting of SEQ ID NO:62,

LCDR2 comprising or consisting of SEQ ID NO: 63, and

LCDR3 comprising or consisting of SEQ ID NO:64.

A heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is at least 80% identical to any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 , 105, 107 and 109; preferably, the heavy chain variable region has a CDRs region amino acid sequence identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109. sequence, and the amino acid sequence of the framework region of the heavy chain variable region is at least 80% identical to the framework region of the amino acid sequence SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109 ; Preferably, the heavy chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109; and/or

A light chain variable region, the amino acid sequence of the light chain variable region is at least 80% identical to any one of the amino acid sequences selected from the group consisting of the following amino acid sequences: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110; preferably, the amino acid sequence of the light chain variable region has at least 80% identity to the amino acid sequence of SEQ ID NO: NO:2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and the framework region of the light chain variable region is at least 80% identical to the framework region of sequence SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110; preferably, the light chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO:2,4,6,8,10,12,14,16,66,68,70,72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110.

Preferably, the antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence pair of the heavy chain variable region/light chain variable region is selected from any one of the groups consisting of the following amino acid sequence pairs:

SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO:81 and SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96 ID NO:96, SEQ ID NO:97 and SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:106, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110;

Preferably, the amino acid sequence pair of the heavy chain variable region/light chain variable region is selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, SEQ ID NO:87 and SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:106, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110.

In one of the technical solutions, it further comprises a constant region, which is derived from an IgG antibody, an IgM antibody, an IgA antibody, an IgD antibody or an IgE antibody. Preferably, the constant region is derived from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody.

In one of the technical solutions, it comprises a heavy chain constant region and/or a light chain constant region, preferably it comprises a murine or humanized heavy chain constant region and/or a light chain constant region; preferably, the amino acid sequence of the humanized heavy chain constant region is as shown in SEQ ID NO: 111 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence SEQ ID NO: 111, and the amino acid sequence of the humanized light chain constant region is as shown in SEQ ID NO: 112 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence SEQ ID NO: 112. EQ ID NO:112 has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; preferably, the amino acid sequence pair of the heavy chain/light chain of the antibody or its antigen-binding fragment is selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NO:129 and SEQ ID NO:130, SEQ ID NO:131 and SEQ ID NO:132, SEQ ID NO:133 and SEQ ID NO:134.

In one of the technical solutions, each CDR region is defined according to the Kabat definition scheme, the Chothia definition scheme, the Abm definition scheme, the IMGT definition scheme and/or the Contact definition scheme; preferably, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

In one embodiment, the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (preferably scFv) or (Fab') ₂ , single domain antibody, diabody (dAb) or linear antibody.

The second aspect of the present invention provides a nucleic acid encoding the Antibodies or antigen-binding fragments thereof. The nucleic acid molecules may be synthetic, recombinant or isolated. Due to the degeneracy of the nucleic acid code, multiple nucleic acids will encode the same amino acid and all are encompassed herein.

The third aspect of the present invention provides an expression vector comprising the nucleic acid described in the second aspect.

The fourth aspect of the present invention provides a host cell comprising the expression vector described in the third aspect.

The fifth aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, which comprises culturing the host cell described in the fourth aspect, and recovering the antibody or antigen-binding fragment expressed thereby from the culture.

The antibodies or antigen-binding fragments thereof described herein can be produced from hybridomas that secrete the antibodies, or from recombinantly produced cells that have been transformed or transfected with one or more genes encoding the antibodies or antigen-binding fragments thereof. Antibodies or antigen-binding portions thereof are produced by culturing host cells under conditions that express nucleic acids to produce antibodies, followed by recovery of the antibodies.

Recombinant expression utilizes the construction of an expression vector comprising a polynucleotide encoding an antibody or its antigen binding portion thereof. Once the polynucleotide is obtained, a vector for producing the antibody can be produced by recombinant DNA techniques well known in the art. The expression vector may include appropriate transcription and translation control signals. This can be accomplished using in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

Optionally, the host cell is a prokaryotic cell or a eukaryotic cell.

Optionally, the host cell is an Escherichia coli cell, a yeast cell, an insect cell, a plant cell or a mammalian cell.

Optionally, the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell or a NSO cell. The cell lines include VERO, BHK, HeIa, COS, MDCK, 293F, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and HsS78Bst cells. However, the cell lines used in the present invention include but are not limited to the above cell lines.

Once the antibody or antigen-binding portion of an antibody has been produced by recombinant expression, it can be purified by any method known in the art for purification of immunoglobulin molecules. It is purified, for example, by chromatography, centrifugation, differential solubility, or by any other standard technique for purification of proteins.

Another aspect of the present invention provides a pharmaceutical composition comprising at least one of the antibodies or antigen-binding fragments thereof described in the first aspect, and a pharmaceutically acceptable excipient, for example, the antibody or antigen-binding fragment thereof is combined with water for injection, or with saline.

In one embodiment, it further comprises a second antibody or an antigen-binding fragment thereof capable of neutralizing RSV virus, wherein the amino acid sequence of the second antibody or an antigen-binding fragment thereof is different from the amino acid sequence of the antibody or an antigen-binding fragment thereof described in the first aspect.

Another aspect of the present invention provides a pharmaceutical composition comprising:

1) a first antibody or an antigen-binding fragment thereof that can bind to the site II epitope of RSV-F protein, preferably, the first antibody or an antigen-binding fragment thereof is any one of the antibodies or antigen-binding fragments thereof as described in any one of claims 1 to 11; and

2) A second antibody or an antigen-binding fragment thereof that can neutralize RSV virus, wherein the amino acid sequence of the second antibody or the antigen-binding fragment thereof is different from the amino acid sequence of the first antibody or the antigen-binding fragment thereof.

In one embodiment, the RSV-related protein bound by the second antibody or antigen-binding fragment thereof is the same as or different from the RSV-F protein bound by the first antibody or antigen-binding fragment thereof;

Preferably, the RSV-related protein is RSV-F protein or RSV-G protein;

Preferably, the RSV-related protein is RSV-F protein.

In one embodiment, the antigenic epitope of the RSV-F protein bound by the second antibody or antigen-binding fragment thereof is the same as or different from the site II epitope of the RSV-F protein bound by the first antibody or antigen-binding fragment thereof;

Preferably, the antigenic epitope of RSV-F protein bound by the second antibody or its antigen-binding fragment is selected from any one of site φ epitope, site I epitope, site II epitope, site III epitope, site IV epitope and site V epitope;

Preferably, the antigenic epitope of the RSV-F protein to which the second antibody or antigen-binding fragment thereof binds is selected from any one of a site φ epitope, a site I epitope, a site III epitope, a site IV epitope and a site V epitope;

Preferably, the antigenic epitope of the RSV-F protein to which the second antibody or antigen-binding fragment thereof binds is a siteφ epitope.

In one embodiment, the second antibody or antigen-binding fragment thereof is selected from Motavizumab, Palivizumab, Nirsevimab, Suptavumab, Clesrovimab, and Felvizumab.

In one embodiment, the second antibody or its antigen-binding fragment comprises: three heavy chain complementary determining regions (CDRs) HCDR1', HCDR2' and HCDR3' contained in the peptide segment with the amino acid sequence of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150 or 152; and/or three light chain complementary determining regions (CDRs) LCDR1', LCDR2' and LCDR3' contained in the peptide segment with the amino acid sequence of SEQ ID NO: 137, 139, 141, 143, 145, 147, 149, 151, 153, 164, 165, 166, 167, 168, 169, 170 or 171.

In one embodiment, the second antibody or antigen-binding fragment thereof comprises:

a) a HCDR3′ functional region having any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 174, 180, 186, 192, 198, 204, 210, 216, and 222; and

b) LCDR3’ functional region, wherein the LCDR3’ functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 177, 183, 189, 195, 201, 207, 213, 219, and 225.

In one embodiment, the second antibody or antigen-binding fragment thereof further comprises:

c) a HCDR1′ functional region, wherein the HCDR1′ functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 172, 178, 184, 190, 196, 202, 208, 214, and 220;

d) LCDR1' functional region, wherein the LCDR1' functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 175, 181, 187, 193, 199, 205, 211, 217, 223, 244, 245, 246, 247, 248, 249, 250 and 251;

e) a HCDR2' functional region, wherein the HCDR2' functional region has any one of the amino acid sequences selected from the group consisting of the following amino acid sequences: SEQ ID NO: 173, 179, 185, 191, 197, 203, 209, 215, and 221; and

f) LCDR2’ functional region, wherein the LCDR2’ functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 176, 182, 188, 194, 200, 206, 212, 218, and 224.

In one embodiment, the second antibody or antigen-binding fragment thereof comprises: 1) three heavy chain complementary determining regions (CDRs) contained in a peptide segment with an amino acid sequence of SEQ ID NO: 148, and three light chain complementary determining regions (CDRs) contained in a peptide segment with an amino acid sequence of SEQ ID NO: 149, 164, 165, 166, 167, 168, 169, 170, or 171; or

2) three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 136, and three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 137; or

3) three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 138 and three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 139; or

4) three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 140, and three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 141; or

5) three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 142, and three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 143; or

6) three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 144, and three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 145; or

7) The three heavy chain complementary determinants contained in the peptide segment with the amino acid sequence of SEQ ID NO: 146 regions (CDRs), and three light chain complementary determining regions (CDRs) contained in a peptide segment having an amino acid sequence of SEQ ID NO: 147; or

8) the three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 150, and the three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 151; or

9) three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:152, and three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:153.

1) HCDR1′ comprising or consisting of SEQ ID NO: 172,

HCDR2' comprising or consisting of SEQ ID NO:173,

HCDR3' comprising or consisting of SEQ ID NO:174,

LCDR1' comprising or consisting of SEQ ID NO:175,

LCDR2' comprising or consisting of SEQ ID NO:176, and

LCDR3' comprising or consisting of SEQ ID NO:177; or

2) HCDR1′ comprising or consisting of SEQ ID NO: 178,

HCDR2' comprising or consisting of SEQ ID NO:179,

HCDR3' comprising or consisting of SEQ ID NO: 180,

LCDR1' comprising or consisting of SEQ ID NO: 181,

LCDR2' comprising or consisting of SEQ ID NO: 182, and

LCDR3' comprising or consisting of SEQ ID NO:183; or

3) HCDR1′ comprising or consisting of SEQ ID NO: 184,

HCDR2' comprising or consisting of SEQ ID NO:185,

HCDR3' comprising or consisting of SEQ ID NO:186,

LCDR1' comprising or consisting of SEQ ID NO: 187,

LCDR2' comprising or consisting of SEQ ID NO: 188, and

LCDR3' comprising or consisting of SEQ ID NO: 189; or

4) HCDR1′ comprising or consisting of SEQ ID NO: 190,

HCDR2' comprising or consisting of SEQ ID NO:191,

HCDR3' comprising or consisting of SEQ ID NO:192,

LCDR1' comprising or consisting of SEQ ID NO: 193,

LCDR2' comprising SEQ ID NO: 194 or consisting thereof, and

LCDR3' comprising or consisting of SEQ ID NO:195; or

5) HCDR1′ comprising or consisting of SEQ ID NO: 196,

HCDR2' comprising or consisting of SEQ ID NO:197,

HCDR3' comprising or consisting of SEQ ID NO:198,

LCDR1' comprising or consisting of SEQ ID NO: 199,

LCDR2' comprising or consisting of SEQ ID NO: 200, and

LCDR3' comprising or consisting of SEQ ID NO:201; or

6) HCDR1′ comprising or consisting of SEQ ID NO:202,

HCDR2' comprising or consisting of SEQ ID NO:203,

HCDR3' comprising or consisting of SEQ ID NO:204,

LCDR1' comprising or consisting of SEQ ID NO:205,

LCDR2' comprising or consisting of SEQ ID NO:206, and

LCDR3' comprising or consisting of SEQ ID NO:207; or

7) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:211,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

8) HCDR1′ comprising or consisting of SEQ ID NO: 214,

HCDR2' comprising or consisting of SEQ ID NO:215,

HCDR3' comprising or consisting of SEQ ID NO:216,

LCDR1' comprising or consisting of SEQ ID NO:217,

LCDR2' comprising or consisting of SEQ ID NO:218, and

LCDR3 comprising or consisting of SEQ ID NO: 219; or

9) HCDR1′ comprising or consisting of SEQ ID NO: 220,

HCDR2' comprising or consisting of SEQ ID NO:221,

HCDR3' comprising or consisting of SEQ ID NO:222,

LCDR1' comprising or consisting of SEQ ID NO:223,

LCDR2' comprising or consisting of SEQ ID NO:224, and

LCDR3' comprising or consisting of SEQ ID NO:225; or

10) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:244,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

11) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:245,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

12) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:246,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

13) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:247,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

14) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:248,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

15) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:249,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

16) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:250,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

17) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:251,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213.

In one embodiment, the second antibody or antigen-binding fragment thereof comprises: a heavy chain variable region, the amino acid sequence of the heavy chain variable region having at least 80% identity to any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, and 162; preferably, the heavy chain variable region has an amino acid sequence of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, and 162. 160, or 162, and the non-CDRs region of the heavy chain variable region has a sequence that is at least 80% identical to the non-CDRs region of the peptide segment whose amino acid sequence is SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162; preferably, the heavy chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, and 162; and/or a light chain variable region, the amino acid sequence of the light chain variable region has at least 80% identity to any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO:137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, and 171; preferably, the light chain variable region has a CDRs region that is the same as the peptide segment with an amino acid sequence of SEQ ID NO:137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, or 171, and the non-CDRs region of the light chain variable region is the same as the peptide segment with an amino acid sequence of SEQ ID NO: NO:137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, or 171. The non-CDRs region of the peptide fragment has at least 80% identity; preferably, the light chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO:137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, and 171.

In one embodiment, the second antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence pair of the heavy chain variable region/light chain variable region is selected from the group consisting of the following amino acid sequence pairs: SEQ ID NO: 136/SEQ ID NO: 137, SEQ ID NO: 138/SEQ ID NO: 139, SEQ ID NO: 140/SEQ ID NO: 141, SEQ ID NO: 142/SEQ ID NO: 143, SEQ ID NO: 144/SEQ ID NO: 145, SEQ ID NO: 146/SEQ ID NO: 147, SEQ ID NO: 148/SEQ ID NO: 149, SEQ ID NO: 150/SEQ ID NO: 151, SEQ ID NO: 152/SEQ ID NO: 153, SEQ ID NO: 154/SEQ ID NO: 155, SEQ ID NO: 156/SEQ ID NO: 157, SEQ ID NO: 158/SEQ ID NO: 159, SEQ ID NO: 160 NO:160/SEQ ID NO:167, SEQ ID NO:156/SEQ ID NO:168, SEQ ID NO:156/SEQ ID NO:169, SEQ ID NO:156/SEQ ID NO:170, and SEQ ID NO:156/SEQ ID NO:171;

Preferably, the amino acid sequence pairs of the heavy chain variable region/light chain variable region are selected from the group consisting of the following amino acid sequence pairs: SEQ ID NO: 148/149, 154/155, 156/157, 158/159, 160/161, 162/163, 156/164, 156/165, 156/166, 156/167, 156/168, 156/169, 156/170, and 156/171.

In one embodiment, the second antibody or its antigen-binding fragment comprises a heavy chain constant region and/or a light chain constant region, preferably it comprises a murine or humanized heavy chain constant region and/or a light chain constant region; preferably, the amino acid sequence of the humanized heavy chain constant region is as shown in SEQ ID NO:252, and the amino acid sequence of the humanized light chain constant region is as shown in SEQ ID NO:253.

In one embodiment, the amino acid sequence pairs of the heavy chain/light chain of the second antibody or antigen-binding fragment thereof are selected from the group consisting of the following amino acid sequence pairs: SEQ ID NO: 254/255, 256/257 and 258/259

Preferably, the amino acid sequence pairs of the heavy chain/light chain of the first antibody or its antigen-binding fragment of the pharmaceutical composition are selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 129 and SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134, and the amino acid sequence pairs of the heavy chain/light chain of the second antibody or its antigen-binding fragment of the composition are selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 254 and SEQ ID NO: 255, SEQ ID NO: 256 and SEQ ID NO: 257, SEQ ID NO: 258 and SEQ ID NO: 259.

Another aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof or the pharmaceutical composition described above in the preparation of a medicament for treating and/or preventing respiratory syncytial virus infection (RSV) or symptoms associated with respiratory syncytial virus (RSV) infection.

The present invention also provides the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned The pharmaceutical composition is used for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with respiratory syncytial virus (RSV) infection.

The present invention also provides a method for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with respiratory syncytial virus (RSV) infection, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof described above or the pharmaceutical composition described above to a subject in need thereof.

The present invention also provides a use of an active ingredient, wherein the active ingredient is selected from the antibody or antigen-binding fragment thereof described in the first aspect, and the active ingredient is used to prepare a detection plate or a kit, wherein the detection plate or the kit is used to detect respiratory syncytial virus (RSV).

The present invention also provides a detection plate, which comprises a substrate and a test strip, wherein the test strip contains the antibody or antigen-binding fragment thereof selected from the first aspect.

The present invention also provides a kit, which comprises:

(1) a first container, wherein the first container contains an antibody or an antigen-binding fragment thereof selected from the first aspect; and/or

(2) a second container, wherein the second container contains a secondary antibody selected from the antibodies described in the first aspect;

Alternatively, the kit contains the above-mentioned detection plate.

Another aspect of the present invention further provides an antibody preparation capable of neutralizing respiratory syncytial virus (RSV), comprising:

Any pharmaceutical composition as described above; preferably, the amino acid sequence pair of the heavy chain/light chain of the first antibody or antigen-binding fragment thereof of the pharmaceutical composition is selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 129 and SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134, and/or the amino acid sequence pair of the heavy chain/light chain of the second antibody or antigen-binding fragment thereof of the composition is selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 254/255, 256/257 and 258/259;

Buffer;

Preferably, the buffer is selected from citrate buffer, histidine buffer, phosphate buffer, One or more of acetate buffer, succinate buffer, lactate buffer, tromethamine buffer, aspartate buffer, glutamate buffer or adipic acid buffer; preferably, the buffer is selected from citric acid buffer and/or phosphate buffer; preferably, the buffer is selected from citric acid buffer and phosphate buffer.

In one embodiment, the pH value of the antibody preparation is 5.6-6.5; preferably, the pH value of the antibody preparation is 5.7-6.3; preferably, the pH value of the antibody preparation is 5.7, 5.9, 6.1, 6.3.

In one embodiment, the concentration of the citrate buffer in the buffer is 10-20 mM, and the concentration of the phosphate buffer in the buffer is 6-12 mM. Preferably, the concentration of the citrate buffer is 13-17 mM, and the concentration of the phosphate buffer is 7-11 mM.

In one embodiment, the antibody formulation further comprises a stabilizer and/or a surfactant.

In one embodiment, the stabilizer is selected from one or more of sucrose, trehalose, sorbitol, arginine hydrochloride or mannitol;

Preferably, the stabilizer is selected from sorbitol and/or arginine hydrochloride;

Preferably, the stabilizer is selected from sorbitol and arginine hydrochloride.

In one embodiment, the concentration of sorbitol in the stabilizer is 250-300 mM, and the concentration of arginine hydrochloride is 60-100 mM; preferably, the concentration of sorbitol in the stabilizer is 260-280 mM, and the concentration of arginine hydrochloride is 70-90 mM.

In one embodiment, the surfactant is selected from Tween 80 or Tween 20; preferably, the surfactant is Tween 80.

In one embodiment, the concentration of the surfactant is 0.05-0.2 mg/ml; preferably, the concentration of the surfactant is 0.1 mg/ml.

In one embodiment, the total concentration of the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof in the composition is 40-200 mg/ml; preferably, the total concentration is 80-160 mg/ml; preferably, the total concentration is 100-140 mg/ml;

In one embodiment, the mass ratio of the first antibody or antigen-binding fragment to the second antibody or antigen-binding fragment in the composition is 1:0.2-5; preferably, the mass ratio is 1:0.4-2.5.

In one embodiment, the antibody formulation comprises:

20-100 mg/ml first antibody or antigen-binding fragment, 20-100 mg/ml second antibody or antigen-binding fragment, 2-6 mM citric acid, 9-13 mM sodium citrate, 7-11 mM Na ₂ HPO ₄ , 250-300 mM sorbitol, 60-100 mM arginine hydrochloride, 0.05-0.2 mg/ml Tween 80, pH about 5.6-6.5.

In one embodiment, the antibody formulation comprises:

40-80 mg/ml first antibody or antigen-binding fragment, 40-80 mg/ml second antibody or antigen-binding fragment, 2-6 mM citric acid, 9-13 mM sodium citrate, 7-11 mM Na ₂ HPO ₄ , 250-300 mM sorbitol, 60-100 mM arginine hydrochloride, 0.05-0.2 mg/ml Tween 80, pH about 5.6-6.5.

In one embodiment, the antibody formulation comprises: 60 mg/ml first antibody or antigen-binding fragment, 60 mg/ml second antibody or antigen-binding fragment, 4.09 mM citric acid, 11.225 mM sodium citrate, 9.659 mM Na ₂ HPO ₄ , 274 mM sorbitol, 80 mM arginine hydrochloride, 0.1 mg/ml Tween 80, pH about 5.7-6.2.

In one embodiment, the antibody formulation comprises: 40 mg/ml first antibody or antigen-binding fragment, 80 mg/ml second antibody or antigen-binding fragment, 4.09 mM citric acid, 11.225 mM sodium citrate, 9.659 mM Na ₂ HPO ₄ , 274 mM sorbitol, 80 mM arginine hydrochloride, 0.1 mg/ml Tween 80, pH about 5.7-6.2.

Another aspect of the present invention provides use of the antibody preparation described above in the preparation of a medicament for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection.

Another aspect of the present invention provides the antibody preparation described above, which is used for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection.

Another aspect of the present invention provides a method for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection, comprising administering a therapeutically effective amount of the antibody preparation described above to a subject in need thereof.

The RSV antibody provided by the present invention can bind to RSV-F protein with high affinity after humanization, and experiments show that it is significantly better than palivizumab in providing protection against RSV attack, and has an effect comparable to that of MEDI8897.

In addition, the antibody composition provided by the present invention combines two antibodies targeting different epitopes of RSV virus, which can reduce the use of a single drug compared to antibodies that recognize a single epitope. The drug resistance and limitations are reduced, achieving better neutralization effect and improving protective efficacy. Experiments have shown that it is superior to palivizumab and MEDI8897 in providing protection against RSV attacks.

definition

Unless otherwise indicated, any polypeptide chain is described herein as having an amino acid sequence starting at the N-terminus and ending at the C-terminus.

The term "antibody and antigen-binding portion thereof" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

The term "antibody" as used herein refers to an immunoglobulin molecule composed of four polypeptide chains, namely two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further divided into highly variable regions called complementarity determining regions (CDR(s)), interspersed with more conserved regions called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino acid terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, of which the three CDRs of VH are HCDR1, HCDR2 and HCDR3, and the three CDRs of VL are LCDR1, LCDR2 and LCDR3. The amino acid distribution of each domain is usually consistent with the following definition: Kabat Sequences of Proteins of Immunological Interest (Kabat protein sequences with immunological significance) (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al., Nature, 342: 878-883 (1989).

There are five major antibody types: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The amino acid sequence at the C-terminus of an antibody molecule is relatively stable, and this region is called the constant region. The constant region of the same antibody is the same. The constant region of the antibody light chain is the same. The constant region is composed of one Ig domain; the constant region of the heavy chain is composed of 3-4 tandem Ig domains and a hinge region for increased flexibility. IgA, IgE, and IgG have three domains (CH1, CH2, and CH3), and IgD and IgM have four domains (CH1, CH2, CH3, and CH4).

The heavy chain constant domains or heavy chain constant regions corresponding to different types of immunoglobulins are called α, δ, ε, γ and μ respectively. IgG molecules can be degraded into two Fab segments and one Fc segment under the action of papain. The Fab segment is composed of the variable region of the antibody light chain, the constant region of the light chain, the variable region of the heavy chain and the constant region of the heavy chain. The variable region is the site that binds to the antigen, so the Fab segment is also called the antigen binding segment. The Fc segment contains protein sequences common to all antibody molecules and determinants unique to each category. The Fc segment has multiple biological activities, including binding to complement, binding to Fc receptors, and passing through the placenta.

As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion" or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as RSV-F protein). It has been shown that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody. The binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, i.e., a monovalent fragment consisting of VL, VH, CL1, and CH1 domains; (ii) a F(ab') ₂ fragment, i.e., a bivalent fragment consisting of two F(ab) fragments linked by a disulfide bond in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the single-armed VL and VH domains of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) CDRs. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected together by a synthetic linker through a recombinant method to form a single connected chain, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)). Such single-chain antibodies are also included in the term "antigen-binding portion" of the antibody. Other forms of single-chain antibodies, such as bispecific antibodies, are also included.

Different analyses can be used to determine or roughly estimate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition. The Kabat definition is a standard for numbering residues in antibodies and is often used to determine CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition is more specific. The positions of certain structural loop regions are taken into account. See, e.g., Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group to model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbM™, A Computer Program for Modeling Variable Regions of Antibodies" Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from the primary sequence using known databases and ab initio methods, such as those described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). Contact definitions are based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

The antibody of the present invention is not limited to its origin, and can be an antibody from any animal such as a human antibody, a mouse antibody, a rat antibody, etc. It can also be a recombinant antibody such as a chimeric antibody or a humanized antibody. Preferably, it is a humanized antibody.

The term "antibody composition" refers to a combination of two or more antibodies or antigen-binding portions thereof. The antibody composition can be monoclonal (i.e., composed of the same antibody or antigen-binding portion molecules) or polyclonal (i.e., composed of two or more different antibodies or antigen-binding portions that react with the same or different epitopes on the same antigen or even with different antigens).

The ratios between the individual antibodies in the antibody compositions of the invention are generally that the antibodies are administered in equal amounts, but this is not necessarily the case. Thus, a composition of the invention comprising two anti-RSV antibodies or antigen-binding portions thereof generally contains them in a ratio of about 1:1, but depending on the characteristics of the individual antibodies, it may be desirable to use unequal amounts of antibodies or portions. For example, the ratio of one antibody or portion relative to another antibody or portion in a double antibody composition can be, for example, between 5 and 95%, between 10 and 90%, between 20 and 80%, between 30 and 70%, between 40 and 60%, or between 45 and 55%.

The term "epitope" refers to a portion (determinant) of an antigen that specifically binds to a related molecule such as a bispecific binding molecule. Epitope determinants are usually composed of chemically active surface groupings of molecules, such as amino acids or carbohydrates or sugar side chains, and usually have specific three-dimensional structural features and specific charge characteristics. An epitope can be "linear" or "conformational". In a linear epitope, all interaction points between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur along the primary amino acid sequence of the protein. In a conformational epitope, the point at which the interaction point occurs is between the amino acid residues on the protein, and the amino acid residues are separated from each other in the primary amino acid sequence. Once the desired epitope on the antigen is determined, antibodies to the epitope can be generated using techniques well known in the art. In addition, the generation and characterization of antibodies can generate information about the desired epitope. Based on this information, antibodies that bind to the same or similar epitopes can be competitively screened, such as by competitive studies to find antibodies that compete to bind to the antigen.

The size of the antigen epitope is suitable for the antigen binding site of the corresponding antibody. Generally, a polypeptide epitope contains 5 to 6 amino acid residues; a polysaccharide epitope contains 5 to 7 monosaccharides; and an epitope of a nucleic acid hapten contains 6 to 8 nucleotides. The specificity of an antigen epitope is determined by all the residues that make it up, but some of the residues play a greater role than other residues when binding to the antibody, and these residues are called immunodominant groups. The term "chimeric antibody" refers to an antibody that contains the variable regions of the heavy and light chains of mammals other than humans, such as mouse antibodies, and the constant regions of the heavy and light chains of human antibodies. Chimeric antibodies can be prepared using known methods. For example, chimeric antibodies can be prepared by inserting the genes of hybridoma clones into appropriate vectors and then introducing them into hosts. Specifically, reverse transcriptase is used to synthesize cDNA of the variable region (V) of the antibody from the mRNA of the hybridoma. When the DNA encoding the V region of the target antibody is obtained, it is connected to the DNA encoding the desired human antibody constant region (C region) and then inserted into the expression vector. Alternatively, the DNA encoding the antibody V region can be inserted into a DNA expression vector containing the human antibody C region. Inserting it into the expression vector allows it to be expressed under the regulation of the expression regulatory region. Next, the expression vector is used to transform a host cell to express the chimeric antibody.

RSV-F protein: RSV-F protein is a type I integral membrane protein consisting of 574 amino acids. Its inactive precursor (F0) consists of three F0s forming a trimer. When transported through the Golgi apparatus, the host's furin protease is activated at amino acids 109 and 110 of F0. and between amino acids 136 and 137. After cleavage, the short peptide (P27) of the middle 27 amino acids is released, and the remaining two segments F2 and F1 are connected by disulfide bonds (Cys69–Cys212 and Cys37–Cys439) to form a mature F protein structure. There is a highly hydrophobic fusion peptide (FP) at the N-terminus of the F1 protein, which is located in the hydrophobic cavity of the protein and is protected from the influence of the external hydrophilic environment. When the F protein appears on the surface of the virion or the cell surface, its structure is not stable, but is in a high-energy metastable Pre-F structure. Subsequently, the N-terminus of the F1 protein undergoes a series of drastic structural changes, which induces the occurrence of membrane fusion, thereby achieving the purpose of viral infection of cells. At the same time, the process also causes the F protein to transform from a high-energy metastable Pre-F structure to a stable post-fusion F protein structure (postfusion glyprotein, Post-F). The RSV-F protein described in the present invention comprises a metastable structure and a stable structure.

Terminology Site I epitope: Antibodies that recognize the Site I epitope include 131-2a, which recognizes a cysteine-rich region of the F protein. These antibodies block RSV infection by up to 50%, suggesting that the epitope has post-translational heterogeneity or that these antibodies neutralize through indirect effects such as viral aggregation. In addition, these antibodies partially block viral attachment to target cells. The Site I epitope is close to the viral membrane in the pre-F protein conformation, but is located at the apex in the post-F protein conformation.

SiteⅡ epitope: Antibodies targeting SiteⅡ epitope include the marketed preventive monoclonal antibody Synagis and its equivalent derivatives motavizumab and 47F; they mainly recognize aa255-275 of F protein. McLellan et al. (JSMcLellan, M. Chen, JS Chan, et al. J Virol, 84 (2010) 12236-12244) confirmed that this region forms a "helix-turn-helix" secondary structure by analyzing the crystal structure of the complex of motavizumab monoclonal antibody and F protein peptide aa254-277. The crystal structure shows that motavizumab monoclonal antibody binds to one end of the "helix-turn-helix" structure, and makes hydrogen bonds and ionic bonds act on Asn at position 268 and Lys at position 272. Further research results show that mutations at these two points can cause antibody escape. The structure of the SiteⅡ epitope bound by motavizumab is very intact in the post-F conformation, and the antibody binding site is fully exposed. The structure of motavizumab and post-F protein reveals the mechanism by which Synagis and motavizumab monoclonal antibodies have neutralizing activity. The simulated structure of RSV pre-F protein shows that the epitope is inside the pre-F protein conformation. Graham et al. confirmed that Synagis and motavizumab monoclonal antibodies can only inhibit RSV fusion with cells, but cannot inhibit RSV adsorption (JSMcLellan, Y. Yang, et al. J Virol, 85 (2011) 7788-7796; JSMcLellan, M. Chen, A. Kim, et al. Nat Struct Mol Biol, 17 (2010) 248-250).

Site Ⅳ epitope: Site Ⅳ epitope is the target of monoclonal antibodies such as 19 and 101F, which mainly involves aa422-438 of F protein. This epitope is located in the relatively conservative conformation of F protein. McLell lan et al. (J.S.McLell lan, Y.Yang, et al. J Virol, 85 (2011) 7788-7796) have solved the crystal structure of the complex of 101F and F protein peptide (aa 422-438). The results showed that the core area of Site Ⅳ epitope is aa427-437.

Siteφ epitope: Siteφ epitope is the target of pre-F specific antibodies D25, AM22 and 5C4. McLellan et al. (McLellan JS, Chen M, et al. Science 2013, 340: 1113-1117) analyzed the structure of the complex of pre-F specific antibody and pre-F and found that the epitope involves the loose region (aa 62-69) and the α4 helix (aa 196-209) in the F protein. In addition, the results also showed that when the F protein changes from pre-F to post-F conformation, the epitope shifts at least, and the α4 helix shifts 180°. Therefore, the antibody that recognizes this epitope is a pre-F specific antibody and cannot recognize the post-F protein.

Previous research results (McLellan JS, Chen M, et al. Science 2013, 340: 1113-1117) have shown that the Siteφ epitope has a high neutralizing activity and is mainly distributed in the pre-F conformation; the neutralizing activity of the Site II epitope and Site IV epitope is relatively weak and is distributed in both the pre-F and post-F conformations.

The term "humanized antibody" refers to an antibody containing at least one, usually two, substantially complete variable regions, wherein correspondingly all or substantially all CDR regions are from non-human antibodies, and wherein all or substantially all FR regions are from human antibodies.

The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity of a 1:1 interaction between members of a reactive binding pair (e.g., an antibody and an antigen). The affinity of X for its partner Y can generally be represented by an equilibrium dissociation constant ( _KD ), a dissociation constant (Kd), or an association constant (Ka). Affinity can be measured by general methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

The term "surface plasmon resonance" refers to an optical phenomenon that allows real-time interactive analysis by detecting changes in protein concentration in a biosensor matrix, for example using a Biacore™ system.

The term "neutralizing" or "blocking" antibody means an antibody whose binding to the RSV-F protein causes inhibition of the biological activity of the RSV-F protein. The inhibition of the biological activity of the RSV-F protein can be evaluated by measuring one or more RSV-F protein biological activity indicators well known in the art, such as RSV-F protein-induced cell activation and antibody binding to the RSV-F protein (see the following examples).

The sequence similarity of polypeptides is also referred to as sequence identity/origin, and is usually measured by sequence analysis software. Protein analysis software utilizes various substitutions, deletions and other modifications, including similarity measurements of conservative amino acid substitutions to match similar sequences, for example, GCG software includes programs such as Gap and Bestfit, and default parameters can be used to determine the sequence homology or sequence identity of closely related polypeptides, such as homologous polypeptides from different biological species, see GCG version 6.1. The FASTA program in GCG version 6.1 can also be used to compare polypeptide sequences with default or suggested parameters. FASTA (such as FASTA2 and FASTA3) provides comparisons and percentages of sequence identity of the best overlapping regions between the sequences queried and searched (Pearson (2000) as above). When the sequence of the present invention is compared with a database comprising a large number of sequences derived from different organisms, another preferred computing program is the computer program BLAST, especially BLASTP or TBLASTN, which uses default parameters when comparing.

The terms "substantial identity" or "substantially identical" when referring to nucleic acids or fragments thereof, mean that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide substitutions, insertions or deletions, the nucleotide sequence has nucleotide identity in at least about 80%, more preferably at least about 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases as calculated using any of the following sequence identity calculation programs such as FASTA, BLAST or Gap.

When applied to polypeptides, the term "substantially similar" or "substantially similar" means that two peptide sequences have at least 80% sequence identity, more preferably at least 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when optimally aligned using programs such as Gap or BESTFIT with default gap weights. Non-identical residue positions may differ by amino acid substitutions, deletions or insertions, and more preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) of similar chemical properties (such as charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. In the case where two or more amino acid sequences differ due to conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. Examples of groups of amino acids containing side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl groups: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution can be any change that has a positive value in the PAM250 log-likelihood matrix disclosed by Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change with a non-negative value in the PAM250 log-likelihood matrix.

The term "vector" is intended to refer to any molecule or entity (eg, nucleic acid, plasmid, phage or virus) used to transfer protein coding information into a host cell.

The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and/or regulates the expression of one or more heterologous coding regions operably linked thereto. An expression vector may include, but is not limited to, sequences that affect or regulate transcription, translation, and, if introns are present, sequences that affect RNA splicing of the coding region operably linked thereto.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including progeny of the cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the original cell are included herein.

The term "transfection" means the uptake of foreign or exogenous DNA by a cell. A cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Various transfection techniques are well known in the art. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. The techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

The term "treatment" includes therapeutic treatment, prophylactic treatment, and use in reducing the risk of a subject developing a disease or other risk factors. Treatment does not require a complete cure of the disease, but includes embodiments in which symptoms are alleviated or potential risk factors are mitigated.

The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it means that the likelihood of an event occurring is reduced in the presence of the compound or method.

The term "secondary antibody" refers to the second antibody, which is used to bind to the antibody (primary antibody), that is, the antibody of the antibody. Its main function is to detect the presence of the antibody and amplify the signal of the primary antibody. The secondary antibody uses the antigenic nature of the antibody as a large molecule protein to immunize xenogeneic animals, and the immunoglobulin produced by the immune system of xenogeneic animals against this antibody. The secondary antibody is reactive against all antibodies (such as IgG, IgM or IgA, etc.) of a specific species (such as mice).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Results of RSV B9320 attack in mice treated with humanized antibodies. The horizontal axis is the group number, which is: PBS, R6-5, R6-1.5, R6-0.5, R9-5, R9-1.5, R9-0.5, PALI-15, PALI-5, PALI-1.5, where PBS is the blank control group, and PALI-15, PALI-5 and PALI-1.5 are administered at doses of 15 mg/kg, The positive control palivizumab groups were 5 mg/kg and 1.5 mg/kg, R6-5, R6-1.5 and R6-0.5 were the experimental group R6 group with dosages of 5 mg/kg, 1.5 mg/kg and 0.5 mg/kg respectively; R9-5, R9-1.5 and R9-0.5 were the experimental group R9 group with dosages of 5 mg/kg, 1.5 mg/kg and 0.5 mg/kg respectively; the vertical axis was the virus titer (unit: log10 (PFU/g)).

Figure 2: Results of RSV B18537 attack in mice treated with humanized antibodies. The horizontal axis is the group number, which is: PBS, R6-5, R9-5, R9-1.5, R9-0.5, PALI-5, PALI-1.5, PALI-0.5, where PBS is the blank control group, PALI-15, PALI-5 and PALI-1.5 are the positive control palivizumab groups with dosages of 5 mg/kg, 1.5 mg/kg and 0.5 mg/kg respectively, R6-5 is the experimental group R6 with dosage of 5 mg/kg; R9-5, R9-1.5 and R9-0.5 are the experimental group R9 with dosages of 5 mg/kg, 1.5 mg/kg and 0.5 mg/kg respectively; the vertical axis is the virus titer (unit: log10 (PFU/g)).

Figure 3: Results of RSV BWV attack in mice treated with humanized antibodies. The horizontal axis is the group number, which is: PBS, PALI-5, R6-5, R6-0.5, R6-1.5, where PBS is the blank control group, PALI-5 is the positive control palivizumab group with a dosage of 5 mg/kg, R6-5, R6-0.5 and R6-1.5 are the experimental group R6 group with dosages of 5 mg/kg, 0.5 mg/kg and 1.5 mg/kg respectively, 8897-0.5, 8897-1.5 and 8897-5 are the positive control groups of MEDI8897 drugs with dosages of 0.5 mg/kg, 1.5 mg/kg and 5 mg/kg respectively, and the vertical axis is the virus titer (unit: log10 (PFU/g)).

Figure 4: Results of RSV A2 challenge in mice treated with humanized antibody compositions, the horizontal axis is the group number, in order: PBS, PALI-15, PALI-5, PALI-0.5, A2-G+R6 0.5+0.5, A2-G+R6 0.15+0.15, A2-G+R6 0.05+0.05, 8897-0.5, 8897-0.15, 8897-0.05, wherein PBS is the blank control group, PALI-15, PALI-5 and PALI-0.5 are the positive control palivizumab groups administered at doses of 15 mg/kg, 5 mg/kg and 0.5 mg/kg, respectively, A2-G+R6 0.5+0.5, A2-G+R6 0.15+0.15 and A2-G+R6 0.05+0.05, is the experimental group in which A2-G and R6 were administered with 0.5 mg/kg, 0.15 mg/kg and 0.05 mg/kg respectively; 8897-0.5, 8897-0.15 and 8897-0.05 are the positive control groups of MEDI8897 drug administered with doses of 0.5 mg/kg, 0.15 mg/kg and 0.05 mg/kg respectively. The vertical axis is the virus titer (unit: log10 (PFU/g)).

Figure 5: Results of RSV B9320 challenge in mice treated with humanized antibody compositions, the horizontal axis is the group number, in order: PBS, PALI-15, PALI-5, PALI-1.5, A2-G+R6 5+5, A2-G+R6 1.5+1.5, A2-G+R6 0.5+0.5, 8897-1.5, 8897-0.5, 8897-0.05, where PBS is the blank control group, PALI-15, PALI-5 and PALI-1.5 are administered at doses of 15 mg/kg, 5 mg/kg, 1. The positive control palivizumab group of 5 mg/kg, A2-G+R6 5+5, A2-G+R6 1.5+1.5 and A2-G+R6 0.5+0.5 are the experimental groups in which A2-G and R6 were administered with 5 mg/kg, 1.5 mg/kg and 0.5 mg/kg respectively, 8897-1.5, 8897-0.5 and 8897-0.05 are the positive control groups of MEDI8897 drugs administered with doses of 1.5 mg/kg, 0.5 mg/kg and 0.05 mg/kg respectively. The vertical axis is the virus titer (unit: log10 (PFU/g)).

Figure 6: Results of RSV BWV challenge in mice treated with humanized antibody compositions, the horizontal axis is the group number, in order: PBS, PALI-15, PALI-5, PALI-1.5, A2-G+R6 5+5, A2-G+R6 1.5+1.5, A2-G+R6 0.5+0.5, 8897-15, 8897-5, 8897-1.5, PBS is the blank control group, PALI-15, PALI-5 and PALI-1.5 are administered at doses of 15 mg/kg, 5 mg/kg, 1 .5mg/kg positive control palivizumab group, A2-G+R6 5+5, A2-G+R6 1.5+1.5 and A2-G+R6 0.5+0.5 are experimental groups where A2-G and R6 were administered with 5mg/kg, 1.5mg/kg and 0.5mg/kg respectively, 8897-15, 8897-5 and 8897-1.5 are MEDI8897 drug positive control groups administered with doses of 15mg/kg, 5mg/kg and 1.5mg/kg respectively. The vertical axis is the virus titer (unit: log10 (PFU/g)).

Figure 7: is a graph showing the blood drug concentrations of the antibody R6 and A2-G combination in mice, wherein the horizontal axis represents time (unit: h) and the vertical axis represents blood drug concentration (unit: μg/ml).

Figure 8: is a graph showing the blood drug concentration curve of the antibody R6 and A2-G combination in cynomolgus monkeys BM01 and cynomolgus monkeys BM02, with the horizontal axis representing time (unit: h) and the vertical axis representing blood drug concentration (unit: μg/ml).

Figure 9: is a graph showing the blood drug concentration curve of antibody R6 and A2-G combination in cynomolgus monkey BM01 and cynomolgus monkey BM02, with the horizontal axis representing time (unit: h) and the vertical axis representing blood drug concentration (unit: μg/ml).

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

Abbreviations

PBS refers to Phosphate Buffer Solution.

PBST refers to the addition of Tween-20 to PBS solution.

BSA refers to bovine serum albumin. BSA blocking solution is made from bovine serum albumin.

TMB refers to 3,3’,5,5’-Tetramethylbenzidine solution.

MEM medium refers to Minimum Essential Medium, which is a commonly used medium in animal cell culture.

FBS refers to fetal bovine serum.

ND (Not Detected) means that under experimental conditions, the antibody does not exhibit in vitro cell neutralization activity.

Reagents and instruments

RSV B9320 virus was purchased from ATCC catalog number VR-955.

RSV A2 virus was purchased from ATCC with catalog number VR-1540P.

RSV BWV viruses were purchased from ATCC with catalog number VR-1400.

RSV B18537 virus was purchased from ATCC catalog number VR-1580.

Sources of biological materials

The pCDNA3.1(+) expression vector was purchased from Invitrogen with the catalog number V79020.

Mouse myeloma cells SP2/0 were purchased from the American Type Culture Collection with the catalog number CRL-1581.

HEp-2 cells were purchased from ATCC with catalog number CCL-23.

293F cells were purchased from ATCC with catalog number CRL-1573.

Example 1. Screening of hybridoma cell lines

1. Production of recombinant RSV-F protein

According to the gene sequence of RSV B9320 strain fusion (F) protein in the NCBI database, the expression gene after adding the His tag was chemically synthesized, and the expression plasmid of RSV-F protein was constructed with the pCDNA3.1(+) expression vector. After transfection into 293F cells, the supernatant of cell expression was cultured and collected, concentrated and purified by nickel column (Cytiva) to obtain RSV-F protein, whose amino acid sequence is shown in SEQ ID NO:135.

2. Construction of hybridoma cells

40 6-8 week old BALB/C female mice were immunized after one week of feeding. RSV-F protein was used for the first immunization. Before immunization, Sigma Adjuvant System adjuvant (Sigma) was resuspended in 1 ml PBS, and then 1 ml RSV-F protein (1 mg/ml) was added as an antigen. The mixture was emulsified in equal volumes and injected intraperitoneally and subcutaneously. Each mouse was injected with 200 μL each time. 21 days (second immunization) and 42 days (third immunization) after the first immunization were boosted with the same immunization method as the first. Blood was collected on the 14th day after the second and third immunizations to detect the titer. When the titer reached 1:80000, the mouse spleen was taken for fusion. 72 hours before fusion, the immunization was boosted again, and RSV-F antigen protein was injected intraperitoneally once, 200 μL/mouse.

Take the spleen cells of the immunized mice and fuse them with the mouse myeloma cells SP2/0. First, grind the spleen to obtain a spleen cell suspension, mix it with the SP2/0 mouse myeloma cells in the logarithmic growth phase at a ratio of 1:1, fuse the two cells together by electrofusion to obtain hybridoma cells, and then dilute the fusion cell solution to 5000-10000 cells/ml and evenly spread it in a 96-well plate. The fusion medium is DMEM complete screening medium containing HAT and 20% FBS.

3. Screening of hybridoma mother cells using ELISA and Biacore methods

(1) The ELISA method was used to evaluate the immune effect (i.e., screening positive hybridoma mother cell lines). The specific steps were as follows: a. The RSV-F recombinant protein was diluted to 1 μg/mL with a coating solution (50 mM carbonate, pH 9.6), and 100 μL/well was coated on the ELISA plate and incubated at 4°C overnight; b. The plate washer was set to wash twice, and after washing twice with PBST (10 mM PBS + 0.05% Tween 20), 200 μL of 2% BSA was added to each well to block the ELISA plate, and the plate was incubated at 37°C for 2 h; c. The plate washer was set to wash twice, and after washing twice with PBST (10 mM PBS + 0.05% Tween 20), mouse serum or hybridoma supernatant diluted in 2% BSA blocking solution was added, 100 μL per well, and two replicate wells were set up, and a negative control was added, and the plate was incubated at 37°C for 1 h; d. After washing three times with PBST, the secondary antibody (Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP) (Invitrogen) diluted 10,000 times, per well Add 100 μL and incubate at 37°C for 1 hour; e. After washing four times with PBST, add 100 μL of single-component TMB colorimetric solution (Solarbio) to each well and color for 5-15 minutes in the dark; f. Stop the reaction: add 2M H ₂ SO ₄ to stop the reaction and add 50 μL to each well; g. Read: put the ELISA plate into the ELISA reader and read the results at a detection wavelength of 450 nm.

(2) Determine the Biacore binding activity of positive hybridoma mother cell lines. The specific steps are as follows: a. Centrifuge the positive hybridoma mother cell supernatant at 12000rpm for 10min and set aside; b. The equipment used is: Biacore 8K control software, the chip is CM5 chip; c. Write the detection program: set three cycles, then set the capture time (capture the antibody in the supernatant) to 300s, 30μL/min; the analysis sample concentration is 40nM, the binding time is 120s, 30μL/min; the dissociation time is 120s, and the regeneration (10mM glycine-HCl) is 30μL/min, 30s; then fill in the sample number and the protein concentration of the corresponding protein in the list, add the corresponding reagent according to the reagent position given by the program, cover the membrane, put the 96-well plate into the sample chamber, and start the program. d. Analyze the results, and obtain hybridoma mother cell lines with an affinity higher than nM ( ^10-9 ) level. The results are shown in Table 1.

Table 1 Hybridoma cell lines with affinity higher than nM (10 ^-9 ) level

4. Obtaining mouse antibodies

The above 6 hybridoma mother cell lines were subcloned and purified to obtain 8 mouse antibodies. The monoclonal cell lines of the 6 hybridoma mother cells were sequenced, and two of them The CDR region of the monoclonal cell of the hybridoma mother cell had individual amino acid mutations, and thus 8 mouse antibodies were obtained.

The subclone domestication steps are as follows:

1. Subcloning of hybridoma mother cells to establish monoclonal cell lines

(1) Resuscitate positive hybridoma mother cells and, after the cells are growing well, use the limiting dilution method for cloning.

(2) Take 140 μL of cells with good growth, of which 100 μL is used for dilution and 40 μL is used for counting. Dilute to 5 cells per ml with culture medium containing 20% FBS, OPI, and HAT, and plate 200 μL/well.

(3) Place in a 37°C 5% _CO2 incubator. After 7 days, take out the plate and observe it under an inverted microscope. If a monoclonal clone is visible to the naked eye, the antibody can be detected. Mark the plate cover, record it, and count the results.

(4) Indirect ELISA was used for detection. The positive wells with only a single clone growing were marked. The three single clone wells with the highest positive values were selected and transferred to a 24-well plate for expansion culture.

2. Serum-free suspension culture and acclimatization of monoclonal cell lines

(1) The positive monoclonal cells cultured in the 96-well plate were gently pipetted and transferred to a 24-well plate for culture (culture medium: DMEM+20% FBS+2% HAT+1% OPI).

(2) The cells were expanded to 12-well plates (culture medium: DMEM + 20% FBS + 1% OPI). When the cells were in good condition, they were transferred to T25 flasks for expansion.

(3) When the cell confluence in the T25 culture flask reaches more than 80%, gently tap the cells to mix well and then transfer them to a T75 culture flask. Add 20 mL of culture medium (DMEM + 5% FBS) and culture them statically.

(4) When the cell confluence in the T75 culture flask reaches more than 80%, tap the flask vigorously. After the cells in the flask are suspended, transfer all the cells to a 50 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add a small amount of culture medium (KD-Hybri + 2% FBS) to resuspend the cells and transfer them to a 125 mL shake flask. Add 20 mL of culture medium and place in a 37°C, 5% _CO2 incubator for shaking culture. Measure the cell viability and density after 36 hours.

(5) Count the cells that have been cultured in a shaking flask 36 hours after inoculation. If the cell density increases significantly and is between 2×10 ⁶ and 3.5×10 ⁶ cells/mL, transfer all the cells to a 50L centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and resuspend the cells in KD-Hybri (100×ITSplus added before use), dilute the cells to 0.5×10 ⁶ cells/mL, inoculate them into a 500 mL shaking flask, and place in a 50L centrifuge tube. The cells were cultured in an incubator with shaking at 110 rpm, 37°C, and 5% CO ₂ .

(6) Count the cells after shaking culture for 2-3 days. If the total cell density increases to 2×10 ⁶ cells/mL, centrifuge the cells at 8000 rpm for 20 min, collect the supernatant, and filter it through a 0.45 μm filter for later use.

5. In vitro cell activity assay of mouse antibodies

The mouse antibody was added to the HEp-2 cell culture medium of a 96-well microtiter plate in a volume of 50 μL/well in three-fold serial dilutions. Subsequently, 50 μL of samples containing RSV virus (including RSV A2, RSV B9320, RSV BWV, RSV B18537) diluted in a certain proportion were added to each well at 50 μL, and the virus without antibody was set as a positive control, and the antibody without virus was set as a negative control, and incubated at 37°C, 5% _CO2 incubator for 2h. 100 μL of the prepared Hep-2 cell suspension (2×10 ⁵ /mL) was added to each well, and the plates were incubated at 37°C in a 5% _CO2 incubator for 5 days. The neutralizing activity of the antibody was determined by indirect ELISA.

The calculation formula for the neutralizing activity (%) of neutralizing antibodies at different concentrations is as follows:

Neutralization activity (%) = 100 - (test well reading - cell control average) / (virus control average - cell control average) × 100

GraphPad Prism (version 5) was then used for nonlinear fitting analysis, and the _IC50 value of the pure neutralizing antibody was obtained based on the curve.

The results of in vitro cell activity of mouse antibodies are shown in Table 2. The data show that the eight mouse antibodies exhibited better protective efficacy than palivizumab under RSV virus attack.

Table 2 IC ₅₀ results of mouse antibodies neutralizing RSV A2, RSV B9320, RSV B WV and RSV B 18537

6. Sequencing of hybridoma subclone cell lines

The positive hybridoma cells obtained by the above method are cultured, and after the cells grow well, they are cloned by the limiting dilution method.

The cloned cell lines were sequenced, and the sequencing steps were as follows: a. Collect cells, treat with Trizol (Tiangen Biochemical Technology Co., Ltd.), and extract total RNA; b. Reverse transcription: Take 8μL RNA (50ng-5ug) and 1μL primer Oligo (dT) 18 and add them to a 0.2mL PCR tube, incubate at 65℃ for 5min, and ice bath for 2min. Add 10μL 2×ES Reaction Mix, 1μL RT Enzyme Mix, incubate at 42℃ for 30min, and 85℃ for 5 seconds to terminate transcription; c. Perform PCR amplification under the following conditions: 94℃ for 5min; 94℃ for 30s, 55℃ for 30s, 72℃ for 45s, 30 cycles; 72℃ for 10min; 4℃ for 5min; d. The amplified products were sequenced.

Results: The sequencing results of the subcloned hybridoma cell lines are shown in Table 3. In addition, the amino acid sequences of the hypervariable regions (CDRs) of the subcloned hybridoma cell lines in this embodiment are defined by Kabat.

Table 3 Sequencing results of subcloned hybridoma cell lines

Example 2. Preparation of chimeric antibodies

1. Construction and expression of chimeric antibodies

A chimeric antibody was constructed using a fusion PCR method: the variable region of the mouse antibody in Example 1 above was connected to the coding gene of the human antibody constant region (heavy chain constant region amino acid sequence: SEQ ID NO: 111, light chain constant region amino acid sequence: SEQ ID NO: 112) to construct a fusion gene, the fusion gene was amplified, and cloned into the pCDNA3.1(+) expression vector to construct an expression plasmid for the chimeric antibody, and after transfection of 293F cells, the supernatant of the cell expression was cultured and collected, concentrated, and purified by Protein A (Cytiva) to obtain the corresponding chimeric antibody.

2. Affinity determination of chimeric antibodies

The antibody was captured at a flow rate of 10 μl/min for 60 s. The antigen was bound at a flow rate of 30 μl/min for 120 s, and then dissociated at a flow rate of 30 μl/min for 1800 s. The flow rate was regenerated at 30 μl/min for 30 seconds. After the experiment was completed, the results were processed using analysis software. The results are shown in Table 4. The above chimeric antibodies all had significant high affinity for RSV-F protein.

Table 4 Affinity results of chimeric antibodies binding to RSV-F protein

3. In vitro cell activity assay of chimeric antibodies

Detection of the cell neutralization activity of the chimeric antibodies shown in Table 4 (see Example 1 for the detection method)

The results of the in vitro cell activity of the chimeric antibodies are shown in Table 5. The data show that the antibodies provided by the present invention have the ability to neutralize RSV A2, RSV B9320, RSV B WV and RSV B18537. Among them, SQ-1, SQ-2, SQ-3, SQ-4, and SQ-8 showed obvious specificity for RSV B virus strains (such as RSV B9320, RSV B WV, and RSV B18537), especially SQ-1 (heavy chain amino acid sequence: SEQ ID NO: 129, light chain amino acid sequence: SEQ ID NO: 130) had lower IC ₅₀ than palivizumab in neutralizing RSV A2, RSV B9320, RSV B WV, and RSV B18537.

Table 5 IC ₅₀ results of chimeric antibodies neutralizing RSV A2, RSV B9320, RSV B WV and RSV B18537

Example 3. Humanization of Antibody S-Q-1

1. Preparation of humanized antibodies

The mouse CDR region of antibody S-Q-1 is retained, and the Fr region of S-Q-1 is humanized and transplanted. The steps of humanization and transplantation of the Fr region are as follows:

(1) The Prediction function in the Bioluminate software was used to perform homology modeling on the heavy and light chain sequences of the variable regions of mouse antibodies, and the reliability of the homology modeling was evaluated using the Reliable report function. Based on this evaluation, the model was further optimized to obtain the optimized antibody variable region model.

(2) The variable regions of the humanized antibodies were compared using NCBI Ig Blast, and the human germline sequence with the highest homology was selected for CDR transplantation. In the first round of humanization, all the mouse antibody FR sequences were replaced with humanized ones.

(3) Through the advanced option in CDR grafting, key sites such as Canonical Structure Region and Vernier Zone in the framework region FR are predicted.

The sequences of the heavy and light chain variable regions after humanization are shown in Table 6:

Table 6 Heavy and light chain variable regions of chimeric antibodies after humanization

2. Affinity determination of humanized antibodies

Capture the antibody at a flow rate of 10 μl/min for 60 s. Bind the antigen at a flow rate of 30 μl/min for 120 s, and then dissociate at a flow rate of 30 μl/min for 1800 s. Regenerate at a flow rate of 30 μl/min for 30 seconds. After the experiment is over, use the analysis software to process the results. The results are shown in Table 7. The above humanized antibodies bind to RSV-F protein with high affinity. The experimental data show that the affinity of humanized antibodies binding to antigens is higher than nM (10 ^-9 ).

Table 7 Affinity results of humanized antibodies binding to RSV-F protein

3. Cellular activity assay of humanized antibodies

The cell neutralization activity of the humanized antibodies in Table 6 was detected (see Example 1 for the detection method). The results are shown in Table 8. The data show that the humanized modified antibodies based on SQ-1 developed by the present invention have lower IC ₅₀ than palivizumab in neutralizing RSV A2, RSV B9320, RSV BWV, and RSV B18537.

Table 8 IC ₅₀ results of humanized antibodies based on SQ-1 neutralizing RSV A2, RSV B9320, RSV BWV and RSV B18537

Example 4. Animal efficacy experiment of humanized antibodies

1. RSV B9320 virus infection related experiments

Mice were grouped and injected with antibodies: 7-9 week old mice were prepared and randomly divided into 5 mice per group. After 1 week of adaptive feeding, they were divided into positive control palivizumab group (15 mg/kg, 5 mg/kg, 1.5 mg/kg, 0.5 mg/kg, 0.15 mg/kg); experimental group R6 (heavy chain amino acid sequence: SEQ ID NO: 131, light chain amino acid sequence: SEQ ID NO: 132) group (dose 5 mg/kg, 1.5 mg/kg, 0.5 mg/kg); experimental group R9 (heavy chain amino acid sequence: SEQ ID NO: 133, light chain amino acid sequence: SEQ ID NO: 134) group (dose 5 mg/kg, 1.5 mg/kg, 0.5 mg/kg); blank control PBS group. One day before virus infection (-1d), 100 μL of antibody was injected intraperitoneally.

Nasal drops: On the second day (0 d), mice were anesthetized with isoflurane, and 100 μL of RSV B9320 virus (10 ⁷ PFU/mL) was dropped into the nasal cavity of each mouse.

Lung testing: 5 days after infection (5d), mouse lung tissue was taken and placed in 1mL MEM culture medium (Gibco). The lung tissue was made into tissue homogenate using a cell grinder and a mesh sieve. After centrifugation at 2000rpm at 4℃ for 2.5h, the supernatant was collected and the virus titer in the tissue homogenate supernatant was detected. After the first well was diluted 2 times, 100μL was added to a 96-well plate and the sample to be tested was diluted in a 1:2 gradient. Then 100μL 2.5× ¹⁰⁵ /mL Hep2 cells were added and cultured in a _CO2 incubator for 5-6 days. After observing the cell pathological changes, the RSV pathological positive wells were detected by ELISA.

The virus titer [log10 (PFU/g)] in the supernatant of lung tissue homogenate was calculated according to the number of positive wells.

The results are shown in Figure 1. R6 and R9 showed significant superiority against RSV attack. Protective effect of palivizumab.

2. RSV B18537 virus infection related experiments

The experimental procedures were similar to those for RSV B9320 virus infection of mice, with the only difference being that 100 μL of RSV B18537 virus (10 ⁷ PFU/mL) was dripped into the nose.

The results are shown in Figure 2. R6 and R9 showed significantly better protective effects than palivizumab against RSV virus attack.

3. RSV BWV virus infection related experiments

The experimental steps refer to the experimental steps related to RSV B9320 virus infection in mice, except that 100 μL RSV BWV virus (10 ⁷ PFU/mL) was dripped into the nose, and the groups were designed as positive control palivizumab group (5 mg/kg); positive control MEDI8897 group (doses of 5 mg/kg, 1.5 mg/kg, 0.5 mg/kg), experimental group R6 group (doses of 5 mg/kg, 1.5 mg/kg, 0.5 mg/kg); blank control PBS group. MEDI8897 is Nirsevimab, a preventive monoclonal antibody drug developed by AstraZeneca and Sanofi for RSV.

The results are shown in Figure 3. R6 showed a significantly better protective effect than palivizumab and MEDI8897 against RSV attack, which indicates that the antibody sequence provided by the present invention has good drugability.

Example 5. Synthesis of Secondary Antibodies

The antibodies described in Tables 9 to 11 are the second antibodies of the present invention, which are synthesized with reference to Examples 1-4 of patent application WO2023025253.

First, the mouse antibodies shown in Table 9 were obtained by screening hybridoma cell lines, and then the constant regions of the mouse antibodies were replaced with the constant regions of the human antibodies (heavy chain constant region amino acid sequence: SEQ ID NO: 252, light chain constant region amino acid sequence: SEQ ID NO: 253) to obtain chimeric antibodies. Finally, the R10-19B2QH chimeric antibody with good results in in vitro cell activity experiments was selected, the mouse CDR region was retained, and the Fr region of R10-19B2QH was humanized and transplanted to obtain the humanized antibodies shown in Table 10. Then, the humanized antibody A2 with good results in animal experiments was selected to perform site-directed saturation mutagenesis on the cysteine sites in its light chain CDR1 region to obtain the CDR-modified antibodies based on the humanized antibody A2 shown in Table 11.

The amino acid sequence of the light chain of humanized antibody A2 is as follows:

The amino acid sequence of the humanized antibody A2 heavy chain is as follows:

The light chain amino acid sequence of A2-G obtained after mutation of humanized antibody A2 is:

The heavy chain amino acid sequence of A2-G obtained after mutation of humanized antibody A2 is:

The light chain amino acid sequence of A2-A obtained after mutation of humanized antibody A2 is:

The heavy chain amino acid sequence of A2-A obtained after mutation of humanized antibody A2 is:

Table 9 Mouse Antibodies

Table 10 Humanized Antibodies

Table 11 CDR-modified antibodies based on humanized antibody A2

Example 6. Epitope determination experiment of antibodies R6 and A2-G

The epitopes of antibodies R6 and A2-G were determined as follows:

a. Coating: Prepare CBS buffer, dissolve 1 μg/mL antibody MEDI8897 and Palivizumab in CBS buffer, add 100 μL/well to the ELISA plate using a multi-channel pipette, and incubate overnight at 4°C.

b. Washing: After coating, discard the coating solution and wash with PBST, 300 μL/well, wash twice, and pat dry on paper towels.

c. Blocking: 2% BSA (VETEC) dissolved in PBST was used as blocking solution. Blocking was performed after coating, 200 μL/well, and incubated at 37°C for 1 h.

d. Washing: The specific steps are the same as b. Keep at -20℃ when not in use.

e. Antibody-conjugated HRP: Prepare R6-HRP and A2-G-HRP according to the kit instructions.

f. Sample reaction: 100 ng antigen A2/B9320 and gradient dilution R6-HRP (100ng-0.0457ng), A2-G-HRP (250ng-1.54ng) were incubated at 37°C for 2h.

g. Add samples: Add the co-incubated antigen + R6/A2-G mixture to the ELISA plate in d and incubate at 37°C for 1 hour.

h. Washing: The specific steps are the same as b.

i. Color development: Add 100 μL/well TMB color development solution, color development at room temperature or 37°C

j. Stop: Add 50 μL/well of 10% H ₂ SO ₄ stop solution to stop color development, and read the OD450 data using a plate reader.

The results are shown in Table 12. When the plate was coated with MEDI8897, the OD450 reading was correlated with the R6 content, indicating that MEDI8897 did not affect the binding of R6 to antigens A2 and B9320. The OD450 reading on the palivizumab plate was significantly lower, and palivizumab had a competitive inhibitory effect on the binding of R6 to the antigen. Therefore, it was concluded that the epitopes of R6 and palivizumab were consistent, both of which were site Ⅱ. The results of the A2-G epitope competition experiment are shown in Table 13, and it can be concluded that the A2-G epitope was consistent with MEDI8897, which was site Φ.

Table 12 R6 epitope competition

Table 13 A2-G epitope competition

Example 7. Experiments on neutralization of RSV virus by antibody composition

1. RSV A2 virus infection related experiments

Mice were divided into groups for antibody injection: 7-9 week old mice were prepared and randomly divided into 5 mice per group. After 1 week of adaptive feeding, they were divided into positive control palivizumab group (15 mg/kg, 5 mg/kg, 0.5 mg/kg); experimental group R6+A2-G group (two antibodies were mixed in a ratio of 1:1, and the dosage was 0.5mg/kg, 0.15mg/kg, 0.05mg/kg); positive control MEDI 8897 group (dose 0.5mg/kg, 0.15mg/kg, 0.05mg/kg), blank control PBS group. One day before virus infection (-1d), 100μL antibody was injected intraperitoneally.

Nasal drops: On the second day (0 d), mice were anesthetized with isoflurane, and 100 μL of RSV A2 virus (10 ⁷ PFU/mL) was dropped into the nasal cavity of each mouse.

As shown in Figure 4, the efficacy of the antibody combination R6+A2-G against RSV A2 is comparable to that of MEDI 8897 and palivizumab to a certain extent .

2. RSV B9320 virus infection related experiments

Mice were grouped and injected with antibodies: 7-9 week old mice were prepared and randomly divided into 5 mice per group. After 1 week of adaptive feeding, they were divided into positive control palivizumab group (15 mg/kg, 5 mg/kg, 1.5 mg/kg); experimental group R6+A2-G group (two antibodies were mixed in a ratio of 1:1, and the dosage was 5 mg/kg, 1.5 mg/kg, and 0.5 mg/kg); positive control MEDI 8897 group (doses of 1.5 mg/kg, 0.5 mg/kg, and 0.05 mg/kg), and blank control PBS group. One day before virus infection (-1d), 100 μL of each group of antibodies was intraperitoneally injected.

Lung testing: 5 days after infection (5d), mouse lung tissue was taken and placed in 1mL MEM medium (Gibco), and the lung tissue was made into tissue homogenate using a cell grinding rod and a mesh sieve. After centrifugation at 4℃2000rpm for 2.5h, the supernatant was collected and the virus titer in the tissue homogenate supernatant was detected. After the first well was diluted 2 times, 100μL was added to a 96-well plate and the sample to be tested was diluted in a gradient of 1:2. Then 100μL 2.5× ¹⁰⁵ /mL Hep2 cells were added and cultured in a _CO2 incubator for 5-6 days. After observing the cytopathic condition, RSV virus was detected by ELISA. Positive hole.

The virus titer [log10 (PFU/g)] in the supernatant of lung tissue homogenate was calculated based on the number of positive wells. The results are shown in Figure 5 , and the efficacy of the antibody composition R6+A2-G against RSV B9320 is comparable to that of MEDI 8897 and palivizumab.

3. RSV BWV virus infection related experiments

Mice were grouped and injected with antibodies: 7-9 week old mice were prepared and randomly divided into 5 mice per group. After 1 week of adaptive feeding, they were divided into positive control palivizumab group (15 mg/kg, 5 mg/kg, 1.5 mg/kg); experimental group R6+A2-G group (two antibodies were mixed in a ratio of 1:1, and the dosage was 5 mg/kg, 1.5 mg/kg, and 0.5 mg/kg); positive control MEDI 8897 group (doses of 15 mg/kg, 5 mg/kg, and 1.5 mg/kg), and blank control PBS group. One day before virus infection (-1d), 100 μL of each group of antibodies was intraperitoneally injected.

Nasal drops: On the second day (0 d), mice were anesthetized with isoflurane, and 100 μL of RSV BWV virus (10 ⁷ PFU/mL) was dropped into the nasal cavity of each mouse.

The virus titer in the lung tissue homogenate supernatant was calculated based on the number of positive wells [log10 (PFU/g)]. The results are shown in Figure 6, and the antibody combination R6+A2-G is more effective against RSV BWV virus than MEDI8897.

Example 8. Pharmacokinetic (PK) experiment of antibody composition

1. PK experiment in mice

(1) 6-8 week old BALB/c female mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., about 0.02 kg) were divided into two groups, 5 mice in each group, and injected with A2-G + R6 antibody combination (dosage: antibody A2-G 10 mg/kg + antibody R6 10 mg/kg) through the tail vein. 10 μL of blood was collected from the tip of the tail and diluted with 90 μL of PBS, and centrifuged at 5000 rpm for 5 min. The blood was collected at 0.5 h, 1 h, 4 h, 8 h, 24 h, 48 h, 96 h, 168 h, 336 h, 504h, 672h.

(2) Take the diluted blood supernatant for ELISA detection: Take 100μL of the serially diluted serum sample and add it to the ELISA plate coated with RSV-A2/RSV-B9320 recombinant protein 100ng/well, and incubate for 1h. After washing twice with 1×PBST, add 100μL of secondary antibody Mouse Anti-Human IgG Fc Antibody [HRP] (1:10000) and incubate at 37℃ for 1h. After washing 3 times with 1×PBST, add 100μL of single-component TMB colorimetric solution to each well and color for 5-15min in the dark; stop the reaction: add 50μL 2M H2SO4 to each well to stop the reaction; read: put the ELISA plate into the ELISA reader and read the reading. The detection wavelength is OD450, and the antibody concentration in the serum sample is calculated based on the standard curve.

The following pharmacokinetic parameters were calculated using the non-compartmental statistical moment method of DAS software: AUC _0-t , AUC _0-∞ , MRT _0-∞ , C _max , T _max , and t _1/2 . The experimental results are shown in Table 14 , and the drug-time curve is shown in Figure 7 .

Table 14

Conclusion: The results in Table 14 and Figure 7 show that the two antibodies in the A2-G+R6 antibody composition provided by the present invention do not produce antagonistic effects in mice.

2. PK experiment in cynomolgus monkeys

(1) 2-2.5 years old male cynomolgus monkeys (2.0-4.5kg, two, numbered BM01 and BM02) were injected intramuscularly with A2-G+R6 (dosage: A2-G 30mg/kg, R6 30mg/kg). Whole blood samples (about 1.0mL) were taken from the vein of the non-administered limb of the cynomolgus monkey, and taken from the blood collection needle into a labeled procoagulant tube, and immediately placed in an ice box for storage. The blood was allowed to coagulate naturally and then centrifuged (centrifugation conditions: temperature: 2-8℃, centrifugal force: 1500-2500×g, time: 10-15min). The serum was separated into labeled low-adsorption EP tubes. The serum samples were stored in a refrigerator at -60 to -90℃. The blood samples were collected within 4 hours from the time of sample storage. The blood collection time points were 0h before administration, 1h, 4h, 1d, 2d, 3d, 4d, 6d, 8d, 10d, 12d, 14d, 16d, 20d, 24d, 31d, 41d, 55d.

The following pharmacokinetic parameters were calculated using the non-compartmental statistical moment method of DAS software: AUC _0-t , AUC _0-∞ , MRT _0-∞ , Cmax, Tmax, and t _1/2 . The experimental results are shown in Table 15 , and the drug-time curves are shown in Figures 8 and 9 .

Table 15

Conclusion: The results in Table 15, Figure 8 and Figure 9 show that the two antibodies in the A2-G+R6 antibody composition provided by the present invention do not produce antagonistic effects in cynomolgus monkeys.

Example 9: Preparation formula screening

1. Prepare the formulation according to Table 16

Table 16 Preparation formula

2. Formulation screening

Recipe 1:

Conclusion: After the formulation was stored at 40°C for 2 weeks, the content of single antibodies in the antibody preparation did not change much, and the growth of aggregates was not obvious, indicating that the antibodies could be stably stored under this formulation and the stability of the preparation was relatively strong.

Recipe 2:

Conclusion: After the formulation was stored at 40°C for 8 weeks, the single antibody content in the formulation could still maintain more than 90%. After accelerated storage at 4°C for 8 weeks, the single antibody content only decreased by about 1%, indicating that the antibody can be stably stored under the formulation and the formulation is relatively stable.

The present invention has been described through the above embodiments, but it should be understood that the above embodiments are only for the purpose of example and description, and are not intended to limit the present invention to the scope of the described embodiments. In addition, it can be understood by those skilled in the art that the present invention is not limited to the above embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the scope of the protection claimed by the present invention. The protection scope of the present invention is defined by the attached claims and their equivalents.

Sequence Listing

Claims

An antibody or antigen-binding fragment thereof comprising:

The three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment with the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109; and/or

The three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment with the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110.
An antibody or antigen-binding fragment thereof comprising:

1) HCDR3, wherein the HCDR3 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 19, 25, 31, 37, 43, 49, 55, and 61; and

2) LCDR3, wherein LCDR3 comprises any amino acid sequence in the group consisting of the following amino acid sequences or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 22, 28, 34, 40, 46, 52, 58, 64, and 70.
The antibody or antigen-binding fragment thereof according to claim 2, comprising:

1) HCDR1, wherein the HCDR1 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 17, 23, 29, 35, 41, 47, 53, and 59; and

2) LCDR1, wherein LCDR1 comprises or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 20, 26, 32, 38, 44, 50, 56 and 62;

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

3) HCDR2, wherein the HCDR2 comprises or consists of any one of the group consisting of the following amino acid sequences: SEQ ID NO: 18, 24, 30, 36, 42, 48, 54 and 60; and

4) LCDR2, wherein LCDR2 comprises or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 21, 27, 33, 39, 45, 51, 57 and 63.
The antibody or antigen-binding fragment thereof according to claim 1, comprising:

1) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 1, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:2; or

2) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 3, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:4; or

3) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO:5, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:6; or

4) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO:7, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:8; or

5) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO:9, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:10; or

6) The amino acid sequence is the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 11, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:12; or

7) The amino acid sequence of the three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the peptide segment shown in SEQ ID NO: 13, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown in SEQ ID NO:14; or

8) The amino acid sequence is the three heavy chains contained in the peptide segment shown in SEQ ID NO: 15. complement determining regions HCDR1, HCDR2 and HCDR3, and

The amino acid sequence is the three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the peptide segment shown as SEQ ID NO:16.
The antibody or antigen-binding fragment thereof according to claim 4, comprising:

1) HCDR1 comprising or consisting of SEQ ID NO: 17,

HCDR2 comprising or consisting of SEQ ID NO:18,

HCDR3 comprising or consisting of SEQ ID NO:19,

LCDR1 comprising or consisting of SEQ ID NO:20,

LCDR2 comprising or consisting of SEQ ID NO:21, and

LCDR3 comprising or consisting of SEQ ID NO:22; or

2) HCDR1 comprising or consisting of SEQ ID NO: 23,

HCDR2 comprising or consisting of SEQ ID NO:24,

HCDR3 comprising or consisting of SEQ ID NO:25,

LCDR1 comprising or consisting of SEQ ID NO:26,

LCDR2 comprising or consisting of SEQ ID NO:27, and

LCDR3 comprising or consisting of SEQ ID NO:28; or

3) comprising HCDR1 represented by or consisting of SEQ ID NO: 29,

HCDR2 comprising or consisting of SEQ ID NO:30,

HCDR3 comprising or consisting of SEQ ID NO:31,

LCDR1 comprising or consisting of SEQ ID NO:32,

LCDR2 comprising or consisting of SEQ ID NO:33, and

LCDR3 comprising or consisting of SEQ ID NO:34; or

4) HCDR1 comprising or consisting of SEQ ID NO:35,

HCDR2 comprising or consisting of SEQ ID NO:36,

HCDR3 comprising or consisting of SEQ ID NO:37,

LCDR1 comprising or consisting of SEQ ID NO:38,

LCDR2 comprising or consisting of SEQ ID NO:39, and

LCDR3 comprising or consisting of SEQ ID NO:40; or

5) HCDR1 comprising or consisting of SEQ ID NO:41,

A HCDR2 comprising or consisting of SEQ ID NO:42,

HCDR3 comprising or consisting of SEQ ID NO:43,

LCDR1 comprising or consisting of SEQ ID NO:44,

LCDR2 comprising or consisting of SEQ ID NO:45, and

LCDR3 comprising or consisting of SEQ ID NO:46; or

6) HCDR1 comprising or consisting of SEQ ID NO:47,

HCDR2 comprising or consisting of SEQ ID NO:48,

HCDR3 comprising or consisting of SEQ ID NO:49,

LCDR1 comprising or consisting of SEQ ID NO:50,

LCDR2 comprising or consisting of SEQ ID NO:51, and

LCDR3 comprising or consisting of SEQ ID NO:52; or

7) HCDR1 comprising or consisting of SEQ ID NO:53,

HCDR2 comprising or consisting of SEQ ID NO:54,

HCDR3 comprising or consisting of SEQ ID NO:55,

LCDR1 comprising or consisting of SEQ ID NO:56,

LCDR2 comprising or consisting of SEQ ID NO:57, and

LCDR3 comprising or consisting of SEQ ID NO:58; or

8) HCDR1 comprising or consisting of SEQ ID NO:59,

HCDR2 comprising or consisting of SEQ ID NO:60,

HCDR3 comprising or consisting of SEQ ID NO:61,

LCDR1 comprising or consisting of SEQ ID NO:62,

LCDR2 comprising or consisting of SEQ ID NO:63, and

LCDR3 comprising or consisting of SEQ ID NO:64.
An antibody or antigen-binding fragment thereof comprising:

A heavy chain variable region, the amino acid sequence of the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109; preferably, the heavy chain variable region is selected from the group consisting of SEQ ID NO: NO:1,3,5,7,9,11,13,15,65,67,69,71,73,75,77,79,81,83,85,87,89,91, 93, 95, 97, 99, 101, 103, 105, 107 and 109, compared with an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions), preferably, the amino acid mutations do not occur in the heavy chain complementary determining region; preferably, the heavy chain variable region has a CDRs region amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, and the amino acid sequence of the framework region of the heavy chain variable region is identical to the amino acid sequence of SEQ ID NO: 1, 3, 5 NO:1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109 has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical; preferably, the heavy chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109; and/or a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the amino acid sequences selected from the group consisting of: SEQ ID ... NO:2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110; preferably, the light chain variable region is selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110, compared with an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions), preferably, the amino acid mutations do not occur in the complementary determining region of the light chain; preferably, the amino acid sequence of the light chain variable region has the same amino acid sequence as SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110, and the framework region of the light chain variable region is the same as the sequence of SEQ ID NO:2, 4, 6, 8, 10, The framework regions of the light chain variable region of the present invention have at least 80% identity with those of SEQ ID NO: 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110; preferably, the light chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110.
The antibody or antigen-binding fragment thereof according to claim 6, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence pair of the heavy chain variable region/light chain variable region is selected from any one of the group consisting of the following amino acid sequence pairs:

SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO:81 and SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:85 and SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:96 ID NO:96, SEQ ID NO:97 and SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:106, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110;

Preferably, the amino acid sequence pair of the heavy chain variable region/light chain variable region is selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: : 94, SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102, SEQ ID NO:103 and SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:106, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112, SEQ ID NO:113 and SEQ ID NO:114, SEQ ID NO:115 and SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118, SEQ ID NO:119 and SEQ ID NO:120 NO:104, SEQ ID NO:105 and SEQ ID NO:106, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO:110.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, further comprising a constant region, wherein the constant region is derived from an IgG antibody, an IgM antibody, an IgA antibody, an IgD antibody or an IgE antibody, preferably, the constant region is derived from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which comprises a heavy chain constant region and/or a light chain constant region, preferably it comprises a murine or humanized heavy chain constant region and/or a light chain constant region; preferably, the amino acid sequence of the humanized heavy chain constant region is as shown in SEQ ID NO: 111 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence SEQ ID NO: 111, and the amino acid sequence of the humanized light chain constant region is as shown in SEQ ID NO: 112. An amino acid sequence as shown or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence SEQ ID NO:112; preferably, the amino acid sequence pair of the heavy chain/light chain of the antibody or its antigen-binding fragment is selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NO:129 and SEQ ID NO:130, SEQ ID NO:131 and SEQ ID NO:132, SEQ ID NO:133 and SEQ ID NO:134.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein each CDR region is defined according to the Kabat definition scheme, the Chothia definition scheme, the Abm definition scheme, the IMGT definition scheme and/or the Contact definition scheme; preferably, wherein The antibody is a murine antibody, a chimeric antibody or a humanized antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (preferably scFv) or (Fab') 2 , single domain antibody, diabody (dAb) or linear antibody.
A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.
An expression vector comprising the nucleic acid according to claim 12.
A host cell comprising the expression vector according to claim 13.
A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to claim 14, and recovering the antibody or the antigen-binding fragment thereof expressed thereby from the culture.
The method of claim 15, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
The method according to claim 15 or 16, wherein the host cell is an Escherichia coli cell, a yeast cell, an insect cell, a plant cell or a mammalian cell.
The method of claim 15, wherein the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell or a NSO cell.
A pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and a pharmaceutically acceptable excipient.
A pharmaceutical composition according to claim 19, further comprising a second antibody or antigen-binding fragment thereof capable of neutralizing RSV virus, wherein the amino acid sequence of the second antibody or antigen-binding fragment thereof is different from the amino acid sequence of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11. 21. A pharmaceutical composition comprising:

1) a first antibody or an antigen-binding fragment thereof that can bind to the site II epitope of RSV-F protein, preferably, the first antibody or an antigen-binding fragment thereof is any one of the antibodies or antigen-binding fragments thereof as described in any one of claims 1 to 11; and

2) A second antibody or an antigen-binding fragment thereof that can neutralize RSV virus, wherein the amino acid sequence of the second antibody or the antigen-binding fragment thereof is different from the amino acid sequence of the first antibody or the antigen-binding fragment thereof.
The pharmaceutical composition of claim 20 or 21, characterized in that the RSV-related protein bound by the second antibody or antigen-binding fragment thereof is the same as or different from the RSV-F protein bound by the first antibody or antigen-binding fragment thereof or any one of the antibodies or antigen-binding fragments of any one of claims 1 to 11;

Preferably, the RSV-related protein is RSV-F protein or RSV-G protein;

Preferably, the RSV-related protein is RSV-F protein.
The pharmaceutical composition according to any one of claims 20 to 22, characterized in that the antigenic epitope of the RSV-F protein bound by the second antibody or its antigen-binding fragment is the same as or different from the site II epitope of the RSV-F protein bound by the first antibody or its antigen-binding fragment or any antibody or its antigen-binding fragment according to any one of claims 1 to 11;

Preferably, the antigenic epitope of RSV-F protein bound by the second antibody or its antigen-binding fragment is selected from any one of site φ epitope, site I epitope, site II epitope, site III epitope, site IV epitope and site V epitope;

Preferably, the antigenic epitope of the RSV-F protein to which the second antibody or antigen-binding fragment thereof binds is selected from any one of the site φ epitope, the site I epitope, the site III epitope, the site IV epitope and the site V epitope;

Preferably, the antigenic epitope of RSV-F protein bound by the second antibody or antigen-binding fragment thereof is a siteφ epitope.
The pharmaceutical composition according to any one of claims 20 to 23, wherein the second antibody or antigen-binding fragment thereof is selected from Motavizumab, Palivizumab, Nirsevimab, Suptavumab, Clesrovimab, and Felvizumab.
The pharmaceutical composition according to any one of claims 20 to 23, wherein the second antibody or antigen-binding fragment thereof comprises:

The amino acid sequence of the three heavy chain complementary determining regions (CDRs) HCDR1', HCDR2' and HCDR3' contained in the peptide segment of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150 or 152; and/or

The three light chain complementary determining regions (CDRs) LCDR1', LCDR2' and LCDR3' contained in the peptide segment with the amino acid sequence of SEQ ID NO: 137, 139, 141, 143, 145, 147, 149, 151, 153, 164, 165, 166, 167, 168, 169, 170 or 171.
The pharmaceutical composition according to any one of claims 20 to 23, wherein the second antibody or antigen-binding fragment thereof comprises:

a) a HCDR3′ functional region having any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 174, 180, 186, 192, 198, 204, 210, 216, and 222; and

b) LCDR3’ functional region, wherein the LCDR3’ functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 177, 183, 189, 195, 201, 207, 213, 219, and 225.
The pharmaceutical composition of claim 26, wherein the second antibody or antigen-binding fragment thereof further comprises:

c) a HCDR1′ functional region, wherein the HCDR1′ functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 172, 178, 184, 190, 196, 202, 208, 214, and 220;

d) LCDR1' functional region, wherein the LCDR1' functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 175, 181, 187, 193, 199, 205, 211, 217, 223, 244, 245, 246, 247, 248, 249, 250 and 251;

e) a HCDR2′ functional region having any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 173, 179, 185, 191, 197, 203, 209, 215, and 221; and

f) LCDR2’ functional region, wherein the LCDR2’ functional region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 176, 182, 188, 194, 200, 206, 212, 218, and 224.
The pharmaceutical composition according to any one of claims 25 to 27, wherein the second antibody or antigen-binding fragment thereof comprises:

1) three heavy chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO: 148, and

The three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:149, 164, 165, 166, 167, 168, 169, 170, or 171; or

2) The three heavy chains contained in the peptide segment with the amino acid sequence of SEQ ID NO: 136 are complementary determining regions (CDRs), and

The three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:137; or

3) Three heavy chain complementary determining regions (CDRs) and

The three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:139; or

4) three heavy chain complementary determining regions (CDRs) contained in the peptide segment having the amino acid sequence of SEQ ID NO: 140, and

The three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:141; or

5) three heavy chain complementary determining regions (CDRs) contained in the peptide segment having the amino acid sequence of SEQ ID NO: 142, and

The three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:143; or

6) three heavy chain complementary determining regions (CDRs) contained in the peptide segment having the amino acid sequence of SEQ ID NO: 144, and

The three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:145; or

7) three heavy chain complementary determining regions (CDRs) contained in the peptide segment having the amino acid sequence of SEQ ID NO: 146, and

The three light chain complementary determining regions (CDRs) contained in the peptide segment with the amino acid sequence of SEQ ID NO:147; or

8) three heavy chain complementary determining regions (CDRs) contained in the peptide segment having the amino acid sequence of SEQ ID NO: 150, and

The amino acid sequence is the three light chain complementary determining regions (CDRs) contained in the peptide segment of SEQ ID NO:151; or

9) three heavy chain complementary determining regions (CDRs) contained in the peptide segment having the amino acid sequence of SEQ ID NO: 152, and

The three light chain complementary sequences contained in the peptide segment with the amino acid sequence of SEQ ID NO: 153 CDRs.
The pharmaceutical composition of claim 28, wherein the second antibody or antigen-binding fragment thereof comprises:

1) HCDR1′ comprising or consisting of SEQ ID NO: 172,

HCDR2' comprising or consisting of SEQ ID NO:173,

HCDR3' comprising or consisting of SEQ ID NO:174,

LCDR1' comprising or consisting of SEQ ID NO:175,

LCDR2' comprising or consisting of SEQ ID NO:176, and

LCDR3' comprising or consisting of SEQ ID NO:177; or

2) HCDR1′ comprising or consisting of SEQ ID NO: 178,

HCDR2' comprising or consisting of SEQ ID NO:179,

HCDR3' comprising or consisting of SEQ ID NO: 180,

LCDR1' comprising or consisting of SEQ ID NO: 181,

LCDR2' comprising or consisting of SEQ ID NO: 182, and

LCDR3' comprising or consisting of SEQ ID NO:183; or

3) HCDR1′ comprising or consisting of SEQ ID NO: 184,

HCDR2' comprising or consisting of SEQ ID NO:185,

HCDR3' comprising or consisting of SEQ ID NO:186,

LCDR1' comprising or consisting of SEQ ID NO: 187,

LCDR2' comprising or consisting of SEQ ID NO: 188, and

LCDR3' comprising or consisting of SEQ ID NO:189; or

4) HCDR1′ comprising or consisting of SEQ ID NO: 190,

HCDR2' comprising or consisting of SEQ ID NO:191,

HCDR3' comprising or consisting of SEQ ID NO:192,

LCDR1' comprising or consisting of SEQ ID NO: 193,

LCDR2' comprising SEQ ID NO: 194 or consisting thereof, and

LCDR3' comprising or consisting of SEQ ID NO:195; or

5) HCDR1′ comprising or consisting of SEQ ID NO: 196,

HCDR2' comprising or consisting of SEQ ID NO:197,

HCDR3' comprising or consisting of SEQ ID NO: 198,

LCDR1' comprising or consisting of SEQ ID NO: 199,

LCDR2' comprising or consisting of SEQ ID NO: 200, and

LCDR3' comprising or consisting of SEQ ID NO:201; or

6) HCDR1′ comprising or consisting of SEQ ID NO:202,

HCDR2' comprising or consisting of SEQ ID NO:203,

HCDR3' comprising or consisting of SEQ ID NO:204,

LCDR1' comprising or consisting of SEQ ID NO:205,

LCDR2' comprising or consisting of SEQ ID NO:206, and

LCDR3' comprising or consisting of SEQ ID NO:207; or

7) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:211,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

8) HCDR1′ comprising or consisting of SEQ ID NO: 214,

HCDR2' comprising or consisting of SEQ ID NO:215,

HCDR3' comprising or consisting of SEQ ID NO:216,

LCDR1' comprising or consisting of SEQ ID NO:217,

LCDR2' comprising or consisting of SEQ ID NO:218, and

LCDR3 comprising or consisting of SEQ ID NO:219; or

9) HCDR1′ comprising or consisting of SEQ ID NO: 220,

HCDR2' comprising or consisting of SEQ ID NO:221,

HCDR3' comprising or consisting of SEQ ID NO:222,

LCDR1' comprising or consisting of SEQ ID NO:223,

LCDR2' comprising or consisting of SEQ ID NO:224, and

LCDR3' comprising or consisting of SEQ ID NO:225; or

10) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO: 210,

LCDR1' comprising or consisting of SEQ ID NO:244,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

11) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:245,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

12) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:246,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

13) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:247,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

14) HCDR1′ comprising or consisting of SEQ ID NO: 208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:248,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

15) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO: 210,

LCDR1' comprising or consisting of SEQ ID NO:249,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

16) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:250,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213; or

17) HCDR1′ comprising or consisting of SEQ ID NO:208,

HCDR2' comprising or consisting of SEQ ID NO:209,

HCDR3' comprising or consisting of SEQ ID NO:210,

LCDR1' comprising or consisting of SEQ ID NO:251,

LCDR2' comprising or consisting of SEQ ID NO:212, and

LCDR3' comprising or consisting of SEQ ID NO:213.
The pharmaceutical composition according to any one of claims 20 to 23, wherein the second antibody or antigen-binding fragment thereof comprises:

A heavy chain variable region, the amino acid sequence of which is at least 80% identical to any one of the amino acid sequences selected from the group consisting of the following amino acid sequences: SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, and 162; preferably, the heavy chain variable region has a CDRs region that is identical to a peptide segment having an amino acid sequence of SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162, And the non-CDRs region of the heavy chain variable region has a sequence that is at least 80% identical to the non-CDRs region of the peptide fragment whose amino acid sequence is SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162; preferably, the heavy chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, and 162; and/or

The light chain variable region has an amino acid sequence selected from the group consisting of The light chain variable region has at least 80% identity to any amino acid sequence in the group consisting of SEQ ID NO: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, and 171; preferably, the light chain variable region has a CDRs region that is the same as the peptide segment with an amino acid sequence of SEQ ID NO: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, or 171, and the non-CDRs region of the light chain variable region is identical to the amino acid sequence of SEQ ID NO: NO:137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, or 171. The non-CDRs region of the peptide fragment has at least 80% identity; preferably, the light chain variable region has any amino acid sequence selected from the group consisting of the following amino acid sequences: SEQ ID NO:137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, and 171.
The pharmaceutical composition of claim 30, wherein the second antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence pair of the heavy chain variable region/light chain variable region is selected from the group consisting of the following amino acid sequence pairs: SEQ ID NO: 136/SEQ ID NO: 137, SEQ ID NO: 138/SEQ ID NO: 139, SEQ ID NO: 140/SEQ ID NO: 141, SEQ ID NO: 142/SEQ ID NO: 143, SEQ ID NO: 144/SEQ ID NO: 145, SEQ ID NO: 146/SEQ ID NO: 147, SEQ ID NO: 148/SEQ ID NO: 149, SEQ ID NO: 150/SEQ ID NO: 151, SEQ ID NO: 152/SEQ ID NO: 153, SEQ ID NO: 154/SEQ ID NO: 155 Q ID NO:155、SEQ ID NO:156/SEQ ID NO:157、SEQ ID NO:158/SEQ ID NO:159、SEQ ID NO:160/SEQ ID NO:161、SEQ ID NO:162/SEQ ID NO:163、SEQ ID NO:156/SEQ ID NO:164、SEQ ID NO:156/SEQ ID NO:165 O:165, SEQ ID NO:156/SEQ ID NO:166, SEQ ID NO:156/SEQ ID NO:167, SEQ ID NO:156/SEQ ID NO:168, SEQ ID NO:156/SEQ ID NO:169, SEQ ID NO:156/SEQ ID NO:170, and SEQ ID NO:156/SEQ ID NO:171;

Preferably, the amino acid sequence pairs of the heavy chain variable region/light chain variable region are selected from the group consisting of the following amino acid sequence pairs: SEQ ID NO: 148/149, 154/155, 156/157, 158/159, 160/161, 162/163, 156/164, 156/165, 156/166, 156/167, 156/168, 156/169, 156/170, and 156/171.
The pharmaceutical composition according to any one of claims 25 to 31, characterized in that the second antibody or its antigen-binding fragment comprises a heavy chain constant region and/or a light chain constant region, preferably it comprises a murine or humanized heavy chain constant region and/or a light chain constant region; preferably, the amino acid sequence of the humanized heavy chain constant region is as shown in SEQ ID NO: 252, and the amino acid sequence of the humanized light chain constant region is as shown in SEQ ID NO: 253.
The pharmaceutical composition of claim 32, wherein the amino acid sequence pair of the heavy chain/light chain of the second antibody or antigen-binding fragment thereof is selected from the group consisting of the following amino acid sequence pairs: SEQ ID NO: 254/255, 256/257 and 258/259;

Preferably, the amino acid sequence pairs of the heavy chain/light chain of the first antibody or its antigen-binding fragment of the pharmaceutical composition are selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 129 and SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134, and the amino acid sequence pairs of the heavy chain/light chain of the second antibody or its antigen-binding fragment of the composition are selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 254 and SEQ ID NO: 255, SEQ ID NO: 256 and SEQ ID NO: 257, SEQ ID NO: 258 and SEQ ID NO: 259.
Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the pharmaceutical composition according to any one of claims 19 to 33 in the preparation of a medicament for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the pharmaceutical composition according to any one of claims 19 to 33, for use in treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection.
A method for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in any one of claims 1-11 or the pharmaceutical composition described in any one of claims 19-33.
Use of an active ingredient, wherein the active ingredient is selected from the group consisting of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the active ingredient is used to prepare a test plate or a kit, wherein the test plate or the kit is used to detect respiratory RSV.
A detection plate, comprising: a substrate and a test strip, wherein the test strip contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.
An antibody preparation capable of neutralizing respiratory syncytial virus (RSV), comprising:

Any pharmaceutical composition according to any one of claims 20 to 33; preferably, the amino acid sequence pair of the heavy chain/light chain of the first antibody or antigen-binding fragment thereof of the pharmaceutical composition is selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 129 and SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134, and/or the amino acid sequence pair of the heavy chain/light chain of the second antibody or antigen-binding fragment thereof of the composition is selected from any one of the group consisting of the following amino acid sequence pairs: SEQ ID NO: 254/255, 256/257 and 258/259;

Buffer;

Preferably, the buffer is selected from one or more of a citric acid buffer, a histidine buffer, a phosphate buffer, an acetate buffer, a succinic acid buffer, a lactic acid buffer, a tromethamine buffer, an aspartic acid buffer, a glutamic acid buffer or adipic acid buffer; preferably, the buffer is selected from a citric acid buffer and/or a phosphate buffer; preferably, the buffer is selected from a citric acid buffer and a phosphate buffer.
The antibody preparation of claim 39, wherein the pH value of the antibody preparation is 5.6-6.5; preferably, the pH value of the antibody preparation is 5.7-6.3; preferably, the pH value of the antibody preparation is 5.7, 5.9, 6.1, 6.3.
The antibody preparation according to claim 39 or 40, wherein the concentration of the citrate buffer in the buffer is 10-20 mM, and the concentration of the phosphate buffer in the buffer is 6-12 mM, preferably, the concentration of the citrate buffer is 13-17 mM, and the concentration of the phosphate buffer is 7-11 mM.
The antibody formulation of any one of claims 39 to 41, wherein the antibody formulation further comprises a stabilizer and/or a surfactant.
The antibody preparation according to claim 42, wherein the stabilizer is selected from one or more of sucrose, trehalose, sorbitol, arginine hydrochloride or mannitol;

Preferably, the stabilizer is selected from sorbitol and/or arginine hydrochloride;

Preferably, the stabilizer is selected from sorbitol and arginine hydrochloride.
The antibody preparation according to claim 43, wherein the concentration of sorbitol in the stabilizer is 250-300 mM, and the concentration of arginine hydrochloride is 60-100 mM; preferably, the concentration of sorbitol in the stabilizer is 260-280 mM, and the concentration of arginine hydrochloride is 70-90 mM.
The antibody preparation according to claim 42, wherein the surfactant is selected from Tween 80 or Tween 20; preferably, the surfactant is Tween 80.
The antibody preparation according to claim 45, wherein the concentration of the surfactant is 0.05-0.2 mg/ml; preferably, the concentration of the surfactant is 0.1 mg/ml.
The antibody preparation according to any one of claims 39 to 46, characterized in that the total concentration of the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof in the composition is 40-200 mg/ml; preferably, the total concentration is 80-160 mg/ml; preferably, the total concentration is 100-140 mg/ml;
The antibody preparation of claim 47, wherein the mass ratio of the first antibody or antigen-binding fragment to the second antibody or antigen-binding fragment in the composition is 1:0.2-5; preferably, the concentration ratio is 1:0.4-2.5.
The antibody preparation according to any one of claims 39 to 42, characterized in that it comprises:

20-100 mg/ml first antibody or antigen-binding fragment, 20-100 mg/ml second antibody or antigen-binding fragment, 2-6 mM citric acid, 9-13 mM sodium citrate, 7-11 mM Na 2 HPO 4 , 250-300 mM sorbitol, 60-100 mM arginine hydrochloride, 0.05-0.2 mg/ml Tween 80, pH about 5.6-6.5.
The antibody preparation according to claim 49, characterized in that it comprises:

40-80 mg/ml first antibody or antigen-binding fragment, 40-80 mg/ml second antibody or antigen-binding fragment, 2-6 mM citric acid, 9-13 mM sodium citrate, 7-11 mM Na 2 HPO 4 , 250-300 mM sorbitol, 60-100 mM arginine hydrochloride, 0.05-0.2 mg/ml Tween 80, pH about 5.6-6.5.
The antibody preparation according to any one of claims 39 to 42, characterized in that it comprises: 60 mg/ml first antibody or antigen-binding fragment, 60 mg/ml second antibody or antigen-binding fragment, 4.09 mM citric acid, 11.225 mM sodium citrate, 9.659 mM Na 2 HPO 4 , 274 mM sorbitol, 80 mM arginine hydrochloride, 0.1 mg/ml Tween 80, and pH is about 5.7-6.2.
The antibody preparation according to any one of claims 39 to 42, characterized in that it comprises: 40 mg/ml first antibody or antigen-binding fragment, 80 mg/ml second antibody or antigen-binding fragment, 4.09 mM citric acid, 11.225 mM sodium citrate, 9.659 mM Na 2 HPO 4 , 274 mM sorbitol, 80 mM arginine hydrochloride, 0.1 mg/ml Tween 80, and pH is about 5.7-6.2.
Use of the antibody preparation according to any one of claims 39 to 52 in the preparation of a medicament for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection.
The antibody preparation of any one of claims 39-52, which is used for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection.
A method for treating and/or preventing respiratory syncytial virus (RSV) infection or symptoms associated with RSV infection, comprising administering a therapeutically effective amount of the antibody preparation of any one of claims 39-52 to a subject in need thereof.