Anti-p24 antibodies and uses thereof

Abstract

The invention discloses an anti-P24 antibody and application thereof, and relates to the field of antibodies. The anti-P24 antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for P24 detection, and has excellent affinity or activity.

Description

[0001] Cross - reference to related applications

[0002] This disclosure claims the priority of a Chinese patent application with the application number 202311718489.1, titled "Anti - P24 Antibody and Its Use", filed with the Chinese Patent Office on December 13, 2023, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention belongs to the technical field of antibodies. Specifically, the present invention relates to a P24 antibody and its use. More specifically, the present invention relates to an antibody against P24, a nucleic acid molecule, a vector, a cell or a host, a method for preparing an antibody, a conjugate, a reagent or a kit and its use, a method for detecting P24, a method for screening a P24 antibody, and a mutant library. Background art

[0004] The Human Immunodeficiency Virus (HIV), that is, the AIDS virus (AIDS), also known as the Acquired Immunodeficiency Syndrome virus, is a virus that causes defects in the human immune system. It is a lentivirus that infects human immune system cells and belongs to a type of retrovirus. HIV is the pathogen of AIDS and is mainly transmitted through sexual contact, blood, and mother - to - child transmission. In recent years, the number of HIV - infected patients has been on the rise. Currently, the routine laboratory method for diagnosing HIV infection is to measure serum HIV antibodies, but there are limitations in measuring HIV antibodies: more than 70% of HIV - infected individuals can only be detected for antibodies 6 months after infection, and the antibody - detection method increases the risk of transmission during the "window period" of HIV; in addition, newborns produce antibodies 1 year after birth, and the HIV antibodies from the mother can cause false positives; due to the continuous presence of HIV antibodies during the disease process, they only disappear in the late stage of AIDS and cannot be used as a stable indicator for treatment monitoring.

[0005] P24 is the main structural protein of the HIV virus particle, which is the product of the structural gene GAG and plays an important role in the packaging and maturation of the virus. The amino acid sequence of the P24 protein is highly conserved among different HIV strains, and the deletion of P24 will cause the virus to be unable to assemble normally. The P24 protein has strong specificity and has no cross - reaction with most other retroviruses. When the human body is infected with HIV, the first virus marker to appear in the blood of the infected person is the viral P24 protein. There is a long window period between virus infection and the detection of HIV antibodies. Therefore, the detection of HIV - P24 antigen has played an important role in the early diagnosis of HIV infection, the prognosis judgment of patients, the screening and evaluation of anti - HIV drugs, and the discovery of mother - to - child transmission, etc.

[0006] The detection of HIV-1 p24 antigen uses serological diagnostic methods, mainly including double antibody sandwich ELISA, immunocomplex cleavage detection method, ultrasensitive EIA, enzyme-linked immunofluorescence method, etc. Currently, the double antibody sandwich method is commonly used to detect human immunodeficiency virus p24 antigen, and obtaining an antibody against p24 is the key to realizing the double antibody sandwich detection method. Therefore, there is a strong demand in this field for antibodies that are effective, bind to p24, and detect it. SUMMARY OF THE INVENTION

[0007] The present invention aims to provide an antibody against p24, a reagent or a kit for detecting p24.

[0008] In one aspect of the present invention, the present invention provides an anti-p24 antibody, which includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are as follows:

[0009] HCDR 1: SYAIS;

[0010] HCDR 2: GSIPIFGTTHYAQKFQG;

[0011] HCDR 3: AATAIFGVPX 1 NNFYAMEV, wherein X 1 is R or S;

[0012] LCDR 1: RX 2 SQSLLHSNGYX 3 YLD, wherein X 2 is S, R or W, X 3 is N or L;

[0013] LCDR 2: IGSTRAS;

[0014] LCDR 3: MQAVQTF.

[0015] In the second aspect of the present invention, the present invention provides an anti-p24 antibody, which includes a heavy chain variable region and / or a light chain variable region; the heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO: 17 or its variant, and compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 includes an R108S mutation; the light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 18 or its variant, and compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 includes a mutation at at least one of the following sites: S25R / W, N36L.

[0016] In the third aspect of the present invention, an anti-P24 antibody is provided, which comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3. The HCDR1, HCDR2, HCDR3 are the HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody in the second aspect; and the LCDR1, LCDR2, LCDR3 are the LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody in the second aspect.

[0017] In the fourth aspect of the present invention, a nucleic acid molecule is provided, which encodes the antibody described in the first aspect, the second aspect or the third aspect.

[0018] In the fifth aspect of the present invention, a vector is provided, which comprises the nucleic acid molecule described in the fourth aspect.

[0019] In the sixth aspect of the present invention, a cell or host is provided, which comprises: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect or the third aspect.

[0020] In the seventh aspect of the present invention, a method for preparing the antibody described in the first aspect, the second aspect or the third aspect is provided, which comprises culturing the cell or host described in the sixth aspect.

[0021] In the eighth aspect of the present invention, a conjugate is provided, which comprises: the antibody described in the first aspect, the second aspect or the third aspect and a conjugate part conjugated thereto.

[0022] In the ninth aspect of the present invention, a reagent or kit is provided, which comprises: the antibody described in the first aspect, the second aspect or the third aspect or the conjugate described in the seventh aspect.

[0023] In the tenth aspect of the present invention, the use of the antibody described in the first aspect, the second aspect or the third aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect in detecting P24, preparing a product for detecting P24, or preparing a product for diagnosing HIV infection is provided.

[0024] In the eleventh aspect of the present invention, a method for detecting P24 is provided, which comprises: contacting a sample to be detected with the antibody described in the first aspect, the second aspect or the third aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect to form an immune complex.

[0025] In a twelfth aspect of the present invention, a method for screening P24 antibodies is proposed, the method comprising: a) designing primers for amino acid substitution at the sites in X 1 , X 2 and X 3 defined in the antibody of the first aspect, or at the mutation sites defined in the antibody of the second aspect; b) using the nucleic acid molecule of the fourth aspect, the vector of the fifth aspect or the cell of the sixth aspect as a template to construct a mutant library with the primers in a); c) screening P24 antibodies from the mutant library.

[0026] In a thirteenth aspect of the present invention, a mutant library is proposed, the mutant library comprising the antibody of the first aspect, the second aspect or the third aspect.

[0027] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Detailed Embodiments

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0029] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, "a plurality of" means two or more.

[0030] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0031] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0032] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0033] In this text, the terms "optionally", "optional", "alternatively", "alternative", or "alternate" generally mean that the subsequent described event or condition can but does not necessarily occur, and this description includes the cases where the event or condition occurs, as well as the cases where the event or condition does not occur.

[0034] In this text, the term "antibody" is used in the broadest sense, which may include full-length monoclonal antibodies, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, and the specific structure is not limited as long as they exhibit the required antigen-binding ability.

[0035] In this text, the terms "full-length antibody", "full-length monoclonal antibody", or "full-length monoclonal antibody" are all composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0036] In this text, the terms "polyclonal antibody" and "multispecific antibody" are synonymous, and both refer to antibodies that can recognize multiple antigen epitopes, such as antibodies that can recognize two antigen epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that can recognize three antigen epitopes, or antibodies that can recognize four antigen epitopes. It is understood in a broad sense, and the specific structure is not limited as long as it can recognize multiple antigen epitopes. In the present invention, at least one of the multiple antigen epitopes is derived from P24.

[0037] In this text, the term "antigen-binding fragment of an antibody" is a fragment that contains a part or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but still has the functional activity of specifically binding to an antigen. For example, this fragment may contain a part or all of the CDR of the antibody. Such a fragment binds to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, scFv-Fc fusion proteins, scFv-Fv fusion proteins, single-domain antibodies, or minimal recognition units. Such fragments can be produced by recombinant nucleic acid techniques or can be produced by enzymatic cleavage or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0038] In this text, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment that contains only the Fab molecule, which is composed of VH and CH of the heavy chain 1 and the complete light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0039] As used herein, the term "F(ab') 2 antibody" or "F(ab') 2 fragment" has two antigen-binding F(ab') portions linked together by disulfide bonds.

[0040] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a variable light chain (VL) and a variable heavy chain (VH) linked by non-covalent bonds, which is the smallest functional fragment of an antibody molecule that retains the complete antigen-binding site.

[0041] As used herein, the terms "single-chain antibody", "scFv fragment" are antibodies or fragments formed by linking the variable heavy chain and variable light chain of an antibody with a short peptide.

[0042] As used herein, the terms "minimal recognition unit" and "MRU" both refer to an antibody or fragment consisting of only one CDR, and its molecular weight is very small, accounting for only about 1% of the complete antibody.

[0043] As used herein, the terms "complementary determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, which refer to the regions containing one or more or even all of the main amino acid residues that contribute to the binding affinity of the antibody for the antigen or epitope it recognizes.

[0044] In this text, the heavy chain complementarity determining regions (CDRs of the heavy chain variable region) are denoted by "HCDRs" or "HCDR", which include HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3); the light chain complementarity determining regions (CDRs of the light chain variable region) are denoted by "LCDRs" or "LCDR", which include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2), and LCDR3 (also known as CDR-L3). Commonly used CDR definition systems in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is referred to Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. The definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that the CDRs defined in the present invention include, but are not limited to, the CDRs defined by the methods in Table 1. CDRs defined by other methods disclosed in the art based on the heavy chain variable region and light chain variable region disclosed in this application also fall within the scope of protection of this disclosure.

[0045] Table 1: CDR Definitions 1

[0046] CDR Kabat <![CDATA[AbM 2 > IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..35 5 > <![CDATA[H26~H32..34 4 > HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0047] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number".

[0048] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0049] 3 If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0050] 4 When neither H35A nor H35B is present, CDR-H1 ends at position 32; when only H35A is present, CDR-H1 ends at position 33; when both H35A and H35B are present, CDR-H1 ends at position 34.

[0051] 5 When neither H35A nor H35B is present, CDR-H1 ends at position 33; when only H35A is present, CDR-H1 ends at position 34; when both H35A and H35B are present, CDR-H1 ends at position 35.

[0052] Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. A person of ordinary skill in the art can clearly map the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "Kabat numbering" refers to numbering using the numbering system described in "Sequence of Proteins of Immunological Interest" (1983) by Kabat et al., U.S. Dept. of Health and Human Services. The HCDRs and LCDRs of the antibodies in this application are numbered using the above numbering system. For the specific numbering results, see Table 1. It should be noted that the polypeptide sequences of the present invention are not numbered according to the Kabat numbering system. However, a person of ordinary skill in the art can fully convert the sequence numbers in the sequence listing into Kabat numbers.

[0053] As used herein, the term "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the regions other than the CDRs in the heavy chain variable region (which can be denoted as VH) and the light chain variable region (which can be denoted as VL) of an antibody; among them, the heavy chain framework region is denoted as "HFR" and can be further divided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region is denoted as "LFR" and can be further divided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0054] As used herein, the heavy chain variable region is obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0055] In this text, when the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods. For example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.)). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8th edition, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0056] Without substantially affecting the antibody activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody sequences. They are all considered to be included within the scope of protection of the present invention. Amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention can have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequences. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner.

[0057] It should be noted that the positions or numbers of the mutation sites defined in the specification and claims of the present invention also need to be adjusted according to the number and positions of the added and / or deleted amino acids.

[0058] In this article, the term "at least 80% homology" means at least 80% with each reference sequence, and can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology. The term "at least 90% homology" means at least 90% with each reference sequence, and can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology.

[0059] In this article, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule containing one or more nucleotide or amino acid mutations.

[0060] In this article, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating after insertion into a suitable host, which transfers the inserted nucleic acid molecule into cells or hosts and / or between cells or hosts. The vector can include vectors mainly for inserting DNA or RNA into cells, vectors mainly for replicating DNA or RNA, and expression vectors mainly for transcription and / or translation of DNA or RNA. The vector also includes vectors with multiple above-mentioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Usually, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0061] In this text, the term "cell" generally refers to a cell obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques, and having unique traits with stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant vector can be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of nucleic acid (such as a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0062] The present invention provides an anti-P24 antibody, a nucleic acid molecule, a vector, a cell or a host, a method for preparing an antibody, a conjugate, a reagent or a kit and their uses, a method for detecting P24, a method for screening an anti-P24 antibody, and a mutant library, which will be described in detail below respectively.

[0063] Antibody

[0064] In the first aspect of the present invention, the present invention provides an anti-P24 antibody. According to an embodiment of the present invention, the antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are as follows:

[0065] HCDR 1: SYAIS;

[0066] HCDR 2: GSIPIFGTTHYAQKFQG;

[0067] HCDR 3: AATAIFGVPX 1 NNFYAMEV, wherein X 1 is R or S;

[0068] LCDR 1: RX 2 SQSLLHSNGYX 3 YLD, wherein X 2 is S, R or W, and X 3 is N or L;

[0069] LCDR 2: IGSTRAS;

[0070] LCDR 3: MQAVQTF.

[0071] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0072] From the definition principles of the foregoing CDRs, it can be seen that when different systems are used for definition, even for the same heavy chain variable region or the same light chain variable region, the obtained CDRs are different. "HCDR 1:SYAIS" in the present application means that the amino acid sequence of HCDR 1 includes or is SYAIS, and it can also be adjusted according to different systems to obtain an HCDR 1 with a sequence shorter or longer than SYAIS.

[0073] In some alternative embodiments of the present invention, the X 1 is R;

[0074] In some alternative embodiments of the present invention, the X 1 is S;

[0075] In some alternative embodiments of the present invention, the X 2 is S;

[0076] In some alternative embodiments of the present invention, the X 2 is R;

[0077] In some alternative embodiments of the present invention, the X 2 is W;

[0078] In some alternative embodiments of the present invention, the X 3 is N;

[0079] In some alternative embodiments of the present invention, the X 3 is L;

[0080] In some alternative embodiments of the present invention, X 1 is R, X 2 is S, and X 3 is N do not hold simultaneously;

[0081] In some alternative embodiments of the present invention, the X 1 , X 2 and X 3 are selected from any one of the following combinations:

[0082]

[0083] The antibody according to the first aspect of the present invention has excellent activity, affinity, stability, or specificity.

[0084] The antibody according to the first aspect of the present invention has improved activity, affinity, stability or specificity.

[0085] In the second aspect of the present invention, there is provided an anti-P24 antibody, comprising a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises an R108S mutation; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 comprises a mutation at at least one of the following sites: S25R / W, N36L.

[0086] It should be noted that the numbering of the above-mentioned heavy chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 17 from the N-terminus to the C-terminus. For example, the 108th position refers to the 108th position of the amino acid sequence shown in SEQ ID NO: 17 starting from the N-terminus; the "R108S" means that the arginine at the 108th position of the amino acid sequence shown in SEQ ID NO: 17 is replaced by serine.

[0087] The numbering of the above-mentioned light chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 18 from the N-terminus to the C-terminus. For example, the 25th position refers to the 25th position of the amino acid sequence shown in SEQ ID NO: 18 starting from the N-terminus; the "S25R" means that the serine at the 25th position of the amino acid sequence shown in SEQ ID NO: 18 is replaced by arginine; the "S25R / W" means that the serine at the 25th position of the amino acid sequence shown in SEQ ID NO: 18 can be replaced by arginine or tryptophan.

[0088] According to an embodiment of the present invention, the above-mentioned antibody may further comprise at least one of the following additional technical features:

[0089] In some alternative embodiments of the present invention, the mutation at the 108th position is R108S;

[0090] In some alternative embodiments of the present invention, the mutation at the 25th position is S25R;

[0091] In some alternative embodiments of the present invention, the mutation at the 25th position is S25W;

[0092] In some alternative embodiments of the present invention, the mutation at the 36th position is N36L;

[0093] In some alternative embodiments of the present invention, it is not simultaneously true that the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 17 and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 18;

[0094] In some alternative embodiments of the present invention, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

[0095]

[0096] It should be noted that the "," in the above table represents "and", that is, combinatorial mutation. For example, "S25W,N36L" represents a combinatorial mutation of two mutations, S25W and N36L.

[0097] In a third aspect of the present invention, the present invention provides an anti-P24 antibody. According to the embodiments of the present invention, the antibody includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1, HCDR2, HCDR3 are the HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody in the second aspect; the LCDR1, LCDR2, LCDR3 are the LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody in the second aspect.

[0098] It should be noted that the HCDR1, HCDR2 and HCDR3 in the antibody described in the third aspect are the HCDR1, HCDR2, HCDR3 of the same heavy chain variable region defined by the antibody in the second aspect, and the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2, LCDR3 of the same light chain variable region defined by the antibody in the second aspect.

[0099] For example, when the heavy chain variable region defined by the antibody in the second aspect has no mutation compared to SEQ ID NO: 17, the HCDR1, HCDR2, HCDR3 of this heavy chain variable region are respectively HCDR 1: SYAIS; HCDR 2: GSIPIFGTTHYAQKFQG; HCDR 3: AATAIFGVPRNNFYAMEV; then the HCDR1, HCDR2, HCDR3 contained in the antibody described in the third aspect are also respectively HCDR 1: SYAIS; HCDR 2: GSIPIFGTTHYAQKFQG; HCDR 3: AATAIFGVPRNNFYAMEV.

[0100] For example, when there is only a mutation at the S25R site in the light chain variable region defined in the antibody described in the second aspect compared to SEQ ID NO: 18, the LCDR1, LCDR2, and LCDR3 of this light chain variable region are respectively LCDR 1: RRSQSLLHSNGYNYLD; LCDR 2: IGSTRAS; LCDR 3: MQAVQTF; then the LCDR1, LCDR2, and LCDR3 contained in the antibody described in the third aspect are also respectively LCDR 1: RRSQSLLHSNGYNYLD; LCDR 2: IGSTRAS; LCDR 3: MQAVQTF.

[0101] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0102] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

[0103] According to an embodiment of the present invention, the antibody described in the first aspect or the third aspect further includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0104] In an alternative embodiment of the present invention, at least a part of at least one of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0105] According to an embodiment of the present invention, the HFR1 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology thereto; the HFR2 comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology thereto; the HFR3 comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology thereto; the HFR4 comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology thereto; the LFR1 comprises the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 80% homology thereto; the LFR2 comprises the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 80% homology thereto; the LFR3 comprises the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80% homology thereto; the LFR4 comprises the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 80% homology thereto.

[0106] According to an embodiment of the present invention, the antibody described in the first aspect, second aspect or third aspect above may further comprise at least one of the following technical features:

[0107] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -9 M.

[0108] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -10 M.

[0109] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -11 M.

[0110] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -12 M.

[0111] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -13 M.

[0112] According to an embodiment of the present invention, the antibody further comprises a constant region; wherein, the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

[0113] According to an embodiment of the present invention, at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody or a mutant thereof.

[0114] According to an embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0115] In an alternative embodiment of the present invention, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0116] In an alternative embodiment of the present invention, the IgG is selected from IgG1, IgG2, IgG3, IgG4.

[0117] According to an embodiment of the present invention, the light chain constant region includes a κ-type or λ-type light chain constant region.

[0118] In an alternative embodiment of the present invention, the heavy chain constant region includes or is the heavy chain constant region shown in SEQ ID NO:15 or an amino acid sequence having at least 80% identity therewith; or the light chain constant region includes or is the light chain constant region shown in SEQ ID NO:16 or an amino acid sequence having at least 80% identity therewith.

[0119] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0120] Herein, the division of variable region and constant region sequences refers to the IMGT division method, see Lefranc, the international ImMunoGeneTics database. Nucl.Acids Res., 29(1):207-209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: housemouse (Mus musculus) IGHC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. There will be some amino acid differences between the variable regions divided by different methods and the C-terminus of the variable region or the N-terminus of the constant region divided by IMGT. The variable regions or constant regions divided by other methods well-known in the art are also within the protection scope of the present invention.

[0121] Those skilled in the art can understand that the features and advantages described for the antibodies in the first aspect also apply to the antibodies in the second aspect or the third aspect, and will not be repeated here.

[0122] Nucleic acid molecule, vector, cell or host, method for preparing antibody

[0123] In the process of preparing or obtaining the antibodies described in the first aspect, the second aspect or the third aspect, nucleic acid molecules expressing these antibodies can be used, connected to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0124] In the fourth aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody described in the first aspect, the second aspect or the third aspect. The nucleic acid molecule according to the embodiment of the present invention can encode and obtain the above-mentioned antibody.

[0125] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0126] It should be noted that for the nucleic acid molecules mentioned in this article, those skilled in the art should understand that it actually includes either any one of the complementary double strands or both. For convenience, in this article, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the molecular sequences in the present invention include DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0127] In the fifth aspect of the present invention, the present invention provides a vector. According to the embodiments of the present invention, the vector includes the nucleic acid molecule described in the fourth aspect. When connecting the above-mentioned nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can come directly from the vector itself or be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements are operably connected. As used herein, "operably connected" means connecting an exogenous gene to a vector such that the control elements in the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned antibody can be effectively achieved, and thus a large amount of the antibody can be obtained in vitro.

[0128] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage.

[0129] In an alternative embodiment of the present invention, the expression vector is a plasmid expression vector.

[0130] In the sixth aspect of the present invention, the present invention provides a cell or a host. According to the embodiments of the present invention, the cell or host includes: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect or the third aspect. Using this cell under suitable conditions, the aforementioned antibody can be effectively expressed intracellularly.

[0131] According to the embodiments of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0132] It should be noted that the cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0133] In an alternative embodiment of the present invention, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0134] It should be noted that the "suitable conditions" described in the present invention refer to the conditions suitable for the expression of the antibodies of the present invention. It is easily understood by those skilled in the art that the conditions suitable for the expression of the antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies according to the specific environment of the laboratory.

[0135] In a seventh aspect of the present invention, there is provided a method for preparing the antibody according to the first, second or third aspect. According to an embodiment of the present invention, the method includes culturing the cells or hosts according to the sixth aspect. The methods according to some specific embodiments of the present invention can effectively obtain a large amount of the antibody.

[0136] Based on the amino acid sequence of the antibody of the present disclosure, it is easily conceivable by those skilled in the art to prepare the antibody by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, separating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody as described in any one of the above, which is easily achievable for those skilled in the art. Based on this, no matter what technique is used to prepare the antibody of the present disclosure, it falls within the protection scope of the present disclosure.

[0137] Those skilled in the art can understand that the features and advantages described above for the antibody according to the first, second or third aspect also apply to the nucleic acid molecule, vector, cell or host, and the method for preparing the antibody, which will not be elaborated herein.

[0138] Conjugates, reagents or kits and their uses

[0139] In the eighth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate includes: an antibody as described in the first aspect, the second aspect or the third aspect, and a conjugate moiety conjugated thereto. The conjugate according to the embodiment of the present invention can specifically bind to P24, and can be used for qualitative or quantitative detection of P24, or for indicating P24-related diseases.

[0140] According to an embodiment of the present invention, the above conjugate may further include at least one of the following additional technical features:

[0141] According to an embodiment of the present invention, the conjugate moiety includes at least one of a purification tag, an affinity substance, a marker, and a solid-phase carrier.

[0142] According to an embodiment of the present invention, the purification tag includes at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag.

[0143] As used herein, the affinity substance may be, for example, biotin, a biotin derivative, or streptavidin, or one of nucleic acids of a sense strand and an antisense strand that are complementary to each other.

[0144] In some alternative embodiments of the present invention, the affinity substance includes at least one selected from biotin, a biotin derivative, or streptavidin.

[0145] As used herein, a "marker" refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of a corresponding target can be achieved through this characteristic.

[0146] According to an embodiment of the present invention, the marker includes at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based markers.

[0147] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter what marker is used, it falls within the protection scope of the present invention.

[0148] According to an embodiment of the present invention, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0149] According to an embodiment of the present invention, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0150] According to an embodiment of the present invention, the radioisotopes include, but are not limited to 212 Bi 131 I 111 In 90 Y 186 Re 211 At 125 I 188 Re 153 Sm 213 Bi 32 P 94 mTc 99 mTc 203 Pb 67 Ga 68 Ga 43 Sc 47 Sc 110 mIn 97 Ru 62 Cu 64 Cu 67 Cu 68 Cu 86 Y 88 Y 121 Sn 161 Tb 166 Ho 105 Rh177 Lu 172 Lu and 18 F.

[0151] According to an embodiment of the present invention, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosalic acid and its derivatives, and peroxyoxalate and its derivatives.

[0152] According to an embodiment of the present invention, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0153] According to an embodiment of the present invention, the colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latex.

[0154] According to an embodiment of the present invention, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0155] In this context, the solid-phase carrier can be a substance capable of being suspended or dispersed in a liquid phase (e.g., solid-phase carriers such as particles, magnetic beads, etc.), or a solid phase capable of accommodating or carrying a liquid phase (e.g., supports such as plates, membranes, test tubes, etc., and containers such as microtiter plates, microfluidic channels, glass capillaries, nano-columns, monolithic columns, etc.).

[0156] According to an embodiment of the present invention, the solid-phase carrier includes at least one selected from microspheres, plates, and membranes.

[0157] In a ninth aspect of the present invention, the present invention provides a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit includes: the antibody according to the first aspect, the second aspect, or the third aspect, or the conjugate according to the eighth aspect. As mentioned above, the antibodies in some specific embodiments or examples of the present invention can bind to P24. Therefore, the reagent or the kit containing the antibody can effectively perform qualitative or quantitative detection of P24. By applying the reagent or the kit provided by the present invention, for example, it can be used for detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of P24 and its antibody. As mentioned above, the mutant antibodies of the present invention have improved P24 binding activity, affinity, stability, or specificity. Therefore, the reagent or the kit containing the antibody has improved detection sensitivity or specificity.

[0158] The above-mentioned kit may include any one or more of the following: treatment solution, anti-P24 antibody, P24 quality control product, anti-IgG antibody, instruction manual or literature, etc. The anti-P24 antibody can be used in different types of diagnostic tests, for example, it can detect the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, relevant diseases can be tested by detecting the serum or blood of the subject.

[0159] In the tenth aspect of the present invention, the present invention provides the use of the antibody described in the first aspect, the conjugate described in the eighth aspect, the reagent or kit described in the ninth aspect in detecting P24, diagnosing HIV infection, preparing a product for detecting P24, or preparing a product for diagnosing HIV infection.

[0160] According to the embodiments of the present invention, the "product" includes but is not limited to reagents, test strips, reagent plates, or kits.

[0161] In the eleventh aspect of the present invention, the present invention provides a method for detecting P24. According to the embodiments of the present invention, the method includes: contacting the sample to be detected with the antibody described in the first aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect to form an immune complex.

[0162] According to the embodiments of the present invention, based on the signal of the immune complex, it is determined whether the sample to be detected contains P24 or the content of P24.

[0163] According to the embodiments of the present invention, the immune complex further includes a second antibody that binds to the antibody.

[0164] According to the embodiments of the present invention, the immune complex further includes a second antibody that binds to P24.

[0165] According to the embodiments of the present invention, the signal includes a fluorescence signal.

[0166] Method, mutant library

[0167] In the twelfth aspect of the present invention, the present invention provides a method for screening anti-P24 antibodies. According to the embodiments of the present invention, the method includes: a) designing primers for amino acid substitution at 1, 2, or 3 sites among X 1 、X 2 and X 3 defined in the antibody described in the first aspect, or at the mutant sites defined in the antibody described in the second aspect; b) using the mutant library constructed with the primers in a) with the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, or the cell or host described in the sixth aspect as a template.

[0168] c) Screen for P24 antibodies from the mutant library.

[0169] According to an embodiment of the present invention, the mutant library is a single-site saturation mutant library.

[0170] According to an embodiment of the present invention, the P24 antibody includes or is the antibody described in the first aspect, the second aspect or the third aspect.

[0171] In the thirteenth aspect of the present invention, the present invention provides a mutant library, which includes the antibody described in the first aspect, the second aspect or the third aspect.

[0172] According to an embodiment of the present invention, the mutant library is obtained by the method described in the twelfth aspect.

[0173] In the fourteenth aspect of the present invention, the present invention provides a method for diagnosing HIV infection. According to an embodiment of the present invention, the method includes: contacting a sample to be tested from a subject with the antibody described in the first aspect, the second aspect or the third aspect, the conjugate described in the eighth aspect, or the reagent or kit described in the ninth aspect to form an immune complex.

[0174] According to an embodiment of the present invention, the method described in the above fourteenth aspect may further include at least one of the following technical features:

[0175] According to an embodiment of the present invention, the immune complex further includes a second antibody that binds to the antibody;

[0176] According to an embodiment of the present invention, the immune complex further includes a second antibody that binds to the P24.

[0177] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0178] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, mice, apes, humans, livestock, sports animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0179] The amino acid sequences involved herein are shown in Table 2:

[0180] Table 2: Amino acid sequences

[0181]

[0182]

[0183] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications are followed. Those reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchases.

[0184] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise noted, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0185] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 2011), each of which is hereby expressly incorporated by reference.

[0186] In this example, restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, Taq DNA polymerase was purchased from TaKaRa, the gel extraction kit and plasmid extraction kit were commercially available, and primer synthesis and gene sequencing were completed by an outsourcing company. The sequence of the P24 monoclonal antibody (hereinafter referred to as the WT antibody) was derived from phage display antibody sequencing.

[0187] Example 1: Construction and screening of a mutant library

[0188] 1. Construction of a wild-type (WT) P24 antibody (abbreviated as WT antibody) template plasmid

[0189] (1) Synthesis of WT antibody gene:

[0190] The nucleotide sequences of VH and VL of the WT antibody were optimized for E. coli codons, and then the antibody gene sequences were synthesized. The amino acid sequences of VH and VL of the WT antibody are shown in SEQ ID NO:17 and SEQ ID NO:18 respectively, and the amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO:19 and SEQ ID NO:20 respectively.

[0191] (2) Amplification of WT antibody gene fragments:

[0192] The nucleotide sequences of the synthesized antibody VH and VL in step (1) were amplified by PCR using DNA polymerase, and then the antibody bands were separated by agarose gel electrophoresis. Subsequently, the antibody gene fragments were purified using a gel extraction kit.

[0193] (3) Enzyme digestion and ligation of WT antibody gene fragments:

[0194] The antibody gene fragments obtained in step (2) and the V01 vector plasmid (including the nucleotide sequence of the constant region) were digested with restriction endonucleases simultaneously, and then the antibody gene fragments and the V01 vector with sticky ends were purified using a gel extraction kit. Subsequently, the antibody gene fragments and the V01 vector were ligated with T4 DNA ligase at 22°C for 4 hours. The ligation reaction product was then recovered and purified, and the DNA concentration was measured. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells to obtain a bacterial solution. Then, the entire bacterial solution was spread on a plate containing ampicillin resistance and cultured overnight at 37°C.

[0195] (4) Extraction and sequencing verification of the WT template plasmid

[0196] Ten monoclonal colonies from the overnight culture in step (3) were selected, and colony PCR and gel electrophoresis were performed using Taq DNA polymerase. The bacteria with the correct insertion of the antibody gene sequence were selected for culture and amplification. The WT template plasmid was obtained using a plasmid extraction kit and sent to a sequencing company for gene sequencing verification.

[0197] 2. Construction of the single-point mutation library

[0198] In this part of the experiment, single-point saturation mutagenesis was performed on the entire CDR regions of VH and VL of the WT antibody obtained in step 1 to construct a single-point mutation library.

[0199] (1) Primer design and synthesis

[0200] Using degenerate base codons, 70 pairs of upstream and downstream primers for single-site saturation mutagenesis of amino acid sites (70 amino acid sites) in the full CDR regions of VH and VL were designed and submitted to an outsourcing company for primer synthesis.

[0201] (2) PCR amplification of single-site saturation mutagenesis plasmids

[0202] Using the primers obtained in step (1), the single-site saturation mutagenesis plasmids were amplified by PCR. The reaction system was configured according to Table 3, and then the amplification preparation of the single-site saturation mutagenesis library plasmids was carried out using the PCR reaction conditions in Table 4. Finally, the WT template plasmid obtained in step 1 was digested with a restriction endonuclease at 37 °C for 1 hour to obtain plasmids of 70 mutant libraries.

[0203] Table 3: Reaction system for PCR amplification

[0204] WT template plasmid 50 ng DNA polymerase 1 μl DNA polymerase buffer 10 μl dNTP (2.5 mM) 4 μl Forward primer (10 μM) 1 μl Reverse primer (10 μM) 1 μl ddH2O Volume made up to 50 μl

[0205] Table 4: PCR reaction conditions

[0206] Step1 Step2 Step3 Step4 Step5 Step6 Temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ Time 5 min 30s 30s 2 min 5 min ∞

[0207] Note: Steps 2 to 4 are carried out for 22 cycles.

[0208] (3) Transformation of single-site saturation mutagenesis plasmids:

[0209] Take 10 μl of the reaction products of the plasmids of the mutant library of amino acid sites in the full CDR regions obtained in step (2) and transform them into 100 μl of TG1 Escherichia coli competent cells to obtain a bacterial solution, and then spread all the bacterial solution on a plate containing ampicillin resistance and culture overnight at 37 °C.

[0210] 3. Screening of single-site mutant libraries

[0211] (1) Expression of antibodies in the mutant library

[0212] Add 500 μl of culture medium to a 96-well culture plate in advance. For each single-site mutant library, select 92 monoclonal colonies, and set up WT, negative control (i.e., colonies without inserting VH\VL genes), and blank control (i.e., only culture medium without colonies) colonies. After culturing at 37 °C for 5 - 6 hours, transfer the bacterial solution to a new 96-well culture plate, and then, after culturing at 37 °C for 1 - 2 hours, finally add the induction medium and culture overnight at 37 °C to express antibodies, obtaining the antibody expression supernatants of 70 mutant libraries.

[0213] (2) Screening and sequencing of the mutant library

[0214] The commercially available P24 was added to 70 ELISA plates at a dose of 0.0078 μg / ml and 100 μl / well, and incubated overnight at 4 °C for coating. The next day, it was blocked with 1-2% skim milk powder. The antibody expression supernatants of the 70 mutant libraries obtained in step (1) were added to the ELISA plate wells at a dose of 100 μl / well, and WT, negative control (i.e., colonies without inserted VH\VL genes) and blank control (i.e., only medium without colonies) were set. Incubate at room temperature for 2 hours, and use the detection method of conventional ELISA for subsequent washing, color development and reading; finally, organize and analyze the data results. The results show that the activity of the mutants is significantly better than that of WT. The clones with improved activity were sent for sequencing, and finally the sequencing results were analyzed, and the mutation sites of 4 unique mutant candidate clones were selected (see Table 5) for constructing a combinatorial mutant library. (The Ratio value in Table 5 represents the degree of affinity improvement. When the Ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of WT).

[0215] Table 5: Screening results and mutation sites of candidate clones

[0216]

[0217] Note: In this article, WT always represents no mutation relative to the wild-type sequence (the same as Tables 6 and 7); the positions of the mutant amino acids in this article were obtained by sequentially numbering the amino acid sequences of VH or VL of WT from the N-terminus to the C-terminus.

[0218] 4. Construction of combinatorial mutant library

[0219] (1) Design and synthesis of library primers:

[0220] According to the mutation sites on VH and VL of the P24 antibody obtained in step 3-(2), amplification primers for the combinatorial mutant library were designed and primers were synthesized.

[0221] (2) Fragment amplification and ligation

[0222] According to the PCR system in Table 3 and the PCR reaction conditions in Table 4, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel extraction. Using the method of Overlap PCR, the antibody mutant fragments were spliced into complete antibody fragments (heavy chain variable region or light chain variable region).

[0223] Finally, the antibody fragment was inserted into the V01 vector by restriction enzyme digestion and ligation to form a complete antibody expression plasmid (for the specific steps, see the steps of "Restriction Enzyme Digestion and Ligation of WT Antibody Gene Fragment" in Step 1-(3)). 100 ng of the plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells, and all the bacterial solution was spread on a plate containing ampicillin resistance and cultured overnight at 37 °C.

[0224] 5. Screening of the combinatorial mutant library

[0225] Randomly select 1 combinatorial mutant antibody for supernatant expression, ELISA binding screening detection, and positive clone sequencing analysis. The mutant site information and Ratio values are shown in Table 6. The results show that the binding activity of the mutant antibody is significantly better than that of WT.

[0226] Table 6: Information on combinatorial mutant candidate clones

[0227]

[0228] Example 2: Expression of mutant P24 antibody

[0229] In this example, the mutant P24 antibody screened in Example 1 was expressed, and the specific experimental operations are as follows:

[0230] 1. Construction of eukaryotic recombinant expression plasmid

[0231] pcDNA TM The pcDNA vector is the constructed recombinant antibody eukaryotic expression vector. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI have been introduced into this expression vector, and it is named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the variable region gene sequences of the 5 candidate clones screened in Example 1, VL and VH gene-specific amplification primers for the corresponding antibody sequences and constant region overlap primers were designed. The two ends of the primers respectively carried HindIII and EcoRI enzyme digestion sites and protective bases, and a 0.73 KB light chain gene fragment and a 1.40 kb heavy chain gene fragment were amplified by PCR amplification.

[0232] The heavy chain and light chain gene fragments were digested with HindIII / EcoRI double enzymes, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. The digested antibody light and heavy chain gene fragments and the vector were purified and recovered. Then, the gene fragments encoding the antibody light and heavy chains were respectively ligated into the 3.4A expression vector and transformed into DH5α Escherichia coli competent cells. After colonies grew, single colonies were picked respectively for PCR identification of positive clones, and the positive clones were picked for sequencing to determine the correctness of the sequence. The clones with correct sequencing were selected for plasmid extraction for standby.

[0233] 2. Preparation of recombinant antibody samples

[0234] Resuscitate HEK293 cells in advance, subculture them to a 200 ml system until the cell density reaches (3 - 5)×10 6 cells / ml and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml as the cell dilution. Prepare plasmid DNA and transfection reagent dilutions with the medium respectively. Add the transfection reagent dilution to the plasmid DNA dilution, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell dilution within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture at a rotation speed of 120 rmp and a CO 2 content of 8%, and collect the samples by centrifugation after 13 days. Purify the antibody by affinity chromatography using a protein A affinity column.

[0235] Example 3: Affinity analysis

[0236] The affinity of some of the obtained mutant antibodies was detected and analyzed. The specific steps were as follows: The binding and dissociation curves of the antigen and antibody were tested on a Biacore8K+ device, and the instrument automatically fit to obtain the affinity constant, binding rate, and dissociation rate. In the affinity detection results, KD represents the equilibrium dissociation constant, that is, the affinity constant. The smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate. The results showed that the mutant antibodies had a better affinity for P24.

[0237] Table 7: Affinity detection data

[0238]

[0239]

[0240] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0241] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-P24 antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are the amino acid sequences shown below: HCDR 1: SYAIS; HCDR 2:GSIPIFGTTHYAQKFQG; HCDR 3:AATAIFGVPX1NNFYAMEV, wherein X1 is R or S; LCDR 1: RX2SQSLLHSNGYX3YLD, where X2 is S, R or W, and X3 is N or L; LCDR 2:IGSTRAS; LCDR 3: MQAVQTF; Optionally, X1 is R; Optionally, X1 is S; Optionally, X2 is S; Optionally, X2 is R; Optionally, X2 is W; Optionally, X3 is N; Optionally, X3 is L; Optionally, X1 is R, X2 is S, and X3 is N are not simultaneously true; Optionally, X1, X2 and X3 are selected from any one of the following combinations:

2. An anti-P24 antibody, characterized in that: include: a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, wherein compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises a mutation at the R108S site; The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, wherein compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 comprises a mutation in at least one of the following sites: S25R / W, N36L; Optionally, the mutation at position 108 is R108S; Optionally, the mutation at position 25 is S25R; Optionally, the mutation at position 25 is S25W; Optionally, the mutation at position 36 is N36L; Optionally, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 17 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 18 are not simultaneously true; Optionally, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

3. An anti-P24 antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region defined by the antibody according to claim 2; the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region defined by the antibody according to claim 2; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems; Optionally, the antibody comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of mouse antibody, human antibody, primate antibody, bovine antibody, horse antibody, dairy cow antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting cock antibody or mutants thereof; Optionally, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.

4. The antibody according to any one of claims 1 to 3, characterized in that The antibody further comprises a constant region; Wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type; Optionally, the heavy chain constant region comprises or is the heavy chain constant region shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto; or The light chain constant region comprises or is the light chain constant region shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity thereto.

5. A nucleic acid, a vector, a cell or a method for preparing the antibody according to any one of claims 1 to 4, characterized in that: The nucleic acid encodes the antibody according to any one of claims 1 to 4; the vector contains the above-mentioned nucleic acid; the cell contains the above-mentioned nucleic acid or vector; and the method comprises culturing the above-mentioned cell.

6. A conjugate, characterized in that: include: The antibody according to any one of claims 1 to 4 and the conjugated part thereof; Optionally, the coupling part includes at least one of a purification tag, an affinity substance, a marker and a solid phase carrier; Optionally, the purification tag includes at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag; Optionally, the solid support comprises at least one selected from microspheres, plates and membranes; Optionally, the label comprises at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the affinity substance includes at least one selected from biotin, a biotin derivative or streptavidin.

7. A reagent or a kit, characterized in that: include: The antibody according to any one of claims 1 to 4 or the conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 4, the conjugate according to claim 6, the reagent or the kit according to claim 7 in detecting P24, preparing a product for detecting P24 or preparing a product for diagnosing HIV infection.

9. A method for detecting P24, characterized in that: include: a) contacting the antibody according to any one of claims 1 to 4, the conjugate according to claim 6, or the reagent or kit according to claim 7 with a sample to be detected to form an immune complex; b) determining whether the sample to be tested contains P24 or the content of P24 based on the signal of the immune complex; Optionally, in step a), the immune complex further comprises a second antibody, which binds to the antibody; Optionally, in step a), the immune complex further comprises a second antibody, and the second antibody binds to the P24.

10. A method for screening P24 antibodies, characterized in that: include: a) designing primers for performing amino acid substitution at the sites of X1, X2 and X3 defined in the antibody of claim 1, or at the mutation sites defined in the antibody of claim 2; b) using the nucleic acid, vector or cell of claim 5 as a template and the primers described in a) to construct a mutation library; c) screening P24 antibodies from the mutant library; Optionally, the mutation library is a single-site saturation mutation library; Optionally, the P24 antibody includes or is the antibody according to any one of claims 1 to 4.

11. A mutation library, characterized in that The mutant library comprises the antibody according to any one of claims 1 to 4; Optionally, the mutant library is obtained by the method of claim 10.

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