Anticoagulant factor XI antibodies or antigen binding fragments thereof and uses thereof

Abstract

The invention provides an antibody or an antigen binding fragment thereof. The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HFR1 as shown in an amino acid sequence SEQ ID NO: 7, an HFR2 as shown in an amino acid sequence SEQ ID NO: 8, an HFR3 as shown in an amino acid sequence SEQ ID NO: 9 or a mutant thereof, and an HFR4 as shown in an amino acid sequence SEQ ID NO: 10; the light chain variable region comprises an LFR1 shown as an amino acid sequence of SEQ ID NO: 13, an LFR2 shown as an amino acid sequence of SEQ ID NO: 14 or a mutant thereof, an LFR3 shown as an amino acid sequence of SEQ ID NO: 15 or a mutant thereof, and an LFR4 shown as an amino acid sequence of SEQ ID NO: 16 or a mutant thereof. The antibody or the antigen binding fragment thereof can improve the expression quantity of the antibody while ensuring the antigen binding activity.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, specifically, the present invention relates to an anti-coagulation factor XI antibody or an antigen-binding fragment thereof and uses thereof, more specifically, the present invention relates to an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a recombinant cell, an anticoagulant, a pharmaceutical composition and uses thereof and a method for increasing the expression amount of an antibody. Background Art

[0002] Thrombosis is an often overlooked danger for patients. When there is one or more blood clots inside a blood vessel, the clot can block blood flow where it forms, or it can break free and travel to other parts of the body. If the transferred clot gets stuck in a vital area, it can be fatal.

[0003] The coagulation cascade in vivo consists of the intrinsic and extrinsic pathways. The intrinsic pathway, also known as the contact activation pathway, is triggered by surface contact, leading to the activation of coagulation factor XII (FXII for short). The intrinsic pathway involves FXI, FIX, and FVIII pathways, also known as the tissue factor (TF) pathway, which is triggered by vascular injury, leading to the formation of the TF-FVIIa activation complex. These two pathways can intersect and activate a common pathway, leading to the conversion of prothrombin to thrombin and ultimately the formation of a cross-linked fibrin clot. An ideal anticoagulant should effectively prevent thrombosis without impairing hemostasis. Evidence suggests that the intrinsic coagulation pathway is important for the amplification phase of coagulation, while the extrinsic and common pathways are more involved in the initiation and diffusion phases. These findings suggest that the intrinsic pathway plays a minor role in normal hemostasis and that inhibition of the intrinsic pathway may provide an antithrombotic effect in thrombus formation with a lower risk of bleeding. FXI is a component of the intrinsic pathway that has only recently been recognized as a potential inhibitor of thrombosis, as it may have the potential to induce an antithrombotic effect without affecting bleeding.

[0004] The most commonly used anticoagulants in clinical practice include: parenteral anticoagulants (such as heparin), coumarin anticoagulants (such as warfarin), antiplatelet coagulation drugs (such as aspirin) and azacitidine (Bolivar), etc. However, all of these drugs have the risk of bleeding.

[0005] Factor XI (FXI) deficiency is a rare congenital bleeding disorder. FXI is a member of the contact activation pathway that, together with the factor VII-tissue factor pathway, leads to the formation of thrombin and subsequent thrombosis. Individuals with FXI deficiency rarely bleed spontaneously and only bleed after surgery or trauma. Experimentally induced bleeding in animals is not affected by FXI deficiency or inhibition, suggesting that the FXI amplification pathway may not be important for normal hemostasis. In contrast, FXI inhibition has antithrombotic effects in animal models of arterial, venous, and cerebral thrombosis, and elevated plasma FXI levels may increase the incidence of thromboembolic disease in humans. There is also some evidence that patients with severe FXI deficiency suffer less from ischemic stroke or venous thrombosis. Therefore, FXI has a unique role in thrombosis and hemostasis, and the development of an anti-factor XI antibody is urgently needed. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides an antibody or an antigen-binding fragment thereof, which can increase the expression amount of the antibody or the antigen-binding fragment thereof while ensuring the antigen-binding activity.

[0007] In the first aspect of the present invention, the present invention proposes an antibody or an antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises HCDRs and HFRs; the HFRs comprises: HFR1 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:7, HFR2 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:8, HFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:9, HFR4 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:10; the light chain variable region comprises LCDRs and LFRs; the LFRs comprises: LFR1 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:13, LFR2 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:14, LFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:15, LFR4 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:16. The antibody or the antigen-binding fragment thereof according to the embodiment of the present invention has the advantages of high expression amount.

[0008] In a second 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 or antigen-binding fragment thereof described in the first aspect.

[0009] In the third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect.

[0010] In a fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: the nucleic acid molecule of the second aspect or the expression vector of the third aspect; or expresses the antibody or antigen-binding fragment thereof of the first aspect.

[0011] In a fifth aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises: the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect.

[0012] In the sixth aspect of the present invention, the present invention provides a use of the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, and the composition described in the fifth aspect in preparing a product, wherein the product is used to detect antigens or prevent and / or treat diseases, such as detecting coagulation factor XI or preventing and / or treating diseases caused by blood clots.

[0013] In the seventh aspect of the present invention, the present invention provides a method for increasing the expression of an antibody, wherein the HCDRs of the antibody are non-human HCDRs, and the LCDRs of the antibody are non-human LCDRs. According to an embodiment of the present invention, the method comprises: making the HFRs and LFRs of the antibody consistent with the HFRs and LFRs described in the first aspect of the present invention.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0016] Figure 1 The results of the binding of the 3G12 chimeric antibody and the humanized 3G12 antibody to the antigens FXI and FXIa detected by ELISA in Example 2 of the present invention are shown.

[0017] Figure 2 The APTT results of 3G12 chimeric antibody and humanized 3G12 antibody detected by APTT detection method in Example 3 of the present invention are shown.

[0018] Figure 3In Example 3 of the present invention, the effects of mab3G12zhIgG1-b4, mab3G12 zhIgG1-b5 and three different positive controls on APTT were compared in human serum in vitro by the APTT detection method.

[0019] Figure 4 The APTT and PT activities of mab3G12zhIgG1-b5 and mab3G12 zhIgG1-b5 were detected in vitro in human serum by the APTT detection method in Example 3 of the present invention.

[0020] Figure 5 The SPR results of humanized mab3G12zhIgG1-b5 and mab3G12 zhIgG1-b5 antibodies binding to different concentrations of FXI by SPR detection method in Example 4 of the present invention are shown in FIG.

[0021] Figure 6 The SPR results of the binding of humanized mab3G12zhIgG1-b5 and mab3G12 zhIgG1-b5 antibodies to different concentrations of FXIa were determined by the SPR detection method in Example 4 of the present invention.

[0022] Figure 7 The SPR results and parameters of the humanized mab3G12zhIgG1-b5 and mab3G12 zhIgG1-b5 antibodies binding to different concentrations of FXI (A) or FXIa (B) determined by the SPR detection method in Example 4 of the present invention.

[0023] Figure 8 These are the SPR results of the binding of humanized mab3G12zhIgG1 antibodies b0, b4, b5, b6, b7, and b8 to different concentrations of FXI determined by the SPR detection method in Example 4 of the present invention.

[0024] Fig. 9 These are the SPR results of the binding of humanized mab3G12zhIgG1 antibodies b0, b4, b5, b6, b7, and b8 to different concentrations of FXIa determined by the SPR detection method in Example 4 of the present invention. DETAILED DESCRIPTION

[0025] 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 understood as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0027] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0028] In order to make the present invention more easily understood, 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 belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0029] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0030] As used herein, the terms "optionally", "optional", "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0031] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and 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 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. 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. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize 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, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0032] Under the premise of not substantially affecting the activity of the antibody (retaining at least 80% or at least 90% of the activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2 or 3) amino acids in the sequence of the present invention to obtain variants of the antibody or its antigen-binding fragment sequence. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0033] As used herein, the term "at least 80% homology" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology to each reference sequence.

[0034] As used herein, the term "at least 90% homology" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% homology to each reference sequence.

[0035] As used herein, the term "variant" or "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0036] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred to a cell or host and / or between cells or hosts. The expression vector may include an expression vector that is mainly used to insert DNA or RNA into a cell, an expression vector that is mainly used to replicate DNA or RNA, and an expression vector that is mainly used to transcribe and / or translate DNA or RNA. The expression vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, the vector can produce a desired expression product by culturing a suitable cell or host that contains the vector.

[0037] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of 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 terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells 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. The present invention proposes an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a recombinant cell, an anticoagulant, a pharmaceutical composition, and their uses and a method for increasing the expression of an antibody, which will be described in detail below.

[0038] Antibodies or antigen-binding fragments thereof

[0039] In the first aspect of the present invention, the present invention proposes an antibody or an antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises HCDRs and HFRs; the HFRs comprises: HFR1 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:7, HFR2 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:8, HFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:9, HFR4 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:10; the light chain variable region comprises LCDRs and LFRs; the LFRs comprises: LFR1 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:13, LFR2 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:14, LFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:15, LFR4 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:16. The antibody or the antigen-binding fragment thereof according to the embodiment of the present invention has the advantages of high antibody expression while ensuring antigen binding activity.

[0040] In this article, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited as long as they show the desired biological activity. Exemplarily, for full-length monoclonal antibodies, it generally includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl terminal (C terminal) is relatively stable and changes very little, which is called the constant region (C region). The V regions of the L chain and the H chain are respectively called VL and VH.

[0041] Herein, the heavy chain complementary determining regions (heavy chain variable region CDR) are represented 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 complementary determining regions (light chain variable region CDR) are represented 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).

[0042] Commonly used CDR definition schemes in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table A below. The definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that the CDRs in the present invention include but are not limited to CDRs defined by other methods in Table A. CDRs determined based on the heavy chain variable region and light chain variable region disclosed in the present invention using other rules disclosed in the art also fall within the scope of protection of the present disclosure.

[0043] Table A: CDR Definition 1

[0044] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33 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

[0045] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number". 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0046] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0047] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0048] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0049] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. A person of ordinary skill in the art can clearly correspond the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As described herein, "Kabat numbering" refers to numbering using the numbering system described in "Kabat et al., U. S. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983)". The above numbering system is used to number the HCDRs and LCDRs of the antibodies or their functional fragments of the present application, and the specific numbering results are shown in Table 1. It should be noted that the polypeptide sequences in the sequence table and Table 2 of the present invention are not numbered according to the Kabat numbering system. However, a person of ordinary skill in the art is fully capable of converting the sequence numbers of the sequence table into Kabat numbers.

[0050] Herein, "framework region" or "FR" region includes heavy chain framework region and light chain framework region, and refers to the region other than CDR in antibody heavy chain variable region (which can be expressed as VH) and light chain variable region (which can be expressed as VL); wherein, heavy chain framework region (heavy chain variable region FR) is expressed as "HFRs" or "HFR", and can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; light chain framework region (light chain variable region FR) is expressed as "LFRs" or "LFR", and can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0051] As used herein, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0052] It should be noted that the framework region in the present invention can be determined based on CDR, that is, when CDR is defined using the Kabat system (see Table A for details), the four parts separated by CDR in the light chain variable region or the heavy chain variable region correspond to the HFR1, HFR2, HFR3 and HFR4 framework regions, respectively.

[0053] According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment thereof may further include at least one of the following technical features:

[0054] According to an embodiment of the present invention, the HFRs include: HFR1 as shown in the amino acid sequence of SEQ ID NO:7, HFR2 as shown in the amino acid sequence of SEQ ID NO:8, HFR3 or its mutants as shown in the amino acid sequence of SEQ ID NO:9, and HFR4 as shown in the amino acid sequence of SEQ ID NO:10.

[0055] According to an embodiment of the present invention, the LFRs include: LFR1 as shown in the amino acid sequence of SEQ ID NO:13, LFR2 or its mutant as shown in the amino acid sequence of SEQ ID NO:14, LFR3 or its mutant as shown in the amino acid sequence of SEQ ID NO:15, and LFR4 or its mutant as shown in the amino acid sequence of SEQ ID NO:16.

[0056] According to an embodiment of the present invention, compared with HFR3 shown in the amino acid sequence of SEQ ID NO: 9, the mutant of HFR3 has at least one of the following mutation sites or its equivalent sites: N76, Y91 and H94.

[0057] According to an embodiment of the present invention, compared with HFR3 shown in the amino acid sequence of SEQ ID NO: 9, the mutant of HFR3 has at least one of the following mutation sites or its equivalent site: N76D, Y91F and H94R.

[0058] It should be noted that the amino acid numbers of the heavy chain variable region or the light chain variable region in this article are defined using the Kabat system. For example, "N76D" means that the asparagine at position 76 in the heavy chain variable region defined by the Kabat system is replaced by aspartic acid.

[0059] In this article, the term "equivalent site" refers to a site where the position and amino acid type of a certain specific site are different due to different antibody sources (e.g., antibodies derived from different sources or species), types (e.g., IgG, IgM, IgA, etc.), subclasses (e.g., IgG can be divided into subclasses such as IgG1 and IgG2), or the existence of alleles. The equivalent site of the specific site can be determined by sequence and structural homology comparison.

[0060] According to an embodiment of the present invention, the mutant of HFR3 has an amino acid sequence as shown in SEQ ID NO: 11 or 12.

[0061] According to an embodiment of the present invention, compared with LFR2 shown in the amino acid sequence of SEQ ID NO: 14, the mutant of LFR2 has a mutation site of Y36 or an equivalent site thereof.

[0062] According to an embodiment of the present invention, compared with LFR2 shown in the amino acid sequence of SEQ ID NO: 14, the mutant of LFR2 has a mutation site of Y36F or an equivalent site thereof.

[0063] According to an embodiment of the present invention, the mutant of LFR2 has an amino acid sequence as shown in SEQ ID NO:17.

[0064] According to an embodiment of the present invention, compared with LFR3 shown in the amino acid sequence of SEQ ID NO: 15, the mutant of LFR3 has a mutation site of Y87 or an equivalent site thereof.

[0065] According to an embodiment of the present invention, compared with LFR3 shown in the amino acid sequence of SEQ ID NO: 15, the mutant of LFR3 has a mutation site of Y87F or an equivalent site thereof.

[0066] According to an embodiment of the present invention, the mutant of LFR3 has an amino acid sequence as shown in SEQ ID NO:18.

[0067] According to an embodiment of the present invention, compared with LFR4 shown in the amino acid sequence of SEQ ID NO: 16, the mutant of LFR4 has a mutation site of Q100 or an equivalent site thereof.

[0068] According to an embodiment of the present invention, compared with LFR4 shown in the amino acid sequence of SEQ ID NO: 16, the mutant of LFR4 has a mutation site of Q100G or an equivalent site thereof.

[0069] According to an embodiment of the present invention, the mutant of LFR4 has an amino acid sequence as shown in SEQ ID NO:19.

[0070] According to an embodiment of the present invention, the HFRs include any one of the following groups:

[0071] Group HFR1 HFR2 HFR3 HFR4 1 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:10 2 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:11 SEQ ID NO:10 3 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:10 .

[0072] According to an embodiment of the present invention, the LFRs include any one of the following groups:

[0073] Group LFR1 LFR2 LFR3 LFR4 1 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 2 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:15 SEQ ID NO:16 3 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:16 4 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:19 5 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:16 6 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:15 SEQ ID NO:19 7 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:19 8 SEQ ID NO:13 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 .

[0074] According to an embodiment of the present invention, the HCDRs are non-human HCDRs, and / or the LCDRs are non-human LCDRs.

[0075] According to an embodiment of the present invention, the HCDRs include HCDR1, HCDR2 and HCDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively.

[0076] According to an embodiment of the present invention, the LCDRs include LCDR1, LCDR2 and LCDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

[0077] According to an embodiment of the present invention, the antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, or an amino acid sequence having at least 90% homology thereto; and / or a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or an amino acid sequence having at least 90% homology thereto.

[0078] In an optional embodiment of the present invention, the antibody or antigen-binding fragment thereof is selected from any one of the following combinations or a heavy chain variable region and a light chain variable region having more than 90% identity therewith:

[0079] Group Heavy chain variable region Light chain variable region 1 SEQ ID NO:20 SEQ ID NO:23 2 SEQ ID NO:21 SEQ ID NO:23 3 SEQ ID NO:22 SEQ ID NO:23 4 SEQ ID NO:20 SEQ ID NO:24 5 SEQ ID NO:21 SEQ ID NO:24 6 SEQ ID NO:22 SEQ ID NO:24 7 SEQ ID NO:20 SEQ ID NO:25 8 SEQ ID NO:21 SEQ ID NO:25 9 SEQ ID NO:22 SEQ ID NO:25 10 SEQ ID NO:20 SEQ ID NO:26 11 SEQ ID NO:21 SEQ ID NO:26 12 SEQ ID NO:22 SEQ ID NO:26 13 SEQ ID NO:20 SEQ ID NO:27 14 SEQ ID NO:21 SEQ ID NO:27 15 SEQ ID NO:22 SEQ ID NO:27 16 SEQ ID NO:20 SEQ ID NO:28 17 SEQ ID NO:21 SEQ ID NO:28 18 SEQ ID NO:22 SEQ ID NO:28 19 SEQ ID NO:20 SEQ ID NO:29 20 SEQ ID NO:21 SEQ ID NO:29 21 SEQ ID NO:22 SEQ ID NO:29 22 SEQ ID NO:20 SEQ ID NO:30 23 SEQ ID NO:21 SEQ ID NO:30 24 SEQ ID NO:22 SEQ ID NO:30 .

[0080] The antibodies or antigen-binding fragments thereof according to the embodiments of the present invention can maintain a higher degree of humanization and increase the expression level of the antibodies or antigen-binding fragments thereof by introducing as few back mutations as possible.

[0081] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof 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.

[0082] According to an embodiment of the present invention, 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.

[0083] According to an embodiment of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0084] According to an embodiment of the present invention, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type.

[0085] In an optional embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from human antibodies or mutants thereof.

[0086] Herein, the "heavy chain constant region" includes at least one of a CH1 fragment, a hinge region, a CH2 fragment, and a CH3 fragment.

[0087] In a preferred embodiment of the present invention, the heavy chain constant region is derived from the Fc fragment (hinge region, CH2 fragment and CH3 fragment) of human IgG1.

[0088] In an optional embodiment of the present invention, the light chain constant region includes a light chain constant region selected from κ type.

[0089] 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.

[0090] In an optional embodiment of the present invention, the heavy chain constant region includes or is a heavy chain constant region as shown in SEQ ID NO:42 or an amino acid sequence having at least 80% identity thereto; or the light chain constant region includes or is a light chain constant region as shown in SEQ ID NO:43 or an amino acid sequence having at least 80% identity thereto.

[0091] In an optional embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: a heavy chain of an amino acid sequence as shown in any one of SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, or an amino acid sequence having at least 80% homology thereto; and / or a light chain of an amino acid sequence as shown in any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, or an amino acid sequence having at least 80% homology thereto;

[0092] In an optional embodiment of the present invention, the antibody or antigen-binding fragment thereof is selected from any one of the following combinations or a heavy chain and a light chain having more than 80% identity therewith:

[0093]

[0094]

[0095] According to an embodiment of the present invention, the antibody includes at least one selected from a polyclonal antibody, a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a F(ab)2 antibody, a Fv antibody, a single-chain antibody, a single-domain antibody and a minimum recognition unit; or, the antigen-binding fragment includes at least one selected from a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

[0096] As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

[0097] In this article, the terms "polyantibody" and "multispecific antibody" are synonymous and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that can recognize three antigenic epitopes, or antibodies that can recognize four antigenic epitopes. This is understood in a broad sense and the specific structure is not limited as long as it can recognize multiple antigenic epitopes.

[0098] In this article, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen proteins through the heavy chain variable region.

[0099] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.

[0100] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0101] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.

[0102] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0103] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, and their molecular weight is very small, accounting for only about 1% of the complete antibody.

[0104] The above-mentioned antibodies or antigen-binding fragments thereof generally have the same binding specificity as the antibody from which they are derived. It is easy for a person skilled in the art to understand from the contents of the disclosure that the above-mentioned antibodies or antigen-binding fragments thereof can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction splitting disulfide bonds. Based on the structure of the complete antibody in the disclosure, it is easy for a person skilled in the art to obtain the above-mentioned antibodies or antigen-binding fragments thereof. The above-mentioned antibodies or antigen-binding fragments thereof can also be obtained by recombinant genetics techniques known to a person skilled in the art or by, for example, automatic peptide synthesizers, such as those sold by Applied BioSystems, etc.

[0105] Nucleic acid molecules, expression vectors, recombinant cells

[0106] In the process of preparing or obtaining the antibodies or antigen-binding fragments thereof described in the first aspect, nucleic acid molecules expressing these antibodies or antigen-binding fragments thereof can be connected to different expression vectors and then expressed in different recombinant cells to obtain the corresponding antibodies or antigen-binding fragments thereof.

[0107] In a second 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 or antigen-binding fragment thereof described in the first aspect. The nucleic acid molecule according to an embodiment of the present invention can encode the above-mentioned antibody or antigen-binding fragment thereof.

[0108] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0109] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0110] In the third aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector includes the nucleic acid molecule described in the second aspect. When the above-mentioned nucleic acid molecule is connected to the expression vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, they are not from the expression vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. Herein, "operably connected" means connecting the exogenous gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, the expression of the aforementioned antibody or its antigen-binding fragment can be effectively achieved under the mediation of the regulatory system, thereby achieving a large amount of in vitro acquisition of the antibody or its antigen-binding fragment.

[0111] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0112] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector.

[0113] In the fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expresses the antibody or antigen-binding fragment thereof described in the first aspect. The recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment thereof in the recombinant cell under suitable conditions.

[0114] According to an embodiment of the present invention, the cell is obtained by introducing the expression vector described in the third aspect into a recombinant cell.

[0115] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0116] In an optional embodiment of the present invention, the recombinant cell is a mammalian cell, including a BHK cell, a CHO cell, a NSO cell or a COS cell, and does not include an animal germ cell, a fertilized egg or an embryonic stem cell.

[0117] It should be noted that the "suitable conditions" described in the present invention refer to conditions suitable for the expression of the antibody or antigen-binding fragment thereof of the present invention. It is easy for those skilled in the art to understand that conditions suitable for the expression of the antibody or antigen-binding fragment thereof include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody or antigen-binding fragment thereof according to the specific environment of the laboratory.

[0118] Composition and use thereof, method for increasing antibody expression

[0119] In the fifth aspect of the present invention, the present invention proposes a composition. According to an embodiment of the present invention, the composition comprises: the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. Thus, the composition containing the aforementioned antibody or antigen-binding fragment thereof can specifically bind to an antigen, and can be used to detect an antigen or prevent and / or treat a disease. For example, it can bind to coagulation factor XI to treat and prevent diseases caused by blood clots.

[0120] According to an embodiment of the present invention, it further includes a pharmaceutically acceptable carrier or excipient.

[0121] According to an embodiment of the present invention, the composition is a pharmaceutical composition.

[0122] According to an embodiment of the present invention, the pharmaceutical composition is an anticoagulant drug, so that the anticoagulant drug can specifically bind to an antigen, for example, can specifically bind to FXI.

[0123] According to an embodiment of the present invention, the composition is an anticoagulant.

[0124] In this article, the term "anticoagulant drugs" refers to a class of pharmaceutical compositions that can be used to prevent or reduce the formation of blood clots. The types of anticoagulant drugs mainly include: antiplatelet drugs, antithrombin drugs, direct thrombin inhibitors or direct coagulation factor Xa inhibitors. In addition to the active ingredients of the drug, anticoagulant drugs also include drug carriers and auxiliary ingredients.

[0125] It should be noted that an anticoagulant refers to an agent that can inhibit or delay the formation of blood clots. This agent can regulate blood coagulation and prevent thrombosis.

[0126] The administration of the pharmaceutical composition of the present invention can be carried out by any acceptable mode of administration. The pharmaceutical composition of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations, such as capsules, tablets, injections, lyophilized powders, and the current methods for preparing these dosage forms are known or obvious to those skilled in the art. The auxiliary components of the pharmaceutical composition may include solvents, thickeners, buffers or surfactants. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes.

[0127] In the sixth aspect of the present invention, the present invention proposes a use of the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, and the composition described in the fifth aspect in preparing a product, wherein the product is used to detect antigens or prevent and / or treat diseases.

[0128] According to an embodiment of the present invention, the product is a medicine or a kit.

[0129] According to an embodiment of the present invention, the medicine is used for preventing and / or treating a disease.

[0130] According to an embodiment of the present invention, the disease is a disease caused by blood clot.

[0131] According to an embodiment of the present invention, the kit is used for detecting antigens.

[0132] According to an embodiment of the present invention, the antigen is coagulation factor XI.

[0133] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Additionally, for purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or condition.

[0134] In the seventh aspect of the present invention, the present invention proposes a method for increasing the expression of an antibody, wherein the HCDRs of the antibody are non-human HCDRs, and the LCDRs of the antibody are non-human LCDRs. According to an embodiment of the present invention, the HFRs and LFRs of the antibody are consistent with the HFRs and LFRs described in the first aspect of the present invention. According to an embodiment of the present invention, the amino acid sequence of the antibody is consistent with the amino acid sequence of the antibody or its antigen-binding fragment described in the first aspect of the present invention. The sequence description involved in the present invention is shown in the following table:

[0135]

[0136]

[0137]

[0138] Note: In the above table, the reversion mutation site of the heavy chain HFR3 mutant of the 3G12 antibody as shown in SEQ ID NO:11 and SEQ ID NO:12 amino acid sequences is relative to the heavy chain HFR3 of the 3G12 antibody as shown in SEQ ID NO:9 amino acid sequence; the reversion mutation site of the heavy chain LFR2 mutant of the 3G12 antibody as shown in SEQ ID NO:17 amino acid sequence is relative to the heavy chain LFR2 of the 3G12 antibody as shown in SEQ ID NO:14 amino acid sequence; the reversion mutation site of the heavy chain LFR3 mutant of the 3G12 antibody as shown in SEQ ID NO:18 amino acid sequence is relative to the heavy chain LFR3 of the 3G12 antibody as shown in SEQ ID NO:15 amino acid sequence; the reversion mutation site of the heavy chain LFR4 mutant of the 3G12 antibody as shown in SEQ ID NO:19 amino acid sequence is relative to the heavy chain LFR4 of the 3G12 antibody as shown in SEQ ID NO:16 amino acid sequence.

[0139] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0140] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0141] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0142] Example 1: Humanized design of hybridoma 3G12 antibody variable region sequence

[0143] Humanization of antibodies can reduce the immunogenicity of monoclonal antibodies and reduce their activation of the human immune system by replacing non-human antibody frameworks with human antibody frameworks, which is a very important step in the discovery process of therapeutic antibodies. Therefore, in this embodiment, humanization transformation was performed based on the VH of the 3G12 chimeric antibody (such as the amino acid sequence shown in SEQ ID NO: 34) and the VL of the 3G12 chimeric antibody (such as the amino acid sequence shown in SEQ ID NO: 35) to obtain 3G12 antibody VH.h0 (such as the amino acid sequence shown in SEQ ID NO: 20) and 3G12 antibody VL.g0 (such as the amino acid sequence shown in SEQ ID NO: 23). Then, the 3G12 antibody VH.h0 was reversed, and the reverse mutation site included at least one of N78D, Y96F and H99R; and the 3G12 antibody VL.g0 was reversed, and the reverse mutation site included at least one of Y40F, Y91F and Q104G. The back mutation sites were freely combined to obtain 3G12 antibody VH.h1 (amino acid sequence as shown in SEQ ID NO:21, mutation sites are N76D, H94R), 3G12 antibody VH.h2 (amino acid sequence as shown in SEQ ID NO:22, mutation sites are N76D, Y91F and H94R), 3G12 antibody VL.g1 (amino acid sequence as shown in SEQ ID NO:24, mutation site is Y36F) and 3G12 antibody VL.g2 (amino acid sequence as shown in SEQ ID NO:25, mutation sites are Y36F, Y87F and Q100G).

[0144] Furthermore, the heavy chain constant region of the above antibody adopts the human IgG1Fc sequence, and the light chain constant region is a Kappa chain sequence. Therefore, the heavy chain corresponding to 3G12 antibody VH.h0 is such as the amino acid sequence shown in SEQ ID NO:31 (referred to as 3G12 antibody heavy chain h0 or 3G12 heavy chain h0), the heavy chain corresponding to 3G12 antibody VH.h1 is such as the amino acid sequence shown in SEQ ID NO:32 (referred to as 3G12 antibody heavy chain h1 or 3G12 heavy chain h1), and the heavy chain corresponding to 3G12 antibody VH.h2 is such as the amino acid sequence shown in SEQ ID NO:33 (referred to as 3G12 antibody heavy chain h2 or 3G12 heavy chain h2); the light chain corresponding to 3G12 antibody VL.g0 is such as the amino acid sequence shown in SEQ ID NO:34 (referred to as 3G12 antibody light chain g0 or 3G12 light chain g0), the light chain corresponding to 3G12 antibody VL.g1 is such as the amino acid sequence shown in SEQ ID NO:35 (referred to as 3G12 antibody light chain g1 or 3G12 light chain g1), and the light chain corresponding to 3G12 antibody VL.g2 is such as the amino acid sequence shown in SEQ ID NO:36 (referred to as 3G12 antibody light chain g1 or 3G12 light chain g1). The amino acid sequence of SEQ ID NO:36 (referred to as 3G12 antibody light chain g2 or 3G12 light chain g2). The heavy chain of the 3G12 chimeric antibody is the amino acid sequence of SEQ ID NO:46, and the light chain of the 3G12 chimeric antibody is the amino acid sequence of SEQ ID NO:47. The combination of the above light chain and heavy chain is shown in Table 1.

[0145] Table 1: Composition of light and heavy chains in each antibody and antibody name

[0146]

[0147]

[0148] In the above table, the content in “()” means “the number of back mutation sites in the light chain + the number of back mutation sites in the heavy chain = the total number of back mutation sites in the antibody”.

[0149] The antibodies prepared above were purified and identified as follows: After determining the cloned sequence, the variable region sequence was cloned into the pTT5 vector by PCR technology, and then the ExpiCHO cells were transfected to express the antibody. The cells were cultured in a shaking incubator at 32°C for 12 days. The cell culture supernatant was harvested and clarified by centrifugation at 2000rpm for 10 minutes, and then filtered through a 0.22um filter. The clarified supernatant was purified using AKTA and MabselectSure (1mL) columns and eluted with 0.2M Tris-Glycine (pH3.4) buffer. After concentration, the eluted antibody was further purified by Superdex 200 (GE) chromatography. The purified protein was tested for quality and purity using an ultra-micro UV-visible spectrophotometer, and the test results are shown in Table 2.

[0150] All antibodies in this example have a molecular size of about 144KD in a non-reduced state. After reduction, the heavy chain is about 50KD, and the light chain is about 25KD. Among them, the purity of the humanized antibodies (mab3G12 zhIgG1-b0 to mab3G12zhIgG1-b8) is above 95%.

[0151] The production of all humanized antibodies (mab3G12zhIgG1-b0 to mab3G12 zhIgG1-b8) was greater than that of 3G12 chimeric antibodies. Specifically, the production of 3G12 chimeric antibodies was 23.7 mg / L, while the production of humanized antibodies was above 100 mg / L, which was more than 5 times that of chimeric antibodies. The three humanized antibodies with the highest production were mab3G12zhIgG1-b2 (207.7 mg / L), mab3G12 zhIgG1-b5 (203.4 mg / L), and mab3G12 zhIgG1-b3 (188 mg / L).

[0152] Therefore, it can be further explained that the expression level of the antibody can be further increased by using the framework region of the humanized antibody in the present application to humanize the framework region of antibodies of other species.

[0153] Table 2: Molecular weight and expression level of each antibody

[0154]

[0155]

[0156] Example 2: Binding analysis of humanized 3G12 antibody to FXI and FXIa proteins by ELISA

[0157] As described in the present invention, the binding of the 3G12 chimeric antibody and the humanized 3G12 antibody prepared in Example 1 to human FXI and FXIa proteins was analyzed by ELISA. The specific test steps are as follows: the plate is coated with 100 μL of coating solution per well, then the lid is closed, and the incubation solution (1×PBS with 100 ng / well human FXI or human FXIa; ERL, Cat No. HFXI1111and HFXIa 1111a) is added and incubated at 2-8°C for 12-18 hours. The wells are aspirated, washed once with >200 μL of washing buffer (PBST (1×PBS solution containing 0.05% (v / v) Tween 80) per well), inverted and tapped on absorbent paper to remove excess liquid. The plates were blocked with 200 μL of blocking buffer (1×PBS+4% milk; BD, 23200) per well for 1 hour at room temperature, aspirated, inverted, and tapped on absorbent paper to remove excess liquid.

[0158] The 3G12 chimeric antibody and humanized 3G12 antibody (mab3G12zhIgG1-b0 to mab3G12 zhIgG1-b8) prepared in Example 1 were diluted with blocking buffer (i.e., primary antibody), and the primary antibody dilution was 5, 10, and 50 ng / ml. 100 μL of primary antibody solution was added to each well and incubated for 2 hours at room temperature with continuous gentle shaking (500 rpm). The wells were aspirated and washed 3 times with >200 μL of washing buffer per well, inverted and tapped on absorbent paper to remove excess liquid. The secondary antibody (mouse anti-human IgG1 Fc antibody HRP (1:2,500, Invitrogen, MH1715)) was diluted 2500 times with blocking buffer to prepare a secondary antibody working solution. 100 μL of secondary antibody working solution was added to each well and incubated at room temperature for 30 minutes. Wash 5 times with >200 μL of wash buffer per well, invert and tap on absorbent paper to remove excess liquid. Add 100 μL of TMB substrate solution (Biopanda, TMB-S-004) to each well and incubate at room temperature for 30 minutes. Add 100 μL of stop solution (Solarbio, C1058) to each well. Measure the absorbance at 450 nm within 30 minutes after adding the stop solution. For the binding ability of each antibody to hFXI and hFXIa proteins, see Figure 1 Among them, FXIa is the active form of FXI.

[0159] Depend on Figure 1 It can be seen that at the concentrations of 10 and 50 ng / ml, all humanized 3G12 antibodies have strong binding ability with FXI and FXIa. Therefore, it can be further explained that the humanized modification of the antibody framework region of the present invention still maintains good binding ability with FXI and FXIa antigens.

[0160] Example 3: Evaluation of the anticoagulant activity of the antibody of the present invention in human plasma using activated partial thromboplastin time (APTT) and prothrombin time (PT) detection indicators

[0161] In this example, the anticoagulant activity of the 3G12 chimeric antibody and humanized 3G12 antibody prepared in Example 1 and the Fc of the wild type of human IgG1 in human plasma was evaluated by APTT and PT detection methods. In this example, NOV1401 (its amino acid sequence and preparation method are shown in US20170022292A1), AB023 (its amino acid sequence and preparation method are shown in WO2020154234A1) and BAY1213790 (BAY for short, its amino acid sequence and preparation method are shown in US9783614B2) were used as positive controls. All antibodies (3G12 antibody and positive control antibody) in this example were expressed using the ExpiCHO cell system. The specific detection steps are as follows:

[0162] Standard human plasma (purchased from Siemens Inc.) and different concentrations of the antibody to be tested (concentration gradient of 0-300 nM, 3G12 chimeric antibody prepared in Example 1, humanized 3G12 antibody (mab3G12 zhIgG1-b0 to mab3G12zhIgG1-b8) or positive control antibody) were mixed in equal volumes, incubated for 5 minutes, and then detected using a CA600 analyzer to obtain a mixture of plasma and antibody.

[0163] In the APTT assay, 50 μL of the above plasma and antibody mixture was added with 25 μL of APTT reagent (SMN10445709, Symens Inc.), mixed, and incubated at 37°C for 4 min. 25 μL of CaCl2 solution (25 mM, SMN10446232, Symens Inc.) was added, and the clotting time was recorded.

[0164] In the PT assay, 50 μL of the above plasma and antibody mixture was added with an equal volume of PT reagent (SMN10446442, Symens Inc.), mixed, and the clotting time was measured at 37°C.

[0165] from Figure 2 A's APTT test results show that at the working concentrations of 3μg / ml and 5μg / ml, mab3G12zhIgG1-b0 to mab3G12 zhIgG1-b8 all have similar properties of delaying APTT time. Five humanized antibodies, including mab3G12zhIgG1-b4 to mab3G12 zhIgG1-b8, were selected and mixed with human plasma at 8 working concentrations of 0 to 300nM. Figure 2As can be seen from the results in B, all five antibodies showed properties similar to those of the chimeric 3G12 antibody in delaying APTT.

[0166] from Figure 3 From the results of experiments A and 3B, it can be seen that the ability of mab3G12zhIgG1-b4 and mab3G12 zhIgG1-b5 to delay APTT is greater than that of the three positive control antibodies, indicating that mab3G12 zhIgG1-b4 and mab3G12 zhIgG1-b5 antibodies have better anticoagulant activity.

[0167] And, by Figure 4 It can be seen that mab3G12zhIgG1-b4 and mab3G12 zhIgG1-b5 have no effect on the PT of human serum, which further shows that mab3G12 zhIgG1-b5 and mab3G12 zhIgG1-b5 antibodies only affect the APTT pathway in human serum.

[0168] Example 4: Affinity measurement by SPR

[0169] In this example, the affinity of the 3G12 chimeric antibody and the humanized 3G12 antibody prepared in Example 1 was determined by the SPR detection method. The specific detection steps are as follows:

[0170] SPR interaction analysis of captured antibodies (3G12 chimeric antibodies prepared in Example 1, humanized 3G12 antibodies (mab3G12zhIgG1-b0 to mab3G12 zhIgG1-b8), AB023, BAY) and FXI / FXIa was performed on a Biacore T200 system (GE Healthcare) using a high immobilization human antibody capture kit (Cytiva). Immobilization of anti-human IgG (Fc) antibodies was performed on a CM5 chip using a standard amine coupling kit (Cytiva) with 10 mM sodium acetate at pH 5.0 and 0.5 mg / ml anti-human IgG (Fc) antibody solution. The immobilization level of anti-human IgG (Fc) reached >9000RU. Using running buffer HBS-EP, each 1ug / ml antibody solution was prepared, and the capture immobilization level was 50RU at a flow rate of 10 microliters / min. Subsequently, 0.005-0.33ug / ml of FXI / FXIa was applied in HBS-EP at a flow rate of 30 μl / min for 60-180 s. The dissociation phase was monitored for 1000 s. A 30-s wash step with 3M magnesium chloride at a flow rate of 30 μl / min was performed to regenerate the surface. Data analysis was performed using Biacore T200 evaluation software.

[0171] The results show that the humanized 3G12 antibodies (mab3G12zhIgG1-b0 to mab3G12 zhIgG1-b8) of the present invention have high affinity for FXI or FXIa, and the KD values ​​are all at the level of 10-12. For details of the specific values ​​and experimental setting parameters, see Figure 7 , Figure 8 and Fig. 9 , SPR curve is shown in Figure 5 , Figure 6 , Figure 8 and Fig. 9 .

[0172] Therefore, it can be seen from the experimental results of the above examples that the antibodies obtained in the present invention are all capable of binding to coagulation factor XI, and have the potential to be developed as potential drugs for preventing and treating thrombosis.

[0173] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0174] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that: including a heavy chain variable region and a light chain variable region; Wherein, the heavy chain variable region includes HCDRs and HFRs; The HFRs include: HFR1 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:7, HFR2 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 8, HFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:9, HFR4 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 10; The light chain variable region includes LCDRs and LFRs; The LFRs include: LFR1 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 13, LFR2 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 14, LFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 15, LFR4 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:

16.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The HFRs include: HFR1 as shown in the amino acid sequence of SEQ ID NO:7, HFR2 as shown in the amino acid sequence of SEQ ID NO: 8, HFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO:9, HFR4 as shown in the amino acid sequence of SEQ ID NO: 10; Optionally, the LFRs include: LFR1 as shown in the amino acid sequence of SEQ ID NO: 13, LFR2 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 14, LFR3 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 15, LFR4 or a mutant thereof as shown in the amino acid sequence of SEQ ID NO: 16; Optionally, compared with the HFR3 shown in the amino acid sequence of SEQ ID NO: 9, the mutant of HFR3 has at least one of the following mutation sites or its equivalent site: N76, Y91 and H94; Optionally, compared with the HFR3 shown in the amino acid sequence of SEQ ID NO: 9, the mutant of HFR3 has at least one of the following mutation sites or its equivalent site: N76D, Y91F and H94R; Optionally, the mutant of HFR3 has an amino acid sequence as shown in SEQ ID NO: 11 or 12; Optionally, compared with LFR2 as shown in the amino acid sequence of SEQ ID NO: 14, the mutant of LFR2 has a mutation site of Y36 or an equivalent site thereof; Optionally, compared with LFR2 as shown in the amino acid sequence of SEQ ID NO: 14, the mutant of LFR2 has a mutation site of Y36F or an equivalent site thereof; Optionally, the mutant of LFR2 has an amino acid sequence as shown in SEQ ID NO: 17; Optionally, compared with LFR3 as shown in the amino acid sequence of SEQ ID NO: 15, the mutant of LFR3 has a mutation site of Y87 or an equivalent site thereof; Optionally, compared with LFR3 as shown in the amino acid sequence of SEQ ID NO: 15, the mutant of LFR3 has a mutation site of Y87F or an equivalent site thereof; Optionally, the mutant of LFR3 has an amino acid sequence as shown in SEQ ID NO: 18; Optionally, compared with LFR4 as shown in the amino acid sequence of SEQ ID NO: 16, the mutant of LFR4 has a mutation site of Q100 or an equivalent site thereof; Optionally, compared with LFR4 as shown in the amino acid sequence of SEQ ID NO: 16, the mutant of LFR4 has a mutation site of Q100G or an equivalent site thereof; Optionally, the mutant of LFR4 has an amino acid sequence as shown in SEQ ID NO: 19; Optionally, the HFRs include any one of the following groups: ； Optionally, the LFRs include any one of the following groups: 。 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The HCDRs are non-human HCDRs, and / or the LCDRs are non-human LCDRs; Optionally, the HCDRs include HCDR1, HCDR2 and HCDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; Optionally, the LCDRs include LCDR1, LCDR2 and LCDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively; Optionally, the antibody or antigen-binding fragment thereof comprises: The heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, or an amino acid sequence having at least 90% homology thereto; and / or A light chain variable region of an amino acid sequence as shown in any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, or an amino acid sequence having at least 90% homology thereto; Optionally, the antibody or antigen-binding fragment thereof comprises any one of the following groups or a heavy chain variable region and a light chain variable region having more than 90% identity therewith:

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The antibody or antigen-binding fragment thereof 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 comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from a κ type or a λ type; Optionally, the light chain constant region and the heavy chain constant region are both from human antibodies or mutants thereof; Optionally, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or The N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region; Optionally; the heavy chain constant region comprises the heavy chain constant region shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% identity thereto; and / or The light chain constant region comprises the light chain constant region shown in SEQ ID NO:43 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody or antigen-binding fragment thereof comprises: The heavy chain of an amino acid sequence as shown in any one of SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, or an amino acid sequence having at least 80% homology thereto; and / or A light chain of an amino acid sequence as shown in any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, or an amino acid sequence having at least 80% homology thereto; Optionally, the antibody or antigen-binding fragment thereof comprises any one of the following groups or a heavy chain or a light chain having more than 80% identity therewith: ； Optionally, the antibody comprises at least one selected from polyclonal antibody, full-length monoclonal antibody, Fab antibody, Fab' antibody, F(ab')2 antibody, F(ab)2 antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit; or The antigen binding fragment comprises at least one selected from the group consisting of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

5. A nucleic acid molecule, characterized in that comprising encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; Optionally, the nucleic acid molecule is DNA.

6. An expression vector, characterized in that: comprising the nucleic acid molecule of claim 5; Optionally, the expression vector comprises a vector selected from a eukaryotic expression vector or a prokaryotic expression vector; Preferably, the expression vector is a plasmid expression vector.

7. A recombinant cell, characterized in that include: comprising expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; or the nucleic acid molecule according to claim 5 or the expression vector according to claim 6; Optionally, the recombinant cell is obtained by introducing the expression vector of claim 6 into a host cell; Optionally, the recombinant cell is a eukaryotic cell, preferably the recombinant cell is a mammalian cell.

8. A composition, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, or the recombinant cell according to claim 7; Optionally, further comprising a pharmaceutically acceptable carrier or excipient; Optionally, the composition is a pharmaceutical composition; Optionally, the pharmaceutical composition is an anticoagulant drug; Optionally, the composition is an anticoagulant.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the recombinant cell according to claim 7 or the composition according to claim 8 in the preparation of a product for detecting an antigen or preventing and / or treating a disease; Optionally, the product is a drug or a kit; Optionally, the medicament is for preventing and / or treating a disease; Optionally, the disease is a disease caused by blood clot formation; Optionally, the kit is used to detect antigens; Optionally, the antigen is coagulation factor XI.

10. A method for increasing the expression level of an antibody, wherein the HCDRs of the antibody are non-human HCDRs, and the LCDRs of the antibody are non-human LCDRs, characterized in that: include: The HFRs and LFRs of the antibody are consistent with the HFRs and LFRs of any one of claims 1 to 2; Optionally, the amino acid sequence of the antibody is consistent with the amino acid sequence of the antibody or antigen-binding fragment thereof according to any one of claims 3 to 4.

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