CN116589588B

CN116589588B - Antibodies that bind to coagulation factor X

Info

Publication number: CN116589588B
Application number: CN202310403360.5A
Authority: CN
Inventors: 刘爽; 刘原伍; 刘成亮; 刘云菲; 郝维维; 卫科科; 张新静; 陈宪; 朱鹿燕; 周婷婷; 苏鸿声
Original assignee: Beijing Jike Shengsi Pharmaceutical Technology Co ltd
Current assignee: Beijing Jike Shengsi Pharmaceutical Technology Co ltd
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-12-22
Anticipated expiration: 2043-04-14
Also published as: CN116589588A

Abstract

The present application provides antibodies or antigen-binding fragments thereof that specifically bind to coagulation factor X, pharmaceutical compositions comprising the antibodies or antigen-binding fragments, and methods of treatment using the antibodies.

Description

Antibodies that bind to coagulation factor X

Technical Field

The present application is in the field of bioengineering, and in particular relates to antibodies that specifically bind to coagulation factor X and pharmaceutical formulations comprising such antibodies as active ingredients.

Background

"factor X" or "FX" is a vitamin K-dependent factor that has structural similarity to factor VII, prothrombin, FIX and protein C. Factor X consists of 448 amino acid residues, which upon activation releases a peptide stretch, forming two peptide chains linked by disulfide bonds. Activated factor X (Xa) is located at the junction of the intrinsic and extrinsic coagulation pathways and mainly catalyzes the conversion of factor II to factor IIa, and a factor Xa inhibitor can inhibit the physiological effect of 138 prothrombin molecules due to the amplification of biological signals present in the coagulation process. Thus, direct inhibitors of factor xa are more potent than direct thrombin inhibitors. Factor Xa inhibitors that have been marketed are advantageously valsartan, apixaban, edoxaban, and the like, for use in the treatment of thrombotic disorders. Antibody drugs for inhibiting the activity of blood coagulation factor X or blood coagulation factor Xa have not been reported.

Disclosure of Invention

The object of the present application is to provide an antibody or an antigen-binding fragment thereof that specifically binds to coagulation Factor X (FX), and a pharmaceutical preparation comprising the antibody or the antigen-binding fragment thereof as an active ingredient, and use of such a pharmaceutical preparation as an anticoagulation drug.

In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to coagulation factor X, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of the heavy chain variable region as shown in SEQ ID No. 1, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of the light chain variable region as shown in SEQ ID No. 2.

In some embodiments, an antibody or antigen binding fragment thereof that specifically binds to coagulation factor X of the present application comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to the Kabat numbering system, wherein:

the HCDR1 comprises or consists of an amino acid sequence as shown in SEQ ID NO. 4,

the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 6,

the HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 8,

The LCDR1 comprises or consists of an amino acid sequence as shown in SEQ ID NO. 11,

the LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 13 and

the LCDR3 comprises or consists of an amino acid sequence as shown in SEQ ID NO. 15.

In some embodiments, an antibody or antigen binding fragment thereof that specifically binds to coagulation factor X of the present application is according to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the IMGT numbering system, wherein:

the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO. 18,

the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 20,

the HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 22,

the LCDR1 comprises or consists of an amino acid sequence as shown in SEQ ID NO. 25,

the LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO 27 and

the LCDR3 comprises or consists of an amino acid sequence as set forth in SEQ ID NO. 29.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to coagulation factor X of the present application is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, or a human antibody or antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to coagulation factor X of the present application is a murine antibody or antigen-binding fragment thereof. In some specific embodiments, an antibody or antigen-binding fragment thereof that specifically binds to coagulation factor X of the present application is a murine antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein: the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 2, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 1. In some specific embodiments, an antibody or antigen-binding fragment thereof that specifically binds to coagulation factor X of the present application is a murine antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises the amino acid sequence shown in SEQ ID No. 2 and the VH comprises the amino acid sequence shown in SEQ ID No. 1.

In some embodiments, the antibody or antigen binding fragment thereof that specifically binds to coagulation factor X of the present application is an isotype selected from IgG, igA, igM, igE and IgD. In some embodiments, an antibody or antigen binding fragment thereof that specifically binds to coagulation factor X of the present application is a subtype selected from the group consisting of IgG1, igG2, igG3, and IgG 4. In a specific embodiment, an antibody or antigen binding fragment thereof that specifically binds to coagulation factor X of the present application is an IgG4 subtype.

In some embodiments, the antigen binding fragment of the present application that specifically binds to coagulation factor X is selected from the group consisting of Fab, fab ', F (ab') ₂ Fv, scFv, dAb, nanobodies, fd and Fd'.

In some embodiments, an antibody or antigen binding fragment thereof of the present application that specifically binds to coagulation factor X comprises a heavy chain constant region and a light chain constant region. In some preferred embodiments, an antibody or antigen binding fragment thereof that specifically binds to factor X of the present application comprises a heavy chain constant region that is an IgG4 constant region and a light chain constant region that is a human kappa light chain constant region. In some specific embodiments, an antibody or antigen binding fragment thereof that specifically binds to factor X of the present application comprises a heavy chain constant region that is an IgG4 constant region and a light chain constant region that is a human kappa light chain constant region comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:31 and/or the light chain constant region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 32.

In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to coagulation factor X of the present application comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO 33; the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 34.

In some embodiments, the antibodies that specifically bind to coagulation factor X of the present application are monoclonal antibodies.

In some embodiments, the antibody that specifically binds to coagulation factor X of the present application is a bispecific antibody or a multispecific antibody.

In a second aspect, the present application provides a kit comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof or a bispecific antibody or antigen-binding fragment thereof of the present application.

In a third aspect, the present application provides a vector comprising a nucleic acid of the present application.

In a fourth aspect, the present application provides a host cell comprising a nucleic acid or vector of the present application.

In a fifth aspect, the present application provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof of the present application, or a bispecific antibody or antigen-binding fragment thereof of the present application; and (ii) a pharmaceutically acceptable carrier or excipient.

In a sixth aspect, the present application provides the use of an antibody or antigen-binding fragment thereof of the present application, a bispecific antibody or antigen-binding fragment thereof of the present application, a pharmaceutical composition of the present application in the manufacture of an anticoagulant drug.

The antibody specifically combined with the blood coagulation factor X can be specifically combined with the blood coagulation factor X, and further, the antibody can block the activation of the blood coagulation factor X after being combined with the blood coagulation factor X, so that the antibody can be used as a blood coagulation factor X inhibitor, and further can be used for preparing an anticoagulant drug to reduce thrombosis.

Detailed Description

The above features and advantages of the present application and additional features and advantages thereof will be more clearly understood hereinafter from the following detailed description of embodiments. The embodiments herein are illustrative, explanatory, and are used for general understanding of the present application. The embodiments should not be construed as limiting the scope of the application.

Terminology and definitions

Unless defined otherwise herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and the like.

Unless otherwise indicated or defined, the terms "comprises," "comprising," and variations thereof such as "comprises" and "comprising" are to be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically comprise a variable region and a constant region in each of the heavy and light chains. The variable regions of the heavy and light chains of antibodies comprise binding domains that interact with the antigen. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and components of the complement system, such as the first component C1q of the classical pathway of complement activation. Thus, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the portion of the antibody that binds to an antigen.

The "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region interspersed with three "complementarity determining regions" or "CDRs". The framework regions are used to modulate the CDRs for specific binding to the epitope. CDRs comprise amino acid residues in antibodies that are primarily responsible for antigen binding. From amino-terminus to carboxy-terminus, both VL and VH domains comprise the following Framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDRs 1, 2 and 3 of the VL domain are also referred to herein as LCDR1, LCDR2 and LCDR3, respectively; CDRs 1, 2 and 3 of the VH domain are also referred to herein as HCDR1, HCDR2 and HCDR3, respectively.

Amino acid assignments for each VL and VH domain are according to any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, MD,1987 and 1991)); chothia definitions (Chothia & Lesk, J.mol. Biol.196:901-917,1987; chothia et al, nature 342:878-883,1989); chothia Kabat CDR, wherein CDR-H1 is a complex of Chothia and Kabat CDRs; abM definition used by Oxford Molecular antibody modeling software; IMGT (ImMunoGeneTics) (Lefranc, M.P. et al, dev. Comp. Immunol.27:55-77 (2003)); and the CONTACT definition by Martin et al (web bioinfo. Org. Uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are given the same numbering the present application may use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined by Kabat or IMGT.

The term "antibody" is used herein in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. Antibodies may contain additional modifications such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins comprising an epitope of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibodies exhibit the desired biological activity.

As used herein, the term "antigen-binding fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen (e.g., factor X or activated factor X). It has been shown that the antigen binding function of an antibody can be performed by fragments of full length antibodies.

Examples of antigen-binding fragments included within the term "antigen-binding portion" of an antibody include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) A F (ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bond; (iii) Fab ' fragments, which are essentially Fab but have a partial hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed.,3.Sup. Rd. 1993); (iv) Fd fragments, which consist of VH and CH1 domains, (v) Fd ' fragments, which have VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain, (vi) Fv fragments, which consist of VL and VH domains of a single arm of an antibody, (vii) dAb fragments (Ward et al (1989) Nature 341: 544-546), which consist of VH domains, (viii) separate Complementarity Determining Regions (CDRs), and (ix) nanobodies, which are heavy chain variable regions comprising a single variable domain and two constant domains, (v) Fd ' fragments, which in addition comprise a "linear antibody" comprising a pair of tandem Fd fragments (VH-CH 1-VH-CH 1) which together with a modified version of any of the foregoing fragments that retains antigen binding activity form antigen binding domains, such that an antigen binding region together with a complementary light chain polypeptide, and a modified version of any of the foregoing fragment can form an antigen binding domain, whereby the same antigen binding domain can be obtained by a single chain binding domain as that of the scFv fragment or a single chain binding domain comprising the scFv fragment can be obtained by conventional techniques, and whereby the fragment binding to the scFv fragment can be screened for the same heavy chain variable domain as the VH domain, and the scFv fragment can be obtained by conventional techniques, pp.269-315.

As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, for example, between an antibody and its target antigen. The binding specificity of an antibody may be determined based on affinity and/or avidity. Affinity is expressed by the equilibrium constant (KD) for antigen-to-antibody dissociation, a measure of the strength of binding between an epitope and the antigen binding site of an antibody: the smaller the KD value, the stronger the binding strength between the epitope and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1/KD. Avidity is a measure of the strength of binding between an antibody and the antigen of interest. Avidity relates to the affinity between an epitope and the antigen binding site of an antibody and the number of relevant binding sites present on the antibody.

Specific binding of the antibody to the antigen or antigenic determinant may be determined by any suitable means known per se, including for example scatchard analysis and/or competitive binding assays, such as Radioimmunoassays (RIA), enzyme Immunoassays (EIA) and sandwich competition assays and different variants known per se in the art. Typically, the antibody will be at 10 ^-5 To 10 ^-12 Dissociation constant (KD) binding of M or less, preferably at 10 ^-7 To 10 ^-12 M or less, more preferably at 10 ^-8 To 10 ^-12 Dissociation constant (KD) binding of M, and/or with a dissociation constant (KD) of at least 10 ⁷ M ^-1 Preferably at least 10 ⁸ M ^-1 More preferably at least 10 ⁹ M ^-1 For example at least 10 ¹² M ^-1 Is bound to the substrate with binding affinity. Generally considered to be any greater than 10 ^-4 K of M _D Values represent non-specific binding.

The term "epitope" refers to the site on an antigen to which an antibody binds. Epitopes can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed by consecutive amino acids (also referred to as linear epitopes) are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost in the treatment of denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. An epitope defines the smallest binding site of an antibody and is therefore a specific target for an antibody or antigen binding fragment thereof.

As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at identical positions after alignment. For example, "the amino acid sequence is X% identical to SEQ ID NO: Y" refers to the percent identity of the amino acid sequence to SEQ ID NO: Y and is stated as the X% of the residues in the amino acid sequence being identical to the sequence residues disclosed in SEQ ID NO: Y. Such calculations are typically performed using a computer program. Exemplary procedures for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman,1988; pearson, 1990), gapped BLAST (Altschul et al, 1997), BLASTP, BLASTN or GCG (Devereux et al, 1984).

Furthermore, in determining the degree of sequence identity between two amino acid sequences, the skilled artisan may consider so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g. WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and, preferably, the types and/or combinations of these substitutions may be selected in accordance with the relevant teachings from WO 04/037999 and WO 98/49185, and the additional references cited therein.

Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is substituted by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) polar, positively charged residues: his, arg and Lys; (d) large aliphatic, nonpolar residues: met, leu, he, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.

Particularly preferred conservative substitutions are as follows: ala to Gly or to Ser; arg to Lys; asn to Gln or to His; asp to Glu; cys to Ser; gln to Asn; glu to Asp; gly to Ala or to Pro; his to Asn or to Gln; lie to Leu or to Val; leu to Ile or to Val; lys to Arg, to gin, or to Glu; met to Leu, to Tyr or to Ile; phe to Met, to Leu, or to Tyr; ser to Thr; thr to Ser; trp to Tyr; tyr to Trp; and/or Phe to Val, to Ile or to Leu.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, each antibody that makes up the population is identical except for a small number of mutations that may occur naturally. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies produced by any particular method.

The term "bispecific antibody" is to be understood in the context of the present application as an antibody having two different antigen binding regions defined by different antibody sequences. This is understood to be binding to different targets, but also includes binding to different epitopes of one target.

The term "coding sequence" means a polynucleotide encoding an amino acid sequence of a protein or polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame beginning with a start codon (e.g., ATG, GTG or TTG) and ending with a stop codon (e.g., TAA, TAG or TGA). The coding sequence may be derived from genomic DNA, or synthetic DNA, or a combination thereof.

Due to the degeneracy of the genetic code, several nucleic acids may encode polypeptides having the same amino acid sequence. For example, both codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at each position where the codon is determined to be alanine, the codon can be replaced with any other codon encoding alanine without altering the encoded polypeptide. One of ordinary skill in the art will recognize that modifications can be made to codons in a nucleic acid (except AUG, which is typically methionine-only, and TGG, which is typically tryptophan-only), without altering the amino acid sequence of the protein or polypeptide it encodes. Thus, codons in the coding sequence of the protein can be modified using a codon usage table appropriate for the host cell of interest to obtain optimal expression in a particular host cell (e.g., a prokaryotic cell or eukaryotic cell). Codon preference in various hosts is known in the art.

The term "expression" refers to the step of converting genetic information of a polynucleotide into RNA by enzymatic transcription such as RNA polymerase, and converting the above genetic information into a protein or polypeptide by translation of mRNA on a ribosome. In this context, the term "expression" includes any step involving the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "vector" refers to a vector that is autonomously replicable in a host cell, preferably a multicopy vector. The term "vector" as used herein is capable of transporting a nucleic acid molecule of another nucleic acid to which it is linked. In addition, vectors often have markers such as antibiotic resistance genes for selection of transformants. In addition, the vector may have a promoter and/or terminator for expressing the introduced gene. The vector may be, for example, a bacterial plasmid-derived vector, a viral vector, a yeast plasmid-derived vector, a phage-derived vector, a cosmid, a phagemid, or the like.

The term "expression vector" refers to a vector that enables expression of a gene of interest in a cell, and is typically a linear or circular DNA molecule that includes a polynucleotide encoding a protein or polypeptide and is operably linked to expression control sequences.

Nucleic acids, such as vectors or expression vectors, can be delivered into prokaryotic and eukaryotic cells by a variety of methods known in the art. Methods for delivering nucleic acids into cells include, but are not limited to, various chemical, electrochemical and biological methods, such as heat shock transformation, electroporation, transfection such as liposome-mediated transfection, DEAE-Dextran-mediated transfection or calcium phosphate transfection, and the like. In addition, methods such as treatment of the recipient cells with calcium chloride to increase their permeability to DNA, and methods of preparing competent cells from cells at the growth stage followed by transformation with DNA can be used. It is also possible to use a method of preparing a DNA receptor cell into a protoplast or a spheroplast (which can easily ingest recombinant DNA) and then introducing the recombinant DNA into the DNA receptor cell. The transformation method is not particularly limited, and a person skilled in the art can select an appropriate transformation method according to, for example, the host cell used and the type of vector or expression vector to be transformed.

As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not deleterious to the recipient thereof and/or that such carrier or adjuvant is approved or available for inclusion in a pharmaceutical composition for parenteral administration to a human.

As used herein, the terms "treat," "treatment," and the like refer to the administration of an agent or the performance of a procedure in order to obtain an effect. These effects may be prophylactic in terms of preventing the disease or symptoms thereof, either entirely or in part, and/or may be therapeutic in terms of affecting a partial or complete cure of the disease and/or disease symptoms. As used herein, "treating" may include treating a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) Preventing the occurrence of a disease or disease symptom in a subject susceptible to the disease but not yet diagnosed as having the disease (e.g., including diseases that may be associated with or caused by a primary disease); (b) inhibiting the disease, i.e., arresting its development; (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; relief; reducing symptoms or making the disease condition more tolerable to the patient; slowing the rate of deterioration or decay; or to reduce end point debilitation of deterioration. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of the physician's examination. Thus, the term "treating" includes administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or prevent or inhibit the development of symptoms or disorders associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, disease symptom or disease side effect in a subject.

The term "effective amount" as used herein refers to an amount sufficient to effect treatment of a disease when administered to a subject to treat such disease.

As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment or therapy is desired. "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic animals, and laboratory animals, zoo animals, sports animals, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

"factor X" or "FX" is a vitamin K-dependent factor that has structural similarity to factor VII, prothrombin, FIX and protein C. The preproprotein is converted to the mature double-stranded form of the zymogen FX by cleavage of the tripeptide RKR. Human FX zymogen comprises four distinct domains, including an N-terminal gamma-carboxyglutamic acid (Gla) rich domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. FX circulates in plasma as a double-stranded zymogen comprising residues 1-139 of SEQ ID NO:35 (light chain) and residues 143-448 of SEQ ID NO:35 (heavy chain). Activation of FX occurs by limited proteolysis at Arg194, which results in release of the activation peptide (Aa 143-194). Thus, activated FX (FXa) consists of residues 1-139 of SEQ ID NO:35 (light chain) and residues 195-448 of SEQ ID NO:35 (activated heavy chain). Thus, the circulating FX molecule comprises a zymogen FX and an activated form of FX, which are referred to herein as FX and FXa, respectively, with reference to SEQ ID NO: 35. In this application, FX is intended to cover all natural variants of FX. The term "FX and/or its activated form (FXa)" may also be referred to as "FX/FXa" or "FX (a)".

Amino acid sequence of factor X preproprotein (SEQ ID NO: 35):

ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSSPLK

in the context of functional variants, the number of amino acids inserted, deleted and/or substituted preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is from 1% to 33%, more preferably is from 5% to 30%, more preferably is from 10% to 25%, more preferably is from 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of amino acids inserted, deleted and/or substituted may be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, most preferably 1 to 2. In preferred embodiments, the number of amino acids inserted, deleted and/or substituted is 1, 2, 3, 4, 5, 6 or 7.

In some embodiments, insertions, deletions, and/or substitutions may be in the Framework (FR) regions, such as FR1, FR2, FR3, and/or FR4; and/or constant regions, e.g., CL, CH1, CH2, and/or CH 3.

In some embodiments, the substitution of one or more amino acids may be conservative substitutions of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is substituted by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) polar, positively charged residues: his, arg and Lys; (d) large aliphatic, nonpolar residues: met, leu, he, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.

Immunoglobulin molecules can be divided into five classes (isotypes) according to the amino acid sequence of the antibody heavy chain constant region: igA, igD, igE, igG and IgM, and can be further divided into different subtypes such as IgG1, igG2, igG3, igG4, igA1, igA2, etc. The light chain of an antibody can be classified into a lambda (lambda) chain and a kappa (kappa) chain according to the amino acid sequence of the light chain. The antibodies disclosed herein may be of any of the classes or subtypes described above.

In some embodiments, the Fc region may be of any isotype, including but not limited to IgG1, igG2, igG3, and IgG4, and may comprise one or more mutations or modifications. In one embodiment, the Fc region is of or derived from an IgG1 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

In one embodiment, the Fc region is functionally deficient. For example, the Fc region may be of the IgG1 isotype, or of a non-IgG 1 type, such as IgG2, igG3 or IgG4, which has been mutated such that its ability to mediate effector functions such as ADCC is reduced or even eliminated. Such mutations are described in Dall' Acqua WF et al, J Immunol.177 (2): 1129-1138 (2006) and Hezareh M, J virol; 75 (24): 12161-12168 (2001).

In one embodiment, the Fc region comprises a mutation that removes the receptor site for Asn linked glycosylation or is otherwise manipulated to alter the glycosylation characteristics. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove Asn-linked glycosylation sites. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence having an N297Q mutation.

In a further embodiment, the Fc region is glycoengineered to reduce fucose and thereby enhance ADCC, e.g., by administering a peptide of formula (i) to a subject in need thereofCompounds were added to the medium during antibody production as described in US2009317869 or as described in van Berkel et al (2010) biotechnol. Bioeng.105:350, or by using FUT8 knockout cells, such as Yamane-Ohnuki et al (2004) biotechnol. Bioeng 87: 614. Alternatively, one can use Et al (1999) Nature Biotech 17:176 to optimize ADCC. In another embodiment, the Fc region is engineered to enhance complement activation, for example, at Natsume et al (2009) Cancer sci.100: 2411.

In yet another aspect, the present application provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof disclosed herein.

In another aspect, the present application provides vectors comprising the nucleic acids disclosed herein.

Any carrier may be suitable for use in the present application. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. The recombinant expression vector may be any suitable recombinant expression vector.

In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-Puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (puroIRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, ttRMPVIR, pMSCV-IRES-mCherry FP, pRetrox GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

In some embodiments, the vector is a plasmid vector. For example, the plasmid vector may be selected from the pUC series (Fermentas Life Sciences, glen Burnie, md.), the pBluescript series (Stratagene, laJolla, calif.), the pET series (Novagen, madison, wis.), the pGEX series (Pharmacia Biotech, uppsala, sweden) and the pEX series (Clontech, palo Alto, calif.). Phage vectors such as λGT10, λGT11, λ ZapII (Stratagene), λEMBL4, and λNM1149 can also be used.

Examples of plant expression vectors useful in the present application include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the present application include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques, such as Sambrook et al, molecular Cloning: a Laboratory Manual, third edition, cold Spring Harbor Press, cold Spring Harbor, n.y.2001; and Ausubel et al Current Protocols in Molecular Biology, greene Publishing Associates and John Wiley & Sons, N.Y., 1994. Circular or linear expression vector constructs can be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems may be derived from, for example, COlEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, etc.

In another aspect, the present application provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

Any cell can be used as a host cell for the nucleic acids or vectors of the present application. In some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae (Enterobacterhaceae), such as Escherichia, such as E.coli; enterobacter (Enterobacter); erwinia (Erwinia); klebsiella (Klebsiella); proteus (Proteus); salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); serratia (Serratia), such as Serratia marcescens (Serratia marcescans); and Shigella (Shigella); bacillus (bacillus), such as bacillus subtilis (b. Subtilis) and bacillus licheniformis (b. Lichenifermis); pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (P.aeromonas); and Streptomyces (Streptomyces). In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, host cells include, for example, CHO cells, such as CHOs cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T and HEK293FS.

In yet another aspect, the present application provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

In some embodiments, carriers or excipients used with the compositions disclosed herein include, but are not limited to, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and the surfactant polyoxyethylene-sorbitan monooleate.

In some embodiments of the pharmaceutical compositions disclosed herein, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent may be selected from antibodies, chemotherapeutic agents, and small molecule drugs.

In yet another aspect, the present application provides a conjugate comprising an antibody or antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto. In some embodiments of the conjugates disclosed herein, the chemical moiety is selected from the group consisting of a therapeutic agent and a detectable moiety.

In some embodiments, the detectable moiety may be selected from biotin, streptavidin, an enzyme or a catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

In another aspect, the present application provides a method of preventing and/or treating a thrombotic disorder comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, or an immunoconjugate, pharmaceutical composition, nucleic acid, vector, or host cell comprising or encoding an antibody or antigen-binding fragment thereof disclosed herein.

In some embodiments, the thrombotic disorders include, but are not limited to, prevention of post-operative (e.g., orthopedic) venous embolism (e.g., deep vein thrombosis and pulmonary embolism), prevention or acute phase treatment of ischemic cerebral infarction, treatment of chronic arterial occlusion (e.g., thromboangiitis obliterans, arteriosclerosis obliterans, etc.), and the like.

In some embodiments, an antibody or antigen-binding fragment thereof disclosed herein, or an immunoconjugate, pharmaceutical composition, nucleic acid, vector, or host cell disclosed herein comprising or encoding an antibody or antigen-binding fragment thereof disclosed herein, is administered intravenously or subcutaneously.

In some embodiments, the dosage administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject being treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician may determine an effective amount to administer to a human or other subject to treat a medical condition. The precise amount required for therapeutic effectiveness may depend on a number of factors, such as the activity of the antibody and the route of administration.

The dosage of the antibodies, compositions or conjugates described herein may be administered to the mammal once within a suitable period of time or in a series of sub-doses, for example once daily, every half-week, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annual or yearly, as desired. Dosage units comprising an effective amount of the antibody, composition or conjugate may be administered in a single daily dose, or the total daily dose may be administered in two, three, four or more divided doses administered daily, as desired.

The appropriate mode of administration may be selected by the physician. The route of administration may be parenteral, for example by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration may be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition, or conjugate is selected for parenteral delivery, inhalation, or delivery through the digestive tract, e.g., oral. The administration dosage and method may vary depending on the weight, age, condition, etc. of the subject, and may be appropriately selected.

In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In certain embodiments, the binding agent is administered prior to, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents, and small molecule drugs.

In yet another aspect, the present application provides a pharmaceutical package or kit comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as antibodies or antigen binding fragments disclosed herein. Optionally, associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notification reflects approval of the manufacture, use or sale by the government agency for human administration.

In a specific embodiment, the kit comprises a first container comprising an antibody or antigen binding fragment disclosed herein. In a specific embodiment, the kit comprises a first container that is a vial containing the antibody or antigen binding fragment as a lyophilized sterile powder under vacuum, and the kit further comprises a second container containing a pharmaceutically acceptable fluid.

In a specific embodiment, the present application provides an injection device comprising an antibody or antigen binding fragment disclosed herein. In a specific embodiment, the injection device comprises the antibody in the form of a sterile solution. In a specific embodiment, the injection device is a syringe.

In another aspect, the present application provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein, in the preparation of an anticoagulant.

In some embodiments, the anticoagulants provided herein are useful in inhibiting thrombosis, and thus may be used in the treatment and/or prevention of thrombotic disorders.

Examples

The content of the present application will be further described below in connection with examples. It should be understood that the following examples are illustrative only and should not be construed as limiting the scope of the present application.

EXAMPLE 1 FX monoclonal antibody hybridoma screening

Immunization of mice: male Balb/c mice, 2/group, 6 weeks old at the time of first immunization. Primary immunization and secondary immunization: FX antigen (HFX 1010, enzyme Research Laboratories) was diluted as described, and the diluted antigen was mixed rapidly with a mixed rapid immune adjuvant (QuickAntibody-Mouse 5W, KX0210041, beijing Boolon immunization technique) at a volume ratio of 1:1, and the immunization dose was 40. Mu.g FX/Mouse, and 100. Mu.l/Mouse were injected into the calf muscle of the hind leg. Boosting: FX antigen was diluted with PBS and the immunization dose was 100. Mu.g/mouse, and 100. Mu.l/mouse was intraperitoneally injected.

Cell fusion:

obtaining feeder cells: one healthy, non-immunized ICR mouse was taken the day before fusion, sacrificed for cervical dislocation, after body surface disinfection and fixation, skin was cut from the thigh, the peritoneum was exposed, and the peritoneum was sterilized with alcohol cotton balls. The abdomen was gently massaged with a 5ml syringe, a 12# needle, 5ml HAT medium was injected into the abdomen, the syringe was fixed with the right hand, the left hand alcohol cotton ball was held, the fluid in the abdomen was withdrawn, and the prepared vessel was filled. The extracted cell suspension was added to 50ml of HAT medium (containing 1% diabody, 10% fbs), 96-well plates were plated, 50 μl per well (calculated as 10 plates), and cultured until the next day.

Feeder cell count: 5ml of HAT medium was injected into the abdominal cavity, and about 3ml was aspirated back into 45ml of HAT medium.

Preparation of myeloma cells SP 2/0: myeloma cells SP2/0 were inoculated into T25 flasks containing DMEM+10% foetal calf serum and cultured at 37℃for resuscitation. And carrying out passage amplification after the condition is good. Passaging was performed at a density of around 80%. Most of the primary culture supernatant was discarded, fresh medium was added, and the cells were directly blown down without digestion, and subjected to 1-time separation and 2-time expansion culture. If the growth state is good, the culture is expanded when the density is as high as 80%. And amplifying to 8-10 bottles when the cells are fused. On the day of fusion, SP2/0 cells with good growth state were selected, the supernatant was discarded when the density was as high as 80%, 8 vials were taken and the cells were gently blown down with 30ml of serum-free streptavidin-containing DMEM medium.

Obtaining a spleen cell suspension: taking the mice after the immunity enhancement in the first step, removing eyeballs, taking blood, separating serum, killing the mice by cervical dislocation, soaking the mice in 75% alcohol, disinfecting the body surface for 5min, then placing the mice on a dissecting plate of the mice in an ultra-clean bench, and fixing limbs by using an injection needle. The spleen was removed from the abdominal cavity by aseptically opening, placed in a dish containing a basal medium of streptavidin, and the connective tissue attached around was carefully removed with forceps and scissors. The spleen was then transferred to another plate containing a small amount of basal medium. Pressing spleen with elbow needle, punching hole with small needle, and squeezing with elbow needle to release spleen cell fully to obtain spleen cell suspension.

Fusion: the myeloma cells and spleen cells (cell number ratio 1:5-1:10) prepared above were mixed in a 50ml centrifuge tube with a cover, centrifuged at 1000rpm for 10min, and the supernatant was decanted and thoroughly discarded to avoid affecting the effect of PEG. The fusion tube was placed in the palm and the bottom was gently shaken to mix the two cells thoroughly. 1ml of PEG preheated at 37℃was added dropwise to the fusion tube at a constant rate over 45-60s with a pipette while gently shaking (one drop every 2 seconds). Immediately drop-in of 37℃preheated basal medium to dilute PEG to lose its effect by pipette 1ml of preheated basal medium (drop every 3 seconds) in the first minute2ml (2 drops every 3 seconds) was added in two minutes, 8ml was added in the third minute, and the mixture was allowed to stand at 37℃for 10 minutes and centrifuged at 1000rpm for 10 minutes, and the supernatant was discarded. Add 5ml of HAT medium, gently suspend the pelleted cells, and finally add HAT to about 200 ml. Split charging into macrophage cell culture plate with 96 holes, 180 μl/hole, placing the culture plate at 37deg.C, 5% CO ₂ Culturing in an incubator. When the cell culture supernatant turns yellow or clones are distributed to more than 1/10 of the bottom area of the hole, a proper amount of cell supernatant is sucked for antibody titer detection. The hybridoma cell with the better antibody titer was selected and designated as 2F5.

Example 2 neutralization detection of antibodies

Sample preparation: hybridoma cell 2F5 was cultured in DMEM+3% FBS medium at 37℃with 5% CO ₂ The cells were cultured for 4 days, and the supernatant was purified by Protein A and then dissolved in 1 XPBS to obtain a sample to be measured, and the concentration (initial concentration) was measured by a spectrophotometer.

Firstly, phosphatide is addedSP FVIII kit, chromagenix, cat: 82408663 With FIXa+FX solution (+.>SP FVIII kit) is evenly mixed according to the ratio of 1:5 and placed on ice for standby; the sample to be tested is diluted 1:5 (dilution:>buffer working-solution of SP FVIII kit; the blank control was: and (3) diluting the liquid. Taking 12.5 mu l of diluted sample to be tested or blank control, adding 50 mu l of mixed solution of phospholipid, FIXa and FX into an ELISA plate, uniformly mixing, and incubating for 1h at 4 ℃; diluting FVIII (Ren Jie, hui's pharmaceutical Co., ltd.) with diluent to 3IU/ml, adding 2.5 μl into 200 μl of diluent, adding diluted FVIII 12.5 μl/hole into the plate hole, shaking 30s in a microplate reader, incubating at 37deg.C for 5min, and adding CaCl preheated to 37deg.C ₂ 25 μl/well of substrate preheated to 37℃and the substrate(s) (A)>SP FVIII kit), 50. Mu.l/well, incubation for 5min at 37℃and finally 20% acetic acid, 25. Mu.l/well, termination of the reaction, detection of absorbance (OD) values at 405nm/490nm with a microplate reader.

Table 1.2F5 neutralization test results of hybridoma supernatants

Experimental grouping	Initial concentration (μg/mL)	OD value
			2F5 hybridoma supernatant	696	0.4282
Blank control	/	1.1664

From the results, it can be seen that the amount of FXa produced was significantly reduced after the addition of the antibodies of the present application, indicating that the antibodies of the present application were able to effectively block FX activation to FXa.

Example 3 antibody affinity detection

Preparing a sample: hybridoma cell 2F5 was cultured in DMEM+3% FBS medium at 37℃with 5% CO ₂ The cells were cultured for 4 days, and the supernatant was purified by Protein A and then dissolved in 1 XPBS to obtain a sample to be measured, and the concentration (initial concentration) was measured by a spectrophotometer.

The anti-FX antibody functional affinity constants were determined by a non-competitive ELISA method, which comprises the following steps: 1) Coating liquid [ Na ] ₂ CO ₃ 0.795g，NaHCO ₃ 1.465g，KH ₂ PO ₄ 0.125g in 500mL purified water, pH 9.6]Diluting FX antigen, coating concentration of 400, 200, 100 and 50ng/mL, and coating at 4 ℃ overnight with blank control as coating liquid; 2) PBST is washed for 4 times; 3) PBS containing 4% BSA was blocked at 37℃for 2h in 250. Mu.l per well; 4) 2F5 hybridoma supernatant dilution from 1000ng/mL two-fold dilution, 50 u l/hole, 37 degrees C, 1h; 5) PBST is washed for 4 times; 6) The secondary antibody (goat anti-mouse IgG-HRP) was diluted 10000-fold with PBS containing 2% BSA or 1% BSA, 50 μl/well, 37℃for 0.5h; 7) PBST is washed for 6 times; 8) TMB 100. Mu.l, development 10min, 50. Mu.l 2M H ₂ SO ₄ The reaction was terminated.

Affinity result calculation scheme:

1) Calculation of OD 100%: OD100% is the antibody plateau. In the case of monovalent antibodies, the antigen and antibody bind in a 1:1 format. The concentration of the antibody is so great that the antigen on the solid support is all bound to the antibody, i.e. the concentration of the antibody is equal to the concentration of the coating antigen. Substituting the calculated value into a fitting equation to obtain the OD100%.

2) OD50% calculation and conversion unit: OD50% = OD100%/2; OD50% at this time unit is ng/ml, converted to 10-6 g/L, the conversion process is as follows: hybridoma supernatant anti-FX antibody 150kDa, molar mass 1.5X10-5 g/mol, antibody concentration (mol/L) = (g/L)/(g/mol).

3) Affinity constant calculation: the affinity constant (L/moL) was calculated by substituting the formula k= (n-1)/2 (nAb '-Ab), where Ab and Ab' represent the concentration of antibody (moL/L) that gives half the absorbance when the antigen concentration is Ag and Ag ', respectively, and n=ag/Ag'. Then, when n=2, 3K values are obtained, when n=4, 2K values are obtained, when n=8, 1K value is obtained, and the average of 6K values is obtained as a final result, and the affinity constant of the antibody against hybridoma 2F5 is 3.76×10≡9l/mol.

The murine antibody obtained by ELISA method is of the IgG1 subtype.

EXAMPLE 4 chimeric antibody preparation

1. Preparation of DNA fragments encoding antibody variable regions from mouse hybridoma cells

Total RNA was extracted from anti-FX antibody-producing hybridoma 2F5 by Trizol method, and 30-50. Mu.l of DEPC treated water was used to dissolve the RNA. Single-stranded cDNA was synthesized by reverse transcription using 1-2. Mu.g of the extracted RNA as a template and M-MLV reverse transcriptase (NEB). The single-chain cDNA obtained by reverse transcription is used as a template, and the light and heavy chain variable region genes of the antibody are amplified by a 5' RACE method and sequenced.

The sequences of the heavy and light chain variable regions of monoclonal antibody 2F5 that specifically bind to coagulation factor X are shown in table 2.

TABLE 2 amino acid sequences of the heavy and light chain variable regions of anti-FX antibodies produced by hybridoma 2F5

TABLE 3 amino acid sequences of the heavy and light chain CDRs of anti-FX antibody produced by hybridoma 2F5 (according to the CDR numbering system of Kabat and IMGT)

2. Chimeric antibody production

The light and heavy chain variable regions of the above murine antibody were grafted to the heavy chain constant region (g 4 Fc) and the human kappa light chain constant region (KC) of the human IgG4 antibody as described below to obtain the heavy and light chains of chimeric antibody 2F 5-02. The transiently transfected expression plasmid for chimeric antibody 2F5-02 was constructed according to conventional methods known in the art.

Heavy chain constant region sequence:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：31)

heavy chain sequence:

EVQLQQSGPELVKPGASVKMSCKASGYTFTSNVMHWVKQKPGQGLEWIGYINPYNDDTKYNEKFKGKATLTSDKSSNTAYMELSSLTSEDSTVYYCARDYGSSAWFAHWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：33)

light chain constant region sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：32)

light chain sequence:

DIVMSQSPSSLAVSVGEKVTLSCKTSQSLLYRSNQKNYLAWYQQKPGQSPKVLIYWASTRESGVPDRFTGSGSGTDFTLSISSVKAEDLAVYYCQQYYDYPWTFGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：34)

EXAMPLE 5 transient transfection expression of chimeric antibodies

The plasmid encoding chimeric antibody 2F5-02 constructed in example 4 was transferred into HEK 293F cells as a host cell by liposome transfection using the kitDNA transfection kit (/ -A)>SA). Resuscitating HEK 293F cells, subculturing to an adaptation state, and expanding cells for transfection when the cell state is good, wherein the temperature is 37 ℃ and the CO content is 5% ₂ Culturing at 125 rpm. Expi293 was used 1 day prior to transfection ^TM Expression Medium (Life) cells were resuspended to 300ml, cell density 1.0X10 ⁶ Cells/ml, cells continued to be cultured in a carbon dioxide shaker. On the day of transfection, use +.>I Reduced Serum Medium the plasmid was diluted to 240. Mu.g to 30ml and mixed well and the plasmid dilution was added to 240. Mu.l +.>In Reagent, immediately mix and incubate at room temperature for 10min. 30ml of the plasmid mixture was added to 300ml of the cell suspension and incubated in a carbon dioxide shaker. After 4 hours of transfection, 135 μ lFectoPRO Booster reagent was added to the cell suspension, immediately mixed and the flask was kept in incubator. Counted daily, supernatants were collected by centrifugation at 4000rpm for 20min 3 days post-transfection and used for the next Protein A purification to prepare the corresponding antibodies.

Example 6 neutralization assay and affinity detection of chimeric antibodies

1. Neutralization test

The chimeric antibody 2F5-02 prepared in example 4 was tested by the neutralization test described above, and the results are shown in Table 4.

TABLE 4 chimeric antibody neutralization assay results

Experimental grouping	Concentration (μg/mL)	OD value
			2F5-02	396	0.3606
Blank control (Diluent)	/	0.9947

2. Affinity detection

Using the affinity assay method of example 3, the affinity constant of the chimeric antibody 2F5-02 was 3X 10 ⁹ L/mol。

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to coagulation factor X, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein:

the VH comprises HCDR1, HCDR2 and HCDR3 according to the Kabat numbering system, and the VL comprises LCDR1, LCDR2 and LCDR3 according to the Kabat numbering system, wherein:

the HCDR1 consists of an amino acid sequence shown in SEQ ID NO. 4,

the HCDR2 consists of an amino acid sequence shown in SEQ ID NO. 6,

the HCDR3 consists of an amino acid sequence shown in SEQ ID NO. 8,

the LCDR1 consists of an amino acid sequence shown in SEQ ID NO. 11,

the LCDR2 consists of the amino acid sequence shown in SEQ ID NO. 13 and

the LCDR3 consists of an amino acid sequence shown in SEQ ID NO. 15;

or alternatively

The VH comprises HCDR1, HCDR2 and HCDR3 according to the IMGT numbering system, and the VL comprises LCDR1, LCDR2 and LCDR3 according to the IMGT numbering system, wherein:

The HCDR1 consists of an amino acid sequence shown in SEQ ID NO. 18,

the HCDR2 consists of an amino acid sequence shown in SEQ ID NO. 20,

the HCDR3 consists of an amino acid sequence shown in SEQ ID NO. 22,

the LCDR1 is composed of an amino acid sequence shown in SEQ ID NO. 25,

the LCDR2 consists of the amino acid sequence shown in SEQ ID NO. 27 and

the LCDR3 consists of the amino acid sequence shown in SEQ ID NO. 29.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, wherein:

the VL comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 2, and the VH comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 1.

4. The antibody or antigen-binding fragment thereof according to claim 3,

the VL comprises an amino acid sequence as shown in SEQ ID NO. 2, and the VH comprises an amino acid sequence as shown in SEQ ID NO. 1.

5. The antibody or antigen binding fragment thereof of any one of claims 1 to 4, wherein the antibody is an isotype selected from IgG, igA, igM, igE and IgD.

6. The antibody or antigen binding fragment thereof of any one of claims 1 to 5, wherein the antibody is a subtype selected from IgG1, igG2, igG3 and IgG 4.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') ₂ Fv and scFv.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the heavy chain constant region is an IgG4 constant region and the light chain constant region is a human kappa light chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 31 and/or the light chain constant region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 32.

11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 33; the light chain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 34.

12. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13.

15. A pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11; and (ii) a pharmaceutically acceptable carrier or excipient.

16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the pharmaceutical composition of claim 15, in the manufacture of a medicament for the prevention of post-operative venous embolism.

17. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the pharmaceutical composition of claim 15, in the manufacture of a medicament for the prevention or acute phase treatment of ischemic cerebral infarction.

18. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the pharmaceutical composition of claim 15, in the manufacture of a medicament for the treatment of chronic arterial occlusive disease.