CN110343181B - Single domain antibodies against coagulation Factor IX (FIX) - Google Patents
Single domain antibodies against coagulation Factor IX (FIX) Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
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- C12N15/09—Recombinant DNA-technology
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Abstract
The invention relates to the field of medical biology and discloses a single-domain antibody aiming at coagulation Factor IX (FIX). In particular, the invention discloses a FIX binding molecule derived from the single domain antibody and application thereof, in particular application in detecting FIX and preparing FIX-poor plasma.
Description
Technical Field
The invention relates to the field of medical biology and discloses a single-domain antibody aiming at coagulation Factor IX (FIX). In particular, the invention discloses a FIX binding molecule derived from the single domain antibody and application thereof, in particular application in detecting FIX and preparing FIX-poor plasma.
Background
Coagulation factors are various protein components involved in the process of blood coagulation. Its physiological role is to be activated when a blood vessel bleeds, to bind platelets together and to fill leaks in the blood vessel. This process is called coagulation. They are produced in part by the liver. Can be inhibited by coumarin. For uniform nomenclature, the world health organization is numbered with Roman numerals in the order in which they were discovered, with coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, and the like.
Hereditary coagulation dysfunctional diseases caused by deficiency of coagulation factors are generally classified into type A and type B, and belong to the accompanying inheritance. The prevalence of newborn males is approximately 1: 5000. Generally, the more severe the condition, the earlier the blood stasis and bleeding will occur. There are approximately 40 million patients worldwide, calculated as incidence. The number of patients in China should be 10 thousands, but unfortunately most patients are not diagnosed, and the current treated patients are estimated to be only 8 percent (about 8000 people).
Hemophilia b is a coagulopathy caused by a deficiency in coagulation nine Factor (FIX) in the body, characterized by spontaneous or trauma-related bleeding, the site of bleeding being primarily in the joints, soft tissues and muscles. According to the information of the diagnosis and treatment center of thrombus hemostasis in the hematological hospital (hematology institute) of the Chinese academy of medicine, 9804 hemophiliacs are registered nationwide at present, but the number of actual patients is far more than that, wherein the number of hemophiliacs B is about 15 to 20 percent.
Currently, there is no radical treatment for hemophilia. The best treatment is replacement therapy, i.e. timely infusion of the relevant coagulation factors to the patient. Otherwise, once a wound appears on a patient, the patient loses blood and dies because of the loss of the blood coagulation function. The frequency of administration is higher if the patient has already bled. At present, most of the medical coagulation nine factors for China depend on blood plasma products, namely the blood plasma products are limited by supply sources and have potential safety hazards; the only recombinant product is BeneFIX from Hewlett-packard, USA, which is expensive. BeneFIX prices of 2009 Wyeth quote $ 0.93 per international unit, patients report actual medical costs per unit of $ 4, and one dose of 250-. Temporary treatment for bleeding costs approximately 15-20 ten thousand dollars per year for a hemophiliac patient, and is doubled if prophylactic treatment is required. In view of the market effect of FIX and FIX, the only effective drugs for treating hemophilia B at present, FIX imitation drugs and long-acting FIX have been developed and produced by several companies internationally.
For the development of recombinant FIX, a key ring is to try to maintain its biological activity and bioavailability. FIX contains a variety of post-translational modifications and has a very important impact on its biological activity. The current analysis means can only carry out posttranslational modification and specific activity analysis on highly purified recombinant FIX at the later development stage. If a simpler detection method is available, the purity can be analyzed simply and rapidly at an early stage, even the activity of the recombinant FIX in cell feed liquid can be greatly helped for project development, early process screening and the like.
In the activity detection of the recombinant blood coagulation factor, nine-factor-poor plasma is required. At the same time, the diagnosis of coagulation disorders also requires the examination of the coagulation factor activity of the patient. There is also a need in the art for an efficient, low cost method for producing factor-poor (e.g., factor IX) plasma.
Summary of The Invention
In a first aspect, the present invention provides a coagulation Factor Ix (FIX) binding molecule capable of binding FIX and comprising at least one immunoglobulin single variable domain comprising a CDR1, a CDR2 and a CDR3 selected from the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, CDR3 shown in SEQ ID NO. 6;
(3) CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO. 9;
(4) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
(5) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15; and
(6) CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17, and CDR3 shown in SEQ ID NO. 18.
In some embodiments, the immunoglobulin single variable domain is a VHH.
In some embodiments, the VHH comprises the amino acid sequence of any one of SEQ ID NOS 19-24.
In some embodiments, the FIX binding molecule further comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region. In some embodiments, the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO 25.
In some embodiments, the FIX-binding molecule binds FIX with a KD value of less than 1X 10-7M, preferably less than 1X 10-8M, more preferably less than 1X 10-9M, more preferably less than 1X 10-10M, particularly more preferably less than 1X 10-11M。
In a second aspect, the invention provides a nucleic acid molecule encoding a FIX binding molecule of the invention.
In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the second aspect of the invention operably linked to an expression control element.
In a fourth aspect, the invention provides a host cell comprising a nucleic acid molecule of the invention or transformed with an expression vector of the invention and capable of expressing said FIX binding molecule.
In a fifth aspect, the invention provides a method of producing a FIX binding molecule of the invention, comprising:
a) culturing the host cell of the invention under conditions that allow expression of the FIX binding molecule;
b) recovering the FIX binding molecule expressed by the host cell from the culture from step a); and
c) optionally further purifying and/or modifying the FIX binding molecule resulting from step b).
In a sixth aspect, the present invention provides a kit for detecting the presence and/or quantifying FIX in a target sample and/or detecting the level of carboxylation of FIX in a target sample, comprising a FIX binding molecule of the invention. In some embodiments, the kit further comprises a control sample comprising a predetermined amount of FIX or FIX comprising a predetermined level of carboxylation.
In a seventh aspect, the present invention provides a method of detecting the presence and/or quantifying FIX in a sample and/or detecting the level of carboxylation of FIX in a sample, the method comprising:
a) contacting the target sample and the control sample with a FIX binding molecule of the invention, respectively, under conditions enabling the formation of a complex between the FIX binding molecule and FIX;
b) detecting the formation of the complex by detecting the presence of the complex,
wherein the difference in complex formation between the target sample and the control sample is indicative of the presence and/or amount of FIX and/or its level of carboxylation in the target sample, preferably the control sample contains a predetermined amount of FIX or FIX containing a predetermined level of carboxylation.
In an eighth aspect, the invention provides a method of preparing a FIX-poor blood sample comprising
a) Contacting a blood sample with a FIX binding molecule of the invention, whereby FIX in the blood sample forms a complex with the FIX binding molecule,
b) separating said complex from said blood sample, and
c) FIX-depleted blood samples were harvested.
In some embodiments, wherein the FIX binding molecule is immobilized on a solid support. In some embodiments, wherein the blood sample is plasma.
In a ninth aspect, the present invention provides a FIX affinity chromatography medium comprising a solid support having fixed thereon a FIX binding molecule of the invention. In some embodiments, wherein the solid support is made of a material selected from the group consisting of: polyethylene, polystyrene, polypropylene, polysulfone, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, cellulose acetate, cross-linked dextran, cross-linked agarose, chitin, chitosan, cross-linked dextran, cross-linked alginic acid, silicone, fluoropolymers, magnetic media, and other synthetic polymers. In some embodiments, the FIX affinity chromatography medium is an agarose medium or magnetic bead on which FIX binding molecules of the invention are immobilized.
In a tenth aspect, the present invention provides a device for preparing a FIX-depleted blood sample comprising a FIX affinity chromatography medium of the present invention.
Drawings
FIG. 1 shows the sequence of an anti-FIX single domain antibody.
FIG. 2 shows the binding curves of anti-FIX single domain antibody-Fc fusion protein to antigen FIX.
FIG. 3 shows the Western blot results of FIX and FVII with anti-FIX single domain antibody-Fc fusion proteins.
FIG. 4 shows Western blot results of recombinant human FIX and anti-FIX single domain antibody-Fc fusion protein.
FIG. 5 shows Western blot results of different carboxylated recombinant human FIX proteins with anti-FIX single domain antibody-Fc fusion protein.
Detailed Description
Definition of
Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art that will be understood by those skilled in the art. Reference is made, for example, to standard manuals, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual"; lewis, "Genes VIII"; and Roitt et al, "Immunology" (8 th edition), and the general prior art cited herein; moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically recited may be and have been performed in a manner known per se to those of skill in the art. Reference is also made, for example, to standard manuals, the general prior art mentioned above and to other references cited therein.
Unless otherwise indicated, the terms "antibody" or "immunoglobulin" used interchangeably herein, whether referring to a heavy chain antibody or to a conventional 4 chain antibody, are used as general terms to include full-length antibodies, individual chains thereof, as well as all portions, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" as used herein (e.g. in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence" etc.) should generally be understood to include both the relevant amino acid sequences and the nucleic acid or nucleotide sequences encoding the sequences, unless a more limited interpretation is required herein.
As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, domains are responsible for individual functional properties of proteins, and in many cases may be added, removed, or transferred to other proteins without loss of function of the rest of the protein and/or domain.
The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or to a polypeptide consisting essentially of such a globular region. The immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristics of an antibody molecule, consisting of a 2-layer sandwich of about 7 antiparallel beta sheet strands arranged in two beta sheets, optionally stabilized by conserved disulfide bonds.
The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain consisting essentially of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR 1", "framework region 2" or "FR 2", "framework region 3" or "FR 3", and "framework region 4" or "FR 4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR 1", "complementarity determining region 2" or "CDR 2", and "complementarity determining region 3" or "CDR 3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Immunoglobulin variable domains confer specificity for an antigen to an antibody by virtue of having an antigen binding site.
The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that is capable of specifically binding an epitope of an antigen without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody", e.g. immunoglobulin single variable domains VH and VL (VH and VL domains). Another example of an immunoglobulin single variable domain is a camelidae "VHH domain" (or simply "VHH") as defined below.
"VHH domain", also called heavy chain single domain antibody, VHH, VHH domains, VHH antibody fragments and VHH antibodies, variable for antigen-binding immunoglobulins, referred to as "heavy chain antibodies" (i.e. "antibodies lacking a light chain")(ii) a domain (Hamers-Casterman C, Atarhouch T, Muylermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: Nature encapsulation antibodies dehanid of light chains "; Nature 363,446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from a heavy chain variable domain (which is referred to herein as a "VH domain") present in conventional 4 chain antibodies, and a light chain variable domain (which is referred to herein as a "VL domain") present in conventional 4 chain antibodies. The VHH domain specifically binds to an epitope without the need for an additional antigen binding domain (as opposed to the VH or VL domain in conventional 4 chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed from a single immunoglobulin domain.
In the context of the present invention, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH domain, VHH antibody fragment, VHH antibody and
and "
Domain "(" Nanobody "is a trademark of Ablynx n.v. company, Ghent, Belgium) is used interchangeably.
For example, as shown in FIG. 2 of Riechmann and Muylermans, J.Immunol.methods 231,25-38(1999), the amino acid residues employed for the VHH domains in the family Camelidae are numbered according to the general numbering of the VH domains given by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication No. 91). According to this numbering process,
FR1 contains the amino acid residues at positions 1-30,
-CDR1 comprises amino acid residues at positions 31-35,
FR2 contains the amino acids at positions 36-49,
-CDR2 comprises amino acid residues at positions 50-65,
FR3 contains the amino acid residues at positions 66-94,
-CDR3 comprises amino acid residues at positions 95 to 102, and
-FR4 comprises the amino acid residue at position 103-113.
It should be noted, however, that the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering, as is well known in the art for VH and VHH domains (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
Other systems for numbering the amino acid residues of VH domains are known in the art, such as the Chothia numbering system. Chothia has the same amino acid numbering as Kabat, but will divide the CDR regions based on loop (loop) regions in the antibody variable region structure, and thus will differ from Kabat in the amino acid regions that the CDR regions comprise. Further, an AbM coding system and the like are available. The other coding systems may also be similarly applied to VHH domains. However, unless otherwise indicated, in the present specification, claims and drawings, numbering according to Kabat and as appropriate for the VHH domain as described above, or a combination of Kabat and Chothia, will be followed.
The total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.
Other structural and functional properties of VHH domains and polypeptides comprising the same may be summarized as follows:
the VHH domain, which has been naturally "designed" to functionally bind to an antigen in the absence and without interaction with a light chain variable domain, can be used as a single and relatively small functional antigen binding unit, domain or polypeptide. This distinguishes VHH domains from VH and VL domains of conventional 4 chain antibodies, which are themselves generally unsuitable for practical application as single antigen binding proteins or immunoglobulin single variable domains, but need to be combined in some form or another to provide a functional antigen binding unit (e.g. in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain).
Because of these unique properties, the use of VHH domains-alone or as part of a larger polypeptide-offers many advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g., Fab-or F (ab')2-fragments) of the protein: only a single domain is required to bind antigen with high affinity and high selectivity, so that neither two separate domains need be present, nor is it required to ensure that the two domains are present in the proper spatial conformation and configuration (e.g., scFv's typically require the use of specially designed linkers); the VHH domain may be expressed from a single gene and does not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats (formatting); the VHH domain is highly soluble and has no tendency to aggregate; the VHH domain is highly stable to heat, pH, proteases and other denaturants or conditions, and therefore refrigeration equipment may not be used in preparation, storage or transport, thereby achieving cost, time and environmental savings; VHH domains are easy to prepare and relatively inexpensive, even on the scale required for production; the VHH domain is relatively small compared to conventional 4 chain antibodies and antigen binding fragments thereof (about 15kDa or 1/10 of conventional IgG in size), and therefore shows higher tissue permeability and can be administered at higher doses compared to conventional 4 chain antibodies and antigen binding fragments thereof; VHH domains may exhibit so-called cavity-binding properties (especially due to their extended CDR3 loops compared to conventional VH domains) allowing access to targets and epitopes not accessible by conventional 4-chain antibodies and antigen-binding fragments thereof.
Methods for obtaining VHHs that bind to a particular antigen or epitope have been previously disclosed in the following references: van der Linden et al, Journal of Immunological Methods,240(2000) 185-195; li et al, J Biol chem, 287(2012) 13713-13721; deffar et al, African Journal of Biotechnology Vol.8(12), pp.2645-2652,17June, 2009; WO 94/04678; US 7790367, 2006-09-14, METHOD FOR SCREENING A LIBRARY OF VHH POLYPEPTIDES, Casterman, Cecil, Hamers, Raymond; and US 7786047, 2006-02-10, IMMUNOGLOBULINS DEVOID OF LIGHT CHAINS, Casterman, Cecil, Hamers, Raymond.
In addition, those skilled in the art will also appreciate that it is possible to "graft" one or more of the above CDRs onto other "scaffolds," including but not limited to human scaffolds or non-immunoglobulin scaffolds. Scaffolds and techniques suitable for such CDR grafting are known in the art.
As used herein, the term "epitope" or the interchangeably used term "antigenic determinant" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Antigenic determinants generally comprise chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be a "linear" epitope or a "conformational" epitope. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). In a linear epitope, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction exists across protein amino acid residues that are separated from each other.
Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). For example, a linear epitope can be determined by, for example, the following methods: a plurality of peptides are simultaneously synthesized on a solid support, wherein the peptides correspond to portions of a protein molecule, and the peptides are reacted with an antibody while still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al (1984) Proc.Natl.Acad.Sci.USA 81: 3998-; geysen et al (1986) molecular. Immunol.23: 709-715. Similarly, conformational epitopes can be identified by determining the spatial configuration of amino acids, such as by x-ray crystallography and 2-dimensional nuclear magnetic resonance, for example. See, e.g., Epitope Mapping Protocols (supra).
Antibodies can be screened for binding competition with the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen. A high throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is described in International patent application WO 03/48731. Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecule of the invention for binding to the same epitope on FIX can be obtained using conventional techniques known to those skilled in the art.
In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the invention) molecule can bind. Specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity. The affinity, expressed by the dissociation equilibrium constant (KD) of an antigen to an antigen binding protein, is a measure of the strength of binding between an epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen-binding molecule (alternatively, affinity can also be expressed as the association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be determined in a known manner depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen binding molecule (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and an associated antigen. Affinity is related to both: affinity to its antigen binding site on the antigen binding molecule, and the number of relevant binding sites present on the antigen binding molecule.
As used herein, the term "FIX binding molecule" means any molecule capable of binding FIX with high affinity. The FIX binding molecule may comprise an antibody as defined herein or a conjugate thereof directed against FIX. FIX binding molecules also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.
"FIX-binding molecule" may alternatively refer to monovalent molecules that bind FIX (i.e., molecules that bind one epitope of FIX), as well as bivalent or multivalent binding molecules (i.e., binding molecules that bind more than one epitope). A "FIX binding molecule" of the invention may comprise at least one immunoglobulin single variable domain that binds FIX, such as VHH. In some embodiments, a "FIX binding molecule" of the invention may comprise two immunoglobulin single variable domains that bind FIX, such as VHH.
Generally, the FIX binding molecules of the invention will be measured preferably at 10 as measured by Biacore or KinExA or biofilm interference techniques (BLI)-7To 10-11Mole/liter (M), more preferably 10-8To 10-11Mole/liter, even more preferably 10-9To 10-11Even more preferably 10-10To 10-11Or a dissociation constant (KD) of at least 10, and/or7M-1Preferably at least 108M-1More preferably at least 109M-1More preferably at least 1010M-1E.g. at least 1011M-1Binds to the antigen to be bound (i.e., FIX). Binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, Surface Plasmon Resonance (SPR) assays, Scatchard assays, biofilm interference (BLI) assays, and/or competitive binding assays (e.g., Radioimmunoassays (RIA), Enzyme Immunoassays (EIA), and sandwich competitive assays).
A polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been separated from at least one other component with which it is normally associated in the source or medium (culture medium), such as another protein/polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component, as compared to the reaction medium or culture medium from which it is naturally derived and/or from which it is obtained. In particular, a polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold and up to 1000-fold or more than 1000-fold. The "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).
An "affinity matured" anti-FIX antibody, in particular a VHH or domain antibody, has one or more changes in one or more CDRs which result in an increased affinity for FIX compared to its respective parent anti-FIX antibody. Affinity matured anti-FIX antibodies can be prepared by methods known in the art, for example, as described below: marks et al, 1992, Biotechnology 10: 779-; shier et al, 1995, Gene 169: 147-; yelton et al, 1995, Immunol.155: 1994-2004; jackson et al, 1995, J.Immunol.154(7): 3310-9; and Hawkins et al, 1992, J.MoI.biol.226(3): 889896; KS Johnson and RE Hawkins, "Affinity mapping of antibodies using phase display", Oxford University Press 1996.
FIX binding molecules of the invention
In a first aspect, the present invention provides a FIX binding molecule comprising at least one immunoglobulin single variable domain capable of binding FIX. In some embodiments, the FIX binding molecule comprises an immunoglobulin single variable domain that binds FIX. In some embodiments, the FIX binding molecule comprises two or more immunoglobulin single variable domains that bind FIX. In some embodiments, the amino acid sequence of FIX of the present invention is shown in SEQ ID No. 32, which is the full-length amino acid sequence of mature human FIX.
In some embodiments, the at least one immunoglobulin single variable domain comprises a CDR1, a CDR2, and a CDR3 selected from the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3 (corresponding to CDR of antibody strain nFN 50);
(2) CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, CDR3 shown in SEQ ID NO. 6 (corresponding to CDR of antibody strain nFN 52);
(3) CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO. 9 (corresponding to CDR of antibody strain nFN 62);
(4) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12 (corresponding to CDR of antibody strain nFN 64);
(5) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15 (corresponding to CDR of antibody strain nFN 65); and
(6) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18 (corresponding to the CDR of antibody strain nFN 69).
In some embodiments, at least one immunoglobulin single variable domain in a FIX binding molecule of the invention is a VHH. In some embodiments, the VHH comprises the amino acid sequence of any one of SEQ ID NOs 19-24. In other embodiments, the VHH in a FIX binding molecule of the invention comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to any one of SEQ ID NOs 19-24. Alternatively, the amino acid sequence of said VHH comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NO 19-24. For example, 1,2, 3,4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions are included.
In some embodiments, the FIX binding molecules of the invention are obtained by affinity maturation. Affinity matured FIX binding molecules may have one or more alterations in one or more CDRs that result in an increased affinity for FIX compared to the parent FIX binding molecule.
In some embodiments, a FIX binding molecule of the invention comprises an immunoglobulin Fc region in addition to at least one immunoglobulin single variable domain capable of specifically binding FIX. The inclusion of an immunoglobulin Fc region in the FIX binding molecules of the present invention allows the binding molecules to form dimers. Fc regions useful in the present invention may be from different subtypes of immunoglobulin, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.
The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, more preferably an Fc region of human IgG 1. In some embodiments, the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO 25.
In some embodiments, in the FIX binding molecules of the invention, the immunoglobulin Fc region (e.g., the Fc region of human IgG 1) is linked directly or indirectly via a linker (e.g., a peptide linker) to the C-terminus of the immunoglobulin single variable domain (e.g., VHH).
In another aspect, the FIX binding molecules of the invention also encompass anti-FIX antibody molecules capable of binding to the same epitope on FIX as a VHH consisting of the amino acid sequence of any one of SEQ ID NOs 19-24.
The FIX-binding molecules of the invention may have a KD value for binding to FIX of less than 1X 10-7M, preferably less than 1X 10-8M, more preferably less than 1X 10-9M, more preferably less than 1X 10-10M, particularly preferably less than 1X 10-11M。
In some embodiments, the FIX binding molecules of the invention specifically bind FIX, but not other coagulation factors such as FVII, FVIII or with lower affinity.
In some embodiments, FIX-binding molecules of the invention bind more strongly to FIX that is more gamma carboxylated and more active.
Furthermore, the FIX binding molecules of the present invention are resistant to acid, base and/or salt treatment treatments. For example, the activity of the FIX-binding molecules of the invention remains substantially unchanged after about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 16 hours, about 24 hours, about 32 hours, or more, treatment with an acid (e.g., 0.1M acetic acid), and/or treatment with a base (e.g., 0.1M sodium bicarbonate) for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 16 hours, about 24 hours, about 32 hours, or more, and/or treatment with a salt (e.g., 0.5M sodium chloride) for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 16 hours, about 24 hours, about 32 hours, or more.
Nucleic acids, vectors, host cells
In another aspect, the invention relates to a nucleic acid molecule encoding a FIX binding molecule of the invention. The nucleic acid of the present invention may be RNA, DNA or cDNA. According to one embodiment of the invention, the nucleic acid of the invention is a substantially isolated nucleic acid. In some embodiments, the nucleic acid molecule encoding a FIX binding molecule of the invention comprises the nucleotide sequence of any of SEQ ID NOs 26-31.
The nucleic acid of the invention may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid or YAC. The vector may especially be an expression vector, i.e. a vector providing for the expression of the FIX binding molecule in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, and the like). The selection of the elements and their sequences for expression in a particular host is within the knowledge of one skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for expression of the FIX binding molecules of the invention, such as promoters, enhancers, terminators, integration factors, selection markers, leaders, reporters.
The nucleic acids of the invention may be prepared or obtained in a known manner (e.g., by automated DNA synthesis and/or recombinant DNA techniques) based on information regarding the amino acid sequence of the polypeptides of the invention given herein, and/or may be isolated from a suitable natural source.
In another aspect, the invention relates to a host cell expressing or capable of expressing one or more FIX binding molecules of the invention and/or comprising a nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
Suitable bacterial cells include cells of gram-negative bacterial strains, such as Escherichia coli, Proteus and Pseudomonas strains, and gram-positive bacterial strains, such as Bacillus (Bacillus), Streptomyces, Staphylococcus and Lactococcus strains.
Suitable fungal cells include cells of species of the genera Trichoderma (Trichoderma), Neurospora (Neurospora) and Aspergillus (Aspergillus); or cells of species including Saccharomyces (Saccharomyces) such as Saccharomyces cerevisiae, Schizosaccharomyces (Schizosaccharomyces pombe), Pichia (Pichia) such as Pichia pastoris and Pichia methanolica, and Hansenula.
Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins may also be used in the present invention.
The invention also provides a method of producing a FIX binding molecule of the invention, the method generally comprising the steps of:
-culturing the host cell of the invention under conditions allowing the expression of the FIX binding molecule of the invention; and
-recovering from the culture the FIX binding molecule expressed by the host cell; and
-optionally further purifying and/or modifying the FIX binding molecule of the invention.
In a preferred embodiment, the FIX binding molecules of the invention are produced using mammalian cells. The FIX binding molecules of the invention can be highly expressed in mammalian cells. For example, the expression level may be up to about 100mg/L, preferably about 150mg/L, preferably about 200mg/L, preferably about 300mg/L, more preferably about 400mg/L or more preferably about 500mg/L or more.
FIX-binding molecules of the invention can be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, followed by isolation from the host cell and optionally further purification; or it may be produced extracellularly (e.g. in the medium in which the host cell is cultured), followed by isolation from the medium and optionally further purification.
Methods and reagents for recombinant production of polypeptides, such as specifically adapted expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, and the like, are known in the art. Similarly, protein isolation and purification techniques suitable for use in the methods of making FIX binding molecules of the invention are well known to those skilled in the art.
However, FIX-binding molecules of the invention may also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.
FIX detection
In another aspect, the present invention provides a method of detecting the presence and/or quantifying FIX in a sample and/or detecting the level of carboxylation of FIX in a sample, the method comprising:
a) contacting the target sample and the control sample with a FIX binding molecule of the invention, respectively, under conditions enabling the formation of a complex between the FIX binding molecule and FIX;
b) detecting the formation of the complex by detecting the presence of the complex,
wherein the difference in complex formation between the target sample and the control sample is indicative of the presence and/or amount of FIX and/or its level of carboxylation in the target sample, preferably the control sample contains a predetermined amount of FIX or FIX containing a predetermined level of carboxylation.
In some embodiments, the carboxylation is gamma carboxylation. In some embodiments, the biological activity of FIX is positively correlated with its level of carboxylation. Thus, detecting the level of carboxylation of FIX in a sample can be used to assess the biological activity of FIX in a sample. The invention also encompasses methods of assessing the biological activity of FIX in a sample by detecting the level of carboxylation of FIX in the sample.
In some embodiments, the method further comprises the step of obtaining a standard curve by serial gradient dilution of a control sample containing a predetermined amount of FIX or a predetermined level of carboxylation of FIX.
In some embodiments, FIX binding molecules of the present invention can be conjugated with fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, tags, and the like that can be used for detection or can be detected by other reagents. In one embodiment, the detection is performed by methods known in the art for immunodetection, such as western blotting or ELISA. In some embodiments, FIX-binding molecules of the invention may have a human Fc region and thus may be paired with commercial secondary antibodies for detection of FIX content at a greatly reduced cost compared to commercial FIX detection kits.
In some embodiments, the target sample is a FIX product prepared by a different method.
Preparation of blood plasma of blood coagulation factor IX lacking
In another aspect, the present invention provides a method for preparing a FIX-poor blood sample comprising
a) Contacting a blood sample with a FIX binding molecule of the invention, whereby FIX in the blood sample forms a complex with the FIX binding molecule,
b) separating said complex from said blood sample, and
c) FIX-depleted blood samples were harvested.
In some embodiments, wherein the FIX binding molecule is immobilized on a solid support. In some embodiments, the solid support is selected from the group consisting of: polyethylene, polystyrene, polypropylene, polysulfone, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, cellulose acetate, cross-linked dextran, cross-linked agarose, chitin, chitosan, cross-linked dextran, cross-linked alginic acid, silicone, fluoropolymers and other synthetic polymers, or magnetic media (e.g., magnetic beads).
The surface of the solid support may be functionalized or otherwise carry functional groups to allow covalent or non-covalent attachment of FIX binding molecules as described herein. In some embodiments, the solid support is agarose activated with CNBr. In other embodiments, the solid support is a Streptavidin (SA) labeled magnetic bead.
In some embodiments, wherein the blood sample is plasma. The FIX-poor plasma prepared by the method is particularly suitable for clinically detecting the FIX activity. Methods for detecting FIX activity using FIX-poor plasma are well known in the art.
In another aspect, the invention provides a FIX affinity chromatography medium comprising a solid support having fixed thereon a FIX binding molecule of the invention. In some embodiments, wherein the solid support is made of a material selected from the group consisting of: polyethylene, polystyrene, polypropylene, polysulfone, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, cellulose acetate, cross-linked dextran, cross-linked agarose, chitin, chitosan, cross-linked dextran, cross-linked alginic acid, silicone, fluoropolymers and other synthetic polymers, or magnetic media (e.g., magnetic beads). The solid support may be in the form of a filter, membrane, solid fiber, hollow fiber, or bead. The FIX affinity chromatography media can be used for the separation and preparation of FIX, or in particular for the preparation of FIX-poor blood samples such as FIX-poor plasma.
In another aspect, the invention also provides a device for preparing a FIX-depleted blood sample comprising a FIX affinity chromatography medium of the invention. The device may be, for example, a chromatography column.
Reagent kit
Also included within the scope of the invention are kits comprising a FIX binding molecule of the invention, a FIX affinity chromatography medium of the invention, or a device of the invention. The kit typically includes a label indicating the intended use of the contents of the kit (e.g., for detecting FIX or its activity, or for preparing FIX-depleted plasma). The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
Examples
The invention will now be further illustrated by way of the following examples, without thereby limiting the invention to the scope of the examples described.
Example 1: screening of anti-FIX heavy chain Single Domain antibodies
1.1. Construction of the library:
the lymphocytes of 14 camels which were not immunized were selected and the spleen of 5 camels and the lymph node of 8 camels were extracted, total RNA of lymphocytes and tissues was extracted using an RNA extraction kit provided by QIAGEN, all of the extracted RNA was reverse-transcribed into cDNA using a Super-Script III FIRST STRANDSUPERMIX kit according to the instructions, and the nucleic acid fragment encoding the variable region of the heavy chain antibody was amplified by nested PCR.
The heavy chain single domain antibody nucleic acid fragment of interest was recovered and cloned into the phage display vector pMECS using restriction enzymes (purchased from NEB) PstI and NotI. The product was then electro-transformed into E.coli electro-competent cells TG1, a non-immune single domain antibody phage display library was constructed and the library was assayed. The size of the reservoir was calculated to be 1.4X 10 by gradient dilution plating9. To examine the insertion rate of the library, 100 clones were randomly selected for sequencing examination, and 99 clones with correct foreign fragment insertion were found with a correct rate of 99%. By analyzing and aligning the DNA and amino acid sequences of the sequenced clones respectively, it was confirmed that all the sequences were completely different, were the expected camelid VHH sequences with a diversity of 100%.
1.2 anti-FIX heavy chain single domain antibody panning:
plates were coated with recombinant FIX in an amount of 0.5. mu.g/well and left overnight at 4 ℃. The next day, after 2 hours of room temperature blocking with 2% skim milk, 100. mu.l phage (. about.10) per well was added10×1013pfu, from a 1.1 bactrian camel non-immune single domain antibody display library), at room temperature for 2 hours. After that, the phage were washed 25 times with PBST20 (containing 0.05% Tween 20 in PBS) to wash away unbound phage. Finally, the phage specifically bound to FIX were dissociated with glycine (100mM, pH 2.06) and infected with E.coli TG1 in log phase of growth, generated and purifiedPhage were used for the next round of screening. The above screening process was repeated for 3 rounds. Thus, positive clones are enriched, and thus FIX-specific antibodies in the antibody library are screened using phage display technology.
1.3 screening of specific single positive clones by enzyme-linked immunosorbent assay (ELISA):
after 3 rounds of panning, the obtained FIX bound positive phages to infect blank e.coli and were plated. Subsequently, 95 single colonies were picked and inoculated into 2TY-AG, each cultured to an OD600 of about 0.8, and then IPTG was added to a final concentration of about 1mM to induce expression, overnight at 25 ℃. The single domain antibody is expressed in the periplasm of the escherichia coli, the thalli are harvested the next day, TES is added to crack the thalli by an osmotic pressure impact method, and the supernatant is used for ELISA detection. Plates were coated with FIX overnight at 4 ℃ and the supernatant (control was a blank E.coli lysis supernatant) was added and allowed to react at room temperature for 2 hours. After washing, a secondary antibody, Goat anti-HA tag HRP (from abcam) was added and reacted at room temperature for 2 hours. After washing, TMB developing solution was added and the absorbance at a wavelength of 405nm was read. When the OD value of the sample well is more than 2 times larger than that of the control well, the positive clone well is identified. Positive clones were sequenced.
The amino acid sequence of each clone was analyzed according to the sequence alignment software Vector NTI. Clones with > 90% sequence homology of CDR1, CDR2, and CDR3 were considered as identical antibody strains. The final 6 different antibodies were obtained in total, the sequences are shown in FIG. 1, and the CDR regions are boxed according to the rules of Kabat and Chothia.
Example 2: preparation of anti-FIX Single Domain antibody-Fc fusion protein by expression in mammalian cells
2.1. Preparation of vector expressing FIX Single Domain antibody-Fc fusion protein
Designing primers to perform PCR amplification on FIX single domain antibody VHH fragments, fusing with DNA fragments (amino acid sequences are shown in figure 1) encoding human IgG1-Fc, and cloning to a conventional mammal expression vector to obtain a recombinant plasmid for expressing FIX single domain antibody-Fc fusion protein in mammals. Wherein the amplification of different VHH fragments uses universal primers as follows:
upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC (SEQ ID NO:33)
Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG (SEQ ID NO:34)
2.2. Fc fusion protein for preparing FIX single-domain antibody
The 2.1 construction vector was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmids were diluted with Freestyle293 medium and added with Polyethyleneimine (PEI) solution required for transformation, and each set of plasmid/PEI mixture was added separately to HEK293 cell suspension and incubated at 37 ℃, 5% CO2, 130 rpm. After four hours, the CELLs were supplemented with EX-CELL293 medium and suspension cultured at 130 rpm. After 24 hours 3.8mM VPA was added and after 72 hours 4g/L glucose was added. And after culturing for 5-6 days, collecting transient expression culture supernatant, and purifying the target FIX single-domain antibody-Fc fusion Protein by a Protein A affinity chromatography. The purity of the obtained protein was checked by SDS-PAGE and SEC-HPLC. The nFN50-FC was mostly found to exist as a polymer when examined by SDS-PAGE. Therefore, nFN50-FC was excluded from subsequent experiments. Transient results for the remaining proteins are shown in Table 1.
Table 1: the obtained anti-FIX single domain antibody-Fc fusion protein is subjected to transient transformation and then is purified in one step.
| Antibodies | Amount of expression (mg/L) | Purity of SDS-PAGE | Proportion of monomer% |
| nFN 65-FC | 318 | >95％ | 98.751 |
| nFN 62-FC | 402 | >95％ | 99.713 |
| nFN 52-FC | 351 | >95％ | 99.710 |
| nFN 64-FC | 409 | >95％ | 95.242 |
| nFN 69-FC | 428 | >95％ | 95.319 |
The expression quantity of FIX single domain antibody-Fc fusion Protein is above 300mg/L, and the target Protein with stable concentration and high purity is obtained after one-step purification by Protein A affinity chromatography column.
Example 3: binding curve of anti-FIX single domain antibody-Fc fusion protein and FIX
The binding curve of the anti-FIX single domain antibody-Fc fusion protein to FIX was studied by ELISA.
Plates were coated with recombinant human FIX protein in an amount of 0.2. mu.g/well overnight at 4 ℃. The anti-FIX single domain antibody-Fc fusion protein obtained in example 2 was diluted in a gradient (10 ×) and reacted with the coated plate at room temperature for 2 hours. After washing, a secondary antibody (horseradish peroxidase-labeled goat anti-human IgG-Fc antibody (Sigma)) was added and reacted at room temperature for 2 hours. After washing, adding a developing solution, reading the absorption values of 450nm and 650nm, and subtracting the absorption value of 650nm from the absorption value of 450nm to obtain the final absorption value.
Data processing and mapping analysis were performed using the software SotfMax Pro v 5.4. The binding curve of the anti-FIX single domain antibody-Fc fusion protein and the recombinant human FIX protein and the EC50 value (the EC50 value of all antibodies is about 50ng/mL) are obtained by four-parameter fitting. Reflecting the affinity of the antibody for FIX.
The results are shown in FIG. 2, in which the ordinate is OD value and the abscissa is concentration (in ng/mL) of anti-FIX single domain antibody-Fc fusion protein; circles, squares, right triangles, diamonds, circles represent anti-FIX single domain antibody-Fc fusion proteins, respectively: nFN52-FC, nFN64-FC, nFN69-FC, nFN62-FC, nFN 65-FC. The five proteins all have high affinity for FIX and their affinity is comparable.
Example 4: selectivity of anti-FIX single domain antibody-Fc fusion proteins
The selection specificity of the anti-FIX single domain antibody-Fc fusion protein was investigated by ELISA.
The plates were coated with human FIX, FVII, FVIII, and TFPI proteins in an amount of 0.5. mu.g/well, respectively, overnight at 4 ℃ and 0.5. mu.g/well of the anti-FIX single domain antibody-Fc fusion protein obtained in example 2 (control was blank buffer) was added thereto, and reacted at room temperature for 2 hours. After washing, a secondary antibody (horseradish peroxidase-labeled goat anti-human IgG-Fc antibody (Sigma)) was added and reacted at room temperature for 2 hours. After washing, a developing solution was added to the solution, and the absorbance at a wavelength of 405nm was read. The results are shown in Table 2.
Table 2: binding of anti-FIX single domain antibody-Fc fusion proteins to human FIX and its cognate proteins.
| Antibodies | OD (human FIX) | OD (human FVII) | OD (human FVIII) | OD (human TFPI) |
| nFN52-FC | 2.272 | 1.045 | 0.046 | 0.034 |
| nFN62-FC | 2.285 | 0.798 | 0.04 | 0.028 |
| nFN64-FC | 2.379 | 2.048 | 0.113 | 0.077 |
| nFN65-FC | 2.312 | 0.083 | 0.046 | 0.036 |
| nFN69-FC | 1.975 | 0.881 | 0.054 | 0.032 |
| Blank space | 0.042 | 0.037 | 0.031 | 0.022 |
It can be seen that all the obtained FIX single domain antibody Fc fusion proteins do not bind human FVIII and TFPI proteins, wherein nFN52/62/64/69-Fc binds both human FIX and FVII proteins, and nFN65-Fc binds only human FIX protein and not human FVII protein.
Example 5: detection of non-specific binding of FIX single-domain antibody-Fc fusion protein to empty cells
The CHOK1 empty cells and 293F empty cells were resuspended in 3% BSA-PBS and the cell number was adjusted to 6X 106cells/mL, the FIX single domain antibody-Fc fusion protein obtained in example 2 was added to the cells respectively at a final concentration of 20. mu.g/mL, while negative and blank controls were set, and the cells were incubated in ice for 60 min. After washing, a secondary APC anti-human IgG-Fc antibody (Biolegend) was added and the mixture was incubated in ice for 30 min. After washing, the cells were resuspended in 500. mu.L of PBS buffer containing 1% BSA and detected by flow cytometry. The results are shown in Table 3.
Table 3: binding of anti-FIX single domain antibody-Fc fusion protein to CHOK1 and 293F null cells.
| Antibodies | MFI (for CHOK1 cells) | MFI (for 293F cells) |
| nFN52-FC | 5.86 | 5.16 |
| nFN64-FC | 6.86 | 5.82 |
| nFN69-FC | 4.57 | 4.6 |
| nFN62-FC | 4.88 | 4.36 |
| nFN65-FC | 5.12 | 5.16 |
| Blank control | 4.28 | 4.01 |
| Negative control | 4.25 | 4.12 |
As can be seen, the mean fluorescence intensity values of the anti-FIX single domain antibody-Fc fusion protein bound to the CHOK1 and 293F null cells were not significantly different from those of the blank control and the negative control, indicating that the anti-FIX single domain antibody-Fc fusion protein did not bind non-specifically to both CHOK1 and 293F null cells.
Example 6: detection of affinity of anti-FIX Single Domain antibody-Fc fusion protein
The binding kinetics of the anti-FIX single domain antibody-Fc fusion protein obtained in example 2 to recombinant human FIX was examined by biofilm interference (BLI) technique. The detection is performed using a molecular interaction device.
The anti-FIX single domain antibody-Fc fusion proteins nFN52-FC, nFN64-FC, nFN69-FC were diluted to a final concentration of 20. mu.g/mL, immobilized on an AHC biosensor, and the kinetics were measured. Human FIX, FVII proteins were diluted to 5 concentrations with 0.02% PBST20, respectively: 1000nM, 500nM, 250nM, 125nM, 62.5 nM. And injecting samples for 120s, wherein the dissociation time is 300 s. Regeneration was carried out with 10mM glycine-HCl (pH1.7) for 5 s. The association rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Languir association model (Octet K2 Data analysis software version 9.0 (Data analysis 9.0)). The equilibrium dissociation constant (kD) was calculated as the ratio kdis/kon.
The measured affinities for binding of the fusion proteins to FIX are shown in table 4 and to FVII in table 5. The results showed that the anti-FIX single domain antibody-Fc fusion proteins nFN52-FC, nFN62-FC, nFN64-FC, nFN69-FC bound FIX with higher affinity than FVII, and nFN65-FC bound FIX only.
Table 4: affinity of anti-FIX single domain antibody-Fc fusion protein to bind FIX.
| Antibodies | KD(M) | kon(1/Ms) | kdis(1/s) |
| nFN52-FC | 3.02E-09 | 8.55E+04 | 2.58E-04 |
| nFN64-FC | 5.73E-09 | 7.18E+04 | 4.11E-04 |
| nFN69-FC | 7.91E-10 | 6.03E+04 | 4.77E-05 |
| nFN62-FC | 4.77E-09 | 1.08E+05 | 5.16E-04 |
| nFN65-FC | 6.24E-09 | 6.94E+04 | 4.33E-04 |
Table 5: affinity of anti-FIX single domain antibody-Fc fusion protein binding to FVII.
| Antibodies | KD(M) | kon(1/Ms) | kdis(1/s) |
| nFN52-FC | 7.62E-09 | 8.31E+04 | 6.33E-04 |
| nFN64-FC | 1.58E-08 | 6.71E+04 | 1.06E-03 |
| nFN69-FC | 7.94E-09 | 2.63E+04 | 2.09E-04 |
| nFN62-FC | 1.50E-08 | 3.58E+04 | 5.38E-04 |
| nFN65-FC | NA | NA | NA |
The recognition of FIX or FVII by the anti-FIX single domain antibody-Fc fusion protein is detected by western blotting.
Adding the recombinant human FIX and FVII into non-reducing loading buffer respectively, and then carrying out boiling water bath treatment to fully denature the protein. SDS-PAGE was performed to obtain a loading of 0.5. mu.g protein. Proteins were transferred to a PVDF membrane activated with methanol by a semi-dry transfer method. After washing the membrane with 0.05% TBST20, the membrane was blocked with 5% skim milk powder at room temperature for 2 h. After washing the membrane with 0.05% TBST 20. The anti-FIX single domain antibody-Fc fusion proteins nFN52-FC, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC obtained in example 2 were each diluted in 0.05% TBST20 to a final concentration of 2.5. mu.g/mL. And (3) incubating the membrane and the diluted fusion protein for 2h at room temperature, washing the membrane, adding a secondary antibody marked by HRP, and incubating for 2h at room temperature. After washing the membrane, ECL substrate developing solution is added for developing color, and a gel imaging system is exposed for imaging, and the result is shown in figure 3.
FIG. 3 shows that antibody nFN65-FC binds only to FIX, and the remaining antibodies nFN52-FC, nFN64-FC, nFN69-FC, nFN62-FC bind both FVII and FIX, consistent with the results of the previous assays.
Comparison of FVII and FIX amino acid sequences shows that the homology of the carboxylated region is high. Considering that FIX activity correlates with the degree of carboxylation, western blot further verifies that antibodies bind to FIX with different activities.
BeneFIX drugs, as well as the inventors self-produced, varying degrees of modification of recombinant human FIX proteins 293DS-1, CH0DS-1, were tested by the APTT (activated partial thromboplastin time) method. The results were obtained: when the activity of the control BeneFIX is 150% of the standard, the activity of the recombinant human FIX protein 293DS-1 and CH0DS-1 is 89.3% and 138.6% respectively. The recombinant human FIX protein 293DS-1 and CH0DS-1 are added into a non-reducing loading buffer solution and then treated in a boiling water bath to fully denature the protein. SDS-PAGE was performed, and the amount of the sample was 0.5. mu.g. Proteins were transferred to a PVDF membrane activated with methanol by a semi-dry transfer method. After washing the membrane with 0.05% TBST20, the membrane was blocked with 5% skim milk powder at room temperature for 2 h. After washing the membrane with 0.05% TBST 20. The anti-FIX single domain antibody-Fc fusion proteins nFN52-FC, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC obtained in example 2 were each diluted in 0.05% TBST20 to a final concentration of 2.5. mu.g/mL. And (3) incubating the membrane and the diluted fusion protein for 2h at room temperature, washing the membrane, adding a secondary antibody marked by HRP, and incubating for 2h at room temperature. After washing the membrane, ECL substrate developing solution is added for developing color, and a gel imaging system is exposed for imaging, and the result is shown in figure 4.
FIG. 4 shows that nFN69-FC binds significantly differently to FIX of different activities, better to FIX CH0DS-1 of better degree of modification and activity, and worse to FIX 293DS-1 of poorer degree of modification and activity.
nFN69-FC was then further verified to bind to different carboxylated FIX.
When the control BeneFIX carboxylation modification is 12.451/mol, the carboxylation modifications of other recombinant human FIX proteins KN020Ko-A14, KN020Ko-A21 and KN020BAP150619 are 12.046/mol, 10.785/mol and 7.488/mol respectively. Adding the recombinant human FIX proteins KN020Ko-A14, KN020Ko-A21 and KN020BAP150619 with different carboxylation levels into a non-reducing sample buffer solution, and then carrying out boiling water bath treatment to fully denature the proteins. SDS-PAGE was performed, and the amount of the sample was 0.5. mu.g. Proteins were transferred to a PVDF membrane activated with methanol by a semi-dry transfer method. After washing the membrane with 0.05% TBST20, the membrane was blocked with 5% skim milk powder at room temperature for 2 h. After washing the membrane with 0.05% TBST 20. The anti-FIX single domain antibody-Fc fusion proteins nFN52-FC, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC obtained in example 2 were each diluted in 0.05% TBST20 to a final concentration of 2.5. mu.g/mL. And (3) incubating the membrane and the diluted fusion protein for 2h at room temperature, washing the membrane, adding a secondary antibody marked by HRP, and incubating for 2h at room temperature. After washing the membrane, adding ECL substrate color developing solution for color development, and exposing and imaging by a gel imaging system, wherein the result is shown in figure 5.
FIG. 5 shows that nFN69-FC binds with different carboxylated FIX with a clear difference, the higher the degree of carboxylation, the better the binding and the darker the color development.
Example 8: acid-base stability research of FIX-resistant single-domain antibody-Fc fusion protein
The anti-FIX single domain antibody-Fc fusion protein obtained in example 2 was treated under the following three conditions, respectively, and its tolerance to acid and base and stability were examined: (1)0.1M NaHCO3+0.5M NaCl, pH8.3, at 4 ℃ overnight, (2)0.1M acetic acid +0.5M NaCl, pH4.0, at room temperature for 3h, (3)0.1M acetic acid +0.5M NaCl, pH4.0, at room temperature for 3h, followed by 0.1M NaHCO3+0.5M NaCl, pH8.3, at 4 ℃ overnight.
The treated fusion protein was exchanged into 1 × PBS, and the concentration of the fusion protein in PBS was determined with Nanodrop. The purity of the treated fusion protein was checked by SEC.
Its binding activity to antigen FIX was detected by ELISA. Specifically, plates were coated with recombinant human FIX in an amount of 0.2. mu.g/well overnight at 4 ℃. The treated fusion protein was diluted in gradient and reacted with the coated plate for 2 hours at room temperature. After washing, a secondary antibody (horseradish peroxidase-labeled goat anti-human IgG-Fc antibody (Sigma)) was added and reacted at room temperature for 2 hours. After washing, adding a developing solution, reading the absorption values of 450nm and 650nm, and subtracting the absorption value of 650nm from the absorption value of 450nm to obtain the final absorption value. Data processing and mapping analysis are carried out by using software SotfMax Pro v5.4, and a binding curve and an EC50 value of the binding of the processed anti-FIX single domain antibody-Fc fusion protein and FIX are obtained through four-parameter fitting. Reflecting the affinity of the antibody for FIX. The results are shown in Table 6.
Table 6: binding of the treated anti-FIX single domain antibody-Fc fusion protein to FIX.
| Antibodies | EC50 (original) | EC50(pH 4.0 3h) | EC50(pH 8.3 overnight) | EC50(pH 8.3 overnight after pH 4.03 h) |
| nFN52-Fc | 71ng/mL | 62.9ng/mL | 66ng/mL | 65.5ng/mL |
| nFN64-Fc | 66.6ng/mL | 73.2ng/mL | 74.8ng/mL | 75.4ng/mL |
| nFN69-Fc | 95ng/mL | 78.5ng/mL | 78.9ng/mL | 72.4ng/mL |
| nFN62-Fc | 67ng/mL | 68.5ng/mL | 69.3ng/mL | 67.8ng/mL |
| nFN65-Fc | 69.5ng/mL | 72ng/mL | 70ng/mL | 70.3ng/mL |
As can be seen, the binding activity of the acid and/or base treated antibodies nFN52-Fc, nFN64-Fc, nFN69-Fc, nFN62-Fc, nFN65-Fc to FIX was good, with no significant difference compared to before treatment.
The treated anti-FIX single domain antibody-Fc fusion proteins were filtered with 0.22 μm water films, respectively. The filtered fusion protein was subjected to SEC-HPLC analysis. SEC-HPLC parameters were as follows: the sample amount is 50 μ G, the chromatographic column is TOSOH G3000SWXL 7.8 × 300mM, 5 μm and the protective column is TOSOH TSK gel guard column SWXL 6.0 × 40mM, 7 μm, and the mobile phase is 20mM Na2HPO4+300mM NaCl pH7.4, flow rate of 0.5mL/min, elution time of 45min, detection wavelength of 280 nm. The results are shown in Table 7.
Table 7: SEC-HPLC analysis of treated anti-FIX single domain antibody-Fc fusion proteins.
According to SEC results, the percentage of the main peak of the antibody treated under acid-base conditions is not obviously changed from that before treatment, which shows that the fusion antibody has strong acid-base resistance and good stability.
Example 9: preparation of plasma lean in FIX
9.1. Preparation of anti-FIX single domain antibody-Fc fusion protein affinity purification medium
The inventors tried two different affinity purification media.
I. FIX single domain antibody-Fc fusion proteins were coupled using agarose medium.
The anti-FIX single domain antibody-Fc fusion proteins nFN52-Fc, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC obtained in example 2 were exchanged to a solution of 0.2M NaHCO 30.5M NaCl, pH8.3 using a 3kDa ultrafiltration tube.
The fusion protein was coupled to CNBr pre-activated agarose medium at a loading of 3mg/mL overnight at 4 ℃. Then, the coupled medium was alternately eluted with Tris-HCl 0.05M, pH8.0 and acetate buffer 0.2M, pH4.0, and then equilibrated with PBS to obtain an anti-FIX single domain antibody-Fc fusion protein affinity chromatography medium.
Immobilization of biotin-labeled FIX single domain antibody-Fc fusion protein using streptavidin (streptavidin) -labeled microspheric beads.
The anti-FIX single domain antibody-Fc fusion proteins nFN52-Fc, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC obtained in example 2 were subjected to EZ-LinkTMThe Sulfo-NHS-Biotin kit (Thermo) labeled Biotin.
The biotin-labeled fusion protein was mixed with PBS-treated Dynabeads at a loading of 0.1mg/mLTMM-280Streptavidin magnetic beads (Thermo) are incubated at room temperature for half an hour, and then the unconjugated protein in the supernatant is removed by a magnetic frame to obtain FIX single domain antibody-Fc fusion protein labeled magnetic beads.
9.2. FIX-poor plasma is prepared by treating whole human plasma with FIX single-domain antibody-Fc fusion protein-agarose medium or FIX single-domain antibody-Fc fusion protein-magnetic bead, and verification is carried out
Human standard plasma was loaded on the fusion protein-agarose medium obtained above, or magnetic beads, and incubated at room temperature for 2h with gentle shaking. Plasma was also treated with unconjugated agarose medium or magnetic beads as negative controls (labeled negative control 1, negative control 2, respectively). The media or magnetic beads are then removed by centrifugation or magnetic holder to obtain a treated plasma supernatant.
The activity of FIX in the treated plasma supernatants was measured using the method of APTT. The treated plasma supernatant was loaded into a fully automatic coagulometer, automatically mixed and incubated with Dade Actin Activated cytoloplastic Reagent, Calcium Chloride Solution and FACTOR IX deficent Reagent, and the absorbance was measured at 660nm, while a standard curve (log clotting time versus log concentration percentage) was set with standards. The results are shown in Table 8.
Table 8:
| sample (I) | Clotting time(s) | Relative standard substance activity% |
| |
68.3 | 91.2 |
| FIX-poor plasma treated with nFN52-FC agarose | 103.2 | 4.6 |
| FIX-poor plasma treated with nFN62-FC agarose | 99.7 | 6.3 |
| FIX-poor plasma treated with nFN64-FC agarose | 102.6 | 4.8 |
| FIX-poor plasma treated with nFN65-FC agarose | 102.8 | 4.8 |
| FIX-poor plasma treated with nFN69-FC agarose | 104.5 | 4.1 |
| |
66.5 | 97.1 |
| FIX-depleted plasma treated with nFN52-FC magnetic beads | 100.1 | 5.8 |
| FIX-depleted plasma treated with nFN62-FC magnetic beads | 101.3 | 5.1 |
| FIX-depleted plasma treated with nFN64-FC magnetic beads | 98.3 | 7.5 |
| FIX-depleted plasma treated with nFN65-FC magnetic beads | 99.1 | 6.8 |
| FIX-depleted plasma treated with nFN69-FC magnetic beads | 102.8 | 4.8 |
The results show that the APTT time of plasma treated with nFN52-FC, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC labeled affinity chromatography media or magnetic beads is greatly prolonged compared to the control group, indicating that endogenous FIX in plasma is substantially captured and removed by the media or magnetic beads. Therefore, the invention proves that the anti-FIX single domain antibody-Fc fusion protein can be used for preparing the blood coagulation factor IX-lacking plasma.
A sequence table:
>SEQ ID NO:1
GYTYSRYCMG
>SEQ ID NO:2
AICTGGGSTYYADSVKG
>SEQ ID NO:3
YVGSDCGNAGRAY
>SEQ ID NO:4
GDISKVASMA
>SEQ ID NO:5
GLSIGTGRTYYADSVKG
>SEQ ID NO:6
VVAGVKY
SEQ ID NO:7
GDISKVASMA
>SEQ ID NO:8
GLSIGTGRTYYADSVKG
>SEQ ID NO:9
KGDQRFGGYLD
>SEQ ID NO:10
GDISKVASMA
>SEQ ID NO:11
GLSIGTGRTYYADSVKG
>SEQ ID NO:12
VTRWPPLAGGNWPAKY
>SEQ ID NO:13
LHNTNINAMA
>SEQ ID NO:14
ALLTRGGNTWYDDSVKG
>SEQ ID NO:15
VRRRDGYKY
>SEQ ID NO:16
GDISKVASMA
>SEQ ID NO:17
GLSIGTGRTYYADSVKG
>SEQ ID NO:18
NDQRRARY
>SEQ ID NO:19 nFN50
>SEQ ID NO:20 nFN52
>SEQ ID NO:21 nFN62
>SEQ ID NO:22 nFN64
>SEQ ID NO:23 nFN65
>SEQ ID NO:24 nFN69
>SEQ ID NO:25 IgG1-FC
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
>SEQ ID NO:26 nFN50
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTAGGCTCTGGATACACCTACAGTAGGTACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTATTTGTACTGGCGGTGGTAGTACGTACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAAGTCGAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCCTGTATTACTGTAAATTGTACGTTGGTAGTGATTGTGGAAACGCTGGCCGTGCTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC
>SEQ ID NO:27nFN52
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGCCGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAATACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTATATTACTGCGCCACAGTGGTAGCTGGCGTGAAGTACCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC
>SEQ ID NO:28nFN62
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGCCGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAATACGCTGTACCTGCAGATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAAAAGGCGACCAGCGTTTTGGAGGATATCTTGACTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCATC
>SEQ ID NO:29nFN64
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGCCGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAATACGCTGTACCTGCAGATGAACAGCCTGAAAACTGAGGACACTGCCATATACTACTGTGCGCTCGTGACGAGGTGGCCTCCTCTCGCCGGTGGCAACTGGCCAGCTAAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC
>SEQ ID NO:30nFN65
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATCGGTGCAGGCTGGAGGATCTCTGACGCTCTCCTGTGCATACTCTCTGCACAACACCAATATCAACGCCATGGCCTGGTTCCGCCAGACTCCAGGCAAGGAGCGCGAGGGGGTCGCTGCTCTTCTCACTCGTGGTGGCAATACATGGTATGACGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAACACGGTGTATCTACAAATGGATGACCTGAAACCTGAGGACACTGCCGTGTACTACTGTGCGGCAGTCCGGAGAAGGGATGGATATAAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC
>SEQ ID NO:31nFN69
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGCCGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAATACGCTGTACCTGCAGATGAACAGCCTGAAAACTGAGGACACTGCCATATACTACTGTGCGTATAATGATCAGCGTCGTGCACGGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC
>SEQ ID NO:32mature FIX
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT
33 upstream primer of SEQ ID NO
cccACCGGTCAGGTGCAGCTGCAGGAGTC
34 downstream primer of SEQ ID NO
cccGGATCCTGAGGAGACGGTGACCTGG
Sequence listing
<110> Suzhou kang ning Jie Rui Biotech Co., Ltd
<120> Single Domain antibody against coagulation Factor IX (FIX)
<130> I2018TC2110CB
<160> 34
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 1
Gly Tyr Thr Tyr Ser Arg Tyr Cys Met Gly
1 5 10
<210> 2
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 2
Ala Ile Cys Thr Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 3
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 3
Tyr Val Gly Ser Asp Cys Gly Asn Ala Gly Arg Ala Tyr
1 5 10
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 4
Gly Asp Ile Ser Lys Val Ala Ser Met Ala
1 5 10
<210> 5
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 5
Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 6
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 6
Val Val Ala Gly Val Lys Tyr
1 5
<210> 7
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 7
Gly Asp Ile Ser Lys Val Ala Ser Met Ala
1 5 10
<210> 8
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 8
Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 9
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 9
Lys Gly Asp Gln Arg Phe Gly Gly Tyr Leu Asp
1 5 10
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 10
Gly Asp Ile Ser Lys Val Ala Ser Met Ala
1 5 10
<210> 11
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 11
Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 12
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 12
Val Thr Arg Trp Pro Pro Leu Ala Gly Gly Asn Trp Pro Ala Lys Tyr
1 5 10 15
<210> 13
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 13
Leu His Asn Thr Asn Ile Asn Ala Met Ala
1 5 10
<210> 14
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 14
Ala Leu Leu Thr Arg Gly Gly Asn Thr Trp Tyr Asp Asp Ser Val Lys
1 5 10 15
Gly
<210> 15
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 15
Val Arg Arg Arg Asp Gly Tyr Lys Tyr
1 5
<210> 16
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 16
Gly Asp Ile Ser Lys Val Ala Ser Met Ala
1 5 10
<210> 17
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 17
Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 18
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 18
Asn Asp Gln Arg Arg Ala Arg Tyr
1 5
<210> 19
<211> 122
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 19
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Tyr Thr Tyr Ser Arg Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Cys Thr Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Lys Leu Tyr Val Gly Ser Asp Cys Gly Asn Ala Gly Arg Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 20
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 20
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Asp Ile Ser Lys Val Ala
20 25 30
Ser Met Ala Trp Phe Arg Gln Ala Pro Leu Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Val Val Ala Gly Val Lys Tyr Arg Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser
115
<210> 21
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 21
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Asp Ile Ser Lys Val Ala
20 25 30
Ser Met Ala Trp Phe Arg Gln Ala Pro Leu Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Lys Gly Asp Gln Arg Phe Gly Gly Tyr Leu Asp Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 22
<211> 125
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 22
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Asp Ile Ser Lys Val Ala
20 25 30
Ser Met Ala Trp Phe Arg Gln Ala Pro Leu Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Leu Val Thr Arg Trp Pro Pro Leu Ala Gly Gly Asn Trp Pro Ala
100 105 110
Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 23
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 23
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Tyr Ser Leu His Asn Thr Asn Ile Asn
20 25 30
Ala Met Ala Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Leu Leu Thr Arg Gly Gly Asn Thr Trp Tyr Asp Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asp Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Val Arg Arg Arg Asp Gly Tyr Lys Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 24
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 24
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly Asp Ile Ser Lys Val Ala
20 25 30
Ser Met Ala Trp Phe Arg Gln Ala Pro Leu Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Leu Ser Ile Gly Thr Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Tyr Asn Asp Gln Arg Arg Ala Arg Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 25
<211> 232
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 25
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 26
<211> 368
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 26
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgtag gctctggata cacctacagt aggtactgca tgggctggtt ccgccaggct 120
ccagggaagg agcgcgaggg ggtcgcagct atttgtactg gcggtggtag tacgtactat 180
gccgactccg tgaagggccg attcaccatc tcccaagaca agtcgaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccctgt attactgtaa attgtacgtt 300
ggtagtgatt gtggaaacgc tggccgtgct tactggggcc aggggaccca ggtcaccgtc 360
tcctcatc 368
<210> 27
<211> 350
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 27
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag gctctggaga catcagtaag gtagcttcaa tggcctggtt ccgccaggct 120
ccattgaagg agcgcgaggg ggtcgccggt ttaagtattg gtacaggtag gacatactac 180
gccgactccg tgaagggccg attcaccatc tcccgagaca acgccaagaa tacgctgtat 240
ctgcaaatga acagcctgaa acctgaggac acagccgtat attactgcgc cacagtggta 300
gctggcgtga agtaccgggg ccaggggacc caggtcaccg tctcctcatc 350
<210> 28
<211> 362
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 28
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag gctctggaga catcagtaag gtagcttcaa tggcctggtt ccgccaggct 120
ccattgaagg agcgcgaggg ggtcgccggt ttaagtattg gtacaggtag gacatactac 180
gccgactccg tgaagggccg attcaccatc tcccgagaca acgccaagaa tacgctgtac 240
ctgcagatga acagcctgaa acctgaggac actgccatgt actactgtgc ggcaaaaggc 300
gaccagcgtt ttggaggata tcttgactgg ggtcagggga cccaggtcac cgtctcctca 360
tc 362
<210> 29
<211> 377
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 29
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag gctctggaga catcagtaag gtagcttcaa tggcctggtt ccgccaggct 120
ccattgaagg agcgcgaggg ggtcgccggt ttaagtattg gtacaggtag gacatactac 180
gccgactccg tgaagggccg attcaccatc tcccgagaca acgccaagaa tacgctgtac 240
ctgcagatga acagcctgaa aactgaggac actgccatat actactgtgc gctcgtgacg 300
aggtggcctc ctctcgccgg tggcaactgg ccagctaagt actggggcca ggggacccag 360
gtcaccgtct cctcatc 377
<210> 30
<211> 356
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 30
caggtgcagc tgcaggagtc tgggggagga tcggtgcagg ctggaggatc tctgacgctc 60
tcctgtgcat actctctgca caacaccaat atcaacgcca tggcctggtt ccgccagact 120
ccaggcaagg agcgcgaggg ggtcgctgct cttctcactc gtggtggcaa tacatggtat 180
gacgactccg tgaagggccg attcaccatc tcccgagaca acgccaagaa cacggtgtat 240
ctacaaatgg atgacctgaa acctgaggac actgccgtgt actactgtgc ggcagtccgg 300
agaagggatg gatataagta ctggggccag gggacccagg tcaccgtctc ctcatc 356
<210> 31
<211> 353
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 31
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag gctctggaga catcagtaag gtagcttcaa tggcctggtt ccgccaggct 120
ccattgaagg agcgcgaggg ggtcgccggt ttaagtattg gtacaggtag gacatactac 180
gccgactccg tgaagggccg attcaccatc tcccgagaca acgccaagaa tacgctgtac 240
ctgcagatga acagcctgaa aactgaggac actgccatat actactgtgc gtataatgat 300
cagcgtcgtg cacggtactg gggccagggg acccaggtca ccgtctcctc atc 353
<210> 32
<211> 415
<212> PRT
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 32
Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg
1 5 10 15
Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe
20 25 30
Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly
35 40 45
Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp
50 55 60
Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys
65 70 75 80
Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu
85 90 95
Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr
100 105 110
Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val
115 120 125
Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr
130 135 140
Arg Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu
145 150 155 160
Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn
165 170 175
Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe
180 185 190
Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly
195 200 205
Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu
210 215 220
Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu
225 230 235 240
Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His
245 250 255
His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu
260 265 270
Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile
275 280 285
Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser
290 295 300
Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala
305 310 315 320
Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys
325 330 335
Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly
340 345 350
Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro
355 360 365
His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser
370 375 380
Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys
385 390 395 400
Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr
405 410 415
<210> 33
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 33
cccaccggtc aggtgcagct gcaggagtc 29
<210> 34
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial sequence
<400> 34
cccggatcct gaggagacgg tgacctgg 28
Claims (22)
1. A coagulation Factor Ix (FIX) binding molecule capable of binding FIX and comprising at least one immunoglobulin single variable domain comprising a CDR1, a CDR2 and a CDR3 selected from the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, CDR3 shown in SEQ ID NO. 6;
(3) CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO. 9;
(4) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
(5) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15; and
(6) CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17, and CDR3 shown in SEQ ID NO. 18.
2. FIX binding molecule according to claim 1, wherein said immunoglobulin single variable domain is a VHH.
3. FIX binding molecule according to claim 2, wherein the VHH comprises the amino acid sequence of any of SEQ ID NO 19-24.
4. FIX binding molecule according to any one of claims 1-3, further comprising an immunoglobulin Fc-region.
5. FIX binding molecule of claim 4, wherein the immunoglobulin Fc region is a human immunoglobulin Fc region.
6. FIX binding molecule according to claim 5, wherein the amino acid sequence of the immunoglobulin Fc-region is shown in SEQ ID NO 25.
7. FIX-binding molecule according to any of claims 1 to 3, which binds FIX with a KD-value of less than 1 x 10-7M。
8. A nucleic acid molecule encoding a FIX binding molecule of any of claims 1-7.
9. An expression vector comprising the nucleic acid molecule of claim 8 operably linked to an expression control element.
10. A host cell comprising the nucleic acid molecule of claim 8 or transformed with the expression vector of claim 9 and capable of expressing the FIX binding molecule.
11. A method of producing the FIX binding molecule of any of claims 1-7 comprising:
a) culturing the host cell of claim 10 under conditions that allow expression of the FIX binding molecule;
b) recovering the FIX binding molecule expressed by the host cell from the culture from step a); and
c) optionally further purifying and/or modifying the FIX binding molecule resulting from step b).
12. A kit for detecting the presence and/or quantifying FIX in a target sample and/or detecting the level of carboxylation of FIX in a target sample comprising a FIX binding molecule according to any one of claims 1 to 7.
13. The kit of claim 12, further comprising a control sample comprising a predetermined amount of FIX or FIX comprising a predetermined level of carboxylation.
14. A non-diagnostic method for detecting the presence of FIX in a target sample and/or quantifying FIX in a sample and/or detecting the level of carboxylation of FIX in a sample, the method comprising:
a) contacting the target sample and the control sample with a FIX binding molecule according to any of claims 1-7, respectively, under conditions enabling the formation of a complex between the FIX binding molecule and FIX;
b) detecting the formation of the complex by detecting the presence of the complex,
wherein a difference in complex formation between the target sample and the control sample is indicative of the presence and/or amount of FIX and/or its level of carboxylation in the target sample.
15. The method of claim 14, wherein the control sample contains a predetermined amount of FIX or FIX containing a predetermined level of carboxylation.
16. A method of preparing a blood sample lacking FIX comprising
a) Contacting a blood sample with FIX binding molecule of any of claims 1-7, whereby FIX in said blood sample forms a complex with said FIX binding molecule,
b) separating said complex from said blood sample, and
c) blood samples lacking FIX were harvested.
17. The method of claim 16, wherein said FIX binding molecule is immobilized on a solid support.
18. The method of claim 16 or 17, wherein the blood sample is plasma.
19. A FIX affinity chromatography medium comprising a solid support having fixed thereon a FIX binding molecule of any of claims 1-7.
20. FIX affinity chromatography media according to claim 19, wherein said solid support is made of a material selected from the group consisting of: polyethylene, polystyrene, polypropylene, polysulfone, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, cellulose acetate, cross-linked dextran, cross-linked agarose, chitin, chitosan, cross-linked dextran, cross-linked alginic acid, silicone, fluoropolymers, and magnetic media.
21. FIX affinity chromatography medium according to claim 19, which is an agarose medium or magnetic beads on which FIX binding molecules according to any of claims 1-7 are immobilized.
22. A device for preparing a blood sample lacking FIX comprising the FIX affinity chromatography medium of any one of claims 19-21.
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| CN201810305368.7A CN110343181B (en) | 2018-04-08 | 2018-04-08 | Single domain antibodies against coagulation Factor IX (FIX) |
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| CN116284422B (en) * | 2023-05-25 | 2023-07-21 | 北京和颐林生物科技有限公司 | Anti-superoxide dismutase antibody and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396494A (en) * | 2013-04-27 | 2013-11-20 | 江苏省疾病预防控制中心 | Monoclonal antibody of blood coagulation factor IX |
| CN106397592A (en) * | 2015-07-31 | 2017-02-15 | 苏州康宁杰瑞生物科技有限公司 | Single-domain antibody directed at programmed death ligand (PD-L1) and derived protein thereof |
| CN107400166A (en) * | 2016-05-19 | 2017-11-28 | 苏州康宁杰瑞生物科技有限公司 | for CTLA4 single domain antibody and its derived protein |
| CN109563154A (en) * | 2016-05-16 | 2019-04-02 | 拜科赛尔塔公司 | Anti- Padua factors IX antibody |
| CN110023339A (en) * | 2016-09-23 | 2019-07-16 | Csl有限公司 | Coagulation factor binding protein and its application |
-
2018
- 2018-04-08 CN CN201810305368.7A patent/CN110343181B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396494A (en) * | 2013-04-27 | 2013-11-20 | 江苏省疾病预防控制中心 | Monoclonal antibody of blood coagulation factor IX |
| CN106397592A (en) * | 2015-07-31 | 2017-02-15 | 苏州康宁杰瑞生物科技有限公司 | Single-domain antibody directed at programmed death ligand (PD-L1) and derived protein thereof |
| CN109563154A (en) * | 2016-05-16 | 2019-04-02 | 拜科赛尔塔公司 | Anti- Padua factors IX antibody |
| CN107400166A (en) * | 2016-05-19 | 2017-11-28 | 苏州康宁杰瑞生物科技有限公司 | for CTLA4 single domain antibody and its derived protein |
| CN110023339A (en) * | 2016-09-23 | 2019-07-16 | Csl有限公司 | Coagulation factor binding protein and its application |
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