ANTI-IL-4R SINGLE-DOMAIN ANTIBODY AND USE THEREOF
20220411519 · 2022-12-29
Inventors
Cpc classification
C07K16/2866
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
A61K47/6849
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
Abstract
Disclosed is an anti-IL-4R single-domain antibody and the use thereof. In particular, disclosed are a IL-4R single-domain antibody and a VHH chain thereof, a coding sequence encoding the above-mentioned single-domain antibody or the VHH chain thereof, a corresponding expression vector, host cells capable of expressing the single-domain antibody, and a method for producing the single-domain antibody. The single-domain antibody can specifically recognize human and marmoset IL-4R, but does not recognize mouse IL-4R, and the specificity is good; the single-domain antibody can effectively inhibit the proliferation of TF-1 cells and the activation of a pSTAT6 signaling pathway in cells.
Claims
1-10. (canceled)
11. An anti-IL-4R single-domain antibody, which comprises a VHH chain having the following 3 complementarity determining regions or CDRs: CDR1 as shown in SEQ ID No: 1, CDR2 as shown in SEQ ID No: 2, and CDR3 as shown in SEQ ID No: 3.
12. The anti-IL-4R single-domain antibody of claim 11, wherein the single-domain antibody comprises monomer, bivalent antibody, tetravalent antibody, and/or multivalent antibody.
13. The anti-IL-4R single-domain antibody of claim 11, wherein the VHH chain comprises 4 framework regions or FRs.
14. The anti-IL-4R single-domain antibody of claim 13, wherein the four framework regions are selected from the group consisting of: (a) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 6, and FR4 as shown in SEQ ID NO: 7; and (b) FR1 as shown in SEQ ID NO: 10, FR2 as shown in SEQ ID NO: 11, FR3 as shown in SEQ ID NO: 12, and FR4 as shown in SEQ ID NO: 13.
15. The anti-IL-4R single-domain antibody of claim 11, wherein the single-domain antibody comprises a VHH chain whose amino acid sequence is as shown in SEQ ID NO: 8 or 14.
16. The anti-IL-4R single-domain antibody of claim 11, wherein the single-domain antibody is selected from the group consisting of animal derived antibody, chimeric antibody, and humanized antibody.
17. The anti-IL-4R single-domain antibody of claim 11, wherein the anti-IL-4R antibody comprises two VHH chains of amino acid sequences as shown in SEQ ID NO: 8 or SEQ ID NO: 14.
18. The anti-IL-4R single-domain antibody of claim 17, wherein the two VHH chains of amino acid sequences as shown in SEQ ID NO: 8 or 14 are linked via a linker.
19. The anti-IL-4R single-domain antibody of claim 18, wherein the linker is selected from the group consisting of GGGGSGGGS (SEQ ID NO: 18).
20. The anti-IL-4R single-domain antibody of claim 11, wherein the amino acid sequence of the anti-IL-4R antibody is shown as SEQ ID NO: 16.
21. The anti-IL-4R single-domain antibody of claim 11, wherein the anti-IL-4R antibody is a tetravalent antibody whose amino acid sequence is as shown in SEQ ID NO:19.
22. An anti-IL-4R single-domain antibody Fc fusion protein, wherein the fusion antibody has a structure as shown in Formula Ia or Ib from N-terminus to C-terminus:
A-L-B (Ia);
B-L-A (Ib); wherein A is an anti-IL-4R single-domain antibody of claim 11; B is an Fc fragment of the IgG; and L is none or a flexible linker.
23. A polynucleotide encoding an anti-IL-4R single-domain antibody of claim 11, or an Fc fusion protein thereof.
24. An expression vector containing a polynucleotide of claim 23.
25. A host cell whose genome has been incorporated a polynucleotide of claim 23 or which contains an expression vector containing the polynucleotide.
26. A method for producing an anti-IL-4R single-domain antibody, which comprises the steps of: (a) cultivating the host cell of claim 25 under a condition suitable for the production of a single-domain antibody, thereby obtaining a culture containing the anti-IL-4R single-domain antibody; and (b) isolating or recovering the anti-IL-4R single-domain antibody from the culture; and (c) optionally purified and/or modified the anti-IL-4R single-domain antibody from step (b).
27. An immunoconjugate comprising: (a) an anti-IL-4R single-domain antibody of claim 11, and (b) a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, gold nanoparticles/nanorods, magnetic nanoparticles, viral coat proteins or VLP, and a combination thereof.
28. A kit comprising an anti-IL-4R single-domain antibody of claim 11 or a conjugate thereof.
29. A pharmaceutical composition which comprises an anti-IL-4R single-domain antibody of claim 11 or a conjugate thereof and a pharmaceutically acceptable carrier.
Description
DESCRIPTION OF DRAWINGS
[0137]
[0138]
[0139]
[0140]
[0141]
[0142]
[0143]
[0144]
[0145]
[0146]
[0147]
[0148]
[0149]
[0150]
[0151]
[0152]
[0153]
DETAILED DESCRIPTION OF INVENTION
[0154] After extensive and intensive researches and lots of screening, the present inventors have successfully obtained a class of anti-IL-4R single-domain antibodies. The experimental results show that the single-domain antibody of the present invention can specifically recognize IL-4R, and has good specificity. It can effectively recognize human and marmoset IL-4R, but not mouse IL-4R. It also can effectively inhibit the proliferation of the TF-1 cells and the activation of the pSTAT6 signal pathway in cells. The singe-domain antibody of the present invention is easy to generate. Based on these, the invention is completed.
[0155] Specifically, the present inventors utilized human-derived IL-4 antigen protein to immunize camels to obtain a high-quality immune single-domain antibody gene library. Then the IL-4R protein molecule was coupled to the ELISA plate to display the correct spatial structure of IL-4R protein and was used as an antigen to screen the immune single domain antibody gene library (camel heavy chain antibody phage display gene library) via phage display technology, thereby obtaining the IL-4R specific single-domain antibody gene. The gene was then transferred into mammalian cells to obtain a single domain antibody strain that could be efficiently expressed in mammalian cells and had high specificity. Thereafter, the anti-IL-4R single-domain antibody with blocking activity was identified by ELISA, flow cytometry and luciferase reporter gene detection system, etc.
Terms
[0156] As used herein, the terms “single-domain antibody of the present invention”, “anti-IL-4R single-domain antibody of the present invention”, “IL-4R single-domain antibody of the present invention”, “anti-IL-4R single-domain antibody”, and “IL-4R single-domain antibody” have the same meaning and can be used interchangeably, and each refers to a single-domain antibody that specifically recognize and bind to IL-4R (including human IL-4R). Preferably, the variable region of the single-domain antibody of the present invention has CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3. More preferably, the framework region of the single-domain antibody of the present invention has (a) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 6, and FR4 as shown in SEQ ID NO: 7; or (b) FR1 as shown in SEQ ID NO: 10, FR2 as shown in SEQ ID NO: 11, FR3 as shown in SEQ ID NO: 12, and FR4 as shown in SEQ ID NO: 13.
[0157] As used herein, the term “antibody” or “immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 Daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced disulfide bonds in the chain. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end. The constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.
[0158] As used herein, the terms “single-domain antibody”, “VHH”, “nanobody”, “single domain antibody” (sdAb, or nanobody) have the same meaning and can be used interchangeably, and refer to a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions, wherein the VHH is constructed via cloning of the variable region of an antibody heavy chain. Usually, the antibody that naturally lacks the light chain and the heavy chain constant region 1 (CH1) is obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
[0159] As used herein, the term “variable” means that certain parts of the variable region in an antibody differ in sequence, which forms the binding and specificity of various specific antibodies for their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light chain variable regions and heavy chain variable regions. The more conserved part of the variable region is called the framework region (FR). The variable regions in the natural heavy and light chains each contain four FR regions, which are roughly in the β-fold configuration, connected by the three CDRs that form the connecting loop, and in some cases part of the β-folded structure may be formed. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain to form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669) (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity involved in antibodies.
[0160] As known to those skilled in the art, immunoconjugates and fusion expression products include: conjugates formed by combining drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules with the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the anti-IL-4R antibody or fragments thereof.
[0161] As used herein, the terms “heavy chain variable region” and “VH” can be used interchangeably.
[0162] As used herein, the terms “variable region” and “complementarity determining region (CDR)” can be used interchangeably.
[0163] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
[0164] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above heavy chain variable region and heavy chain constant region.
[0165] In the present invention, the terms “antibody of the present invention”, “protein of the present invention”, or “polypeptide of the present invention” can be used interchangeably, and refer to a polypeptide that specifically binds to the IL-4R protein, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initial methionine.
[0166] The present invention further provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product having a heavy chain containing a variable region (i.e., immunoconjugate and fusion expression product), as long as the variable region is the same as the heavy chain variable region of the antibody of the present invention or has at least 90% homology with that, preferably at least 95% homology with that.
[0167] In general, the antigen-binding properties of antibodies can be described by three specific regions located in the variable region of the heavy chain, called variable regions (CDRs). The segment is divided into 4 framework regions (FRs), the amino acid sequences of the four FRs are relatively conservative, and do not directly participate in the binding reaction. These CDRs form a circular structure, and the β-pleated sheet formed by the FRs in between are close to each other in space structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
[0168] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least parts of them are involved in binding antigens. Therefore, the present invention includes those molecules having a CDRs-containing antibody heavy chain variable region, as long as their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.
[0169] The present invention includes not only whole antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
[0170] As used herein, the terms “fragment”, “derivative” and “analog” refer to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by the fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing the additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.
[0171] The antibody of the present invention refers to a polypeptide having IL-4R protein binding activity and containing the above-mentioned CDR regions. The term also includes variant forms of polypeptides containing the above CDR regions that have the same function as the antibodies of the present invention. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions, insertions and/or substitutions, and one or several (usually less than 20, preferably less than 10, and more preferably less than 5) amino acids addition to the C-terminal and/or N-terminal. For example, in the art, the substitution of amino acids with close or similar properties usually does not change the function of the protein. As another example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.
[0172] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with DNA encoding the antibody of the present invention under highly or lowly stringent conditions, and polypeptides or proteins obtained using antiserum against antibodies of the present invention.
[0173] The present invention further provides other polypeptides, such as fusion proteins comprising single-domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of single-domain antibodies of the present invention. Generally, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.
[0174] In the present invention, “conservative variant of the antibody of the present invention” refers to that compared with the amino acid sequence of the antibody of the present invention, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or close properties to form a polypeptide. These conservative variant polypeptides are best produced by amino acid substitution according to Table 1.
TABLE-US-00001 TABLE 1 The initial Preferred residues Representative substitution substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu
[0175] The present invention further provides polynucleotide molecules encoding the above antibodies or fragments thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.
[0176] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence encoding the mature polypeptide with various additional coding sequences; a coding sequence encoding the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.
[0177] The term “polynucleotide encoding a polypeptide” may include a polynucleotide encoding the polypeptide, or a polynucleotide further containing additional coding and/or non-coding sequences.
[0178] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity to the above-mentioned sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2) denaturing agent, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42° C., etc. is added during hybridization; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, and more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
[0179] The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can generally be obtained by PCR amplification method, recombination method or artificial synthesis method. A feasible method is to use synthetic methods to synthesize the relevant sequences, especially when the fragment length is short. Generally, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
[0180] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
[0181] At present, the DNA sequence encoding the protein (or a fragment or a derivative thereof) of the present invention can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
[0182] The present invention also relates to vectors containing the appropriate DNA sequence as described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins. The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COST, 293 cells, etc.
[0183] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl.sub.2) method. The procedures used are well known in the art. Another method is to use MgCl.sub.2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
[0184] The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture can be selected from various conventional mediums. The cultivation is carried out under conditions suitable for the growth of host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
[0185] The recombinant polypeptide in the above method may be expressed in a cell or on a cell membrane, or secreted out of the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, disruption of bacteria through penetration, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
[0186] The antibody of the present invention may be used alone, or may be combined or coupled with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modified portion, or a combination or coupling of any of above these substances.
[0187] Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electronic computer X-ray tomography) contrast agents, or an enzyme capable of producing a detectable product.
[0188] Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxin; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. magnetic nanosphere; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), etc.
[0189] Interleukin-4 (IL-4)
[0190] Interleukin-4 (IL-4, also known as B cells stimulating factor or BSF-1) is a cytokine mainly produced by activated T cells, monocytes, basophils, mast cells, and eosinophils. IL-4 can respond to low concentration of antibodies against the surface immunoglobulin and stimulate the proliferation of B cells. IL-4 has been shown to have a wide range of biological activities, including stimulating the growth of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. IL-4 induces the expression of class II major histocompatibility complex molecules in resting B cells and enhances the secretion of IgE and IgG1 isotypes in stimulated B cells.
[0191] Interleukin-4 Receptor α (IL-4Rα)
[0192] The human IL-4Rα subunit is a 140 kDa type I membrane protein that binds human IL-4 with high affinity. Il-4Rα is expressed in low quantities in many cell types, such as peripheral blood T cells, monocytes, airway epithelial cells, B cells, and lung fibroblasts. Interleukin-4 receptor α subunit (IL-4Rα) locates in chromosome 16P12.1-PI1.2 region, which is also an asthma susceptibility region. Gene polymorphisms in this region are associated with hypersensitivity and elevated serum IgE levels. In addition, IL-4Rα is a common component of the IL-4 and IL-13 gene receptor complex. Studies have shown that IL-4Rα gene polymorphisms are associated with asthma, elevated serum IgE levels, and atopic dermatitis.
[0193] Pharmaceutical Composition
[0194] The present invention further provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above antibody or an active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the substance being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.
[0195] The pharmaceutical composition of the present invention can be directly used to bind IL-4R protein molecules, and thus can be used to treat asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, etc. In addition, other therapeutic agents can be used simultaneously.
[0196] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above single domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerin, ethanol, and a combination thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by a conventional method using a physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. In addition, the polypeptide of the present invention can be used together with other therapeutic agents.
[0197] When using a pharmaceutical composition, a safe and effective amount of an immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is about from 10 μg/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of skilled physicians.
[0198] Anti-IL-4R Single-Domain Antibody
[0199] In the present invention, the anti-IL-4R single-domain antibody includes monomer, bivalent antibody, tetravalent antibody, and/or multivalent antibody.
[0200] In a preferred embodiment of the present invention, the anti-IL-4R single-domain antibody comprises one, two or more VHH chains of amino acid sequence as shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
[0201] Typically, the anti-IL-4R single-domain antibody comprises two VHH chains of amino acid sequence as shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
[0202] In another preferred embodiment, the anti-IL-4R single-domain antibody comprises four VHH chains of amino acid sequence as shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
[0203] Typically, the anti-IL-4R single-domain antibody comprises VHH chains of amino acid sequence as shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
[0204] Typically, the anti-IL-4R single-domain antibody comprises two VHH chains of amino acid sequence as shown in SEQ ID NO: 14.
[0205] Typically, the anti-IL-4R single-domain antibody comprises four VHH chains of amino acid sequence as shown in SEQ ID NO: 14.
[0206] In a preferred embodiment of the present invention, the two VHH chains containing amino acid sequence as shown in SEQ ID NO: 8 are linked via a linker.
[0207] In a preferred embodiment of the present invention, the two VHH chains containing amino acid sequence as shown in SEQ ID NO: 14 are linked via linkers.
[0208] In a preferred embodiment of the present invention, the linker is selected from the following sequences: (G.sub.aS.sub.b).sub.x-(G.sub.mS.sub.n).sub.y, wherein each of a, b, m, n, x, and y is 0 or 1 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (more preferably, a=4, while b=1, and m=3 while n=1).
[0209] In a preferred embodiment of the present invention, the linker is selected from the group consisting of GGGGSGGGS (SEQ ID NO: 18).
[0210] In a preferred embodiment of the present invention, the amino acid sequence of the bivalent anti-IL-4R single-domain antibody is shown as SEQ ID NO: 16.
[0211] In a preferred embodiment of the present invention, the bivalent anti-IL-4R single-domain antibody is shown as SEQ ID NO: 19.
[0212] Labeled Single-Domain Antibody
[0213] In a preferred embodiment of the present invention, the single-domain antibody has a detectable label. More preferably, the label is selected from the group consisting of: isotopes, colloidal gold labels, colored labels or fluorescent labels.
[0214] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-IL-4R single-domain antibody is labeled with colloidal gold to obtain a colloidal gold labeled single-domain antibody.
[0215] The new anti-IL-4R single-domain antibody of the present invention has good specificity and high titer.
[0216] Detection Method
[0217] The present invention further relates to a method for detecting IL-4R protein. The steps of the method are roughly as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; and detecting the level of IL-4R protein in the dissolved sample.
[0218] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
[0219] Kit
[0220] The present invention further provides a kit containing the antibody (or a fragment thereof) or a detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction for use, a buffer, and the like.
[0221] The present invention further provides a detection kit for detecting the level of IL-4R, which includes an antibody that recognizes the IL-4R protein, a lysis medium for dissolving the sample, general reagents and buffers required for the detection, such as various buffers, detection markers, detection substrates, etc. The detection kit may be an in vitro diagnostic device.
[0222] Application
[0223] As described above, the single-domain antibody of the present invention has a wide range of biological application value and clinical application value, and its application involves the diagnosis and treatment of IL-4R-related diseases, basic medical research, biological research and other fields. A preferred application is for clinical diagnosis and targeted therapy for IL-4R.
[0224] The main advantages of the present invention include:
[0225] (a) The single-domain antibody of the present invention specifically binds to IL-4R protein with correct spatial structure.
[0226] (b) The single-domain antibody of the present invention can recognize the human and marmoset IL-4R, but not mouse IL-4R.
[0227] (c) The single-domain antibody of the present invention has a stronger binding and blocking activity than that of the commercially available monoclonal antibody Dupilumab.
[0228] (d) The single-domain antibody of the present invention has a good inhibitory effect on the proliferation of TF-1 cell, which is superior to that of the commercially available monoclonal antibody Dupilumab.
[0229] (e) The single-domain antibody of the present invention can effectively inhibit the activation of pSTAT6 signal pathway in cells, which is superior to that of the commercially available monoclonal antibody Dupilumab.
[0230] (f) The production of the single-domain antibody of the present invention is simple.
[0231] The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples without particular conditions, they are performed under routine conditions (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual (third edition) (2001, CSHL Press) or as instructed by the manufacturer. Unless otherwise specified, all percentages or parts are by weight.
Example 1: Screening and Expression of Anti-IL-4R Single-Domain Antibody
[0232] In order to obtain a single-domain antibody specific for human IL-4R, firstly, human IL-4R protein was transiently expressed by a mammalian cell HEK293F, and then used for immunization in a camel after affinity purification. For specific methods, please refer to the method described in Example 1 and Example 2 of the patent CN2018101517526. Briefly, one Xinjiang Bactrian camel was immunized with purified IL-4R protein. Total RNA was isolated from camel peripheral blood after 7 times of immunization. Then VHH gene was amplified by reverse transcription and PCR, and cloned into phage vector pMECS, and transformed into TG1 to construct the phage display library. The capacity of the constructed library capacity is 1.1×10.sup.9 CFU as shown in
Example 2: Screening of Anti-IL-4R Single-Domain Antibody with Blocking Activity by FACS
[0233] (1) A certain number of 293F/IL-4R cells that were transitioned for 48 h were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were re-suspended and washed with PBS for once, and counted. The cells were divided into 96-well plates at 100 ul/well, and then centrifuged at 3000 rpm at 4° C. for 4 min. (2) The supernatant was aspirated, and the single-domain antibody with different sequences in gradient diluted in example 1 was added (two-fold gradient dilution starting from 40 ug/mL). 50 uL was added to each well, and then 50 uL of 5 ug/mL IL-4-biotin was added to mix, and incubated at 4° C. for 20 min. (3) Centrifugation was performed at 3000 rpm at 4° C. for 4 min, and the supernatant was discarded. 200 uL PBS was added to each well to wash the cells, and the supernatant was discarded by centrifugation again. Diluted SA-PE staining solution was added and incubated at 4° C. for 20 min. (4) Centrifugation was performed at 3000 rpm at 4° C. for 4 min, and the supernatant was discarded. 200 uL PBS was added to each well to wash the cells, and the supernatant was discarded by centrifugation again. 200 uL PBS was added to re-suspend the cells, and the PE signal of the samples was detected by flow cytometry. The results are shown in
Example 3: The Proliferation Inhibitory Effect on the TF-1 Cell of the Candidate Single-Domain Antibody by FACS
[0234] The detection method of the proliferation inhibitory effect on the TF-1 cell induced by IL-4 of the candidate single-domain antibody was as follows: (1) A certain number of TF-1 cells were taken, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. PBS was added to resuspend the cells, and the cells were centrifuged at 1000 rpm for 5 min and washed once. After that, an appropriate amount of PBS was added to resuspend and count the cells, and the concentration of the cell solution was diluted to 6×10.sup.5/mL, and the cells were divided into 96-well plates. (2) 50 uL gradient diluted (three times gradient dilution starting from 20 ug/mL) IgG1 (negative control), Dupilumab (positive control) and IL-4Ra Nb103 antibody were mixed with 50 uL 200 ng/mL IL-4 protein, and the collected cells were resuspended. At the same time, 100 uL PBS was added to all wells around the cells to prevent evaporation of the solution in the wells containing cells. (4) The cells were cultured in the incubator for 3 days. After 3 days, the cells were taken out and added with CCK8 solution at 10 uL/well. The cells were placed at 37° C. for 4 h for developing. (5) After developing, the OD450 of each well was read with a microplate reader. The results are shown in
[0235] The detection method of the inhibitory effect of candidate single-domain antibody on the proliferation of TF-1 cells induced by IL-13 was the same as above. The diluted antibody to be tested was mixed with IL-13 at a concentration of 10 ng/mL, and added into the cells. The results of detection are shown in
Example 4: The Species Specificity of Candidate Single-Domain Antibody Detected by ELISA
[0236] ELISA was used to verify whether the candidate antibody could cross-react with IL-4R proteins of different species, and the method was as follows: (1) 1 ug/mL human IL-4R, mouse IL-4R and cynomolgus monkey IL-4R were added into the plate and coated overnight at 4° C. with 100 uL/well. (2) After washing with PBST for 5 times, 300 uL 1% BSA was added to each well and sealed for 2 hours at room temperature. (3) After washing with PBST for 5 times, 100 uL of 2 ug/mL antibody to be tested was added and incubated at 37° C. for 1 hour. (4) After washing with PBST for 5 times, 100 uL diluted anti-HA antibody (diluted at 1:2000) was added and incubated at 37° C. for 1 hour. (4) After washing with PBST for 5 times, 100 uL diluted anti-mouse IgG antibody (diluted at 1:2000) was added and incubated at 37° C. for 30 minutes. (5) After washing with PBST for 5 times, 100 uL PA chromogenic solution was added and developing at 37° C. for 10 minutes, and the absorption value was measured at 405 nm wavelength with a microplate reader. The results are shown in
[0237] ELISA was used again to verify whether the candidate antibody could cross-react with IL-4R protein of different monkey types. The detection method was the same as above. The results are shown in
Example 5: Humanization of Candidate Single-Domain Antibody
[0238] The candidate antibody was humanized wherein the variable region was kept unchanged, and humanized design was carried out for the sequence of the four framework regions. The huamanization method refers to the method of Example 4 in patent application CN2018101517526. Then, the humanized antibody sequence was constructed on pFUSE vector to fuse the humanized single-domain antibody with Fc sequence and to form huNb103 (the fusion protein sequence refers to SEQ ID NO:16, and the encoding nucleotide sequence thereof refers to SEQ ID NO:17), and expressed by HEK293F system. The expressed protein could be used for subsequent verification. The expression method refers to Example 3 in patent application CN2018101517526. The humanized antibody sequences are as shown in the following Table 2:
TABLE-US-00002 TABLE 2 Sequence number (SEQ ID NO:) Antibody region Before humanization After humanization FR1 4 10 CDR1 1 1 FR2 5 11 CDR2 2 2 FR3 6 12 CDR3 3 3 FR4 7 13 Complete amino acid 8 14 sequence Complete nucleotide 9 15 sequence
Example 6: Binding Activity of the Humanized Antibody Detected by FACS
[0239] (1) HEK293F cells with high IL-4Rα expression were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were re-suspended with 5 mL PBS and centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then the cells were re-suspended with 2 mL PBS, and counted. The cells were divided into 96-well plates with 3×10.sup.5 cells per well; (2) Humanized antibody huNb103 (amino acid sequence refers to SEQ ID NO:16) and Dupilumab were diluted by 2-fold gradient dilution (40 ug/mL, 20 ug/mL, 10 ug/mL, 5 ug/mL, 2.5 ug/mL, 1.25 ug/mL, 0.625 ug/mL, 0.313 ug/mL, 0.156 ug/mL, 0.078 ug/mL). The cells in the 96-well plate were resuspended with the diluted antibody and incubated at 4° C. for 20 min. (3) Centrifugation was performed at 3000 rpm at 4° C. for 4 min, and 200 ul PBS was added to each well. After resuspension, centrifugation was performed at 3000 rpm at 4° C. for 4 min. (4) Diluted anti-human Fc-FITC antibody (diluted at 1:200) was added and incubated at 4° C. for 20 min. (5) Centrifugation was performed at 3000 rpm at 4° C. for 4 min, and the supernatant was discarded, 200 uL PBS was added into each well to wash cells for two times, then 200 uL PBS was added to re-suspend the cells. The cells were transferred to flow tube to detect FITC signal of each sample via flow cytometry.
[0240] The results are shown in
Example 7: Blocking Activity of the Humanized Antibody Detected by FACS
[0241] (1) HEK293F cells with high IL-4Rα expression were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were re-suspended with 5 mL PBS and centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then the cells were re-suspended with 2 mL PBS, and counted. The cells were divided into 96-well plates with 3×10.sup.5 cells per well. (2) Humanized antibody huNb103 (amino acid sequence refers to SEQ ID NO:16) and Dupilumab were diluted by 2-fold gradient dilution (40 ug/mL, 20 ug/mL, 10 ug/mL, 5 ug/mL, 2.5 ug/mL, 1.25 ug/mL, 0.625 ug/mL, 0.313 ug/mL, 0.156 ug/mL, 0.078 ug/mL). The cells in the 96-well plate were resuspended with the diluted antibody and incubated at 4° C. for 20 min. (3) Centrifugation was performed at 3000 rpm at 4° C. for 4 min, and 200 ul PBS was added to each well. After resuspension, centrifugation was performed at 3000 rpm at 4° C. for 4 min. (4) Diluted SA-PE staining solution (diluted at 0.3:100) was added and incubated at 4° C. for 20 min. (5) Centrifugation was performed at 3000 rpm at 4° C. for 4 min, and the supernatant was discarded, 200 uL PBS was added into each well to wash cells for two times, then 200 uL PBS was added again to re-suspend the cells. The cells were transferred to flow tube to detect PE signal of each sample via flow cytometry.
[0242] The results are shown in
Example 8: Construction and Preparation of Humanized Tetravalent Antibody
[0243] In order to further improve the activity of the antibody, the multivalent antibody was designed for preparation. The structure of the constructed tetravalent single-domain antibody is shown in
Example 9: Inhibitory Effect of Humanized Tetravalent Antibody on TF-1 Cell Proliferation
[0244] (1) The resuscitated TF-1 cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with 5 mL PBS, and centrifuged at 1000 rpm for 5 min. The cells were resuspended with 5 mL PBS and counted. The concentration of cell solution was diluted to 6×10.sup.5/mL, and divided into a 96-well plate at 60 uL/well. (2) After 50 uL of IL-4Ra antibody in gradient dilution (50 ug/mL, 12.5 ug/mL, 3.125 ug/mL, 0.781 ug/mL, 0.195 ug/mL, 0.049 ug/mL, 0.012 ug/mL, 0.003 ug/mL) were mixed with 50 uL of 5 ng/mL IL-4 protein respectively, 40 uL of the each mixture was taken out and added to the cell solution. (3) At the same time, 200 uL PBS was added to all wells around the cells to prevent evaporation of the solution in the wells containing cells, and cultured at 37° C. in a 5% CO2 incubator for 72 h. (4) The cell culture plate was taken out, and added with 10 uL CCK8 solution to the wells, then placed at 37° C. for 4 h for developing. (5) After developing, OD450 value of each well was read with a microplate reader.
[0245] The results are shown in
[0246] Similarly, the inhibitory effect on the proliferation of TF-1 cells induced by IL-13 of the tetravalent antibody is also significant. The concentration of IL-13 added during detection was 10 ng/mL, and the other steps were the same as above. The results are shown in
Example 10: Detection of the Inhibitory Effect of Humanized Tetravalent Antibody on pSTAT6 Signal Pathway in HEK-Blue™ IL-4/IL-13 Cells
[0247] (1) HeK-IL-4/IL13™ cells were cultured at 37° C. with 5% CO2. After growing to a certain number, the cells were collected, washed with 20 mL PBS twice, and then resuspended with 10 mL DMEM complete medium. 50 uL Trypan blue staining solution and 50 uL cell solution were mixed for cell counting. The concentration of HEK-IL-4/IL-13™ cell solution was diluted to 3×10.sup.5/mL, and the cells were divided into a 96-well culture plate at 160 uL/well. (2) The antibody to be tested in gradient dilution (40 ug/mL, 10 ug/mL, 0.625 ug/mL, 0.156 ug/mL, 0.039 ug/mL, 0.010 ug/mL, 0.002 ug/mL) was mixed with an equal volume of 5 ng/mL IL-4 protein respectively. (3) 40 uL of the mixture was added to the packed cell plate, and 200 uL of PBS was added to the all wells around the cells to prevent evaporation of the solution in the wells containing cells. Incubation was performed at 37° C. in a 5% CO2 incubator for 22 h. (4) Quanti-Blue reagent was added to the 96-well plate, 180 uL/well, and then 20 uL cultured cell supernatant was added and incubated at 37° C. for 2 h. The reading value at 655 nm was detected by a microplate reader.
[0248] The results are shown in
[0249] Similarly, humanized tetravalent antibodies significantly inhibit the pSTAT6 signaling pathway in HEK-Blue™ IL-4/IL-13 cells induced by IL-13. The concentration of IL-13 added during detection was 10 ng/mL, and the other steps were the same as above. The result is shown in
Example 11: The Efficacy of Humanized Tetravalent Antibody Verified by HIL4/hIL4Ra Transgenic Mouse OVA Model
[0250] According to body weight, fifteen animals were randomly divided into 3 groups with 5 animals in each group. Group 1 was negative control group, group 2 was low dose group of the test article (5 mpK), and group 3 was high dose group of the test article (25 mpk). The animals in groups 1-3 underwent OVA sensitization and administration: 200 μg/mL OVA was prepared and injected intraperitoneally, 200 μL per animal. The sensitization was performed on days 0, 7 and 14. On days 21-25, 2% OVA was atomized for 30 min each time for 5 consecutive days. The test article was administrated on days 20 and 23, and samples were collected for analysis on the next day after the stimulation operation on day 25.
[0251] The results are shown in
[0252] As shown in
[0253] All literatures mentioned in the present application are incorporated by reference herein, as though individually incorporated by reference. Additionally, it should be understood that after reading the above teaching, many variations and modifications may be made by the skilled in the art, and these equivalents also fall within the scope as defined by the appended claims.
TABLE-US-00003 Sequence information of the present invention: SEQ ID NO: 1 GSTSYRYCMA SEQ ID NO: 2 IRPRSGRA SEQ ID NO: 3 AASDNDGNCQDY SEQ ID NO: 4 QVQLQESGGGSVQAGGSLRVSCAAS SEQ ID NO: 5 WFRQAPGKEREAVAS SEQ ID NO: 6 YYADSVKGRFTISLDNAKNTLYLQMNSLKPEDTAMY SEQ ID NO: 7 YCWGKGTQVTVSS SEQ ID NO: 8 QVQLQESGGGSVQAGGSLRVSCAASGSTSYRYCMA WFRQAPGKEREAVASIRPRSGRAYYADSVKGRFTI SLDNAKNTLYLQMNSLKPEDTAMYYCAASDNDGNC QDYWGKGTQVTVSS SEQ ID NO: 9 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGT GCAGGCTGGAGGGTCTCTGAGAGTCTCCTGTGCAG CCTCTGGATCCACCTCCTATAGATACTGTATGGCC TGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGC GGTCGCATCCATTCGCCCACGTAGTGGTAGGGCAT ACTATGCCGACTCCGTGAAGGGCCGATTCACCATC TCCCTAGACAACGCCAAGAACACGCTGTATCTGCA AATGAACAGTCTGAAACCTGAGGACACTGCCATGT ACTACTGTGCGGCCTCCGACAACGACGGTAATTGC CAGGACTACTGGGGCAAAGGAACCCAGGTCACCGT CTCCTCA SEQ ID NO: 10 EVQLVESGGGLVQPGGSLRLSCAAS SEQ ID NO: 11 WFRQAPGKGLEAVAS SEQ ID NO: 12 YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YC SEQ ID NO: 13 WGKGTLVTVSS SEQ ID NO: 14 EVQLVESGGGLVQPGGSLRLSCAASGSTSYRYCMA WFRQAPGKGLEAVASIRPRSGRAYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAASDNDGNC QDYWGKGTLVTVSS SEQ ID NO: 15 CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGT GCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCG CCAGCGGCAGCACCAGCTACAGGTACTGCATGGCC TGGTTCAGGCAGGCCCCCGGCAAGGGCCTGGAGGC CGTGGCCAGCATCAGGCCCAGGAGCGGCAGGGCCT ACTACGCCGACAGCGTGAAGGGCAGGTTCACCATC AGCAGGGACAACAGCAAGAACACCCTGTACCTGCA GATGAACAGCCTGAGGGCCGAGGACACCGCCATGT ACTACTGCGCCGCCAGCGACAACGACGGCAACTGC CAGGACTACTGGGGCAAGGGCACCCTGGTGACCGT GAGCAGC (SEQ ID NO: 16 = SEQ ID NO: 14 Fc fragment) SEQ ID NO: 16 EVQLVESGGGLVQPGGSLRLSCAASGSTSYRYCMA WFRQAPGKGLEAVASIRPRSGRAYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAASDNDGNC QDYWGKGTLVTVSSESKYGPPCPPCPAPEFLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 17 (nucleotide sequence encoding huNb103) GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGT GCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCG CCTCCGGCTCCACCTCCTACAGGTACTGCATGGCC TGGTTCAGGCAGGCCCCCGGCAAGGGCCTGGAGGC CGTGGCCTCCATCAGGCCCAGGTCCGGCAGGGCCT ACTACGCCGACTCCGTGAAGGGCAGGTTCACCATC TCCAGGGACAACTCCAAGAACACCCTGTACCTGCA GATGAACTCCCTGAGGGCCGAGGACACCGCCGTGT ACTACTGCGCCGCCTCCGACAACGACGGCAACTGC CAGGACTACTGGGGCAAGGGCACCCTGGTGACCGT GTCCTCCGAGTCCAAGTACGGCCCCCCCTGCCCCC CCTGCCCCGCCCCCGAGTTCCTGGGCGGCCCCTCC GTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCT GATGATCTCCAGGACCCCCGAGGTGACCTGCGTGG TGGTGGACGTGTCCCAGGAGGACCCCGAGGTGCAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAA CGCCAAGACCAAGCCCAGGGAGGAGCAGTTCAACT CCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTG CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG CAAGGTGTCCAACAAGGGCCTGCCCTCCTCCATCG AGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGG GAGCCCCAGGTGTACACCCTGCCCCCCTCCGAGGA GATGACCAAGAACCAGGTGTCCCTGACCTGCCTGG TGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAG TGGGAGTCCAACGGCCAGCCCGAGAACAACTACAA GACCACCCCCCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTGTACTCCAGGCTGACCGTGGACAAGTCC AGGTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGT GATGCACGAGGCCCTGCACAACCACTACACCCAGA AGTCCCTGTCCCTGTCCCTGGGC SEQ ID NO: 18 GGGGSGGGS (SEQ ID NO: 19 = SEQ ID NO: 14 + SEQ ID NO: 18 + SEQ ID NO: 14 + Fcg fragment) SEQ ID NO: 19 EVQLVESGGGLVQPGGSLRLSCAASGSTSYRYCMA WFRQAPGKGLEAVASIRPRSGRAYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAASDNDGNC QDYWGKGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGSTSYRYCMAWFRQAPGKGLEA VASIRPRSGRAYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAASDNDGNCQDYWGKGTLVTV SSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG SEQ ID NO: 20 GAAGTGCAGCTGGTGGAGTCTGGAGGCGGCCTGGT GCAGCCTGGAGGCTCTCTGAGACTGTCTTGTGCTG CCTCTGGCAGCACATCTTACAGATACTGCATGGCC TGGTTCAGACAGGCTCCTGGCAAGGGCCTGGAGGC CGTGGCCTCTATCAGACCTAGATCTGGAAGGGCCT ACTATGCTGACAGCGTGAAGGGCAGGTTCACAATC TCTAGAGATAACTCTAAGAACACTCTGTACCTGCA GATGAACTCTCTGAGAGCAGAGGATACCGCCGTGT ACTACTGTGCCGCCAGCGATAACGATGGAAACTGT CAGGATTATTGGGGCAAGGGAACACTGGTGACAGT GTCTAGCGGCGGAGGCGGAAGCGGCGGCGGAAGCG AGGTGCAGCTGGTGGAGTCCGGAGGCGGCCTGGTG CAGCCAGGAGGCAGCCTGAGACTGAGCTGCGCCGC CAGCGGCAGCACATCTTACAGGTACTGCATGGCCT GGTTTAGGCAGGCTCCAGGAAAGGGCCTGGAGGCC GTGGCCAGCATTAGACCCAGGAGCGGCAGAGCTTA CTACGCCGACTCTGTGAAGGGCAGATTCACCATCA GCAGAGATAACAGTAAGAACACCCTGTACCTGCAG ATGAATAGCCTGAGAGCTGAGGATACCGCTGTGTA TTACTGTGCTGCCTCTGACAACGACGGCAACTGTC AGGATTACTGGGGAAAGGGCACACTGGTGACAGTG TCTTCTGAGTCTAAGTACGGCCCACCTTGTCCTCC TTGCCCTGCCCCCGAGTTTCTGGGAGGCCCATCTG TGTTTCTGTTCCCTCCTAAGCCTAAGGACACACTG ATGATTTCTAGAACACCTGAGGTGACTTGTGTGGT GGTGGACGTGAGCCAGGAGGACCCTGAGGTGCAGT TTAACTGGTACGTGGACGGCGTGGAGGTGCACAAC GCCAAGACCAAGCCTAGAGAGGAGCAGTTCAACAG CACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGC ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTGAGCAACAAGGGACTGCCATCTAGCATCGA GAAGACCATCTCTAAGGCCAAGGGCCAGCCAAGAG AGCCACAGGTGTACACCCTGCCTCCTTCTGAGGAG ATGACCAAGAACCAGGTGTCTCTGACCTGTCTGGT GAAGGGCTTCTACCCTAGCGACATCGCCGTGGAGT GGGAGTCCAACGGCCAGCCTGAGAACAACTACAAG ACAACCCCACCTGTGCTGGATAGCGACGGCAGCTT CTTTCTGTACAGCAGACTGACCGTGGATAAGTCTA GGTGGCAGGAGGGAAACGTGTTTAGCTGTTCTGTG ATGCACGAGGCCCTGCACAACCACTACACACAGAA GAGCCTGTCTCTGAGCCTGGGC