KR20220131717A - A MULTIVALENT ANTIBODY SPECIFIC FOR BROAD SPECTRUM OF Neisseria gonorrhoeae AND USE OF THE SAME - Google Patents

Abstract

The present invention relates to a gonorrhoeae-specific multivalent antibody or a functional fragment thereof, which comprises: a single side chain variable fragment (scFv) including a heavy chain and a light chain in the direction from the N-terminus to the C-terminus, wherein the heavy chain includes a CDR-H1 region represented by SEQ ID NO: 1, a CDR-H2 region represented by SEQ ID NO: 2, and a CDR-H3 region represented by SEQ ID NO: 3, and the light chain includes a CDR-L1 region represented by SEQ ID NO: 4, a CDR-L2 region represented by SEQ ID NO: 5, and a CDR-L3 region represented by SEQ ID NO: 6; a hinge portion; heavy chain no-fragment region 2 (CH_2); heavy chain no-fragment region 3 (CH_3); and a single side chain variable fragment (scFv) including a heavy chain and a light chain wherein the heavy chain includes a CDR-H1 region represented by SEQ ID NO: 7, a CDR-H2 region represented by SEQ ID NO: 8, and a CDR-H3 region represented by SEQ ID NO: 9, and the light chain includes a CDR-L1 region represented by SEQ ID NO: 10, a CDR-L2 region represented by No. 11 and a CDR-L3 region represented by SEQ ID NO: 12. A gonorrhoeae-specific multivalent antibody or a functional fragment thereof can be used as an important basic material capable of diagnosing, treating, and preventing the infection of the gonorrhoeae, rapidly and correctly.

Description

Gonorrhea-specific multivalent antibody that specifically binds to various gonorrhea and its application

The present invention relates to a gonorrhea-specific multivalent antibody specifically binding to various gonorrhea and its application.

Gonorrhea is one of the sexually transmitted diseases caused by Neisseria gonorrhoeae (Neisseria gonorrhoeae) gram-negative bacteria. More than 78 million new patients are infected every year, and strains that are particularly resistant to the third-generation antibiotics of the cephalosporin class are emerging. The number of cases of treatment failure is increasing. This is currently a major global public health problem as treatment options for the treatment of gonorrhea are limited.

Therefore, there is a need for research on the development of fast and accurate diagnostic products that can diagnose, manage and control gonorrhea at an early stage. to be.

In the prior art, the diagnosis of gonorrhea gonorrhea (gonococcus) is mainly performed by culturing a patient sample or by gram staining and gene amplification. If a large number of characteristic Gram-negative dicocci are observed in neutrophils by Gram staining, it can be diagnosed according to the clinical picture. Molecular diagnostic methods (PCR and multiplex PCR), which are currently widely used, require time and equipment to culture and PCR methods, so a highly reliable antibody diagnostic method is required.

However, the immunological diagnostic method has disadvantages in that it has low specificity for Gonorrhea and cross-reactivity with other Nigerian species (eg, Nigeria meningitidis), which requires additional testing. Therefore, there is a need for an antibody that has high sensitivity and that specifically binds to gonorrhea.

Currently, in clinical practice, antibiotic resistance is determined based on the minimal inhibitory concentration (MIC) in which the growth of gonococci in a culture medium containing a specific antibiotic is suppressed. A detectable immunochromatography-based diagnostic product is not commercially available.

In addition, as a solution to the current antibiotic-resistant strain, new antibacterial agents such as solithromycin (CEM-101), gepotidacin (GSK2140944), and zoliflodacin (AZE0914/ETX0914) are in the clinical development stage. However, there is a need to develop a detection method capable of rapidly detecting Gonorrhea bacteria as well as a therapeutic agent and vaccine for Gonorrhea.

In particular, an antibody for applying an immunochemical diagnostic method to POCT should show high affinity for antigen and binding sensitivity to a wide variety of strains in order to facilitate rapid diagnosis.

[Prior Patent Literature]

Korean Patent Publication No. 2001-0072121 (20010731)

The present invention was devised in response to the above needs, and an object of the present invention is to provide a gonorrhea-specific antibody having high affinity for antigen and binding sensitivity to various and extensive strains in order to facilitate rapid diagnosis.

Another object of the present invention is to provide a method for producing a gonorrhea-specific antibody having a high affinity for an antigen and binding sensitivity to a diverse and wide range of strains to facilitate rapid diagnosis.

Another object of the present invention is to provide a kit for diagnosing gonorrhea, which has high affinity for antigen and binding sensitivity to various and extensive strains in order to facilitate rapid diagnosis.

In order to achieve the above object, the present invention is from the N-terminus to the C-terminal direction,

A heavy chain comprising a CDR-H1 region set forth in SEQ ID NO: 1, a CDR-H2 region set forth in SEQ ID NO: 2 and a CDR-H3 region set forth in SEQ ID NO: 3 and a CDR-L1 region set forth in SEQ ID NO: 4, SEQ ID NO: a single side chain variable fragment (scFv) comprising a light chain comprising a CDR-L2 region set forth in SEQ ID NO: 5 and a CDR-L3 region set forth in SEQ ID NO: 6;

hinge region;

heavy chain constant region 2 (CH ₂ );

heavy chain constant region 3 (CH ₃ );

A heavy chain comprising a CDR-H1 region set forth in SEQ ID NO: 7, a CDR-H2 region set forth in SEQ ID NO: 8 and a CDR-H3 region set forth in SEQ ID NO: 9 and a CDR-L1 region set forth in SEQ ID NO: 10, SEQ ID NO: A single side chain variable fragment (scFv) comprising a light chain comprising a CDR-L2 region set forth in 11 and a CDR-L3 region set forth in SEQ ID NO: 12

Provided is a gonorrhea-specific multivalent antibody or functional fragment thereof.

In one embodiment of the present invention,

The antibody preferably includes a disulfide bond linker, but is not limited thereto.

In another embodiment of the present invention,

The functional fragment is preferably a single side chain variable fragment (scFv), but is not limited thereto.

In another embodiment of the present invention,

The antibody preferably includes one of the amino acid sequences set forth in SEQ ID NOs: 13 to 17, but is not limited thereto.

Hereinafter, the present invention will be described.

The present invention is a follow-up patent to the Korean Patent Application No. 10-2018-0008883 of the inventors of the present invention registered as a domestic patent of the inventor of the present invention,

Antibodies for practical application to POCT to which immunochemical diagnostic methods are applied should show high affinity for antigens and binding sensitivity to various and extensive strains in order to facilitate rapid diagnosis.

As can be seen from FIG. 7a, clone 1 and clone 4, which were the most effective in the patent application No. 10-2018-0008883, showed binding to a wide range of clinical isolates of Gonorrhea, but were analyzed as having different epitopes. (FIG. 7A, Table 3), as can be seen from FIG. 8, a multivalent maxibody with high sensitivity and specificity for gonorrhea was prepared by not showing binding force to some clinical isolates of Gonorrhea.

Hereinafter, terms used in the present invention will be described.

As used herein, the term "antibody" includes immunoglobulin molecules having immunological reactivity with a specific antigen, and is a concept including both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and double-binding antibodies (eg, bispecific antibodies).

As used herein, the term "monoclonal antibody" is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Unlike polyclonal antibodies, which typically include different antibodies directed against different epitopes (epitopes), monoclonal antibodies are directed against a single determinant on an antigen.

Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage of not being contaminated by other immunoglobulins because they are synthesized by hybridoma culture.

Typically, immunoglobulins have heavy and light chains, each heavy and light chain comprising a constant region and a variable region (these regions are also known as “domains”). The variable regions of the light and heavy chains comprise three variable regions called "complementarity determining regions" (denoted as "CDRs") and four "framework regions". The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain (chain) are typically called CDR1, CDR2, CDR3 sequentially, starting from the N-terminus, and are also identified by the chain in which the specific CDR is located.

The term "phage display technology" used in the present invention refers to a technology for selecting scFvs exhibiting antigen-binding ability by selecting only phages exhibiting significant binding ability with a target antigen from a phage library.

In the above technique, "panning" refers to a peptide having the property of binding to a target molecule (antibody, enzyme, cell surface receptor, etc.) from a phage library that expresses the peptide on the coat of phage. It refers to the process of selecting only the pies that are expressed in By repeating this process 3 to 10 times, scFvs exhibiting significant binding ability to the target antigen can be selected, and finally the selected scFvs can be prepared as gonorrhea-specific antibodies.

In the present invention, the multivalent maxibody protein structure consists of scFv of Clone 1 antibody - hinge - CH2- CH3 - (G ₄ S) ₂ linker - scFv of clone 4 antibody.

In the case of mouse Fc antibody, it was indicated as clone-1/4-muFc.

Hereinafter, the present invention will be described in detail.

The present invention provides a gonorrhea-specific multivalent antibody, or a functional fragment thereof, that specifically binds to Neisseria gonorrhoeae.

In the present invention, nine kinds of phage-nanoantibodies were selected by repeating biopanning (see FIG. 2) to screen and select gonorrhea-specific nanoantibodies (see FIG. 3).

Specifically, refer to the previously published Patent Publication No. 10-2019-0090247 of the present inventors for information on the nine types of antibodies.

In addition, the antibody comprises a sequence in which the variable regions of the heavy and light chains comprise the amino acid sequence of the CDR region or have at least 80%, preferably at least 90%, more preferably at least 95% homology with these sequences. can

The antibody may be a monoclonal antibody or a polyclonal antibody.

The present invention also provides a cell line for producing the antibody. Specifically, the antibody can be expressed by amplifying the scFv (single-chain variable fragment) portion by a method such as PCR, subcloning it into a backbone vector, and transfecting animal cells. . In addition, the functional fragments (Fab, scFc, Vh/Vl) can be expressed in E. coli in the same manner as above.

Antibodies having an antibody light chain variable region (VL) and an antibody light chain variable region (VL) containing the complementarity determining region (CDR) of the specific sequence are WHO L strains that show resistance to the third-generation antibiotics of the cephalosporin class. It was confirmed that it specifically binds (see FIG. 4), and does not show cross-reactivity to Neisseria meningitidis and a negative control strain, which causes meningitis known as gonorrhea related species (see FIG. 5).

In addition, the present invention is the gonorrhea-specific multivalent antibody; Or it provides a composition for detecting gonorrhea comprising a conjugate in which a label is bound to the gonorrhea-specific antibody.

In one embodiment of the present invention, the gonorrhea may be an antibiotic-resistant gonorrhea, for example, it may be a gonorrhea resistant to one or more antibiotics selected from the group consisting of penicillin, tetracycline, ciprofloxacin and ceftriaxone. In one embodiment,

The gonorrhea may be a WHO L standard strain, which is one of 14 standard strains providing quality assurance by performing phenotype, genetic characteristics and genome analysis of each strain in the World Health Organization (WHO). Gonorrhoeae (N gonorrhoeae) WHO L standard strain has the characteristics of showing resistance to penicillin, tetracycline, ciprofloxacin and ceftriaxone of the third-generation cephalosporin antibiotic series, and is used as an indicator of antibiotic-resistant gonorrhea.

In one embodiment, the labeling material may be any one selected from the group consisting of enzymes, luciferases, magnetic particles, fluorescent materials, and radioactive isotopes. However, in addition to those exemplified above, any one can be used as long as it can be used in an immunological assay.

The composition for detection according to the present invention may include the monoclonal antibody of the present invention and a reagent used for immunological analysis. Reagents used for immunological analysis include suitable carriers, labels capable of generating a detectable signal, solubilizing agents, and detergents used in all known quantitative assay methods based on antigen-antibody binding as a principle. Examples of the quantitative analysis method include, but are not limited to, immunoblotting, immunoprecipitation, enzyme immunoassay, protein chip, rapid assay, and microarray method.

Suitable carriers include, but are not limited to, a soluble carrier, such as any one of physiologically acceptable buffers known in the art (eg, PBS) or an insoluble carrier;

For example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, glass, metal, agarose, and combinations thereof may be used.

The present invention also provides a composition for treating gonorrhea comprising the gonorrhea-specific multivalent antibody or the nucleic acid molecule of the present invention.

In one embodiment, the composition for treating gonorrhea may further include a pharmaceutically acceptable excipient, carrier, diluent, and the like.

Specifically, the composition may include slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virus particles as carriers.

salts of inorganic acids such as, for example, hydrochloride, hydrobromide, phosphate and sulfate; pharmaceutically acceptable salts such as salts of organic acids such as acetate, propionate, malonate and benzoate; Water, saline, liquids such as glycerol and ethanol, and auxiliary substances such as wetting agents, emulsifying agents or pH buffering substances may be used.

In addition, the composition is formulated in a dosage form suitable for administration in a patient's body according to a conventional method in the pharmaceutical field, preferably a formulation useful for administration of a protein drug, and administration commonly used in the art. Oral, or intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes using methods It may be administered by a parenteral administration route, including, but not limited to.

Formulations suitable for this purpose include various preparations for oral administration such as tablets, pills, dragees, powders, capsules, syrups, solutions, gels, suspensions, emulsions, and microemulsions; and preparations for parenteral administration such as injections such as ampoules for injection, injections and sprays such as hypospray.

Formulations for injection or infusion may take the form of suspensions, solutions or emulsions, and may contain formulation agents such as suspending agents, preservatives, stabilizing agents and/or dispersing agents. In addition, the antibody molecule may be formulated in a dried form that can be used by reconditioning an appropriate sterile liquid prior to use.

Since the composition of the present invention contains an antibody molecule that is easily degraded in the gastrointestinal tract as an active ingredient, when the composition is to be administered by a route using the gastrointestinal tract, the antibody is protected from degradation, and after the antibody is released, it is absorbed into the gastrointestinal tract. It is preferable to include a drug that is

As an active ingredient of the composition or pharmaceutical formulation of the present invention, the antibody is administered by dividing 0.01 to 50 mg/kg of body weight per day, preferably 0.1 to 20 mg/kg of body weight, once or several times for mammals including humans. can be administered.

However, the actual dosage of the active ingredient is determined in light of several related factors such as the disease to be prevented or treated, the severity of the disease, the route of administration, the patient's weight, age and sex, drug combination, reaction sensitivity, and resistance/response to treatment. It is to be understood that this has to be determined and, therefore, the above dosages are not intended to limit the scope of the present invention in any way.

The present invention also provides the gonorrhea-specific multivalent antibody; Alternatively, a composition for diagnosing gonorrhea comprising a conjugate or a nucleic acid molecule of the present invention binding a label to the gonorrhea-specific antibody, and a kit for diagnosing gonorrhea comprising the composition for diagnosing gonorrhea.

The present invention also provides a method for detecting gonorrhea, comprising the step of detecting the presence of gonorrhea in a sample using the gonorrhea-specific multivalent antibody.

In addition, there is provided an analysis method for providing information necessary for diagnosing gonorrhea, which includes detecting the presence of gonorrhea in a biological sample isolated from an individual using the gonorrhea-specific antibody.

Examples of functional antibody fragments of the present invention include Fab, Fab', F(ab') 2 , scFv, Diabody, Tribody, dsFv, and CDR-containing peptides.

Fab is a fragment obtained by treating IgG with the protease papain (cleaved to the 224th amino acid residue of the H chain). It is an antibody fragment having an antigen-binding activity of about 50,000 molecular weight.

The Fab of the present invention can be obtained by treating the antibody of the present invention with the protease papain. Alternatively, the Fab can be prepared by inserting DNA encoding the Fab of the antibody into an expression vector for prokaryotes or eukaryotes, and introducing the vector into prokaryotes or eukaryotes to express them.

F(ab')2 is a fragment obtained by treating IgG with the protease pepsin (cleaved to the 234th amino acid residue of the H chain), slightly larger than that of Fab bound through the S-S bond of the hinge region. It is an antibody fragment having an antigen-binding activity with a molecular weight of about 100,000.

F(ab') 2 of the present invention can be obtained by treating the antibody of the present invention with the proteolytic enzyme pepsin. Alternatively, the following Fab' can be constructed by thioether bond or S-S bond. Fab' is an antibody fragment having an antigen-binding activity of about 50,000 in molecular weight by cleaving the S-S bond of the hinge region of F(ab')2.

scFv is a VH-P-VL to VLPVH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (P) of 12 or more residues, and is an antibody fragment having antigen-binding activity.

The scFv of the present invention is obtained by obtaining cDNA encoding the VH and VL of the antibody of the present invention, constructing DNA encoding the scFv, and inserting the DNA into an expression vector for prokaryotes or expression vectors for eukaryotes to obtain the expression vector It can be produced by expression by introduction into prokaryotes or eukaryotes.

A diabody is an antibody fragment in which scFvs having the same or different antigen-binding specificities form a dimer, and an antibody fragment having bivalent antigen-binding activity to the same antigen or bispecific antigen-binding activity to different antigens.

Diabody of the present invention, for example, obtain cDNA encoding VH and VL of the antibody of the present invention, construct DNA encoding scFv having a polypeptide linker of 3 to 15 residues, and express the DNA for prokaryotes Diabody can be produced by inserting it into a vector or an expression vector for eukaryotes and introducing the expression vector into prokaryotes or eukaryotes.

In addition, when the linker P length is 3-10, a tribody is formed and may be included as a tribody.

dsFv refers to a polypeptide in which one amino acid residue in VH and VL is substituted with a cysteine residue, and is bound through an S-S bond between the cysteine residues. Amino acid residues substituted with cysteine residues can be selected based on the prediction of the conformational structure of the antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697 (1994)).

Various antibodies can be displayed on the surface of phage by expressing human antibody genes in Ecoli in the form of fusions with phage surface proteins.

This technology is called phage display, and it is possible to produce antibody libraries of various combinations existing in the human body. From this library, a human monoclonal antibody that binds to a specific antigen can be selected by a panning method.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the light chain variable region of the antibody against gonorrhea or antigen-binding fragment thereof.

As used herein, the term "nucleic acid molecule" has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules. also includes modified analogs (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584) heavy and light chain variable regions of the present invention The sequence of the encoding nucleic acid molecule may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

According to one embodiment of the present invention, the nucleic acid molecule encoding the antibody of the present invention comprises a nucleotide encoding one of the amino acid sequences set forth in SEQ ID NOs: 13 to 17 in SEQ ID NO: 13 of the present invention.

The nucleic acid molecule of the present invention is construed to include a nucleotide sequence exhibiting substantial identity to the above-described nucleotide sequence. The substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. of homology, in one specific example, at least 90% homology, in another specific example, at least 95% homology.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the antibody of the present invention.

As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, including a plasmid vector; cozmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, and the like.

According to one embodiment of the present invention, in the vector of the present invention, a nucleic acid molecule encoding a light chain variable region and a nucleic acid molecule encoding a heavy chain variable region are operatively linked to a promoter.

As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, wherein By this, the regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

Vectors of the invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is a host, a strong promoter capable of propagating transcription (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter) , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and transcription/translation termination sequences. When E coli (eg, HB101, BL21, DH5α, etc.) is used as a host cell, promoter and operator sites of the E coli tryptophan biosynthesis pathway (Yanofsky, C J Bacteriol. 158:1018-1024 (1984)) and phage λ leftward A promoter (pLλ promoter, Herskowitz, I and Hagen, D Ann Rev Genet. 14:399-445 (1980)) can be used as a regulatory region. When Bacillus bacteria is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl Environ Microbiol. 64:3932-3938 (1998); Mol Gen Genet. 250:734-741 (1996)) or Bacillus bacteria Any promoter that can be expressed in .

On the other hand, the recombinant vector of the present invention is a plasmid often used in the art (eg, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phage (eg, λgt4·λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle) creatine promoter) or promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 75K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, The LTR promoter of HIV, the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Loose Sacoma virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The recombinant vector of the present invention may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA); maltose binding protein (NEB, USA); FLAG (IBI, USA); tag sequences such as 6x His (hexahistidine; Quiagen, USA), Pre-S1, c-Myc; There are leading sequences such as OmpA and PelB. In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.

On the other hand, the recombinant vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a resistance gene to tetracycline.

The vector expressing the antibody of the present invention may be a vector system in which a light chain and a heavy chain are simultaneously expressed in one vector or a system in which a light chain and a heavy chain are expressed in separate vectors. In the latter case, both vectors are introduced into the host cell through co-transformation and targeted transformation. Simultaneous transformation is a method of selecting cells expressing both light and heavy chains after simultaneously introducing each vector DNA encoding the light and heavy chains into a host cell. Target transformation involves selecting cells transformed with a vector containing a light chain (or heavy chain) and re-transforming the selected cells expressing the light chain with a vector containing a heavy chain (or light chain) to express both the light chain and the heavy chain. It is a method for final selection of cells. In the following examples, antibodies were prepared using a vector system in which light chains (VL and CL) and heavy chains (VH and CH1) were simultaneously expressed in one vector.

According to another aspect of the present invention, the present invention provides a host cell transformed with the recombinant vector of the present invention.

A host cell capable of stably and continuously cloning and expressing the vector of the present invention is known in the art and any host cell can be used, for example, Escherichia coli, Bacillus subtilis and Bacillus thuringien. Bacillus sp. strains such as cis, Streptomyces, Pseudomonas (eg Pseudomonas putida), Proteus mirabilis or Staphylococcus (eg Pseudomonas putida) Examples include, but are not limited to, prokaryotic host cells such as Staphylocus carnosus).

Suitable eukaryotic host cells of the vector include fungi such as Aspergillus species, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces ) and cells of higher eukaryotes such as yeast, other lower eukaryotes, insect-derived cells, such as Neurospora crassa, and cells derived from plants or mammals can be used.

As used herein, "transformation" and/or "transfection" into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue or organ, and as is known in the art, a suitable standard depending on the host cell. It can be done by choosing a technique. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation with silicon carbide fibers, agrobacterial mediated transformation, PEG, dextran sulfate, and lipofectamine. and drying/suppression mediated transformation methods, and the like.

According to another aspect of the present invention, the present invention comprises the steps of (a) culturing a host cell transformed with the recombinant vector of the present invention; and (b) expressing the anti-gonococcal antibody or antigen-binding fragment thereof in the host cell.

The culture of the transformed host cells in the antibody production can be made according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. These various culture methods are disclosed in various literatures (eg, James M Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture is divided into suspension culture and adherent culture according to the cell growth method, and batch, fed-batch and continuous culture methods according to the culture method. The medium used for culture should suitably satisfy the requirements of a particular strain.

In animal cell culture, the medium contains various carbon sources, nitrogen sources and trace elements. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL) and soybean wheat and inorganic such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. nitrogen source. These nitrogen sources may be used alone or in combination. The medium may contain, as phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salt. It may also contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (eg, air) is injected into the culture. The temperature of the culture is usually from 20°C to 45°C, preferably from 25°C to 40°C.

Antibodies obtained by culturing transformed host cells may be used in an unpurified state, and may be further purified with high purity using various conventional methods, for example, dialysis, salt precipitation, and chromatography. Among them, the method using chromatography is the most used, and the type and order of the column can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc. depending on the characteristics of the antibody and culture method.

The present invention also provides a single side chain variable fragment (scFv) comprising a heavy chain comprising a CDR-H1 region, a CDR-H2 region and a CDR-H3 region, and a light chain comprising a CDR-L1 region, a CDR-L2 region and a CDR-L3 region );

hinge region;

heavy chain constant region 2 (CH ₂ );

heavy chain constant region 3 (CH ₃ ); and

a heavy chain comprising a CDR-H1 region, a CDR-H2 region and a CDR-H3 region; and

Provided is a composition for diagnosing a disease comprising a multivalent antibody or a functional fragment thereof comprising a single side chain variable fragment (scFv) comprising a light chain comprising a CDR-L1 region, a CDR-L2 region and a CDR-L3 region.

Antibodies applicable to immunochemical method-based diagnostic POCT are important to select antibodies with high sensitivity and specificity for a wide variety of strains.

It was confirmed that the gonorrhea multivalent maxibody antibody developed in the present invention specifically detected various and extensive Gonoria clinical isolates and showed no cross-reaction to the Nigerian negative control strain.

Therefore, since the present invention can react and bind to gonorrhea very sensitively and specifically, it will be used as an important basic material for quickly and accurately screening, treating and preventing gonorrhea infection.

In addition, the multivalent maxibody antibody platform was evaluated for its potential applicability for diagnosis of other infectious diseases and cancers.

1 is a photograph of cultured gonorrhea strain.
2 is a schematic diagram of a method for discovering gonorrhea-specific antibodies using cell-based phage display technology.
3 is a graph showing the results of screening phage-nano-antibodies specifically binding to gonorrhea cell lines.
FIG. 4 is a result of screening a phage-nano antibody that specifically binds to the gonorrhea WHO L (standard strain) cell line.
5 is a graph evaluating the cross-reaction of gonorrhea-specific phage-nano antibodies.
6 is a graph analyzing the binding affinity of the gonorrhea-specific maxibody antibody;
Figure 7 is a figure showing the binding properties of the maxi body to a wide range of strains of N. gonorrhoeae,
(A) Binding of maxibody clones 1 and 4 shows maximal cross-reactivity with various N. gonorrhoeae strains (top panel). A450 ratios of maxi-body to Ab1 (left Y-axis) and maxi-body to Ab2 (right Y-axis) are shown in the lower panel.
(B) Affinity of N. gonorrhoeae maxibodies was determined by ELISA using WHO L standard strains. N. gonorrhoeae standard strain was fixed and serially diluted maxi-bodies were reacted, followed by reaction with HRP-enzyme-conjugated secondary antibody. EC50 was calculated using GraphPad Prism. clone 1 (group 1); clones 2 and 8 (group 4); clone 3 (group 3); Clone 4 (Group 2).
8 shows anti-N. Binding of gonorrhoeae multivalent maxibodies and analysis of multivalent maxibody specificity of N. gonorrhoea. Commercially available anti-N. gonorrhoeae antibodies (Ab1 and Ab2) were used as positive controls. A mouse isotype control antibody was included as a negative control antibody. Bound antibody was detected with HRP-anti-mouse antibody.
9 is a diagram of optimal maxi-body pair analysis for diagnosis;
(A) Schematic of a sandwich ELISA to determine the optimal combination of maxibody capture and detection for gonorrhea. Clone 4 maxibodies containing muFc domains and multivalent maxibodies (clone-1/4-muFc and clone1/4(ds)-muFc) were used to capture N. gonorrhoeae, and multivalent clone 1/4(ds)- The huFc maxi-body detects antigen bound to the capture maxi-body.
(B) Identification of optimal capture maxibodies using WHO L reference strains and N. gonorrhoeae clinical isolate mixtures. N. meningitidis, N. lactamica and N. sicca were used as controls for irrelevant Neisseria strains.
(C) A wide range of patient-derived clinical isolates of N. gonorrhoeae were used as antibody antigens. Multivalent clone-1/4 (ds)-huFc was used as detection maxibody with HRP-conjugated anti-huFc antibody. A450 values obtained in the absence of antigen were used to determine A450 ratio values expressed as mean ± standard deviation (SD) of three independent experiments. Clinical isolates not detected by maxibody clones 1 or 4 are shown: Nos. 1481 (***), 834 (*) and 1442 (**).
Figure 10 is the VH and VL sequences of clones 1 to 4 of the present invention, CDR-H1,2,3 is shown in green and CDR-L1,2,3 is shown in light blue in the figure;
11 is a total of five protein sequences of the multivalent maxibody of the present invention;
The hinge-CH2-CH3 portion of the multivalent maxibody is of two types, human Fc 1 and mouse Fc 1,
The multivalent maxibody protein structure is as follows, and it is the scFv of the Clone 1 antibody - the hinge-CH2-CH3- (G4S)2 linker - the scFv of the clone 4 antibody,
'scFv of Clone 1 antibody' is yellow, 'hinge' is green, 'CH2-CH3' is light blue and purple, respectively, '(G4S)2 linker' is blue, and 'scFv of clone 4 antibody' is gray. ,
In the case of a combination mouse Fc antibody, it was denoted as clone-1/4-muFc.
12 is a table showing anti- N. gonorrhoeae maxibody properties according to antibody specificity for clinical isolates. The sensitivity and 95% CI value for detection of N. gonorrhoeae were calculated with MedCalc statistical software, and crossed with control Neisseria spp. in the table. In the reflection category, 'No' means that no cross-reactivity was observed with N. sicca, N. meningitidis and N. lactamica.
13 is an overall manufacturing schematic diagram of the present invention;

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1: Culture of strains of the genus Neisseria

Nigerian genus strains [N. gonorrhoeae (gonorrhoeae)], N. sicca (negative control strain), N. meningitidis (meningitis bacilli, related strains), N. lactamica (negative control strain) and N. gonorrhoeae WHO L isolated from patients The standard strain was cultured on a chocolate agar plate, and the colonies were scraped and washed with PBS buffer (phosphate buffer). Thereafter, the strain was inactivated by heat treatment at 56° C. for 30 minutes. The inactivated strain was cultured again on a chocolate agar plate to confirm death (see FIG. 1).

Example 2: Screening and selection of gonorrhea cell-specific nanoantibodies using phage display technology

Biopanning was performed to screen and select gonorrhea-specific nanoantibodies. In order to proceed with biopanning, the nanoantibody library was placed in N. sicca (negative control strain) and stirred for 1 hour to deplete the nonspecifically binding antibody.

The depleted nanoantibody library was put into a mixture of 8 clinical isolates of gonorrhea and reacted for 2 hours. After washing and removing the non-specific binder, 100 mM glycine (glycine, pH 2.1) was added to obtain a specific phage-nano antibody bound to the gonorrhea mixed strain, and incubated with 1 M Tris-hydrochloride (Tris-HCl, The eluate was neutralized with pH 8). To amplify the phage nanoantibody, the eluted solution was put into TG1 cells, and then the phage nanoantibody was expressed and the phage titer was measured, and used for the next round. A single colony was obtained by performing phage display up to three times (R1, R2, R3) in total (see FIG. 2).

Example 3: Selection of gonorrhea cell-specific small molecule antibody

In order to select gonorrhea cell-specific nanoantibodies, phage-ELISA was performed as follows. Experiments were conducted on 576 colonies obtained through gonorrhea biopanning.

Gonorrhea phage-nanoantibodies were expressed in TG1 the day before the experiment. Gonorrhea and a negative control strain lysate and BSA (bovine serum albumin) were coated on an immunoplate. The next day, the coated plate was washed 3 times with PBST (0.05%) (Phosphate Buffered Saline with 0.05% Tween 20), and then MPBST (3% milk PBST) was added thereto, followed by incubation at 37° C. for 1 hour. The expressed phage-nano antibody was blocked with 3% MPBST while stirring for 1 hour.

Thereafter, the phage-nano antibody was placed in a blocking plate and reacted at room temperature for 1 hour. After the reaction, the plate was washed with PBST (0.05%) 6 times, and HRP-conjugated anti-M13 (horseradish peroxidase conjugated anti-M13) antibody was added to the plate and reacted at room temperature for 1 hour. After incubation, washing was performed 6 times, and TMB (Tetramethylbenzidine) was added to observe color development, and absorbance was measured at 450 nm.

80 kinds of gonorrhea cell-specific phage-nano antibodies were identified among a total of 576 clones by the experiment as described above. In addition, the specific binding of the nine finally selected phage-nanoantibodies having a single DNA sequence among 80 kinds to the gonorrhea cell line is shown in FIG. 3 .

Example 4: Selection of gonorrhea WHO L standard strain-specific nanoantibodies

The World Health Organization (WHO) selects 14 standard strains of N gonorrhoeae and performs phenotype, genetic characteristics and genome analysis of each strain to provide quality assurance. Among them, gonorrhoeae (N. gonorrhoeae) WHO L standard strain is characterized by resistance to penicillin, tetracycline, ciprofloxacin, and ceftriaxone of the third-generation cephalosporin antibiotic series. Therefore, the specific binding of the nanoantibody to the WHO L standard strain was analyzed.

For 80 kinds of phage-nanoantibodies showing specific binding to the gonorrhea strain, the binding to the gonorrhea WHO L standard strain was confirmed by the phage-ELISA method. Gonorrhea WHO L standard strain and negative control strain lysate was coated on a 96-well immunoplate to react with phage-nano antibody, and then, binding was confirmed by developing color with a TMB substrate.

63 kinds of phage-nano antibodies that specifically bind to the gonorrhea WHO L standard strain were confirmed by the experiment as described above. The results of specific binding to the gonorrhea WHO L standard strain of the finally selected 9 types of phage-nano antibodies having a single DNA sequence among 63 types are shown in FIG. 4 .

Example 5: Evaluation of cross-reactivity of gonorrhea-specific screening antibody

Among the 11 species of Nigerian genus that colonize humans, two strains known as pathogens are Gonorrhea (N. gonorrhoeae) and Bacillus meningitis (N. meningitidis).

Therefore, we evaluated the cross-reactivity of nanoantibodies against a closely related species of N. meningitidis that causes meningitis.

To evaluate the screening antibody cross-reactivity, 63 kinds of phage-nanoantibodies showing specific binding to the gonorrhea WHO L standard strain were analyzed for binding to N. meningitidis related species. N. gonorrhoeae, N. gonorrhoeae WHO L (standard strain), negative control strain and N. meningitidis (relative) lysate were coated on an immune plate to react with phage-nanoantibody, and then ABTS[2,2'- azino-di-(3-ethylbenzthiazoline sulfonic acid)] substrate was used, and absorbance was measured at 405 nm to confirm binding.

In the above experiment, a total of 59 types of phage-nano antibodies were selected except for 4 types of phage-nano antibodies that bind to N. meningitidis related species. FIG. 5 shows the cross-reactivity evaluation results of the nine finally selected phage-nano-antibodies having a single DNA sequence among 59 species.

Example 6: Anti-expression in animal cell expression system N. gonorrhoeae Binding force analysis of 8 individual clinical isolates of 9 maxibody types

Binding of anti- N. gonorrhoeae maxibody to individual clinical isolates isolated from patients was analyzed.

Gonorrhea lysate isolated from individual patients was coated on an immunoplate to react with each maxibody, followed by color development with TMB substrate and absorbance measurement at 450 nm to confirm binding.

It was confirmed that 9 types of anti- N. gonorrhoeae maxibody specifically bind to 8 individual clinical isolates and do not cross-react with N. sicca and N. meningitidis negative control strains.

According to the type of strain to which they bind, maxibody antibodies were classified into 5 groups.

Maxibody clone 1 (group 1) was bound to all strains except clinical isolates #1481, and maxibody clone 4 (group 2) was bound to all strains except clinical isolates #834 and 1446. This suggests that maxibody clone 4 and clone 1 are capable of binding to different epitopes for the gonorrhea antigen.

Group 3 (maxibody clone 3) and group 4 (maxibody clone 2, 6, 8, 11, 12) showed up to 75% and 50% sensitivity to clinical isolates, respectively, and maxibody clone 9 (group 5) and commercial antibody Ab2 showed the lowest sensitivity to clinical isolates, ~25%. In contrast, the commercial antibody Ab1 showed binding to all 11 clinical isolates, but showed significant cross-reactivity with the negative control strains N. sicca and N. meningitidis strains, confirming that it was not suitable as a diagnostic antibody (Fig. 6 and Figure 7A).

The affinity of the Gonoria Maxibody antibody was confirmed by ELISA. After coating the Gonorrhea WHO L standard strain on an immunoplate, the maxibody antibody was serially diluted 2-fold to react and bind. maxibody clone 4 (group 2) showed the highest affinity for antigen with an EC50 value of 3 nM, confirming that the affinity was 6.3 times higher than that of the commercial antibody Ab1 (19 nM). In contrast, clones 1 and 8 maxibody showed similar affinity to the commercial antibody Ab1. Therefore, maxibody clones 1 and 4 were selected and reformatted based on the sensitivity and affinity for Gonorrhea to produce a multivalent maxibody.

Example 7: Gonorrhea-specific multivalent maxibody production and binding force analysis

Antibodies for applying immunochemical diagnostic methods to POCT should show high affinity for antigens and binding sensitivity to a wide variety of strains to facilitate rapid diagnosis.

Maxibody clone 1 and clone 4 showed binding to a wide range of clinical isolates of Gonorrhea, and were analyzed to have different epitopes (FIG. 7A).

Therefore, by combining maxibody clone 1 and clone 4, we tried to construct a multivalent maxibody with higher sensitivity and specificity.

Specifically, by reformatting two types of selectable maxibody antibodies (clone 1 and clone 4), four types of multivalent maxibody of scFv-Fc-scFv format, clone-1/4, clone-1/4(ds), and clone-4 /1 and clone-4(ds)/1 were constructed.

Each scFv fragment was amplified by PCR and sub-cloned into pCEP4 mammalian expression vector using AgeI and HidIII or AflII and NheI restriction enzymes.

To further stabilize the scFv domain, we included an antibody clone with a disulfide bond added as “ds (disulfied stabilized)”, such as clone-1/4 (ds).

Gonorrhea-specific multivalent maxibody binding assay method :

The prepared multivalent maxibody antibody was evaluated whether it was possible to detect the Gonorrhea strain using the ELISA technique. After coating individual clinical isolates of gonorrhea on an immunoplate, the gonorrhea-specific maxibody and multivalent maxibody antibodies are added to react. After washing, the HRP-enzyme-conjugated secondary antibody is added to react, and then TMB or ABTS substrate is added to develop color. Measure the absorbance value at A450nm or 405nm.

It was confirmed that the clinical isolates #1446 and #1481, which were not detected by maxibody clone 1 and clone 4 antibodies, respectively, were detectable in both multivalent maxibody clone-1/4 and clone-1/4(ds). However, in the case of the multivalent maxibody clone-4/1 and clone-4(ds)/1 antibodies, clinical isolate #1446 could not be detected (FIG. 8).

Therefore, the possibility of applying the multivalent maxibody clone-1/4 and clone-1/4(ds) to immunochemical diagnostic products was evaluated.

Example 8: Selection and evaluation of maxibody antibody pair for immunochemical diagnostic product application

POCT products based on immunochemical diagnostics use two antibodies that bind to different sites on an antigen. Therefore, it is essential to select the optimal capture-detection antibody pair to evaluate the application potential of immunodiagnostic products.

The present inventors evaluated the optimal capture-detection antibody pair for the detection of gonorrhea using a sandwich-ELISA method.

Gonorrhea capture antibodies (maxibody clone 4-muFc, multivalent maxibody clone-1/4-muFc and clone-1/4(ds)-muFc) are coated on an immunoplate. The maxibody clone 1-muFc antibody was not used as a capture antibody because its affinity for gonorrhea was lower than that of clone 4. After washing, the WHO L standard strain, a mixture of Gonoria clinical isolates, 19 individual Gonorrhea clinical isolates and negative control strains ( N. sicca, N. meningitidis and N. lactamica ) were put into an immunoplate coated with capture antibody and reacted and combined. make it After washing, the gonorrhea detection antibody (multivalent maxibody clone-1/4(ds)-huFc) is added to the immunoplate for reaction. After that, the HRP-enzyme-conjugated secondary antibody is added to react, and then TMB or ABTS substrate is added to develop color. Measure the absorbance values at A450 nm or 405 nm (Fig. 9A).

Multivalent maxibody detects both the WHO standard strain and the mixture of Gonoria clinical isolates, and it was confirmed that it does not bind to the negative control strain. In addition, compared to maxibody clone 4, when multivalent maxibody clone-1/4 and clone 1/4(ds) were used as capture antibodies, it was analyzed that the binding strength of gonorrhea detection was increased by at least 4 times (FIG. 9B).

In addition, when multivalent maxibody clone-1/4 and clone 1/4(ds) are used as capture antibodies, all 19 clinical isolates of gonorrhea can be detected, and the detection intensity is increased by 2 to 20 times compared to maxibody clone 4 was confirmed (Fig. 9C, Fig. 12).

<110> KANGWON NATIONAL UNIVERSITY RESEARCH & UNIVERSITY-INDUSTRY COOPERATION FOUNDATION <120> A MULTIVALENT ANTIBODY SPECIFIC FOR BROAD SPECTRUM OF Neisseria gonorrhoeae AND USE210 OF THE SAME <130> P21-0018HS <160> 17 <170> 1 KoPatentIn <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Clone 1-CDR H1 <400> 1 Ser Gly Tyr Ser Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213 > Artificial Sequence <220> <223> Clone 1-CDR H2 <400> 2 Gly Ile Tyr Ser Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Clone 1-CDR H3 <400> 3 Arg Thr Asn Gly Trp Phe Asp Tyr 1 5 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220 > <223> Clone 1-CDR L1 <400> 4 Ser Gly Ser Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Thr 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> < 223> Clone 1-CDR L2 <400> 5 Ala Asp Ser His Arg Pro Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Clone 1-CDR L3 <400> 6 Ala Thr Trp Asp Tyr Ser Leu Ser Gly Tyr Val 1 5 10 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Clone 4-CDR H1 <400> 7 Ser Asp Tyr Ala Met Ser 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Clone 4 -CDR H2 <400> 8 Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <223 > Clone 4-CDR H3 <400> 9 Lys His Ser Arg Arg Leu Leu Pro Thr Trp Gly Ser Tyr Asp Asn Gly 1 5 10 15 Met Asp Val <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Clone 4-CDR L1 <400> 10 Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Ser 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220 > <223> Clone 4-CDR L2 <400> 11 Ala Asp Ser Lys Arg Pro Ser 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Clone 4-C DR L3 <400> 12 Gly Ser Trp Asp Tyr Ser Leu Ser Gly Tyr Val 1 5 10 <210> 13 <211> 743 <212> PRT <213> Artificial Sequence <220> <223> Multivalent maxibody clone-4(ds )/1-muFc <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Ser Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Arg Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Asn Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp 210 215 220 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu Gly Lys Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly 465 470 475 480 Ser Gly Gly Gly Gly Ser Leu Lys Glu Val Gln Leu Leu Glu Ser Gly 485 490 495 Gly Gly Leu Val Gln Thr Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 500 505 510 Ser Gly Phe Thr Phe Ser Asp Tyr Ala Met Ser Trp Val Arg Gln Ala 515 520 525 Pro Gly Lys Cys Leu Glu Trp Val Ser Ala Ile Ser Pro Gly Ser Ser 530 535 540 Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 545 550 555 560 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala 565 570 575 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Ser Arg Arg Leu Leu 580 585 590 Pro Thr Trp Gly Ser Tyr Asp Asn Gly Met Asp Val Trp Gly Gln Gly 595 600 605 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 610 615 620 Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala 625 630 635 640 Ser Gly Thr Pro Gly Gln Gly Val Thr Ile Ser Cys Thr Gly Ser Ser 645 650 655 Ser Asn Ile Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly 660 665 670 Thr Ala Pro Lys Leu Leu Ile Tyr Ala Asp Ser Lys Arg Pro Ser Gly 675 680 685 Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu 690 695 700 Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly 705 710 715 720 Ser Trp Asp Tyr Ser Leu Ser Gly Tyr Val Phe Gly Cys Gly Thr Lys 725 730 735 Leu Thr Val Leu Gly Ala Ser 740 <210> 14 <211> 740 <212> PRT <213> Artificial Sequence <220> < 223> Multivalent maxibody clone-4(ds)/1-muFc <400> 14 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Ser Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Arg Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Asn Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp 210 215 220 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu Gly Lys Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro 245 250 255 Pro Cys Pro Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys 260 265 270 Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 275 280 285 Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 290 295 300 Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu 305 310 315 320 Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala 340 345 350 Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg 355 360 365 Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met 370 375 380 Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro 385 390 395 400 Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 405 410 415 Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val 420 425 430 Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr 435 440 445 Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu 450 455 460 Lys Ser Leu Ser His Ser Pro Gly Lys Gly Gly Gly Gly Gly Ser Gly Gly 465 470 475 480 Gly Gly Ser Leu Lys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 485 490 495 Val Gln Thr Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 500 505 510 Thr Phe Ser Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys 515 520 525 Gly Leu Glu Trp Val Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr 530 535 540 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 545 550 555 560 Lys Asn Thr Leu Tyr Leu Gln Met Ser Leu Arg Ala Glu Asp Thr 565 570 575 Ala Val Tyr Tyr Cys Ala Lys His Ser Arg Arg Leu Leu Pro Thr Trp 580 585 590 Gly Ser Tyr Asp Asn Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 625 630 635 640 Pro Gly Gln Gly Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile 645 650 655 Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 660 665 670 Lys Leu Leu Ile Tyr Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp 675 680 685 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 690 695 700 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp 705 710 715 720 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 725 730 735 Leu Gly Ala Ser 740 <210> 15 <211> 742 <212> PRT <213> Artificial Sequence <220> <223> Multivalent maxibody clone-4(ds)/1-muFc <400> 15 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Ser Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Asn Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Arg Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 145 150 155 160 Gly Asn Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp 210 215 220 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu Gly Lys Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro 245 250 255 Pro Cys Pro Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys 260 265 270 Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 275 280 285 Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 290 295 300 Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu 305 310 315 320 Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala 340 345 350 Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg 355 360 365 Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met 370 375 380 Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro 385 390 395 400 Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 405 410 415 Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val 420 425 430 Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr 435 440 445 Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu 450 450 455 460 Lys Ser Leu Ser His Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly 465 470 475 480 Gly Gly Ser Leu Lys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 485 490 495 Val Gln Thr Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 500 505 510 Thr Phe Ser Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys 515 520 525 Cys Leu Glu Trp Val Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr 530 535 540 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 545 550 555 560 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 565 570 575 Ala Val Tyr Tyr Cys Ala Lys His Ser Arg Arg Leu Leu Pro Thr Trp 580 585 590 Gly Ser Tyr Asp Asn Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 625 630 635 640 Pro Gly Gln Gly Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile 645 650 655 Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 660 665 670 Lys Leu Leu Ile Tyr Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp 675 680 685 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 690 695 700 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp 705 710 715 720 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Cys Gly Thr Lys Leu Thr Val 725 730 735 Leu Gly Ala Ser *** *** 740 <210> 16 <211> 740 <212> PRT <213> Artificial Sequence <220> <223> Multivalent maxibody clone-4(ds)/1-muFc <400> 16 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Ser Arg Arg Leu Leu Pro Thr Trp Gly Ser Tyr Asp Asn 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gly Gly Val 145 150 155 160 Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val 165 170 175 Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 180 185 190 Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu 210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu Ser Gly 225 230 235 240 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Lys Leu Glu 245 250 255 Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Cys Pro Val Pro 260 265 270 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 275 280 285 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 290 295 300 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 370 375 380 Val Tyr Thr Ile Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 405 410 415 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 420 425 430 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 435 440 445 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 450 455 460 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 465 470 475 480 Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Lys 485 490 495 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 500 505 510 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 515 520 525 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 530 535 540 Ser Gly Ile Tyr Ser Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 545 550 555 560 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 565 570 575 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 580 585 590 Ala Arg Thr Asn Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 625 630 635 640 Pro Gly Arg Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 645 650 655 Gly Asn Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 660 665 670 Lys Leu Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp 675 680 685 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 690 695 700 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp 705 710 715 720 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 725 730 735 Leu Gly Ala Ser 740 <210> 17 <211> 740 <212> PRT <213> Artificial Sequence <220> <223> Multivalent maxibody clone-4(ds )/1-muFc <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Ser Arg Arg Leu Leu Pro Thr Trp Gly Ser Tyr Asp Asn 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gln Ser 130 135 140 Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Gly Val 145 150 155 160 Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val 165 170 175 Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 180 185 190 Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 195 200 205 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu 210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu Ser Gly 225 230 235 240 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Lys Leu Glu 245 250 255 Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Cys Pro Val Pro 260 265 270 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 275 280 285 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 290 295 300 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330 335 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 340 345 350 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 370 375 380 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 405 410 415 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 420 425 430 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 435 440 445 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 450 455 460 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 465 470 475 480 Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Lys 485 490 495 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 500 505 510 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 515 520 525 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 530 535 540 Ser Gly Ile Tyr Ser Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 545 550 555 560 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 565 570 575 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 580 585 590 Ala Arg Thr Asn Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 625 630 635 640 Pro Gly Arg Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 645 650 655 Gly Asn Asn Tyr Val Thr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 660 665 670 Lys Leu Leu Ile Tyr Ala Asp Ser His Arg Pro Ser Gly Val Pro Asp 675 680 685 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 690 695 700 Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp 705 710 715 720 Tyr Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val 725 730 735Leu Gly Ala Ser 740

Claims (12)

N-terminal to C-terminal direction,
A heavy chain comprising a CDR-H1 region set forth in SEQ ID NO: 1, a CDR-H2 region set forth in SEQ ID NO: 2 and a CDR-H3 region set forth in SEQ ID NO: 3 and a CDR-L1 region set forth in SEQ ID NO: 4, SEQ ID NO: a single side chain variable fragment (scFv) comprising a light chain comprising a CDR-L2 region set forth in SEQ ID NO: 5 and a CDR-L3 region set forth in SEQ ID NO: 6;
hinge region;
heavy chain constant region 2 (CH ₂ );
heavy chain constant region 3 (CH ₃ ); and
A heavy chain comprising a CDR-H1 region set forth in SEQ ID NO: 7, a CDR-H2 region set forth in SEQ ID NO: 8 and a CDR-H3 region set forth in SEQ ID NO: 9 and a CDR-L1 region set forth in SEQ ID NO: 10, SEQ ID NO: A single side chain variable fragment (scFv) comprising a light chain comprising a CDR-L2 region set forth in 11 and a CDR-L3 region set forth in SEQ ID NO: 12
Gonorrhea-specific multivalent antibody or functional fragment thereof.
According to claim 1,
The antibody is a gonorrhea-specific multivalent antibody or functional fragment thereof, characterized in that it comprises a disulfide bond linker. According to claim 1,
The functional fragment is an antibody, or a functional fragment thereof, characterized in that the single side chain variable fragment (scFv).
According to claim 1,
The antibody, or a functional fragment thereof, characterized in that it comprises one of the amino acid sequences set forth in SEQ ID NOs: 13 to 17.
The gonorrhea-specific antibody of any one of claims 1 to 4; Or a composition for detecting gonorrhea comprising a conjugate in which a label is bound to the gonorrhea-specific antibody of any one of claims 1 to 4.
6. The method of claim 5,
The composition for detecting gonorrhea, characterized in that the gonorrhea is antibiotic-resistant gonorrhea.
6. The method of claim 5,
The gonorrhea detection composition for gonorrhea, characterized in that the gonorrhea is resistant to one or more antibiotics selected from the group consisting of penicillin, tetracycline, ciprofloxacin and ceftriaxone.
A nucleic acid molecule encoding the gonorrhea-specific multivalent antibody of claim 1 or a functional fragment thereof.
A composition for treating gonorrhea comprising the gonorrhea-specific antibody or the nucleic acid molecule of claim 8 according to any one of claims 1 to 4.
A kit for diagnosing gonorrhea comprising the antibody of any one of claims 1 to 4 or the nucleic acid molecule of claim 8.
A method of detecting gonorrhea, comprising the step of detecting the presence of gonorrhea in a sample using the gonorrhea-specific antibody of any one of claims 1 to 4 or the nucleic acid molecule of claim 8.
a heavy chain comprising a CDR-H1 region, a CDR-H2 region and a CDR-H3 region; and
a single side chain variable fragment (scFv) comprising a light chain comprising a CDR-L1 region, a CDR-L2 region and a CDR-L3 region;
hinge region;
heavy chain constant region 2 (CH ₂ );
heavy chain constant region 3 (CH ₃ ); and
a heavy chain comprising a CDR-H1 region, a CDR-H2 region and a CDR-H3 region; and
A composition for diagnosing a disease comprising a multivalent antibody or a functional fragment thereof comprising a single side chain variable fragment (scFv) comprising a light chain comprising a CDR-L1 region, a CDR-L2 region and a CDR-L3 region.

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