RU2793255C2 - Multivalent mono- or bispecific recombinant antibodies for analytical purposes - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: method for constructing a non-chimeric or chimeric multivalent recombinant antibody for antigen detection. Disclosed are the use of a multivalent recombinant antibody obtained by the specified method, and a method for detecting an antigen using an antibody obtained by the specified method. Also, a conjugate for detecting an antigen containing an antibody obtained by this method is disclosed.

EFFECT: invention makes it possible to effectively produce antibodies for antigen detection.

19 cl, 9 dwg, 14 tbl, 12 ex

Description

The present description relates to new analytical specific multivalent recombinant antibodies that are particularly useful in immunoassays. In particular, hexavalent, octavalent, and decavalent antibodies, their construction, preparation, characterization, and use in target antigen assays are described.

Prior Art

An immunoassay is a biochemical test that determines the presence or concentration of a macromolecule or small molecule in a solution, usually using an antibody as the specific detection agent. A molecule detected by an immunoassay is referred to as the "test substance" or "target analyte" and in many cases is a protein, although it may be of a different type and size, provided that the antibody or antibodies used in the immunoassay are capable of facilitating specific detection. the substance under study. In clinical diagnosis, immunoassays often detect test substances in biological fluids such as serum, plasma, or urine. More generally, as contemplated by the present invention, immunoassays qualitatively or quantitatively detect a target analyte in some type of sample, provided that the sample is a liquid sample or can be processed to become a liquid sample, and also provided that that the test substance is contained in an aqueous solution, being part of the liquid phase of the sample.

Immunoassays known to those skilled in the art come in a variety of formats and variations. Immunoassay can be carried out in several stages with the addition of reagents and their washing out or separation at different stages of the analysis. Multistage assays are often referred to as separation immunoassays or heterogeneous immunoassays. Some immunoassays can be performed by simply mixing reagents and sample and making physical measurements. Such assays are referred to as homogeneous immunoassays or, more rarely, undivided immunoassays.

Immunoassay is based on the ability of an antibody to recognize and specifically bind to a substance of interest, even if the substance is present in a small amount in a sample in a complex mixture of other molecules. The specific molecular structure recognized by an antibody is called an "antigen" and the specific region on an antigen that an antibody binds to is called an "epitope".

In addition to the specific binding of an antibody to an analyte, another fundamental feature of all immunoassays is the ability to generate a measurable signal in response to binding. Often the antibody is associated with a detectable label. In modern immunoassays, a large number of labels are used, which make it possible to detect binding by various methods. Many labels can be detected because they are able to emit, change the color of the solution, fluoresce when exposed to light caused by induction.

A typical embodiment of a heterogeneous antibody immunoassay involves a so-called "sandwich" format in which two (first and second) different non-overlapping analyte antigens are linked by a first and second antibody, respectively. That is, due to binding to the first and second antigens, the first and second antibodies form a "sandwich" with the test substance. The first antibody is bound to a detectable label, the second antibody is immobilized or capable of being immobilized, allowing the addition and removal of reagents and washing steps. A heterogeneous sandwich immunoassay involves the general steps of (a) contacting a sample containing test substance with a first and a second antibody, after which the test substance becomes bound (clamped) to the first and second antibodies, where the second antibody is or becomes immobilized, after which (b ) carry out the step of determining the amount of immobilized label, which is part of the sandwich. The amount of label detected corresponds to the amount of test substance in the sandwich and therefore corresponds to the amount of test substance in the sample.

In the technical field of preparation of material for immunoassay, oligomers or polymers of antibodies known in this field are used; they are often used to enhance the antigen-binding properties of an antibody. EP 0 955 546 A1 describes a chemically polymerized antibody conjugate which is labeled with a dye. The antibody polymerization product is characterized by a large number of functional antigen binding sites, that is, it is a "multivalent" binder. It is known that the use of a multivalent binder in an immunoassay leads to an improvement in antigen-binding sensitivity. It is described that a polymerized antibody associated with a detectable label is described for use in antigen-antibody reactions for diagnostic purposes.

Using the enzyme as a detectable label, EP 175560 A2 reports a process for preparing a polymeric enzyme/antibody conjugate by covalently linking a prepolymerized enzyme to an antibody or fragment thereof, such as a Fab, Fab' or F(ab')2 fragment. This patent also describes an immunoassay for detecting an analyte in a liquid sample, which includes the steps of (a) complexing the conjugate with the analyte, (b) separating the complex, (c) detecting the enzymatic activity in the complex, and (d) correlating the detected enzymatic activity with the amount of the test substance in the sample. The cited patent also mentions the optimization of the preparation of the conjugate with respect to the stoichiometry of the antibody or its fragments, on the one hand, and the prepolymerized enzyme, on the other hand. Stoichiometry has been shown to have an effect on detection sensitivity and background activity in an immunoassay.

US 20030143638 A1 describes a method for regulating the reactivity of an antigen-antibody interaction. This method includes the steps of (a) obtaining a plurality of antibody polymers having different degrees of polymerization; (b) linking a plurality of antibody polymers to carriers to form a set of antibody/carrier complexes; and (c) selecting from the set of complexes such an antibody/carrier complex that reacts with the antigen to the desired extent.

Each arm of an antibody that binds to a target antigen is called an antigen-binding fragment (=Fab). Fab means "antigen binding fragment"; this is the region of an antibody that binds to antigens, consisting of one constant and one variable domain each of the heavy (Fab _H ) and light chains (Fab _L ). Thus, a Fab fragment contains two aligned polypeptides, a first heavy chain fragment (Fab _H ) and an unfragmented light chain (Fab _L ), which are aligned with the heavy chain fragment and linked through a disulfide bridge. The tail region of an antibody is commonly referred to as the crystallizable fragment (=Fc), which contains in the antibody isotypes IgG, IgA and IgD two additional heavy chain fragments (each called Fc _H ) that are identical and aligned with each other and that are linked to one or more disulfide bridges. Proteolytic processing of an immunoglobulin of an IgG, IgA or IgD isotype can be used to artificially cleave an antibody to form Fc and Fab fragments that can be separated and isolated. The papain enzyme can be used to cleave one immunoglobulin into two Fab fragments and one Fc fragment. The enzyme pepsin cleaves below the hinge region, resulting in an F(ab')2 fragment and a pFc' fragment. Similarly, the IdeS enzyme specifically cleaves IgG at the hinge region.

Antigen-binding antibody fragments without Fc moieties (obtained in isolated form after proteolytic processing, eg, pepsin or papain) are particularly preferred when the test substance sample is whole blood, serum, or plasma. It is known that components contained in such samples can non-specifically bind to Fc portions of conventional antibodies. Non-specific interaction of sample components with Fc moieties can enhance the background signal of the immunoassay. Increasing the background signal has a negative impact on the performance of the analysis because the signal-to-noise ratio decreases.

In the prior art, one embodiment of the immunoassay includes the step of chemically cross-linking fragments of antigen-binding antibodies after removing their Fc portions, thereby forming a polymer from such fragments. Such polymerized antigen-binding antibody fragments are currently preferred in a number of immunoassays that require high sensitivity to the target antigen and low background signal. The crosslinking step is performed to create a multivalent test substance binder with improved binding properties. For a number of commercially available immunoassays, an antibody-derived multivalent analyte-specific binder without an Fc moiety and bound to a label is the preferred detection agent known in the art. In certain cases, chemically cross-linked (ie, polymerized) antibody fragments are superior to antibodies in their natural form, which still includes Fc portions, because the signal-to-noise ratio of the immunoassay can thus be improved.

Naturally occurring non-fragmented antibodies contain two heavy chains, which are linked by disulfide bonds, and two light chains. Each light chain is linked to one of the heavy chains by disulfide bonds. Each Fab _H portion in an immunoglobulin heavy chain has a variable domain (VH) at the N-terminus followed by a number of constant domains (three or four constant domains, CH1, CH2, CH3 and CH4, depending on the antibody class). Each Fab _L light chain has a variable domain (VL) at its N-terminal end and a constant domain (CL) at its other (C-terminal) end; the light chain constant domain is aligned with the first constant domain (CH1) of the heavy chain, and the light chain variable domain (VL) is aligned with the heavy chain variable domain (VH). I believe that certain amino acid residues form the interface between the domains of the light and heavy chains, which provides alignment due to physico-chemical interactions.

The constant domains are not directly involved in the binding of an antibody to its target antigen, but they are involved in various effector functions in vivo. The variable domains of each pair of light and heavy chains are directly involved in the binding of an antibody to its epitope. The variable domains of naturally occurring light (VL) and heavy (VH) chains have the same general structure; each contains four frame regions (framework region, FR) whose sequences are somewhat conserved and connected by three complementarity determining regions (CDRs). The CDRs in each strand are in close proximity to the FR; the epitope binding site is formed by the combined CDRs of the aligned light and heavy chains in the corresponding Fab portion of the antibody.

О., Pathobiology 74, 2007, 3-14; Shen J. с соавт., J. Immunol. Methods 318, 2007, 65-74; Wu С. с соавт., Nature Biotech. 25, 2007, 1290-1297). В таких форматах используют линкеры либо для слияния ядра антитела (IgA, IgD, IgE, IgG или IgM) с другим связывающим белком (например, scFv), либо для слияния, например, двух фрагментов Fab или scFv (Fischer N., Leger О., Pathobiology 74, 2007, 3-14).Recently, many recombinant multispecific antibody formats have been developed, such as quadrivalent bispecific antibodies produced by hybridization, such as the IgG antibody format and single chain domains (see, for example, Coloma MJ et al., Nature Biotech. 15, 1997, 159-163 ; WO 2001/077342; Morrison SL, Nature Biotech 25, 2007, 1233-1234). Several other new formats have also been developed in which the antibody core structure (IgA, IgD, IgE, IgG, or IgM) is no longer preserved; for example, dia-, tria- or tetra-bodies, mini-bodies and several single-stranded formats (scFv, Bis-scFv). A number of them are able to bind two or more antigens (Holliger P. et al., Nature Biotech. 23, 2005, 1126-1136; Fischer N.,

O., Pathobiology 74, 2007, 3-14; Shen J. et al., J. Immunol. Methods 318, 2007, 65-74; Wu C. et al., Nature Biotech. 25, 2007, 1290-1297). Such formats use linkers to either fuse an antibody core (IgA, IgD, IgE, IgG, or IgM) to another binding protein (e.g., scFv) or to fuse, for example, two Fab or scFv fragments (Fischer N., Leger O. , Pathobiology 74, 2007, 3-14).

WO 2001/077342 describes various engineered antibodies. A specific engineered IgG class antibody is described that contains four antigen-binding sites. In particular, the N-terminal CH1-VH portion of each heavy chain is extended by another CH1-VH portion. Accordingly, in a fully assembled antibody, the N-terminal portion of each heavy chain is aligned and associated with not one but two corresponding light chains. Thus, each arm of such a tetravalent antibody contains a first and a second antigen-binding site, with both sites located in tandem. This invention mentions that such engineered antibodies may be useful in diagnostic assays, for example, in the detection of antigens of interest in particular cells, tissues, or serum.

An additional review on engineered antibodies was provided by Chiu M.L., Gillilang G.L. Curr Opin Struct Biol 38, 2016, 163-173; Tiller K.L., Tessier P.M. Annu Rev Biomed Eng 17, 2015, 191-216. A number of different designs of multivalent antibodies are discussed in the publication Deyev S.M., Lebedenko E.N. BioEssays 30, 2008, 904-918.

Hexavalent engineered antibodies are described by Blanco-Toribio A. et al., mAbs 5, 2013, 70-79. Stone E. et al., J. Immunol. Methods 318, 2007, 88-94. The foregoing illustrates the desire in the art to provide modified immunoglobulins as starting material for immunoassays that have reduced background signal. In addition, immunoglobulins are desirable that provide high assay sensitivity for the target analyte.

The production of multivalent antigen-binding macromolecules by chemical linkage or cross-linking (polymerization) of whole antibodies or antibody fragments is already a practical approach to solving such technical problems. But the formation of chemical bonds is a probabilistic process in several respects. First, that chemical cross-linking is not (or only to a limited extent) site-specific. That is, there are always several available amino acid side chains in a polypeptide, which, in principle, can participate in a cross-linking reaction. And for each considered side chain, there is a different probability of its participation in the reaction. A chemical reaction can be quantitative or non-quantitative. In addition, in principle, in a chemical reaction with antibodies or antibody fragments, not one product is formed, but a number of different products. This means that it is impossible to accurately predict which specific amino acid side chains of the first and second polypeptide are involved in the formation of cross-links.

In addition, there is usually a distribution relative to the average number of polypeptides that combine to form a multivalent antigen-binding macromolecule. Chemical cross-linking reactions result in the molecular weight distribution of the products, reflecting the amount of antibodies or antibody fragments that are linked together. Typically, however, only a subset of the products are of practical use and are technically suitable as multivalent antigen-binding macromolecules in immunoassays. Therefore, in order to create a sufficiently standardized and reproducible assay, it is necessary to identify, separate, purify and characterize the necessary sub-fraction of products for further use.

Accordingly, there is a need in the art to improve the production of multivalent antigen-binding macromolecules suitable for use as detection reagents in immunoassays. It is desirable that such macromolecules be biochemically stable and designed in such a way that the design process is convenient. In addition, recombinant constructs of multivalent antigen-binding macromolecules are desirable that can be expressed in transformed host cells where there is a high likelihood of success in expressing and producing the desired product in good yield. The basis of the present description is the unexpected finding that the multivalent recombinant antibodies described in the present invention can advantageously be produced recombinantly in large quantities in stably transformed cells. Expression levels are observed that are comparable to recombinantly expressed conventional immunoglobulins. The multivalent recombinant antibodies described in the present invention are of great advantage as a reagent for the detection of an analyte. In this regard, the disclosed recombinant antibodies improve the signal-to-noise ratio in immunoassays, especially when compared to conventional immunoglobulins.

Brief description of the invention

As a first object related to all other objects and described embodiments of the present invention, the present invention provides a multivalent recombinant antibody that contains a number of p Fab _L light chain polypeptides and a dimer of two heavy chain polypeptides, where each heavy chain polypeptide has the structure of formula I

Where

(a) p is a value selected from the group consisting of 6, 8, and 10, each of m and n is independently selected as an integer from 1 to 3, and each of m and n is selected such that the value of p is (2 +2×(n+m)),

(b) "-" means a covalent bond in the polypeptide chain,

(c) each L designation is optional and, if present, is an independently selected variable linker amino acid sequence,

(d) each designation dd(Fc _H ) is a heavy chain dimerization region of an immunoglobulin region that is not antigen-binding,

(e) in a dimer, two dd(Fc _H ) are aligned relative to each other in physical proximity;

(e) each Fab _H designation is independently selected from A _H and B _H where A _H and B _H are different and A _H and B _H are independently selected from the group consisting of

Where

VH stands for the N-terminal variable domain of an immunoglobulin heavy chain,

VL stands for the N-terminal immunoglobulin light chain variable domain,

CH1 denotes the C-terminal constant domain 1 of the immunoglobulin heavy chain,

CL stands for C-terminal immunoglobulin light chain constant domain,

(a) each Fab _L is independently selected from A _L and B _L where A _L and B _L are different and A _L and B _L are independently selected from the group consisting of

(b) each Fab _H :Fab _L antigen-binding site of an antibody is an aligned pair (the alignment is indicated by ":"), where each aligned pair is independently selected from the group consisting of A _H :A _L and B _H :B _L , where A _H :A _L and B _H :B _L are independently selected from the group consisting of

[VL-CH1] _H :[VH-CL] _L ,

[VL-CL] _H :[VH-CH1] _L ,

[VH-CH1] _H :[VL-CL] _L ,

[VH-CL] _H :[VL-CH1] _L ,

where in each aligned pair, the corresponding CL and CH1 are covalently linked through a disulfide bond.

As a second object related to all other objects and embodiments of the present invention, the present invention uses the multivalent antibody of the present invention in an antigen detection assay. As a third aspect relating to all other aspects and embodiments of the present invention, the present invention uses a kit containing a chimeric or non-chimeric multivalent recombinant antibody described as the first aspect of the present invention.

As a fourth aspect related to all other aspects and described embodiments of the present invention, the present invention provides a method for detecting an antigen comprising the steps of contacting a multivalent recombinant antibody described as a first aspect of the present invention with an antigen, thereby forming a complex of the antigen and multivalent recombinant antibody, followed by detection of the resulting complex, thereby detecting the antigen. In one of the embodiments of the present invention, the method includes the steps of (a) mixing a multivalent recombinant antibody of the present invention with a liquid sample suspected of containing an antigen, (b) incubating the sample and the multivalent recombinant antibody from step (a), resulting in the formation of a complex of antigen and multivalent recombinant antibody, if the antigen is present and available for contact with the multivalent recombinant antibody during incubation, (c) detecting the complex formed in step (b), thereby detecting the antigen.

Detailed description of the invention

The term "antibody" is used in the present specification in the broadest sense, as it refers to various antibody structures, including, but not limited to, structures known as monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) and antibody fragments. provided that they exhibit appropriate antigen-binding activity.

The term "antibody specificity" refers to the selective recognition by an antibody of a particular epitope of an antigen. Natural antibodies, for example, are monospecific. In the context of the present invention, the term "monospecific antibody" means an antibody that has one or more binding sites, each of which binds to the same epitope of the same antigen. Thus, "monospecific" antibodies are antibodies that bind to a single epitope. By way of example, to which the present invention is not limited, monoclonal antibodies are monospecific. More generally, an antibody capable of binding only one epitope is understood to be monospecific. Obviously, for the purposes of the present description, as well as all objects and embodiments of the present invention, more than one binding site may exist or be found in relation to one epitope, and the binding sites are specific to the epitope. Thus, the term "monospecific" covers different binding sites, if they are usually defined by specificity for the same epitope. In this respect, a monospecific antibody may include binding sites that differ in epitope binding kinetics.

"Bispecific", "trispecific", "tetraspecific", "pentaspecific", "hexaspecific" and other antibodies, also called "multispecific" antibodies, bind two or more different epitopes (e.g., two, three, four or more different epitopes) . Epitopes may or may not be identical, they may be on the same or on different antigens. An example of a multispecific antibody is a "bispecific antibody" that binds two different epitopes. Typically, when an antibody has more than one specificity, the recognized epitopes may be associated with one antigen or with more than one antigen.

Used in the present invention, the concept of "valence" means the presence of a certain number of binding sites in the antibody molecule. For example, a natural IgG immunoglobulin antibody has two binding sites and is therefore bivalent. Thus, "trivalent" means there are three binding sites in an antibody molecule, "quadrivalent" means four binding sites, "hexavalent" means six binding sites, and so on.

"Conservative substitutions" refer to both amino acid and nucleic acid sequences. With regard to specific nucleic acid sequences, "conservatively substituted" refers to those nucleic acids that encode identical or substantially identical amino acid sequences, or if the nucleic acid does not encode an amino acid sequence, substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for a particular protein. For example, all codons GCA, GCC, GCG and GCU code for the amino acid alanine. Thus, at each position where alanine is designated by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one type of conservatively modified variations. In the present invention, each nucleic acid sequence that encodes a polypeptide also describes each possible silent variation of the nucleic acid. One skilled in the art will appreciate that every codon in a nucleic acid (with the exception of AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silent variation of the nucleic acid that encodes a polypeptide is latent in each described sequence.

With regard to amino acid sequences, it is known to those skilled in the art that single substitutions in a peptide, polypeptide or protein sequence that change one amino acid or a small percentage of the amino acids in an amino acid sequence are "conservative substitutions" where the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables containing functionally similar amino acids are known to those skilled in the art. Conservative substitution tables containing functionally similar amino acids are known to those skilled in the art. Each of the following eight groups contains amino acids that are conservative substitutions for each other:

The term "conservative amino acid substitutions" refers to all substitutions in which the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the control sequence. For example, conservative amino acid substitutions include replacing one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, isoleucine, methionine, phenylalanine, or tryptophan, with another; replacing one hydroxyl-containing amino acid, such as serine and threonine, with another; replacing one acid residue, such as glutamic acid or aspartic acid, with another; replacing one amide-containing residue, such as asparagine and glutamine, with another; replacing one aromatic residue, such as phenylalanine and tyrosine, with another; replacing one basic residue, such as lysine, arginine and histidine, with another; and replacing one small amino acid, such as alanine, serine, threonine, and glycine, with another.

In the context of the present invention, the terms “deletion” and “insertion” in relation to an amino acid sequence mean the removal or addition of one or more amino acids to the amino terminus, carboxy terminus, internal part of the amino acid sequence, or a combination thereof, for example, the insertion can be made to one of the antibodies , which are the objects of the present invention.

In the context of the present invention, the term "homologous sequences" refers to amino acid sequences that are at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% homologous to the corresponding control sequences. Sequences that are at least 90% identical have no more than 1 change, ie any combination of deletions, insertions or substitutions, per 10 amino acids of the reference sequence. The percent homology is determined by comparing the amino acid sequence of this variant with a control sequence, which is carried out, for example, according to the MEG ALIGN ™ project in the DNA STAR ™ program.

The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or sub-sequences that are the same. Sequences are "substantially identical" if they share a percentage of the same amino acid residues or nucleotides that is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% match and align identity to determine the best fit within a comparison window or defined region as measured using one of the following sequence comparison algorithms (or other algorithms available to those skilled in the art) or by manual alignment and visual inspection. This definition also applies to the addition of a test sequence. An identity may exist in a region that is at least about 50 amino acids or nucleotides in length, or in a region that is 75-100 amino acids or nucleotides in length, or, unless specifically indicated, the entire sequence of a polynucleotide or polypeptide. A polynucleotide encoding a polypeptide of the present invention, including non-human homologues from other species, can be obtained by a method comprising the steps of screening the library under stringent hybridization conditions with a labeled probe having the polynucleotide sequence or fragment thereof of the present invention and isolating full-length cDNA and genomic clones containing the specified polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art.

When comparing sequences, it is common to consider one sequence as a control sequence against which the test sequences are compared. When using the sequence comparison algorithm, the studied and control sequences are entered into the computer, if necessary, the coordinates of the subsequence and the program parameters of the sequence algorithm are indicated. You can use the default program settings or specify alternative settings. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences relative to their control sequences based on the program parameters.

"Antibody fragments" include a portion of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, in Huston JS, Methods in Enzymol. 203, 1991, 46-88. In addition, antibody fragments contain single-chain polypeptides having properties of the V _H domain, namely the ability to assemble with the V _L domain or bind the V _L domain to IGF-1, namely the ability to assemble together with the V _H domain with a functional antigen binding site and thus thus, the properties of the antibody of the present invention are provided.

Used in the present invention, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to the preparation of antibody molecules of a single amino acid composition.

The term "specific binding agent" is used to refer to an agent used that is capable of either specifically binding to, or being specifically associated with, the test substance of interest. Many different analytical approaches for performing immunoassays are known in the art. Depending on the particular assay scheme, various biotinylated specific binding agents may be used. In one embodiment of the present invention, the biotinylated specific binding agent is selected from the group consisting of a biotinylated test substance-specific binding agent, a biotinylated solid-phase-bound test substance, and a biotinylated solid-phase-bound antigen.

The term “test substance-specific binding agent” refers to a molecule that specifically binds to the test substance of interest. An analyte-specific binding agent in the present invention typically includes binding or capturing molecules capable of binding to the analyte (also referred to as the analyte of interest; target molecule). In one embodiment of the present invention, the analyte-specific binding agent exhibits a binding affinity of at least 10 ⁷ l/mol for the respective target molecule, ie the analyte. A test substance-specific binding agent in other embodiments of the present invention exhibits an affinity of 10 ⁸ L/mol or even 10 ⁹ L/mol for the respective target molecule. The person skilled in the art will appreciate that the term "specific" is used to mean that other biomolecules present in the sample do not substantially bind to a binding agent specific to the test substance. In some embodiments of the present invention, the level of binding to a biomolecule other than the target molecule results in a binding affinity of only 10%, more preferably only 5% of the binding affinity to the target molecule or less. In one embodiment of the present invention, the binding affinity for molecules other than the test substance is not measurable. In another embodiment of the present invention, the binding agent specific for the test substance meets both the above minimum criteria for binding affinity (affinity) and specificity.

The term "test substance-specific binding" in the context of the present invention means the immunospecific interaction of an antibody with its target epitope on the test substance, ie the binding of the antibody to the epitope on the test substance. The concept of test substance-specific binding of an antibody through its epitope on the test substance is fully understood by those skilled in the art.

The terms "polypeptide", "peptide" and "protein" refer to a polymer of amino acid residues. These terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues is not a naturally encoded amino acid. In the context of the present invention, the terms cover amino acid chains, where the amino acid residues are linked by covalent peptide bonds. Polypeptides, peptides and proteins are written using standard sequence notation with the nitrogen end on the left and the carboxy end on the right. Standard single-letter designations are used as follows: A-alanine, C-cysteine, D-aspartic acid, E-glutamic acid, F-phenylalanine, G-glycine, H-histidine, S-isoleucine, K-lysine, L-leucine, M -methionine, N-asparagine, P-proline, Q-glutamine, R-arginine, S-serine, T-threonine, V-valine, W-tryptophan, Y-tyrosine. In the present invention, the term "peptide" refers to a polymer of amino acids that is up to 5 amino acids in length. In the present invention, the term "polypeptide" refers to a polymer of amino acids that is 6 or more amino acids in length. The term "protein" means a polypeptide chain or a polypeptide chain with additional modifications such as glycosylation, phosphorylation, acetylation or other post-translational modifications.

"Antibody fragments" include a portion of an intact antibody, preferably containing its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; single chain antibody molecules; scFv, sc(Fv)2; diabody; multispecific antibodies formed from antibody fragments.

When antibodies are digested with papain, two identical antigen-binding fragments are formed, called "Fab" fragments, each of which has one antigen-binding site and a residual "Fc" fragment, the name of which reflects its ability to easily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen binding sites and is still capable of antigen crosslinking.

The Fab fragment contains the heavy and light chain variable domains, as well as the light chain constant domain and the heavy chain first constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. In the present invention, Fab'-SH is a Fab' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments were originally generated as pairs of Fab fragments with hinged cysteines between them. Other chemical combinations of antibody fragments are also known.

In the context of the present invention, the term "monoclonal antibody" refers to an antibody isolated from a population of essentially homogeneous antibodies, i.e. the individual antibodies that make up the population are identical except for possible mutations, for example, naturally occurring mutations that may be present in small amounts. Thus, the term "monoclonal" indicates that such an antibody is not a mixture of individual antibodies. In some embodiments of the present invention, such a monoclonal antibody is usually an antibody containing a polypeptide sequence that binds a target, and the polypeptide sequence that binds to the target is obtained by a method that includes selecting one polypeptide sequence that binds the target from a plurality of polypeptide sequences. For example, the process of selecting such a sequence may be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be noted that the selected target binding sequence can be further modified, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce in vivo immunogenicity, to create a multispecific antibody, etc. .d., and that an antibody containing an altered target-binding sequence is also a monoclonal antibody of the present invention. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally not contaminated with other immunoglobulins.

The term "monoclonal" refers to the property of an antibody derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be obtained by various methods, including, for example, the hybridoma method (see, for example, Kohler and Milstein., Nature, 256, 1975, 495-497; Hongo et al., Hybridoma , 14(3), 1995, 253-260; Harlow et al., Antibodies: A Laboratory Manual, 1988, 2nd ed., Cold Spring Harbor Laboratory Press; Haemmerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 1981, 563-681, Elsevier, New York), DNA recombination methods (see, for example, US 4816567), phage display methods (see, for example, Clackson et al., Nature, 352 , 1991, 624-628; Marks et al., J. Mol. Biol. 222, 1992, 581-597; Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee J Mol Biol 340(5) 2004 1073-1093 Fellouse PNAS USA 101(34) 2004 12467-12472 Lee et al J Immunol Methods 284(1- 2), 2004, 119-132; and technologies for producing human or animal antibodies, similar to human antibodies that have parts or all of human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., PNAS USA 90:2551, 1993; Jakobovits et al., Nature 362, 1993, 255-258; Bruggemann et al., Year in Immunol. 7:33, 1993; US 5545807; 5545806; 5569825; 5625126; 5633425; 5661016; Marks et al., Bio/Technology 10 1992, 779-783; Lonberg et al., Nature 368, 1994, 856-859; Morrison, Nature 368, 1994, 812-813; Fishwild et al., Nature Biotechnol. 14, 1996, 845-851; Neuberger, Nature Biotechnol. 14, 1996, 826; Lonberg and Huszar, Intern. Rev. Immunol. 13, 1995, 65-93).

In the present invention, monoclonal antibodies specifically refer to "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (see, for example, US 4816567 and Morrison et al., PNAS USA 81, 1984, 6851-6855). Chimeric antibodies include PRIMATIZED® antibodies in which the antigen-binding region of the antibody is derived from an antibody generated, for example, by immunizing monkeys with an antigen of interest.

The concept of "hypervariable region (hypervariable region)", "HVR" or "HV" in the context of the present invention refers to regions of the variable domain of an antibody that are hypervariable in sequence and/or form structurally defined loops. Typically, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In native antibodies, H3 and L3 show the most diversity of the six HVRs, and in particular, H3 is considered to play a unique role in conferring fine specificity on antibodies. See, for example, Xu et al., Immunity 13, 2000, 37-45; Johnson and Wu, in: Methods in Molecular Biology 248, 2003, 1-25, ed. Lo, Human Press, Totova, New Jersey. Indeed, natural camelid antibodies, consisting only of a heavy chain, are functional and stable, although there is no light chain. See, for example, Hamers-Casterman et al., Nature 363, 1993, 446-448; Sheriff et al., Nature Struct. Biol. 3, 1996, 733-736.

A number of HVR descriptions are used in the present invention. HVRs, which are Kabat's complementarity-determining regions (CDRs), are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5e ed., 1991, ed. at the Public Health Service, National Institutes of Health, Bethesda, Maryland). Chothia refers instead to the arrangement of structural loops (Chothia and Lesk, J. Mol. Biol. 196, 1987, 901-917). Monoclonal antibody (AbM) HVRs represent a compromise between Kabat CDRs and Chothia structural loops; they are used in Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on the analysis of available complex crystal structures. The remains of each of these HVRs are listed below.

HVRs can contain "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL and 26-35 (H1 ), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al., see above for a description of each of these extended HVRs.

The expressions "Kabat numbering of variable domain residues" or "Kabat numbering of amino acid positions" and variants thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains of the antibody compilation in Kabat et al., see above. Using this numbering system, a certain linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion in the FR or HVR of the variable domain. For example, a heavy chain variable domain may include one amino acid insertion (Kabat residue 52a) after residue 52 in H2 and inserted residues (e.g., Kabat residues 82a, 82b and 82c, etc.) after heavy chain FR residue 82. Kabat residue numbering can be determined for a given antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence.

The concept of "experimental animal" does not apply to humans. In one embodiment of the present invention, the experimental animal is selected from rats, mice, hamsters, rabbit, camels, llamas, primates (excluding humans), sheep, dogs, cows, chickens, amphibians, sharks and reptiles. In one such embodiment of the present invention, the experimental animal is a rabbit.

An epitope is a region of an antigen that is associated with an antibody binding site. The term "epitope" includes any determinant capable of specifically binding to an antibody. In some embodiments, epitope determinants include reactive surface moieties such as amino acids, glycan side chains, phosphoryl, or sulfonyl, and in some embodiments, certain three-dimensional structural properties and/or certain charge characteristics may be present.

As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody to an antigen epitope in an in vitro assay, in particular a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) with purified antigen. In certain embodiments of the present invention, an antibody specifically binds to an antigen when it preferentially recognizes the target antigen in a complex mixture of proteins and/or macromolecules.

The binding affinity of an antibody to an antigen is defined in terms of k _a (rate constant for association of an antibody in an antibody/antigen complex), (dissociation rate constant) and K _D (k _d /k _a ). In one embodiment of the present invention, binding or what specifically binds means an affinity (binding affinity) (K _D ) of 10 ^-7 mol/l or less, in one embodiment of the present invention from 10 ^-7 M to 10 ^-13 mol/ l. Thus, a multispecific antibody, in all of its aspects and embodiments of the present invention, specifically binds to each target antigen for which it is specific with a binding affinity (K _D ) of 10 ^-7 mol/l or less, for example, with binding affinity (K _D ) from 10 ^-7 to 10 ^-13 mol/L. In one embodiment of the present invention, the binding affinity (K _D ) is from 10 ^-8 to 10 ^-13 mol/l. In this regard, the target antigen may be a single molecule or different molecules. In one of the embodiments of the present invention, the target antigen is a complex formed by two or more different molecules, where the multispecific antibody specifically binds to the complex with a binding affinity (K _D ) of 10 ^-7 mol/l or less.

In the present invention, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules of a single amino acid composition. A recombinant antibody according to all provided objects and embodiments of the present invention, refer to the concept of "monoclonal antibody".

The term "chimeric" refers to an antibody in which part of the heavy and/or light chain is from a particular source or species, while the remainder of the heavy and/or light chain is from a different source or species. In one of the embodiments of the present invention, the recombinant antibody of all objects and variants of implementation described in the present invention may contain a part of Fab _H :Fab _L derived from the first species, and the Fc _H part from the second species. In another embodiment of the present invention, the recombinant antibody contains many distinctive features with different sections of Fab _H :Fab _L obtained from different sources or species.

The terms "binding site" or "antigen-binding site" as used in the present invention means the region(s) of an antibody molecule to which a ligand (eg, its antigen or antigenic fragment) actually binds, and which is derived from the antibody. The antigen binding site includes antibody heavy chain variable domains (VH) and/or antibody light chain variable domains (VL) or a VH/VL pair.

Antigen-binding sites that specifically bind to the desired antigen can be derived a) from known antibodies that specifically bind to the appropriate target antigen, or b) from novel antibodies or antibody fragments generated by de novo immunization methods using, inter alia, either the protein of the antigen , or a nucleic acid encoding a protein as a target antigen or fragments thereof, or by phage display.

The antigen-binding site of an antibody, including a recombinant antibody described in all aspects and embodiments of the present invention, may contain six complementarity determining regions (CDRs) that contribute to varying degrees to the binding site's affinity for an antigen epitope. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDRs and framework regions (FRs) is determined by comparison with a compiled database of amino acid sequences in which these regions have been defined according to the variability among these sequences. Also within the scope of the present invention are functional antigen binding sites consisting of fewer CDRs (ie where binding specificity is determined by three, four or five CDRs). For example, less than 6 CDRs may be sufficient for binding. In some cases, a VH or VL domain is sufficient.

"Light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two different types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. A wild-type light chain typically contains two immunoglobulin domains, typically one variable domain (VL), which is important for antigen binding, and a constant domain (CL).

There are several different types of "heavy chains" that define the class or isotype of an antibody. The wild-type heavy chain contains a series of immunoglobulin domains, usually with one variable domain (VH), which is important for antigen binding, and several constant domains (CH1, CH2, CH3, etc.).

The term "domain Fc" is used in the present invention to define the C-terminal region of the heavy chain of an immunoglobulin, which contains at least part of the constant region. For example, in natural antibodies, the Fc domain consists of two identical protein fragments derived from the second and third constant domains of the two antibody heavy chains in the IgG, IgA, and IgD isotypes; The Fc domains of IgM and IgE contain three heavy chain constant domains (CH 2-4 domains) in each polypeptide chain. In the present invention, the term "deprived of the Fc domain" means that the bispecific antibodies of the present invention do not contain a CH2, CH3 and CH4 domain; that is, the constant heavy chain consists solely of one or more CH1 domains.

In the context of the present invention, the terms "variable domains" or "variable region" means each of the pair of light and heavy chains that is directly involved in the binding of an antibody to an antigen. The light chain variable domain is designated "VL" and the light chain variable domain is designated "VH". The variable domains of human light and heavy chains have the same general structure. Each variable domain includes four framework regions (FRs) that are highly sequence conserved. The FRs are linked by three "hypervariable regions" (or "complementarity determining regions, CDRs"). The CDRs in each chain are separated by such framework amino acids. Therefore, the light and heavy chains of antibodies contain in the direction from the N - to the C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The FR adopts a β-sheet conformation and the CDRs can form loops connecting the β-sheet structure. The CDRs in each strand are held in their three-dimensional structure by the FR and, together with the CDRs from the other strand, form an "antigen binding site". The heavy chain CDR3 is the region that most promotes antigen binding. CDR and FR regions are defined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Public Health Service, National Institutes of Health, Bethesda, MD.

The term "constant domains" or "constant region" as used in this application means the sum of antibody domains other than the variable region. The constant region is not directly involved in antigen binding, but exhibits various effector functions.

Depending on the amino acid sequence of the heavy chain constant region, antibodies are divided into classes: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain constant regions that correspond to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The light chain constant regions (CL) that can be found in all five classes of antibodies are denoted κ (kappa) and λ (lambda). As used herein, "constant domains" are human constant domains derived from a heavy chain constant region of a human IgG1, IgG2, IgG3 or IgG4 antibody subclass and/or a light chain kappa or lambda constant region. Such constant domains and regions are known in the art and are described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5e ed., 1991, Public Health Service, National Institutes of Health, Bethesda, Maryland.

In the context of the present invention, the term "tertiary structure" refers to the geometric shape of the antibody of the present invention. The tertiary structure contains the scaffold of the polypeptide chain containing the domains of the antibody, and the amino acid side chains interact and bind in a number of ways.

The multivalent antibody of the present invention is produced by recombination methods. Methods for recombinant production of antibodies are well known in the art and include protein expression in prokaryotic and eukaryotic cells, followed by isolation of the antibody and, as a rule, purification to pharmaceutically acceptable purity. For expression of antibodies in a host cell, nucleic acids encoding the respective light and heavy chains as described in the present invention are inserted into expression vectors by standard methods. Expression occurs in appropriate prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). General methods for the recombinant production of antibodies are well known in the art and are described, for example, in the review articles Makrides S.C., Protein Expr. Purif. 17, 1999, 183-202; Geisse S. et al., Protein Expr. Purif. 8, 1996, 271-282; Kaufman R.J., Mol. Biotechnol. 16, 2000, 151-161; Werner R.G., Drug Res. 48, 1998, 870-880.

In the context of the present invention, the term "polynucleotide" or "nucleic acid" is used interchangeably; it refers to polymers of nucleotides of any length and represents DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthesis reaction. The polynucleotide may contain modified nucleotides such as methylated nucleotides and their analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may contain a modification(s) made after synthesis, for example, obtained by conjugation with a label. Other types of modifications include, for example, "caps", replacement of one or more natural nucleotides with analogs, internucleotide modifications, for example, modifications with uncharged bonds (for example, methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged bonds (for example , phosphorothioates, phosphorodithioates, etc.) containing side fragments, e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.) containing intercalators (e.g., acridine, psoralen etc.), containing chelators (for example, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylators, containing modified bonds (for example, alpha-anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). In addition, any of the hydroxyl groups normally present in sugars may be replaced, for example, with phosphonate groups, phosphate groups, protected with standard protecting groups, or activated to provide additional bonds to additional nucleotides, or may be conjugated to solid or semi-solid carriers. . The 5'- and 3'-terminal OH may be phosphorylated or substituted with amines or fragments of organic capping groups containing from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to provide standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose , carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, those in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate" ), P(O)R, P(O)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing ether ( - O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all bonds in a polynucleotide need to be identical. The previous description applies to all polynucleotides listed here, including RNA and DNA.

The term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or at a location on the chromosome that differs from its natural chromosomal location.

In the context of the present invention, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is associated. The concept includes a vector as a self-replicating nucleic acid structure, as well as a vector integrated into the genome of the host cell into which it has been introduced. The term includes vectors that function primarily to insert DNA or RNA into a cell (for example, chromosomal integration), replication vectors that function primarily to replicate DNA or RNA, and expression vectors whose function is to carry out transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one of the functions described.

An "expression vector" is a vector capable of directing the expression of the nucleic acids to which it is operably linked. When an expression vector is introduced into an appropriate host cell, it can be transcribed and translated into a polypeptide. When transforming host cells, the methods of the present invention use "expression vectors"; thus the term "vector" in connection with the transformation of host cells, as described in the present invention, means "expression vector". "Expression system" generally refers to an appropriate host cell consisting of an expression vector that can function to produce the desired expression product.

In the context of the present invention, expression refers to the process by which a nucleic acid is transcribed into mRNA and/or the process by which a transcribed mRNA (also referred to as a transcriptome) is subsequently translated into peptides, polypeptides or proteins. The transcripts and encoded polypeptides are collectively referred to as a gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may involve mRNA splicing.

In the context of the present invention, the term "transformation" refers to the process of transferring vectors/nucleic acid into a host cell. If cells without insurmountable cell wall barriers are used as host cells, then transfection is carried out, for example, by the calcium phosphate precipitation method as described by Graham and Van der Eh, Virology 52, 1978, 546ff. However, other methods for introducing DNA into cells can also be used, such as nuclear injection or protoplast fusion. If prokaryotic cells or cells that contain a cell wall that is difficult to overcome are used, one of the methods of transfection is, for example, treatment with calcium, namely calcium chloride, as described by Cohen F.N. et al., PNAS 69, 1972, 7110 et seq.

The term "host cell", as used in the present invention, means any cellular system that can be developed to obtain antibodies in accordance with the present invention.

In the present invention, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such designations also refer to the progeny of these cells. Thus, the terms "transformants" and "transformed cells" mean the primary subject cell and cultures derived from it, without regard to the number of transfers. It should also be noted that all offspring may not be completely identical in DNA content due to intentional or accidental mutations. Included within the term are progeny variants that have the same function or biological activity as that found in the originally transformed cell. Where other definitions are implied, it follows from the context.

The expression of NS0 cells is described, for example, in Barnes L.M. et al., Cytotechnology 32, 2000, 109-123; Barnes L.M. et al., Biotech. Bioeng. 73, 2001, 261-270. Temporal expression is described, for example, in Durocher Y. et al., Nucl. Acids. Res. 30, 2002, E9. Cloning of variable domains is described in Orlandi R. et al., Proc. Natl. Acad. sci. USA 86, 1989, 3833-3837; Carter P. et al., Proc. Natl. Acad. sci. USA 89, 1992, 4285-4289; Norderhaug L. et al., J. Immunol. Methods 204, 1997, 77-87. A preferred transient expression system (HEK 293) is described in Schlaeger E.-J. and Christensen K., Cytotechnology 30, 1999, 71-83; Schlaeger E.-J., J. Immunol. Methods 194, 1996, 191-199.

Antibodies produced by host cells may undergo post-translational cleavage of one or more, especially one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a cleaved variant of the heavy chain). For example, this may be when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, Kabat EU numbering).

Therefore, heavy chain amino acid sequences, including CH3 domains, are designated herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated.

The compositions of the present invention, for example, the pharmaceutical compositions described herein, comprise the antibody population of the present invention. The antibody population may include antibodies having a full length heavy chain and antibodies having a cleaved variant heavy chain. In one embodiment of the present invention, the antibody population consists of a mixture of antibodies having a full-length heavy chain and antibodies having a cleaved variant of the heavy chain, where at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies have a cleaved heavy chain variant.

Antibody purification (isolation of antibodies from cultured host cells) is performed to remove cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard methods including alkaline/SDS treatment, CsCl gradient, column chromatography, agarose gel electrophoresis, and others. methods well known in the art. See Current Protocols in Molecular Biology, 1987, ed. Ausubel F. et al., Greene Publishing and Wiley Interscience, New York. Various methods known and widely used for protein purification include microbial protein affinity chromatography (e.g., protein A or protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange (carboxymethyl resins), anion exchange (aminoethyl resins), and in mixed mode), thiophilic adsorption (for example, with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (for example, with phenylsepharose, azaarenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (for example, with using Ni(II)- and Cu(II)-bound material), size exclusion chromatography and electrophoretic methods (e.g. gel electrophoresis, capillary electrophoresis) (Vijayalakshmi M.A., Appl. Biochem. Biotech. 75, 1998,93- 102).

The term "immunoconjugate" means an antibody conjugated to one or more heterologous molecules, including, but not limited to, a detectable label.

Immunoglobulins are glycoproteins that specifically bind to target antigens. Bivalent and monospecific immunoglobulins, such as natural forms of IgG, have a four-chain structure as a backbone. They consist of two identical light chains (L) and two identical heavy chains (H) held together by interchain disulfide bonds and non-covalent interactions. IgM immunoglobulin is an exception in terms of the number of heavy and light chains and is not considered further. The light chain is formed by two domains, variable and constant, and the heavy chain is formed by one variable domain and three constant domains. Immunoglobulin sequences are usually numbered according to a general scheme (Kabat-Chothia) designed to assign the same serial number to topologically equivalent residues (Al-Lazikani B. et al., J. Mol. Biol. 273, 1997, 927-948 ). It is a widely accepted single standard for ordering and numbering antibody residues.

In describing the present invention relating to the modular design of a recombinant antibody, one or more domains or regions of an immunoglobulin can be considered as design elements. In the context of the present invention, one element is selected from the group consisting of "CH1", "CH2", "CH3", "CL", "VH", "VL", "<hinge region>" and "L". Each of these individual elements can also be referred to as the "basic element" of the heavy or light chain when considering the structure of the recombinant antibody as a subject of the present invention. Several basic elements can be combined into a higher order element, for example, "Fc _H ", "Fab _H " and "Fab _L ", but the list is not limited to them, the result is a combination of the main elements presented in formulas II to X, respectively.

It will be apparent to those skilled in the art that recombinant technologies allow the construction of various non-naturally occurring Fab _H and Fab _L elements as shown above, such as, but not limited to, VL-CL _H or VH-CH1 _L . An antibody with a CL-CH1 substitution in the binding arm is an example of the so-called CrossMab. Crossed monoclonal antibodies (CrossMab) are described in detail in WO 2009/080253 and Schaefer W. et al., PNAS, 108, 2011, 11187-11191. As a rule, unless explicitly stated otherwise, the orientation of each group of elements is always from the N-terminus on the left side to the C-terminus on the right side. In all representations of polypeptide chains with basic and/or higher order elements, "-" denotes a covalent bond in the polypeptide chain. It should also be appreciated that in the description, a subset of heavy chain elements may be given as a single entry (e.g., X=[-L-Fab _H ] or Y=[Fab _H -L-], as appropriate) for the purpose of specifically explaining the elements. in a subset and/or its features. Unless otherwise indicated, any heavy chain subgroup discussed in the present invention is considered to be a covalently linked integral part of the full contiguous heavy chain polypeptide. Similarly, a light chain is always referred to as a set of elements, such as formulas VI to IX, ie without additional elements present in the corresponding polypeptide chain, unless otherwise indicated.

One skilled in the art of recombinant immunoglobulin construction will recognize that each of the basic elements "CH1", "CH2", "CH3", "CL", "VH", "VL", "<hinge region>" and "L" reflects a functional entity that does not change with minor changes in the amino acid sequence of the corresponding element. For example, there may be one or more neutral amino acid exchanges, or minimal additions or minimal deletions of amino acids, in particular terminal additions of 1 to 20 amino acids to any of the basic elements, however, provided that, despite the presence of these variations, formed a functional immunoglobulin according to the first aspect of the present invention, ie the alignment of the heavy and light chains is not adversely affected and the functionality of the antigen binding sites is not disturbed. That is, in the presence of neutral amino acid exchanges, or minor additions or minor deletions, the alignment and covalent bonding of the two heavy chains remain unchanged, and the alignment and covalent bonding of Fab _H :Fab _L remain unchanged, and the specificity and sensitivity of antigen binding are unchanged. In this regard, the meanings of "intact" and "unchanged" are in the range of 95% to 100% of each individual respective property compared to the property of the corresponding immunoglobulin without any of these variations.

In the context of the present invention, the basic architecture of a "conventional Ig isotype" is represented by the architecture of a naturally occurring monospecific and divalent isotype antibody selected from the group consisting of IgG, IgA and IgD, wherein the antibody comprises two immunoglobulin heavy chain polypeptides of formula XI

and two Fab _L immunoglobulin light chains, where "" means a covalent bond in the polypeptide chain; where Fc _H is the immunoglobulin heavy chain portion comprising an N-terminal hinge domain (=<hinge region>) followed by a heavy chain constant domain 2 (=CH2) and a C-terminal heavy chain constant domain 3 (=CH3); and wherein each Fab _H is part of an immunoglobulin heavy chain VH-CH1 comprising an N-terminal heavy chain variable domain (=VH) and a C-terminal heavy chain constant domain 1 (=CH1); where each Fab is a VL-CL of an immunoglobulin light chain containing a light chain variable domain at the N-terminus (=VL) and a light chain constant domain at the C-terminus (=CL); where in the antibody, the respective hinge domains, CH2 and CH3, of the two heavy chains are aligned with each other, and the respective hinge domains of the two heavy chains are covalently linked to each other via one or more disulfide bonds; where each Fab _H :Fab _L antigen-binding site of an antibody is an aligned pair of the VH-CH1 immunoglobulin heavy chain portion and the VL-CL immunoglobulin light chain portion, where in each pair the corresponding CL and CH1, and VL and VH are aligned relative to each other, and in each the pair corresponding to VL-CL and VH-CH1 are covalently linked via a disulfide bond.

Recombinantly engineered examples and established variants of a monospecific and divalent common Ig isotype antibody known to one of skill in the art include antibodies with a Fab _H that is selected from the group consisting of

where VH is an immunoglobulin heavy chain variable domain, VL is an immunoglobulin light chain variable domain, CH1 is an immunoglobulin heavy chain constant domain 1, and is an immunoglobulin light chain constant domain;

and each Fab _L is independently selected from the group consisting of

where the Fab _H :Fab _L binding site of the antibody is an aligned pair, the alignment is indicated by ":", where the aligned pair is independently selected from the group consisting of

[VL-CH1] _H :[VH-CL] _L ,

[VL-CL] _H :[VH-CH1] _L ,

[VH-CH1] _H :[VL-CL] _L ,

[VH-CL] _H :[VL-CH1] _L ,

and where in an aligned pair, the respective CL and CH1 are covalently bonded via a disulfide bond.

A particular technical advantage regarding the use of an antibody specific for an analyte to detect an analyte as a target in a complex mixture has been found by experimentally extending the conventional basic architecture by adding/attaching additional analyte-specific Fab _H :Fab _L units. Surprisingly, extension of the antibody by adding additional Fab _H :Fab _L antigen binding sites in the Fc region has been found to provide an additional advantage in terms of the binding properties of the recombinant antibody. In addition, expanding each antibody arm by adding additional Fab _H :Fab _L antigen binding sites also improves binding properties. As a result, compared with a conventional IgG molecule, a single multivalent recombinant antibody has an increased value regarding the ratio of the surface of antigen-binding regions to the surface of non-antigen-binding regions. A particular advantage is the improved signal to noise ratio when an antibody of the present invention is used as a capture and/or detection agent, for example in a sandwich immunoassay for antigen detection.

Thus, a chimeric or non-chimeric polyvalent recombinant antibody is contemplated as a first object relating to all other objects and embodiments of the present invention, wherein the antibody comprises p Fab _L light chain polypeptides and a dimer of two heavy chain polypeptides, wherein each heavy chain polypeptide has structure of formula I

wherein

(i) p is a number selected from 6, 8 and 10,

each value of m and n is chosen independently from integers from 1 to 3,

each value of m and n is chosen such that the value of p is (2+2×(n+m));

(k) the sign "-" means a covalent bond in the polypeptide chain;

(k) each L is optional and, if present, is an independently selected variable linker amino acid sequence;

(m) each dd(Fc _H ) is a heavy chain dimerization region of a non-antigen binding immunoglobulin heavy chain region;

(h) in the dimer, two dd(Fc _H ) are aligned relative to each other in physical proximity;

(o) each Fab _H is independently selected from A _H and B _H where A _H and B _H are different and A _H and B _H are independently selected from the group consisting of

Where

VH is the N-terminal variable domain of an immunoglobulin heavy chain,

VL is the N-terminal variable domain of an immunoglobulin light chain,

CH1 is the C-terminal constant domain 1 of an immunoglobulin heavy chain, and

CL is the C-terminal constant domain of an immunoglobulin light chain;

(p) each Fab _L is independently selected from A _L and B _L , where A _L and B _L are different, and A _L and B _L are independently selected from the group consisting of

(p) each Fab _H :Fab _L antigen-binding site of an antibody is an aligned pair (the alignment is indicated by ":"), where each aligned pair is independently selected from the group consisting of A _H :A _L and B _H :B _L , where A _H :A _L and B _H :B _L are independently selected from the group consisting of

[VL-CH1] _H : [VH-CL] _L ,

[VL-CL] _H :[VH-CH1] _L ,

[VH-CH1] _H :[VL-CL] _L ,

[VH-CL] _H :[VL-CH1] _L ,

and wherein in each aligned pair, the respective CL and CH1 are covalently bonded via a disulfide bond.

In the present invention, the recombinant antibody is not divalent like a conventional antibody of one of the IgA, IgD, IgE, or IgG immunoglobulin classes, but is multivalent. In the recombinant multivalent antibody of the present invention, the light chains are similar or identical to the light chains of natural immunoglobulins. It is important to note that it is the recombinant immunoglobulin heavy chain construct that allows multivalent antigen binding by generating multiple Fab _H elements in each heavy chain. The novel recombinant antibody described in all aspects and embodiments of the present invention is characterized by a modular construct that combines 4 or more antigen binding sites (=Fab=Fab _H :Fab _L ) in a single antibody molecule. More specifically, the present invention provides for a recombinant multivalent antibody having a number of antigen binding sites indicated by the p value given above in formula I, where p is an integer that may be selected from the group consisting of 6, 8 and 10, and, optionally 12. That is, the number of Fab _H elements contained in one heavy chain is p/2, i.e. an integer selected from the group consisting of 3, 4, 5 and optionally 6.

The values of m and n are chosen independently, and each of m and n is chosen such that the value of p is (2+2×(n+m)). In one embodiment of the present invention, the value m is selected from the group of integers consisting of 0, 1, 2, 3, and 4. More specifically, m is selected from the group of integers consisting of 1, 2, 3, and 4. More specifically, m is selected from the group of integers consisting of 1, 2 and 3. In another embodiment of the present invention, n is selected from the group of integers consisting of 0, 1, 2, 3 and 4. More specifically, n is selected from the group of integers consisting of 1, 2, 3 and 4. More specifically, n is selected from the group of integers consisting of 1, 2 and 3.

For example, in an embodiment of the present invention where p is 6, both m and n are 1. In an embodiment of the present invention where p is 8, or n is 1 and m is 2, or n is 2 and m is 2. In the embodiment where p is 10, n is 1, 2, or 3 and m is 3, 2, or 1, respectively. In an embodiment of the present invention, where p is 12, n is 1, 2, 3, or 4, and m is 4, 3, 2, or 1, respectively.

A recombinant antibody, pertaining to all aspects and embodiments of the present invention, comprises (and in one of the embodiments of the present invention consists solely of) immunoglobulin domains, immunoglobulin elements, and immunoglobulin regions, in accordance with the modular design described in the present invention.

The recombinant antibody, pertaining to all aspects and embodiments of the present invention, contains aligned heavy chains of formula I. Each heavy chain is composed of building blocks starting from the N-terminus in a certain order towards the C-terminus of the heavy chain.

In one embodiment of the present invention, a recombinant antibody relating to all the described objects and embodiments of the present invention is chimeric and contains elements from different species. In another embodiment of the present invention, the recombinant antibody relating to all the described objects and embodiments of the present invention is non-chimeric and contains elements derived from the same species.

A recombinant antibody pertaining to all aspects and embodiments of the present invention is characterized by a modular construct that combines the structural elements of immunoglobulin heavy chain Fc _H constant domains that are known in the art. In the immunoglobulin molecule in its native (ie, not engineered) form, the heavy chain Fc _H polypeptide consists of CH2 and CH3 constant domains as basic elements. Attached to the CH2 domain at the N-terminus is a <hinge region> domain providing cysteine-SH groups for heavy chain cross-links. In one embodiment of the present invention, the Fc _H portion of the recombinant antibody comprises an assembly of domains according to formula X, <hinge region>-CH2-CH3 immunoglobulin belongs to a class selected from the group consisting of IgG, IgA and IgD. In a more specific embodiment, the Fc _H portion is an amino acid sequence derived from a mammalian species selected from the group consisting of human, mouse, rat, sheep and rabbit.

More generally, the Fc _H higher order element is a variant of the heavy chain dimerization domain of the non-antigen binding region of an immunoglobulin, referred to herein as dd (FcH). Of primary importance is the fact that dd(Fc _H ) facilitates alignment and joining of two heavy chain polypeptides that are part of a multivalent recombinant antibody as described herein. The bond in this regard may be entirely due to physical forces, for example, in one embodiment of the present invention, dd(Fc _H ) contains one CH3 element capable of forming a CH3/CH3 complex, as in the paired heavy chains of the IgG molecule. In the present invention, the dd(Fc _H ) variant is a domain containing one or more elements selected from the group consisting of CH3, <hinge>-CH3, and <hinge>-CH2-CH3. In another embodiment of the present invention, dd(Fc _H ) is a single element domain selected from the group consisting of CH3, <hinge>-CH3, and <hinge>-CH2-CH3. In the presence of a hinge region, the connection of two heavy chains is carried out not only by physical forces, but also by disulfide bridges between cysteine residues in the hinge region in the first and second heavy chain of a multivalent recombinant antibody according to the description of the present invention.

The Fc _H portion of the heavy chain may be extended at the N-terminus with a linker amino acid sequence L, which is optional and, if present, is an independently selected variable amino acid sequence of the linker.

As a rule, in the context of the present invention, in connection with all objects and variants of its implementation, a "linker amino acid sequence", denoted by "L", is a peptide sequence containing from 1 to 60 amino acid residues that connects two domains of a polypeptide that contains a plurality of domains, in particular, a plurality of different domains. In the heavy chain of the recombinant antibody described in the present invention, the amino acid sequences of the linker are considered as optional elements located in different places, as presented in formula I.

The amino acid sequence of the linker is usually composed of flexible residues such as glycine and serie, such that adjacent domains of the polypeptide can move freely relative to each other. Longer sequences can be especially useful if it is necessary to ensure that two neighboring domains do not sterically interfere with each other. In one embodiment of the present invention, the linker amino acid sequence contains, more specifically, glycine and serine residues. The amino acids glycine and series are zwitterionic and hydrophilic. These properties make them a frequent choice for repetitive linker sequences. Thus, in an even more specific embodiment of the present invention, each amino acid sequence of a linker in the heavy chain of a recombinant antibody, relating to all objects and embodiments that are described in the present invention, includes an independently selected variable amino acid sequence of the linker selected from the group of formula XII,

where u is an integer selected from 1 to 10, q is an integer from 1 to 5 and r is an integer from 1 to 10. In another embodiment of the present invention, each linker amino acid sequence contains an independently selected variable linker amino acid sequence of formula XII where and is 3 or 4, q is 1, and r is selected from the group consisting of 3, 4, 5, and 6. The very specific linker amino acid sequence includes, even more specifically, consists of the amino acid sequence GGGSGGGSGGGSGGGS (SEQ ID NO: 1 ). One skilled in the art will appreciate the advantage of alternative repeat sequences containing glycine and series, which also serve the same technical purpose. In yet another embodiment of the present invention, the selected linker amino acid sequence is (GSAT) ₁ , (GSAT) ₂ , (GSAT) ₃ or (GSAT) ₄ . In yet another specific embodiment of the present invention, the selected linker amino acid sequence is (SEG) ₁ , (SEG) ₂ , (SEG) ₃ or (SEG) ₄ .

In another embodiment of the present invention, the selected linker amino acid sequence is (EAAAR) ₁ , (EAAAR) ₂ , (EAAAR) ₃ , (EAAAR) ₄ or (EAAAR) ₅ (Merutka G. et al., Biochem. 30, 1991, 4245 -4248; Sommese RF et al., Protein Sci. 19, 2010, 2001-2005; Yan W. et al., Biochem. 46, 2007, 8517-8524). In this context, one skilled in the art will appreciate that EAAR elements are examples of a more rigid linker such that the two attached domains at either end do not come close.

Extension of the Fc _H -portion from the N-terminus of the CH1 domain connected to the hinge domain (<hinge region>) is optionally via an L linker. -CH2-CH3 derived from an immunoglobulin isotype selected from the group consisting of IgG, IgA and IgD. In another embodiment of the present invention, the CH1 domain and <hinge region>-CH2-CH3 portion are represented by an amino acid sequence derived from a mammalian species selected from the group consisting of human, mouse, rat, sheep and rabbit. Examples of parts CH1 and <hinge region>-CH2-CH3 include the corresponding amino acid sequences shown in table. A.

Despite differences in amino acids between isotypes and subclasses within an isotype, each CH region in an immunoglobulin heavy chain folds into a permanent structure consisting of a three-stranded beta sheet of four strands linked together by an intrasystemic disulfide bond (Schroeder H.W., Cavacini L. J Allergy Clin Immunol 125, 2010, S41-S52).

In the present invention, the modular structure also contemplates that in the recombinant antibody heavy chain, as provided in all aspects and embodiments of the present invention, one or more CH1 elements may be replaced by a CL element. Representative CL elements include the corresponding amino acid sequences shown in Table B. Immunoglobulin light chains are classified as kappa or lambda according to their serological properties and sequences. Although the table lists amino acids for CL kappa domains, it is understood that CL lambda domains are not excluded from the selection of constant elements to form part of the Fab _H heavy chain.

It is known to those skilled in the art that minor changes to the amino acid sequences representing the CH region are possible without affecting the structural features of these domains.

Variable domains determine the specificity of an antigen. Most of the diversity of variable domains is found in three regions from each (heavy and light) chain, called hypervariable regions or CDRs. They are named according to the chain they belong to and in the order in which they appear in the sequence (L1, L2, L3, H1, H2 and H3). The regions between the CDRs in the variable region are called framework regions (framework region, FW).

The C-terminal portion of the Fc _H of a recombinant antibody, in all aspects and embodiments of the present invention, can be extended with a variable domain that is part of another portion of the Fab _H . Optionally located between the CH3 element of the Fab _H portion and the adjacent variable domain is a linker amino acid sequence L. Details and embodiments regarding the amino acid sequence of the linker are given above.

Within the heavy chain of the recombinant antibody, each CH1 or CL element combines with a VH or VL element, thereby forming part of the Fab _H heavy chain. The VH and VL elements are selected from previously studied and molecularly characterized monoclonal antibodies that are directed against the target antigen. In this context, "molecularly characterized" means that the amino acid sequences of the VH and VL domains of a selected previously known monoclonal antibody have been determined as an important basis for constructing the antibody. Thus, in all aspects and embodiments, the element Fab _H is selected from the group consisting of

In a certain embodiment of the present invention, the two heavy chains are identical or not identical. An example of two non-identical heavy chains is a ridge-to-trough configuration that directs the pairing and alignment of two heavy chains during intracellular assembly of an antibody. In another embodiment of the present invention, a label is attached to the C- or N-terminus of one heavy chain. In a more specific embodiment, the label is an affinity label, such as a histidine label, known in the art. In another embodiment of the present invention, the label is attached at the C-terminus and contains positively charged amino acids. In other specific embodiments, the two heavy chains are identical.

In one embodiment of the present invention, the multivalent antibody is monospecific and the antigen binding sites are identical or different.

In one embodiment of the present invention, the multivalent antibody is monospecific and the antigen binding sites are derived from a single parental monospecific monoclonal antibody and represent the Fab _H :Fab _L antigen binding site of the parent monoclonal antibody. Accordingly, the multivalent recombinant antibody contains either A _H :A _L or B _H :B _L .

In another embodiment of the present invention, the multivalent antibody is monospecific and the A _H :A _L antigen binding site is capable of binding to a first epitope and the B _H : _BL antigen binding site is capable of binding to a second epitope, where the first and second epitopes are identical. In this embodiment, the structural composition of the two antigen binding sites is different, and they are derived from two monospecific monoclonal antibodies of different origin, each of which binds to the same epitope, but with differences in the respective binding pockets. In yet another embodiment of the present invention, the antibody is monospecific and the antigen binding sites are identical or different, and the antigen binding sites are capable of specifically binding to an epitope contained in the same molecule or in different molecules.

In another embodiment of the present invention, the multivalent antibody is bispecific, i.e. the antibody contains A _H :A _L and B _H :B _L and the first antigen binding site is able to specifically bind to the first epitope and the second antigen binding site is able to specifically bind to a different second epitope. epitope, where the first epitope and the second epitope are contained in the same molecule.

In another embodiment of the present invention, the multivalent antibody is bispecific, i.e. the antibody contains A _H :A _L and B _H :B _L and the first antigen binding site is able to specifically bind to the first epitope and the second antigen binding site is able to specifically bind to a different second epitope. epitope, where the first epitope is contained in the first molecule and the second epitope is contained in the second molecule. In one of the embodiments of the present invention, the first and second molecules are identical or not identical. In another embodiment of the present invention, the two molecules are contained in totality or in a complex.

In another specific embodiment of the present invention, the multivalent antibody is linked to a detectable label. In principle, all labels known to those skilled in the art of developing immunoassays are possible. In particular, the detectable label is an enzyme capable of catalyzing a substrate reaction, wherein the substrate to be reacted is a water-soluble or insoluble dye. In another embodiment, the enzyme catalyzes the reaction of the substrate, whereby the substrate generates photon emission. In a preferred embodiment of the present invention, the label is a chemiluminescent agent, namely an electrochemiluminescent compound capable of covalently binding to a multivalent recombinant antibody. A specific electrochemiluminescent compound is a ruthenium compound (ruthenium complex) as described, for example, in Staffilani M. et al., Inorg. Chem. 42, 2003, 7789-7798, and other compounds containing ruthenium or iridium (ruthenium or iridium complex) known in the art.

As a second object related to all other objects and embodiments of the present invention, the use of a multivalent antibody in an antigen detection assay is contemplated, wherein the antibody is a chimeric or non-chimeric multivalent recombinant antibody, wherein the antibody comprises p FabL light chain polypeptides and a dimer of two polypeptides heavy chain, wherein each heavy chain polypeptide has the structure of formula I

Where

(c) p is a value selected from the group consisting of 6, 8 and 10,

each value of m and n is chosen independently from integers from 1 to 3,

each value of m and n is chosen so that the value of p is (2+2×(n+m));

(r) the sign "-" means a covalent bond in the polypeptide chain;

(y) each L is optional and, if present, is an independently selected variable linker amino acid sequence;

(f) each dd(Fc _H ) is a heavy chain dimerization region of an immunoglobulin non-antigen binding region;

(x) in the dimer, two dd(Fc _H ) are aligned relative to each other in physical proximity;

(c) each Fab _H is independently selected from A _H and B _H , where A _H and B _H are different, and A _H and B _H are independently selected from the group consisting of

Where

VH is the N-terminal variable domain of an immunoglobulin heavy chain,

VL is the N-terminal variable domain of an immunoglobulin light chain,

CH1 is the C-terminal constant domain 1 of an immunoglobulin heavy chain,

CL is the C-terminal constant domain of an immunoglobulin light chain;

(h) each Fab _L is independently selected from A _L and B _L where A _L and B _L are different and A _L and B _L are independently selected from the group consisting of

(w) each Fab _H :Fab _L antigen binding site of the antibody is an aligned pair (the alignment is indicated by ":"), where each aligned pair is independently selected from the group consisting of A _H :A _L and B _H :B _L , where A _H :A _L and B _H :B _L are independently selected from the group consisting of

[VL-CH1] _H :[VH-CL] _L ,

[VL-CL] _H :[VH-CH1] _L ,

[VH-CH1] _H :[VL-CL] _L ,

[VH-CL] _H :[VL-CH1] _L ,

and wherein in each aligned pair, the respective CL and CH1 are covalently bonded via a disulfide bond.

In one embodiment, the present invention describes an application in which the method is a sandwich assay in which an antigen is bound by a first capture antibody and a second detection antibody. In one of the embodiments of the present invention, the use, in accordance with all other objects and variants of implementation of the present invention, the multivalent antibody is a capture antibody. In another embodiment, the present invention describes, in accordance with all other objects and the described embodiments, the use of a multivalent antibody, which is a labeled detection antibody.

As a third object associated with all other objects and embodiments of the present invention, provide a kit containing a chimeric or non-chimeric multivalent recombinant antibody described in the first object of the present invention. In one embodiment of the present invention, the kit further comprises a detectable label. In yet another embodiment of the present invention, a detectable label is attached to a multivalent recombinant antibody. In another embodiment of the present invention, the kit further comprises magnetic particles coated with an anti-X specific binding partner and a capture agent capable of binding to an antigen to which the multivalent recombinant antibody also binds, wherein the capture agent is conjugated to X, and X and anti-X able to form a stable complex.

As a fourth aspect associated with all other aspects and embodiments of the present invention, a method for detecting an antigen is provided, comprising the steps of contacting a multivalent recombinant antibody described in the first aspect of the present invention with an antigen, resulting in the formation of a complex of the antigen and the multivalent recombinant antibody, followed by detecting the formed complex, thereby detecting the antigen. In one of the embodiments of the present invention, the method includes the steps of (a) mixing a multivalent recombinant antibody of the present invention with a liquid sample suspected of containing an antigen, (b) incubating the sample and the multivalent recombinant antibody from step (a), thereby forming a complex of antigen and multivalent recombinant antibody if the antigen is present and available for contact with the multivalent recombinant antibody during incubation, (c) detection of the complex formed in step (b), i.e. detection of the antigen. Such detection in another embodiment of the present invention is qualitative, that is, it detects the presence or absence of an antigen in a liquid sample. In another embodiment of the present invention, the detection is quantitative, that is, the amount of antigen in the liquid sample is detected, which in an even more specific embodiment is suspected to contain the antigen.

In another embodiment of the present invention, the liquid sample is an aqueous sample, more specifically a body fluid, even more specifically a body fluid selected from the group consisting of whole blood, serum, hemolyzed blood, plasma, serum, urine, synovial fluid, cerebrospinal fluid, lacrimal fluid, sputum, saliva, exhaled condensate, bronchoalveolar lavage, semen, female ejaculate, vaginal lubrication, breast milk, pulmonary aspirate, amniotic fluid, lymph, interstitial fluid, mucus, stool suspension or purified stool supernatant, cell homogenate or purified supernatant, exudate, sweat, peritoneal fluid, bile, pleural fluid, pericardial fluid and others.

In yet another embodiment of the present invention, the fourth object described in the present invention is a method comprising the steps of (a) mixing a multivalent recombinant antibody of the present invention with a liquid sample suspected of containing an antigen, (b) incubating the sample and the multivalent recombinant antibody from step ( a) thereby forming a complex of the antigen and the multivalent recombinant antibody, if the antigen is present and available for contact with the multivalent recombinant antibody during incubation, (c) immobilizing the complex formed in step (b), and (d) detecting the immobilized complex, thereby most antigen detection, quantitatively or qualitatively.

In another embodiment of the present invention, the fourth object described in the present invention is a method comprising the steps of (a) mixing a labeled multivalent recombinant antibody of the present invention with a liquid sample suspected of containing an antigen, (b) incubating the sample and the labeled multivalent recombinant antibody from step (a), thereby forming a complex between the antigen and the labeled multivalent recombinant antibody, if the antigen is present and available for contact with the labeled multivalent recombinant antibody during incubation, (c) immobilizing the complex formed in step (b), and (d) detecting the immobilized labels, thereby detecting the antigen. In a further embodiment of the present invention, this method is advantageously performed using the kit of the present invention.

In one of the embodiments of the present invention, a detectable label, for example, a label that can be detected using electrochemiluminescence, is attached to a multivalent recombinant antibody; the method comprises the steps of (a) mixing a labeled multivalent recombinant antibody of the present invention with a liquid sample suspected of containing an antigen, (b) incubating the sample and the labeled multivalent recombinant antibody from step (a), magnetic particles coated with an anti-X specific binding partner, and a capture agent capable of binding an antigen to which the multivalent recombinant antibody also binds, wherein the capture reagent is conjugated to X, thereby forming a sandwich complex of coated magnetic particles, the capture reagent, the antigen, and the labeled multivalent recombinant antibody, if the antigen is present and accessible to contact with both the labeled multivalent recombinant antibody and the capture reagent during incubation, (c) immobilizing the sandwich complex formed in step (b), and (d) detecting the immobilized label, thereby detecting the antigen. In yet another embodiment of the present invention, this method is advantageously performed using the kit of the present invention.

In yet another embodiment of the present invention, the described method includes the steps of adding a labeled multivalent recombinant antibody of the present invention to a solid phase suspected of containing an antigen on the surface, incubating the solid phase and the labeled multivalent recombinant antibody from step (a), thereby forming a complex of antigen and labeled multivalent recombinant antibody if the antigen is present and available for contact with the labeled multivalent recombinant antibody during incubation, followed by washing of the solid phase, thereby removing the uncomplexed labeled multivalent recombinant antibody, followed by detection of the label on the solid phase, thus detecting the antigen. In a more specific embodiment of the present invention, the solid phase is capable of capturing the antigen, and prior to step (a), the step of contacting the solid phase with a liquid sample suspected of containing the antigen is performed, where the antigen is captured by the solid phase if the antigen is present and available for capture by the solid phase.

The following examples and figures are provided to assist in understanding the present invention, the true scope of which is set forth in the appended claims. Obviously, modifications can be made to the procedures outlined without departing from the spirit of the present invention.

Description of figures

Fig. 1. A. Schematic representation of an oligomer of antibody fragments chemically linked to each other. B. shows SEC chromatography representing the result of an example crosslinking experiment using F(ab')2 fragments to prepare oligomers. See example 2 for details.

Fig. 2. A. Schematic representation of an oligomer of antibody fragments chemically bonded to each other. B. shows a divalent monoclonal antibody of the IgG isotype -. B. shows a multivalent antibody with four antigen binding sites. G. shows a multivalent antibody with six antigen binding sites. D. shows a multivalent antibody with eight antigen binding sites. E. shows a multivalent antibody with twelve antigen binding sites.

Fig. 3. A. shows a map of a typical expression vector for a multivalent antibody heavy chain. B. shows a map of an example light chain expression vector. See example 3 for more information.

Fig. 4. SEC chromatograms. See examples 4 and 5 for more information.

Fig. 5. SEC chromatograms. See example 5 for more information.

Fig. 6. PAGE electropherogram. See example 5 for more information.

Fig. 7. Normalized signal/noise values of labeled antibodies. See example 9 for more information.

Fig. 8. SEC chromatograms of standard and multivalent TN-T antibodies as described in Example 11.

Fig. 9. SEC chromatogram of a standard and multivalent antibody against HIV p24 antigen as described in Example 12, monospecific 2E7.

Fig. 10. SEC chromatogram of a standard and multivalent antibody against HIV p24 antigen as described in Example 12, monospecific 6D9.

Fig. 11. SEC chromatogram of a standard and multivalent antibody against HIV p24 antigen as described in Example 12, A. marker size, B. monospecific 6D9, C. bispecific 6D9/2E7.

Fig. 12. Number of electrochemiluminescence signals generated by ruthenium labeled antibodies. See example 8 for more information.

Fig. 13. SEC chromatograms of standard and multivalent TN-T antibodies as described in Example 11.

Fig. 14. SEC chromatograms of standard and multivalent antibodies against the HIV p24 antigen described in example 12.

Example 1 General Information, Methods and Techniques

Standard methods known in the art are used. Molecular cloning methods are described, for example, in: Sambrook J. "The condensed protocols from Molecular cloning: A laboratory manual", 2006, Cold Spring Harbor Laboratory Press. Methods for obtaining recombinant antibodies, described, for example, in "Monoclonal Antibodies. Methods in Molecular Biology, ed. Ossipow V. and Fischer N., 2014, volume 1131, ed. Springer. Methods of protein chemistry are described, for example, in: Hermanson G. "Bioconjugate Techniques", 3rd edition, 2013, Academic Press. Methods of bioinformatics are described in the book: "Bioinformatics" ed. Keith J.M., vol. I and vol. II; in "Methods in Molecular Biology", 2017, volume 1525 and volume 1526, published by Springer; Martin A.C.R., Allen J. in "Handbook of Therapeutic Antibodies", 2014, edited by Dubel S. and Reichert J.M., Wiley-VCH Immunoassay and related methods are discussed in the book: "The Immunoassay Handbook", 2013, 4th edition, edited by Wild D., Elsevier. Ruthenium complexes are considered as electrochemiluminescent, for example, in Staffilani M. et al., Inorg Chem 42, 2003, 7789 7798. Typically, electrochemiluminescence (ECL) based immunoassays are performed using an Elecsys 2010 analyzer or a next model instrument, such as a Roche analyzer. (Roche Diagnostics GmbH, Mannheim, Germany), e.g. E170, cobas e 601 module, cobas e 602 module, cobas e 801 module and cobas e 411, and the Roche Elecsys assays that have been developed for these analyzers, each of which used under standard conditions unless otherwise noted.

Example 2 Previously Established Workflow for Obtaining a Specifier for Use in an Immunoassay

Using the developed methods and protocols, an IgG isotype monoclonal antibody with the desired specificity and target-binding properties is obtained by recombination using hybridoma cells or transformed mammalian host cells, where the antibody-producing cells secrete it into the supernatant. Various antibodies are prepared, for example from human, mouse, sheep or rabbit. In each case, the appropriate antibody is purified from the supernatant using chromatographic methods and fractionation.

In one embodiment of the present invention, purified IgG is subjected to enzymatic cleavage to produce Fab fragments or F(ab')2 fragments. The F(ab')2 fragments are purified and thereby separated from the Fc parts. The purified F(ab')2 fragments are chemically cross-linked to form a mixture of oligomers with different molecular weights. Fig. 1A depicts such an oligomer illustrating the randomness with which F(ab')2 are combined in a crosslinking conjugation chemistry. Using chromatographic separation techniques, the mixture is fractionated and fractions containing F(ab')2 oligomers of the desired size are combined.

In another embodiment of the present invention, purified IgG is subjected to enzymatic cleavage to obtain Fab fragments. The Fab fragments are purified and thereby separated from the Fc parts. The purified Fab fragments are chemically cross-linked to form a mixture of oligomers with different molecular weights. Using chromatographic separation techniques, the mixture is fractionated and fractions containing Fab oligomers of the desired size are combined.

Optionally, the IgG molecules are oligomerized without prior enzymatic cleavage.

In FIG. 1B shows a chromatographic representation of the results of an example crosslinking experiment using F(ab')2 fragments to produce the oligomers shown schematically in FIG. 1A. It is important to note that in Fig. 1B, the region between the peaks marked (a) and (b) indicate oligomers of different sizes. In practice, they are collected as fractions, which are tested separately and assessed for their suitability for labeling and use in immunoassays. For this purpose, the workflow to which the oligomers are subjected is similar to that described for the multivalent recombinant antibodies in Example 8. That is, the heterogeneous mixture of resulting oligomers is fractionated by size, and samples of each size fraction are labeled with different average label densities per oligomer. The signal-to-noise ratio is then determined for each labeled oligomer sample, and the best-performing samples (ie, those with the highest signal-to-noise ratio) are selected. Fractions of oligomeric size, corresponding to the selected samples, note at a density in accordance with the values established for the respective samples, which were determined to be optimal.

Purified IgG, F(ab')2 or Fab oligomers of the desired size are conjugated to a detectable label; typically, ruthenium-based tags are used to create detection reagents. Labeled oligomers in the desired size range as described above are used in immunoassays where the detection step is performed by signal generation using electrochemiluminescence (ECL). Example 3

Expression vector for obtaining multivalent IgG-derived antibodies

An example is a construct for the expression of an octavalent IgG(P8) antibody as shown in FIG. 2E. In FIG. 3A shows that a plurality of VH-CH1 sequences flanked by linker sequences (eg, (G ₃ S) ₄ ) are added up and down the chain of sequences encoding Hinge-CH2-CH3, thereby forming heavy chains encoding multiple VH- CH1. On the vector map, the heavy chain coding sequence is depicted twice, firstly as a continuously drawn arrow, and secondly as a combination of several arrows, each representing the modular building block of the entire heavy chain.

The vector in FIG. 3A is co-expressed with the vector shown in FIG. 3B. The light chain expression vector expresses a standard light chain consisting of VL and a constant domain (kappa or lambda).

Fig. 3A and B thus show examples of heavy and light chain vectors. The building blocks shown in formula I may be attached as indicated for those used to express IgG(P8).

Similar constructs are made to co-express heavy chain vectors with corresponding light chains, where the heavy chain vectors encode different structures of formula I

Where

(a) each value of m and n is selected independently as an integer from 1 to 5, and each value of m and n is selected such that the values (2+2×(n+m)) are selected from the group consisting of 6, 8, 10 and 12;

(b) "-" means a covalent bond in the polypeptide chain,

(c) each L is optional and, if present, is an independently selected variable linker amino acid sequence, specifically, but not exclusively, a (G ₃ S) ₄ linker amino acid sequence;

(d) Fc _H is a non-antigen binding immunoglobulin heavy chain containing an N-terminal hinge domain; And

(e) each Fab _H is independently selected from A _H and B _H where A _H and B _H are different and A _H and B _H are independently selected from the group consisting of

Where

VH is the N-terminal variable domain of an immunoglobulin heavy chain,

VL is the N-terminal variable domain of an immunoglobulin light chain,

CH1 is the C-terminal constant domain 1 of an immunoglobulin heavy chain, and

CL is the C-terminal constant domain of an immunoglobulin light chain.

Vectors for the expression of the corresponding light chains encoding Fab _L polypeptides,

Where

(a) "-" means a covalent bond in the polypeptide chain; And

(b) each Fab _L is independently selected from A _L and B _L where A _L and B _L are different and A _L and B _L are independently selected from the group consisting of

It is important that each FabH:FabL antigen binding site of an antibody must be an aligned pair (alignment is indicated by a ":"). Thus, the Fab _H and Fab _L sequences that are co-expressed in the transformed host cell are chosen such that the Fab _H and Fab _L regions can form aligned pairs, and each aligned pair is independently selected from the group consisting of A _H :A _L and B _H :B _L , where A _H :A _L and B _H :B _L are independently selected from the group consisting of

[VL-CH1] _H :[VH-CL] _L ,

[VL-CL] _H :[VH-CH1] _L ,

[VH-CH1] _H :[VL-CL] _L ,

[VH-CL] _H :[VL-CH1] _L .

Also produce the above variations. For example, the higher order element Fc _H in the heavy chain has been reduced to just CH3. In addition, one or more Fab _H , derived from a different species than the Fc _H , are combined into a heavy chain expression vector, thus encoding a chimeric heavy chain. In most cases, the light chain polypeptide expression vector contains a single Fab _L coding sequence. However, light chain vectors with two different light chain expression cassettes have also been developed.

Example 4 Recombinant Expression and Purification of Multivalent Recombinant Anti-TSH Antibodies

Multivalent monospecific anti-TSH antibodies (thyroid-stimulating hormone, TSH = human thyroid stimulating hormone) are produced by recombination. For system comparison purposes, anti-TSH binding sites (A _H :A _L ) were generated using different multivalent recombinant antibody constructs as shown in FIG. 2B-E, which are labeled IgG(P4), IgG(P6), IgG(P8), and IgG(P12), respectively. Recombinant expression is carried out transiently in human embryonic kidney (HEK) cells or transiently or permanently in CHO cells. The transformed cells secrete multivalent monospecific anti-TSH antibodies in the serum-free culture supernatant from which they were isolated.

Like the original divalent anti-TSH monoclonal antibody, multivalent anti-TSH can be obtained in sufficient yield and without significant losses during purification of the culture supernatant by protein A affinity chromatography. In another embodiment, multivalent recombinant antibodies are purified using ion exchange chromatography (ion exchange chromatography, IEX) to which the culture supernatant is subjected.

The percentage of aggregates that are observed in all antibody formats tested is always less than 5% of the total antibody protein. The aggregate associated peak can be seen as a small shoulder to the left of the corresponding main peak, in FIG. 4B and C.

In table. 1 shows expression yields and aggregate numbers observed by GFC300 or TSK4000 gel filtration (after chromatographic purification as indicated). See also example 5 for more details on gel filtration. 1 reflects the data obtained for the original divalent anti-TSH monoclonal antibody (TSH = thyroid stimulating hormone).

Example 5 Analysis of Purified Multivalent Recombinant Antibodies

Multivalent monospecific anti-TSH antibodies were prepared and purified as described in Example 4 above. Protein A preparation/isolation was performed on all antibodies tested. The isolated bivalent and multivalent monospecific anti-TSH antibodies are subjected to analytical size exclusion chromatography (SEC). In addition, isolated bivalent and multivalent monospecific anti-TSH antibodies are subjected to polyacrylamide gel electrophoresis (SDS-PAGE). In each case, anti-TSH IgG monoclonal antibodies are also isolated and used as a reference in analytical experiments.

In FIG. 6 shows the results of PAGE chromatography compared to marker protein sizes. Remarkably, different sizes of heavy chains can be recognized, as well as varying numbers of light chains seen as bands. The gel also demonstrates the purity of the preparations.

In FIG. 4 shows the results of size exclusion chromatography (SEC) experiments with TSKgel QC-PAK GFC 300 chromatography material (Tosoh). In FIG. 4A shows the calibration result of the standard (markers for various molecular weights), in which the six peaks represent the following markers (from left to right): beta-galactosidase dimers, beta-galactosidase (465 kDa), sheep IgG (150 kDa), sheep Fab (50 kDa). ), myosin light chain (17 kDa), glycine-tyrosine dipeptide (233 Da).

With respect to the multivalent recombinant forms of MAB<TSH>clone 1, FIG. 4B-D show the results for IgG (P4), IgG (P6) and IgG (P8), respectively. In FIG. 4B and C, a small peak to the left of the main peak is interpreted as representing small amounts of aggregates of the corresponding recombinant multivalent antibody. Notably, the additional peak is absent in FIG. 4D, indicating that aggregates are not detected in preparations using TSKgel QC-PAK GFC 300 chromatographic material (Tosoh).

The same experiments are repeated with different chromatographic materials. In FIG. 5 shows the results obtained using TSKgel G4000SWx1 chromatographic material (Tosoh). In FIG. 5A shows results for the same standards, beta-galactosidase dimers, beta-galactosidase (465 kDa), sheep IgG (150 kDa), sheep Fab (50 kDa), myosin light chain (17 kDa), glycine-tyrosine dipeptide (233 Da ).

Peaks that correspond to sheep IgG (150 kDa), sheep Fab (50 kDa) are not defined as distinct individual peaks, but show a broad peak with a right shoulder corresponding to Fab.

In FIG. 5B shows the results for MAB<TSH>clone 1 IgG(P8). The shoulder to the left of the main peak indicates the presence of aggregates, however, in very small amounts.

Thus, it was concluded that recombinantly expressed multivalent monospecific antibodies can be generated and purified without significant effort and with high purity.

Example 6 Characterization of Target Binding Kinetics

Multivalent monospecific anti-TSH antibodies were prepared and purified as described in Example 4 above. Kinetic analysis was performed at 37° C. on a GE Healthcare Biacore 4000 instrument. The Biacore CM5 S series probe is installed in the instrument, guided hydrodynamically, and adjusted according to the manufacturer's instructions. HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20) was used as a systemic buffer. The sample buffer was systemic buffer supplemented with 1 mg/ml carboxymethyldextran (CMD, Carboxymethyldextran; Fluka).

The following capture system is installed on the biosensor. Anti-human IgG Fc capture monoclonal antibody is immobilized according to manufacturer's instructions using NHS/EDC chemistry. Subsequently, the sensor is saturated with 1 M ethanolamine, pH 8.5. The immobilized antibodies are saturated with the appropriate recombinant multivalent antibody. Various capture points are used to measure interactions and to control. Recombinantly prepared rat TSH antigen is diluted in varying proportions in sample buffer and injected at a flow rate of 30 μl/min for 1 minute. The capture level (Capture Level, CL) of recombinant antibodies is monitored in response units (response unit, RU).

In table. 3 shows the results obtained for various multivalent recombinant antibodies and IgG for comparison. KD means the equilibrium dissociation constant between antibody and antigen, the value of which is expressed in [nM]. The parameter k _on reflects the rate of association, expressed as [1/msec], and K _off reflects the rate of dissociation, expressed as [1/sec]. The parameter t/2 _diss describes the half-life of the test substance bound to the antibody, expressed in [min]. The ratio of the molar amount of antigen associated with a given molar amount of a multivalent recombinant antibody is expressed as the ratio AG/AB.

The designation "E+05" means known to experts in the value of 10 ⁵ .

Notably, the established mole ratios correlate very closely with the number of antigen binding sites of the corresponding antibody. From this result, it can be concluded that the multivalent recombinant antibodies of the present invention indeed provide as many functional antigen binding sites as their design. Thus, the design allows simple and reproducible combination of multivalent target binding functions (antigen binding sites) and actually reflects predictions based on molecular design. This is in sharp contrast to the multivalent oligomers obtained using already known methods (see example 2).

Example 7 Labeling of Multivalent Recombinant Antibodies

Ruthenium conjugates are obtained with an increase in label inclusion. Purified multivalent recombinant antibodies are used in labeling experiments. Recombinant antibodies are used, such as those shown in FIG. 2B-E, which are IgG(P4), IgG(P6), IgG(P8), and IgG(P12), respectively. In labeling experiments, varying amounts of the appropriate antibody are reacted with either the tris-bipyridyl-ruthenium labeling reagent or its sulfonate form (also referred to as "sBPRu") using standard NHS ester addition chemistry. Under these conditions, the ruthenium label covalently attaches to functional groups of lysine amino acid residues in the antibody backbone.

Average ruthenium label incorporation can be defined as the number of protein-bound label molecules per antibody. This amount can be measured by separately determining the amount of protein and the amount of ruthenium label in the labeled antibody sample. Examples of methodological approaches are mass spectrometry of photometric determinations.

Tab. 4 is a comparison of various multivalent recombinant antibodies and IgG with respect to the rate of incorporation of sBPRu.

Surprisingly, it was found that the specific antibodies P6 and P8 show particularly high values. Thus, P6 and P8 provide a particularly effective marking.

Similar results are observed with other labeling reactions using other labels using standard NHS ester addition chemistry (N-hydroxy-succinimide).

Example 8 Counting Electrochemiluminescence Signals. generated by antibodies labeled with ruthenium

Multivalent monospecific anti-TSH antibodies were prepared and purified as described in Example 4 above.

Anti-TSH Roche Cobas Elecsys assay by Roche catalog number 07028091190 is carried out in variations as described. Only the ruthenium labeled detection agent (Ru labeled antibody oligomeric fragments) in the Cobas assay was replaced by a recombinant multimeric antibody with a specific labeling density of ruthenium previously determined. Similarly, IgG is used as a reference. The results for IgG and IgG (P8) are exemplified in Figure 12A for calibration (no target antigen, null measurements) and Figure 12B for measurements with a target antigen concentration of 5 μU TSH/mL. In each diagram, the ordinate represents the strength of the electrochemiluminescent signal, that is, the amount of light from ruthenium detected by the Elecsys instrument; the abscissa represents the average density of incorporated ruthenium label per antibody unit.

The data show that in background experiments (no TSH antigen), the increase in signal as a function of increased label density is higher for IgG compared to the multivalent IgG(P8) construct. The signal obtained in the absence of antigen is also referred to as "noise".

Similar results are observed when the TSH target is measured at a concentration of 5 uU TSH/mL. The value measured based on the antigen actually present is referred to as the "signal".

It has been found that IgG(P8) and IgG can produce the same noise or signal, with the result that, under test conditions, IgG(P8) always has a higher label density than IgG, which generates about an equivalent amount of electrochemiluminescent light.

This result is interpreted as an indication that labeled IgG(P8) is able to generate a technically superior signal to noise ratio compared to IgG.

Table 5 presents the measurement values obtained for IgG, which are the basis for the graphs in FIG. 12A.

S/N: signal to noise ratio

The inclusion of ruthenium means the average amount of label Ru per antibody. Table 6 presents the measurement values obtained for IgG(P8) which are the basis for the graphs in FIG. 12B.

S/N: signal to noise ratio

Ruthenium incorporation means average amount of Ru label per antibody

Example 9 Determination of Optimal Label Density for Recombinant Antibody to Obtain Optimal Signal-to-Noise Ratio in Immunoassays

Multivalent monospecific anti-TSH antibodies were prepared and purified as described above in Example 4.

First, the required amount of TSH antigen for calibration is obtained (5 μU TSH/ml, as determined by the Roche Cobas Elecsys assay, Roche catalog 07028091190, Roche Diagnostics GmbH, Mannheim, Germany). Multivalent recombinant anti-TSH antibodies are also prepared as shown in FIG. 2B-E, labeled IgG(P4), IgG(P6), IgG(P8), and IgG(P12), respectively. TSH-specific IgG is used as a reference. It is important that each antibody is contained in different samples, and the samples differ in the average label density for the corresponding antibody.

In the series of experiments described in Example 8, each antibody with a predetermined average label density was used in Elecsys runs and the number of signals corresponding to 5 μU TSH/ml was recorded. Each labeled antibody signal-to-noise ratio measured is then normalized to the corresponding value determined using the original Roche Cobas Elecsys anti-TSH assay, catalog number 07028091190.

Accordingly, each of the diagrams in FIG. 7A-E, illustrating the results, contains an ordinate with a scale indicating percentages with a mark of 100% corresponding to the measurement obtained using the original Roche Cobas Elecsys anti-TSH assay. Each measured value, thus normalized with respect to the 100% reference value of the original assay, indicates the detection capability of the corresponding labeled antibody with which the normalized value was obtained. If the normalized value is below 100%, the corresponding labeled density antibody is technically less preferred; on the other hand, a normalized value above 100% represents an antibody with a more favorable signal-to-noise ratio that outperforms the labeled oligomers of the original assay.

It is important to consider that in experiments with labeled antibodies, they are the only component that has been modified in the original assay, replacing labeled oligomers of chemically bound antibody fragments.

In FIG. 7A show the results obtained for IgG, FIG. 7B - results obtained for IgG(P4), FIG. 7B - results obtained for IgG(P6), FIG. 7D shows the results obtained for IgG(P8) and FIG. 7E - results obtained for IgG(P12). In FIG. 7E shows an overlay from A to E combining all results. It became apparent that specific IgG(P6) and IgG(P8) labels with certain label loadings could outperform the original analysis. Thus, average preferred label densities determined using normalized data allow the determination of the most effective labeled conjugates suitable for use in the required immunoassays, in particular in diagnostic immunoassays. These recombinant multivalent antibodies with label densities leading to the highest relative values are selected for further experiments. The same was done for labeled IgG.

In this regard, it should be noted that the process of determining the optimal label densities, as described above, is also performed for each newly synthesized batch of chemically linked antibodies or fragments thereof, as described in example 2. That is, the sizes of oligomer fractions are usually labeled to give different densities so that find out which combination of oligomer size and label density is capable of producing equal results as the original assay.

Therefore, the selection process for determining the optimal label density is generally an established standard practice in the art.

Example 10 Evaluation of a multivalent anti-TSH antibody to detect different concentrations of TSH antigen. and comparison with standard oligomers of antibody fragments

The original anti-TSH Roche Cobas Elecsys assay, Roche catalog 07028091190, including chemically bound IgG fragments labeled with ruthenium as detection reagent, is used to measure a dilution series of TSH antigen, where each aliquot containing a dilution of TSH antigen is prepared in a universal commercially available diluent , Roche part number 1173277122 (Roche Diagnostics GmbH, Mannheim, Germany). The range of TSH concentrations obtained from the dilution aliquots is chosen to represent the range of physiological concentrations of at least 95% of the patient population. Subsequently, the detection reagent is replaced with multivalent anti-TSH antibodies with optimal label density (see example 9). In table. 7 and 8 summarize the results in which the signal-to-noise ratios are tabulated as percentages relative to the standard assay with the original detection reagent, ie containing chemically bound ruthenium labeled antibody fragments. For the data in the table. 8, the measurements are taken after the additional prewash step has been switched on. In other words, before the detection complexes are placed in the measurement cell of the Elecsys instrument, the detection complexes are magnetically immobilized and washed with an additional volume of buffer, thereby more effectively removing unwanted components. In table. 7 shows the measurement data without the prewash step, which results in a slightly lower signal-to-noise ratio.

Measurement without prewash

Pre-flush measurements

Signal to noise ratio (S/N) values are calculated and normalized according to current commercial analysis TSH Elecsys part number 07028091190 from Roche catalog. The results show that multivalent IgG(P6) and IgG(P8) conjugates show the best signal-to-noise ratio results, with over 160% better values at high and low TSH concentrations than in the original assay, which included the standard reagent for detection with covalently chemically cross-linked antibody fragments.

Example 11 Anti-Troponin-T (TN-T) Multivalent IgG(P8) Antibody

The light and heavy chain expression vectors for the octavalent antibody are constructed and transiently expressed in HEK293F host cells that secrete the antibody into the serum-free culture medium supernatant. The antibody is isolated from the supernatant using protein A affinity chromatography. Purified antibody can be obtained with a yield of 61 ml/l of supernatant. GFC300 analytical SEC chromatography shows that the purified multivalent anti-TNT antibodies are pure and low in aggregates.

Fig. 13B. shows the results of the SEC analysis, FIG. 13A shows the same dimensional standards as in FIG. 10A.

The IgG(P8) ruthenium conjugate was prepared to replace the original standard chemically crosslinked ruthenium conjugate of the original Roche Elecsys assay, catalog number 05092744190 (Roche Diagnostics GmbH, Mannheim, Germany). At all tested concentrations of target TN-T antigen in the range of 4.5-4000 ng/ml, the IgG (P8)-Ru conjugate showed excellent performance in the Elecsys TN-T assay.

The data in the table. 9 were obtained by the same method as in example 10. A prewash step was included.

Pre-flush measurements

Example 12 Anti-HIV Antigen Multivalent Antibodies

Finally, the new multivalent format antibodies described herein were tested in the HIV antigen assay, catalog number 11971611122 (Roche Diagnostics GmbH, Mannheim, Germany). This assay uses two separate oligomers of a rutenylated anti-p24 antibody fragment. That is, the first and second oligomers are specific for the detection of the first and second epitope of the target p24 protein. Both anti-p24 IgG monoclonal clones (E and D) were used to construct the IgG(P4), IgG(P6) and IgG(P8) formats. Additionally, an IgG(P8) variant with four antigen-binding sites from clone E and four binding sites from clone D was generated, resulting in an octagonal and bispecific antibody. The bispecific property was generated using human and mouse _kappa CH1 and C sequences, respectively. All molecules are expressed in HEK293 cells and purified by protein A chromatography. Analytical-SEC shows that all E, D multivalent anti-p24 antibodies and the E/D biclonal molecule can be produced in high purity and low aggregation in IgG formats. (P4), IgG(P6) and IgG(P8). All constructs were expressed in HEK293 cells, purified by protein A chromatography. All proteins were ruthenium-conjugated with optimal labeling.

Fig. 14A shows a size standard similar to the chromatogram shown in FIG. 10A; in fig. 14B-D show monospecific antibodies P4, P6 and P8, respectively, for E-specificity. For D-specific constructs, FIG. 14E show standard dimensions similar to the chromatogram shown in FIG. 10A; fig. 14E and 14G show monospecific antibodies P4, P6, respectively, for D-specificity.

Figure 143 shows a size standard similar to the chromatogram shown in FIG. 10A, but with a different SEC material, namely Superose 6. Using the same SEC material, the P8 monospecific construct was analyzed for specificity D, as shown in Figure 14K. In FIG. 14K shows the P8 bispecific constructor having four E antigen binding sites and four D antigen binding sites, also analyzed using Superose 6 SEC.

Evaluation of these conjugates in the HIV-Ag assay (FIG. 7) showed that for E and D IgG(P6) ruthenium conjugates were superior to ruthenium conjugates from chemically polymerized antibody fragments (ori), IgG(P4) and IgG(P8). The signal-to-noise ratios for 6D7 and E IgG (P6)-Ru are 31% and 86% higher than in the assay with the original reagent. In addition, the assay equipped with a bispecific multivalent IgG(P8) reagent with four antigen binding sites from clone D and four from E showed an 18% better S/N ratio than the original, which contained oligomerized antibody fragments from D and E.

IgG(P4), IgG(P6) and IgG(P8) antibodies from the HIV-ag Elecsys assay are labeled with ruthenium at the optimal label to protein ratio. Elecsys-HIV-Ag assays are run with the IgG(P4), IgG(P6) and IgG(P8)-ruthenium conjugates of these antibodies in parallel with the corresponding assay with the current parent conjugates (chemically cross-linked oligomers). The HIV-Ag assay consists of two ruthenium-labeled and polymerized antibodies (D and D). Each of these is tested and compared to the new multivalent variants separately (A and B), or both have been replaced by an E/D multivalent biclonal variant. Signal to noise ratio (S/N) values are calculated and normalized.

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SEQUENCE LIST

<110> F.HOFFMANN-LA ROSCH AG

<120> MULTIVALENT MONO-OR BISPECIFIC

RECOMBINANT ANTIBODIES

FOR ANALYTICAL PURPOSES

<130> P34395-WO

<150>EP17192532.4

<151> 2017-09-22

<160> 20

<170>PatentIn version 3.5

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Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

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<223> Murine IgG1, CAmino acid sequence for CH1 domain

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Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

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Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

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Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

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Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

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Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile

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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

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Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp

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Ser Leu Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Ile

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Cys Asn Val Ala His Pro Ala Ser Lys Thr Glu Leu Ile Lys Arg Ile

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<210> 7

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<223> Murine IgG3, Amino acid sequence for <hinge>-CH2-CH3 portion

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Val Asp Asn Lys Glu Val His Thr Ala Trp Thr Gln Pro Arg Glu Ala

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225 230

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<211> 104

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

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Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

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Lys Val Glu Pro Lys Ser Cys Asp

100

<210> 9

<211> 226

<212> PRT

<213> Artificial Sequence

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

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145 150 155 160

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

165 170 175

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

180 185 190

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

195 200 205

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

210 215 220

Gly Lys

225

<210> 10

<211> 102

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG2, Amino acid sequence for CH1 domain

<400> 10

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys

100

8

<210> 11

<211> 224

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG2, Amino acid sequence for <hinge>-CH2-CH3 portion (FcH)

<400> 11

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

1 5 10 15

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

20 25 30

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

35 40 45

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

50 55 60

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

65 70 75 80

Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

85 90 95

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

100 105 110

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

115 120 125

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

130 135 140

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

145 150 155 160

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

165 170 175

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

180 185 190

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

195 200 205

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210> 12

<211> 112

<212> PRT

9

<213> Artificial Sequence

<220>

<223> Human IgG3, Amino acid sequence for CH1 domain

<400> 12

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105 110

<210> 13

<211> 265

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG3, Amino acid sequence for <hinge>-CH2-CH3 portion (FcH)

<400> 13

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

1 5 10 15

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

20 25 30

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

35 40 45

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

50 55 60

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

65 70 75 80

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

85 90 95

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

10

100 105 110

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

115 120 125

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

130 135 140

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

145 150 155 160

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

165 170 175

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

180 185 190

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

195 200 205

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

210 215 220

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

225 230 235 240

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

245 250 255

Lys Ser Leu Ser Leu Ser Pro Gly Lys

260 265

<210> 14

<211> 103

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG4, Amino acid sequence for CH1 domain

<400> 14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

eleven

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Ser Pro Asn Met Val

100

<210> 15

<211> 224

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG4, Amino acid sequence for <hinge>-CH2-CH3 portion (FcH)

<400> 15

Pro His Ala His His Ala Gln Ala Pro Glu Phe Leu Gly Gly Pro Ser

1 5 10 15

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

20 25 30

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

35 40 45

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

50 55 60

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

65 70 75 80

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

85 90 95

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

100 105 110

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

115 120 125

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

130 135 140

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

145 150 155 160

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

165 170 175

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

180 185 190

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

12

195 200 205

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210> 16

<211> 101

<212> PRT

<213> Artificial Sequence

<220>

<223> Rabbit IgG, Amino acid sequence for CH1 domain

<400> 16

Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly

1 5 10 15

Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn

35 40 45

Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys

65 70 75 80

Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala

85 90 95

Pro Ser Thr Cys Ser

100

<210> 17

<211> 222

<212> PRT

<213> Artificial Sequence

<220>

<223> Rabbit IgG, Amino acid sequence for <hinge>-CH2-CH3 portion (FcH)

<400> 17

Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

1 5 10 15

Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

20 25 30

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val

35 40 45

Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro

50 55 60

13

Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr

65 70 75 80

Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys

85 90 95

Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

100 105 110

Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro

115 120 125

Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile

130 135 140

Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly

145 150 155 160

Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp

165 170 175

Gly Ser Tyr Phe Leu Tyr Asn Lys Leu Ser Val Pro Thr Ser Glu Trp

180 185 190

Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His

195 200 205

Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys

210 215 220

<210> 18

<211> 103

<212> PRT

<213> Artificial Sequence

<220>

<223> Murine IgG, Amino acid sequence for CL kappa domain

<400> 18

Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser

1 5 10 15

Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp

20 25 30

Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val

35 40 45

Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met

50 55 60

Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser

65 70 75 80

14

Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys

85 90 95

Ser Phe Asn Arg Asn Glu Cys

100

<210> 19

<211> 103

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG, Amino acid sequence for CL kappa domain

<400> 19

Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser

1 5 10 15

Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp

20 25 30

Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val

35 40 45

Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met

50 55 60

Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser

65 70 75 80

Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys

85 90 95

Ser Phe Asn Arg Asn Glu Cys

100

<210> 20

<211> 100

<212> PRT

<213> Artificial Sequence

<220>

<223> Rabbit IgG, Amino acid sequence for CL kappa domain

<400> 20

Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr

1 5 10 15

Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val

20 25 30

Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu

35 40 45

Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser

15

50 55 60

Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr

65 70 75 80

Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn

85 90 95

Arg Gly Asp Cys

100

<---

Claims (52)

1. A method of constructing a non-chimeric or chimeric multivalent recombinant antibody for antigen detection, comprising cross-linking Fab fragments, which contains p Fab _L light chain polypeptides and a dimer of two heavy chain polypeptides, each heavy chain polypeptide having the structure of formula I _N-terminus [Fab _H -L-] _n Fab _H -L-dd(Fc _H )[-L-Fab _H ] _{m C-terminus} (formula I), Where (a) p is a value selected from the group consisting of 6, 8 and 10, the value of p is (2+2*(n+m)), and each value of m and n is independently selected from integers from 1 to 3; (b) the designation "-" is a covalent bond in the polypeptide chain; (c) each L is optional and, if present, is an independently selected amino acid sequence of the variable linker; (d) each dd(Fc _H ) region is a heavy chain dimerization region in the heavy chain non-antigen binding region of an immunoglobulin; (e) in a dimer, two dd(Fc _H ) are aligned with each other in physical proximity; (e) each Fab _H is independently selected from A _H and B _H , where A _H and B _H are different and A _H and B _H are independently selected from the group containing

Where VH stands for immunoglobulin heavy chain variable domain, VL stands for immunoglobulin light chain variable domain, CH1 stands for immunoglobulin heavy chain constant domain 1 and CL means light chain constant domain; (g) each Fab _L is independently selected from A _L and B _L , where A _L and B _L are different and A _L and B _L are independently selected from the group consisting of

(h) each Fab _H :Fab _L antigen binding site of an antibody is an aligned pair whose alignment is indicated by ":", each aligned pair being independently selected from the group consisting of A _H :A _L and B _H :B _L , where A _H :A _L and B _H :B _L are independently selected from the group consisting of [VL-CH1] _H :[VH-CL] _L , [VL-CL] _H :[VH-CH1] _L , [VH-CH1] _H :[VL-CL] _L , [VH-CL] _H :[VL-CH1] _L , where in each aligned pair, the corresponding CL and CH1 are covalently linked through a disulfide bond. 2. The method of claim 1 wherein the antibody is bispecific or monospecific. 3. The method of claim 1 or 2, wherein the A _H :A _L antigen binding site is capable of binding to the first epitope and the B _H :B _L antigen binding site is capable of binding to the second epitope, wherein the first and second epitopes are different. 4. The method of claim 3, wherein the antibody is bispecific, the first antigen binding site is able to specifically bind to the first epitope, and the second antigen binding site is able to specifically bind to a different second epitope, wherein the first epitope and the second epitope are contained in the same molecule. 5. The method of claim 3, wherein the antibody is bispecific and the first antigen binding site is able to specifically bind to the first epitope and the second antigen binding site is able to specifically bind to a different second epitope, wherein the first epitope is contained in the first molecule and the second epitope is contained in the second molecule. 6. The method according to claim 5, wherein the first and second molecules are identical or not identical. 7. The method according to paragraphs. 5 and 6, in which two molecules are contained in an aggregate or complex. 8. The method according to paragraphs. 1 and 2, wherein the A _H :A _L antigen binding site is capable of binding to the same epitope as the B _H :B _L antigen binding site. 9. The method of claim 8, wherein the antibody is monospecific and the antigen binding sites are identical or different. 10. The method of claim 9, wherein the antigen binding sites are capable of specifically binding to an epitope contained in the same molecule or in different molecules. 11. The use of a multivalent recombinant antibody obtained by the method according to paragraphs. 1-10 and conjugated to a detectable label, in an antigen detection assay. 12. Use according to claim 11, wherein the assay is a sandwich assay. 13. A conjugate for antigen detection, containing a multivalent recombinant antibody obtained by the method according to any one of paragraphs. 1-10, which is conjugated to a detectable label. 14. A method for detecting an antigen, comprising the steps of contacting a multivalent recombinant antibody obtained by the method according to any one of paragraphs. 1-10 with an antigen to form a complex of the antigen and a multivalent recombinant antibody, followed by detection of the formed complex, thereby detecting the antigen. 15. The method according to p. 14, including the steps (a) mixing a multivalent recombinant antibody obtained by the method according to any one of paragraphs. 1-10, with a liquid sample suspected of containing an antigen, (b) incubating the sample and the multivalent recombinant antibody from step (a), thereby forming a complex of the antigen and the multivalent recombinant antibody, if the antigen is present and available for contact with the multivalent recombinant antibody during incubation, (c) detecting the complex formed in step (b), thereby detecting the antigen. 16. The method according to p. 14, which includes the steps (a) mixing a multivalent recombinant antibody obtained by the method according to any one of paragraphs. 1-10, with a liquid sample suspected of containing an antigen, (b) incubating the sample and the multivalent recombinant antibody from step (a), thereby forming a complex of the antigen and the multivalent recombinant antibody, if the antigen is present and available for contact with the multivalent recombinant antibody during incubation, (c) immobilizing the complex formed in step (b), and (d) detecting the immobilized complex, thereby detecting the antigen. 17. The method according to p. 14, which includes the steps (a) mixing the labeled multivalent recombinant antibody obtained according to any one of paragraphs. 1-10, with a liquid sample suspected of containing an antigen, (b) incubating the sample and the labeled multivalent recombinant antibody from step (a), thereby forming a complex of the antigen and the labeled multivalent recombinant antibody, if the antigen is present and available for contact with the labeled multivalent recombinant antibody during incubation, (c) immobilizing the complex formed in step (b), and (d) detecting the immobilized label, thereby detecting the antigen. 18. The method according to p. 14, which includes the steps (a) adding a labeled multivalent recombinant antibody obtained by the method according to any one of paragraphs. 1-10, to a solid phase, presumably containing an antigen on the surface, (b) incubating the solid phase and the labeled multivalent recombinant antibody from step (a), thereby forming a complex of the antigen and the labeled multivalent recombinant antibody, if the antigen is present and available for contact with the labeled multivalent recombinant antibody during incubation, then (c) washing the solid phase to remove the uncomplexed labeled multivalent recombinant antibody, then (d) detecting the label on the solid phase, thereby detecting the antigen. 19. The method of claim 18 wherein the solid phase is capable of capturing the antigen and prior to step (a) the step of contacting the solid phase with a liquid sample suspected of containing the antigen is performed, wherein the antigen is captured by the solid phase if the antigen is present and available for capture by the solid phase.

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