Patent Description:
Classical antibodies [<NUM>, <NUM>] are large multimeric proteins (IgG ~ <NUM> kDa), which comprise two identical heavy H-chains (one variable VH domain, three constant domains CH1, CH2 and CH3, and a hinged region between CH1 and CH2 domains) and two identical light L-chains (consisting of the variable domain VL and constant domain CL). A four-chain molecule has non-covalent and covalent (disulfide) bonds connecting the chains. Papain protease can be used to break down an antibody molecule into two fragments: Fab (Fragment antigen binding) and Fc (Fragment crystallizable). Therefore, one region of the molecule (Fab) defines its antigen-related specificity, and another region (Fc) exercises the effector functions targeted at antigen elimination [<NUM>, <NUM>]. CH1 and CH2 domains of H-chain are divided by a hinge region that assures mobility of Fab-region and interaction of IgG molecule with Ig effector receptors located on the cells. CH2 domain contains the regions binding both FcY receptors that mediate cell activation (ADCC and ADCP) and complement system molecules (CDC). In addition, this domain contains the site that is an attach point for carbohydrates of all immunoglobulin isotypes. CH3-domain largely determines the stability of IgG dimer and interacts with FcRn-receptor on the cell surface establishing pharmacokinetic properties of antibodies as well as their metabolism and distribution in the body. The combination of complementarity determining regions (CDR) of the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) forms an antigen-binding fragment, while the framework regions of the variable domains and the constant domains are not directly involved in antigen recognition. A minimized Fab-derivative for classical antibodies is a single-chain structure in which variable domains of the heavy and light chains are connected with a linker sequence (scFv).

Finding significant amounts of specific non-classical antibodies of simplified structure in the blood of Camelidae animals (camels, llamas, vicunas) was a valuable discovery [<NUM>]. Such antibodies (heavy chain antibody, HCAb) consist of a dimeric single shortened heavy chain (without CH1 domain) with no light chain at all. Antigen-binding fragment of HCAb is formed by only one heavy chain variable domain (VHH), which is connected through the hinged region to Fc-domain. VHH is often called a single-domain antibody, "nanobody", "mini-antibody" or "nano-antibody". It appeared that in addition to small size (<NUM>-<NUM> kDa), such isolated mono-domain structure has a number of advantages as compared to classical IgG antibodies, namely aggregation, chemical and thermal stability. VHH antibodies can be successfully cloned and expressed in bacterial and yeast cells. With such properties, these antibodies were developed in therapeutic field by Ablynx Company and in laboratory and industrial chromatography (CaptureSelect affinity products).

Non-classical antibodies (HCAb) including a dimer of just a single Ig heavy chain were first discovered by electrophoretic analysis of immunoglobulins in the serum obtained from various representatives of Camelidae bloodline [<NUM>]. The relative fraction of HCAb varies from about <NUM>-<NUM>% (of all IgG) in llamas and vicunas to about <NUM>-<NUM>% in camels [<NUM>].

It is assumed that non-classical antibodies (HCAb), at least in case of Camelidae, resulted from relatively recent evolution of the genes of classical antibodies. Two heavy chain constant domains, CH2 and CH3, in case of HCAb and classical antibodies are highly conserved. In HCAb there is no domain corresponding to the first constant domain CH1 of classical antibodies. The genome of the one-humped camel (Dromedary) contains a cluster of about fifty VH- and forty VHH-generative genes followed by multiple genes of D-segments, J-segments and genes encoding the constant regions (Cµ, Cγ, Cε and Cα). It is clear that some of Cγ-genes serve to form HCAb (mutations result in the loss of CH1-domain), and other serve to form classical antibodies (with sustained CH1-domain). The same genes of D- and J-segments may randomly connect to either one of VH-genes or one of VHH-genes. This indicates that VH- and VHH-genes are located in the same gene locus [<NUM>-<NUM>].

The organization of variable domains of non-classical antibodies (VHH) and variable domains of classical antibodies (VH) is very similar, as human VH-domains of IgG3 subclass have most evident homology to VH and VHH of Camelidae. In both cases, V-domains comprise four conservative framework regions FR surrounding three hypervariable complementary-determining regions (CDR). In addition, in both cases a <NUM>-D structure typical of immunoglobulin V-domain of two β-layers is formed, one of which comprises <NUM> amino acid sequences and another comprises <NUM> amino acid sequences [<NUM>, <NUM>]. All three hypervariable regions in this structure form a cluster on one side of V-domain, where they participate in antigen recognition and are located in the loops connecting β-structures. However, there are several important distinctions related to the functioning of single-domain VHH. Thus, CDR1 and CDR3 of VHH are significantly enlarged. Complementary-determining regions of VHH often contain cysteine residues in two sections at once (usually in CDR1 and CDR3, less frequently - in CDR2 and CDR3). The studies of VHH crystal structures have shown that these cysteine residues form disulfide bonds and provide additional stability to the loop structure of these antibodies [<NUM>]. The strongest and the most reproducible distinguishing feature of VHH is represented by four substitutions of hydrophobic amino acid residues with hydrophilic ones in the second framework region (Val37Phe, Gly44Glu, Leu45Arg, Trp47Gly, according to the numbering of Kabat et al [<NUM>]). This framework region of VH-domain is highly conservative, enriched with hydrophobic amino acid residues and is essential for linking to the variable domain of the light chain VL. VHH-domain differs greatly in this aspect: substitutions of hydrophobic amino acids with hydrophilic makes the association of VHH and VL impossible. These substitutions also explain high solubility of VHH (nano-antibody), when it is obtained as a recombinant protein [<NUM>].

It appears that the repertoires of possible paratopes (antigen-binding parts of an antibody) of HCAb and classical antibodies may be significantly different. Since these two antibody types co-exist in the same organism, it can be assumed that they do not compete but are complementary to each other. For example, it was repeatedly noted that both antibody types could occur simaltaneously, exclusively or in different ratios with regard to various epitopes of the antigen material upon immunization of the very same animal. Despite the suspected lower variety of paratopes possible for single-domain antibodies, as compared to classical two-domain antibodies, many publications have clearly demonstrated that HCAb can be obtained against the most diverse epitopes of a rather wide range of antigens [<NUM>]. Apparently, this is due to enlarged CDR1 and CDR3 regions. We also should note a surprisingly large (as compared to V-domains of classical antibodies) number of somatic hypermutations in VHH that are likely to accumulate in the course of affine maturation of the antibody during immunization [<NUM>]. X-ray diffraction analysis revealed that antigen-binding loop regions of VHH are able to form structures unusual for classical V-domains [<NUM>, <NUM>]. While in case of VH- and VL-domains of classical antibodies, all six CDRs contribute almost the same to antigen binding, in case of VHH, CDR3 is usually the most important for the formation of a paratope. It has been shown that CDR3 in VHH (but not in VH or VL) is capable of forming uncommonly long finger-structures that can deepen into the antigen structure and, in particular, detect active enzyme centers [<NUM>]. Small size of the antigen-binding region of VHH and its ability to form unusual emerging paratopes explain how HCAb capable of epitopes inaccessible for classical antibodies can be obtained (for example, production of antibodies that efficiently inhibit enzymes) [<NUM>].

With all its high potential of specificity, unique, as compared to classical IgG antibodies, the therapeutic use of a single-domain VHH is sometimes limited due to its rapid elimination from the organism. There are several solutions designed to improve the pharmacokinetics of VHH structures, including chemical conjugation with PEG and covalent binding to polypeptides mediating the reduced clearance from the blood, such as Human serum albumin (HSA) or Fc-fragment classical human antibody as fusion proteins that possess a half-life of up to three weeks [<NUM>,<NUM>,<NUM>]. Small peptides attached to VHH by genetic engineering methods and capable of high-affinity non-covalent interaction with said components (HSA and IgG) in human blood have been successfully used [<NUM>]. However, the technological efficiency and immunogenicity of these approaches remain questionable, and the feasibility of using thereof in either clinical or earlier study phases is now on trial.

In addition, the greatest limitation of using antibodies as medicinal agents for the treatment of various diseases is due to their aggregation and chemical stability, affinity and immunogenicity. Since the majority of monoclonal antibodies are obtained on the basis of mice, the regular use of such antibodies in humans causes development of immune response to antibody therapy (for example, allergic reactions). These types of immune response finally result at least in lack of efficiency, and in potential anaphylactic reactions at the worst. On the other hand, aggregation or chemically unstable therapeutic antibodies reduce therapeutic properties of the drug product over time and may increase its immunogenicity upon administration to human patients.

In reference with the above, it is important to develop VHH-based antibodies with improved (in comparison to previously known antibodies) functional and therapeutic features, particularly increased aggregation, chemical and thermal stability and improved affinity, which would at the same time be easily and simply produced, including on industrial scale.

The background of the invention includes various antibody structures containing VHH domain.

Patent application <CIT> describes antibodies that comprise separate variable domains linked with Fc-fragment. Nano-bodies can be used as variable domains with Fc obtained from IgE antibodies. The said domain and Fc fragments can be connected through a linker located in the hinge region.

Patent application <CIT> disclosed antibodies comprising complete VHH antibodies or parts thereof directly connected to the constant regions of human antibodies.

Patent application <CIT> discloses llama and humanized antagonistic VHH antibodies (Nanobodies) and Fc fusions thereof, which bind IL-17A and/or IL-17F.

In addition, there are amino acid substitutions known to affect the physical-chemical and biological properties of antibodies.

For example, application <CIT> describes the antibody heavy chain variable domains wherein amino acid substitutions were introduced in positions <NUM>, <NUM>, <NUM>, <NUM>- <NUM> and 100a to improve the hydrophilic properties of the antibody obtained.

Now, the methods have been developed to optimize the structure of isolated VHH and VH mono-domains in order to reduce the immunogenicity and improve the aggregation stability thereof.

Thus, Vincke at al. [<NUM>] have found that Glu-<NUM>→Gly and Arg-<NUM>→Leu substitutions in specific amino acids result in a separate domain that is more stable, though less soluble. Other substitutions in the framework region FR-<NUM> Phe-<NUM>→Val and Gly/Ala-<NUM>→Trp are crucial for antibody affinity to the antigen due to reorientation of H3-loop, raising dissociation constant <NUM>-<NUM>-fold (<NUM>·<NUM>-<NUM> <NUM>/s). Phe-<NUM>→Val substitution caused the reduced stability of the antibodies obtained. The substitution of Gly-<NUM> and Leu-<NUM> in VH-sequence resulted in lower stability of the domain, while Glu-<NUM> and Arg-<NUM> humanization in VHH allows obtaining stable variable domains.

It is also known from literature, that in the presence of short HCDR3 regions neutralizing the shading effect of the conformation of classical VHH, and upon introduction of VH-characteristic Trp-<NUM>→Gly-<NUM> substitution as well as Tyr-<NUM>→Val-<NUM>, Glu-<NUM>→Gly-<NUM> and Arg-<NUM>→Leu-<NUM>, the isolated VHH domains can regenerate the ability to bind with VL domain [<NUM>].

The relationship between the increased aggregation stability of therapeutic antibodies of classical IgG structure and the reduced immunogenicity thereof was demonstrated in multiple studies and summarized in the review by Hermeling et al. , <NUM> [<NUM>]. Yet they did not reveal any antibodies comprising derivative VHH domains, but linked to the variable domains of the light chains within full-size human IgG.

Therefore, there is a need for development of a new format of antibodies that would have improved stability and affinity, good expression and low immunogenicity.

Besides, no approaches were earlier described with regard to the development of such molecules that would be easy to obtain, have improved aggregation stability, increased affinity and high expression level in the mammal cell culture.

With reference to the above, this disclosure is the first to describe antibodies comprising VHH-derivatives that are able to bind to variable domains of the light chains of the full-size human IgG, which results in the formation of a structure that is similar to the natural one (and, hence, having low immunogenicity), but has improved aggregation stability, increased affinity, and a structure of a therapeutic monoclonal antibody.

"Monoclonal antibody" as used herein relates to an antibody obtained from llama, chimeric antibody, humanized antibody or fully human antibody, unless otherwise is stated in the present application. Monoclonal antibodies, according to the invention, can be produced using, for example, recombinant technology, phage display technology, synthetic technology or combinations of these or other technologies well known from the prior art.

"Monoclonal antibody" refers to an antibody obtained from a single copy or a clone including, for example, any eukaryotic, prokaryotic or phage clone, rather than to production method thereof. "Monoclonal antibody" can be an intact antibody (with full or full-length Fc-region), actually intact antibody, an antibody part or fragment comprising an antigen-binding region, for example, Fab-fragment, Fab'-fragment or F(ab')<NUM>-fragment from llama or chimeric, humanized or human antibody. "Fab"-fragment comprises a variable light chain domain and a constant light chain domain as well as a variable heavy chain domain and the first constant heavy chain domain (CH1). "F(ab')<NUM>" antibody fragment contains a pair of Fab-fragments, which are mostly covalently bound by hinged cysteine residues at C-terminal regions. Other chemical bonds between antibody fragments are also well known from the prior art.

In addition, "monoclonal antibody" as used herein can be a single-chain Fv that can be obtained by binding DNA encoding VHH and VL with a linker sequence. As long as the protein keeps its ability of specific or preferable binding to the target (for example, epitope or antigen), it is covered by the term "antibody". Antibodies can be either glycosylated or not and are within the framework of the invention.

The term "derivative" or antibody "variant", as used herein, refers to a molecule, the amino acid sequence of which differs from the parental sequence by adding, deletion and/or substitution of one or more amino acid residues in the sequence of parental antibody. In the preferred embodiment, an antibody contains at least one (for example, from one to about ten, preferably <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>) amino acid substitutions in FR- or CDR-regions of the parental antibody. This application defines the identity or homology regarding the sequence of a variant antibody as the percentage of amino acid residues in a variant antibody sequence that are identical to residues in parental antibody after aligning the sequences and, if needed, introducing gaps in order to achieve the maximum percentage identical sequence.

An antibody derivative (from parental one) keeps its ability to bind the same antigen or, preferably, epitope with which the parental antibody binds, or, preferably, exhibits at least one property or biological activity exceeding that of the parental antibody. For example, the antibody preferably has a better aggregation stability, more strong affinity, improved pharmacokinetics or increased ability to inhibit the antigen biological activity, as compared to parental antibody.

The term "VHH-derivative", as used herein, refers to the derivatives of VHH antibodies, the amino acid sequence of which differs from the sequence of parental VHH antibody by substitution of one or more amino acid residues in the sequence of parental antibody. In the preferred embodiment, VHH antibody contains at least one (for example, from one to about ten preferably <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>) amino acid substitutions in FR- or CDR-regions of the parental antibody.

An antibody derivative keeps its ability to bind the same antigen or, preferably, epitope with which the parental antibody binds, or, preferably, exhibits at least one property or biological activity exceeding that of the parental antibody. For example, the antibody preferably has a better aggregation stability, stronger affinity, improved pharmacokinetics or increased ability to inhibit the antigen biological activity, as compared to parental antibody.

"Parental VHH antibody" or "initial VHH antibody", or "wild VHH antibody" as used herein refers to VHH antibody isolated from an immunized or non-immunized Camelidae animal encoded with amino acid sequence that is used to produce a VHH variant. Parental antibody can have a framework sequence originating from Camelidae (with respect to VHH variable domain), but preferably the frame sequence of the light chain variable domain is of completely or substantially human origin.

"Parental" or "initial", or "wild" antibody, as used herein, refers to an antibody encoded with amino acid sequence that is used to produce a variant. Parental antibody can have a framework sequence originating from Camelidae (with respect to VHH variable domain), but preferably the frame sequence of the light chain variable domain is of completely or substantially human origin.

As used herein, the term "specifically binds" refers to such a situation in which one party involved in the process of specific binding does not significantly bind molecules different from the ones of its specific binding partner (partners). This term also applies if, for example, an antigen-binding site of the antibody according to the invention is specific for particular epitope that is carried by a number of antigens; in this case, the specific antibody with an antigen-binding domain will be able to bind specifically with various epitope-carrying antigens. Thus, the monoclonal antibody, according to the invention, specifically binds to human IL-<NUM> (IL-17A), while it does not specifically bind human IL-17B, IL-17C, IL-17D or IL-17E. Moreover, a monoclonal antibody of the invention specifically binds human IL-<NUM> and IL-<NUM> of cynomolgus monkey, but it does not specifically bind neither rat IL-<NUM> nor murine IL-<NUM>.

As used herein, the term "preferably binds" refers to such a situation in which an antibody binds a specific antigen at least by <NUM>% more, preferably by about <NUM>%, or <NUM>-fold, <NUM>-fold, <NUM>-fold or <NUM>-fold more than it binds any other antigen, as measured according to the procedures known from the prior art (for example, competitive ELISA or KD measurements obtained with Octet apparatus). Antibody can preferably bind one epitope within an antigen but not bind another epitope of the same antigen. Thus, an antibody of the invention preferably binds human IL-<NUM>, but not rabbit IL-<NUM>.

As used herein, the term "epitope" refers to the molecule part that can be recognized by and bind an antibody via one or several antigen-binding sites of an antibody. Epitopes often comprise chemically surface-active groups of molecules such as amino acids or sugar side chains, and have specific <NUM>-D structural characteristics. "Inhibiting epitope" and/or "neutralizing epitope" means an epitope that in the context of an intact antigen molecule and while binding with an antibody specific to the said epitope, causes in vivo or in vitro loss or reduction of activity of the molecule or organism that contains the molecule.

As used herein, the term "epitope" also refers to a polypeptide fragment, having antigenic and/or immunogenic activity in animals, preferably in mammals such as mice and humans. The term "antigenic epitope" as used herein is a polypeptide fragment which can specifically bind the antibody and can be detected by any technique well known from the prior art (for example, by means of standard immunoassay). Antigen epitopes are not necessary immunogenic, but they can possess immunogenicity. "Immunogenic epitope", as used herein, is defined as a polypeptide fragment that evokes an antibody response in animals, as determined by any method of the prior art. "Nonlinear epitope" or "conformational epitope" contains nonadjacent polypeptides (amino acids) within the antigen protein, which binds with epitope-specific antibody.

The words "functional activity" or "functional characteristics" or the terms "biological activity" or "activity" referring to an antibody, according to the invention, are interchangeable as used herein, including, but not limited to: epitope/antigen affinity and specificity; ability to neutralize or to be an antagonist to IL-<NUM> in vivo or in vitro; IC<NUM>; antibody stability and in vivo immunogenicity of the antibody. Other biological properties or antibody characteristics identified from the prior art include, for example, cross-reactivity (i.e. reaction with non-human homologs of the target peptide or with other proteins or targets) and ability to retain high levels of protein expression in mammal cells. The aforementioned properties or characteristics may be observed, measured or evaluated using procedures recognized in the prior art, including but not limited to ELISA, competitive ELISA, Octet analysis, neutralization assay in vitro or in vivo without limitation, receptor binding, production and/or release of cytokine or growth factor, signal transduction and immune histochemical study of tissue sections obtained from various sources including humans, primates or any other source.

The population of "monoclonal antibodies" as used herein refers to homogenous or essentially homogeneous antibody population (i.e. at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>%, but more preferably no less than about <NUM> or <NUM>%, or further preferably, at least <NUM>% of antibodies in the population will compete for the same antigen/epitope in ELISA, or further preferably, antibodies are identical in their amino acid sequence).

A full-size antibody existing in nature is represented by immunoglobulin molecule comprising four polypeptide chains (two heavy full length H chains of about <NUM>-<NUM> kDa, and two light full length L chains of about <NUM> kDa) linked via disulfide bonds. Amino-terminal part of each chain comprises a variable domain of about <NUM>-<NUM> or more amino acids that are responsible for binding an antigen. Carboxyl-terminal domain of each chain determines the constant region that is mostly responsible for the effector function. Light chains are classified as kappa and lambda and have specific constant regions. Each light chain consists of a variable N-terminal light chain region (hereafter referred to as VL or VK) and a constant light chain region consisting of a single domain (CL or CK). Heavy chains are classified as γ, δ, α, µ and ε and determine antibody isotype, such as IgG, IgM, IgE, IgA and IgD respectively; some of them can be additionally divided into sub-classes (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1. Each heavy chain type is characterized by specific constant region. Each heavy chain comprises a variable N-terminal region (hereafter referred to as VH) and a constant heavy chain region. Constant heavy chain region consists of three domains (CH1, CH2 and CH3) for IgG, IgD and IgA, and of <NUM> domains (CH1, CH2, CH3 and CH4) for IgM and IgE. Variable domains VH, VHH and VL can also be divided into the so-called hypervariable regions (complementarity determining regions, CDR) interspersing with more conservative framework regions (FR). Each variable domain comprises three CDRs and FRs located in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

In this application, <NUM> heavy chain CDRs are referred to as "HCDR1, HCDR2 and HCDR3", while <NUM> light chain CDR are referred to as "LCDR1, LCDR2 and LCDR3". CDRs contain the majority of amino acid residues specifically interacting with an antigen. CDR-residues are numbered and positioned in compliance with Kabat Numbering Scheme.

The term "antigen" refers to an antigen target against which an antibody can be reactive; it is used herein in the same way as specialists use it in this technical field, including, but not limited to, polypeptides, peptides, polysaccharides, glycoproteins, polynucleotides (for example, DNA), or chemical antigens, receptors or interleukins. Interleukins can include interleukins of various groups, such as interleukin <NUM> (alfa and beta), interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM>, interleukin <NUM> and interleukin <NUM>.

The term "antigen" can also be used to describe the material that is used for immunization of animals (for example, llama) with the purpose of production of antibodies, according to the invention. In this context, "antigen" can have a broader meaning and may cover purified forms of an antigen as well as non-purified or not fully isolated, or purified antigen products such as cells, cell lysates, or supernatants, cell fractions, for example, cell membranes etc. with added haptens conjugated with a protein-carrier. Antigen used for immunization does not necessary mean an antigen structurally identical to an antigen target to which, finally, an antibody of the invention is able to bind. Usually, antigen used for immunization is a downsized version of an antigen target, for example, a fragment comprising an immunogenic epitope. More details about antigens used for immunization are described in literature and may be familiar to a specialist in this technical field.

Variable regions of each light/heavy chain pair form antigen-binding sites of the antibody. Thus, an intact IgG antibody has two binding sites. Except for bifunctional or bi-specific antibodies, two binding sites are identical. According to this application, "antigen-binding region" or "antigen-binding site", or "antigen-binding domain", are interchangeable, as used herein, and refer to an antibody molecule fragment comprising amino acid residues interacting with an antigen and giving the antibody its specificity and affinity to an antigen. This antibody fragment includes frame amino acid residues necessary for maintaining the proper conformation of the antigen-binding residues.

The CDR of VHH antigen-binding region and the entire antigen-binding region of the antibodies covered by the invention fully originate from Camelidae bloodline or are substantially of Camelidae origin, and comprise specific changed amino acid residues, in order to improve the particular properties of an antibody (for example, KD, koff, IC<NUM>). The amino acid sequences of the CDRs of the antibody of the invention are defined in claim <NUM>.

Preferably, the antibody framework regions, in accordance with the invention, are of Camelidae origin or of human origin, or substantially of human origin (at least by <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>% human origin), and comply with Kabat numbering.

"Antibody fragment" may represent an antibody fragment or antibody fragment that has the activity of a full-size antibody. The said antibody fragment may be represented by F(ab')<NUM>, F(ab)<NUM>, Fab', Fab Fv and scFv.

"Interleukin <NUM>", also referred to as "IL-<NUM>" or "IL-17A", is a <NUM>-<NUM> kD homo-dimeric glycoprotein. The gene of human IL-17A encodes the protein consisting of <NUM> amino acids and having a <NUM> amino acid signal sequence and <NUM> amino acid mature segment. Amino acid sequence of human IL-17A is by <NUM>%, <NUM>% and <NUM>% similar to amino acid sequences of a rabbit, mouse and rat, respectively. Amino acid sequence of human IL-17A is by <NUM>% identical to IL-17A of cynomolgus monkey.

The term "antibody", when applied to anti-IL-<NUM> monoclonal antibody of the invention (hereafter referred to as an "antibody of the invention"), as used herein, means a monoclonal IgG antibody.

As used herein, the terms "inhibit" or "neutralize" regarding the activity of an antibody of the invention shall mean the ability to block, prevent, restrict, slow down, stop, reduce or reverse significantly, for example, the development or severity of inhibition subject, including but not limited to biological activity (such as activity of IL-<NUM>) or property, disease or condition. Binding the antibody, according to the invention, with IL-<NUM> results in inhibition or neutralization of IL-<NUM> activity preferably of at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>% or higher.

The term "separated" or "isolated" with regard to nucleic acids or protein products (such as an antibody) refers to the nucleic acid molecule or protein molecule that is identified and separated from at least one of contaminating substances to which it is usually combined in the natural source. Preferably, an "isolated antibody" is an antibody that substantially contains no other antibodies that have particular antigenic specificity (for example, pharmaceutical compositions of the invention contain an isolated antibody that specifically binds IL-<NUM> and substantially contain no antibodies that specifically bind antigens different from IL-<NUM>).

The term "Kabat numbering scheme" or "numbering according to Kabat" as used herein refers to a system of numbering amino acid residues that are more variable (i.e. hypervariable) than other amino acid residues in variable regions of heavy and light antibody chains (<NPL>); <NPL>)).

Polynucleotide is "functionally bound", if it has functional links to other polynucleotides. For example, promoter or enhancer is functionally bound to coding sequence if it affects the sequence transcription. Polypeptide is "functionally bound" to another polypeptide if polynucleotides coding thereof are functionally bound, preferably if they are located in the same open reading frame.

The term "DNA structure", as used herein, refers to DNA or its fragment coding an antibody of the invention. Generally, DNA or its fragment that encodes an antibody (for example, an antibody of the invention) is functionally (operably) bound, within an open reading frame, to at least one other DNA fragment that encodes an additional polypeptide (for example, domain or region of a receptor for another cytokine, such as IL-<NUM>-receptor), and then is inserted into the appropriate expressing vector. Normally, DNA structures are formed in such a way that several DNA fragments encoding certain antibody sites are functionally associated within a reading frame to obtain a solid structure that encodes either an entire antibody or its functional fragment. For example, DNA structure would encode an antibody from N-terminus to C-terminus. Such antibodies can be expressed, isolated and evaluated regarding their activity.

The term "vector", "plasmid" refers to nucleic acids obtained synthetically and via biotechnology containing a certain sequence of functional elements known in this field. Certain vectors can autonomously replicate in host cells to which they were introduced, while other vectors can be integrated into host cell genome and replicate together with the host genome. Moreover, some vectors can mediate the expression of genes to which they are functionally bound. In this application such vectors are called "recombinant expression vectors" (or "expression vectors"); exemplary vectors are well known from the prior art.

As used herein, the terms "cell", "host cell", "cell line" and "cell culture" are interchangeable and refer to an individual cell or cell culture that is a recipient of any isolated polynucleotide, according to the invention, or any recombinant vector (recombinant vectors) that contains the sequence of an antibody of the invention. Host cells involve offspring obtained from an individual host cell; offspring may not necessary be completely identical (regarding the morphology or full DNA complement) to the original host cell due to natural, accidental or intended mutations and/or variations. A host cell includes cells that were transformed, transduced or infected with recombinant vector, or a monoclonal antibody that expresses a polynucleotide covered by the invention or its heavy or light chain. Host cell that contains a recombinant vector covered by the invention (either incorporated into host chromosomes or not) can also be called "recombinant host cell". Preferable host cells to be used in the invention are CHO cells (for example, ATCC CRL-<NUM>), NS0 cells, SP2/<NUM> cells, COS cells (ATCC, for example, CRL-<NUM>, CRL-<NUM>) and HeLa (ATCC CCL-<NUM>). Additional host cells to be used in the invention include plant cells, yeast cells, other mammalian cells and prokaryotic cells.

The term "Specific binding" between an antibody and an antigen target (antigen) refers to immunological specificity. Antibody can specifically bind an antigen target if it binds an antigen epitope stronger than other antigen epitopes. Specific binding does not exclude cross-reactivity with other antigens that carry similar antigen epitopes.

VL domains in antibodies of the invention can be of either VL lambda type or VL kappa type. The term "VL domain" covers both VL lambda and VL kappa isotypes that contain one or more amino acid substitutions, insertions or deletions.

The term "pharmaceutical composition" covers the formulation and/or composition containing a therapeutically efficient amount of the antibody, according to the invention, and adjuvants (excipients, diluents, filling materials, solvents and other adjuvants, such as).

The term "use" or "treatment" applies to the ability of using an antibody of the invention or a pharmaceutical composition containing thereof to treat, alleviate the course of the disease, expedite the remission or reduce the recurrence rate for the disease or disorders mediated by receptors with which an antibody of the invention can bind.

The present invention proposes humanized monoclonal antibodies of IgG type wherein variable domains are represented by a combination of VHH-derivative with a variable domain of the light chain VL, as defined in claim <NUM>.

In one embodiment, the heavy chain variable domain of the antibody according to the invention may comprise amino acid substitutions at positions 44X<NUM>45X<NUM>, with X<NUM> = G, A, V, S, T; and X<NUM> = A, V, T, H; or combinations thereof (<NUM> and <NUM> designate the positions of amino acid substitutions). Hereinafter the position of amino acid substitution is indicated using Kabat numbering scheme (http://www.

Another embodiment involves an antibody of the invention that has improved aggregation stability of the VHH-domain, as compared to the native VHH antibody VHH isolated from an immunized animal as indicated in claim <NUM>, wherein the immunized animal is from Camelidae family.

Another embodiment of the invention involves an antibody as specified in claim <NUM>, which comprises VHH-derivative that is a variable domain of the heavy chain of an antibody isolated from an immunized animal from Camelidae family. Herein, VHH-derivative can have additional amino acid substitutions typical of humans at any positions, except for the following substitutions at positions <NUM> and <NUM>:.

Another embodiment involves an antibody as specified in claim <NUM>, which comprises VHH-derivative that can be represented by a heavy chain variable domain isolated from non-immunized animal from Camelidae family, and wherein the VHH-derivative can have additional amino acid substitutions typical of humans at any positions, except for the following substitutions at positions <NUM> and <NUM>:.

The antibody of the invention comprises a light chain variable domain VL that is a derivative of a human antibody or a light chain variable domain VL of a humanized antibody.

Another embodiment involves an antibody of the invention that comprises a VHH-derivative that contains cysteine-<NUM> (Kabat numbering scheme), and a light chain variable domain VL that contains cysteine-<NUM> (Kabat numbering scheme).

Another embodiment of the invention involves an antibody of any of the following isotypes: IgG1, IgG2, IgG3 or IgG4.

Another embodiment involves an antibody of the invention that comprises a non-native modified Fc as a part of IgG.

Another embodiment involves an antibody of the invention that has such aggregation stability under which the content of aggregates increases by not more than <NUM>% of their initial content in the solution, when used in concentrations over <NUM>/ml and stored for ><NUM> months at a temperature of <NUM>. In additional embodiment of the invention, an antibody has such aggregation stability under which the content of aggregates increases by not more than <NUM>% of their initial content in the solution, when used in concentrations over <NUM>/ml and stored for ><NUM> weeks at a temperature of <NUM>. Another additional embodiment of the invention involves an antibody that has such aggregation stability under which the content of aggregates increases by not more than <NUM>% of their initial content in the solution, when used in concentrations over <NUM>/ml and stored for ><NUM> at a temperature of <NUM>. One more additional embodiment of the invention involves an antibody that has such aggregation stability under which the content of aggregates increases by not more than <NUM>% of their initial content in the solution, when used in concentrations over <NUM>/ml and stored for > <NUM> at a temperature of <NUM>.

One embodiment involves an antibody with dissociation constant KD ≤ <NUM>-<NUM> M. Another embodiment involves an antibody of the invention that has an antibody-antigen interaction association constant kon(<NUM>/Ms) ≥ <NUM><NUM> <NUM>/Ms. One more embodiment of the invention involves an antibody that has a antigen-antibody dissociation constant dis(<NUM>/s) ≤ <NUM>-<NUM> <NUM>/s.

The antibodies of the invention can be produced by a method which involves phases selected from the following: directed mutagenesis, display methods, genetic engineering, biochemistry and high-performance biotechnology methods well known from the prior art, which can include the methods for directed mutagenesis in different positions of VHH domain of Camelidae antibodies.

In addition, the invention provides a DNA construct encoding antibodies, according to the invention and an expression vector comprising one or several DNA constructs of the invention.

Moreover, a cell line comprising the said expression vector or DNA construct is suggested.

In addition, the invention suggests a method of production of a humanized monoclonal antibody according to claim <NUM>, which involves incubation of the host cell in a culture medium under the conditions sufficient to obtain the said antibody , followed by isolation and purification of the obtained antibody.

The inventive antibody can be used in a pharmaceutical composition that contains the antibody in combination with one or several pharmaceutically suitable excipients, diluents or carriers. Details of the techniques to produce the composition are described in special biotechnology guidelines, for example in [<NUM>].

The antibody of the invention as defined in claim <NUM> specifically binds human IL-17A and comprises a derivative of the heavy chain variable domain (VHH) comprising <NUM> hypervariable regions HCDR1, HCDR2 and HCDR3 wherein:.

In a preferred embodiment, the inventive antibody specifically binds human IL-17A and comprises a derivative of the heavy chain variable domain (VHH) comprising an amino acid sequence of SEQ ID NO: <NUM> and a variable domain of the light chain (VL) of a human antibody or a variable domain of the light chain of a humanized antibody.

In a alternative further preferred embodiment, the antibody specifically binds human IL-17A and contains a VHH-derivative comprising an amino acid sequence of SEQ ID NO: <NUM>; and a variable domain of the light chain (VL) of a human antibody comprising an amino acid sequence of SEQ ID NO: <NUM>.

In a further preferred embodiment, the antibody that specifically binds human IL-17A and contains a heavy chain comprising SEQ ID NO: <NUM> amino acid sequence, and a variable domain of a human antibody light chain (VL) comprising SEQ ID NO: <NUM> sequence.

An alternative embodiment involves an antibody of the invention that specifically binds human IL-17A, wherein the said antibody has a binding affinity to human IL-17A characterized by KD of ≤ <NUM>-<NUM> M. Another embodiment involves an antibody that specifically binds human IL-17A, wherein the kinetic association constant kon(<NUM>/Ms) for human IL-17A is at least <NUM><NUM> <NUM>/Ms. Another embodiment involves an antibody that specifically binds human IL-17A, wherein the kinetic dissociation constant dis(<NUM>/s) for human IL-17A is not more than <NUM>-<NUM> <NUM>/s. An alternative embodiment involves an antibody that specifically binds human IL-17A and inhibits the activity of human IL-17A by no less than <NUM>% with respect to any parameter examined by any specific activity testing.

An alternative embodiment involves an antibody that specifically binds human IL-17A, wherein the said antibody is produced by mammalian, yeast or bacterial cells.

An alternative embodiment involves an antibody that specifically binds human IL-17A and contains one or more additional amino acid substitutions in Fc-region, as compared to natural Fc, wherein the said substitutions improve physical-chemical and pharmacokinetic properties of an antibody, as compared to the antibody with natural Fc, and do not result in loss of antibody's ability to bind IL-17A.

An alternative embodiment suggests a DNA construct encoding an antibody according to the invention that specifically binds human IL-17A. Moreover, the invention suggests an expression vector comprising one or more DNA constructs encoding an antibody that specifically binds human IL-17A according to the invention. In addition, a host cell was suggested comprising a vector for obtaining an antibody that specifically binds human IL-17A according to the invention.

In addition, the invention suggests a method for the production of an antibody according to the invention that specifically binds human IL-17A, based on culturing host cells comprising a DNA construct in a culture medium under conditions suitable to obtain the said antibody, and further isolation and purification of the said antibody.

A pharmaceutical composition comprising an antibody that specifically binds human IL-17A can be administered in a therapeutically effective amount to treat an IL-17A-mediated disease or disorder.

The following examples illustrate the present invention, yet they are not intended to limit the present invention to those examples per se.

<FIG> demonstrates a scheme for production and optimization of VHH-based antibody.

Antibodies and antigens were generated in established cell line obtained from Chinese hamster ovary cells (CHO-K1) according to the published protocols [<NUM>; <NUM>]. Cells constitutively expressing the gene of EBNA1 protein (Epstein-Barr virus nuclear antigen <NUM>) were used. Suspension cultivation was conducted in flasks on orbital shaker using serum-free media from Life Technologies Corporation, in accordance with manufacturer's guidelines. For transient expression, cells in concentration of <NUM>*<NUM><NUM>/ml were transfected by means of linear polyethyleneimine (PEI MAX, Polysciences). DNA/PEI ratio was <NUM>:<NUM>-<NUM>:<NUM>. <NUM>-<NUM> days after transfection, the cell culture was centrifuged under <NUM> for <NUM> and filtered through <NUM> filter. Target proteins from culture liquid were isolated by affine HPLC.

Recombinant IL-17A protein containing <NUM> His amino acids in C-terminal region was isolated and purified from culture liquid with Profinity IMAC Ni-charged resin (Bio-Rad). Prior to purification procedures, NiCl<NUM> was added to culture liquid to reach the concentration of <NUM>. Then <NUM> of Profinity IMAC Ni-charged was added to culture liquid and mixed on a shaker for <NUM> at a room temperature. Sorbent was transferred to <NUM> Thermo scientific Polypropylene columns and washed with <NUM> column volumes of PBS to remove non-specifically bound components. The bound antigen was eluted with <NUM> imidazole (pH <NUM>) and <NUM> NaCl. Then the protein was dialyzed into PBS (pH <NUM>) by means of SnakeSkin Dialysis Tubing technique, filtered (<NUM>), transferred into tubes and stored at -<NUM>. The purity of the protein obtained was evaluated by SDS-PAGE (<FIG>, <FIG>, <FIG>).

The tested and control IgG1 antibodies were purified on <NUM> Hi Trap rProteinA FF column (GE Healthcare) in accordance with the aforementioned procedure for IL-17A-Fc. The purity of the protein obtained was evaluated by SDS-PAGE (<FIG>, <FIG>, <FIG>).

Lama Glama animal was immunized <NUM> times in succession by means of subcutaneous administration of antigen material mixed with an equal volume of full (first injection) or partial (all injections except for the first one) Freund's adjuvant. A mixture of recombinant proteins (<NUM> of each protein per injection) one of which was human IL-17A (Kit from R&D Systems) was used as an antigen. The second injection (immunization stage) was performed <NUM> weeks after the first one; three more immunizations were performed with a <NUM>-week interval. Blood samples (<NUM>) were collected <NUM> days after each injection starting from the third one.

The selected llama blood was diluted two times with PBS solution containing <NUM> EDTA. Then <NUM> of diluted blood was layered over <NUM> of Histopaque® - <NUM> medium (Sigma, density of <NUM>/ml) and centrifuged for <NUM> under <NUM>. Mononuclear cells (lymphocytes and monocytes) were selected from plasma/Histopaque medium interphase zone and washed with PBS containing <NUM> EDTA.

The total amount of RNA from mononuclear llama cells was isolated using RNeasy Mini Kit in accordance with the protocol (QIAGEN). RNA concentration assay was performed using Nanovue (GE Healthcare); the quality of isolated RNA was tested by means of <NUM>% agarose gel electrophoresis.

The reverse transcription reaction was conducted using MMLV RT kit (Evrogen), according to the recommended protocol, with MMuLV reverse transcriptase and random hexamer primers.

The reverse transcription products were used as a matrix in a two-stage polymerase chain reaction to obtain the genes of variant domains flanked with restriction sites; the reaction was performed using oligonucleotide kit and records of the authors [<NUM>; <NUM>; <NUM>]. Further, genes encoding variable domains of the light and heavy chains were put together in one fragment by means of sequential reactions of restriction, ligation and amplification as shown at <FIG>. Heavy chain genes were attached separately to kappa and lambda light chain genes. In this case, the estimated count of matrix molecules in all reactions was no less than <NUM><NUM>. The DNA product obtained (VL-CK-VH) was treated with Nhel /Eco91I restriction enzymes and ligated into original phagemid pH <NUM>. Phagemid structure is presented at <FIG>. Ligation products were transformed into SS320 electrocompetent cells prepared in accordance with protocols [<NUM>]. The repertoire of structureed kappa and lambda Fab libraries was <NUM>*<NUM><NUM> and <NUM>*<NUM><NUM>, respectively. The product of native phage-display library was prepared in accordance with the procedure described above [<NUM>].

Specific anti-IL1 7A phage-display Fab-antibodies were selected from a phage Fab-display library using a recombinant human IL-17A (a kit from R&D Systems); a series of selection cycles was performed, as described above [<NUM>; <NUM>; <NUM>]. In order to perform the selection process by panning method, human IL-17A in <NUM> carbonate buffer (pH <NUM>) was adsorbed overnight at <NUM> on the surface of HighSorb tubes (Nunc). Further, tubes were washed with PBS (pH <NUM>) and then blocked with solution containing PBS (pH <NUM>) - fat-free milk (<NUM>% weight/volume) for <NUM> hour. Then, <NUM>-<NUM> of phage solution (<NUM><NUM> phage particles per ml) in PBS (pH <NUM>) - fat free milk (<NUM>% w/vol) was transferred to the tube with the antigen, and the system was incubated for <NUM> under stirring. Unbound phages were removed by a series of washing cycles with PBS (pH <NUM>) - Tween <NUM> (<NUM>% vol. The number of washing cycles was increased from the first round to the third one by <NUM>-<NUM>-<NUM> times, respectively. Phage particles that remained bound were eluted with <NUM> Gly-HCl solution (pH <NUM>) during <NUM> under stirring, and then neutralized with <NUM> TRIS-HCl (pH <NUM>). coli TG1 bacteria were infected with the phages obtained; further, then the phages were isolated and used in the next cycle.

After the second and the third selection round, ELISA performed for the polyclonal phage product has shown significant enrichment. Pooled clones enriched with human Fab were re-cloned to expression plasmid LL comprising myctag and His6 tag on C-terminus of CHlgene of the heavy chain.

ELISA was used to measure the binding of Fab-fragments under research with human IL-17A. Fab with published AIN457 sequence (Novartis) was used as a positive control. ELISA plate wells (Nunc ImmunoMaxisorp) were coated with <NUM>µl (<NUM>µg/ml in IX coating carbonate buffer) IL-17A-Fc, hermetically closed and incubated overnight at <NUM>. All further stages were conducted in accordance with the standard ELISA protocol with high-performance systems such as GenetixQ-pix2xt (Molecular Devices) and Tecan Freedom EVO <NUM> (Tecan). Non-specific binding was blocked by adding the blocking buffer BB (<NUM>µl <NUM>% fat-free milk in PBS). Plates were incubated on a shaker for <NUM> at a room temperature. After washing with PBS-Tween, each cell was coated with <NUM>µl of test Fab-containing cell supernatant mixed with equal volume of BB. Plates were incubated on a shaker for <NUM> hour at a room temperature; further, each plate well was washed <NUM> times with PBS-Tween buffer. After washing, each well was coated (<NUM>µl/well) with anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) in PBS-Tween (<NUM>:<NUM>). Plates were transferred to rotation shaker (<NUM> at room temperature) and then washed <NUM> times with PBS-Tween buffer as described above. Colorimetric signal was obtained by adding TMB (<NUM>µl/well) until saturation (the average of <NUM>-<NUM>); further development was blocked by adding stop solution (<NUM>µl/well, <NUM>% sulfuric acid). Absorbance was measured at <NUM> using the appropriate Tecan-Sunrise plate reader (Tecan). Antibody binding was proportional to the signal produced. Those clones for which the color signal exceeded the baseline signal by more than <NUM> times were tested in competitive ELISA in order to reveal antagonistic Fab blocking the interaction between IL-17A ligand and receptor.

Competitive ELISA technique was used to test the antagonistic capacity of previously selected specific Fab against human IL-17A. Fab with published AIN457 sequence (Novartis) was used as a positive antagonist control. ELISA plates wells (Nunc Immuno Maxisorp) were covered with <NUM>µl/well IL-17RA-Fc receptor (R&D Systems; <NUM>µg/ml solution in IX coating carbonate buffer) and incubated overnight at <NUM>. All further stages were performed in accordance with standard ELISA protocols with high-performance systems such as GenetixQ-pix2xt (Molecular Devices) and Tecan Freedom EVO <NUM> (Tecan). Non-specific binding was suppressed by adding the blocking buffer BB (<NUM>µl <NUM>% fat-free milk in PBS). Plates were incubated for <NUM> hour on a shaker at a room temperature.

At the same time, <NUM>µl of test Fab-containing cell supernatant in non-binding <NUM>-well plate was mixed with <NUM>µl of IL-17A-His6-Flag (<NUM>µg/ml in <NUM>% milk diluted with PBS-Tween). The plate was incubated for <NUM> hour at <NUM> on a shaker under <NUM> rpm.

After the plate containing IL-17RA-Fc receptor was washed from BB solution, it was coated with the reaction mixture of Fab and IL-17A-His6-Flag in the amount of <NUM>µl per well. Plates were incubated and shaked for <NUM> at a room temperature, and each well was washed <NUM> times with PBS-Tween buffer. Further, <NUM>µl /well of <NUM>µg/ml anti-FLAG murine M2 antibody (Sigma) was added, and plates were incubated for <NUM> at a room temperature. After incubation, each plate well was washed <NUM> times with PBS-Tween, then coated with <NUM>µl/well of antimurine-IgG HRP-conjugated secondary antibody (Pierce-ThermoScientific) <NUM>:<NUM> diluted with PBS-Tween. The plates were incubated on rotation shaker for <NUM> at a room temperature and washed <NUM> times with PBS-Tween, as mentioned above. Colorimetric signal was obtained by adding TMB (<NUM>µl/well) until saturation (the average of <NUM>-<NUM>); further development was blocked by adding stop solution (<NUM>µl/well, <NUM>% sulfuric acid). The absorbance was measured at <NUM> using an appropriate Tecan-Sunrise plate reader (Tecan). The antibody binding was proportional to the color signal produced.

Those clones that demonstrated suppression at the level corresponding to that of control Fab antibody AIN457 were marked as positive and used in further tests. Genes of the variable domains of positive clones were subject to sequencing in accordance with standard protocols on Applied Biosystems <NUM> Genetic Analyzer (Applied Biosystems) followed by the appropriate analysis. Clones comprising <NUM> VHHFab variable domains were selected for further studies (<FIG>). In addition, it was found that 3VHHFab clone is represented in combination with <NUM> light chain domains of various sequences, three of which are shown at Figure 7A. This indicates its relative structural resistance and the fact that this is VHH domain and not the light chain that contributes to IL-17A binding.

The comparative koff screening for anti-IL-17A Fab-candidates was performed using Pall Forte Bio Octet Red <NUM> system. Anti-FABCH1 biosensors were rehydrated for <NUM> in a working buffer comprising <NUM> PBS (pH <NUM>-<NUM>), <NUM>% Tween-<NUM> and <NUM>% BSA. 10x working buffer was added to test the samples of E. coli supernatants up to 1x of the final concentration. Then anti-FABCH1 biosensors were steeped into E. coli supernatants containing Fab-fragments of candidate antibodies and incubated for <NUM> hours at a temperature of <NUM>. Sensors coated with Fab-fragments were transferred to wells with working buffer, and a baseline was registered (<NUM> sec). Then sensors were transferred to wells with analyte solution (IL-17A, <NUM>µg/ml) to achieve the antigen-antibody association (<NUM> sec). After that, sensors were returned into wells with working buffer for further dissociation (<NUM> sec). The used sensors were subject to regeneration after each test: they were placed three times into regenerating buffer (Gly-HCl, pH <NUM>) and then were applicable for use in further experiments. The curves obtained were analyzed with Octet Data Analysis (version <NUM>) according to the standard procedure with <NUM>:<NUM> interaction model.

The results obtained for koff-screening of anti-IL-17A Fab candidates are presented at Figure 7B and Table <NUM>. Specific and high-affine binding of all unique VHHFab with human IL-17A was demonstrated, wherein 3VHHFab showed very fast kon and very slow kdis(<NUM>/s) that was beyond the detection limit of the device.

Thus, 3VHHVK4B11 candidate was selected for further investigation based on the analytical results obtained.

Genes of the variable domain of the light and heavy VHH chains of 3VHHVK4B11 candidate were cloned in pEE-Hc <IMG> pEE-Lc plasmid for joint transient expression in CHO-EBNA cells as described in Example <NUM>. Further, substitutions at positions <NUM> and <NUM> (Kabat numbering scheme) were introduced by means of oligonucleotide-directed mutagenesis using PfuUltraHS polymerase (Stratagene) in accordance with Protocol [Q5® Site-Directed Mutagenesis Kit (NEB)] and procedure described in [<NUM>]. Plasmid pEE-3VHH was used as a matrix. PCR products were fractioned on low-melting agarose and purified on appropriate columns. After ligation, DNA was transformed into E. Upon selection of mutant clones with correct sequences, plasmids with mutations in 3VHH were co-transfected with pEE-LcVK4B11 (refer to <FIG> and Table <NUM>).

The comparative koff screening for anti- IL-17A VHHFab candidates was performed according to the standard protocol with Pall Forte Bio Octet Red <NUM> system (refer to Example <NUM>). A significant reduction of koff was found for mut1, mut2 and mut4, and less significant reduction for mut3, as compared to natural 3VHHFab (<FIG>, <FIG>).

The comparative analysis of aggregation characteristics for anti-IL-17A VHHIgG1 candidates was performed using the following procedure. The preparation of VHHIgG1 antibody (<NUM>/ml) in PBS buffer was heated for <NUM> hours at <NUM>. The agregation induced by thermal stress was evaluated by means of size-exclusion HPLC. The test was performed on <NUM> HPLC System (Agilent) using Tosoh TSKGel G3000SWXL column, <NUM> x <NUM>, Cat. No. <NUM> with Tosoh TSKgel Guard SWXL pre-column, <NUM> x <NUM> (particles of <NUM>, Cat. No. <NUM>). Isocratic elution with mobile phase containing <NUM> sodium phosphate buffer and <NUM> NaCl (pH <NUM>) was performed under <NUM>/min flow rate with detection at <NUM> and <NUM> wavelengths. The antibody samples were diluted with PBS (pH <NUM>) to the concentration of ~<NUM>/ml. The injection volume was <NUM>µl. Gel filtration standard mixture (Bio-Rad, Cat. No. <NUM>-<NUM>) was used to calibrate the column prior to the test.

Chromatograms presented at <FIG>, <FIG> and summary Table <NUM> demonstrate that all mutants aggregate to various extents under thermal stress, wherein the minimum stability was observed for the natural variant and the maximum stability was revealed for mut1 comprising E44G+R45L substitutions typical of classical VH structure.

In addition, the comparative study of thermal stability of the obtained preparations was performed using Thermofluor procedure (also referred to as Thermal shift assay) that determines the protein melting point measuring changes in the fluorescence of a specific dye SYPROOrange that binds to hydrophobic surfaces of the denatured protein [<NUM>]. StepOneReal-TimePCRSystem (Applied Biosystems) apparatus and recommended protocol were used to study the mutant products. The study results are shown in Table <NUM>. They rather correlate to the results obtained under thermal stress test, which confirm the stability of mut1 and mut4 in comparison to the initial variant 3VHHIgG1VK4B11.

The ability of IL-<NUM> to induce the production of IL-<NUM> by HT1080 human cells (ATCC:CCL-<NUM>) was used to analyze the neutralizing activity of VHHIgG1 candidates mut1 and mut4 regarding human recombinant IL-17A. Cells were grown on DMEM culture medium with added <NUM>% inactivated fetal serum, gentamycin and glutamine. Aliquots of <NUM>*<NUM><NUM> cells per well were seeded in <NUM>-well culture plates. Cells were allowed for attachment for <NUM> hours. The mixture of <NUM> ng/ml recombinant IL-<NUM> and <NUM> ng/ml TNF-α was incubated with VHHIgG1 dilutions for <NUM> hour at <NUM>. Then cytokine/antibody mixture was added to the cells and left overnight. The production of IL-<NUM> by HT1080 cell culture was proportional to the amount of IL-<NUM> added. The amount of the released IL-<NUM> in cell supernatant samples was evaluated by ELISA technique using DuoSet ELISA Development System Human IL6 (RD System, Cat. No. DY206). The results of evaluation of the antagonistic properties of VHHIgG1 candidates are presented at <FIG> in comparison with AIN457 (anti-IL-17A antibody by Novartis). The IC<NUM> value for mut1 was almost <NUM> times higher than that for the natural variant, while mut4 variant almost completely maintained its inhibiting capacity. The value of IC<NUM> for mut4 candidate was <NUM>±<NUM> pM. Following the results of the present study and the overall physical-chemical and biological characteristics, the said candidate was selected for further development and optimization.

The total RNA of B-lymphocytes collected from <NUM> human donors was isolated with RNeasy Mini Kit, in accordance with the appropriate protocol (QIAGEN). RNA concentration was measured with Nanovue kit (GE Healthcare), and the quality of isolated RNA was tested by <NUM>% agarose gel electrophoresis.

The reverse transcription reaction was held with MMLV RT kit (Evrogen), according to the recommended protocol, with MMuLV reverse transcriptase and random hexamer primers.

The reverse transcription products were used as a matrix in a two-stage polymerase chain reaction to obtain the genes of variable domains flanked with restriction sites; the reaction was performed using oligonucleotide kit and protocols described in [<NUM>]. Chimeric Fab specific against IL-17A were generated according to the procedure described in <CIT> based on phagemid pH5, as specified above. Genes encoding variable domains of the human light chains and genes encoding the variable domain of mut4VHH were put together in one fragment by means of sequential reactions of restriction, ligation and amplification as shown at <FIG>. In this case, the estimated count of matrix molecules in all reactions was no less than <NUM><NUM>. The DNA product obtained (VL-CK-VH) was treated with Nhel /Eco91I restriction enzymes and ligated into original phagemid pH <NUM>. The ligation products were transformed into SS320 electrocompetent cells prepared in accordance with protocols described in [<NUM>]. The repertoire of chimeric mut4 VHH Fab-library based on mixed kappa and lambda human chains was <NUM>*<NUM><NUM> transformants. Chimeric phage-displayed Fab-libraries were prepared in accordance with the procedure described above [<NUM>].

The selection of phage-displayed chimeric Fab-libraries was performed under conditions described above (refer to Example <NUM>), except for the additional incubation of IL17A-bound phage antibodies at a temperature of <NUM> within <NUM> hours in the presence of <NUM>µg/ml of IL-17A dissolved.

After the second selection round at IL-17A, significant enrichment of the library was observed. The obtained pooled clones of enriched chimeric mut4 VHH Fab-libraries were used in IL-17A screening, according to the standard protocol (refer to Example <NUM>). The final positive clones were subject to sequencing. <FIG> shows four sequences of the variable domains of human light chains VK1A7, VK3c18, VK3cl18 and VK4clE12 from high-affinity mut4 VHHFab. These sequences belong to different kappa human germ lines VK1, VK3 and VK4 comprising the minimum number of somatic mutations. It should be noted that human sequence VK4clE12 shows high homology to the previously selected llama light chain VK4B1, yet it has its specific distinctions.

The genes of the variable domain of the heavy VHH chains of mut4VHH candidate were cloned in pEE-Hc plasmid, and genes of variable domains of human light chains VK1A7, VK3c18, VK3cl18 VK3A4 and VK4E12 were cloned in pEE-Lc plasmid for the joint transient expression in CHO-EBNA cells, as described in Example <NUM>. Further, the obtained antibodies were exposed to thermal stress and their aggregation profile was studied as shown in Example <NUM>. The results obtained are presented in a summary Table <NUM>.

Furthermore, the comparative thermal stability study of the products obtained was conducted using Thermofluor procedure similar to that described in Example <NUM>. Based on the data obtained, the conclusion can be made that the selected mut4VHH pairs comprising human light chains were more stable than the natural variant comprising VVK4B11 llama light chain. Thus, in the comparative study the best aggregation stability parameters were demonstrated by mut4 VHHIgG1VK1A7 and mut4 VHHIgG1VK3c18 combinations.

The ability of IL-<NUM> to induce the production of IL-<NUM> by human HT1080 cells (ATCC:CCL-<NUM>) was used to analyze the neutralizing capacity of VHHIgG1 candidates mut4 VHHIgG1VK3c8 and mut4 VHHIgG1VK1A7 regarding human recombinant IL-<NUM> (refer to Example <NUM>). <FIG> shows the results obtained for the blocking test. It should be noted that mut4 VHHIgG1VK1A7 variant (characterized by maximum stability) demonstrated a significant drop of IC<NUM> value, while mut4 VHHIgG1VK3c8 variant with medium stability exceeded by several times the inhibition observed for more stable mut4 VHHIgG1VK1A7 variant.

For the purpose of further improvement of the aggregation stability, the method of oligonucleotide-directed mutagenesis was used to introduce substitutions at positions <NUM> and <NUM> in FR2 region of VHH for mut4 VHHIgG1VK3c8 candidate (hereafter referred to as m4VHHc8). The study did not include variants with aromatic, aliphatic or positive amino acids at position <NUM>, as they are potentially immunogenic and structurally forbidden. Mutants were transiently expressed as described in Example <NUM> (<FIG>, <FIG>). Though the amount of products for different mutants varies from one experiment to another, it should be noted that yields within the same experiment were of comparable size for all substitutions. The obtained mutant variants were exposed to thermal stress. Table <NUM> demonstrates the results obtained for different mutants. It can be seen from the results that 44G45G and 44G45D are less stable mutants the stability of which is lower than that of the initial 44G45R combination within m4VHHc8. Such variants as 44G45N, 44V45T, 44D45T, 44T45T and 44D45V mainly preserved their stability. All other combinations increased the aggregation stability of m4VHHc8, and the most stable were the variants comprising aromatic or aliphatic groups at position <NUM>. However, the variants comprising small hydrophilic and hydrophobic amino acids at both positions, such as 44G45V, 44G45T, 44V45V, 44A45V, 44T45V, 44A45T and 44S45T, have also demonstrated significant stability.

In order to evaluate the neutralizing activity in the cell test as described in Example <NUM>, experiments were performed with various mutants (<FIG>). It was shown that the small amino acids at VHH FR2 positions <NUM> and <NUM> demonstrated high antagonistic activity, while the large ones (aliphatic and aromatic) introduced at position <NUM> resulted in <NUM>-fold reduction of IC<NUM>. <FIG> presents a diagram of stability and functional properties of mutants comprising various amino acid substitutions that determine interaction of the variable domain of the heavy and light chains.

Mutations of individual positions of candidate's CDRs were inserted by means of NNK randomization technique [<NUM>] with Q5® Site-Directed Mutagenesis Kit (NEB), in accordance with the protocol. Plasmid pLL-Fab was used as a matrix. PCR products were fractioned on low-melting agarose and purified at the columns. After ligation, DNA was transformed into E. coli expression strain BL21gold (Stratagene). The individual clones obtained were grown by Fab expression in <NUM>-well plates, as described above. Supernatants containing mutant Fab arms were analyzed by ELISA under the conditions described above and using high-performance Genetix Q-pix2xt and Tecan Freedom EVO200 systems. The concentration of immobilized IL-17A was <NUM>µg/ml. Bound Fab arms were stained with <NUM>:<NUM> diluted Goat antiHuman IgG (Fab')<NUM> (HRP) conjugate (Pierce) and TMB+H2O2/ H2SO4 dye; absorption was measured at <NUM> wavelength.

The results of scanning mutagenesis are presented in Table <NUM>. The Table shows substitutions within CDR corresponding to ≤<NUM>% reduction of mutant Fab/human II-17A binding signal, when compared to the wild type sequence. Thus, such individual mutants or any combinations thereof are the field of the invention.

Thus, screening was performed for BCD109 antibody CDR amino acid positions that are tolerant to amino acid substitutions. It was demonstrated that the present set of amino acid substitutions does not significantly change the antibody affinity to human IL-17A. The said combination of substitutions can be used to improve various properties of the candidate.

BCD109 antibody was obtained from m4VHHc8 variant comprising 44G45T substitutions (described in Example <NUM>) by introducing humanizing mutations Q5V and R89V to VHH that do not change either the stability or IC<NUM> (no data provided), and three additional mutations in CH2 domain FclgG1, 232Y/234T/236E, intended to improve the antibody pharmacokinetics. The antibody has been transiently expressed.

The affinity of BCD109 binding to human, monkey and rat IL-17A was studied at OctetRed <NUM> system (ForteBio). BCD109 was non-specifically immobilized on the surface of amine reactive second-generation sensors (AR2G) according to the standard protocol described in the manufacturer's manual. The test was conducted at a temperature of <NUM> and using PBS with <NUM>% Tween-<NUM> and <NUM>% BSA as a working buffer.

Human, monkey and rat IL-17A were titrated with the working buffer with concentration from <NUM> to <NUM> with an increment of <NUM>.

Binding curves (after subtracting a reference signal) were analyzed with Octet Data Analysis software (Version <NUM>), in accordance with the standard procedure and <NUM>:<NUM> interaction model. The results are presented at <FIG>.

It can be seen that BCD109 binds to human and monkey IL-17A with picomolar affinity (<FIG>). Moreover, the candidate does not interact with rat IL-17A (no curves are presented).

BCD109 preparation of <NUM>/ml in PBS was heated for <NUM> hours at a temperature of <NUM> in accordance with the protocol described in Example <NUM>.

Results represented at <FIG> show that BCD109 antibody remains stable under thermal stress: the aggregate content was less than <NUM>%.

BCD109 antibody in concentration of <NUM>/ml was dissolved in the required amount of water for injection and pH was brought to <NUM> with citric acid. Solution was filtered (filtration sterilization) and sealed into ampoules.

The product obtained was stable for <NUM> months with no sedimentation.

Claim 1:
A humanized monoclonal IgG antibody which comprises a combination of a VHH-domain derived from an animal from the Camelidae family and a light chain variable domain VL,
wherein said antibody specifically binds to human IL-17A and comprises:
a) a heavy chain variable domain (VHH) comprising <NUM> hypervariable regions HCDR1, HCDR2 and HCDR3, wherein:
HCDR1 comprises the amino acid sequence of G-T-F-A-T-S-P-M-G (SEQ ID NO: <NUM>);
HCDR2 comprises the amino acid sequence of A-I-S-P-S-G-G-D-R-I-Y-A-D-S-V-K-G (SEQ ID NO: <NUM>); and
HCDR3 comprises the amino acid sequence of C-A-V-R-R-R-F-D-G-T-S-Y-Y-T-G-D-Y-D-S (SEQ ID NO: <NUM>); and
b) a variable domain of the light chain (VL) of a human antibody or a variable domain of the light chain of a humanized antibody.