WO2021023860A1

WO2021023860A1 - Improved horseradish peroxidase polypeptides

Info

Publication number: WO2021023860A1
Application number: PCT/EP2020/072235
Authority: WO
Inventors: Tomas Dobransky; Stanislav HRESKO
Original assignee: Db Biotech, As
Priority date: 2019-08-07
Filing date: 2020-08-07
Publication date: 2021-02-11
Also published as: EP4031658A1

Abstract

The present invention relates to polypeptides comprising multiple units of horseradish peroxidase (HRP) which can be used for several labeling detection systems. The present invention further relates to polynucleotides encoding such polypeptides, vectors and host cells comprising such polynucleotides, as well as to uses thereof and methods applying such polypeptides and polynucleotides.

Description

Improved Horseradish Peroxidase Polypeptides

Detection of a target biological marker in a biological sample or specimen may be achieved by contacting the target with a molecule which specifically binds to the target. The molecule may be, for example, a protein or a nucleic acid probe ( e.g . for use in in situ hybridization or Southern or northern blots). The molecule may be linked, either directly or indirectly to a detectable substance, thus permitting the staining and detection of the target contained in a biological sample. Detectable substances that are commonly used include dyes such as Texas red, and FITC, as well as radioactive isotopes, metal particles and enzymes which upon catalysis of a specific substrate permit colormetric detection of the target biological marker (see e.g. Coons et al. 1941, Proc Soc Exp Bio/ Med 47:200; Nakane and Pierce 1966, J Histochem Cytochem 14:929). One system for the detection of biological markers relies on immunologically derived molecules which specifically bind to a desired target biological marker in a sample.

Immunologically based detection of biological markers advantageously exploits the specificity of immune derived proteins such as antibodies for specific biological markers of interest. Typically the antibody will recognize a specific epitope on the target biological marker which permits the target to be distinguished and thus detected from other biological markers contained within a given biological sample or specimen. A variety of formats capable of detecting biological markers of interest are known, including enzyme linked immuno-assays (ELISA), flow cytometry, western blots, radioimmunoassay (RIA) and immunohistochemistry (IHC) (see e.g. Janeway et al. Immunobiology 5th edition; Garland Publishing, N.Y. NY). All of these techniques are useful in research as well as in the detection and diagnosis of a variety of diseases and conditions. IHC specifically provides a method of detecting a biological marker in a sample or tissue specimen in situ (see Mokry 1996, ACTA MEDICA 39:129). The overall-cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the biological marker of interest. Typically a sample is fixed with formalin and paraffin-embedded and cut into sections for viewing by light microscopy. The intensity of the signal obtained correlates with the level of expression of the target molecule in the sample. Early methods for performing IHC relied solely on direct detection. Direct detection means the detectable agent, e.g., a fluorescent dye, was linked directly to the primary antibody ( i.e . the antibody that specifically binds to the target biological marker). The limitation of this method was that biological markers expressed at low levels produced a weak detection signal. Attempts to overcome this limitation included linking multiple copies of the primary antibody to a dextran polymer conjugated with an enzyme such as horse radish peroxidase (HRP) (e.g., EPOS®) (Dakocytomation, Carpenteria, CA) (see Dakocytomation catalog).

Other attempts to overcome this problem included addition of a second antibody. To amplify the detection, the primary antibody was not labeled. Instead, application of a secondary antibody, linked to a detectable substance, which specifically bound to the primary antibody was added to the method. Multiple copies of the secondary antibody could bind the primary antibody thus enhancing the signal. This method is known as the indirect detection.

Several products which exploit indirect detection for use in IHC are commercially available. Examples include EnVision™+ (Dakocytomation, Carpenteria, CA) (see Dakocytomation catalog) which provides secondary antibodies linked to a polymer conjugated with horse radish peroxidase (HRP) or Alkaline Peroxidase (AP). A dual linked version of EnVision™ Systems (EnVision ™+) is also available. The dual link provides for two different secondary antibodies linked to two different polymers, where each of the polymers is conjugated to an enzyme such as HRP. The two secondary antibodies specifically bind to antibodies from different species (e.g. rabbit and mouse), thus providing flexibility in the choice of a primary antibody. Powervision® (Immunovision, Springdale, AZ) also uses a secondary antibody linked to a polymer conjugated with an enzyme. Non-biotin amplification (NBA)™ kit (Zymed Laboratories Inc., South San Francisco, CA) provides for a secondary antibody conjugated to a hapten (fluorescein) and a tertiary antibody, which specifically binds to the hapten where the tertiary antibody is conjugated with HRP. HistoFine® (Nichirei Corp, Tokyo, Japan) provides for an F(ab) linked to a polymer conjugated with an enzyme such as HRP. Also known in the art are detection systems relying on secondary antibodies linked to haptens such as biotin. In this system, the secondary antibody is contacted with a biotin binding partner such as avidin or streptavidin which is conjugated to HRP.

Despite the variety of commercial products available for performing IHC, the need still exists for methods and compositions which provide increased sensitivity in the detection of biological markers normally present at low levels in a sample, e.g., by IHC. Increased sensitivity would be useful in particular, when a condition or a disease is associated with a decrease in the amount of expression of biological markers as compared to normal.

Particularly, the HRP (horseradish peroxidase) micropolymer detection system has some limitations in routine immunohistochemistry applications. The dextran-based backbone with covalently bound HRP (up to 20 molecules of enzyme in one single chain) conjugated with anti-mouse and/or anti-rabbit IgG, together form a complex with molecular weight over a million daltons. This causes protein target markers accessibility issues, as well as potential complex stability concerns.

Accordingly, the technical problem underlying the present invention, was to comply with the disadvantages set out above.

The present invention addresses the technical problem by providing improved polypeptides comprising multiple HRP units as set forth herein below and as defined by the claims.

Thus, the present invention relates to a polypeptide comprising at least 2 horseradish peroxidase (HRP) units, preferably not more than 7, more preferably not more than 6, and most preferably not more than 5 units. In one embodiment, the polypeptide of the present invention comprises 3 or 4 HRP units. In a specific embodiment of the present invention, the polypeptide comprises 3 HRP units. In a preferred embodiment, the polypeptide comprises 3 to 5 horseradish peroxidase (HRP) units.

As defined herein, a horseradish peroxidase (HRP) is a metalloenzyme as known in the art which is able to catalyze the oxidation of various organic substrates by hydrogen peroxide, thereby yielding a characteristic color change which is detectable by spectrophotometric methods known in the art (cf. , e.g., Veitch et al. , Phytochemistry 2004, 65: 249-259; or Akkara et al., J Polymer Sci (1991), 29: 1561-1574). A unit of HRP as used and referred to herein means that a given polypeptide comprises such HRP metalloenzyme. For example, a polypeptide comprising 3 units HRP means that such polypeptide comprises 3 copies of HRP metalloenzyme, each being able to catalyze the oxidation of various organic substrates by hydrogen peroxide as known in the art for HRP. Typical substrates for HRP comprise, e.g., DAB, pyrogallol, TMB, ABTS, OPO, AEC, AmplexRed, Homovallinic acid, and others. There are multiple isotypes known for HRP. In one embodiment of the present invention, HRP refers to C-types of HRP, for example C1A (cf. UniProt entry #P00433 as of April 1, 2019) or variants/ mutants thereof as described and provided herein. As has been surprisingly found in context with the present invention, a polypeptide comprising multiple (2 to 7, preferably 3 or 4, most preferably 3) HRP units in a row exhibits superior sensitivity effect compared to single HRP units or larger HRP chains. This effect can even be improved by applying mutations which increase activity and/or stability of the HRP units as has been shown by specific mutations in context with the present invention. The superior effect of the polypeptide of the present invention over HRP protein detection systems of the prior art is inter alia illustrated in Figure 1. The sensitivity effect of a polypeptide having only, e.g., 3 HRP units can be compared to molecules comprising much higher numbers of HRP, thus not only being more difficult to handle, but also being less efficient in production. Given the small size of the inventive polypeptide, it is further possible to couple multiple molecules to a single target protein (e.g., antibody), thus further increasing sensitivity. Thus, the surprising advantage of the inventive polypeptide described and provided herein lies both in the size - easier accessibility to the target antigen - and stability of the product. Potential applications of the highly active and specific HRP polypeptide of the present invention comprise research and/or clinical diagnostics.

In one embodiment of the present invention, the polymer comprises 2 to 7, 2 to 6, 2 to 5, 3 to 5, with 3 to 5 being preferred, or 3 or 4 (preferably 3) HRP units. Such units may generally be any HRP as known in the art, being capable of catalyzing the oxidation of various organic substrates (e.g., DAB, DAB+, TMB ABTS, OPO, AEC, AmplexRed, Homovallinic acid; DAB or DAB+ being specific examples in accordance with the present invention) by hydrogen peroxide, thereby yielding a characteristic color change which is detectable by spectrophotometric methods known in the art. In one embodiment of the present invention, at least 1 , preferably at least 2, more preferably at least 3, most preferably all of said HRP units has/have an amino acid sequence being at least 95, 96%, 97%, 98%, 99%, or 99.3% similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 9.

In a preferred embodiment, the polypeptide comprises 3 to 5 horseradish peroxidase (HRP) units, wherein at least 1 of said HRP units has an amino acid sequence being at least 95% similar to the amino acid sequence of SEQ ID NO: 9.

In accordance with the present invention, as used herein in context with amino acid sequences, the term “similar” means that a given amino acid sequence comprises identical amino acids or only conservative or highly conservative substitutions compared to the amino acid sequence of the respective SEQ ID NO. As used herein, “conservative” (amino acid) substitutions mean (amino acid) substitutions as listed as “Exemplary Substitutions” in Table I below. “Highly conservative” (amino acid) substitutions as used herein mean (amino acid) substitutions as shown under the heading “Preferred Substitutions” in Table I below. TABLE I Amino Acid Substitutions

In one embodiment of the present invention, the polymer comprises 2 to 7, 2 to 6, 2 to 5, 3 to 5, with 3 to 5 being preferred, or 3 or 4 (preferably 3) HRP units. Such units may generally be any HRP as known in the art, being capable of catalyzing the oxidation of various organic substrates (e.g., DAB, DAB+, TMB ABTS, OPO, AEC, AmplexRed, Homovallinic acid; DAB and DAB+ being specific examples in accordance with the present invention) by hydrogen peroxide, thereby yielding a characteristic color change which is detectable by spectrophotometric methods known in the art. In one embodiment of the present invention, at least 1 , preferably at least 2, more preferably at least 3, most preferably all of said HRP units has/have an amino acid sequence being at least 95, 96%, 97%, 98%, 99%, or 99.3% similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 9 or 12, and comprise at least 1 (preferably exactly 1) amino acid substitution which increases activity of the HRP unit, and/or at least 1 (preferably exactly 1) amino acid substitution which increases stability of the HRP unit (“increased” activity and/or stability compared to the HRP having the amino acid sequence of SEQ ID NO: 9). In a preferred embodiment of the present invention, such HRP unit comprises at least 1 (preferably exactly 1) amino acid substitution which increases activity of the HRP unit, and at least 1 (preferably exactly 1) amino acid substitution which increases stability of the HRP unit (“increased” activity and/or stability compared to the HRP having the amino acid sequence of SEQ ID NO: 9). In this context of the present invention, in one embodiment, said at least 1 (preferably exactly 1) amino acid substitution increasing activity is within amino acid positions 40 to 46 of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12), but not at position 43 (His). In a specific embodiment of the present invention, said at least 1 (preferably exactly 1) amino acid substitution increasing activity is a substitution at position 42 (Phe) of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12), preferably a conservative amino acid substitution as defined herein (see “Exemplary Substitutions” of Table I), i.e. , L, V, I, A or Y, more preferably an F42A substitution. In another specific embodiment of the present invention, said at least 1 (preferably exactly 1) amino acid substitution increasing activity is a substitution at position 45 (Cys) of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12), preferably a conservative amino acid substitution as defined herein (see “Exemplary Substitutions” of Table I), i.e. S or A, e.g. a C45A or C45S substitution, more preferably a C45A substitution. In a more specific embodiment of the present invention, said at least 1 preferably at least 2, more preferably at least 3, most preferably all of said HRP units of the polypeptide of the present invention has/have an amino acid sequence being at least 95, 96%, 97%, 98%, 99%, or 99.3% similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 9 or 12, and comprise a substitution at position 42 (preferably conservative, more preferably F42A) and/or 45 (preferably C45A or C45S, more preferably C45A) as defined above, particularly an F42A, C45S and/or C45A substitution, preferably an F42A substitution alone or in combination with a C45S or C45A substitution, more preferably an F42A substitution alone or in combination with a C45S substitution, most preferably an F42A substitution alone, within amino acid positions 40 to 46 of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12), respectively.

In accordance with the present invention, in one embodiment, said at least 1 (preferably exactly 1) amino acid substitution increasing stability of said at least 1 HRP unit of the polypeptide of the present invention may be at position 176 (Asn) of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12), preferably, a conservative amino acid substitution selected from Q, H, D, K, R or S. In a more specific embodiment of the present invention, said at least 1 (preferably exactly 1) amino acid substitution increasing stability is an N176S substitution. That is, in that embodiment of the present invention, said at least 1 preferably at least 2, more preferably at least 3, most preferably all of said HRP units of the polypeptide of the present invention has/have an amino acid sequence being at least 95, 96%, 97%, 98%, 99%, or 99.3% similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 9 or 12, and comprise a substitution at position 176 of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12), preferably an N176S substitution. In one embodiment of the present invention, the polymer comprises 2 to 7, 2 to 6, 2 to 5, 3 to 5, or 3 or 4 (preferably 3) HRP units. Such units may generally be any HRP as known in the art, being capable of catalyzing the oxidation of various organic substrates (e.g., DAB, TMB ABTS, OPO, AEC, AmplexRed, Homovallinic acid; DAB being a specific example in accordance with the present invention) by hydrogen peroxide, thereby yielding a characteristic color change which is detectable by spectrophotometric methods known in the art. In a more specific embodiment of the present invention, at least 1, preferably at least 2, more preferably at least 3, most preferably all of said HRP units has/have an amino acid sequence being at least 95, 96%, 97%, 98%, 99%, or 99.3% similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 9 or 12, and comprise an amino acid substitution which increases activity of the HRP unit (preferably at position 42 and/or 45 of SEQ ID NO: 9 or corresponding position of SEQ ID NO: 12, more preferably an F42A substitution or a F42A / C45S double substitution, most preferably a F42A substitution), and an amino acid substitution which increases stability of the HRP unit (preferably at position 176 of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12), more preferably an N176S substitution), both activity and stability compared to the HRP having the amino acid sequence of SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12).

Thus, in a preferred embodiment, the polypeptide comprises 3 to 5 horseradish peroxidase (HRP) units, wherein at least 1 of said HRP units has an amino acid sequence being at least 95% similar to the amino acid sequence of SEQ ID NO: 9, wherein said at least 1 HRP unit being at least 95% similar to the amino acid sequence of SEQ ID NO: 9 and comprises at least 1 amino acid substitution at position F42 and/or C45 of SEQ ID NO: 9, wherein said amino acid substitution is a conservative amino acid substitution, which increases the activity of the HRP unit, and/or at least 1 amino acid substitution which increases stability of the HRP unit.

More preferably, the polypeptide comprises 3 to 5 horseradish peroxidase (HRP) units, wherein at least 1 of said HRP units has an amino acid sequence being at least 95% similar to the amino acid sequence of SEQ ID NO: 9, wherein said at least 1 HRP unit being at least 95% similar to the amino acid sequence of SEQ ID NO: 9 and comprises at least 1 amino acid substitution at position F42 and/or C45 of SEQ ID NO: 9, wherein said amino acid substitution is a conservative amino acid substitution, which increases the activity of the HRP unit and at least 1 amino acid substitution which increases stability of the HRP unit.

Preferably, said amino acid substitution increasing stability of the HRP unit is at amino acid position N176. More preferably, said amino acid substitution increasing stability of the HRP unit is a conservative amino acid substitution selected from Q, H, D, K, R or S. Even more preferably, said amino acid substitution increasing stability is N176S.

In a specific embodiment of the present invention, the polypeptide comprises 3 or 4 HRP units (preferably 3 HRP units), at least 1 of these units (preferably all units) has/have an amino acid sequence being at least 95, 96%, 97%, 98%, 99%, or 99.3% similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 9 or 12, and comprise an F42A and an N176S substitution compared to SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12). In a more specific embodiment of the present invention, the polypeptide comprises 3 HRP units, all units have an amino acid sequence being at least 95, 96%, 97%, 98%, 99%, or 99.3% similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 9 or 12, and comprise an F42A and an N176S substitution compared to SEQ ID NO: 9 (or corresponding position of SEQ ID NO: 12).

The polypeptide of the present invention may further comprise a linker between the HRP units. The linker may be any linker which allows flexibility of the single HRP units and is preferably an amino acid linker. In one embodiment of the present invention, the linker comprise about 7 to 20 amino acids, preferably about 8 to 17 amino acids, more preferably about 9 to 15 amino acids. In accordance with the present invention, examples for such linkers comprise those having an amino acid sequences as shown in SEQ ID NO: 10 or 11.

In a specific embodiment of the present invention, the polypeptide described and provided herein comprises or consist of an amino acid being at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, 99.9% or 100% (for SEQ ID NO: 12: preferably at least 98%; for SEQ ID NO: 14: preferably at least 99.9% or 100%) similar or identical (e.g., identical) to the amino acid sequence of SEQ ID NO: 12 or 14. For example, the polypeptide of the present invention comprises or consist of an amino acid sequence as shown in SEQ ID NO: 14.

HRP production is generally known in the art (cf. Kainer FW and Glieder A, 2015, Appl Microbiol Biotechnol. 99; 1611-1625). The polypeptide of the present invention may be producible or produced by any suitable host organism, including bacteria (e.g., E. coif), insect cells, yeast and CHO cells. In one embodiment of the present invention, the polypeptide described and provided herein is producible or produced by yeast or CHO cells, preferably yeast cells. Examples for suitable yeast cells comprise in accordance of the present invention Pichia pastoris (cf. Koliasnikov VO et al, 2011, Acta Naturae. 3, 85-92) and Saccharomyces cerevisiae (cf. Morawski B, Quan S, and Arnold FH, 2001 , Biotech and Bioeng, 76, 2, 99-107), particularly Pichia pastoris. The polypeptide of the present invention may further comprise a tag (e.g., a protein tag) at the N- or the C-terminus (preferably C-terminus) to allow easy purification of the polypeptide. In accordance with the present invention, suitable tags comprise His-Tag (e.g., Ni Sepharose High Performance), FLAG-Tag, Strep-Tag, Myc-Tag, HA-Tag, C-Tag, and others known in the art. In one embodiment of the present invention, the polypeptide described and provided herein comprises a His-Tag at its C-terminus.

The polypeptide of the present invention may further be coupled (e.g., covalently linked) to a binding agent (e.g., an antibody) as described herein. For example, the antigen is an antibody, e.g., a monoclonal antibody. As a further example in context with the present invention, the polypeptide described and provided herein may be linked to the Fc-part of an antibody, e.g., to sugar moieties of the Fc-part.

A “binding agent” as used herein may be a polypeptide which comprises one or more binding domains capable of binding to a target epitope. A recognition molecule, so to say, provides the scaffold for said one or more binding domains so that said binding domains can bind/interact with a given target structure/antigen/epitope. The term "binding domain" characterizes in connection with the present invention a domain of a polypeptide which specifically binds/interacts with a given target epitope. An “epitope” is antigenic and thus the term epitope is sometimes also referred to herein as “antigenic structure” or “antigenic determinant”. Thus, the binding domain is an "antigen-interaction-site". The term "antigen- interaction-site" defines, in accordance with the present invention, a motif of a polypeptide, which is able to specifically interact with a specific antigen or a specific group of antigens, e.g. the identical antigen in different species. Said binding/interaction is also understood to define a "specific recognition".

The term "specifically recognizing" (equally used herein with “specifically binding”, “directed to” or “reacting with”) means in accordance with this invention that the binding agent is capable of specifically interacting with and/or binding to at least two, preferably at least three, more preferably at least four amino acids of an epitope as defined herein. Such binding may be exemplified by the specificity of a "lock-and-key-principle". Thus, the term “specifically” in this context means that the recognition molecule binds to a given target epitope but does not essentially bind to another protein. The term “another protein” includes any protein including proteins closely related to or being homologous to the epitope against which the recognition molecule is directed to. However, the term “another protein” does not include that the recognition molecule cross-reacts with the epitope from a species different from that against which the recognition molecule was generated.

The term “does not essentially bind” as used herein means that the epitope recognition molecule of the present invention does not bind another protein, i.e. shows a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6 or 5% with another protein.

Specific binding is believed to be effected by specific motifs in the amino acid sequence of the binding domain and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure. The specific interaction of the antigen-interaction-site with its specific antigen may result as well in a simple binding of said site to the antigen. Moreover, the specific interaction of the antigen- interaction-site with its specific antigen may alternatively result in the initiation of a signal, e.g. due to the induction of a change of the conformation of the antigen, an oligomerization of the antigen, etc. A preferred example of a binding domain in line with the present invention is an antibody. Typically, binding is considered “specific” when the binding affinity is higher than 10^-6M. Preferably, binding is considered specific when binding affinity is about 10^-11 to 10^-8 M (K_D), preferably of about 10^-11 to 10^-9 M. If necessary, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions. Whether the recognition molecule specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of said recognition molecule with an epitope with the reaction of said recognition molecule with (an) other protein(s).

The term “polypeptide” is equally used herein with the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide" as used herein describes a group of molecules which typically comprise more than 15 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides/proteins wherein the modification is effected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art. A preferred binding agent to be employed in context with the present invention is an antibody. An “antibody” when used herein is a protein comprising one or more polypeptides (comprising one or more binding domains, preferably antigen binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.

In particular, an “antibody” when used herein, is typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1, and lgA2. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons.

The term "amino acid" or "amino acid residue" as used herein typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

When used herein the term "antibody" does not only refer to an immunoglobulin (or intact antibody), but also to a fragment thereof, and encompasses any polypeptide comprising an antigen-binding fragment or an antigen-binding domain. Preferably, the fragment such as Fab, F(ab')₂, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, such fragments would comprise an antigen-binding domain and have the same properties as the antibodies described herein.

The term “antibody” as used herein includes antibodies that compete for binding to the same epitope as the epitope bound by the antibodies of the present invention, preferably obtainable by the methods for the generation of an antibody as described herein elsewhere.

To determine if a test antibody can compete for binding to the same epitope, a cross blocking assay e.g., a competitive ELISA assay can be performed. In an exemplary competitive ELISA assay, epitope-coated wells of a microtiter plate, or epitope-coated sepharose beads, are pre-incubated with or without candidate competing antibody and then a biotin-labeled antibody of the invention is added. The amount of labeled antibody bound to the epitope in the wells or on the beads is measured using avidin-peroxidase conjugate and appropriate substrate.

The term “antibody" also includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific such as bispecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies, with a polyclonal antibody being preferred. Said term also includes domain antibodies (dAbs) and nanobodies.

Furthermore, the term "antibody" as employed in the invention also relates to derivatives or variants of the antibodies described herein which display the same specificity as the described antibodies. Examples of "antibody variants" include humanized variants of non human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al., J Mol Biol (1992), 254, 889-896; and Lowman et al., Biochemistry (1991), 30: 10832- 10837) and antibody mutants with altered effector function (s) (see, e.g., US Patent 5, 648, 260).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature (1975), 256: 495, or may be made by recombinant DNA methods (see, e.g., U. S. Patent No. 4,816, 567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature (1991), 352: 624- 628; and Marks etal., J Mol Biol (1991), 222: 581-597, for example.

The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U. S. Patent No. 4,816, 567; Morrison et al., PNAS USA (1984), 81 : 6851-6855). Chimeric antibodies of interest herein include "primitized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab¹) 2 or other antigen-binding subsequences of antibodies) of mostly human sequences, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, "humanized antibodies" as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature (1986), 321: 522-525; Reichmann et al., Nature (1988), 332: 323-329; and Presta, Curr. Op. Struct Biol (1992), 2: 593-596.

The term "human antibody" includes antibodies having variable and constant regions corresponding substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (See Kabat et al., loc. cit.). The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, CDR3. The human antibody can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.

Numerous methods known to those skilled in the art are available for obtaining antibodies or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods (U.S. Patent 4,816,567). Monoclonal antibodies may also be produced by generation of hybridomas (see e.g., Kohler and Milstein, Nature (1975), 256: 495-499) in accordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof.

One exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in U.S. Patent No. 5,223,409; Smith, Science (1985), 228: 1315-1317; Clackson et al., Nature (1991), 352: 624-628; Marks et al., J Mol Biol (1991), 222: 581-597WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced using recombinant DNA techniques known in the art. A variety of approaches for making chimeric antibodies have been described. See, e.g., Morrison et al., PNAS USA (1985), 81: 6851; Takeda et ai, Nature (1985), 314: 452; U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; EP 171496; EP 173494, GB 2177096. Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison, Science(1985), 229: 1202-1207; Oi et aL, BioTechniques (1986), 4: 214; US 5,585,089; US 5,693,761; US 5,693,762; US 5,859,205; and US 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

In a specific embodiment of the present invention, the polypeptide described and provided herein is covalently linked to 1 to 4, 2 to 4 or 2 to 3 sugar moieties of the Fc part of an antibody, e.g. to SATA-activated antibodies (cf. Dhawan, 2002, Peptides, 23: 2091-2098). Generally, in this context, any antibodies, e.g., anti-mouse, anti-goat, or anti-rabbit (preferably anti-mouse or anti-rabbit), may be applied in accordance with the present invention. The antibody may be of any isotype, e.g., IgG, IgM, IgA, IgD, IgE or any subclass, e.g., lgG1, lgG2, lgG3, or lgG4.

The present invention further relates to a polynucleotide encoding the polypeptide of the present invention as described and provided herein. In this context, the nucleotide sequences encoding the single HRP units of the inventive polypeptide may all have the same nucleotide sequences or have different sequences, preferably at least 1 is different from the others, most preferably they all differ from one another by at least one nucleotide, but may (and preferably do) encode the same amino acid sequence. In one embodiment of the present invention, the polynucleotide of the present invention comprises a nucleotide sequence being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.9% or 100% identical to SEQ ID NOs. 1, 4 or 7. That is, in one embodiment of the present invention, the nucleotide sequence encoding 1 or more of the HRP units comprises or has a nucleotide sequence being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.9% or 100% identical to any of the nucleotide sequences shown in SEQ ID NOs. 1 , 4 or 7. In a preferred embodiment of the present invention, at least 1 nucleotide sequence encoding a single HRP unit of the inventive polypeptide is different from at least 1 other nucleotide sequence encoding another HRP unit of the polypeptide. More preferably, all nucleotide sequences each encoding a HRP unit are different from one another, but may (and preferably do) encode the same HRP amino acid sequence. In one embodiment of the present invention, the nucleotide sequence encoding 1 or more of the HRP units comprises or has a nucleotide sequence being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, or 99.9% identical to any of the nucleotide sequences shown in SEQ ID NOs. 1, 4 or 7, and comprises a substitution at nucleotide triplet 124-126 (preferably resulting in an amino acid substitution F A), and/or (preferably and) at nucleotide triplet 526- 528 (preferably resulting in an amino acid substitution N S).

In this context, it is preferred that the nucleotide sequences still encode the same HRP unit amino acid sequences (e.g., they all encode an amino acid sequence being at least 95%, 96%, 97%, 98%, 99%, or 99.3% similar or identical to SEQ ID NO: 9, preferably with the substitutions as set forth above, e.g., F42 (F42A), and/or N176 (N176S)), i.e. the nucleotide differences between the nucleotide sequences encoding different HRP units are “silent” when it comes to translation. In a specific embodiment of the present invention, the polynucleotide of the present invention comprises or has a nucleotide sequence being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.9% or 100% identical to SEQ ID NO: 8. In a further specific embodiment of the present invention, the polynucleotide of the present invention comprises or has a nucleotide sequence being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, or 99.9% identical to SEQ ID NO: 8, and comprise one or more substitutions at the nucleotide triplets encoding F42 and/or N176 (numeration corresponding to each HRP unit; e.g., F42, F367 and F692, and N176, N501 and N826, corresponding to the encoded amino acid sequence; cf. SEQ ID NO: 12), preferably resulting in an amino acid substation of F42A and/or N176S (numeration corresponding to each HRP unit; e.g., F42A, F367A and F692A, and N176S, N501S and N826S, corresponding to the encoded amino acid sequence; cf. SEQ ID NOs: 12 and 14), most preferably comprising substitutions at all nucleotide triplets encoding F42 (F42A) and N176 (N176S) (numeration corresponding to each HRP unit; e.g., F42 (F42A), F367 (F367A) and F 692 (F692A), and N176 (N176S), N501 (N501S) and N826 (N826S), corresponding to the encoded amino acid sequence; cf. SEQ ID NOs: 12 and 14). For example, the polynucleotide of the present invention comprises or has a nucleotide sequence being at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, or 99.9% identical to SEQ ID NO: 8, and comprise one or more substitutions at nucleotide triplets 124-126 (preferably resulting in an amino acid substitution F A), 526-528 (preferably resulting in an amino acid substitution N S), 1099-1101 (preferably resulting in an amino acid substitution F A), 1501-1503 (preferably resulting in an amino acid substitution N S), 2074-2076 (preferably resulting in an amino acid substitution F A), and/or (preferably and) 2476-2478 (preferably resulting in an amino acid substitution N S).

Furthermore, the polynucleotide of the present invention may also comprise nucleotide sequences encoding a signal peptide and/or a propeptide of a HRP unit.

As used herein, unless specifically defined otherwise, the term “nucleic acid” or “nucleic acid molecule” is used synonymously with “oligonucleotide”, “nucleic acid strand”, or the like, and means a polymer comprising one, two, or more nucleotides. In this context, also the term “target sequence” as used herein comprises nucleic acid molecules.

As used herein, “silent” mutations mean base substitutions within a nucleic acid sequence which do not change the amino acid sequence encoded by the nucleic acid sequence. “Conservative” substitutions mean substitutions as listed as “Exemplary Substitutions” in Table I. “Highly conservative” substitutions as used herein mean substitutions as shown under the heading “Preferred Substitutions” in Table I.

The term "position" when used in accordance with the present invention means the position of an amino acid within an amino acid sequence depicted herein. The term "corresponding" in this context also includes that a position is not only determined by the number of the preceding nucleotides/amino acids.

The level of identity between two or more sequences (e.g., nucleic acid sequences or amino acid sequences) can be easily determined by methods known in the art, e.g., by BLAST analysis. Generally, in context with the present invention, if two sequences (e.g., polynucleotide sequences or amino acid sequences) to be compared by, e.g., sequence comparisons differ in identity, then the term "identity" may refer to the shorter sequence and that part of the longer sequence that matches said shorter sequence. Therefore, when the sequences which are compared do not have the same length, the degree of identity may preferably either refer to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence or to the percentage of nucleotides in the longer sequence which are identical to nucleotide sequence in the shorter sequence. In this context, the skilled person is readily in the position to determine that part of a longer sequence that matches the shorter sequence. Furthermore, as used herein, identity levels of nucleic acid sequences or amino acid sequences may refer to the entire length of the respective sequence and is preferably assessed pair-wise, wherein each gap is to be counted as one mismatch. These definitions for sequence comparisons (e.g., establishment of "identity" values) are to be applied for all sequences described and disclosed herein.

Moreover, the term “identity” as used herein means that there is a functional and/or structural equivalence between the corresponding sequences. Nucleic acid/amino acid sequences having the given identity levels to the herein-described particular nucleic acid/amino acid sequences may represent derivatives/variants of these sequences which, preferably, have the same biological function. They may be either naturally occurring variations, for instance sequences from other varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques. Deviations from the above-described nucleic acid sequences may have been produced, e.g., by deletion, substitution, addition, insertion and/or recombination. The term "addition - refers to adding at least one nucleic acid residue/amino acid to the end of the given sequence, whereas "insertion" refers to inserting at least one nucleic acid residue/amino acid within a given sequence. The term "deletion" refers to deleting or removal of at least one nucleic acid residue or amino acid residue in a given sequence. The term "substitution" refers to the replacement of at least one nucleic acid residue/amino acid residue in a given sequence. Again, these definitions as used here apply, mutatis mutandis, for all sequences provided and described herein.

Generally, as used herein, the terms ..polynucleotide" and ..nucleic acid" or ..nucleic acid molecule" are to be construed synonymously. Generally, nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules. Furthermore, the term "nucleic acid molecule" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 5525711, US 471 1955, US 5792608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribosomal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332 - 4339). Said polynucleotide sequence may be in the form of a vector, plasmid or of viral DNA or RNA. Also described herein are nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present invention. Particularly, such a fragment is a functional fragment. Examples for such functional fragments are nucleic acid molecules which can serve as primers.

The present invention further relates to a vector comprising the polynucleotide described and provided herein

The term "vector" as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In one embodiment of the present invention, the vectors are suitable for the transformation, transduction and/or transfection of host cells as described herein, e.g., prokaryotic cells (e.g., (eu)bacteria, archaea), eukaryotic cells (e.g., mammalian cells, insect cells) fungal cells, yeast, and the like. Examples of bacterial host cells in context with the present invention comprise Gram negative and Gram positive cells. Specific examples for suitable host cells may comprise inter alia CHO cells and yeast cells such as, e.g., S. cerevisiae, Schizosaccharmyces pombe, Micrococcus luteus, or - preferably - Pichia pastoris (today also known as Komagataella pastoris or Komagataella phaffii). In one embodiment of the present invention, said vectors are suitable for stable transformation of the host cells.

Accordingly, in one aspect of the invention, the vector as provided is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is preferably at least one restriction site or a polylinker which enables the insertion of a nucleic acid sequence/molecule desired to be expressed. It is to be understood that when the vector provided herein is generated by taking advantage of an expression vector known in the prior art that already comprises a promoter suitable to be employed in context of this invention. The nucleic acid construct is preferably inserted into that vector in a manner the resulting vector comprises only one promoter suitable to be employed in context of this invention. The skilled person knows how such insertion can be put into practice. For example, the promoter can be excised either from the nucleic acid construct or from the expression vector prior to ligation. In one embodiment of the present invention, the vector is able to integrate into the host cell genome. The vector may be any vector suitable for the respective host cell, preferably an expression vector. A non-limiting example of the vector of the present invention may comprise yeast expression vector pPICZaA (see, e.g., Table 1 and Figure 2) comprising the polynucleotide in context of the present invention.

Table 1 : Features of pPICZaA

The present invention also relates to host cells comprising a polynucleotide as described and provided herein encoding a polypeptide according to the present invention, and/or a vector as described and provided herein. In one embodiment of the present invention, said polynucleotide comprised by said host cell is codon-optimized for said host cell as described herein. In one embodiment of the present invention, said host cell of the present invention is able to stably express the polynucleotide and/or vector.

The host cell of the present invention may generally be any host cell, preferably it allows glycosylation of the polypeptide of the present invention. Such host cells may comprise, inter alia, eukaryotic cells (e.g., mammalian cells, insect cells), fungal cells, yeast, and the like. In one embodiment of the present invention, the host cell is a yeast or CHO cell, preferably a yeast cell. Specific examples for suitable host cells may comprise inter alia CHO, S. cerevisiae, and Pichia pastoris, most preferably Pichia pastoris.

The present invention further relates to the use of a polypeptide of the present invention in immuno-analytical methods. Such immune-analytical methods are known in the art and comprise, inter alia, immunohistochemistry (I HC), ELISA, Western blot, ELIA, immunocytochemistry (ICC), protein arrays, and others.

The present invention further relates to a method for detecting a biological marker in a sample (e.g., biological sample like blood sample, tissue sample, urine sample, salivary sample, or other fluid or solid body sample comprising a biological marker (e.g., antigen, epitope, nucleic acid sequence) of interest to be detected), comprising

(i) contacting the sample with at least one first binding agent (e.g., nucleic acid molecule or antibody; for specific immune-analytical methods e.g. primary antibody) which (specifically) binds to the biological marker and forms a first complex,

(ii) optionally contacting the first complex with a second binding agent (e.g., nucleic acid molecule or antibody; secondary antibody) which (specifically) binds to the first complex to form a second complex; wherein at least one first binding agent or - if step (ii) is applied - at least one second binding agent is covalently linked to the polypeptide of the present invention (preferably at sugar moieties of the Fc part of the respective antibody as also described herein above),

(iii) contacting the polypeptide with a substrate (e.g., DAB, pyrogallol or others as described and exemplified herein) of said polypeptide; and

(iv) detecting binding of said polypeptide with said substrate.

Biological marker, as used herein, refers to any molecule present in a biological sample. The marker may include a protein, including a glycoprotein or lipoprotein, phosphoprotein, methylated protein, or a protein fragment, e.g., a peptide or a polypeptide, a nucleic acid, e.g., DNA, RNA, a lipid, a glyco-lipid, a sugar, a polysaccharide, a starch. The marker may be expressed on the surface of the biological sample, e.g., membrane bound. The marker may be contained in the interior of the biological sample, i.e., within the cell membrane, e.g., within the cytoplasm, within the nucleus, within an intracellular compartment or organelle.

The present invention may also be characterized by the following items:

1. Polypeptide comprising 2 to 5 horseradish peroxidase (HRP) units.

2. Polypeptide of item 1, wherein at least 1 of said HRP units has an amino acid sequence being at least 95% similar to the amino acid sequence of SEQ ID NO: 9.

3. Polypeptide of item 2, wherein said at least 1 HRP unit being at least 95% similar to the amino acid sequence of SEQ ID NO: 9 comprises at least 1 amino acid substitution which increases activity of the HRP unit, and/or at least 1 amino acid substitution which increases stability of the HRP unit.

4. Polypeptide of item 3, wherein said amino acid substitution increasing activity is within amino acid positions 40 to 46 of SEQ ID NO: 9.

5. Polypeptide of item 4, wherein said amino acid substitution increasing activity is at position F42 and/or C45 of SEQ ID NO: 9.

6. Polypeptide of item 5, wherein said acid substitution increasing activity is F42A, C45A, and/or C45S of SEQ ID NO: 9.

7. Polypeptide of item 3, wherein said amino acid substitution increasing stability is at amino acid position N176. 8. Polypeptide of item 7, wherein said acid substitution increasing stability is N176S.

9. Polypeptide of any one of items 1 to 8, comprising a linker of 9 to 15 amino acids between each HRP unit.

10. Polypeptide of any one items 1 to 9, which is producible by yeast or CHO cell.

11. Polypeptide of any one of items 1 to 10, further comprising a protein tag a the N- or the C-terminus for purification of the polypeptide.

12. Polypeptide of any one of items 1 to 11 , which is further covalently linked to a binding agent.

13. Polypeptide of item 12, wherein said binding agent is an antibody and said polypeptide is linked to the sugar moiety of the Fc part of said antibody.

14. Polypeptide of item 12 or 13, wherein said binding agent or antibody is an anti-rabbit, anti-goat, or anti-mouse antibody.

15. Polynucleotide encoding the polypeptide of any one of items 1 to 13.

16. Vector comprising the polynucleotide of item 15.

17. Host cell comprising the polynucleotide of item 15 and/or the vector of item 16.

18. Use of the polypeptide of any one of items 1 to 14 in immuno-analytical methods.

19. Method for detecting a biological marker in a sample, comprising

(i) contacting the sample with at least one first binding agent which (specifically) binds to the biological marker and forms a first complex;

(ii) optionally contacting the first complex with a second binding agent which (specifically) binds to the first complex to form a second complex; wherein at least one first binding agent or - if step (ii) is applied - at least one second binding agent is covalently linked to the polypeptide of any one of items 1 to 11 ,

(iii) contacting the polypeptide with a substrate of said polypeptide; and

(iv) detecting binding of said polypeptide with said substrate. It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

When used herein “consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms "comprisingTcomprise”, “containingTcontains”, "consisting/consist essentially of" and "consisting/consist of may be replaced with either of the other two terms.

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

Unless specified otherwise, any reference made herein to UniProt entries (e.g., UniProt entry #P00433) or other database entries refers to the respective entry as of April 1, 2019.

The embodiments which characterize the present invention are described herein, shown in the Figures, illustrated in the Examples, and reflected in the claims.

The following sequences have been used.

SEQ ID NO: 1

DNA Sequence, nucleotides 1-918 of SEQ ID NO: 8 Armoracia rusticana

HRP 1 (translates into wt HRP - CIA isoform UniProt# P00433 without aa 2-30 and 336-353)

TTT=F42 (numeration corresponding to encoded aa sequence; may be mutated to, e.g., A42: GOT, GCC, GCA or GCG as described in accordance with the present invention)

AAT=N17 6 (numeration corresponding to encoded aa sequence; may be mutated to, e.g., S176: AGC, AGT, TCT, TCC, TCA, TCG as described in accordance with the present invention)

SEQ ID NO: 2

DNA Sequence, nucleotides 919-969 of SEQ ID NO: 8

Artificial

Spacer/ Linker 1

SEQ ID NO: 3

DNA Sequence, nucleotides 970-975 of SEQ ID NO: 8 Artificial

BssHII restriction site (G/CGCGC)

GCGCGC

SEQ ID NO: 4

DNA Sequence, nucleotides 970-1893 of SEQ ID NO: 8 Armoracia rusticana

HRP 2 (translates into wt HRP - CIA isoform UniProt# P00433 without aa 2-30 and 336-353)

TTC=F42 (numeration corresponding to encoded aa sequence may be mutated to, e.g., A42: GCT, GCC, GCA or GCG as described in accordance with the present invention)

AAC=N176 (numeration corresponding to encoded aa sequence; may be mutated to, e.g., S176: AGC, AGT, TCT, TCC, TCA, TCG as described in accordance with the present invention)

SEQ ID NO: 5

DNA Sequence, nucleotides 1894-1944 of SEQ ID NO: 8

Artificial

Spacer/ Linker 2

GCCGAAGCAGCAGGCAAAGAGGCCGCCGGTAAAGAAGCTGCCGGAAAAGCT SEQ ID NO: 6

DNA Sequence, nucleotides 1945-1950 of SEQ ID NO: 8 Artificial

Hindlll restriction site (A/AGCTT)

AAGCTT SEQ ID NO: 7

DNA Sequence, nucleotides 1951-2868 of SEQ ID NO: 8 Armoracia rusticana

HRP 3 (translates into wt HRP - CIA isoform UniProt# P00433 without aa 2-30 and 336-353)

TTC=F42 (numeration corresponding to encoded aa sequence; may be mutated to, e.g., A42: GOT, GCC, GCA or GCG as described in accordance with the present invention)

SEQ ID NO: 8 DNA sequence Artificial

HRP Trimer with aa linkers and restrictions sites (each HRP unit translating into wt HRP - CIA isoform UniProt# P00433 without aa 2-30 and 336-353)

TTT or TTC=F42 (or F367, or F692) (numeration corresponding to encoded aa sequence of each HRP unit; may be mutated to, e.g., Ala: GCT, GCC, GCA or GCG as described in accordance with the present invention)

AAT or AAC=N176 (or N501, or N826) (numeration corresponding to encoded aa sequence of each HRP unit; may be mutated to, e.g., Ser: AGC, AGT, TCT, TCC, TCA, TCG as described in accordance with the present invention)

Underlined : aa spacer/ linker bold, italics underlined: restriction sites

SEQ ID NO: 9

Protein sequence, aa 1-306, 326-631, or 651-956 of SEQ ID NO:

12

Armoracia rusticana

HRP 1, 2 or 3 (wt HRP - CIA isoform UniProt# P00433 without aa 2-30 and 336-353)

F42 (may be mutated to, e.g., A42 as described in accordance with the present invention)

N176 (may be mutated to, e.g., S176 as described in accordance with the present invention)

SEQ ID NO: 12 Protein sequence Artificial

HRP Triplet (with 3 wt HRP units - CIA isoforms UniProt# P00433 without aa 2-30 and 336-353)

HRP units including F42 and N176 (numeration corresponding to each HRP unit)

F42 (or F367, or F692) (numeration corresponding to each HRP unit; may be mutated to Ala as described in accordance with the present invention for single HRP units or, preferably, each HRP unit)

N176 (or N501, or N826) (numeration corresponding to each HRP unit; may be mutated to Ser as described in accordance with the present invention for single HRP units or, preferably, each HRP unit)

Spacer/ linker

Translation of restrictions site

SEQ ID NO: 13 Protein sequence Armoracia rusticana

HRP sequence according to UniProt #P00433: 353aa bold: aa not comprised by HRP units 1-3 of SEQ ID NOs. 10 and

12 (and corresponding nucleotide sequences) aa 1-30: signal peptide aa 336-353: Propeptide

SEQ ID NO: 14 Protein sequence Artificial

HRP Triplet including F42A and N176S substitutions numeration corresponding to each HRP unit)

HRP units according to CIA isoforms UniProt# P00433 without aa 2-30 and 336-353 including F42A and N176S substitutions (numeration corresponding to each HRP unit)

F42A F367A F692A (corresponding to wt HRP units as described in accordance with the present invention)

N176S N501S N826S (corresponding to wt HRP units as described in accordance with the present invention)

Spacer/ linker

Translation of restrictions site

The Figures show:

Figure 1 Comparative study of HRP specific activities (when reported to g of anti mouse/rabbit IgG + corresponding units of HRP), of HRP-micropolymer detection system, 3xHRP-wt recombinant detection system, and 3xHRP-F42- A/N176-S double mutant. The comparative study shows that 3xHRP-F42- A/N176-S double mutant gives at least 3x higher activity when compared to 3xHRP-wt, and enhanced signal (HRP activities) at least by 65%, when compared to HRP-micropolymer detection system from DAKO/Agilent dual detection system (Dako EnVision®+ Dual Link, Agilent); termed “other supplier” in Figure 1.

Figure 2 pPICZaA

3593 nucleotides:

5’ AOX1 promoter region: bases 1-941

5’ AOX1 priming site: bases 855-875 a-factor signal sequence: bases 941-1207 a-factor priming site: bases 1144-1164

Multiple cloning site: bases 1208-1276 c-myc epitope: bases 1275-1304

Polyhistidine (6xHis) tag: bases 1320-1337

3’ AOX1 priming site: bases 1423-1443

AOX1 transcription termination region: bases 1341-1682

TEF1 promoter: bases 1683-2093

EM7 promoter: bases 2095-2162

Sh ble ORF: bases 2163-2537

CYC1 transcription termination region: bases 2538-2855 pUC origin: bases 2866-3539 (complementary strand)

Figure 3 Repeats of HRP

Profile of activities corresponding to monomer, dimer, and consequent HRP oligomers (3-7 repeats of HRP), expressed as a compact one single product, with amino acid linker between each HRP unit (UniProt#: P00433; aa 31 - 335). Each measure represents the mean + standard deviation of five independent experiments (n=5).

Enzyme activity was measured using pyrogallol as a substrate by standard procedure: Blank - ultrapure water, phosphate buffer, hydrogen peroxide solution, pyrogallol solution.

Sample - ultrapure water, phosphate buffer, hydrogen peroxide solution, pyrogallol solution, rHRP solution.

After 2 min reaction time, the absorbance was taken using the 420nm wavelength. The minimum absorbance for the measure acceptance is 0.5.

The HRP was expressed in yeast cells as a monomer (inserts were composed of amino acids aa 31-335 of HRP isoform C1A - column 1), dimer (double HRP with a 19aa spacer - column 2), trimer (column 3), tetramer (column 4), pentamer (column 5), hexamer (column 6), and heptamer (column 7). The specific activities of recombinant proteins were measured using pyrogallol as a substrate and absorbance was taken at 420mn under standard conditions. Specific activities (U/mg) of described constructs were compared, where 3 and 4 repeats of HRP showed the highest and similar specific activities of HRP. More than 5 (5, 6, and 7) repeats of HRP units lower the enzyme performance (lower specific activities compared to 3 or 4 repeats), which is based most probably on structural/spherical interference of more than 4 units of HRP units expressed in one single chain.

Figure 4 Specific activity of wt and mutant of single chain HRP

Single chain of HRP isoform C1A (UniPRot#: P00433; first amino acid in Met, HRP insert contained the sequence without Signal peptide - aa 1-30, Propeptide - aa339-353, and without 3 amino acids at the C-terminal, 335Asn- Ser-Asn337) was expressed in Pichia pastoris yeast cells as a wild type protein (wt), and in mutated forms, with different mutations (amino acid substitution was made by site-directed mutagenesis) - single mutants: F42A, C45-A, C45-S, and double mutants: F42-A/C45-A, and F42-A/C45-S. The related protein extracts were tested for HRP specific activities in order to demonstrate the influence of mutation in a proximity of catalytic center of HRP. Based on the repeated measures (7 independent experiments), the mutation of Phe42 to alanine enhanced the activity by -95% compared to wt HRP. The activity of HRP was measured by pyrogallol as a substrate, and the absorbance was taken after 2 min of reaction time, at 420 nm. The present invention is further illustrated by the following examples. Yet, the examples and specific embodiments described therein must not be construed as limiting the invention to such specific embodiments.

Examples

Transformation and cultivation of P. pastoris and expression of HRP in P. pastoris

The plasmid DNA (pPICZaA containing the 3xHRP construct) was linearized at Pme I site in the 5ΆOC1 promoter region to integrate into the AOX loci of the P. pastoris genome. The P. pastoris X-33 strain (Invitrogen) was transformed by 10 mg of linearized plasmid via electroporation using Gene Pulser Xcell Electroporation system (Bio-Rad) and Pre-set protocol for P. pastoris (Pulse type: Exponential decay; 25 pF; 200 ohm; 2000 V; Cuvette 0.2 cm). Transformed cells were spread on YPDS plates (1% yeast extract, 2% Peptone, 2% D- glucose, 1M Sorbitol, and 100 mg/ml_ Zeocine™) to select the Zeo^R transformants (Koliasnikov et al, 2011). Integration of the 3xHRP into Pichia genome was confirmed by sequencing.

The selected transformant was cultivated overnight in BMGY/Zeocin (1% yeast extract; 2% peptone; 100 mM potassium phosphate buffer, pH 6.0; 1.34% YNB; 4x10^-5% biotin; 1% glycerol; 50 mg/ml_ Zeocine™) at 30 °C and 230 rpm (Gmeiner and Spadiut, 2015). To induce the production of recombinant 3xHRP, the cell suspension was transferred into BMMY/Zeocin (1% yeast extract; 2% peptone; 100 mM potassium phosphate buffer, pH 6.0; 1.34% YNB; 4x10^-5% biotin; 0.5% methanol; 50 mg/ml_ Zeocine™) containing 30 pM of hemin and cultivated at 30 °C for four days in the Lambda MINIFOR Bioreactor. To maintain the recombinant protein production, 0.5% (v/v) of pure methanol was pulsed. The medium was checked for the highest HRP activity and the culture was collected and centrifuged. The supernatant containing the extracellularly expressed 3xHRP was desalted using Vivaflow 200 Crossflow concentrator to allow the 6xHis purification of the recombinant protein. The recombinant 3xHRP was purified via affinity chromatography using HisTrap™ High Performance columns.

His6 tagged recombinant HRP purification

The C-terminal His6 tagged HRP was purified using the standard protocols as suggested by supplier of His Trap histidine-tagged protein purification columns (Sigma Aldrich/GE Healthcare 29-0510-21; https://www.sigmaaldrich.com/catalog/product/sigma/ge29051021? lang=en&region=SK). Briefly, the column was equilibrated by 5 volumes of loading buffer (50 mM sodium phosphate, 100 mM NaCI, 10 mM imidazole, pH8.0). The concentrated yeast supernatant containing crude HRP enzyme was diluted with loading buffer 5x, and loaded onto the column at the flow rate of 2ml/min. After the loading, the column was washed with washing buffer (50 mM sodium phosphate, 250 mM NaCI, 25 mM imidazole, pH 8.0) - 5 column volumes. The enzyme was eluted from the column with elution buffer (50 mM sodium phosphate, 200 mM imidazole, pH8.0). Average fold of purification was 30-40x, with the yield of 85-90% of total enzymatic activity, after metal affinity purification and consequent desalting (Sigma Aldrich/GE Healthcare 17-0852-01; https://www.sigmaaldrich.com/catalog/product/sigma/ge17085201 ?lang=en& region=SK).

Desalting of pure HRP preparation on NAP25 columns

Column was equilibrated with the 10 volumes buffer of interest (the final buffer in which the sample should be after desalting and original buffer change). The sample was loaded (2-2,5 ml max volume), and after entering completely the Sephadex resin, the sample was eluted with 3.5 ml of final buffer. The 3.5 volume represents desalted sample in the required buffer for further purposes (e.g. HRP-antibody conjugation purposes).

Measurement of HRP activity

HRP enzymatic activity was measured by formation of purpurgallin at 420 nm, as described by supplier of HRP (EC 1.11.1.7) substrate (pyrogallol) (https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of- peroxidase.html).

Conjugation of pure HRP with anti-mouse and anti-rabbit IgG

The standard conjugation protocol was performed based on periodate method of enzyme:antibody conjugation, initially described by Tissej and Kurstak (1984).

Briefly, the 3x HRP product was desalted on NAP25 column and eluted in 1mM acetate buffer, pH 4.4. The resulting concentration was ~70 mg of pure enzyme in 3.5 ml of buffer. Freshly prepared 0.1 M sodium periodate was added to the pure enzyme preparation (700 pi), and slightly rotated for 20 min at room temperature, in dark. Periodate activated HRP was purified from the excess of periodate using NAP25 column (two runs) with 1mM acetated buffer, pH4.4.

15-20 mg solution of goat anti-mouse or anti-rabbit IgG was prepared in 2.5 ml of 50 mM sodium carbonate buffer, pH 9.5. The pH of purified enzyme solution was adjusted with 50mI of 200 mM sodium carbonate buffer, pH 9.5, and immediately mixed with IgG solution. The mixture was stirred for 2hours at room temperature. 250 mI of freshly prepared sodium borohydride solution (4 mg/ml) was added to the conjugate, and stirred for 2 hours at 4 °C.

The final product was stored in 20 mM Tris-HCI, ph 7.5-8.0 (NAP25 column), and stabilized with 5 mg/ml of BSA and 0.015% Thiomersal and 0.04% of potassium ferricyanide. The stability of final conjugated product is given for at least 24 months when stored at 2-8 °C.

Claims

1. Polypeptide comprising 3 to 5 horseradish peroxidase (HRP) units, wherein at least 1 of said HRP units has an amino acid sequence being at least 95% similar to the amino acid sequence of SEQ ID NO: 9, wherein said at least 1 HRP unit being at least 95% similar to the amino acid sequence of SEQ ID NO: 9 and comprising at least 1 amino acid substitution at position F42 and/or C45 of SEQ ID NO: 9, wherein said amino acid substitution is a conservative amino acid substitution which increases the activity of the HRP unit.

2. Polypeptide of claim 1, wherein said amino acid substitution increasing activity is within amino acid positions 40 to 46 of SEQ ID NO: 9.

3. Polypeptide of claim 1 or 2, wherein said conservative amino acid substitution at position F42 is selected from L, V, I, A or Y.

4. Polypeptide of claim 1 or 2, wherein said conservative amino acid substitution at position C45 is selected from S or A.

5. Polypeptide of any one of claims 1 to 4, wherein said amino acid substitution increasing activity is at position F42 and/or C45 of SEQ ID NO: 9.

6. Polypeptide of claim 5, wherein said acid substitution increasing activity is F42A, C45A, and/or C45S of SEQ ID NO: 9.

7. Polypeptide of any one of claims 1 to 6, wherein said polypeptide further comprises at least 1 amino acid substitution, which increases the stability of the HRP unit.

8. Polypeptide of claim 7, wherein said amino acid substitution increasing stability is at amino acid position N176.

9. Polypeptide of claim 7 or 8, wherein said amino acid substitution is a conservative amino acid substitution selected from Q, H, D, K, R or S.

10. Polypeptide of any one of claims 7 to 9, wherein said amino acid substitution increasing stability is N176S.

11. Polypeptide of any one of claims 1 to 10, comprising a linker of 9 to 15 amino acids between each HRP unit.

12. Polypeptide of any one claims 1 to 11 , which is producible by yeast or CHO cell.

13. Polypeptide of any one of claims 1 to 12, further comprising a protein tag a the N- or the C-terminus for purification of the polypeptide.

14. Polypeptide of any one of claims 1 to 13, which is further covalently linked to a binding agent.

15. Polypeptide of claim 14, wherein said binding agent is an antibody and said polypeptide is linked to the sugar moiety of the Fc part of said antibody.

16. Polypeptide of claim 14 or 15, wherein said binding agent or antibody is an anti-rabbit, anti-goat, or anti-mouse antibody.

17. Polynucleotide encoding the polypeptide of any one of claims 1 to 162.

18. Vector comprising the polynucleotide of claim 17.

19. Host cell comprising the polynucleotide of claim 17 and/or the vector of claim 184.

20. Use of the polypeptide of any one of claims 1 to 16 in immuno-analytical methods.

21 . Method for detecting a biological marker in a sample, comprising

(ii) optionally contacting the first complex with a second binding agent which (specifically) binds to the first complex to form a second complex; wherein at least one first binding agent or - if step (ii) is applied - at least one second binding agent is covalently linked to the polypeptide of any one of claims 1 to 16,

(iii) contacting the polypeptide with a substrate of said polypeptide; and

(iv) detecting binding of said polypeptide with said substrate.