AU2024292159A1 - Dsfv as an igg fragment format and methods of production and labelling thereof - Google Patents

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof, in particular (ds)Fv fragments. The invention also relates to methods of producing these antibodies and fragments thereof as well as their uses and corresponding polynucleotides and kits comprising the same.

Description

dsFv as an IgG fragment format and methods of production and labelling thereof

The present invention relates to antibodies and antigen-binding fragments thereof, in particular (ds)Fv fragments. The invention also relates to methods of producing these antibodies and fragments thereof as well as their uses and corresponding polynucleotides and kits comprising the same.

INTRODUCTION

The variable domains of the antibody heavy (VH) and light (VL) chains (together called the Fv part) constitute the minimal subunit of the antibody (Ab) that retains full antigen binding properties. The small size of such an active Ab-fragment is of interest in a number of applications. For example, the small size of such construct allows its access to challenging epitopes, increases its tissue penetration, and the lack of the Fc region reduces unintended immune responses; all of which could be useful for different applications.

Current prior art offers four options for the design and production of such a small Fv fragment (Bates, A. & Power, C. A. “David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments”. Antibodies (Basel) 8, doi:10.3390/antib8020028 (2019)). The first and the most common is via designing a single-chain version of such a construct (a single-chain Fv fragment; scFv), where the VH and the VL are genetically fused with an appropriate-length flexible peptide linker and are expressed from one gene (Huston et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences 85:5879-5883, 1988; and, Bird et al. Single-chain antigen-binding proteins. Science 242(4877) :423-6, 1988).

However, such an approach may require the optimization of the peptide linker, in terms of both length and sequence, for each new clone. The peptide linker can also interfere with the antigen-Ab binding and may hinder the correct cross-orientation of the VH/VL domain and the correct formation of the binding pocket; affecting the Ab-antigen affinity. Furthermore, such scFvs are often challenging to express in a soluble manner leading to potentially low yields of correctly folded scFvs and high tendency for aggregation and reshuffling even after the removal of the aggregates.

Another reported, but less common option, is the expression of the VH and VL from two distinct genes as inclusion bodies in bacterial cytoplasm; either co- or separate expression in two separate cultures. This is then followed by the denaturation and oxidative refolding of the overexpressed protein from the inclusion bodies and the subsequent mixing and co-folding of the VH and VL fragments in case of separate culture expressions (Buchner J, Pastan I, Brinkmann U. A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal Biochem. 205(2):263-70, 1992and, Brinkmann et al. A recombinant immunotoxin containing a disulfide- stabilized Fv fragment. Proc. Natl. Acad. Sci. 90(16):7538-42, 1993)). However, this process is lengthy and inefficient (low yields) and possess the risk of incorrect folding of the Fv fragment and the loss of its binding activity.

A third option, which only works for some rare cases (where the IgG clones possess an unusually strong VH/VL interactions) is the co-expression of the VH and VL and their secretion into the bacterial periplasm, where they co-assemble spontaneously to form an active Fv (Skerra A, Pluckthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988 May 20;240(4855): 1038-41 , 1988), however even when this works, it typically suffers from low yields of correctly assembled Fvs.

Another underlying issue with the above-outlined processes for producing Fv fragments is instability. The non-covalent interactions at the VH/VL interface are inherently too weak to keep them stably bound. Within the full immunoglobulin (e.g. IgG) context, this is normally enforced by the strong covalent and non-covalent interactions between the, now lacking, constant IgG domains at the C termini of the VH and VL (namely the CH1 and CL). The peptide linker between the VH and VL in the scFv goes some distance towards stabilizing it, but this is far from sufficient, and the scFvs have the tendency to engage in intermolecular associations forming multimers and aggregates.

A reported way to stabilize the Fv has been to engineer in an inter-chain disulfide bond by mutating the amino acids at two structurally conserved opposite framework sites of the VH and VL (namely the VH44 and VL100 (Kabat numbering scheme)) to yield a disulfide- stabilised-Fv or -scFv fragment (dsFv or dsscFv) (Brinkmann U, Reiter Y, Jung SH, Lee B, Pastan I. A recombinant immunotoxin containing a disulfide-stabilized Fv fragment. Proc Natl Acad Sci U S A. 90(16):7538-42, 1993; and, Brinkmann, U. Disulfide-stabilized fv fragments. In Kontermann, R., Dubel, S. (eds). Antibody Engineering. Berlin Heidelberg: Springer-Verlag, 2: 181 -9, 2010).

A fourth option, which was only recently reported, is to fuse not only one N-terminus of the VH/VL region to the C-terminus of the corresponding VL/VH, but to fuse both N/C-termini of both variable regions to form a cyclic scFv (W02020/013126). However, while this may go some way towards stabilizing the scFv monomers, one could imagine this could interfere with the binding of the antibody fragment to its target.

Accordingly, there is a need in the art for improved ways of producing correctly folded, active Fv fragments in high yield, with minimal aggregation and shuffling. SUMMARY OF THE INVENTION

The present invention provides a new method for the production and optionally labelling of any Fv fragment, wherein enzyme motifs (e.g. for sortase or protease) are engineered into the elbow regions of the immunoglobulin (e.g. IgG) heavy (the VH-CH1 loop) and light (the VL-CL loop) chains. These motifs (enzyme recognition motifs or sequences) provide targets for the site-specific processing of an antibody molecule (e.g. full length IgG, fragments thereof or fusions comprising an antibody moiety) following its expression and purification to yield a correctly folded active Fv fragment (Example 1). This ensures the correct folding of the VH/VL within their ‘native’ immunoglobulin (e.g. IgG) context, stabilized by the inherent covalent and non-covalent interactions of the heavy and light chain constant domains, before the Fv fragment is enzymatically extracted out of the immunoglobulin (e.g. IgG) molecule. This, in turn, reduces the risk of misfolding or loss in affinities, which can accompany the current methods outlined above. The proposed concept provides a generic way to produce Fv fragments; that does not require peptide linker or refolding conditions optimization for each new candidate and yields a stable product at a good yield that is less likely to suffer from aggregation. Furthermore, the method provides an opportunity for the labelling of the Fv fragment concomitantly in the process. The engineering of a disulfide (also spelled disulphide) inter-chain bond between the VH and VL (via point mutations at conserved positions (Brinkmann. Disulfide-Stabilized Fv Fragments. In: R. Kontermann and S. Duebel, ed., Antibody Engineering, 2nd ed. 2010; 2:181-189)), can optionally complement the described concept to yield a stable dsFv fragment with superior characteristics to scFv or ds-scFvs (Example 2). Cysteines at VH44 and VL100 positions are the most widely used to create a disulfide bond, but other examples are possible; for example VH55 and VL108; VH56 and VL106; or VH L46C and VL D101C (Rodrigues ML, Presta LG, Kotts CE, Wirth C, Mordenti J, Osaka G, Wong WL, Nuijens A, Blackburn B, Carter P. Development of a humanized disulfide- stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug. Cancer Res. 1995 Jan 1 ;55(1):63-70. PMID: 7805042.).

With regard to the tested application for dsFv conjugates, e.g. as a capture biotinylated antibody in an immunoassay (e.g. on Roche Elecsys® immunoassay platform), the small size of the dsFv fragment provides it with a diffusion advantage which surprisingly leads to enhanced assay performance (superior signal-dynamics) (Example 4). The dsFv construct also possesses a superior stability profile, when compared to other IgG fragments tested (Example 4). Furthermore, the removal of the antibody constant domains reduces the risk of assays interference by heterophilic antibodies in the serum directed at constant antibody domains of animal sequence origin (e.g. Human anti-mouse antibodies (HAMA) or Human anti-rabbit antibodies (HARA)) or rheumatoid factors (in case of human IgG constant domains). In immunoassays, such as on the Roche Elecsys® immunoassay system, the use of such a small antibody format (especially on the Reagent 1 side (R1 ; the biotinylated reagent side)) can offer many benefits in terms of higher signal (owing to its diffusion advantage) and less interference (due to the absence of the antibody constant regions).

An illustration of the generic concept and process for making an Fv fragment is shown in Figure 1.

Accordingly, in one aspect an antibody or antigen-binding fragment thereof comprising a Fab domain, said Fab domain comprising an Fv fragment that binds to an antigen of interest, is provided, wherein said Fab domain comprises:

(i) a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and

(ii) a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop).

Suitably, the first recognition motif may be a recognition motif for a transpeptidase and the second recognition motif is a recognition motif for a site-specific protease or vice versa, or the first and second recognitions motifs are both for a transpeptidase or both for a site-specific protease.

Suitably, the recognition motif for the site-specific protease in the VH-CH1 loop and/or the recognition motif for the site-specific protease in the VL-CL loop may be independently selected from the group consisting of: TEV-protease recognition motif, IgA protease recognition motif and 3C protease recognition motif.

Suitably, the recognition motif for a transpeptidase in the VH-CH1 loop and/or the recognition motif for a transpeptidase in the VL-CL loop may be a recognition motif for a sortase or sortase- type transpeptidase.

Suitably, the first recognition motif and/or the second recognition motif may be flanked by an N-terminal linker sequence and/or a C-terminal linker sequence, optionally wherein the linker sequence has a length of at least 2 amino acids and/or is composed of amino acids selected from the group consisting of Glycine, Serine, Alanine, Threonine, Glutamate and Proline. Suitably, the first recognition motif may be positioned anywhere between amino acids 109 and 121 of the heavy chain according to Kabat numbering scheme. In a preferred embodiment the first recognition motif may be positioned anywhere between amino acids 112 and 119 of the heavy chain according to Kabat numbering scheme.

Suitably, the second recognition motif may be positioned anywhere between amino acids 105 and 115 of the light chain according to Kabat numbering scheme.

Suitably, the heavy chain sequence and/or the light chain sequence may comprise an affinity tag.

Suitably, a cysteine residue may be engineered into the VH and VL of the Fab domain at locations to permit disulfide bond formation between the two introduced cysteines.

Suitably, the Fv fragment may be capable of binding a Troponin, in particular Troponin T, in particular cardiac Troponin T (c-Troponin T).

The invention also provides the use of the antibody or antigen-binding fragment as precursor for generating an Fv fragment.

The invention also provides a method for producing an Fv fragment capable of binding to an antigen of interest comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, said contacting is optionally conducted in the presence of a nucleophile for said transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention. Suitably, the antibody or antigen-binding fragment may be an antibody or antigen-binding fragment, wherein the Fv fragment specifically recognizes Troponin T, in particular cardiac Troponin T.

The invention also provides an Fv fragment capable of binding to an antigen of interest obtained by a method of the invention. Suitably, the Fv fragment contains one or more residual amino acid residues at the C-terminal end derived from the recognition motif and, in case a linker was used, from the N-terminal linker.

The invention also provides a method for detecting and/or quantifying an analyte of interest in a sample comprising contacting the sample with the Fv fragment specifically binding said analyte of interest as described herein under conditions allowing for binding of the Fv fragment to the analyte comprised in the sample; and detecting and/or quantifying the analyte in the sample by detecting the complex between the Fv fragment and the analyte.

The invention also provides a method for detecting and/or quantifying troponin T (in particular cardiac Troponin T) in a sample comprising contacting the sample with the Fv fragment described herein under conditions allowing for binding of the Fv fragment to troponin T comprised in the sample; and detecting and/or quantifying troponin T in the sample by detecting the complex between the Fv fragment and troponin T.

Various aspects of the invention are described in further detail below.

DETAILED DESCRIPTION

The present invention provides a novel antibody or antigen-binding fragment thereof, in particular an Fv fragment. Further, a method for producing the same and their uses are provided.

The inventors have surprisingly identified a way to produce Fv fragments with improved properties with regards to stability, folding, yield, etc.

Anti cardiac-troponin T binding Fv fragments with improved properties and improved related assays are also provided.

General definitions

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole.

An antibody is an immunoglobulin molecule capable of specific binding to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site, located in the variable domain of the immunoglobulin molecule. An antibody target could also be a hapten or small molecule.

As used herein the term “antibody or antigen-binding fragment thereof’ refers to any antibody structure that comprises a Fab or an Fv fragment at least.

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to chimeric antibodies, humanized antibodies, monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), conjugated or fused antibodies and antibody fragments so long as they exhibit the desired antigen-binding activity. Preferably, the antibody is a polyclonal antibody. More preferably, the antibody is a monoclonal antibody. The antibody can be from any species. Suitably the antibody is a human antibody, a monkey antibody, a rabbit antibody, a sheep antibody, a mouse antibody or a rat antibody; or the antibody is a chimeric antibody (e.g. mouse/human, rabbit/human chimeras. An antibody can be in any of a variety of forms comprising a Fab domain, including a whole immunoglobulin, an antibody fragment such as Fab itself, and similar fragments, all of which fall under the broad term "antibody", as used herein. In preferred embodiments, an antibody or fragment thereof is used that is specific for an antigen or epitope of interest.

Naturally occurring antibodies are generated by the assembly of heavy chains only or heavy and light chains. Each heavy chain is composed of four domains: the variable domain (VH), CH1 , CH2, and CH3. The light chain is composed of variable domain (VL) and constant domain (CL). In case heavy and light chain being present, the light chain pairs with a cognate heavy chain Fab-fragment comprising the VH and CH1 domains. The associated light chain and heavy chain Fab fragment together are denoted as Fab fragment. The heavy chain CH2 and CH3 domains, together denoted as heavy chain Fc-region, dimerize with additional heavy chain CH2 and CH3 domains from a second chain to form the Fc-region. The Fc-region is connected to the Fab-frag ment(s) via a flexible hinge region. The hinge region comprises several disulfide bridges that covalently link two heavy chains Fc-regions together. In the Fab- fragment, the light chain and the heavy chains are also connected by one disulfide bridge. However, the connectivity differs among the IgG subclasses. The overall structure of full- length IgGs resembles a Y-shape, with the Fc-region forming the base while the two Fab- fragments form the arms and are available for binding to the antigen.

Within the variable domains reside loops called complementarity determining regions (CDRs). These are mainly responsible for the direct interaction of the antibody with its antigen. Because of the significant variability in the number of amino acids in these CDRs, there are multiple numbering schemes for the variable regions. As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and is referred to as “numbering according to Kabat” herein.

The term "antibody fragment" as used herein refers to a portion of a full-length antibody, that comprises an antigen binding site. Examples of antibody fragments include Fab, Fab', and F(ab')2 fragments. Such fragments can be produced form particular IgG species by reduction of hinge-region disulfides or digestion with papain, pepsin, or ficin proteolytic enzymes. For example, papain digestion of whole intact human IgG antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. With human lgG1 , pepsin treatment yields an F(ab')2 fragment that has two antigen binding fragments that are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'); Additionally, certain antibody fragments, e.g. Fab, can be produced recombinantly. Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Antigen-binding immunoglobulin (antibody) fragments are well known in the art. Such fragment need not have a functional Fc receptor binding site. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')2 fragments.

Some types of antibody fragments are defined as follows:

Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. It can be produced from IgG and IgM, and consists of the VH, CH1 and VL, CL regions, linked by an intramolecular disulfide bond. A Fab fragment can be produced by digestion of whole intact antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain. It can also be produced recombinantly.

Fab' is another fragment of an antibody molecule which can be obtained by treating whole intact antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule. It can also be produced recombinantly.

Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. They can be formed by the reduction of F(ab')2 fragments and so may contain a small portion of Fc. The Fab' fragment contains a free sulfhydryl group that may be alkylated or utilized in conjugation with an enzyme, toxin or other protein of interest.

(Fab')2 is the fragment of an antibody that can be obtained by treating whole intact antibody with the enzyme pepsin without subsequent reduction. (Fab')2 can also be produced recombinantly. F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds. This fragment is void of most, but not all, of the Fc region.

Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three complementarity determining regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab.

A single chain antibody (SCA) is defined herein as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also referred to as "single-chain Fv" or "sFv" or “scFv” antibody fragments.

Unless indicated otherwise, the numbering of the residues in an immunoglobulin heavy or light chain used herein is that of the EU index of Kabat (Kabat, E.A., et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242, expressly incorporated herein by reference), also referred to herein as “Kabat numbering” or “according to Kabat” or the like. The term "position" denotes the location of an amino acid residue in the amino acid sequence of a polypeptide. Positions may be numbered sequentially, or according to an established format, for example the EU index of Kabat for antibody numbering. The EU numbering as used herein is based on Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969 (see also http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html).

The term "full length antibody" or “whole intact antibody” denotes an antibody that has a structure and amino acid sequence substantially identical to a native antibody.

The term "full length antibody heavy chain" denotes a polypeptide comprising in N- to C- terminal direction an antibody variable domain (= VH-domain), a first constant domain (= CH1 -domain), an antibody heavy chain hinge region, a second constant domain (= CH2-domain), and a third constant domain (= CH3-domain).

The term "full length antibody light chain" denotes a polypeptide comprising in N- to C-terminal direction an antibody variable domain (= VL-domain) and a constant domain (= CL-domain).

The term "hinge region" denotes the part of an antibody heavy chain polypeptide that joins in a wild-type antibody heavy chain the CH1 domain and the CH2 domain (i.e. between the Fab and Fc portions), e. g. for human I gG 1 from about position 216 to about position 238 according to the EU number system, or from about position 226 to about position 243 of Kabat numbering. Position 226 according to Kabat corresponds to position 99 of a human lgG1 heavy chain constant region. The hinge regions of other IgG subclasses can be determined by aligning with the hinge-region cysteine residues of the human lgG1 subclass sequence.

The hinge region is normally dimeric consisting of two polypeptides with identical amino acid sequence. The hinge region generally comprises about 25 amino acid residues and is flexible allowing the antigen binding regions to move independently. The hinge region can be subdivided into three domains: the upper, the middle, and the lower hinge domain (see e.g. Roux, et al, J. Immunol. 161(1998) 4083).

The term "upper hinge region" of an Fc-region, e. g. for human lgG1 , denotes the stretch of amino acid residues N-terminal to the middle hinge region, i.e. residues 216 to 225 of the Fc- region according to the Ell numbering. The term "middle hinge region", e. g. for human lgG1 , i.e. residues 226 to 230 of the Fc-region according to the Ell numbering, denotes the stretch of amino acid residues comprising the cross-linking cysteine residues, is rich in prolines and cysteines, and it is located between the upper and the lower hinge region.

The term "lower hinge region" of an Fc-region, e. g. for human lgG1 , denotes the stretch of amino acid residues immediately C-terminal to the middle hinge region, i.e. residues 231 to 238 of the Fc-region according to the Ell numbering.

The term “epitope” refers to an antigenic determinant in a polypeptide that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes may be either linear or conformational. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain.

The term "monoclonal antibody" (“mAb”) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody or fragment thereof, as not being a mixture of discrete antibodies or antigenbinding fragments. A mAb is typically highly specific, being directed against a single antigenic site/epitope, however a monoclonal antibody can also refer to a population of a substantially homogeneous bispecific antibody molecule.

The preparation of monoclonal antibodies is conventional and well known to persons of ordinary skill in the art. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ionexchange chromatography. In an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with an antigen of interest to thereby isolate immunoglobulin library members that bind said antigen. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurJZAP™. Phage Display Kit, Catalog No. 240612). Routinely, monoclonal antibodies can also be recombinantly expressed in eukaryotic hosts cells, such as CHO, CV1 , NSO, YO, Sp2/0 or HEK, typically, by co-expression of an antibody heavy chain and an antibody light chain.

While it is possible to use in vitro translation to produce a recombinant immunoglobulin heavy chain as disclosed herein, cellular expressions system will be used in the routine. Suitable host cells for cloning or expression/secretion of polypeptide-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, polypeptides may be produced in bacteria, in particular when glycosylation is not needed (see, e.g., US 5,648,237, US 5,789,199 and US 5,840,523, Charlton, Methods in Molecular Biology 248 (2003) 245-254 (B.K.C. Lo, (ed.), Humana Press, Totowa, NJ), describing expression of antibody fragments in E. coli . After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction or may be isolated from the insoluble fraction so called inclusion bodies which can be solubilized and refolded to bioactive forms. Thereafter the polypeptide can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern (see e.g. Gerngross, Nat. Biotech. 22 (2004) 1409- 1414, and Li, et al, Nat. Biotech. 24 (2006) 210- 215).

Suitable host cells for the expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts (see, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants)).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the COS-7 cell line (monkey kidney CVI cell transformed by SV40); the HEK293 cell line (human embryonic kidney); the BHK cell line (baby hamster kidney); the TM4 mouse Sertoli cell line (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23 (1980) 243-251); the CVI cell line (monkey kidney cell); the VERO-76 cell line (African green monkey kidney cell); the HELA cell line (human cervical carcinoma cell); the MDCK cell line (canine kidney cell); the BRL-3A cell line (buffalo rat liver cell); the W138 cell line (human lung cell); the HepG2 cell line (human liver cell); the MMT 060562 cell line (mouse mammary tumor cell); the TRI cell line (e.g. described in Mather, et al, Anal. N.Y. Acad. Sci. 383 (1982) 44-68); the MRC5 cell line; and the FS4 cells-line. Other useful mammalian host cell lines include the CHO cell line (Chinese hamster ovary cell), including DHFR negative CHO cell lines (see e.g. llrlaub, et al, Proc. Natl. Acad. Sci. USA 77 (1980) 4216), and myeloma cell lines such as Y0, NSO and Sp2/0 cell line. For a review of certain mammalian host cell lines suitable for polypeptide production, see, e.g., Yazaki, and Wu, Methods in Molecular Biology, Antibody Engineering 248 (2004) 255-268 (B.K.C. Lo, (ed.), Humana Press, Totowa, NJ).

Co-expression of an antibody heavy chain and an antibody light chain represents one embodiment according to the present disclosure. Using such co-expression an antibody comprising both, a heavy and a light chain can be obtained, e.g. an IgG- class antibody consisting of two recombinant heavy chains according to the present invention and two light chains.

An antibody or antibody fragment “homolog,” as used herein, means that a relevant amino acid sequence (preferably for example in the CDRs and/or variable domains VH and/or VL) of a protein or a peptide is at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or 100% identical to a given sequence. By way of example, such sequences may be variants derived from various species, or the homologous sequence may be recombinantly produced. The sequence may be derived from the given sequence by truncation, deletion, amino acid substitution, or addition. Percent identity between two amino acid sequences is typically determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) and other alignment algorithms and methods of the art.

As used herein, the term “% sequence identity” in connection with amino acid sequences of polypeptides/peptides and/or nucleic acid sequences or nucleic acid molecules describes the number of matches of identical amino acid or nucleic acid residues of two or more aligned sequences as compared to the number of residues making up the overall length of the compared sequences (or the overall compared portions thereof). Using an alignment of two or more sequences or sub-sequences, the percentage of residues that are the same may be determined when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. Non-limiting examples of algorithms for use in determining sequence identity include, for example, those based on the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res 25(1997), 3389-3402), CLUSTALW computer program (Thompson, Nucl. Acids Res. 2(1994), 4673-4680) or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 85(1988), 2444). Although the FASTA algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e. gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % sequence identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available are the BLAST and BLAST 2.0 algorithms (Altschul et al., Nucl Acids Res., 25(1977), 3389).

Antibodies and their antigen-fragments can be produced using techniques well known in the art. For example, the antibodies can be produced recombinantly in cells.

For recombinant production, a polynucleotide sequence encoding the antibody protein is inserted into an appropriate expression vehicle, such as a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. The expression vehicle is then transfected into a suitable target cell which will express the peptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation and electroporation. A variety of host-expression vector systems may be utilized to express the antibodies described herein.

The presently disclosed invention further provides isolated nucleic acids encoding the antibodies or antigen-binding fragments disclosed or otherwise enabled herein. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which uracil (U) is substituted for thymine (T), unless context requires otherwise.

Standard methods can be used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Methods to modify nucleic acids encoding the disclosed antibodies in order to include the desired cleavage site(s) are well known to a person skilled in the art.

In another embodiment, the nucleic acid molecules which encode the antibodies of the presently disclosed inventive concepts also comprise nucleotide sequences that are, for example, at least 50% identical to the sequences disclosed herein. Also contemplated are embodiments in which a sequence is at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91 % identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical, to a sequence disclosed herein and/or which hybridize to a sequence of the presently disclosed inventive concepts under conditions of high or moderate stringency. The percent identity may be determined by visual inspection of sequence reads and/or mathematical calculation optionally including computer alignment using an appropriate software.

Stringency, including “high stringency,” as used herein, includes conditions readily determined by the skilled artisan based on, for example, the length of the DNA. Generally, such conditions are defined as hybridization conditions of 50% formamide, 6xSSC at 42°C (or other similar hybridization solution, such as, e.g., Stark's solution, in 50% formamide at 42°C), and with washing at approximately 68°C with 0.2xSSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.

“Moderate stringency,” as used herein, includes conditions that can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA. The basic conditions are set forth by Sambrook et al. (79) and include use of a prewashing solution for the nitrocellulose filters 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6xSSC at 42°C (or other similar hybridization solution, such as Stark's solution, in 50% formamide at 42°C), and washing conditions of 60°C, 0.5xSSC, 0.1% SDS.

The monoclonal antibodies include "chimeric" antibodies 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 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).

Antibodies typically bind reversibly to unique regions or epitopes within specific antigens (target molecule) through weak non-covalent interactions which include hydrogen, ionic, hydrophobic, and Van der Waals bonds. The strength or affinity of antibody binding is determined by the net force of weak interactions between a single antibody binding site and its epitope.

As used herein, the “affinity” of the antibody or antigen-binding fragment for the antigen of interest is characterized by its KD, or equilibrium dissociation constant. A stronger affinity is represented by a lower KD while a weaker affinity is represented by a higher KD. AS such, an antibody of the present invention preferably has an affinity for the antigen of interest represented by a Kd s 1000nM, or < 500nM, or < 100nM, or < 50nM, or more preferably by a KD S 25nM, and still more preferably by a KD 10nM, and even more preferably by a K_D 5nM, or < 1nM, or < 0.1nM.

As used herein, the phrase “specifically binds” in the context of an antibody or antibody antigen binding fragment indicates that the respective antigen is bound to the antibody or antibody antigen binding fragment via an antigen-antibody reaction. The term “specifically binding” also expresses that the antibody or antigen binding fragment binds to the indicated structure preferentially over other structures that may show cross reactivity. In other words, specifically binding” also means that the antibody or antigen binding fragment discriminates its antigen from other structures. . For example, a molecule is said to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular molecule (e.g. cell, protein or substance) than it does with alternative equivalent molecules (e.g. cells, proteins or substances). In embodiments, specifically binding means that the molecule (e.g. antibody or antigen-binding fragment) binds its target antigen with an affinity that is at least 3-fld, preferably at least 5-fold, preferably at least 10-fold, preferably at least 20-fold, even more preferably at least 50-fold or at least 100-fold affinity than other non-target antigens.

A variety of assay formats may be used to select an antibody that specifically binds a molecule of interest. For example, solid-phase ELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with an antigen or a receptor, or ligand binding portion thereof, that specifically binds with a cognate ligand or binding partner. Typically, a specific or selective reaction will be at least twice the background signal or noise, more typically more than 10 times background, even more typically, more than 50 times background, more typically, more than 100 times background, yet more typically, more than 500 times background, even more typically, more than 1000 times background, and even more typically, more than 10,000 times background.

In a preferred embodiment surface plasmon resonance spectroscopy (e.g. Biacore ™) is used to identify the required antibody.

The expressions “recognition motif”, “motif”, “cleavage motif”, “cleavage sequence” or “recognition sequence” are used interchangeable herein and as used herein refer to a certain sequence, which is an amino acid sequence that is recognized by a certain enzyme (e.g. proteases or transpeptidases). In the case of proteases and transpeptidases the recognition of such a motif results in the cleavage of the motif/sequence at a certain position. The recognition motif can vary in its number of residues. For example, the recognition motif can consist of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. Some enzymes may have various recognition motifs, which might differ in their specificity and/or catalytic efficiency. In order to qualify as a recognition motif as used herein the motif has to be cuttable by an enzyme described herein under appropriate conditions which are known to a person skilled in the art. A person skilled in the art would be able to identify a recognition motif as qualifying for the intended purpose according to the invention. Qualifying recognition motifs could be identified by literature search or empirically via genetically inserting the motif into the desired IgG framework, proceeding with a reaction such as in Example 1 and judging progress on HPLC, or via observing the sizes of the products on protein gel, or using mass spectrometry. Alternatively, peptides or peptide libraries designed with the motif sequence(s) could be used in a test reaction to assess the motif-enzyme suitability, for example when coupled with mass spectrometry analysis of the products or any other suitable method.

As used herein the term “capable of cleaving” refers to the general ability and capability of an enzyme (e.g. protease, transpeptidase, etc.) to cleave at a certain recognition motif under appropriate conditions. Therefore, it can also be understood as “thereby cleaving” or “cleaving by incubating”.

The first recognition motif as disclosed herein is positioned in the variable heavy (VH) - constant heavy (CH) 1 loop (VH-CH1 loop). The VH-CH1 loop (or elbow/loop region) consists of the amino acids that are positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (in N- to C-terminal direction). The VH-CH1 loop connects the two beta sheets of the C domain with the two beta sheets of the V domain.

The second recognition site is positioned in the variable light (VL) - constant light (CL) loop (VH-CL loop). The VL-CL loop (or elbow loop region) consists of the amino acids that are positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (in N- to C- terminal direction). The VL-CL loop connects the two beta sheets of the constant (C) domain with the two beta sheets of the variable (V) domain.

The VH-CH1 loop, i.e. the loop between the variable region and the constant region of the heavy chain ranges from amino acids 110 to 120 according to the Kabat numbering scheme.

The VL-CL loop, i.e. the loop between the variable region and the constant region of the light chain ranges from amino acids 106 to 114 according to the Kabat numbering scheme. The term "amino acid mutation" denotes a modification in the amino acid sequence of a parent amino acid sequence. Exemplary modifications include amino acid substitutions, insertions, and/or deletions.

The term "amino acid mutations at the position" denotes the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. The term "insertion adjacent to a specified residue" denotes the insertion within one to two residues thereof. The insertion may be N- terminal or C-terminal to the specified residue.

The term "amino acid substitution" denotes the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different "replacement" amino acid residue. The replacement residue or residues may be a "naturally occurring amino acid residue" (i.e. encoded by the genetic code) and selected from the group consisting of: alanine (Ala, A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine (Cys, C); glutamine (Gin, Q); glutamic acid (Glu, E); glycine (Gly, G); histidine (His, H); isoleucine (lie, I): leucine (Leu; L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (Vai, V).

In one embodiment, the amino acid substitution is a replacement by a naturally occurring amino acid. In one embodiment, the replacement residue is not cysteine.

Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein. A "non- naturally occurring amino acid residue" denotes a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non- naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, a-Aminoisobutyric acid (Aib) and other amino acid residue analogues such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301-336. To generate such non-naturally occurring amino acid residues, the procedures of Noren, et al. (Science 244 (1989) 182) and/or Ellman, et al. (supra) can be used. Briefly, these procedures involve chemically activating a suppressor tR A with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA.

The term "amino acid insertion" denotes the incorporation of one or more additional amino acid residue(s) into a predetermined parent amino acid sequence. An "inserted amino acid sequence" according to the present invention will usually consist of at least five amino acids. The inserted amino acid residue(s) may be naturally occurring or non-naturally occurring as defined above. In one embodiment the inserted amino acid residues are naturally occurring amino acid residues. In one embodiment the inserted amino acid residues are naturally occurring amino acid residues but are not cysteine. In one embodiment the inserted amino acid residues are naturally occurring amino acid residues and are neither cysteine nor proline.

The term "amino acid deletion" denotes the removal of at least one amino acid residue at a predetermined position in an amino acid sequence. Within this application whenever an amino acid alteration is mentioned it is a deliberated amino acid alteration and not a random amino acid modification.

Herein single amino acid codes are used to recite peptide or polypeptide sequences such as cleavage motifs.

The term “site-specific” as used herein refers to the specific recognition of a certain amino acid sequence by a certain enzyme, like proteases or transpeptidases. The enzymes recognize and bind specifically to the corresponding “sites”, i.e. sequence of amino acids, resulting in a “site-specificity” for each enzyme.

Troponins used herein refers to troponins in general, the troponin complex or a troponin subunit. Troponin is a complex of three regulatory proteins (troponin C (TnC), troponin I (Tnl), and troponin T (TnT)) that are integral to muscle contraction in skeletal muscle and cardiac muscle, but not smooth muscle. Measurements of cardiac-specific troponins I and T are extensively used as diagnostic and prognostic indicators in the management of myocardial infarction and acute coronary syndrome. Cardiac troponins are sensitive and specific biomarkers of cardiac injury. In particular, cardiac troponin T (cTnT) is highly cardiac specific, and it is not present in serum following non-myocardial muscle or other tissue damage. In addition, cTnT has been shown to be a more persistent and sensitive biomarker than others used for diagnosing myocardial infarction. Thus, cardiac troponins generally are generally useful for diagnosing acute myocardial ischemia, and cTnT is especially useful. Cardiac troponin T is a widely used biomarker in patients with cardiac disease. Its utility in patients with cardiac diseases has recently been reviewed by Westermann et al. (Nature Reviews / Cardiology, vol 14 (2017) 473-483. The use of cTnT is well established in patients with suspected acute myocardial infarction (AMI), but troponin measurement is also used in other acute and nonacute settings. In patients with suspected AMI, early decision-making is crucial to allow rapid treatment and further diagnostic evaluation.

In both cardiac and skeletal muscles, muscular force production is controlled primarily by changes in intracellular calcium concentration. In general, when calcium rises, the muscles contract and, when calcium falls, the muscles relax. T roponin is a component of thin filaments (along with actin and tropomyosin), and is the protein complex to which calcium binds to trigger the production of muscular force. T roponin has three subunits, TnC, Tnl, and TnT, each playing a role in force regulation. Under resting intracellular levels of calcium, tropomyosin covers the active actin sites to which myosin (a molecular motor organized in muscle thick filaments) binds in order to generate force. When calcium becomes bound to specific sites in the N-domain of TnC, a series of protein structural changes occurs, such that tropomyosin is rolled away from myosin-binding sites on actin, allowing myosin to attach to the thin filament and produce force and shorten the sarcomere.

Individual subunits serve different functions:

• Troponin C binds to calcium ions to produce a conformational change in Tnl

• Troponin T binds to tropomyosin, interlocking them to form a troponin-tropomyosin complex

• Troponin I binds to actin in thin myofilaments to hold the actin-tropomyosin complex in place

TnT is a tropomyosin-binding subunit which regulates the interaction of troponin complex with thin filaments; Tnl inhibits ATP-ase activity of acto-myosin; TnC is a Ca2+-binding subunit, playing the main role in Ca2+ dependent regulation of muscle contraction.

T nT and T nl in cardiac muscle are presented by forms different from those in skeletal muscles. Two isoforms of Tnl and two isoforms of TnT are expressed in human skeletal muscle tissue (skTnl and skTnT). Only one tissue-specific isoform of Tnl is described for cardiac muscle tissue (cTnl), whereas the existence of several cardiac specific isoforms of TnT (cTnT) are described in the literature.

Antibody or antigen-binding fragment as precursor for Fv production

According to a first aspect of the invention an antibody or antigen-binding fragment thereof is provided comprising a Fab domain, said Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises

(i) a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and

(ii) a light chain sequence comprising a second recognition motif for a sitespecific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop).

By including the recognition motifs in the VH-CH1 and VL-CL loop it is possible to express and purify the complete antibody or antigen-binding fragment (e.g. Fab fragment) before cleaving off and releasing the Fv fragment. The complete antibody or antigen-binding fragment (e.g. Fab fragment) so produced is therefore a precursor for the inventive method of producing an Fv described herein. An illustration of the generic concept/process for making an Fv fragment based on such a precursor antibody or antigen-binding fragment thereof is shown in Figure 1 .

These recognition motifs (enzyme recognition motifs or sequences) provide targets for the site-specific processing of an antibody molecule (e.g. full length IgG, fragments thereof or fusions comprising an antibody moiety) following its expression and purification to yield a correctly folded active Fv fragment (Example 1). This ensures the correct folding of the VH/VL within their ‘native’ immunoglobulin (e.g. IgG) context, stabilized by the inherent covalent and non-covalent interactions of the heavy and light chain constant domains, before the Fv fragment is enzymatically extracted out of (released from) the immunoglobulin (e.g. IgG) molecule. This, in turn, reduces the risk of misfolding or loss in affinities, which can accompany the current methods outlined above. The newly proposed concept provides a generic way to produce Fv fragments more efficiently; e.g. without the need to include long optimized peptide linkers to link VH/VL or refolding conditions optimization for each new candidate, providing a stable product at a good yield that is less prone to aggregation.

The heavy chain constant region of the antibody or antigen-binding fragment may be selected from the group consisting of: IgG, IgA, IgD, IgE, and IgM. Immunoglobulin constant regions may be further classified into isotypes. The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY, preferably IgG), any subclass (e.g., lgG1 , lgG2, lgG3, lgG4 for human IgG; lgG1 , lgG2a, lgG2b, lgG3 for mouse IgG; lgG1 , lgG2 for sheep IgG and Rb- IgG for rabbit IgG). Light chains can further be of kappa or lambda type. An antibody can be human, humanized, chimeric and/or affinity matured as well as an antibody from other species, for example, goat, mouse, rat, rabbit or sheep.

In one embodiment the present disclosure relates to an antibody (of any of these isotypes or the corresponding isotypes from other species, or their corresponding subclasses), comprising at least two recombinant immunoglobulin heavy chains as disclosed in the present application.

In one embodiment, the antibody or antigen-binding fragment thereof is selected from IgG, IgE and IgD.

In a preferred embodiment, the antibody or antigen-binding fragment thereof is IgG. In another embodiment, the antibody is a mouse/human chimeric antibody or a rabbit/human chimeric antibody.

The antigen of interest that the antibody or antigen-binding fragment binds to can be any antigen or epitope allowing the antibody or antigen-binding fragment to bind thereto. It can be an antigen whereto the antibody or antigen-binding fragment has a high, intermediate or low affinity and/or specificity.

In a preferred embodiment, the Fv fragment is capable of binding a Troponin, in particular cardiac Troponin T (cTNT).

The first recognition motif is positioned in the variable heavy (VH) - constant heavy (CH) 1 loop (VH-CH1 loop). The VH-CH1 loop (or elbow/loop region) consists of the amino acids that are positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (in N- to C-terminal direction). The VH-CH1 loop connects the two beta sheets of the C domain with the two beta sheets of the V domain.

The second recognition site is positioned in the variable light (VL) - constant light (CL) loop (VH-CL loop). The VL-CL loop (or elbow loop region) consists of the amino acids that are positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (in N- to C- terminal direction). The VL-CL loop connects the two beta sheets of the constant (C) domain with the two beta sheets of the variable (V) domain.

The VH-CH1 loop, i.e. the loop between the variable region and the constant region of the heavy chain ranges from amino acids 110 to 120 according to the Kabat numbering scheme.

The VL-CL loop, i.e. the loop between the variable region and the constant region of the light chain ranges from amino acids 106 to 114 according to the Kabat numbering scheme.

In one embodiment the first and/or second recognition motif is artificially introduced into the VH-CH1- and/or VL-CL-loop. A person skilled in the art is familiar with methods to introduce a corresponding recognition motif. For example, standard methods can be used to manipulate DNA as described in Sambrook, J.et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, 20 Cold Spring Harbor, New York, 1989. Alternatively, corresponding sequences may be synthesized. Commercial providers and methods for gene syntheses are known in the art.

The desired gene segments of both light and heavy chains of recombinant immunoglobulin variable chains (VH or VL) comprising the sequence encoding for the enzyme recognition motif and homologous ends to a backbone vector can be prepared by standard chemical synthesis. The synthesized gene fragments can then, e.g., be cloned into a backbone E. coli shuttle vector carrying the immunoglobulin constant domains using overlap-directed DNA assembly methods. The assembled vector can then be used for propagation/amplification of DNA in E. coli. A recombinant heavy chain vector and a recombinant light chain vector are cloned for each construct. Both vectors can then be used for co-transient expression in suitable host cells, e.g., HEK293 cells. The DNA sequences of subcloned gene fragments can also be verified by DNA sequencing. For the expression of a desired gene/protein (e.g. full length antibody heavy chain comprising enzyme recognition motif, full length antibody light chain comprising enzyme recognition motif) a transcription unit comprising, e.g., the following functional elements can be used: the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV), an immunoglobulin heavy chain signal sequence including an intron, a gene/protein to be expressed, the bovine growth hormone polyadenylation sequence (BGH pA). Beside the expression unit/cassette including the desired gene to be expressed, a basic/standard mammalian expression plasmid contains an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.

In one embodiment, the antibody or antigen-binding fragment is recombinant, in particular due to the insertion of the first and/or second recognition motif.

In one embodiment, the first recognition motif is a recognition motif for a transpeptidase and the second recognition motif is a recognition motif for a site-specific protease.

In another embodiment, the first recognition motif is a recognition motif for a site-specific protease and the second recognition motif is a recognition motif for a transpeptidase.

In one embodiment, the the first recognition motif is a recognition motif for a transpeptidase and the second recognition motif is a recognition motif for a transpeptidase.

In one embodiment, the first recognition motif is a recognition motif for a site-specific protease and the second recognition motif is a recognition motif for a site-specific protease.

In one embodiment, the recognition motif for the site-specific protease in the VH-CH1 loop and/or the recognition motif for the site-specific protease in the VL-CL loop are independently selected from the group consisting of: TEV-protease recognition motif, IgA protease recognition motif and 3C protease recognition motif.

TEV protease (Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a highly sequencespecific cysteine protease from Tobacco Etch Virus (TEV). It is a member of the PA (Proteases of mixed nucleophile, superfamily A) clan of chymotrypsin-like proteases. TEV protease and many optimized variants thereof are widely used for affinity tag removal from recombinant proteins and are readily commercially available. An example of a suitable E.coli expression vector can also be obtained from addgene.org ((pcDNA3.1 TEV (full-length), Plasmid #6427).

In one embodiment the TEV protease recognition motif comprises or consists of EXXYXQX, (where X is any amino acid and wherein the protease cleaving/cutting site is between Q and X represented by ^A, i.e. EXXYXQ^AX.).

In another embodiment the TEV protease recognition motif comprises or consists of ENLYFQX1 (SEQ ID NO: 1), wherein X1 is S, G, A, M, C or H, wherein the cutting site is between Q and X, i.e. ENLYFQ^AX1.

IgA proteases have been identified from various organisms like Neisseria gonorrhoeae, Neisseria meningitis, Haemophilus influenzae, Streptococcus pneumonia, Streptococcus sanguis, Streptococcus oralis. Streptococcus mitis, Streptococcus suis, Streptomyces griseus, Ureaplasma ureolyticum, Bacillus licheniformis, Clostridium ramosum, Prevotella melaninogenica, Bacteroides melaninogenicus. The natural substrate for IgA protease is human immunoglobulin A1 , which is one of the most important and most frequently produced antibodies in the human serum and mucus. The enzymes recognize a cleavage sequence 223-240 in the hinge region of the immunoglobulin heavy chain. IgA proteases derived from different strains of bacteria cleave the amino acid chain in the hinge chain of IgA at different positions.

The IgA protease used in the Examples herein is the one from N. gonorrhoeae. This enzyme and an exemplary production procedure for the same is described in European patent publication No. 0495398 (Roche), which is incorporated herewith by reference.

In one embodiment the IgA protease recognition motif comprises or consists of PX, wherein X is S, T, A, V, or G (using standard single letter amino acid code).

In another embodiment the IgA protease recognition motif comprises or consists of X1X2PPX3P, wherein X1 is P or S, X2 is R or T, X3 is T, S or A (using standard single letter amino acid code), with the cutting site being between P and X3 (X1X2PP^AX3P).

The most general consensus of the recognition motif of IgA proteases is P^AX (where X is preferably S, T, A, V, G (using standard single letter amino acid code) and ^A represents the cutting site, i.e. here between P and X). PX as a recognition motif usually occurs in a proline rich sequence context. “Proline rich” may, for example, mean, wherein three proline residues occur within a stretch of ten amino acids around PX (four amino acids upstream and four amino acids downstream of PX). A more specific recognition motif for IgA proteases is PRPPXP (SEQ ID NO: 2), with X = S, T, A, V, G with the cutting site being between P and X (PRPP^AXP).

Human rhinoviruses (HRVs) translate their genetic information into a polyprotein precursor that is mainly processed by a virally encoded 3C protease (3Cpro) to generate functional viral proteins and enzymes. It has been shown that the enzymatic activity of HRV 3Cpro is essential to viral replication. The 3Cpro is distinguished from most other proteases by the fact that it has a cysteine nucleophile but with a chymotrypsin-like serine protease folding. 3C protease is widely used for the removal of affinity tags and is readily available from a number of commercial suppliers. For example, an E.coli expression vector can also be obtained from addgene.org (pSRK2706; Plasmid #78571).

In one embodiment the 30 protease recognition motif comprises or consists of LFQGP (SEQ ID NO: 3) (using standard single letter amino acid code), with the cutting site being between Q and G (LFQ^AGP).

In another embodiment the 30 protease recognition motif comprises or consists of LEVLFQGP (SEQ ID NO: 4) (using standard single letter amino acid code), with the cutting site being between Q and G (LFQ^AGP).

In order to serve as a recognition motif as described herein the recognition motif should enable a corresponding enzyme (protease or transpeptidase) to cleave the amino acid sequence between the corresponding two amino acid residues resulting in a separation of the amino acid sequence into (in the case of one recognition site) two sequences. A person skilled in the art would be well aware of ways to identify and select appropriate pairs of recognition motifs and site-specific proteases or transpeptidases. The method described in Example 6 could be used to test the suitability of appropriate pairs of recognition motifs and site-specific proteases or transpeptidases for use in the method of the invention.

Qualifying recognition motifs can be identified by literature search or empirically via genetically inserting the motif into the desired IgG framework, proceeding with a reaction, such as in Example 1 or Example 6, and assessing progress using an appropriate analytical method, e.g. on HPLC, or via observing the sizes of the products on protein gel, or using mass spectrometry. Alternatively, peptides or peptide libraries designed with the motif sequence(s) could be used in a test reaction to judge the motif-enzyme suitability using an appropriate analytical method, e.g. mass spectrometry analysis of the products or any other suitable method. In brief: a reference precursor recombinant IgG clone (e.g., the 11-7 anti-TnT clone used in the Examples herein could be used or any other suitable IgG clone) is engineered to insert a test enzyme recognition sequence into the heavy chain after amino acid number 114 (Kabat Numbering) / 118 (Ell Numbering) flanked by flexible linkers such as GSG, the same or different enzyme recognition sequence flanked by flexible linkers such as GSG can be engineered into the light chain with insertion after amino acid number 110 (Kabat Numbering) / 110 (Ell Numbering). Optionally, a tag (e.g., 6-Histidine tag) can be added at the C-termini of the heavy and light chains to facilitate purification. The engineered nucleic acid construct can be cloned into and expressed from a suitable cell, for example CHO cell. The IgG (dsFv- precursor) is purified from the culture supernatant using affinity chromatography techniques, for example Protein A. Following the IgG purification a one or two step reaction with the test enzyme(s) of choice if performed according to the supplier instructions in the preferred buffer and in the presence of any required co-reactants. The success of the enzyme and insertion site/sequence within the IgG precursor being tested can be judged by assessing the conversion rate of the IgG to a dsFv/Fv. This could be done by following the reaction progress on an HPLC (i.e. injecting a sample of the reaction mixture on a GFC-column that provides sufficient resolution (e.g. Superdex 75 10/300 from Cytiva)). The percentage of the dsFv peak area as a percentage of the total peak area ca then be calculated.

The following Table 1 shows non-limiting examples of proteases and their recognition motifs that can be used for the purposes described herein.

Table 1 : Proteases and recognition motifs; “^A” represents the cutting site

As used herein the term “any amino acid” means any of the 20 naturally occurring amino acids or any non-naturally occurring amino acids. Examples of non-naturally occurring amino acids include: norleucine, ornithine, norvaline, homoserine, a-Aminoisobutyric acid (Aib) and other amino acid residue analogues such as those described in Ellman, et al., (Meth. Enzym. 202: 301-336, 1991).

In a preferred embodiment the term “any amino acid” refers to any of the 20 naturally occurring amino acids.

In one embodiment the recognition motif for a transpeptidase in the VH-CH1 loop and/or the recognition motif for a transpeptidase in the VL-CL loop is a recognition motif for a sortase or sortase-type transpeptidase. In one embodiment the sortase transpeptidase is one from classes A-F or the sortase-type transpeptidase is an exosortase or Archaesortase.

Transpeptidase is an enzyme class that refers to specific enzyme function, of which several subclasses exist. One subclass is protein-sorting transpeptidase: an enzyme that natively cleaves a C-terminal sorting signal from its target protein(s) and then covalently attaches the remainder to the cell surface. Protein sorting transpeptidases or a sortase-type transpeptidases can be split into two groups. One includes enzymes that are homologous to one another (sortases A-F). The other includes enzymes that are not homologous to sortase A-F but perform same function (e.g. arachaeosortases, exosortase).

Hence, sortase is a specific example of a transpeptidase, namely a protein-sorting transpeptidase. A sortase or protein-sorting transpeptidase (as well as a sortase-type transpeptidase) is an enzyme, such as the sortase A (SrtA) of Staphylococcus aureus, that can cleave one or more target proteins (e.g. produced by the same cell), as part of a specialized pathway of protein targeting. The typical prokaryotic protein-sorting transpeptidase is characterized as a protease, but does not simply hydrolyze a peptide bond. Instead, the larger, N-terminal portion of the cleaved polypeptide is transferred onto another molecule, such as a precursor of the peptidoglycan cell wall in Gram-positive bacteria.

Sortases are a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. For most substrates of sortase enzymes, the recognition signal consists of the motif LPXTG (Leu-Pro-X-Thr-Gly; whereX is any amino acid), then a highly hydrophobic transmembrane sequence, followed by a cluster of basic residues such as arginine. Cleavage occurs between the Thr and Gly, with transient attachment through the Thr residue to the active site Cys residue, followed by transpeptidation that attaches the protein covalently to cell wall components.

Protein-sorting transpeptidases can be roughly categorized in two major categories. The first and by far the most common and well-studied is the group of Sortase Transpeptidases. This is a group of homologous enzymes from different species, classified in classes A-F, which do the same thing, but have different recognition sequences and can fulfil different ultimate purposes in the organisms. The most studied out of these is the Sortase A enzyme from Staphylococcus aureus. The Table 2 below summarizes the recognition motif for the classes A-F.

Table 2: Listing of sortase classes with recognition motifs

In addition, there is another group of single enzymes that are not homologous to Sortase A-F, but can perform a similar protein-sorting reaction. As used herein the term “sortase-type transpeptidase” refers to these enzymes. One report consider them as a second category clustered around/homologous to the enzyme Exosortase. Table 3 lists a few examples with their recognition motif and reference (but this is not an exhaustive list)

Table 3: Sortase-type transpeptidases and their recognition motif

In one embodiment the sortase transpeptidase is a sortase A. For the enzymatic conjugation in vitro in one embodiment a soluble truncated sortase A lacking the membrane-spanning region (SrtA; amino acid residues 60- 206 of Staphylococcus aureus SrtA) is used; see also Ton-That, H., et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12424-12429; llangovan, H., et al., Proc. Natl. Acad. Sci. USA 98 (2001) 6056-6061).

Sortase A (SrtA) is a membrane bound enzyme which attaches proteins covalently to the bacterial cell wall. The wild-type Staphylococcus aureus sortase A (SrtA) is a polypeptide of 206 amino acids with an N-terminal membrane-spanning region. The specific sortase recognition motif on the SrtA substrate is LPX1TX2 (wherein X1 can be any amino acid residue and X2 is G or A). The sortase enzyme cleaves between the residues threonine (T) and X2 (G or A). The ligation motif on the sortase nucleophile (also referred to as sortase substrate), i.e. the polypeptide part of the peptidoglycan that becomes attached to the substrate on the bacterial cell wall, naturally is a pentaglycine motif.

It has been shown that a triglycine and even a diglycine motif on the N-terminus of the peptidoglycan is sufficient to support the SrtA reaction (Clancy, K.W., et al, Peptide Science 94 (2010) 385-396). The reaction proceeds through a thioester acyl-enzyme intermediate, which is resolved by the attack of an amine nucleophile from the oligoglycine, thereby covalently linking the peptidoglycan to a protein substrate and regenerating SrtA.

In one embodiment the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A.

In another embodiment, the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue.

In one embodiment the sortase recognition motif is LPX1TX2 wherein X2 is G. In one embodiment X1 is a naturally occurring amino acid. In one embodiment X1 is not a cysteine residue. In one embodiment X1 is a naturally occurring amino acid and is not a cysteine residue.

In one embodiment the sortase recognition motif is a sortase B recognition motif and comprises or consists of NPX1X2X3, wherein X1 can be any amino acid residue, X2 can be T or S, and X3 can be N, G or S.

In one embodiment the sortase recognition motif is a sortase C recognition motif and comprises or consists of X1X2X3TG, wherein X1 can be I or L, X2 can be P or A and X3 can be any amino acid residue.

In one embodiment the sortase recognition motif is a sortase D recognition motif and comprises or consists of LPXTA (SEQ ID NO: 6), wherein X can be any amino acid residue. In one embodiment the sortase recognition motif is a sortase E recognition motif and comprises or consists of LAXTG (SEQ ID NO: 7), wherein X can be any amino acid residue.

In one embodiment the sortase recognition motif is a sortase F recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue.

In one embodiment the sortase recognition motif is a Exosortase A recognition motif and comprises or consists of PEP.

In one embodiment the sortase recognition motif is a Archaesortase recognition motif and comprises or consists of PGF.

In one embodiment the first recognition motif and/or the second recognition motif is flanked by an N-terminal linker sequence and/or a C-terminal linker sequence.

The term “linker” as used herein refers to any amino acid sequence able to link the recognition motif to the amino acid sequence of the antibody or antigen binding fragment. Thereby the linker is chosen in a way to support the cleavage process, for example by exposing the recognition motif and thereby make it easier accessible for the respective enzyme. The whole construct consisting of the recognition motif and the N-terminal and/or C-terminal linker should be chosen in a way that undesired aggregation and misfolding of the protein is avoided and in a way that it supports the cleavage process by the enzyme, for example by presenting the cleavage site. Further, the construct should still allow the expression of the antibody (in a soluble manner). A person skilled in the art would be well aware of how to choose a construct consisting of the recognition motif and at least one linker accordingly. Suitable linker sequences could be identified by literature search or empirically via genetically inserting the motif and flanking linker into the desired IgG framework, proceeding with reaction, such as in Example 1 , and judging progress using an appropriate analytical technique, for example HPLC ,or via observing the sizes of the products on protein gel, or using mass spectrometry.

In one embodiment, the N-terminal linker sequence has a length of at least 2 amino acids, preferably at least 3 amino acids.

In one embodiment, the C-terminal linker sequence has a length of at least 2 amino acids, preferably at least 3 amino acids.

In one embodiment, the first recognition motif and the second recognition motif are flanked at both ends (N-terminal and C-terminal) by a linker sequence.

In one embodiment, the N-terminal and/or C-terminal linker sequence is composed of amino acids selected from the group consisting of Glycine, Serine, Alanine, Threonine, Glutamate and Proline. In one embodiment, the N-terminal and/or C-terminal linker sequence does not comprise a cysteine (C).

In one embodiment the N-terminal linker comprises or consists of a sequence selected from the group consisting of: GSG, AP, APAP (SEQ ID NO: 8), ESGS (SEQ ID NO: 9), GGGS (SEQ ID NO: 10), GGGGS (SEQ ID NO: 11), and GSGGSG (SEQ ID NO: 12). As elsewhere, standard single letter amino acid codes are used.

In one embodiment the C-terminal linker comprises or consists of a sequence selected from the group consisting of: GSG, GSGGSG (SEQ ID NO: 13), AP, APAP (SEQ ID NO: 14), ESGS (SEQ ID NO: 15), GGGS (SEQ ID NO: 16) and GGGGS (SEQ ID NO: 17). As elsewhere, standard single letter amino acid codes are used.

As used herein the terms “cleavable insert domain” or “insertion sequence” are used interchangeably herein and refer to the sequence introduced into the parental antibody or antigen-binding fragment and comprises the recognition motif alone or the recognition motif flanked by an N-terminal and/or C-terminal linker. The length of the cleavable insert domain can vary in size. For example, its size can be in the range from 2 to 50 amino acids. For example the cleavable insert domain can have a length of 5, 8, 9, 11 , or 12 amino acids. In one embodiment the length of the insert construct is 9 to 15 amino acids for the heavy chain and 3 to 16 for the light chain. As would be clear to a person skilled in the art the total length of the insert construct is chosen in a way to obtain most beneficial conditions for the process, i.e. with regards to folding, aggregation, catalytic, cleavage, accessibility of the cleavage sequence etc.

In one embodiment the cleavable insert domain is 11 , 12 or 13 amino acids long.

In one embodiment the cleavable insert domain is at least 4, 5, 6, 7, 8, 9,10, 11 , 12, 13, 14, 15, 16, 17 amino acids long.

In one embodiment the cleavable insert domain comprises at least 3 amino acids C-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 4 amino acids C-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 5 amino acids C-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 6 amino acids C-terminal to the first and/or second recognition motif. In one embodiment the cleavable insert domain comprises at least 7 amino acids C-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 3 amino acids N-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 4 amino acids N-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 5 amino acids N-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 6 amino acids N-terminal to the first and/or second recognition motif.

In one embodiment the cleavable insert domain comprises at least 7 amino acids N-terminal to the first and/or second recognition motif.

The VH-CH1 loop, i.e. the loop between the variable region and the constant region of the heavy chain ranges from amino acids 110 to 120 according to the Kabat numbering scheme. The recognition motif with or without N- and/or C-terminal linkers can be positioned somewhere between these amino acids either by inclusion or by deletion/replacement/substitution of any number of these amino acids. Examples 5 and 6 demonstrate the ability to locate the recognition motif at locations within the loop between the variable region and the constant region of the heavy chain in the range from amino acids 110 to 120 according to the Kabat numbering scheme. Examples 5 and 6 also demonstrate the ability to use different types of enzyme cleavage.

In an embodiment the recognition motif must not be present in VH and VL outside the VH- CH1 loop and VL-CL loop.

In one embodiment the first recognition motif is positioned anywhere between amino acids 109 and 121 of the heavy chain according to Kabat numbering scheme.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 109 and 121 of the heavy chain according to Kabat numbering scheme. Suitably the LPETG (SEQ ID NO: 35) is flanked by N- and/or C- terminal linker.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 109 and 121 of the heavy chain according to Kabat numbering scheme flanked by GSG or APAP (SEQ ID NO: 8) as N-terminal linker and flanked by GSG or APAP (SEQ ID NO: 8) as C-terminal linker.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 109 and 121 of the heavy chain according to Kabat numbering scheme.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 109 and 121 of the heavy chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

In one embodiment the first recognition motif may be positioned anywhere between amino acids 112 and 119 of the heavy chain according to Kabat numbering scheme.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 112 and 119 of the heavy chain according to Kabat numbering scheme. Suitably the LPETG (SEQ ID NO: 35) is flanked by N- and/or C- terminal linker.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 112 and 119 of the heavy chain according to Kabat numbering scheme flanked by GSG or APAP (SEQ ID NO: 8) as N-terminal linker and flanked by GSG or APAP (SEQ ID NO: 8) as C-terminal linker.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 112 and 119 of the heavy chain according to Kabat numbering scheme.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 112 and 119 of the heavy chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

In one embodiment the first recognition motif is positioned between amino acids 111 and 117 of the heavy chain according to Kabat numbering scheme.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 111 and 117 of the heavy chain according to Kabat numbering scheme. Suitably the LPETG (SEQ ID NO: 35) is flanked by N- and/or C- terminal linker. In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 111 and 117 of the heavy chain according to Kabat numbering scheme flanked by GSG or APAP (SEQ ID NO: 8) as N-terminal linker and flanked by GSG or APAP (SEQ ID NO: 8) as C-terminal linker.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 111 and 117 of the heavy chain according to Kabat numbering scheme.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 111 and 117 of the heavy chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

In one embodiment the first recognition motif is positioned between amino acids 112 and 116 of the heavy chain according to Kabat numbering scheme.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 112 and 116 of the heavy chain according to Kabat numbering scheme. Suitably the LPETG (SEQ ID NO: 35) is flanked by N- and/or C- terminal linker.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 112 and 116 of the heavy chain according to Kabat numbering scheme flanked by GSG or APAP (SEQ ID NO: 8) as N-terminal linker and flanked by GSG or APAP (SEQ ID NO: 8) as C-terminal linker.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 112 and 116 of the heavy chain according to Kabat numbering scheme.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 112 and 116 of the heavy chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

In one embodiment the first recognition motif is positioned between amino acids 113 and 116 of the heavy chain according to Kabat numbering scheme. In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 113 and 116 of the heavy chain according to Kabat numbering scheme. Suitably the LPETG (SEQ ID NO: 35) is flanked by N- and/or C- terminal linker.

In one embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned between amino acids 113 and 116 of the heavy chain according to Kabat numbering scheme flanked by GSG or APAP (SEQ ID NO: 8) as N-terminal linker and flanked by GSG or APAP (SEQ ID NO: 8) as C-terminal linker.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 113 and 116 of the heavy chain according to Kabat numbering scheme.

In one embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) positioned between amino acids 113 and 116 of the heavy chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

In a preferred embodiment the first recognition motif is positioned directly after amino acid 114 of the heavy chain according to Kabat numbering scheme.

In a preferred embodiment the first recognition motif is a sortase A recognition motif positioned after amino acid 114 of the heavy chain according to Kabat numbering scheme.

In another preferred embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned after amino acid 114 of the heavy chain according to Kabat numbering scheme. Suitably the LPETG (SEQ ID NO: 35) is flanked by N- and/or C- terminal linker.

In another preferred embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned after amino acid 114 of the heavy chain according to Kabat numbering scheme flanked by GSG or APAP (SEQ ID NO: 8) as N-terminal linker and flanked by GSG or APAP (SEQ ID NO: 8) as C-terminal linker.

In one preferred embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) after amino acid 114 of the heavy chain according to Kabat numbering scheme.

In one preferred embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) after amino acid 114 of the heavy chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone. The VL-CL loop, i.e. the loop between the variable region and the constant region of the light chain ranges from amino acids 106 to 114 according to the Kabat numbering scheme. The recognition motif with or without N- and/or C-terminal linkers can be positioned somewhere between these amino acids either by inclusion or by deletion/replacement of any number of these amino acids.

In principle, and as supported by Examples 5 and 6, the variation of the insertion site within the heavy/light chain loop region by a few positions up or downstream is tolerated; as long as it is within the flexible unstructured loop linking the two Fd or light chain domains (VH/CH1 or VL/CL).

In one embodiment the second recognition motif is positioned anywhere between amino acids 105 and 115 of the light chain according to Kabat numbering scheme.

In one embodiment the second recognition motif is positioned anywhere between amino acids

107 and 115 of the light chain according to Kabat numbering scheme. In one embodiment the second recognition motif is positioned anywhere between amino acids 107 and 113 of the light chain according to Kabat numbering scheme.

In one embodiment the second recognition motif is positioned anywhere between amino acids

108 and 113 of the light chain according to Kabat numbering scheme.

In one embodiment the second recognition motif is positioned anywhere between amino acids

109 and 112 of the light chain according to Kabat numbering scheme.

In a preferred embodiment the second recognition motif is positioned after amino acids 108, 110, 111 , 112, or 114 amino acids of the light chain according to Kabat numbering scheme.

In a preferred embodiment the second recognition motif is a sortase A recognition motif positioned after amino acid 108, 110, 111 , 112, or 114 of the light chain according to Kabat numbering scheme.

In another preferred embodiment the first recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned after amino acid 108, 110, 111 , 112, or 114 of the light chain according to Kabat numbering scheme. Suitably the LPETG (SEQ ID NO: 35) is flanked by N- and/or C-terminal linker.

In another preferred embodiment the second recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned after amino acid 108, 110, 111 , 112, or 114 of the light chain according to Kabat numbering scheme flanked by GSG, AP, APAP (SEQ ID NO: 8), or ESGS (SEQ ID NO: 9) as N-terminal linker and flanked by GSG, AP, APAP(SEQ ID NO: 8), ESGS (SEQ ID NO: 9),GSGGSG (SEQ ID NO: 13) as C-terminal linker. In another preferred embodiment the second recognition motif is the sortase A recognition motif LPETG (SEQ ID NO: 35) positioned after amino acid 111 of the light chain according to Kabat numbering scheme without being flanked by any linker.

In one embodiment the second cleavable insert domain comprises the sortase A recognition motif LPETG (SEQ ID NO: 35) flanked by ESGS (SEQ ID NO: 9) as N-terminal linker and flanked by ESGS (SEQ ID NO: 9) as C-terminal linker and replaces amino acids 109 - 112 of the light chain according to Kabat numbering scheme.

In one preferred embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27)after amino acid 110 of the light chain according to Kabat numbering scheme.

In one preferred embodiment the cleavable insert domain is GSGLPETGGSG (SEQ ID NO: 27) after amino acid 110 of the light chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

In a preferred embodiment the second recognition motif is a IgA-protease recognition motif positioned after amino acid 108, 110, 111 , 112, or 114 of the light chain according to Kabat numbering scheme. Preferably, the IgA-protease recognition motif is positioned after amino acid 110 of the light chain according to Kabat numbering scheme.

In another preferred embodiment the first recognition motif is the IgA-protease recognition motif PRPPGP positioned after amino acid 108, 110, 111 , 112 or 114 of the light chain according to Kabat numbering scheme. Preferably, the IgA-protease recognition motif is positioned after amino acid 110 of the light chain according to Kabat numbering scheme. Suitably the PRPPGP is flanked by N- and/or C-terminal linker.

In another preferred embodiment the second recognition motif is the IgA-protease recognition motif PRPPGP positioned after amino acid 108, 110, 111 , 112 or 114 of the light chain according to Kabat numbering scheme flanked by GSG, AP, APAP (SEQ ID NO: 8), or ESGS (SEQ ID NO: 9) as N-terminal linker and flanked by GSG, AP, APAP (SEQ ID NO: 8), ESGS (SEQ ID NO: 9), or GSGGSG (SEQ ID NO: 13) as C-terminal linker. Preferably, the IgA- protease recognition motif is positioned after amino acid 110 of the light chain according to Kabat numbering scheme.

In another preferred embodiment the second recognition motif is the IgA-protease recognition motif PRPPGP positioned after amino acid 110 of the light chain according to Kabat numbering scheme without being flanked by any linker. In one embodiment the second cleavable insert domain comprises the IgA-protease recognition motif PRPPGP flanked by ESGS (SEQ ID NO: 9) as N-terminal linker and flanked by ESGS (SEQ ID NO: 9) as C-terminal linker and replaces amino acids 109 - 112 of the light chain according to Kabat numbering scheme.

In one preferred embodiment the cleavable insert domain is GSGPRPPGPGSG (SEQ ID NO: 36) after amino acid 110 of the light chain according to Kabat numbering scheme.

In one preferred embodiment the cleavable insert domain is GSGPRPPGPGSG (SEQ ID NO: 36) after amino acid 110 of the light chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

In a preferred embodiment the second recognition motif is a TEV-protease recognition motif positioned after amino acid 108, 110, 111 , 112, or 114 of the light chain according to Kabat numbering scheme. Preferably, the TEV-protease recognition motif is positioned after amino acid 110 of the light chain according to Kabat numbering scheme.

In another preferred embodiment the first recognition motif is the TEV-protease recognition motif ENLYFQG positioned after amino acid 108, 110, 111 , 112, or 114 of the light chain according to Kabat numbering scheme. Preferably, the TEV-protease recognition motif is positioned after amino acid 110 of the light chain according to Kabat numbering scheme. Suitably the ENLYFQG is flanked by N- and/or C-terminal linker.

In another preferred embodiment the second recognition motif is the TEV-protease recognition motif ENLYFQG positioned after amino acid 108, 110, 111 , 112, or 114 of the light chain according to Kabat numbering scheme flanked by GSG, AP, APAP, or ESGS (SEQ ID NO: 9) as N-terminal linker and flanked by GSG, AP, APAP(SEQ ID NO: 8), ESGS (SEQ ID NO: 9),GSGGSG (SEQ ID NO: 13) as C-terminal linker. Preferably, the TEV-protease recognition motif is positioned after amino acid 110 of the light chain according to Kabat numbering scheme.

In another preferred embodiment the second recognition motif is the TEV-protease recognition motif ENLYFQG positioned after amino acid 110 of the light chain according to Kabat numbering scheme without being flanked by any linker.

In one embodiment the second cleavable insert domain comprises the TEV-protease recognition motif ENLYFQG flanked by ESGS (SEQ ID NO: 9) as N-terminal linker and flanked by ESGS (SEQ ID NO: 9) as C-terminal linker and replaces amino acids 109 - 112 of the light chain according to Kabat numbering scheme. In one preferred embodiment the cleavable insert domain is GSGENLYFQGGSG (SEQ ID NO: 37) after amino acid 110 of the light chain according to Kabat numbering scheme.

In one preferred embodiment the cleavable insert domain is GSGENLYFQGGSG (SEQ ID NO: 37) after amino acid 110 of the light chain according to Kabat numbering scheme of a human IgG backbone (all constant domains are human) with the VH/VL domains originating from a mouse clone.

The expression “after amino acid” or “directly after amino acid” are used interchangeably herein and refer to the position subsequent to the indicated amino acid. For example, “after amino acid 110” and “directly after amino acid 110” refer to the position between 110 and 111.

In one embodiment the first and/or second cleavable insert domain replaces one or more amino acids between amino acids 109 and 121 of the parent VH domain or amino acids 105 and 115 of the parent VL domain according to Kabat numbering scheme, respectively. This means that the cleavable insert domain can either be inserted between any of the amino acids of the corresponding loop or can replace one or more amino acids of the corresponding loop.

In one embodiment the first and/or second recognition motif and any N- or C-terminal linker sequence attached thereto replaces 1 to 4 amino acids of the parent VH or VL sequence.

In one embodiment the antibody or antigen-binding fragment thereof is of mammalian origin. In particular embodiments the antibody or antigen-binding fragment thereof is of rabbit, mouse, rat, or sheep origin.

In another embodiment the antibody or antigen-binding fragment thereof is from a human.

In one embodiment the heavy chain sequence and/or the light chain sequence comprises an affinity tag. The affinity tag can be used to facilitate the purification of the product.

In one embodiment affinity tag is a His-tag, a FLAG-tag, HA, cMyC, poly-Arg or a Strep-tag. Any further tags that could be used for the purpose described herein are known to a person skilled in the art.

For example, in case of a His-tag, the reaction side products (Fc+CH1 , CL), any uncleaved precursor, the excess label and enzymes can be removed via a 2-step purification on His- column (-Ni) followed by gel filtration chromatography (GFC).

I n one embodiment the affinity tag is positioned C-terminally of the first recognition motif and/or the second recognition motif.

In one embodiment the affinity tag is positioned at the C-terminus of the heavy chain sequence and/or the light chain sequence. In one embodiment the antibody or antigen-binding fragment is recombinantly expressed from a host cell.

In one embodiment the host cell is a eukaryotic or prokaryotic host cell.

In one embodiment the host cell is selected from a: mammalian, fungal, plant, insect or bacterial cell.

For example, antibody or antigen-binding fragment may be produced in bacteria, in particular when glycosylation is not needed (see, e.g., US 5,648,237, US 5,789,199 and US 5,840,523, Charlton, Methods in Molecular Biology 248 (2003) 245-254 (B.K.C. Lo, (ed.), Humana Press, Totowa, NJ), describing expression of antibody fragments in E. coli . After expression, the antibody or antigen-binding fragment may be isolated from the bacterial cell paste in a soluble fraction or may be isolated from the insoluble fraction so called inclusion bodies which can be solubilized and refolded to bioactive forms. Thereafter the antibody or antigen-binding fragment can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody or antigen-binding fragment-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern (see e.g. Gerngross, Nat. Biotech. 22 (2004) 1409- 1414, and Li, et al, Nat. Biotech. 24 (2006) 210-215).

Suitable host cells for the expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts (see, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants)).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful.

Other examples of useful mammalian host cell lines are the COS-7 cell line (monkey kidney CVI cell transformed by SV40); the HEK293 cell line (human embryonic kidney); the BHK cell line (baby hamster kidney); the TM4 mouse Sertoli cell line (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23 (1980) 243-251); the CVI cell line (monkey kidney cell); the VERO- 76 cell line (African green monkey kidney cell); the HELA cell line (human cervical carcinoma cell); the MDCK cell line (canine kidney cell); the BRL-3A cell line (buffalo rat liver cell); the W138 cell line (human lung cell); the HepG2 cell line (human liver cell); the MMT 060562 cell line (mouse mammary tumor cell); the TRI cell line (e.g. described in Mather, et al, Anal. N.Y. Acad. Sci. 383 (1982) 44-68); the MRC5 cell line; and the FS4 cells-line. Other useful mammalian host cell lines include the CHO cell line (Chinese hamster ovary cell), including DHFR negative CHO cell lines (see e.g. llrlaub, et al, Proc. Natl. Acad. Sci. USA 77 (1980) 4216), and myeloma cell lines such as Y0, NSO and Sp2/0 cell line. For a review of certain mammalian host cell lines suitable for polypeptide production, see, e.g., Yazaki, and Wu, Methods in Molecular Biology, Antibody Engineering 248 (2004) 255-268 (B.K.C. Lo, (ed.), Humana Press, Totowa, NJ).

In one embodiment the host cell is a mammalian cell selected from a HEK, NSO, Sp2/0, PER.C6 or CHO cell.

In one embodiment the antibody or antigen binding fragment is a precursor for generating an Fv fragment.

The term “precursor” as used herein refers to the antibody or antigen-binding protein being a starting product or intermediate product of a process or method resulting in the generation of an Fv fragment that has been comprised in the Fab fragment of said antibody or antigenbinding protein.

In one embodiment the precursor is incubated with corresponding enzymes (e.g. proteases and/or transpeptidases) as described above resulting in the specific cleavage of the precursor and releasing several products including the Fv fragment. In this scenario the antibody or antigen-binding fragment could be seen as a starting product. For example, when the antibody is produced by the expression via host cells, then purified and subsequently incubated with the corresponding enzymes it could be considered as an intermediate product of the whole process. Nevertheless, in both scenarios the antibody or antigen-binding fragment can be considered as precursor since it has to be obtained first and serves as the required starting material for being able to obtain the final product, i.e. the Fv fragment.

Suitably, the VH and VL domains (polypeptides of the Fab domain) can be engineered to allow disulfide interchain bond formation. For example, a cysteine residue can be positioned in each of the VH and VL domains so as to allow their direct interaction and permit disulfide bond formation between the two cysteines. This adaptation would allow for production of dsFv.

Thus, in one embodiment the VH and VL domains have been engineered to permit inter-chain disulfide bond formation between the VH and VL domains. Suitably, this is by mutating the amino acids at two structurally conserved opposite framework sites of the VH and VL. In one embodiment, the antibody or antigen-binding fragment VH and VL domains each comprise a cysteine residue at a location capable of forming a disulfide bond between the VH and VL.

Suitable sites in the VH and VL domains for introduction of cysteine residues that can facilitate formation of a disulfide bond are known. Criteria to observe when selecting sites include:

(a) That the distance between VH and VL and the special orientation of the residues shall be close enough and directed toward each other to allow proper disulfide linkage without putting strain on the heterodimeric Fv;

(b) The positions need to tolerate the exchange of residues for cysteines without disturbing the folding, structure, and stability of VH or VL; and

(c) The introduced cysteines should be distant from CDR regions of VH and VL to avoid interference with antigen binding.

Introduction of cysteines at VH44 and VL100 are the most widely used and established from literature, but other examples are also reported in the literature. For example: VH55+VL108; VH56+VL106; VH L46C + VL D101C (e.g. see Brinkmann, U. Disulfide-stabilized Fv fragments. In Kontermann, R., Dubel, S. (eds). Antibody Engineering. Berlin Heidelberg: Springer-Verlag, 2:181 - 189, 2010).

In one embodiment the VH of the Fab domain comprises a cysteine at position 44 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position

100 according to Kabat numbering scheme.

In one embodiment the VH of the Fab domain comprises a cysteine at position 55 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position 108 according to Kabat numbering scheme.

In one embodiment the VH of the Fab domain comprises a cysteine at position 56 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position 106 according to Kabat numbering scheme.

In one embodiment the VH of the Fab domain comprises a cysteine at position 46 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position

101 according to Kabat numbering scheme. Suitably, in each of these embodiments, the cysteine in the VH of the Fab domain and the cysteine in the VL of the Fab domain are capable of forming a disulfide bond.

In one embodiment the cysteine at position 44 of the VH according to Kabat numbering scheme and the cysteine at position 100 of the VL according to Kabat numbering scheme form a disulfide bond.

In one embodiment the cysteine at position 55 of the VH according to Kabat numbering scheme and the cysteine at position 108 of the VL according to Kabat numbering scheme form a disulfide bond.

In one embodiment the cysteine at position 56 of the VH according to Kabat numbering scheme and the cysteine at position 106 of the VL according to Kabat numbering scheme form a disulfide bond.

In one embodiment the cysteine at position 46 of the VH according to Kabat numbering scheme and the cysteine at position 101 of the VL according to Kabat numbering scheme form a disulfide bond.

The engineering of a disulfide inter-chain bond between the VH and VL (via point mutations at the conserved positions) complements the described concept and yields a stable dsFv fragment with superior characteristics to scFv or ds-scFvs. The engineering of a disulfide interchain bond between the VH and VL (via point mutations at conserved positions (Brinkmann. Disulfide-Stabilized Fv Fragments. In: R. Kontermann and S. Duebel, ed., Antibody Engineering, 2nd ed. 2010)), complements the described concept and yields a stable dsFv fragment with superior characteristics to scFv or ds-scFvs (Example 2). With regard to the tested application for dsFv conjugates, e.g. as a capture biotinylated antibody on Roche Elecsys® immunoassay platform, the small size of the dsFv fragment provides it with a diffusion advantage which leads to enhanced assay performance (superior signal-dynamics) (Example 4). The dsFv construct also possesses a superior stability profile, when compared to other IgG fragments tested (Example 4). It is, furthermore, inferred that the lack of the IgG constant domains in the Fv construct will reduce the risk for assay interference.

In order for the protease/transpeptidase cleavage to release functional Fv it is preferable that no additional first or second recognition motifs (recognized by the protease/transaminase used in the method of the invention) are present within the Fv, otherwise cleavage by the protease/ transpeptidase within the Fv region could affect the yield of the desired product, i.e. the yield or Fv fragment could be decreased.

In one embodiment the Fv fragment comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the first recognition motif and wherein the Fv fragment comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the second recognition motif.

In a particular example, the antibody or antigen-binding fragment thereof lacks any further/additional first or second recognition motifs other than

• the first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain (i.e. the VH-CH1 loop) and

• the second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain (i.e. the VL-CL loop) .

In this way, when contacted with the protease and/or transpeptidase to release the Fv, the number of additional polypeptides released is kept to a minimum, the available enzyme only needs to cleave at the site of interest and there will likely be fewer intermediate cleavage (partially cleaved) products.

In another embodiments, the first recognition motif and/or the second recognition motif is present at one or more locations within the polypeptide(s) making up the antibody or antigenbinding fragment thereof, additional to the one located in VH-CH1 loop and the one located in VL-CL loop, but at location(s) outside of the Fv domain. In this way, when contacted with the protease and/or transpeptidase to release the Fv, the unrequired parts of the original antibody are cleaved at one or more sites to release smaller polypeptides.

With either of the above two examples, the person skilled in the art is able to decide where or if any additional cleavages outside of the Fv are desired to be made to the antibody or antigen binding fragment thereof, so as to facilitate optimal purification/isolation of the desired functional Fv; e.g. cleavage releases non-Fv proteins/polypeptides of size/charge that are dissimilar to the size/charge of the desired Fv protein so as to facilitate separation of the desired Fv from the undesired polypeptide/protein fragments.

In one embodiment the antibody and/or antigen binding fragment thereof and/or Fab domain comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the first recognition motif and wherein the Fv fragment comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the second recognition motif. Therefore, it should be avoided to have additional identical recognition motifs of the used enzymes contained in the Fv fragment, Fab fragment or rest of the antibody or antigen-binding fragment.

In one embodiment the Fv fragment is capable of binding a Troponin, in particular cardiac Troponin T (Uniprot P45379; SEQ ID NO: 24).

In one embodiment the VH sequence of the Fv comprises or consists of SEQ ID NO: 18. In another embodiment the VH sequence of the Fv is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3018

In one embodiment the VL sequence of the Fv comprises or consists of SEQ ID NO:19. In another embodiment the VL sequence of the Fv is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19.

In one embodiment the heavy chain sequence of the Fab comprises or consists of SEQ ID NO: 20. In another embodiment the heavy chain sequence of the Fab is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20.

In one embodiment the light chain sequence of the Fab comprises or consists of SEQ ID NO: 21. In another embodiment the light chain sequence of the Fab is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21.

The term "% sequence identity", in the context of this invention, refers to the percentage of sequence identity over the entire length of the amino acid or nucleotide sequence of the gene, protein or polypeptide. The terms "protein" and "polypeptide" are, unless expressly indicated otherwise in the context of this description, interchangeable herein.

In a preferred embodiment an antibody or antigen-binding fragment thereof is provided comprising a Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop), wherein the Fv fragment is a cardiac troponin T binding Fv, fragment comprising SEQ ID NO: 18 and/or SEQ ID NO: 19, or a sequence having a sequence identity of at least 85% with SEQ ID NO: 18 and/or SEQ ID NO: 19, wherein the first recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked by a flexible linker (e.g. GSG) at the N- and C-terminus, positioned directly after amino acid 114 according to Kabat numbering scheme (i.e. between 114 and 115), referring to the amino acid number after which the motif and linkers are inserted, wherein the second recognition motif is a TEV- protease recognition motif (e.g. ENLYFQG) within the elbow/loop region linking the VL and CL domains of the light chain flanked by a flexible linker (e.g. GSG) at the N- and C-terminus, positioned directly after amino acid 110 according to Kabat numbering scheme (i.e. between 110 and 111), referring to the amino acid number after which the motif and linkers are inserted, and wherein the VH and VL domains of the Fab domain comprise a cysteine at a location capable of forming a disulfide bond between the VH and VL (e.g. position 44, 55, 56, or 46 for VH according to Kabat numbering scheme combined with e.g. 100, 108, 106, or 101 , respectively, for VL according to Kabat numbering scheme).

In a preferred embodiment an antibody or antigen-binding fragment thereof is provided comprising a Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop), wherein the Fv fragment is a cardiac troponin T binding Fv, fragment comprising SEQ ID NO: 18 and/or SEQ ID NO: 19 or a sequence having a sequence identity of at least 85% with SEQ ID NO: 18 and/or SEQ ID NO: 19, wherein the first recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked by a flexible linker (e.g. GSG) at the N- and C-terminus, positioned directly after amino acid 114 according to Kabat numbering scheme (i.e. between 114 and 115), referring to the amino acid number after which the motif and linkers are inserted, wherein the second recognition motif is a IgA- protease recognition motif (e.g. PRPPGP) within the elbow/loop region linking the VL and CL domains of the light chain flanked by a flexible linker (e.g. GSG) at the N- and C-terminus positioned directly after amino acid 110 according to Kabat numbering scheme (i.e. between 110 and 111), referring to the amino acid number after which the motif and linkers are inserted, and wherein the VH and VL domains of the Fab domain comprise a cysteine at a location capable of forming a disulfide bond between the VH and VL (e.g. position 44, 55, 56, or 46 for VH according to Kabat numbering scheme combined with e.g. 100, 108, 106, or 101 , respectively, for VL according to Kabat numbering scheme).

In a preferred embodiment an antibody or antigen-binding fragment thereof is provided comprising a Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop), wherein the Fv fragment is a troponin T binding Fv, fragment comprising SEQ ID NO: 18 and/or SEQ ID NO: 19, or a sequence having a sequence identity of at least 85% with SEQ ID NO: 18 and/or SEQ ID NO: 19, wherein the first recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked the same a flexible linker (e.g. GSG or APAP) at both the N- and C-terminus, positioned directly after amino acid 114 according to Kabat numbering scheme (i.e. between 114 and 115), referring to the amino acid number after which the motif and linkers are inserted, wherein the second recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VL and CL domains of the light chain flanked by the same flexible linker (e.g. GSG, AP, APAP, and/or ESGS) on both the N- and C-terminus, positioned directly after one of amino acids 108, 110, 111 , 112 or 114 according to Kabat numbering scheme, referring to the amino acid number after which the motif and linkers are inserted, and wherein the VH and VL domains of the Fab domain comprise a cysteine at a location capable of forming a disulfide bond between the VH and VL (e.g. position 44, 55, 56, or 46 for VH according to Kabat numbering scheme combined with e.g. 100, 108, 106, or 101 , respectively, for VL according to Kabat numbering scheme).

In a preferred embodiment an antibody or antigen-binding fragment thereof is provided comprising a Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop), wherein the Fv fragment is a troponin T binding Fv, fragment comprising SEQ ID NO: 18 and/or SEQ ID NO: 19, or a sequence having a sequence identity of at least 85% with SEQ ID NO: 18 and/or SEQ ID NO: 19, wherein the first recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked the same flexible linker (e.g. GSG or AP) at both the N- and C-terminus, positioned directly after amino acid 114 according to Kabat numbering scheme (i.e. between 114 and 115), referring to the amino acid number after which the motif and linkers are inserted, wherein the second recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VL and CL domains of the light chain flanked by a flexible linker (e.g. GSG, AP, APAP, and/or ESGS) at the N-terminus and flanked by a flexible linker (e.g. GSG, AP, APAP, and/or ESGS) at the C-terminus , positioned directly after one of amino acids 108, 110, 111 , 112 or 114 according to Kabat numbering scheme, referring to the amino acid number after which the motif and linkers are inserted, and wherein the VH and VL domains of the Fab domain comprise a cysteine at a location capable of forming a disulfide bond between the VH and VL (e.g. position 44, 55, 56, or 46 for VH according to Kabat numbering scheme combined with e.g. 100, 108, 106, or 101 , respectively, for VL according to Kabat numbering scheme).

In a preferred embodiment an antibody or antigen-binding fragment thereof is provided comprising a Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop), wherein the Fv fragment is a troponin T binding Fv, fragment comprising SEQ ID NO: 18 and/or SEQ ID NO: 19, or a sequence having a sequence identity of at least 85% with SEQ ID NO: 18 and/or SEQ ID NO: 19, wherein the first recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked by a flexible linker (e.g. GSG) at the N- and C-terminus, positioned directly after amino acid 114 according to Kabat numbering scheme (i.e. between 114 and 115), referring to the amino acid number after which the motif and linkers are inserted, wherein the second recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VL and CL domains of the light chain flanked by a flexible linker (e.g. ESGS) at the N- and C-terminus, positioned directly after amino acid 108 according to Kabat numbering scheme, referring to the amino acid number after which the motif and linkers are inserted thereby deleting amino acids 109 to 112, and wherein the VH and VL domains of the Fab domain comprise a cysteine at a location capable of forming a disulfide bond between the VH and VL (e.g. position 44, 55, 56, or 46 for VH according to Kabat numbering scheme combined with e.g. 100, 108, 106, or 101 , respectively, for VL according to Kabat numbering scheme).

In a preferred embodiment an antibody or antigen-binding fragment thereof is provided comprising a Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop), wherein the Fv fragment is a troponin T binding Fv, fragment comprising SEQ ID NO: 18 and/or SEQ ID NO: 19, or a sequence having a sequence identity of at least 85% with SEQ ID NO: 18 and/or SEQ ID NO: 19, wherein the first recognition motif is a sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked by a flexible linker (e.g. GSG) at the N- and C-terminus, positioned directly after amino acid 114 according to Kabat numbering scheme (i.e. between 114 and 115), referring to the amino acid number after which the motif and linkers are inserted, wherein the second recognition motif is the sortase A recognition motif (e.g. LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VL and CL domains of the light chain without any flexible linker at the N- and C-terminus, positioned directly after one of amino acids 108, 110, 111 or 112 according to Kabat numbering scheme (preferably between 111 and 112), referring to the amino acid number after which the motif and linkers are inserted, and wherein the VH and VL domains of the Fab domain comprise a cysteine at a location capable of forming a disulfide bond between the VH and VL (e.g. position 44, 55, 56, or 46 for VH according to Kabat numbering scheme combined with e.g. 100, 108, 106, or 101 , respectively, for VL according to Kabat numbering scheme).

Preferred embodiments are indicated in Table F.

A chimerisation by grafting the VH/VL domains of mouse or rabbit origins onto the H-IgG constant domain backbone carrying the sortase and/or protease recognition motifs is also possible.

As shown in the Examples a particularly beneficial antibody or antigen binding fragment according to the invention comprises or consists of SEQ ID NO: 20 and SEQ ID NO: 21.

This product is particularly useful for the methods described herein, in particular in combination with a labelled sortase nucleophile (poly-glycine) for the cleavage reaction by the sortase A.

It is noted that all statements made and embodiments disclosed with regards to Fv fragments equally apply to dsFv fragments, which differ by an engineered disulfide bond only.

Definitions for the antibody, antigen-binding fragments, recognition motifs, enzymes, linkers, Fv fragments, nucleophiles etc. are provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

Polynucleotides, vectors, and host cells

According to another aspect of the invention a polynucleotide or set of polynucleotides encoding the heavy and light chain sequence of an antibody or antigen-binding fragment according to the invention is provided.

According to another aspect of the invention a vector or set of vectors comprising the polynucleotide or set of polynucleotides encoding the heavy and light chain sequence of an antibody or antigen-binding fragment according to the invention is provided.

The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule , e.g. double-stranded DNA. In one embodiment, the vector has an exogenous nucleic acid sequence inserted into it. A vector can suitably be used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a suitable host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated. Vectors of the present invention can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to plasmid vectors (e.g. pMA-RQ, plIC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAG), yeast (YAC), or human (HAG)) may be used to accommodate larger inserts. In one particular example, a vector described herein may therefore be a plasmid vector. Such plasmid vectors may be present within a cell. In one embodiment, therefore a cell may be provided which comprises a vector (e.g. a plasmid as described herein) comprising a nucleic acid sequence described herein. A cell may therefore be provided comprising a nucleic acid sequence of the invention.

A vector as defined herein may also be a viral vector. A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV), alphavirus vectors and the like. Typically, but not necessarily, viral vectors are replicationdeficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically or preferentially in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. These viral vectors are referred to herein as “oncolytic viruses”. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.

According to another aspect of the invention a host cell comprising the polynucleotide or set of polynucleotides, or the vector or set of vectors of the invention is provided.

Such a host cell may be in vitro and may be in culture.

Suitable host cells are described above.

The host cell can be treated so as to cause or allow expression of the antibody or antigenbinding fragment thereof of the invention from the polynucleotide(s), e.g. by culturing host cells under conditions for expression of the encoding nucleic acid. The purification of the expressed product may be achieved by methods known to one of skill in the art. Thus, the polynucleotide(s) of the invention, including vector nucleic acids that comprise the polynucleotide sequences that encode the polypeptide(s) capable of forming the antibodies or antigen-binding fragments thereof of the invention, may be present, either transiently or stably, in an isolated host cell. The host cell is typically part of a clonal population of host cells. As used herein, reference to a host cell also encompasses a clonal population of said cell. A clonal population is one that has been grown from a single parent host cell. The host cell can be from any suitable organism. A suitable host cell is selected from a: mammalian, fungal, plant, insect or bacterial cell. Suitably, the host cell is a mammalian cell selected from a HEK, NSO, Sp2/0, PER.C6 or CHO cell.

According to another aspect of the invention a method of producing an antibody or antigenbinding fragment according to the invention is provided, said method comprising culturing the host cell according to the invention and isolating said antibody or antigen-binding fragment.

It is noted that all statements made and embodiments disclosed with regards to Fv fragments equally apply to dsFv fragments, which differ by an engineered disulfide bond only.

Definitions for the antibody, antigen-binding fragments, recognition motifs, enzymes, linkers, Fv fragments, nucleophiles etc. are provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc described herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

Uses

The first aspect of the invention refers to “Antibody or antigen-binding fragment as precursor for Fv production”.

In the same context, according to another aspect of the invention, the antibody or antigenbinding fragment according to the invention can be used as precursor for generating a Fv fragment.

By using the antibody or antigen-binding fragment according to the invention as a precursor for generating a Fv fragment it is possible to obtain an Fv fragment in high yields, with a high protein stability, with correct folding, and a superior performance. In particular, a stable dsFv fragment can be yield with superior characteristics to scFv or ds-scFvs.

In one embodiment the use comprises contacting the precursor antibody or antigen-binding fragment according to the invention with (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment according to the invention; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is cleaved by a transpeptidase, said contacting is optionally conducted in the presence of a transpeptidase nucleophile which optionally comprises a tag.

In one embodiment the use comprises contacting the precursor antibody or antigen-binding fragment according to the invention with (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment according to the invention; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is cleaved by a transpeptidase, said contacting is conducted in the presence of a transpeptidase nucleophile which comprises a tag.

In one embodiment the use comprises contacting the precursor antibody or antigen-binding fragment according to the invention with (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment according to the invention; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is cleaved by a transpeptidase, said contacting is conducted in the presence of a transpeptidase nucleophile.

In one embodiment the use comprises contacting the precursor antibody or antigen-binding fragment according to the invention with (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment according to the invention; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is cleaved by a transpeptidase.

As the skilled artisan will readily appreciate, that the incubation of the antibody or antigenbinding fragment with the corresponding enzyme(s) will be performed using conditions appropriate to achieve the desired result, i.e. the desired cleavage. Such appropriate conditions are well-known to a person skilled in the art. The transpeptidase activity of sortase can be taken advantage of to produce fusion proteins, in particular to tag the polypeptide since the C-terminal portion of the cleaved polypeptide is transferred onto an N-terminal portion of another molecule.

The use of the transpeptidase nucleophile is optional. The reaction can be performed without the nucleophile at all. This would still lead to the cleavage of the antibody or antigen-binding fragment.

In another embodiment a nucleophile is added after a certain amount of time after the transpeptidase(s) have been brought into contact with the antibody or antigen-binding fragment.

In another embodiment the nucleophile is brought in contact with the antibody or antigenbinding fragment from the beginning, i.e. at the same time as the transpeptidase(s). In case two transpeptidases are used, due to two different recognition motifs, the nucleophile can either be added together with the first transpeptidase, together with the second peptidase, or, in case both transpeptidases are added at the same time, the nucleophile can be added with both transpeptidases at the same time.

In one embodiment the transpeptidase nucleophile has N- terminally an oligoglycine and consists of at least two glycine residues as its ligation motif, i.e., the transpeptidase ligation motif consists of at least two consecutive glycine residues.

In one embodiment the use according to the present application is practiced using a transpeptidase nucleophile comprising a tag of interest having the formula (Gly)n-tag, wherein n is at least 2. The transpeptidase ligation motif (Gly)n may be longer and may consist of 3, 4, 5, or 6 glycine residues, i.e. n in (Gly)n-tag is 3, 4, 5, or 6.

In nature the sortase ligation motif on the peptidoglycan is a pentaglycine motif. It has been shown that a triglycine and even a diglycine motif on the N-terminus is sufficient to support the SrtA reaction (Clancy, K.W., et al, Peptide Science 94 (2010) 385-396). The reaction proceeds through a thioester acyl-enzyme intermediate, which is resolved by the attack of an amine nucleophile from the oligoglycine, covalently linking peptidoglycan to a protein substrate and regenerating SrtA.

In one embodiment the transpeptidase nucleophile comprises at least GG (glycine glycine).

In one embodiment the transpeptidase is a sortase or sortase-type transpeptidase.

In nature the sortase ligation motif on the peptidoglycan is a pentaglycine motif. It has been shown that a triglycine and even a diglycine motif on the N-terminus is sufficient to support the SrtA reaction (Clancy, K.W., et al, Peptide Science 94 (2010) 385-396). The reaction proceeds through a thioester acyl-enzyme intermediate, which is resolved by the attack of an amine nucleophile from the oligoglycine, covalently linking peptidoglycan to a protein substrate and regenerating SrtA.

The "tag of interest" or simply the "tag" or the “label” (terms are used interchangeable herein) that can be comprised in the nucleophile can be a partner of a binding pair, a functional group, a therapeutic agent (drug), a cytotoxic agent (e.g. a toxin such as doxorubicin or pertussis toxin) and a label (e.g. a fluorophore such as a fluorescent dye like fluorescein or rhodamine, a chemiluminescent or an electro chemiluminescent label (e.g. ruthenium or iridium), a radioactive label, a metal chelate complex for imaging or radiotherapeutic purposes, an enzyme or a fluorescent protein like GFP). The tag can be or comprise an enzyme, an enzyme substrate, a chromophore, a quencher, a radiolabel, a biotin, a metal, a ruthenium label or a electrochemiluminescent moiety, or a nucleic acid.

In one embodiment the tag is biotin.

In one embodiment the use further comprises isolating the Fv fragment. A person skilled in the art is familiar with methods to isolate the Fv fragment from the remaining components in the reaction mixture.

It is noted that all statements made and embodiments disclosed with regards to Fv fragments equally apply to dsFv fragments, which differ by an engineered disulfide bond only.

Definitions for the antibody, antigen-binding fragments, recognition motifs, enzymes, linkers, Fv fragments, nucleophiles etc. are provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc described herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

Methods

The first aspect of the invention refers to “Antibody or antigen-binding fragment as precursor for Fv production”. The second aspect of the invention refers to its uses.

In the same context, according to another aspect of the invention a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, said contacting is optionally conducted in the presence of a nucleophile for said transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

According to another aspect of the invention a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, said contacting is conducted in the presence of a nucleophile for said transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

According to another aspect of the invention a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention;, and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for at least one of the transpeptidases; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for at least one of the transpeptidases; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for at least one of the transpeptidases; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for at least one of the transpeptidases; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for at least one of the transpeptidases; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first and the second recognition motif are sortase A recognition motifs, and comprise or consist of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first and the second recognition motif are sortase A recognition motifs, and comprise or consist of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motifs are sortase A recognition motifs and comprise or consist of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, and wherein the first recognition motif is flanked by an N-terminal linker sequence and a C-terminal linker sequence(e.g. GSG).

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first and the second recognition motif are sortase A recognition motifs, and comprise or consist of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motifs are sortase A recognition motifs and comprise or consist of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, wherein the first recognition motif is flanked by an N-terminal linker sequence and a C-terminal linker sequence (e.g. GSG) and wherein the first recognition motif with the linkers is positioned directly after amino acid 114 of the heavy chain according to Kabat numbering scheme.

As described elsewhere herein (section “Antibody or antigen-binding fragment as precursor for Fv production”) other combinations of linkers, recognition motifs, cleavage sites within the heavy and/or light chains can be used for the methods disclosed herein as well.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, and wherein the first recognition motif is flanked by an N-terminal linker sequence and a C-terminal linker sequence(e.g. GSG).

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, and wherein the first recognition motif is flanked by an N-terminal linker sequence and a C-terminal linker sequence(e.g. GSG), and wherein the first recognition motif with the linkers is positioned directly after amino acid 114 of the heavy chain according to Kabat numbering scheme.

As described elsewhere herein (section “Antibody or antigen-binding fragment as precursors for FV fragment production”) other combinations of linkers, recognition motifs, cleavage sites within the heavy and/or light chains can be used for the methods disclosed herein as well.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific IgA-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific IgA-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, and wherein the second recognition motif is a IgA-protease recognition motif, and comprises or consists of PX, wherein X is S, T, A, V, or G , preferably wherein the IgA-protease recognition motif comprises or consists of X1X2PPX3P, wherein Xi is P or S, X2 is R or T, X3 is T, S or A, more preferably wherein the IgA protease recognition motif comprises or consists of PRPPXP (SEQ ID NO: 2), with X = S, T, A, V, G.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific IgA-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, wherein the second recognition motif is a IgA-protease recognition motif, and comprises or consists of PX, wherein X is S, T, A, V, or G , preferably wherein the IgA-protease recognition motif comprises or consists of X1X2PPX3P , wherein Xi is P or S, X2 is R or T, X3 is T, S or A, more preferably wherein the IgA protease recognition motif comprises or consists of PRPPXP (SEQ ID NO: 2), with X = S, T, A, V, G, and wherein the first recognition motif and second recognition motif are flanked by an N-terminal linker sequence and a C-terminal linker (e.g. GSG).

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific IgA-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, wherein the second recognition motif is a IgA-protease recognition motif, and comprises or consists of PX, wherein X is S, T, A, V, or G , preferably wherein the IgA-protease recognition motif comprises or consists of X1X2PPX3P, wherein Xi is P or S, X2 is R or T, X3 is T, S or A, more preferably wherein the IgA protease recognition motif comprises or consists of PRPPXP (SEQ ID NO: 2), with X = S, T, A, V, G, and wherein the first recognition motif and second recognition motif are flanked by an N-terminal linker sequence and a C-terminal linker sequence (e.g. GSG), and wherein the first recognition motif with the linkers is positioned directly after amino acid 114 of the heavy chain according to Kabat numbering scheme and the second recognition motif with the linkers is positioned directly after amino acid 110 of the light chain according to Kabat numbering scheme.

As described elsewhere herein (section “Antibody or antigen-binding fragment as precursors for Fv production”) other combinations of linkers, recognition motifs, cleavage sites within the heavy and/or light chains can be used for the methods disclosed herein as well.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific TEV-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific TEV-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, and wherein the second recognition motif is a TEV-protease recognition motif, and comprises or consists of EXXYXQX, wherein X is any amino acid, preferably wherein the TEV-protease recognition motif comprises or consists of ENLYFQX (SEQ ID NO: 1), wherein X is S, G, A, M, C or H.

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific TEV-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, wherein the second recognition motif is a TEV-protease recognition motif, and comprises or consists of EXXYXQX, wherein X is any amino acid, preferably wherein the TEV-protease recognition motif comprises or consists of ENLYFQX(SEQ ID NO: 1), wherein X is S, G, A, M, C or H, and wherein the first recognition motif is flanked by an N-terminal linker sequence and a C-terminal linker sequence (e.g. GSG).

In one embodiment a method for producing an Fv fragment capable of binding to an antigen of interest is provided comprising c) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific TEV-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; said contacting is conducted in the presence of a nucleophile for the transpeptidase; and d) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or the antigen-binding fragment according to the invention, wherein the first recognition motif is a sortase A recognition motif, and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue, wherein the second recognition motif is a TEV-protease recognition motif, and comprises or consists of EXXYXQX, wherein X is any amino acid, preferably wherein the TEV-protease recognition motif comprises or consists of ENLYFQX(SEQ ID NO: 1), wherein X is S, G, A, M, C or H, and wherein the first recognition motif is flanked by an N-terminal linker sequence and a C-terminal linker sequence (e.g. GSG), and wherein the first recognition motif with the linkers is positioned directly after amino acid 114 of the heavy chain according to Kabat numbering scheme and the second recognition motif with the linkers is positioned directly after amino acid 110 of the light chain according to Kabat numbering scheme.

As described elsewhere herein (section “Antibody or antigen-binding fragment as precursors for Fv production”) other combinations of linkers, recognition motifs, cleavage sites within the heavy and/or light chains can be used for the methods disclosed herein as well.

According to another aspect of the present invention, a method for producing a tagged Fv capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

Thus, the method gives an opportunity for the labelling of the Fv fragment concomitantly in the process.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention. In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) a sitespecific protease capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein at least the first recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention. In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein the first recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein the first and second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for at least one of the transpeptidases, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein the first and second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific IgA-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein the first recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention.

In one embodiment a method for producing a tagged Fv fragment capable of binding to an antigen of interest is provided comprising a) contacting the antibody or antigen-binding fragment of the invention with (i) the site-specific transpeptidase sortase A capable of cleaving a peptide bond within the first recognition motif and (ii) the site-specific TEV-protease capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of the invention; wherein the first recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to the invention. All the variations with regards to the recognition motifs for the transpeptidases and proteases, their linkers and their positions within the heavy and light chains described for the aspect of the invention to provide a method for producing an Fv fragment capable of binding to an antigen of interest equally apply to the aspect of the invention to provide a method for producing a tagged Fv fragment capable of binding to an antigen of interest.

As the skilled artisan will readily appreciate, such incubation will be performed using buffer conditions appropriate to achieve the desired result. Such appropriate conditions are well- known from the literature. For example, standard sortase ligation buffers e.g. comprise MgCI2.

In one embodiment the antibody or antigen-binding fragment is an antibody or antigen-binding fragment of the invention, wherein the Fv fragment specifically recognizes cardiac T roponin T.

According to another aspect of the invention a method for detecting and/or quantifying cardiac Troponin T in a sample is provided comprising contacting the sample with the Fv fragment of the invention under conditions allowing for binding of the Fv fragment to troponin T comprised in the sample; and detecting and/or quantifying (cardiac) troponin T in the sample by detecting the complex between the Fv fragment and troponin T.

In one embodiment the (cardiac) troponin T is detected and/or quantified by detecting the captured complex between the Fv fragment and (cardiac) troponin T using a second labelled detection antibody.

Examples for ways to detect (and/or quantify) the captured complex between the Fv fragment and (cardiac) troponin T are methods which employ capture on streptavidin beads and electrochemoluminescence detection system (such as immunodetection using Roche Elecsys® analyzers). The Fv may be labelled with biotin and a second antibody may be labelled with a luminophore such as a ruthenium ester (e.g. [Ru(bpy)s]2+, tris(2,2'- bipyridine)ruthenium(ll) (Ru2+)). In the presence of a target (e.g Troponin T), a sandwich immune complex forms with the analyte, the detection and the capture antibodies. The immune complex is immobilized on the working electrode via magnetic streptavidin-coated beads. A coreactant (e.g. oxalate ion (6204(2-)), peroxydisulfate (persulfate, S20s(2-), tri-n- propylamine (TPrA) and other amine-related derivatives, and hydrogen peroxide (H2O2)) is oxidized at the electrode, generating the radical cation, which rapidly deprotonates, forming the radical. The radical and radical cation react with the luminophore (e.g. the ruthenium ester), which emits photons, which are detected.

It is noted that all statements made and embodiments disclosed with regards to Fv fragments equally apply to dsFv fragments, which differ by an engineered disulfide bond only. Definitions for the antibody, antigen-binding fragments, recognition motifs, enzymes, linkers, Fv fragments, nucleophiles etc. are provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc described herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

(ds)Fv fragment

According to another aspect of the invention an (ds)Fv fragment capable of binding to an antigen of interest obtained by a method for producing an (ds)Fv fragment capable of binding to an antigen of interest or obtained by a method for producing a tagged (ds)Fv capable of binding to an antigen of interest is provided.

In one embodiment the (ds)Fv fragment is capable of binding a Troponin, in particular cardiac Troponin T.

In one embodiment the the VH and/or VL sequence of the (ds)Fv fragment comprise one or more amino acids at the C-terminal ends derived from the cleavable insert domain.

An (ds)Fv fragment produced by the method described herein will typically contain some residual amino acid residues at the C-terminal end. These residual residues derive from the recognition motif and, in case a linker was used, from the N-terminal linker. An (ds)Fv fragment produced by the method according to the invention can be identified as such when comprising these residual amino acid residues. For example, in case of an N-terminal linker GSG and the recognition motif LPETG (SEQ ID NO: 35) for sortase A with a C-terminal linker GSG. Since the sortase A cleaves between T and G the residual amino acids would be GSGLPET.

(ds)Fv fragments produced by the method according to the invention have a higher stability compared to (ds)Fv fragments produced differently. There are several reasons for this:

• ensured correct folding of the VH/VL within their ‘native’ IgG context, stabilized by the inherent covalent and non-covalent interactions of the heavy and light chain constant domains, before the Fv fragment is enzymatically extracted out of the IgG

• in turn, reduced risk of misfoldings or loss in affinities

• no further refolding steps/conditions required

• engineered disulfide inter-chain bond between the VH and VL

A person skilled in the art would be able to test an (ds)Fv fragment for its stability. For example, this could be tested by investigating the monomeric status of the protein via HPLC following room temperature or cooled temperature storage for a specified period of time. Alternatively, the same could be done following an appropriate temperature stress model following Arrhenius principles to extrapolate to other temperatures. Another way would be to use thermal shift assays (e.g. differential scanning fluorimetry (DSF)) or dynamic light scattering technique or any protein-tailored activity assay to assess the stability of the protein.

In one embodiment the VH sequence of the (ds)Fv fragment comprises or consists of SEQ ID NO: 22. In another embodiment the VH sequence of the (ds)Fv fragment is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.

In one embodiment wherein the VL sequence of the (ds)Fv fragment comprises or consists of SEQ ID NO: 23. In another embodiment the VL sequence of the (ds)Fv fragment is at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23.

Suitably, as already described elsewhere herein, the VH and VL domains (polypeptides of the Fab domain) of which the Fv fragment derives, can be engineered to allow disulfide interchain bond formation, resulting in a dsFv fragment (disulfide Fv fragment). For example, a cysteine residue can be positioned in each of the VH and VL domains so as to allow their direct interaction and permit disulfide bond formation between the two cysteines. This adaptation would allow for production of dsFv.

Thus, in one embodiment the VH and VL domains have been engineered to permit inter-chain disulfide bond formation between the VH and VL domains. Suitably, this is by mutating the amino acids at two structurally conserved opposite framework sites of the VH and VL.

In one embodiment, the antibody VH and VL domains each comprise a cysteine residue at a location capable of forming, or so as to facilitate formation of, a disulfide bond between the VH and VL.

Suitable sites in the VH and VL domains for introduction of cysteine residues that can facilitate formation of a disulfide bond are known. Criteria to observe when selecting sites include: (a) That the distance between VH and VL and the special orientation of the residues shall be close enough and directed toward each other to allow proper disulfide linkage without putting strain on the heterodimeric Fv;

(b) The positions need to tolerate the exchange of residues for cysteines without disturbing the folding, structure, and stability of VH or VL; and (c) The introduced cysteines should be distant from CDR regions of VH and VL to avoid interference with antigen binding.

Introduction of cysteines at VH44 and VL100 are the most widely used and established from literature, but other examples are also reported in the literature. For example: VH55+VL108; VH56+VL106; VH L46C + VL D101C (e.g. see Brinkmann, II. Disulfide-stabilized Fv fragments. In Kontermann, R., Dubel, S. (eds). Antibody Engineering. Berlin Heidelberg: Springer-Verlag, 2:181 - 189, 2010).

In one embodiment the VH of the Fab domain comprises a cysteine at position 44 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position

100 according to Kabat numbering scheme.

In one embodiment the VH of the Fab domain comprises a cysteine at position 55 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position 108 according to Kabat numbering scheme.

In one embodiment the VH of the Fab domain comprises a cysteine at position 56 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position 106 according to Kabat numbering scheme.

In one embodiment the VH of the Fab domain comprises a cysteine at position 46 according to Kabat numbering scheme and the VL of the Fab domain comprises a cysteine at position

101 according to Kabat numbering scheme.

Suitably, in each of these embodiments, the cysteine in the VH of the Fab domain and the cysteine in the VL of the Fab domain are capable of forming a disulfide bond.

As described above, with the method according to the invention an opportunity is provided for labelling of the (ds)Fv fragment concomitantly in the process by using a labelled/tagged transpeptidase nucleophile.

In one embodiment the (ds)Fv fragment is or can be used as a capture antibody, for example in a sandwich ELISA to bind a desired analyte.

In another embodiment the (ds)Fv fragment is or can be used as a detection antibody, for example in a sandwich ELISA as the detection reagent.

In one embodiment the (ds)Fv fragment according to the invention provides a superior Elecsys Signal Dynamics Performance (Example 3) compared to IgG fragments (Fab, Fab’, and F(ab’)2) with the same target. In one embodiment the (ds)Fv fragment according to the invention provides a superior stability compared to IgG fragments (Fab, Fab’, and F(ab’)2) with the same target (Example 3).

It is noted that all statements made and embodiments disclosed with regards to Fv fragments equally apply to dsFv fragments, which differ by an engineered disulfide bond only.

Definitions for the antibody, antigen-binding fragments, recognition motifs, enzymes, linkers, Fv fragments, nucleophiles etc. are provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc described herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

Kits

According to another aspect of the invention a diagnostic kit comprising an antibody or antigen-binding fragment of the invention is provided.

According to another aspect of the invention an immunoassay kit comprising an antibody or antigen-binding fragment of the invention is provided.

In one embodiment the diagnostic kit or the immunoassay kit further comprises (i) a sitespecific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and/or (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif and optionally a nucleophile, optionally a tag and/or optionally instructions for use. Suitably, the individual components are housed in separate containers, e.g. tubes.

Said kit optionally comprises instructions regarding the use of the contained components, for example, incubation conditions for the corresponding enzymes, recommended buffers, etc. The components in the kit are preferably for simultaneous use.

According to another aspect of the invention a diagnostic kit or the immunoassay kit comprising a (ds)Fv fragment of the invention is provided.

In one embodiment, the (ds)Fv fragment of the invention is labelled.

In one embodiment the diagnostic kit or the immunoassay kit may comprise a (ds)Fv fragment and a second antibody, wherein the second antibody does not compete for binding to the same target as the (ds)Fv fragment. Preferably, the second antibody is an antibody that can be used to form a sandwich with the (ds)Fv fragment, e.g. in a sandwich ELISA. A skilled person would be able to properly select the second antibody. In one embodiment the antibody or antigen-binding fragment or the (ds)Fv fragment specifically binds (cardial) troponin T.

It is noted that all statements made and embodiments disclosed with regards to Fv fragments equally apply to dsFv fragments, which differ by an engineered disulfide bond only.

Definitions for the antibody, antigen-binding fragments, recognition motifs, enzymes, linkers, Fv fragments, nucleophiles etc. are provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc described herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

Sequences

TEV-protease recognition motif

EXXYXQX

SEQ ID NO: 1: TEV-protease recognition motif (specific)

ENLYFQX

IgA-protease recognition motif X1X2PPX3P

SEQ ID NO: 2: IgA-protease recognition motif (specific) PRPPXP

SEQ ID NO: 3: 3C protease recognition motif

LFQGP

SEQ ID NO: 4: 3C protease recognition motif (specific)

LEVLFQGP

Sortase A recognition motif

LPX1TX2

SEQ ID NO: 5: Sortase A recognition motif (specific)

LPX1TG

Sortase B recognition motif NPX1X2X3

Sortase C recognition motif

X1X2X3TG

SEQ ID NO: 6: Sortase D recognition motif LPXTA

SEQ ID NO: 7: Sortase E recognition motif

LAXTG Further sortase A recognition motif

LPXTX2

Exosortase A recognition motif PEP

Archaesortase recognition motif

PGF

N-terminal linker sequence 1

GSG

N-terminal linker sequence 2

AP

SEQ ID NO: 8 N-terminal linker sequence 3 APAP

SEQ ID NO: 9 N-terminal linker sequence 4 ESGS

SEQ ID NO: 10 N-terminal linker sequence 5 GGGS

SEQ ID NO: 11 N-terminal linker sequence 6 GGGGS

SEQ ID NO: 12 N-terminal linker sequence 7 GSGGSG

C-terminal linker sequence 1

GSG

SEQ ID NO: 13: C-terminal linker sequence 1 GSGGSG

N-terminal linker sequence 2

AP

SEQ ID NO: 14 N-terminal linker sequence 3 APAP

SEQ ID NO: 15 N-terminal linker sequence 4 ESGS

SEQ ID NO: 16 N-terminal linker sequence 5 GGGS

SEQ ID NO: 17 N-terminal linker sequence 6 GGGGS

SEQ ID NO: 18: VH sequence of the Fv fragment binding Troponin T

QIQLVQSGPELKKPGETVKISCKASGYTFTDFSMHWVKQAPGKCLKWMGWINTETGEPTY

ADDFKGRFAFSLETSATTAYLQINNLKNEDTATYFCVRSHWMDYWGQGTSVTVSSA SEQ ID NO: 19: VL sequence of the Fv fragment binding Troponin T

DWMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSG

VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHLYTFGCGTKLEIKRAD

SEQ ID NO: 20: Heavy chain sequence of the Fab binding Troponin T

QIQLVQSGPELKKPGETVKISCKASGYTFTDFSMHWVKQAPGKCLKWMGWINTETGEPTY

ADDFKGRFAFSLETSATTAYLQINNLKNEDTATYFCVRSHWMDYWGQGTSVTVSSAGSGL PETGGSGSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

SEQ ID NO: 21: Light chain sequence of the Fab binding Troponin T

DWMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSG

VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHLYTFGCGTKLEIKRADGSGPRPPG PGSGAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Transpeptidase nucleophile

GG

SEQ ID NO: 22: VH sequence of Fv fragment with linker and recognition motif residues remaining after cleavage (underlined)

QIQLVQSGPELKKPGETVKISCKASGYTFTDFSMHWVKQAPGKCLKWMGWINTETGEPTY ADDFKGRFAFSLETSATTAYLQINNLKNEDTATYFCVRSHWMDYWGQGTSVTVSSAGSGL PET

SEQ ID NO: 23: VL sequence of Fv fragment with linker and recognition motif residues remaining after cleavage (underlined)

DWMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSG

VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHLYTFGCGTKLEIKRADGSGPRPP

SEQ ID NO: 24: human cardial Troponin T (cTnT)

MSDIEEVVEEYEEEEQEEAAVEEEEDWREDEDEQEEAAEEDAEAEAETEETRAEEDEEEE

EAKEAEDGPMEESKPKPRSFMPNLVPPKIPDGERVDFDDIHRKRMEKDLNELQALIEAHFE

NRKKEEEELVSLKDRIERRRAERAEQQRIRNEREKERQNRLAEERARREEEENRRKAEDE ARKKKALSNMMHFGGYIQKQAQTERKSGKRQTEREKKKKILAERRKVLAIDHLNEDQLREK AKELWQSIYNLEAEKFDLQEKFKQQKYEINVLRNRINDNQKVSKTRGKAKVTGRWK

SEQ ID NO: 25: heavy chain of Example 1

QIQLVQSGPELKKPGETVKISCKASGYTFTDFSMHWVKQAPGKCLKWMGWINTETGEPTY

ADDFKGRFAFSLETSATTAYLQINNLKNEDTATYFCVRSHWMDYWGQGTSVTVSSAGSGL

PETGGSGSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

ELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSHHHHHH

SEQ ID NO: 26: light chain of Example 1

DWMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSG

VPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHLYTFGCGTKLEIKRADGSGPRPPG PGSGAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSHHHHHH

SEQ ID NO: 27: insertion sequence/ cleavable insert domain sortase A - 1 GSGLPETGGSG SEQ ID NO: 28: insertion sequence/ cleavable insert domain sortase A - 2 APAPLPETGAPAP

SEQ ID NO: 29: insertion sequence/ cleavable insert domain sortase A - 3

APLPETGAP

SEQ ID NO: 30: insertion sequence/ cleavable insert domain sortase A - 4 ESGSLPETGESGS

SEQ ID NO: 31: insertion sequence/ cleavable insert domain sortase A - 5 GSGLPETGAPAP

SEQ ID NO: 32: insertion sequence/ cleavable insert domain sortase A - 6 GSGLPETGGSGGSG

SEQ ID NO: 33: TEV-protease recognition motif (from table 1)

ENLYFQG

SEQ ID NO: 34: TEV-protease recognition motif (from table 1)

ENLYFQS

SEQ ID NO: 35: sortase A recognition motif (Staphylococcus aureus)

LPETG

SEQ ID NO: 36: cleavable insert domain

GSGPRPPGPGSG

SEQ ID NO: 37: cleavable insert domain

GSGENLYFQGGSG

SEQ ID NO: 38: IgA- protease recognition motif

PRPPGP

SEQ ID NO: 39: Motif insertion site (Homo sapiens)

A^STKGP

SEQ ID NO: 40: Motif insertion site (Mus musculus)

A^KTTPP

SEQ ID NO: 41: Motif insertion site (Oryctolagus cuniculus)

G^QPKAP

SEQ ID NO: 42: Motif insertion site (Ovis Aries)

A^STTPP

SEQ ID NO: 43: Motif insertion site (Homo sapiens)

T APS

SEQ ID NO: 44: Motif insertion site (Mus musculus)

AD APT

SEQ ID NO: 45: Motif insertion site (Oryctolagus cuniculus) DP^VAPT

SEQ ID NO: 46: Motif insertion site (Ovis Aries)

P SAPS

SEQ ID NO: 47: Peptide linker ((GGGS)4)

(GGGS)4 incorporated as GGGSGGGSGGGSGGGS

SEQ ID NO: 48: Thrombin recognition motif (from table 1)

LVPR^AGS

SEQ ID NO: 49: Thrombin recognition motif (from table 1)

L(A,F,G,I,T,V or M)V(F,G,I,L,T,W or A)PR^AGS

LX1VX2PR^AGS

X1 can be A, F, G, I, T, V or M

X2 can be F, G, I, L, T, W or A

SEQ ID NO: 50: Enteropeptidase (Enterokinase) recognition motif (from table 1) DDDDK^AX

X can be any amino acid

SEQ ID NO: 51: Caspase 2 recognition motif (from table 1)

DVAD^AX

X can be any amino acid

SEQ ID NO: 52: Genenase I recognition motif (from table 1)

PGAAH^AY

SEQ ID NO: 53: Tobacco vein mottling virus protease recognition motif (from table 1) ETVRFQG^A S

SEQ ID NO: 54: Exosortase C recognition motif (from table 3)

VPDSG

SEQ ID NO: 55: Exosortase D recognition motif (from table 3)

VPLPA

SEQ ID NO: 56: Exosortase E recognition motif (from table 3)

VPEID

SEQ ID NO: 57: insertion sequence/ cleavable insert domain 3C protease GSGLEVLFQGPGSG

Description of the Figures

Figure 1 : Schematic overview of the precursor IgG design and the dsFv production procedure.

Figure 2: Analytics overview of the dsFv precursor and production procedure progress. A: HPLC analysis (GFC) of dsFv precursor. B: HPLC (GFC) analysis showing the reaction products following overnight incubation of the dsFv-IgG-precursor with sortase and IgA protease enzymes.

C: HPLC analysis (GFC) of the final purified product (dsFv) following the two-step purification on His-column (-Ni) and gel filtration chromatography.

D: SDS-gel analysis of reduced samples of the dsFv-IgG-precursor (lane 2), following the overnight reaction with sortase and IgA-protease (lane 3), analysis of a white precipitate formed after the overnight incubation (10x concentrated (lane 4) and 1x (lane 5)), the flow- through of the -Ni first-step-purification (lane 6), the Ni column elution (lane 7), the -Ni flow- through concentrated (lane 8), the IgA-protease enzyme (lane 9), the sortase enzyme (lane 10), the dsFv product after the second GFC purification step (lane 11 and 12).

Figure 3: Analytics overview of the alternative Fv formats.

A: HPLC analysis (GFC) of scFv product of the clone 11-7 showing an aggregates percent of -70%.

B: HPLC analysis (GFC) of sc-dsFv product of the clone 11-7 showing an aggregates percent of -58%.

C: HPLC analysis (GFC) of IgG with Sortase and IgA-Protease motifs at the VH/CH1 and VL/CL; respectively (dsFv precursor), showing an aggregates percent of -3%.

D: HPLC analysis (GFC) of IgG with Sortase and TEV-Protease motifs at the VH/CH1 and VL/CL; respectively (dsFv precursor), showing an aggregates percent of -3%.

Figure 4: Analytics overview of dsFv-precursors of the selected clones collection.

A: HPLC analysis (GFC) of dsFv-precursor of the Clone A (anti-PTH D1.1) showing an aggregates percent of -2%.

B: HPLC analysis (GFC) of dsFv-precursor of the Clone D (anti-HCG 1F7- 9) showing an aggregates percent of -8%.

C: HPLC analysis (GFC) of dsFv-precursor of the Clone E (anti-IgM DM3A3)showing an aggregates percent of -7%.

D: HPLC analysis (GFC) of dsFv-precursor of the Clone F (anti-PSA 30) showing an aggregates percent of -7%. E: HPLC analysis (GFC) of dsFv-precursor of the Clone G (anti- Tau217 16F12) showing an aggregates percent of -4%.

Figure 5: Analytics overview of dsFv products of the selected clones collection.

A: HPLC analysis (GFC) of dsFv product of the Clone A (anti-PTH D1.1).

B: HPLC analysis (GFC) of dsFv product of the Clone E (anti-IgM DM3A3).

C: HPLC analysis (GFC) of dsFv product of the Clone G (antiTau217 16F12).

D: HPLC analysis (GFC) of dsFv-precursor of the Clone D (anti-HCG 1F7-9). The invention will now be further described with reference to the following Examples and Figures.

Examples

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany). Description of the basic/standard mammalian expression plasmid

For the expression of a desired gene/protein (e.g. full length antibody heavy chain, full length antibody light chain, or an Fv-fragment containing an oligoglycine at its N- terminus) a transcription unit comprising the following functional elements was used:

- the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A,

- a human heavy chain immunoglobulin 5 '-untranslated region (5 'UTR),

- a murine immunoglobulin heavy chain signal sequence,

- a gene/protein to be expressed (e.g. full length antibody heavy chain), and

- the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene to be expressed the basic/standard mammalian expression plasmid contains

- an origin of replication from the vector plIC 18 which allows replication of this plasmid in E. coli, and - a beta-lactamase gene which confers ampicillin resistance in E. coli.

Protein determination

The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide.

Example 1 : Overview of the precursor laG design and the dsFv production procedure

The precursor recombinant IgG (from the 11-7 anti-TnT clone).

The anti-troponin T (TnT) clone is a mouse clone derived from a mouse immunization). Here the original mouse VH/VL domains are cloned onto H-IgG backbone containing the extra enzyme motifs (all constant domains are human; Figure 1). It is designed to contain a sortase enzyme recognition motif (LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked by a flexible linker (GSGLPETGGSG (SEQ ID NO: 27); shown by hatched arrow; insertion after amino acid number 114 (Kabat Numbering) / 118 (Ell Numbering)); alternatively, a protease recognition motif could be used instead of the sortase enzyme recognition motif.

The elbow/loop region linking the VL and CL domains of the light chain is designed to contain a protease enzyme (e.g. TEV- or IgA-protease) recognition motif flanked by a flexible linker (e.g. GSGENLYFQGGSG (SEQ ID NO: 37) or GSGPRPPGPGSG (SEQ ID NO: 36); shown by sprinkled arrow; insertion after amino acid number 110 (Kabat Numbering) / 110 (Ell Numbering)); alternatively, a sortase enzyme recognition motif could be used instead of the protease enzyme recognition motif.

A 6-Histidine tag can be added at the C-termini of the heavy and light chains (shown by sprinkled rectangular), e.g. to facilitate purification. Other affinity tags than the 6-His may be used at the chains’ C-termini and would provide the same benefit in any purification steps.

Additionally, a disulfide bond between the VH and VL can be engineered by introducing two cysteines residues; one in each chain (e.g. at position 44 in the VH chain and position 100 in the VL chain (using Kabat numbering scheme)).

Table A outlines the sequences at the insertion sites used within the human IgG (H-IgG) backbone as well as the homologous sequences in other species. A chimerisation by grafting the VH/VL domains of mouse or rabbit origins onto the H-IgG constant domain backbone carrying the sortase and/or protease recognition motifs is also possible.

Table A (above) outlines the sequences at the insertion sites used within the H-IgG backbone as well as the homologous sequences in other species; * represents the site where recognition motif is inserted. Single amino acid code is used.

The enzyme recognition motifs are inserted at the indicated sites flanked by GSG flexible linker sequence on each end to enhance the enzyme accessibility.

Following the IgG purification, a one-step one-pot enzymatic reaction proceeds by mixing the IgG (dsFv-precursor; at a concentration of 9 mg/ml) with the sortase enzyme (1.44 units per mg IgG), the protease enzyme (10 pg per mg IgG in case of IgA protease and following manufacturer’s instructions in case of TEV-protease), excess of a polyglycine-containing-label (this represents the nucleophile with a tag; 50-fold molar excess to the IgG) in Tris buffer in the presence of 5 mM Calcium Chloride. The reaction mixture is incubated overnight at 37°C.

The sortase enzyme catalyses the ligation between the LPETG (SEQ ID NO: 35) motif and the poly-glycine label; cleaving off the constant domains of the heavy chain. The protease cuts through its respective motif; cleaving off the constant domains of the light chain. This yields a labelled dsFv from the IgG precursor; mono-labelled at the VH C-terminus.

When using a construct with two sortase motifs within both the VH/CH1 and the VL/CL loops, the reaction will yield a dsFv; double-labelled at both C-termini. In case of a construct with two protease motifs within both the VH/CH1 and the VL/CL loops, the reaction will yield an unlabeled dsFv.

The reaction shows an overall molar conversion rate in the range of 80-90% from the IgG precursor to dsFv. The reaction side products (Fc+CH1 , CL), any uncleaved precursor, the excess label and enzymes can be removed via a 2-step purification on His-column (-Ni) followed by gel filtration chromatography (GFC).

Figure 2 shows HPLC chromatograms as well as SDS-gels showing the analytics of the IgG- precursor, the progress of the reaction as well as the final pure dsFv fragment.

Example 2: Comparison of the expression profile for dsFv precursor, scFv and ds- scFv.

The same IgG clone (anti-T nT 11-7) was cloned and expressed side-by-side in four Fv formats.

The first is the common scFv format where the VL C-terminus is fused to the VH N-terminus through a peptide linker ((GGGS)4).

The second is based on the scFv design with two extra cysteine point mutations within the VH and VL (at VH44 and VL100; Kabat numbering).

The third and fourth are dsFv-precursors (IgGs with inserted motifs) based on the design explained in Example 1 , where the IgG heavy chain has a sortase motif inserted within the VH/CH1 elbow region and the IgG light chain has either TEV- or IgA protease motif inserted at the VL/CL elbow region.

In addition to the 6-His tags and the two cysteines point mutations, as described in Example 1. These dsFv precursors are processed to dsFv through the enzymatic action of the sortase and protease enzymes, as described in Example 1 .

Table B below discloses the expression/purification yield of each of the constructs in addition to the aggregates percent.

It is worth nothing that for both of the scFv and sc-dsFv, the monomeric product tends to further re-aggregate even after the purification of the aggregates from the mixture through gel- filtration chromatography. Figure 3 shows the overall analytics of the products described above. Overall, this shows the advantage of the enzymatically-produced dsFv in comparison to scFv and sc-dsFv in terms of yield and proteins stability.

Table B (above): Comparison of the purification yields and tendency to aggregate

(aggregates percent) between different constructs of the 11-7 anti-TnT clone expressed in ExpiHEK cells.

*The monomeric product of these constructs tends to re-aggregate even after the purification of the aggregates from the mixture.

**The establishment of a stable-expressing cell line of this construct was feasible and the expression rate increased several folds to the range of 3 g/l.

***The dsFv is not expressed in this form; hence no expression/purification yield as such, but it is produced from the IgG-precursor (as explained in Example 1) with a molar conversion rate of ~ 82%.

Example 3: Elecsys Performance of the 11-7 anti-TnT dsFv.

The dsFv of the anti-TnT 11-7 clone was produced as described in Example 1 through the action of the sortase and IgA-protease enzymes on the relevant precursor and the purification of the dsFv in two steps; as outlined in Example 1.

The performance of the mono-bi-labelled dsFv (dsFv SRT/PROT) was assessed as capture antibody on the Roche immunoassay platform (Elecsys®). This was compared side-by-side to the state of the art site-specific and non-site-specific biotin-labelled IgG fragments, including: a recombinant Fab labelled using site-specific labelling strategies (Fab SSC Conjl and Fab SSC Conj2); an enzymatically produced Fab’ labelled on the hinge cysteine (Fab’ Conj3 MEA); a recombinantly-produced Fab labelled via O-succinimide chemistry on surface lysines (Fab Conj5 NHS); an enzymatically produced F(ab’)2 labelled via O-succinimide chemistry on surface lysines (F(ab’)2 Conj2 NHS); as well as a F(ab’)2labelled using a sitespecific labelling strategy (F(ab’)2 SSC Conjl).

The conjugates described above were spiked into Elecsys R1 standard recipe and measured in a rackpack together with standard beads and a constant R2 bottle on an e601 Elecsys analyzer (Roche’s high throughput immunoassay platform).

On the analyser, fixed volumes of the R1 reagent (contains biotinylated capture dsFv), R2 reagent (contains ruthenlyted detection antibody), serum sample (calibrator; contains analyte), standard streptavidin-coated beads were pipetted together in a reaction vessel followed by incubation for 9 minutes at 37°C. Following, a washing step of the immune complex on the beads, the entire amount of beads with the immune complex captured on it (beads- dsFv_Biotin-analyte-lgG_Ruthenium) was transferred to the measuring cell, where a magnet brings the magnetic beads close to the electrode. A voltage was applied that elicits the electrochemiluminescence response of the ruthenium label, which in turn was collected on detector.

Two samples were measured; (i) a negative human serum sample (HS) to assess the assay blank value and (ii) a Calibrator 1 sample which contains around 14 pg/ml recombinant TnT (Call). As shown in Table C, the calculated signal to noise (Cal1/HS) shows the superior performance of the bi-labelled dsFv to that of to the state of the art site-specific and non-site- specific bi-labelled IgG fragments. The conjugates stability was then challenged through an accelerated thermal stress experiment by incubating the rackpack at 37°C for 3 or 6 weeks and assessing the signal recovery.

Two samples were measured before and after the incubation: (i) a Calibrator 1 sample which contains around 14 pg/ml recombinant TnT (Call) and (ii) a Calibrator 2 sample with a much higher content of recombinant TnT (around 4200 pg/ml).

Table C (above): Comparison of the Elecsys Signal Dynamics Performance of various constructs of the 11-7 anti-TnT Biotinlyted antibody fragments of different labelling strategies (see text for descriptions). HS: Human Serum (blank). Call: low analyte range test calibrator (TnT cone ~ 14 pg/ml). Cal1/HS: Signal dynamics calculated as Cal1/HS signal to noise ratio.

As shown in Table D, the bi-labelled dsFv shows superior stability to that of to the state of the art site-specific and non-site-specific mono-bi-labelled IgG fragments.

Table D (above): Comparison of the stability of various constructs of the 11-7 anti-TnT Biotinlyted antibody fragments of different labelling strategies. Call: low-analyte range test calibrator (TnT cone ~ 14 pg/ml). Cal2: high-analyte range test calibrator (TnT cone ~ 4200 pg/ml). Recovery % refer to the signal recovery following 3- or 6-week reagents’ stress at 35°C; indicative of reagents stability.

Example 4: Exploring the success rate of the current dsFv-precursor design in different clones

In order to test the universality of the IgG/dsFv precursor design described in Example 1, a selection of 7 clones that bind different analytes were cloned and expressed in ExpiHEK cells (with sortase-motif at VH/CH1 , IgA-protease motif at VL/CL and the engineered disulfide bridge). The expression/purification yield was assessed as well as the aggregate percent of the product. As shown in Table 5 and Figure 4, 3 out of the 7 clones have showed good expression yield with low aggregate content and 2 out of the 7 have shown very low expression with low aggregate content. However, 2 out of the 7 did not express at all. The low- or noexpressing clones could perhaps be improved via an improved design of the construct including the linker sequences flanking the inserted motifs, the position of the insertions or the position of the cysteine point mutations. 4 out of the 5 IgG constructs (dsFv precursors), which have shown expression and were purified from ExpiHEK cultures, were further processed via the reaction with sortase and IgA-protease enzymes followed by the purification of the dsFv product as outlined in Example 1. In all cases the production of the dsFv from its precursor, following the standard process described in Example 1 , was successful with conversion rates similar to that observed for the anti-TnT 11-7 clone and with no aggregates in the final product. Figure 5 provides an overview of analytics of the four dsFv constructs produced showing their successful production and monomeric nature. The fifth IgG construct (Clone F) was not pursued due to the low amount of the precursor material available.

Table E (above): Exploring the success rate of the current dsFv-precursor design in different clones (see text for explanations).

Example 5: Variations of linkers and insertion sites

As described in Example 1 , the lead construct (precursor recombinant IgG for the dsFv production) contains a sortase enzyme recognition motif (LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked by a flexible linker (GSGLPETGGSG (SEQ ID NO: 27); insertion after amino acid number 114 (Kabat Numbering) / 118 (Ell Numbering)); alternatively, this could be designed as a protease motif. Furthermore, the elbow/loop region linking the VL and CL domains of the light chain contains an IgA-Protease recognition motif flanked by a flexible linker (e.g. GSGPRPPGPGSG (SEQ ID NO: 36); insertion after amino acid number 110 (Kabat Numbering) / 110 (Ell Numbering)); alternatively, this could be designed as a sortase motif or any protease’s motif.

While the above indicated insertion sites and sequences have worked for many IgG clones (as shown in Example 4), the location of the insertion site within the heavy/light chain loop region can be modified as described herein, so long as the inserted enzyme recognition sequence is positioned within the flexible unstructured loop linking the two Fd or light chain domains (VH/CH1 or VL/CL). This loop region extend for the heavy chain after the last amino acid of the VH domain’s last beta-strand till the last amino acid before the first CH1 domain’s first beta-strand (comprising the region of amino acid numbers 110 - 120 (Kabat Numbering)). For the light chain this region extends after the last amino acid of the VL domain’s last betastrand till the last amino acid before the first CL domain’s first beta-strand (comprising the region of amino acid numbers 106 - 114 Kabat Numbering)).

Additionally, the sequence of the linker sequence flanking the enzyme motif (within the insertion sequence) can be varied and could comprise any combination of the amino acids that are commonly used in the design of flexible linkers for example; Glycine, Serine, Alanine, Threonine, Glutamate in addition to Proline.

In this Example 5, data on IgG precursor construct (dsFv precursors) is provided which holds variants of the insertion sequence that vary in the flanking linker sequence as well variants of the insertion sites. Table F summarizes the design of these constructs regarding the insertion site and insertion sequence within the IgG heavy and light chain. These constructs were cloned and expressed in HEK cells and the expression/purification yield was assessed. Additionally, the reaction to produce the dsFv from these precursors, using the sortase enzyme, was performed and the conversion rates assessed. Table F summarizes the expression yields as well as the conversion rates for the different constructs; compared to a reference construct. Tolerance is observed in terms of the variation to the insertion site as well as the flanking-linker sequence within the dsFv precursor IgG; as outlined above.

‘According to Kabat Numbering Scheme; insxxx refers to the amino acid number after which the indicated enzyme motif sequence is inserted; delinsxxx refers to a stretch of amino acids which are deleted and replaced by the indicated enzyme motif sequence

**Reference construct comprises insertions of the Sortase enzyme motif flanked with similar linker sequences and at the same insertion sites as in the Lead construct described in Example 1

Example 6

As described in Example 1 , the lead construct (precursor recombinant IgG for the dsFv production) contains a sortase enzyme recognition motif (LPETG; SEQ ID NO: 35) within the elbow/loop region linking the VH and the CH1 domains of the heavy chain flanked by a flexible linker (GSGLPETGGSG (SEQ ID NO: 27); insertion after amino acid number 114 (Kabat Numbering) / 118 (Ell Numbering)); alternatively, this could be designed as a protease motif. Furthermore, the elbow/loop region linking the VL and CL domains of the light chain contains an IgA-Protease recognition motif flanked by a flexible linker (GSGPRPPGPGSG (SEQ ID NO: 36); insertion after amino acid number 110 (Kabat Numbering) / 110 (Ell Numbering)); alternatively, this could be designed as a sortase motif or any protease’s motif.

While the above indicated insertion sites and sequences have worked for many IgG clones (as shown in Example 4), the location of the insertion site within the heavy/light chain loop region can be modified as described herein, so long as the inserted enzyme recognition sequence is positioned within the flexible unstructured loop linking the two Fd or light chain domains (VH/CH1 or VL/CL). For the heavy chain, this loop region extends after the last amino acid of the VH domain’s last beta-strand till the last amino acid before the first CH1 domain’s first beta-strand (comprising the region of amino acid numbers 110 - 120 (Kabat Numbering)). For the light chain this region extends after the last amino acid of the VL domain’s last betastrand till the last amino acid before the first CL domain’s first beta-strand (comprising the region of amino acid numbers 106 - 114 Kabat Numbering)).

Additionally, the inserted enzyme motif sequence can be varied. This system therefore allows the use of a variety of proteases and/or transpeptidases to produce the dsFv product from the precursor IgG.

In this Example 6, data on IgG precursor construct (dsFv precursors) is provided for variants of the insertion sequences that vary in the enzyme motif sequence as well variants of the insertion sites spanning the above described VH/CH1 and VL/CL loop regions. The same IgG clone (anti-TnT 11-7) described in Example 1 was used as the basis IgG construct for the variants described in this Example 6.

Table G summarizes the design of these constructs regarding the insertion site and insertion/enzyme motif sequence within the IgG heavy and light chain. These constructs were cloned and expressed in HEK cells and the expression/purification yield was assessed. Additionally, the reaction to produce the dsFv from these precursors, using the sortase enzyme and/or additionally a protease (e.g. IgA-protease, TEV-protease or 3C-protease) was performed and the conversion rates assessed. Table G summarizes the expression yields as well as the conversion rates for the different constructs; compared to a reference construct. Tolerance is observed in terms of the variation to the insertion site and the insertion sequence within the dsFv precursor IgG as well as the use of a variety of proteases/transpeptidases to produce the product dsFv from the precursor IgG; as outlined above.

Example 5 and Example 6 demonstrate the tolerance inherent to the Fv/dsFv precursor design. This tolerance includes flexibility of the insertion site within the IgG heavy and light chains. For the heavy chain, this loop region extends after the last amino acid of the VH domain’s last beta-strand till the last amino acid before the first CH1 domain’s first betastrand (comprising the region of amino acid numbers 110 - 120 (Kabat Numbering)). For the light chain this region extends after the last amino acid of the VL domain’s last beta-strand till the last amino acid before the first CL domain’s first beta-strand (comprising the region of amino acid numbers 106 - 114 Kabat Numbering)). The tolerance also extends to cover variations in the inserted sequence when it comes to both the enzyme motif sequence and the flanking linker sequence. Example 5 and 6 additionally shows the herein-disclosed method of production of Fv/dsFv fragment as a generic scheme that can interchangeably use different combinations of transpeptidases and proteases.

Example 6 also represents an experimental setup that could be used by the skilled in the art to test whether a given enzyme is suitable for the described scheme or method of Fv/dsFv production from an IgG. This would entail designing an IgG constructs including the to-be- tested enzyme motifs within the VH-CH1 and VL-CL linker regions. The enzyme recognition motifs are inserted at the desired sites flanked by the desired flexible linker sequence, for example GSG, on each end to enhance the enzyme accessibility. Suitably, the enzyme recognition motifs are inserted at the same insertion site as was done in Example 6 and with the same flexible linkers, i.e. , for VH insertion after amino acid number 114 (Kabat Numbering) / 118 (Ell Numbering)) and for VL insertion after amino acid number 110 (Kabat Numbering) / 110 (EU Numbering). A C-terminal His-tag could be added at the IgG C- terminus to facilitate the following removal of impurities from the reaction. This is to be followed by expression in an appropriate host cell, for example HEK or CHO cells. The IgG (dsFv-precursor) is purified from the culture supernatant using affinity chromatography techniques, for example Protein A. Following the IgG purification a one or two step reaction with the enzyme (s) of choice proceeds according to the supplier instructions in the preferred buffer and in the presence of any required co-reactants. The used enzymes (proteases / transpeptidases) would catalyse the cleavage and/or cleavage and re-ligation to a ligand of the IgG within both the VH/CH1 and the VL/CL loops. The success of the enzyme and insertion site/sequence within the IgG precursor could be judged by assessing the conversion rate of the IgG to a dsFv/Fv. This could be done by following the reaction progress on an HPLC (i.e. injecting a sample of the reaction mixture on a GFC-column that provides sufficient resolution (e.g. Superdex 75 10/300 from Cytiva)). The percentage of the dsFv peak area as a percentage of the total peak area could be calculated. The total peak area includes, in addition to the dsFv, any remaining unreacted IgG precursor in addition to the reaction by-product. This calculation could be done using a Chromatography Data System like Chromeleon. This was done to assess the conversion rate for the constructs in Example 5. Here, the calculated percent dsFv peak of the total area was additionally normalized as a percentage of the same value for the reference construct (anti-TnT IgG, 11- 7 clone, that carries Sortase motif after amino acid 114 of heavy chain and Sortase motif after amino acid 110 of the light chain). A normalized value of 2% was used as the cut-off for defining success. Alternatively, success of the enzyme and insertion site/sequence within the IgG precursor through assessing the conversion rate of the IgG to a dsFv could be done at the level of the final purified product dsFv/Fv. The reaction product of interest could be purified from the reaction side products: (Fc+CH1 , CL), any uncleaved precursor, the excess label and enzymes via a 2-step purification on His-column (-N i) followed by gel filtration chromatography (GFC). Then the percentage yield of the reaction (percentage of the product dsFv to the precursor IgG (w/w%)) could be calculated. This was done to assess the conversion rate for the constructs in Example 6. Here, the calculated percent yield was additionally normalized as a percentage of the same value for the reference construct (anti-TnT IgG, 11-7 clone, that carries Sortase motif after amino acid 114 of heavy chain and IgA-protease motif after amino acid 110 of the light chain). A normalized value of 2% was used as the cut-off for defining success. While the both above-described methods to assess the construct design success would give comparable results, the first is preferred as it gives a direct read out after the reaction that is not affected by any further complications downstream (e.g. inadequate purification strategy).

*According to Kabat Numbering Scheme; insxxx refers to the amino acid number after which the indicated enzyme motif (insertion) sequence is inserted

**Reference construct comprises insertions of the Sortase enzyme motif flanked with similar linker sequences and at the same insertion sites as in the

Lead construct described in Example 1

***% Conversion is calculated as the percentage of the dsFv product yield amount to the starting precursor IgG amount then referenced to the ref. construct as the 100% n.d. % Conversion could not be assessed due to low expression yield and low availability of starting material. The expression scale will have to be increased for this construct in order to be able to properly assess the conversion rate

Summary:

The main prior art methods for preparing Fv fragments is based upon expression of VH and VL separately and then mixing to form an Fv. When this separate expression followed by mixing is done with wild type sequences, the success rate is low and variable depending on the inherent non-covalent interactions between the VH and VL, which is different for every IgG-clone. The non-covalent interactions at the VH/VL interface are likely too weak to keep them stably bound. Fusing the VH/VL to interaction coiled-coil domains or interacting protein partners can theoretically go one step towards stabilizing this interaction between VH and VL, but the pitfall remains if these fusion domains/proteins interfere with the formation of the correct VH/VL interface. It remains over all again a more lengthy process that require two separate expressions, two purifications, followed by incubation step to form Fv, then enzymatic processing to remove the interaction partner followed by product purification.

The method and constructs described in the application in hand differ in that it ensures the correct folding of the VH/VL within their ‘native’ immunoglobulin (e.g. IgG) context, stabilized by the inherent covalent and non-covalent interactions of the heavy and light chain constant domains, before the Fv fragment is enzymatically extracted out of the immunoglobulin (e.g. IgG) molecule. This, in turn, reduces the risk of misfolding or loss in affinities, which can accompany the current methods outlined above. The proposed concept provides a generic way to produce Fv fragments; that does not require peptide linker or refolding conditions optimization for each new candidate and yields a stable product at a good yield that is less likely to suffer from aggregation.

Altogether Examples 1 - 6 show the superiority of the described method of Fv/dsFv production to the traditional methods of scFv/ds-scFv production. Examples 1 - 6 also show the robustness of the method and its tolerance to variations in the inserted sequence (enzyme motif + linker sequence), the insertion site within VH-CH1 and VL-CL loops as well as the wide range of proteases and/or transpeptidases that could be used to fulfil the production method.

Claims

1. An antibody or antigen-binding fragment thereof comprising a Fab domain, said Fab domain comprising an Fv fragment that binds to an antigen of interest, wherein said Fab domain comprises

(i) a heavy chain sequence comprising a first recognition motif for a site-specific protease or transpeptidase positioned between the last amino acid of the last beta-strand of the VH domain of the Fab and before the first amino acid of the first beta-strand of the CH1 domain of the Fab (i.e. the VH-CH1 loop); and

(ii) a light chain sequence comprising a second recognition motif for a site-specific protease or a transpeptidase positioned between the last amino acid of the last beta-strand of the VL domain of the Fab and before the first amino acid of the first beta-strand of the CL domain of the Fab (i.e. the VL-CL loop).

2. The antibody or antigen-binding fragment of claim 1 , wherein the first recognition motif is a recognition motif for a transpeptidase and the second recognition motif is a recognition motif for a site-specific protease or vice versa, or the first and second recognitions motifs are both for a transpeptidase or both for a site-specific protease.

3. The antibody or antigen-binding fragment of claim 1 , wherein the first recognition motif is a recognition motif for a transpeptidase and the second recognition motif is a recognition motif for a transpeptidase.

4. The antibody or antigen-binding fragment of claim 1 , wherein the first recognition motif is a recognition motif for a site-specific protease and the second recognition motif is a recognition motif for a site-specific protease.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the recognition motif for the site-specific protease in the VH-CH1 loop and/or the recognition motif for the site-specific protease in the VL-CL loop are independently selected from the group consisting of: TEV-protease recognition motif, IgA protease recognition motif and 3C protease recognition motif.

6. The antibody or antigen-binding fragment of claim 5, wherein the TEV-protease recognition motif comprises or consists of EXXYXQ^AX, wherein X is any amino acid, preferably wherein the TEV-protease recognition motif comprises or consists of ENLYFQX (SEQ ID NO: 1), wherein X is S, G, A, M, C or H.

7. The antibody or antigen-binding fragment of claim 5 or 6, wherein the IgA protease recognition motif comprises or consists of PX, wherein X is S, T, A, V, or G , preferably wherein the IgA protease recognition motif comprises or consists of X1X2PPX3P, wherein Xi is P or S, X2 is R or T, X3 is T, S or A, more preferably wherein the IgA protease recognition motif comprises or consists of PRPP^AXP (SEQ ID NO: 2), with X = S, T, A, V, G.

8. The antibody or antigen-binding fragment of any one of claim 5 to 7, wherein the 3C protease recognition motif comprises or consists of LFQGP (SEQ ID NO: 3), preferably wherein the 3C protease recognition motif comprises or consists of LEVLFQGP (SEQ ID NO: 4).

9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the recognition motif for a transpeptidase in the VH-CH1 loop and/or the recognition motif for a transpeptidase in the VL-CL loop is a recognition motif for a sortase or sortase- type transpeptidase.

10. The antibody or antigen-binding fragment of claim 9, wherein the sortase transpeptidase is one from classes A-F or the sortase-type transpeptidase is an exosortase or Archaesortase.

11. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TX2, wherein X1 can be any amino acid residue and X2 is G or A, preferably wherein the sortase recognition motif is a sortase A recognition motif and comprises or consists of LPX1TG (SEQ ID NO: 5), wherein X1 can be any amino acid residue.

12. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a sortase B recognition motif and comprises or consists of NPX1X2X3, , wherein X1 can be any amino acid residue, X2 can be T or S, and X3 can be N, G or S.

13. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a sortase C recognition motif and comprises or consists of X1X2X3TG, wherein X1 can be I or L, X2 can be P or A and X3 can be any amino acid residue.

14. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a sortase D recognition motif and comprises or consists of LPXTA (SEQ ID NO: 6), wherein X can be any amino acid residue.

15. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a sortase E recognition motif and comprises or consists of LAXTG (SEQ ID NO: 7), wherein X can be any amino acid residue.

16. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a sortase F recognition motif and comprises or consists of LPXTG, wherein X can be any amino acid residue.

17. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a Exosortase A recognition motif and comprises or consists of PEP.

18. The antibody or antigen-binding fragment of claim 10, wherein the sortase recognition motif is a Archaesortase recognition motif and comprises or consists of PGF.

19. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the first recognition motif and/or the second recognition motif is flanked by an N-terminal linker sequence and/or a C-terminal linker sequence.

20. The antibody or antigen-binding fragment of claim 19, wherein the N-terminal linker sequence has a length of at least 2 amino acids, preferably at least 3 amino acids.

21. The antibody or antigen-binding fragment of claim 19 or 20, wherein the C-terminal linker sequence has a length of at least 2 amino acids, preferably at least 3 amino acids.

22. The antibody or antigen-binding fragment of any one of claims 19 - 21 , wherein the first recognition motif and the second recognition motif are flanked at both ends (N- terminal and C-terminal) by a linker sequence.

23. The antibody or antigen-binding fragment of any one of claims 19 to 22, wherein the N-terminal and/or C-terminal linker sequence is composed of amino acids selected from the group consisting of Glycine, Serine, Alanine, Threonine, Glutamate and Proline.

24. The antibody or antigen-binding fragment of any one of claims 19 to 23, wherein the N-terminal linker comprises or consists of a sequence selected from the group consisting of: GSG, AP, APAP (SEQ ID NO: 8), ESGS (SEQ ID NO: 9), GGGS (SEQ ID NO: 10), GGGGS (SEQ ID NO: 11) and GSGGSG (SEQ ID NO: 12),.

25. The antibody or antigen-binding fragment of any one of claims 19 to 23, wherein the C-terminal linker comprises or consists of a sequence selected from the group consisting of: GSG, GSGGSG (SEQ ID NO: 13), AP, APAP (SEQ ID NO: 14), ESGS (SEQ ID NO: 15), GGGS (SEQ ID NO: 16) and GGGGS (SEQ ID NO: 17).

26. The antibody or antigen-binding fragment of any one of claims 1 to 25, wherein the first recognition motif is positioned anywhere between amino acids 109 and 121 , such as anywhere between amino acids 112 and 119 or anywhere between amino acids 111 and 117, of the VH domain according to Kabat numbering scheme.

27. The antibody or antigen-binding fragment of any one of claims 1 to 26, wherein the first recognition motif is positioned between amino acids 113 and 116 of the VH domain according to Kabat numbering scheme.

28. The antibody or antigen-binding fragment of any one of claims 1 to 27, wherein the second recognition motif is positioned anywhere between amino acids 105 and 115 of the VL domain according to Kabat numbering scheme.

29. The antibody or antigen-binding fragment of any one of claims 1 to 28, wherein the second recognition motif is positioned anywhere between amino acids 107 and 113 of the VL domain according to Kabat numbering scheme, such as between amino acids 109 and 112 of the VL domain according to Kabat numbering scheme.

30. The antibody or antigen-binding fragment of any one of claims 1 to 29, wherein the first recognition motif and any N- or C-terminal linker sequence attached thereto replaces one or more amino acids between amino acids 109 and 121 of the parent VH domain according to Kabat numbering scheme.

31. The antibody or antigen-binding fragment of any one of claims 1 to 30, wherein the second recognition motif and any N- or C-terminal linker sequence attached thereto replaces one or more amino acids between amino acids 105 and 115 of the parent VL domain according to Kabat numbering scheme.

32. The antibody or antigen-binding fragment of any one of claims 30 or 31 , wherein the first and/or second recognition motif and any N- or C-terminal linker sequence attached thereto replaces 1 to 4 amino acids of the parent VH or VL sequence.

33. The antibody or antigen-binding fragment of any one of claims 1 to 32, wherein the antibody or antigen-binding fragment thereof is selected from: IgG, IgA, IgD, IgE and IgM, preferably IgG, IgE and IgD.

34. The antibody or antigen-binding fragment of any one of claims 1 to 33, wherein the antibody or antigen-binding fragment thereof is IgG.

35. The antibody or antigen-binding fragment of any one of claims 1 to 30, wherein the antibody or antigen-binding fragment thereof is of mammalian origin, such as from a human, a mouse, a rabbit or a sheep.

36. The antibody or antigen-binding fragment of any one of claims 1 to 35, wherein the heavy chain sequence and/or the light chain sequence comprises an affinity tag.

37. The antibody or antigen-binding fragment of claim 36, wherein the affinity tag is a His- tag, a FLAG-tag, HA, cMyC, poly-Arg or a Strep-tag.

38. The antibody or antigen-binding fragment of claim 36 or 37, wherein the affinity tag is positioned C-terminally of the first recognition motif and/or the second recognition motif.

39. The antibody or antigen-binding fragment of any one of claims 36 to 38, wherein the affinity tag is positioned at the C-terminus of the heavy chain sequence and/or the light chain sequence.

40. The antibody or antigen-binding fragment of any one of claims 1 to 39, wherein the antibody or antigen-binding fragment is recombinantly expressed from a host cell, such as a eukaryotic or prokaryotic host cell.

41 . The antibody or antigen-binding fragment of claim 40, wherein the host cell is selected from a: mammalian, fungal, plant, insect or bacterial cell.

42. The antibody or antigen-binding fragment of claim 40 or 41 , wherein the host cell is a mammalian cell selected from a HEK, NS0, Sp2/0, PER. C6 or CHO cell.

43. The antibody or antigen-binding fragment of any one of claims 1 to 42, wherein the antibody or antigen binding fragment is a precursor for generating an Fv fragment.

44. The antibody or antigen-binding fragment of any one of claims 1 to 43, wherein a cysteine residue has been engineered into the VH and VL of the Fab domain at locations to permit disulfide bond formation between the two introduced cysteines, optionally, wherein the VH of the Fab domain comprises a cysteine at position 44 according to Kabat numbering scheme and wherein the VL of the Fab domain comprises a cysteine at position 100 according to Kabat numbering scheme.

45. The antibody or antigen-binding fragment of claim 44, wherein the cysteine at position 44 of the VH according to Kabat numbering scheme and the cysteine at position 100 of the VL according to Kabat numbering scheme form a disulfide bond.

46. The antibody or antigen-binding fragment of any one of claims 1 to 45, wherein the Fv fragment comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the first recognition motif and wherein the Fv fragment comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the second recognition motif.

47. The antibody or antigen-binding fragment of claim 46, wherein the antibody and/or antigen binding fragment thereof and/or Fab domain comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the first recognition motif and wherein the Fv fragment comprises no further recognition motif for the site-specific protease or transpeptidase capable of cleaving the second recognition motif.

48. The antibody or antigen-binding fragment of any one of claims 1 to 47, wherein the Fv fragment is capable of binding a Troponin, in particular Troponin T.

49. The antibody or antigen-binding fragment of any one of claims 1 to 48, wherein the VH sequence of the Fv comprises or consists of SEQ ID NO: 18

50. The antibody or antigen-binding fragment of any one of claims 1 to 49, wherein the VL sequence of the Fv comprises or consists of SEQ ID NO: 19.

51. The antibody or antigen-binding fragment of any one of claims 1 to 50, wherein the heavy chain sequence of the Fab comprises or consists of SEQ ID NO: 20.

52. The antibody or antigen-binding fragment of any one of claims 1 to 51 , wherein the light chain sequence of the Fab comprises or consists of SEQ ID NO: 21 .

53. A polynucleotide or set of polynucleotides encoding the heavy and light chain sequence of the antibody or antigen-binding fragment of any one of claims 1 to 52.

54. A vector or set of vectors comprising the polynucleotide or set of polynucleotides according to claim 53.

55. A host cell comprising the polynucleotide or set of polynucleotides according to claim 53, or the vector or set of vectors according to claim 54.

56. A method of producing an antibody or antigen-binding fragment according to any one of claims 1 to 52, said method comprising culturing the host cell according to claim 55 and isolating said antibody or antigen-binding fragment.

57. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 52 as precursor for generating a Fv fragment.

58. The use of claim 57 comprising contacting the precursor antibody or antigen-binding fragment of any one of claims 1 to 52 with (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a sitespecific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of any one of claims 1 to 52; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is cleaved by a transpeptidase, said contacting is optionally conducted in the presence of a transpeptidase nucleophile which optionally comprises a tag.

59. The use of claim 58, wherein the transpeptidase nucleophile comprises at least GG (glycine glycine).

60. The use of claim 58 or 59, wherein the transpeptidase is a sortase or sortase-type transpeptidase.

61 . The use of any one of claims 58 to 60, wherein the tag is biotin.

62. The use of any one of claims 58 to 61 , wherein the use further comprises isolating the Fv fragment.

63. A method for producing an Fv fragment capable of binding to an antigen of interest comprising a) contacting the antibody or antigen-binding fragment of any one of claims 1 to 52 with (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of any one of claims 1 to 52; wherein if the first and/or the second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, said contacting is optionally conducted in the presence of a nucleophile for said transpeptidase; and b) isolating the released Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to any one of claims 1 to 52.

64. A method for producing a tagged Fv capable of binding to an antigen of interest comprising a) contacting the antibody or antigen-binding fragment of any one of claims 1 to 52 with (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif, thereby releasing the Fv fragment from the Fab domain comprised in the antibody or antigen-binding fragment of any one of claims 1 to 52; wherein at least the first or second recognition site is a transpeptidase recognition motif that is contacted with the corresponding transpeptidase, wherein said contacting is conducted in the presence of a nucleophile for said transpeptidase, wherein the transpeptidase nucleophile comprises a tag; and b) isolating the tagged Fv fragment as comprised in the Fab fragment of the antibody or antigen-binding fragment according to any one of claims 1 to 52.

65. The method of claim 63 or 64, wherein the antibody or antigen-binding fragment is an antibody or antigen-binding fragment of any one of claims 48 to 52, wherein the Fv fragment specifically recognizes Troponin T.

66. An Fv fragment capable of binding to an antigen of interest obtained by a method of any one of claims 63 to 65, optionally wherein the Fv fragment contains one or more residual amino acid residues at the C-terminal end derived from the recognition motif and, in case a linker was used, from the N-terminal linker.

67. The Fv fragment of claim 66, wherein the Fv fragment is capable of binding a T roponin, in particular Troponin T.

68. The Fv fragment of claim 66 or 67, wherein the VH and/or VL sequence of the Fv fragment comprise one or more amino acids at the C-terminal ends derived from the cleavable insert domain.

69. The Fv fragment of any one of claims 66 to 68, wherein the VH sequence of the Fv comprises or consists of SEQ ID NO: 18 or SEQ ID NO: 20.

70. The Fv fragment of claim 66 or 67, wherein the VL sequence of the Fv comprises or consists of SEQ ID NO: 19 or SEQ ID NO: 21.

71. A method for detecting and/or quantifying troponin T in a sample comprising contacting the sample with the Fv fragment of any one of claims 66 to 70 under conditions allowing for binding of the Fv fragment to troponin T comprised in the sample; and detecting and/or quantifying troponin T in the sample by detecting the complex between the Fv fragment and troponin T.

72. A diagnostic kit comprising an antibody or antigen-binding fragment of any one of claims 1 to 52.

73. A diagnostic kit of claim 73 further comprising (i) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the first recognition motif and/or (ii) a site-specific protease or transpeptidase capable of cleaving a peptide bond within the second recognition motif and optionally a nucleophile, optionally a tag and/or optionally instructions for use.

74. A diagnostic kit comprising an Fv fragment of any one of claims 66 to 70.