Engineered protein m and its use in antibody labeling

Abstract

Engineered version of protein M, which binds to both κ and λ light chains and enables the labeling of all IgGs. The disclosed Engineered version of protein M enables the coupling of a wide range of molecules, such as luciferase domains, epitope tags, fluorescent domains, oligonucleotides, reporter enzymes, imaging tags, and small molecules to not only commonly used IgGs, but also chicken IgYs, Fabs, and scFvs, while still preserving effective antigen binding.

Description

Atty docket TUGE.P2006WO / 00643294ENGINEERED PROTEIN M AND ITS USE IN ANTIBODY LABELINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application 63 / 680,803 filed on August 8, 2024, the content of which is incorporated herein by reference in its entireties for all purposes.BACKGROUND

[0002] Protein G, protein L and protein A are immunoglobulin (Ig)-binding proteins.1 5These proteins are extensively studied and widely utilized in various applications, such as IgG immobilization, purification, detection, and labeling.6-9Protein G and protein A bind non- covalently and site-specifically to IgGs via the fragment crystallizable (Fc) or fragment antigen-binding region (Fab).7 10For some applications, such as IgG purification and immobilization, the non-covalent binding between immunoglobulin-binding protein and antibody is required. However, for developing antibody conjugates used in applications such as diagnostics, drug delivery, or imaging, a covalent bond between the antibody and the molecule of interest is essential. Hence, photo-cross-linkable alternatives of protein G and the protein A-derived Z-domain have been developed by introducing the UV-active non-natural amino acid benzoylphenylalanine (BPA) at their binding sites.11-19Although these photo- cross-linkable variants can be used to covalently couple molecules of interest to different IgGs, such as human IgGs, mouse IgG2 / 3 and rat IgG2c, they are not compatible or have a low affinity for others including most sheep antibodies, chicken IgY and the widely used and produced mouse IgGl subclass.11Furthermore, they bind primary to the Fc region of IgGs, rendering these immunoglobulin-binders incompatible with chicken IgY, IgM, IgA, IgE, Fabs and single-chain variable fragments (scFvs).11,20,21

[0003] An alternative strategy for covalently labeling antibodies with molecules of interest involves chemical conjugation techniques, such as using NHS ester groups to label the amine groups of lysine residues or maleimide moieties to target the thiols of cysteines. However, these nonspecific labeling strategies also result in attaching molecules of interest at locations near the antigen binding Fab domain, potentially hindering antigen binding. Additionally, there is no control over the number of labels per antibody, resulting in heterogeneous products. To increase the range of potential labeled antibodies and to extend their applicability across a broader spectrum of targets, a comprehensive conjugation strategy is necessary. This strategy should enable the efficient and site-specific coupling of molecules ofAtty docket TUOE.P2006WO / 00643294 interest, such as but not limited to luciferase domains, epitope tags, oligonucleotides, reporter enzymes, imaging tags and small molecules, to all IgGs, as well as IgMs, IgAs, IgYs and antibody fragments such as Fabs and scFvs.SUMMARY

[0004] In some embodiments, a non-covalent, albeit essentially irreversible, conjugation strategy is disclosed here, which utilizes truncated protein M to label a broad spectrum of both human and non-human IgGs with molecules of interest (Figure I).21In one aspect, the truncated protein M is derived from a larger protein (~60 kDa) that is displayed on the surface of human mycoplasma (Mycoplasma genitalium).21The truncated protein M binds to the variable region of K and X light chains of antibodies through several conserved interactions including hydrogen bonds, salt bridges and van der Waals interactions.21The high affinity of the truncated protein M for both K and X light chains enables the labeling of all IgGs. Additionally, this high affinity of the truncated protein M for both K and X light chains enables the coupling of a wide range of molecules - including but not limited to luciferase domains, epitope tags, fluorescent domains, oligonucleotides, reporter enzymes, imaging tags, and small molecules - to chicken IgYs, Fabs, and scFvs, while still preserving effective antigen binding.

[0005] In some embodiments, this strategy offers a general method for rapid and straightforward labeling of “off-the-shelf’ immunoglobulins, IgYs and antibody fragments. Because antibody labeling based on truncated protein M can also be applied to IgGs, it provides an attractive alternative to protein G, Z-domain or other strategies based on crosslinking of immunoglobulin-binders. Previously, protein M with antigen blocking site was used to attach fluorescent dyes to an antibody. However, as the binding site of an antibody is blocked, it allows binding of some small molecules and peptides only, hindering its use in assay targeting bigger biomolecules, such as proteins and protein complexes.33Splitting protein M with proteolytic cleavage between the N-terminal antibody binding part and the C- terminal antibody-antigen blocking part can be used to activate antibodies on-demand.34

[0006] In one embodiment, the truncated protein M labeling strategy disclosed herein utilizes the N-terminal part of the protein without the C-terminal amino acids blocking the antigen binding site of an antibody. Therefore, antibody labeling does not impede interactions with the antigen and preserves functional antibodies post-labeling.

[0007] One of the truncated protein M without the antigen blocking site is SEQ ID NO:1, composed of 78-440 amino acids of wild type protein M (pM440). A full-length version containing additional amino acid sequences for Truncated protein M without the antigenAtty docket TUGE.P2006WG / 00643294 blocking site can be labelled directly, e.g. by introducing cysteine residue reacting to maleimide modified fluorescent dyes or maleimide modified DNA oligonucleotides (See for example, SEQ ID 3). Truncated protein M without the antigen blocking site can also be fused to the molecule of interest via variable-length linkers to ensure the optimal function of the fusion partner (Figure 1).

[0008] Furthermore, the labeling protocol with pM440 is straightforward and easy as the only required steps are: 1) mixing the antibody (or antibody fragment) with the protein M-molecule of interest and 2) incubating this mixture (Figure 1), in some cases as little as 15 min is sufficient thanks to the high affinity of the protein for the antibody. The dissociation of truncated protein M is very slow, making the labeling of antibodies or antibody fragments effectively irreversible. Additional equipment like a UV lamp, required for the covalent coupling of protein G or the Z domain via the non-natural amino acid BPA, is unnecessary.

[0009] To demonstrates the potential and innovation of protein M labelling method, the following examples describe in detail the development of a protein M based immunoassay and compare these results to previously used protein G based immunoassay. The coupling of split luciferase domains to truncated protein M allows for homogeneous detection of antibody-specific analytes (Figure 2). The ability to label all IgGs and most of the antibody fragments enables straightforward antibody selection and rapid development of new and sensitive immunoassays for a wide variety of relevant targets.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows an overview of the protein M labeling strategy. The ability of protein M to bind to the variable region of light chains allows the labeling of all IgGs, antigenbinding regions (Fabs), single-chain variable fragments (scFvs) and IgYs. The labeling strategy entails the mixing of antibody (fragment) with truncated protein M and the subsequent incubation of this mixture.

[0011] Figure 2 shows homogeneous immunoassay development using the protein M labeling strategy. Antigen-specific antibodies, that bind to distinct epitopes, are labeled with split luciferase fragments via pM440. Binding of these antibodies to the antigen of interest results in a high local concentration of the split luciferase domains, the reconstitution of the luciferase and subsequently the emission of a bioluminescence signal.

[0012] Figure 3 shows results of affinity measurement between the Adalimumab antibody andAtty docket TUGE.P2006WG / 00643294 the pM440 measured using Surface Plasmon Resonance (SPR). 2 nM of the antibody diluted in the running buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.005% P20 surfactant was flown over the protein G functionalized chip. Next, pM440 was flown over at the rate of 30 pL / min for 180 s, followed by dissociation of 1 h with running buffer. The chip was regenerated by applying 10 mM glycine pH 1.5 in pulses of 30 s and 10 s. Obtained sensograms were analyzed in the Biacore Evaluation software, by removing the bulk shift data points and fitting the blank subtracted measurements in 1 : 1 binding model. The protein M440 did not dissociate more than 5%, therefore the fitted dissociation rate of Kdiss of 6.4x10"6s-1represents an upper limit, which combined with Kassof 4.3xl04M-1s-1, corresponds to a KD = 0.15 nM.

[0013] Figure 4 shows SPR analysis of affinity between between Adalimumab and TNFain the absence and in the presence of protein M440 or protein M468. SPR was used to assess the affinity of an antibody in the presence or absence of protein M. A custom made single cycle SPR program was used. An antibody was immobilized on a protein G chip by introducing 2 nM of Adalimumab for 30 s. Subsequently, 1.29 pM of protein M variants (or buffer) were introduced till the antibody was saturated. To assess the affinity of an antibody to its analyte, a range of 0.25 nM and 64 nM were flown over at a rate of 30 pL / min for 180 s, each followed by a dissociation stage. The bulk shift jumps caused by refractive index difference were removed from the blank subtracted sensograms and the affinity of an antibody towards TNFa was calculated with Biacore Evaluation software using 1:1 kinetic binding model. The maximal injection volume for association phase on Biacore X100 was only 90 pL, and the steady state for Adalimumab preincubated with protein M440 was not reached even at the highest tested concentration of the analyte, (a) The SPR sensogram showing the response of the antibody binding to its target TNFa in the absence of protein M and in the presence of protein M440 or protein M468. The injections with various concentrations of TNFa are marked by vertical arrows, (b) The SPR sensogram showing the binding of TNFa to Adalimumab. (c) The SPR sensogram showing the binding of TNFa to Adalimumab preincubated with protein M440. (d) A comparative table showing kinetics rates: association and dissociation rates, equilibrium constant and the quality parameters such as mass transfer: tc, fitting quality: Chi2, data uniqueness U-value.

[0014] Figure 5 shows SPR analysis of affinity between an R508 and IE-6 union in the absence and presence of pM440. For R508, 10 nM was immobilized for 30 s at a rate of 30 pL / min for 180 s, followed by saturation of 2 pM of protein M variants and theAtty docket TUGE.P2006WO / 00643294 concentrations of 0.2-16.2 (3 fold step dilution) were flown over. Next, the step of 9 min (Adalimumab) or 30 min (R508) dissociation followed. The bulk shift jumps caused by refractive index difference were removed from the blank subtracted sensorgrams and the affinity of the antibody for TNFa or IL-6 was calculated with Biacore Evaluation software using 1 : 1 kinetic binding model.

[0015] Figure 6 shows protein-DNA conjugation of pM440 via two distinct strategies. Reducing SDS-PAGE (4-20%) analysis of protein-DNA conjugation, (a) Amine-modified DNA (IDT) was functionalized with maleimide moiety by mixing with 10 molar equivalents of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sulfo-SMCC), incubated for 2 hours at room temperature with vigorous shaking and subsequently purified by ethanol precipitation. Protein M440 with N-terminal cysteine was mixed with maleimide modified DNA in the ratio 5: 1 and conjugated for 3 hours at room temperature with vigorous shaking. B) Protein M440 fused to DCV HUH endonuclease domain was reacted with 10 molar equivalents of SEQID005 and incubated overnight at room temperature. C) An exemplary sensor composed of DNA architecture attached to an antibody via truncated protein M. An antibody is fluorescently labelled with the donor fluorescent dye and the truncated protein M is labelled with DNA. The complementary DNA strand is functionalized with the molecular competitor (bait) and a fluorescent dye. When the antibody target is absent, the bait is bound to the antibody allowing the energy transfer between donor and acceptor fluorophores. When the target is present, the bait is displaced and no energy transfer is possible.

[0016] Figure 7 shows expression and purification of RAPPID-M. Reducing SDS-PAGE (4- 20%) analysis of protein M-LargeBiT (pM-LB) and protein M-SmallBiT (pM-SB) after expression in E. coli and purification using Ni2+ affinity chromatography and Strep-Tactin chromatography.

[0017] Figure 8 shows comparison of IL-6 RAPPID-G with IL-6 RAPPID-M. (a) Nonreducing SDS-PAGE analysis of the photoconjugated anti-IL-6 antibodies mhK23-pG-SB and R508-pG-LB. Photoconjugations were done according to LASIC method11in PBS (pH 7.4) with 1 pM antibody and 2 pM pG-SB or pG-LB. Dose-response curves of (a) of 0.1 nM R508-pG-LB with 0.5 nM mhK23-pG-SB and (b) 0.1 nM R508-pM-LB with 0.5 nM mhK23- pM-SB. The assays were performed in PBS (pH 7.4), 0.1% (w / v) BSA and the data represents technical replicates, with n - 3 independent preparations of IL-6. The dotted lines connect mean values.

[0018] Figure 9 shows storage of RAPPID assay mixtures at 4 °C. (a) Schematic overview ofAtty docket TUGE.P2006WO / 00643294 the different RAPPID assays that were tested: RAPPID-M, conjugated RAPPID-G and unconjugated RAPPID-G. RAPPID response curves of the different RAPPID assays at (b) day 0, immediately after mixing R508-LB with mhK23-SB, (c) stored at 4 °C for 4 days and (d) stored at 4 °C for 8 days, (e) Background signal of the different components of the RAPPID assays, mhK23-SB, R508-LB and R508-LB with mhK23-SB. The assays were performed in PBS (pH 7.4), 0.1% (w / v) BSA and values in (b), (c) and (d) depict mean ± s.d. of technical replicates, with n - 3 independent preparations of the target, and the lines represent the mean. The colored lines connect mean values. The final concentration in the assays was 0.1 nM R508-LB and 0.5 nM mhK23-SB.

[0019] Figure 10 shows RAPPID-M assay with mouse IgGl antibodies, scFv antibody fragment and Fab fragments, (a) RAPPID-M assay using mouse IgGl antibodies. 9540 and 2063 (R&D systems) mouse monoclonal IgGl antibodies were mixed with 2-fold molar excess of pM440-LB and pM440-SB, respectively, and incubated overnight. Then, the sensor was assembled by mixing 500 pM of each complex in PBS with 1% BSA, mixed with IL-6 in the range of 1 pM to 64 nM and incubated for 1 h. The Nluc luciferase substrate was added to final dilution of 1000 and the bioluminescence signal was measured on MC Spark (Tecan).(b) Antibody fragment scFv of Adalimumab binding domain was expressed in Escherichia Coli and purified using Ni2+affinity chromatography, (c) RAPPID-M assay using scFv shown in Figure 10b. scFv was mixed with a 2-fold molar excess of pM440-LB or pM440-SB and incubated overnight. Then, the sensor was assembled by mixing 500 pM of each component in PBS with 1% BSA, mixed with TNF a in the range of 0.25-256 nM and incubated for 1 h. The Nluc luciferase substrate (final 1000-fold dilution) was added and the bioluminescence signal was measured on MC Spark (Tecan).DETAILED DESCIPTION

[0020] Various embodiments of the instant disclosure are further illustrated by the following Items:

[0021] Item 1. An engineered molecule comprising a polypeptide having an amino acid sequence that is at least 90% identical in amino acid sequence to a peptide from amino acid number 78 to amino acid 440 of wild-type protein M of Mycoplasma genitalium.

[0022] Item 2. The engineered molecule of Item 1, wherein the polypeptide lack a C-terminal domain of wild-type protein M that blocks binding between the Ig or fragment thereof and an antigen.Atty docket TUGE.P2006WO / 00643294

[0023] Item 3. The engineered molecule of any of Items 1-2, wherein the polypeptide has at least 90%, or at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 1.

[0024] Item 4. The engineered molecule of any preceding Items, wherein the polypeptide is capable of binding to a variable region of a light chain of an immunoglobulin (Ig) or fragment thereof, wherein the immunoglobulin (Ig) or fragment thereof is at least one member selected from the group consisting of an IgG, an antigen-binding regions (Fab), an single-chain variable fragments (scFv) and an IgY.

[0025] Item 5. The engineered molecule of any preceding Items, further comprising a second moiety, wherein the second moiety is a labeling molecule selected from the group consisting of a luciferase domain, an epitope tag, a fluorescent tag, an oligonucleotide, a reporter enzyme, an imaging tag, a small molecule chemical and combination thereof.

[0026] Item 6. A composition comprising the engineered molecule of any preceding Items.

[0027] Item 7. A polynucleotide comprising a coding sequence encoding the polypeptide of any of Items 1-5.

[0028] Item 8. An engineered molecule comprising a polypeptide having an amino acid sequence that is at least 90%, or at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 3.

[0029] Item 9. The engineered molecule of Item 8, wherein the polypeptide comprises a cysteine at a position n residues from the N-terminus of the polypeptide, n being an integer between 1 and 20.

[0030] Item 10. A polynucleotide comprising a coding sequence encoding the polypeptide of any of Items 8-9.

[0031] Item 11. A method for labeling an immunoglobulin or fragment thereof in a sample, comprising mixing the molecule of any of Items 1-5 with the sample, allowing the molecule to bind to the Ig or fragment thereof in the sample, wherein the molecule is pre-conjugated with a labeling molecule prior to the mixing.

[0032] Item 12. A method for labeling an immunoglobulin or fragment thereof in a sample, comprising mixing the molecule of any of Items 8-9 with the sample, allowing the molecule to bind to the Ig or fragment thereof in the sample, wherein the molecule is pre-conjugated with a labeling molecule prior to the mixing.

[0033] The disclosure will now be illustrated with working examples, which are intended to illustrate the working of disclosure and not intended to restrict the scope of the present disclosure. Unless otherwise defined in this disclosure, all technical and scientific terms usedAtty docket TUGE.P2006WG / 00643294 herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0034] Example 1 Truncated protein M to label antibodies

[0035] Truncated protein M without the antigen blocking part can be used as an adapter to label antibodies and antibody fragments with molecules of interest. To verify the essentially irreversible interaction between truncated protein M440 and an antibody, surface plasmon resonance (SPR) was applied. An anti-TNFa antibody Adalimumab was immobilized on protein G chip and various concentrations of protein M440 were flown over (Figure 3). Minimal dissociation was observed over a period of 1 hour, which is consistent with a kdiss of < 6.4xl0"6s1which is beyond the measurement capabilities of the employed Biacore X100. Please note, that kdiss represents an upper limit, which when combined with the kassof 4.3xl04M-1s-1, corresponds to a KD = 0.15 nM. To verify that the adaptor with the label does not impede antibody functionality, surface plasmon resonance (SPR) measurement was performed using a Biacore X100 (Figure 4-5).

[0036] The affinity of an unlabeled antibody was compared to an antibody labelled with truncated protein M440 (SEQ ID NO:6) and an antibody labelled with protein M468 encompassing the antigen blocking domain (SEQ ID NO:2). A custom-made single cycle SPR program was used where after immobilization of an antibody on protein G chip, various protein M variants were introduced until the antibody was saturated. Subsequently, various concentrations of an analyte, ranging between 0.25 nM and 64 nM, were flown over the chip, each followed by a dissociation stage. We found that the affinity of the widely used antibody drug Adalimumab towards TNFa was 0.65 nM, with maximal response unit (RU) of 25.85. The affinity of Adalimumab preincubated with pM440 for TNFa was 2.94 nM, with the maximum predicted signal of 25.4 RU (Figure 4D). Furthermore, we observed that preincubating Adalimumab with protein M440 decreased the association rate of the antibody for TNFa. The SPR sensogram (Figure 4a-c) shows that the dissociation of the analyte is minimally affected by the presence of protein M440 bound to Adalimumab. Preincubation with antigen blocking protein M (SEQ ID NO:2) yielded no dose response curve between the antibody and its target at the tested concentrations (max predicted RU signal 3.82) and therefore the binding kinetic rate could not be determined.

[0037] Similarly, the effect of protein M440 on the antibody binding was further tested withAtty docket TUOE.P2006WO / 00643294 an anti-interleukin-6 antibody R508 (Figure 5). For R508, 10 nM was immobilized on protein G chip for 30 s, followed by saturation of 2 pM of protein M 440 (or buffer) and the target was flown over in the concentration range of 0.2-16.2 nM (3-fold step dilution). Next, the step of 30 min dissociation followed (Figure 5a-b). The interaction between R508 and interleukin- 6 (IL-6) displayed a dissociation constant of 160 pM, whereas the interaction between R508- pM440 and IL-6 displayed the similar dissociation constant of 89 pM (Figure 5c). These results show that the antibody preincubated with truncated protein M preserves high affinity towards its target, although the kinetics rates can be altered.

[0038] To label antibodies, truncated protein M without the antigen binding site was prelabeled with a molecule of interest. This can be performed by introducing amino acid residue(s) into the truncated protein M sequence that can undergo chemical reactions with the target molecule, such as a cysteine reacting with maleimide-modified molecules or a lysine interacting with NHS-ester functionalized molecules. Alternatively, truncated protein M can be labelled by genetically fusing it to a polypeptide of interest, such as a light producing luciferase domain or a fluorescent protein. Additionally, the protein can be labelled by introducing a tag into its sequence. Some examples are a spy tag creating the spontaneous isopeptide bond with spy catcher modified molecules30, self-labeling SNAP tag reacting with synthetic probes bearing benzylguanine31or HUH tags creating a covalent bond with DNA containing the specific recognition sequence.32To ensure the optimal functionality of both partner domains, they can be separated by a linker of variable length.

[0039] One of the molecules of interest is an oligonucleotide strand and exemplary approaches are described in detail below. Antibody- DNA conjugates are extensively used in microscopy28and biochemical assays such as proximity ligation assay (PLA).29To show the modularity of the protein M labelling approach, we modified truncated protein M without the antigen blocking part with DNA oligonucleotides using two different methods. First, to make sure that the attached DNA is not in close proximity to the antigen binding site, we cloned the cysteine at the N-terminus of the truncated protein M (SEQ ID NOG). Subsequently, the protein was coupled to maleimide modified DNA. Accordingly, pM440 was mixed in the ratio of 5:1 with the amine-modified DNA (SEQ ID NO:4) previously functionalized with maleimide moiety. The reaction was conducted for 3 hours at room temperature and validated on SDS-PAGE gel (Figure 6a). On SDS-PAGE gel, the conjugate migrates slower due to an increased molecular mass and the reaction yield was estimated at >75%. Secondly, we genetically fused truncated protein M to an HUH tag deriving from Muscovy duck circovirus (DCV) and reacted it overnight with 10 molar equivalents of unmodified DNA with a DCVAtty docket TUGE.P2006WO / 00643294 specific recognition sequence. The protein forms a phosphotyrosine covalent bond with the DNA. Figure 6b shows that the mass of the conjugate increases and the yield of the reaction can be estimated at >90%. Those conjugates can be subsequently simply mixed with an off- shelf antibody of choice and used in functional assays. Protein M440-DNA conjugate can be used to attach the DNA architectures to antibodies and used in a competition assay with Forster resonance energy transfer (FRET) fluorescent facilitating readout, similar to a biosensor presented in ref 34. Figure 6c presents a possible biosensor. Fluorescently labelled antibody (or protein M) is attached to the DNA architecture via protein M labelled with single stranded DNA. The complementary DNA strand is functionalized with the molecular competitor (bait) and a fluorescent dye. Multiple DNA strands and DNA nicks can be incorporated in the architecture to enable higher flexibility and enable conformational change. In the absence of the target, bait is bound to the antibody what allows the energy transfer between fluorophore on an antibody (donor) and on the DNA (acceptor). Acceptor dye is then excited and emits fluorescence. In the presence of the target, the bait is being displaced, hampering the energy transfer and only donor fluorescence is observed. By measuring the ratio between the intensities of the donor and acceptor fluorescence, we can derive the concentration of the target.

[0040] Example 2 Protein M labeling to develop bioluminescent immunoassays

[0041] Protein G (pG) has been adapted for the development of bioluminescent RAPPID-G (Ratiometric Plug-and-Play Immunodiagnostics) sensors.22-25RAPPID-G is a homogenous immunoassay platform that comprise of two pG-compatible IgGs that are covalently linked to split NanoLuc (NLuc) luciferase fragments via pG photo-crosslinking.11 26,27Analyte binding results in the formation of a luminescent ternary complex and the emission of blue light. We successfully developed RAPPID-G biosensors for the quantification of a wide range of relevant biomarkers.

[0042] However, the RAPPID-G platform is limited by the usage of protein G-compatible IgGs and cannot be combined with all IgG subclasses, including most sheep antibodies, chicken IgY and the important mouse IgGl subclass. Furthermore, pG interacts with the Fc region of IgGs, making RAPPID-G incompatible with antibody fragments.11,20,21To increase the adaptability of the RAPPID sensor platform and expand its applicability across a wider range of targets, we applied the protein M labeling strategy to develop RAPPID-M (Figure 2). Accordingly, we first designed two RAPPID-M fusion proteins, each consisting of pM440 connected to either LargeBiT (LB) or SmallBiT (SB, Ka - 2.5 pM)26, the two split NLucAtty docket TUGE.P2006WO / 00643294 fragments, via a semi-flexible linker.22We expressed these RAPPID-M proteins in E. coli and used Ni2+affinity chromatography followed by Strep-Tactin purification to obtain pure proteins (pM-SB and pM-LB, Figure 7) with surprisingly high yields (12 and 13 mg per liter culture, respectively).

[0043] Next, the commercially available anti-IL-6 antibodies R508 and mhK23 were labeled with the LB and SB fragments.22These antibodies are rabbit anti-IL-6 and mouse chimeric monoclonals, respectively. Thus, they can be labeled with both the pG-luciferase and pM440- luciferase domains, enabling accurate comparison of the two RAPPID variants. First, R508 was mixed with pG-LB and mhK23 with pG-SB, and after an incubation step of 45 minutes we irradiated the two mixtures with UV light for 10 minutes. Next, the RAPPID-M components were prepared by simply mixing R508 with pM440-LB and mhK23 with pM440- SB. This mixture was subsequently incubated overnight at 4 °C to allow the binding between pM and the light chains of the anti- IL-6 antibodies. To evaluate the performance of RAPPID- G and RAPPID-M, we incubated the assay mix, consisting of 0.1 nM antibody-LB and 0.5 nM antibody-SB, with increasing concentrations of IL-6. The mixture was incubated for 2 hours to allow the binding of the antibodies to IL-6. Figure 8a shows the dose-response curve of RAPPID-G, with a maximal luminescent signal at 0.74 nM. This demonstrates that the antibodies bind to IL-6 and a luminescent ternary complex is formed. At IL-6 concentrations exceeding 0.74 nM, the luminescent signal decreases again due to the Hook effect.22Next, we evaluated the performance of RAPPID-M and surprisingly observed that RAPPID-M yielded a similar dose-response curve with a maximal signal at 2.22 nM (Figure 8b). The maximal signal of RAPPID-M was 1.5-times lower than the maximal signal of the RAPPID-G variant. However, the signal-to-background ratio of RAPPID-M was 413, compared to 133 for RAPPID-G. This large difference in signal-to-background ratio is because the background (BG) of RAPPID-M was unexpectedly ~5 times lower than the background of RAPPID-G (Figure 8c). This low background signal demonstrates that the pM-LB and pM-SB domains do not exchange to result in antibodies that bind both an LB and an SB fragment.

[0044] Next, the anti- IL-6 antibodies R508 and mhK23 were used to further compare the performance of unconjugated pG-based RAPPID, RAPPID-G and RAPPID-M assay and studied their stabilities over time in the fridge. Accordingly, we first made three different RAPPID assay mixtures: 1) RAPPID-M, consisting of R508-pM-LB and mhK23-pM-SB, 2) RAPPID-G, with R508-pG-LB and mhK23-pG-SB and 3) unconjugated RAPPID-G, consisting of R508-pG-LB and mhK23-pG-SB (Figure 9a). The unconjugated RAPPID-G sensor mixtures were not irradiated with UV light, resulting in pG-LB / SB domains that areAtty docket TUGE.P2006WO / 00643294 not covalently coupled to the anti-IL-6 antibodies. Therefore, these domains can exchange, enabling the binding of pG-SB and pG-LB on the same IgG antibody and generating an increased background signal. After the conjugation step, we made three assay mixtures by mixing the R508-LB and mhK23-SB domains to a concentration of 0.2 nM LB and 1 nM SB. Immediately after mixing, we applied the three sensor mixes (RAPPID-M, RAPPID-G and unconjugated RAPPID-G) to measure a range of IL-6 concentrations.

[0045] Figure 9b displays the dose-response curves of these three different RAPPID assays. Photoconjugated RAPPID-G showed the highest absolute signal at 2.22 nM, while RAPPID- M yielded the highest signal-to-background ratio (S / B = 669) at 6.67 nM. The signal-to- background ratio of the unconjugated RAPPID-G was lowest (S / B = 8.4), mainly due to a high background signal (Figure 8c). The remainder of the assay mixtures were stored at 4 °C. After 4 days, we used these sensor mixtures to again measure a range of IL-6 concentrations (Figure 9c). All three assays displayed an IL-6 dependent increase in luminescent signal, albeit with reduced absolute luminescent signals compared to the fresh sensor mix at day 0 (Figure 9b). The maximum luminescent signal decreased by 12.2%, 32.6% and 19.3% for RAPPID-G, unconjugated RAPPID-G and RAPPID-M, respectively. Furthermore, the signal- to- background ratios decreased to 71.2, 4.3 and 354 (Figure 9c). After storing the samples at 4 °C for 8 days, we performed the RAPPID assays again and compared the results with those from day 4 (compare 9d with 9c). The maximum luminescent signal decreased only by 2.4%, 3.7% and 0.2% for RAPPID-G, unconjugated RAPPID-G and RAPPID-M, respectively.These results were unexpected and suggest that there is minimal loss of activity when storing the RAPPID-M assay mixtures for 4 or 8 days and shows that the LB and SB domains do not exchange over time. Data presented in Figure 9b-d are additionally normalized and presented in Figure 9e-g.

[0046] Next, it is evaluated if the pM440 labeling strategy can be used to develop mouse IgGl RAPPID-M immunoassays. Mouse IgGl monoclonals are widely produced and used in diagnostics, their incompatibility with protein G photo-crosslinking is a major disadvantage of RAPPID-G. To increase the range of antibodies that can be labeled and thus expand the range of possible antigen targets, we labeled two anti-IL-6 mouse IgGl antibodies, 9540 and 2063 (R&D systems), with pM440-LB and pM440-SB, respectively. Similarly, the mixtures were incubated overnight. To evaluate the performance of RAPPID-M with mouse IgGl monoclonals, we mixed 500 pM of each labelled antibody with increasing concentration of IL-6. After 1 hour incubation, the bioluminescent signal was measured (Figure 10a). The obtained dose-response curve showed a maximal luminescent signal at 16 nM. ThisAtty docket TUOE.P2006WO / 00643294 demonstrates that the antibodies bind to IL-6 and the luciferase complex is reconstituted.

[0047] Subsequently, it was tested whether the RAPPID-M immunoassay can be extended to antibody fragments. We purified in-house the single chain variable fragment (scFv) of Adalimumab antibody in Escherichia coli and purified it via Ni2+affinity chromatography (Figure 10b). scFv was incubated with pM440-LB and pM440-SB overnight. TNFa, the target of the antibody is trimeric, thus the same antibody fragment can be used in both complexes. To evaluate the performance of the RAPPID-M with scFv, we mixed 500 pM of each immunoassay component and mixed with increasing concentration of TNFa. The bioluminescent signal was measured after 1 hour incubation. The Figure 10c shows a dose response curve and demonstrates the limit of detection at 250 pM and the maximum signal at 16 nM. Next, Fab fragment was obtained by proteolytic cleavage of Cetuximab antibody and employed to perform a RAPPID-M assay for detection of the anti-Cetuximab antibody (Figure lOd). The obtained Fab fragments were mixed with pM-LB or pM-SB in 1:2 ratio and incubated overnight at 4 °C. Using 1 nM of Fab-pM-LB and 1 nM of Fab-pM-SB, a 7- fold increase in bioluminescence activity was obtained upon addition of increasing concentrations of anti-cetuximab antibody, with a LOD of 62.5 pM.

[0048] The following Sequence information forms part of this disclosure:SEQ ID NO:1NDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGD NFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGY DDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDV KTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGSQLNELQLPESVKKVSLYGDYT GVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVIYDLFASKPFTHIDLTQ VTLQNSDNSAIDANKLKQAVGDIYNYRRFERQFQGYFAGGYIDKYLVKNVNTNK DSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNSEQ ID NO:1 Truncated version of Protein M encompassing amino acids 78-440 of the wild type Protein M, lacking the N-terminal 77 amino acids and the C-terminal 116 amino acids.SEQ ID NO:2MGSSHHHHHHSSGLVPRGSHMSLSLNDGSYQSEIDLSGGANFREKFRNFANELSEA ITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFAtty docket TUOE.P2006WO / 00643294NNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYD KWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGSQLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPL VLGSKTNVIYDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTY YRVNENYYPGASIYENERASRDSEFQNEILKRGGSGGSWSHPQFEKCSEQ ID NO:2 Protein M468 with antigen blocking domain (also bolded). The non bolded sequences are used (among others) for purification.SEQ ID NO:3MGSSHHHHHHKLGSCGASGTLVPRGSHMSLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTS NNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLI IRGLSGNGSQLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANS FGFNPLVLGSKTNVIYDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIYNYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNEFGGSGGSWSHPQFEKSSEQ ID NO:3 Protein M440 with N-terminal cysteine. The non bolded sequences are used (among others) for purification.SEQ ID NO:4AmMo-GTGTGTCTCCCTTGATGTCTGTGTTSEQ ID NO:4 Amine modified DNA.SEQ ID NO:5TATTATTACACTTCTGCTTCAAGGAGGTTASEQ ID NO:5 DCV specific DNA sequence.SEQ ID NO:6MGSSHHHHHHKLSGTLVPRGSHMSLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEAtty docket TUOE.P2006WO / 00643294YFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTY DKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLS GNGSQLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNP LVLGSKTNVIYDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIYNYRRF ERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNEFGGSGGSWSHPQFEKCSEQ ID NO:6 shows Protein M440 encompassing amino acids 78-440 of the wild type protein M (bolded), it also contains non-bolded sequences which are used (as tags or others) to facilitate purification.Atty docket TUGE.P2006WG / 00643294REFERENCESThe following references, along with those cited throughout the disclosure, are hereby incorporated by reference into the instant disclosure.1. Graille, M. et al. Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins. Structure 9, 679-687 (2001).2. Bjdrck, L. Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. The Journal of Immunology 140, 1194-1197 (1988).3. Graille, M. et al. Crystal structure of a Staphylococcus aureus protein a domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci U SA 97, 5399-5404 (2000).4. Gronenborn, A. M. et al. A Novel, Highly Stable Fold of the Immunoglobulin Binding Domain of Streptococcal Protein G. Science (1979) 253, 657-661 (1991).5. Bjorck, L. & Kronvall, G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. The Journal of Immunology 133, 969-974 (1984).6. Stahl, S. & Nygren, P. A. The use of gene fusions to protein A and protein G in immunology and biotechnology. Pathol Biol (Paris) 45, 66-76 (1997).7. Bjdrck, L. & Kronvall, G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. The Journal of Immunology 133, 969-974 (1984).8. Akerstrdm, B., Brodin, T., Reis, K. & Bjdrck, L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. The Journal of Immunology 135, 2589-2592 (1985).9. Hober, S., Nord, K. & Linhult, M. Protein A chromatography for antibody purification. Journal of Chromatography B 848, 40-47 (2007).10. Choe, W., Durgannavar, T. A. & Chung, S. J. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials 9, (2016).11. Hui, J. Z., Tamsen, S., Song, Y. & Tsourkas, A. LASIC: Light Activated Site-Specific Conjugation of Native IgGs. Bioconjug Chem 26, 1456-1460 (2015).12. Hui, J. Z. & Tsourkas, A. Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG. Bioconjug Chem 25, 1709-1719 (2014).13. Perols, A. & Karlstrom, A. E. Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains. Bioconjug Chem 25, 481-488 (2014).14. Chin, J. W. et al. Addition of p -Azido- 1 -phenylalanine to the Genetic Code ofAtty docket TUOE.P2006WO / 00643294Escherichia c oli. J Am Chem Soc 124, 9026-9027 (2002).15. Chung, B. H. et al. Photoactivable antibody binding protein: Site-selective and covalent coupling of antibody. Anal Chem 81, 936-942 (2009).16. Konrad, A., Eriksson Karlstrom, A. & Hober, S. Covalent immunoglobulin labeling through a photoactivable synthetic Z domain. Bioconjug Chem 22, 2395-2403 (2011).17. Perols, A. & Karlstrom, A. E. Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains. Bioconjug Chem 25, 481-488 (2014).18. Kanje, S. & Hober, S. In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies. Biotechnol J 10, 564—574 (2015).19. Lee, Y., Jeong, J., Lee, G., Moon, J. H. & Lee, M. K. Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli. Anal Chem 88, 9503-9509 (2016).20. Bjorck, L. & Kronvall, G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. The Journal of Immunology 133, 969-974 (1984).21. Grover, R. K. et al. A structurally distinct human mycoplasma protein that generically blocks antigen-antibody union. Science (1979) 343, 656-661 (2014).22. Ni, Y. et al. A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays. Nat Commun 12, 4586 (2021).23. Van Aalen, E. A., Wouters, S. F. A., Verzijl, D. & Merkx, M. Bioluminescent RAPPID Sensors for the Single-Step Detection of Soluble Axl and Multiplex Analysis of Cell Surface Cancer Biomarkers. Anal Chem 94, 6548-6556 (2022).24. Li, R. et al. Point-of-care therapeutic drug monitoring of tumour necrosis factor-a inhibitors using a single step immunoassay. Sensors & Diagnostics (2023) doi: 10.1039 / D3SD00131H.25. Aalen, E. A. van et al. Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines. Anal Chem (2023) doi: 10.1021 / ACS. ANALCHEM.3C00745.26. Dixon, A. S. et al. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol 11, 400-408 (2016).27. Hall, M. P. et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7, 1848-1857 (2012).28. Agasti, S.S. et al. DNA- barcoded labelling probes for highly multiplexed Exchange - PAINT imaging. Chem Sci 8, 3080-3091 (2017).Atty docket TUGE.P2006WO / 0064329429 Sano, T. et al. Immuno- PCR : very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258, 120-122 (1992).30 Zakeri, B., Fierer, J.O.F. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. U. S. A. 109, E690-E697 (2012).31 Keppler, A., Gendreizig, S. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21, 86-89 (2003).32 Lovendahl, K. et al. Sequence-directed covalent protein-DNA linkages in a single stepp using HUH-tags. J. Am. Chem. Soc. 139, 7030-7035 (2017).33 Dong, J., et al. PM Q-probe: a fluorescent binding protein that converts many antibodies to a fluorescent sensor. Biosens. Bioelectron. 165, 112425 (2020).34 Thompson, I. A. P., et al. An antibody-based molecular switch for continuous smallmolecule biosensing. Sci. Adv, 9, eadh4978 (2023).

Claims

Atty docket TUOE.P2006WO / 00643294CLAIMS1. An engineered molecule comprising a polypeptide having an amino acid sequence that is at least 90% identical in amino acid sequence to a peptide from amino acid number 78 to amino acid 440 of wild-type protein M of Mycoplasma genitalium.

2. The engineered molecule of claim 1, wherein the polypeptide lack a C-terminal domain of wild-type protein M that blocks binding between the Ig or fragment thereof and an antigen.

3. The engineered molecule of claim 1, wherein the polypeptide has at least 90%, or at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 1.

4. The engineered molecule of claim 1 , wherein the polypeptide is capable of binding to a variable region of a light chain of an immunoglobulin (Ig) or fragment thereof, wherein the immunoglobulin (Ig) or fragment thereof is at least one member selected from the group consisting of an IgG, an antigen-binding regions (Fab), an single-chain variable fragments (scFv) and an IgY.

5. The engineered molecule of claim 1, further comprising a second moiety, wherein the second moiety is a labeling molecule selected from the group consisting of a luciferase domain, an epitope tag, a fluorescent tag, an oligonucleotide, a reporter enzyme, an imaging tag, a small molecule chemical and combination thereof.

6. A composition comprising the engineered molecule of claim 1.

7. A polynucleotide comprising a coding sequence encoding the polypeptide of claim 3.

8. An engineered molecule comprising a polypeptide having an amino acid sequence that is at least 90%, or at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 3.

9. The engineered molecule of claim 8, wherein the polypeptide has a cysteine at a position n residues from the N-terminus of the polypeptide, n being an integer between 1 andAtty docket TUOE.P2006WO / 0064329410. A polynucleotide comprising a coding sequence encoding the polypeptide of claim 8.

11. A method for labeling an immunoglobulin or fragment thereof in a sample, comprising mixing the molecule of claim 1 with the sample, allowing the molecule to bind to the Ig or fragment thereof in the sample, wherein the molecule is pre-conjugated with a labeling molecule prior to the mixing.

12. A method for labeling an immunoglobulin or fragment thereof in a sample, comprising mixing the molecule of claim 8 with the sample, allowing the molecule to bind to the Ig or fragment thereof in the sample, wherein the molecule is pre-conjugated with a labeling molecule prior to the mixing.

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