WO2024121156A1

WO2024121156A1 - Cyclic compounds and their use in assays for detecting antibodies

Info

Publication number: WO2024121156A1
Application number: PCT/EP2023/084359
Authority: WO
Inventors: Aubin MICHALON
Original assignee: Neurimmune Ag
Priority date: 2022-12-05
Filing date: 2023-12-05
Publication date: 2024-06-13

Abstract

Provided are cyclic compounds based on peptides comprising an epitope from an amyloidogenic protein involved in systemic amyloidosis, which are useful for immunoassays and potency assays in antibody drug research and development.

Description

Cyclic compounds and their use in assays for detecting antibodies

TECHNICAL FIELD

The present disclosure generally relates to cyclic compounds comprising peptides containing an epitope of a systemic amyloidogenic protein as well as to their use for detecting, quantifying and validating therapeutically useful antibodies and equivalent binding molecules. The present disclosure also relates to the use of the cyclic compounds in potency assays, which are particularly useful for batch release of a pharmaceutical composition comprising the antibody or like binding molecule, specifically when conducting clinical trials, applying for marketing authorization and for quality control of the approved drug.

BACKGROUND

Monoclonal antibody drugs have been maturing from a research target to an improved technology, from clinical research to commercialization over the past three decades. In recent years, the number of monoclonal antibody drugs approved for marketing has rapidly increased, with the landmark 100^th monoclonal antibody product being approved by the United States Food and Drug Administration (FDA) in 2021. In 2019, nine of twenty top-selling drugs were monoclonal antibody drugs (Mullard, Nature Reviews Drug Discovery 20, 491-495 (2021), DOI: 10.1038/d41573-021 -00079-7).

One promising application for therapeutic antibodies is the treatment of amyloidoses, which occur due to toxic amyloid aggregations. Neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease represent a highly prevalent class of fatal localized amyloidoses in which amyloid deposits form in the nervous system where they induce death of specific neuronal cell types. In systemic amyloidoses, such as immunoglobulin light chain, transthyretin and dialysis-related amyloidosis, several organs are affected as the amyloidogenic protein is distributed in different sites of the body as it travels from the site of synthesis. Antibodies and antibody fragments have been already proven to be effective anti-amyloid molecules. For example, aducanumab shows dose-dependent clearance of amyloid deposits in Alzheimer's patients and has recently been approved by the FDA for use in treatment of Alzheimer's disease. Antibody drug discovery and development typically rely on immunoassays, for example for detecting and quantifying the antibody, wherein the sensitivity of the assay is dependent on the affinity of the antibody to its target protein. Likewise, the sensitivity and reliability of potency assays inter alia depend on the antibody’s affinity to its target, in cellular assays. Therefore, due to the importance of immunoassays at different stages of antibody screening and drug development there is a constant need for improvement of such as assays involving optimizing various parameters and employing different strategies. The goal is to enhance the assay's sensitivity, specificity, accuracy, and reproducibility. Key parameters and means that are typically subject to improvement include optimizing antigen coating, use of effective blocking agents to prevent non-specific binding, antibody dilution, incubation conditions, detection system and signal amplification, to name a few.

Accordingly, the problem underlying the present disclosure is the provision of systems and methods for immunoassays for detecting, isolating and characterizing antibodies.

SUMMARY

The present disclosure generally relates to a cyclic compound comprising a peptide or protein fragment, also referred to as cyclic peptide compound, which comprises an epitope of an antibody or equivalent binding molecule and to the use of such cyclic peptide compound in a method of determining the potency of the antibody or binding molecule as well as in drug discovery and diagnostic field in general. The present disclosure further relates to the use of the cyclic peptide compounds in potency assays, e.g., in determining or comparing potencies of binding molecules such as anti-TTR antibodies. The assay methods of the present disclosure are useful for testing batch release of a pharmaceutical composition comprising an antibody or other binding molecules, for example, in evaluation of drug candidates for clinical trials, applying for marketing authorization and for quality control of the approved drug. More specifically, the cyclic peptide compound comprises an epitope of an amyloidogenic protein and can be used in assays for detecting amyloid specific antibodies and corresponding binding fragments thereof. In this context, the cyclic compound of the present disclosure comprises the epitope of an antibody, and thus the cyclic compound is designed to be bound by the antibody. As explained further below, preferably the epitope of the peptide is selected from an epitope that is accessible to binding by the antibody only in the misfolded and/or aggregated form of the protein, for example the epitope is exposed in the pathological protein aggregate only. As also discussed further below and demonstrated in the appended Examples as well as in Examples of WO 2023/099788 Al, the cyclic compound of the present disclosure preferably provides for a higher binding affinity to the antibody than the amyloidogenic protein or protein aggregate, preferably also higher than a corresponding linear peptide in an ELISA assay.

As shown in the appended Examples, a cyclic peptide comprising a TTR epitope (cyclic TTR peptide) has been used as target antigen in immunological and biological assays for the detection of antibodies and for measuring their potency, respectively.

In particular, as illustrated in Example 1, a cyclic peptide as target antigen provides for higher sensitivity of an ELISA assay for an antibody than the natural antigen, i.e., aggregated TTR, also referred to as misfolded TTR. Similar was observed in reporter gene assays as illustrated in Examples 2 and 5, where a cyclic peptide was shown to provide for an improved ADCP assay. Surprisingly, when using the cyclic peptide as antigen instead of a TTR aggregate, the assay showed a remarkable improvement of sensitivity and reliability. Without intending to be bound by theory, the extraordinary performance of the assay with a cyclic peptide could be due to a very stable conformation adopted by the cyclic peptide since it is constrained by having the two extremities connected together, and thus, mimics the stability of a protein aggregate. However, as shown in Examples 2 and 5, even if such theoretical considerations are taken into account, the assay with the cyclic peptide as the target antigen is an order of magnitude more precise and sensitive than the assay with the target antigen present as protein aggregate. Again, without intending to be bound by theory this could be due to the smaller size of the peptide compared to the protein and the resulting higher epitope density which translates into both higher binding capacity and higher avidity effect. Moreover, the epitope in the cyclic peptide might be better accessible compared to the full-length protein. Nevertheless, since the cyclic TTR peptide including the linker amino acids with a total of 31 amino acids is only four times smaller than the full-length TTR protein, the size of the cyclic peptide cannot a priory account for the observed effect. A further reason could be the better control of the conformation for a synthetic peptide compared to a recombinant protein. In particular, more than 95% of the peptide is cyclized, whereas it is unknown what fraction of the misfolded-aggregated TTR protein adopts the amyloid conformation. Because mis.WT-TTR is a heterogenous mix of conformations, a significant fraction of the protein may form amorphous aggregates instead of amyloid. In summary, although with the results of the experiments described in the appended Examples good explanatory approaches and theories can now be developed in retrospect to envisage further cyclic peptides with suitable epitopes for the detection and identification of potent antibodies against amyloidogenic, in particular systemic amyloidogenic proteins, this was not foreseeable without knowledge of the present results and teaching of the present disclosure. In this context, and again without wishing to be bound to any theory, it is noteworthy that it has recently been shown by cryo-EM studies that the amyloid structures of systemic amyloidogenic proteins such as ATTR and AL amyloidosis caused by misfolding of immunoglobulin light chains (LCs) are on the one hand similar, but on the other hand substantially different from those of local amyloidogenic proteins such as tau; see Figure 5 in Schmidt et al., Nat. Commun. 10 (2019), 5008, https://doi.org/10.1038/s41467-019-13038. Therefore, it is reasonable to assume that the present results for cyclic peptides derived from TTR can also be applied to other systemic amyloidogenic proteins.

Accordingly, the present disclosure relates to the provision of cyclic compounds comprising peptides containing an epitope of a systemic amyloidogenic protein, the epitope preferably being accessible to binding by an antibody only in the misfolded and/or aggregated form of the protein, as in the case of a neoepitope, and/or the epitope being at least not present in the physiologically active form of the protein, e.g. in the case of an epitope accessible in the monomer of the TTR protein, which is hidden in the physiologically active tetramer and is no longer accessible to antibody binding. As illustrated in the appended Examples, the cyclic peptide compound of the present disclosure is particularly useful in immunological assays, such as ELISA assay which can be used for example for drug discovery or diagnostic methods as well as in potency assays for characterizing therapeutically useful antibodies and equivalent binding molecules for which antibody Fc-mediated activities play a critical role in the mechanism of action.

In particular, the outstanding performance of the cyclic peptide compound as target antigen has been first shown in the ELISA assays described in Example 1. ECso values for antibody binding to the cyclic peptide were compared to ECso values for antibody binding to the protein aggregate and to a linear peptide comprising the same epitope as the cyclic peptide. The cyclic peptide performed best, in that it showed the highest binding affinity to the antibody, i.e., the lowest ECso value. As mentioned above, the higher binding affinity between the antibody and the cyclic peptide in comparison to the protein aggregate could be due to the higher epitope density, which results in better apparent binding affinity, due to the higher binding capacity and higher avidity effect, due to the better accessibility of the epitope in the cyclic peptide as compared to the full-length protein, and/or due to the better control of the conformation for a synthetic peptide as compared to a recombinant protein. Accordingly, in one embodiment, the cyclic peptide compound and cyclic peptide of the present disclosure, respectively, provide for a higher binding affinity with an antibody than with the target protein it is derived from and higher than a corresponding linear peptide in an ELISA assay such as described in appended Example 1, preferably at least 2-fold, more preferably at least 3-, 4-, or 5-fold and most preferably at least 6-, 7- 8-, 9- or 10-fold higher compared to the full-length target protein and/or linear peptide.

The therapeutic utility of an antibody, in particular as a drug effective for the treatment of amyloidoses, depends not only on the ability of the antibody to bind the aggregate, but also on antibody Fc-mediated activities, which play a critical role in the mechanism of action. Binding of antibodies to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles (called antibody-dependent cellular phagocytosis, or ADCP). When producing a pharmaceutical composition, it is not sufficient to formulate the drug substance into the drug product, it is also essential that the resulting drug product is approved by the regulatory body in the country in which the pharmaceutical composition is to be used. In the United States, the responsible regulatory body is the FDA (http://www.fda.gov/), and in Europe, it is for instance the European Agency for the Evaluation of Medicinal Products (EMEA) (http://www.emea.eu.int/).

The approval process is thoroughly regulated, and the drug developers are required to submit a substantial amount of information regarding the drug product candidate to the regulatory authorities to obtain approval. This may include information about the potency of the drug product candidate and corresponding assays to determine the potency. Such a potency assay serves to characterize the product, to monitor lot-to-lot consistency and to assure stability of the product. The potency of antibodies for which Fc binding to Fc receptor plays a critical role for the mechanism of action are traditionally measured by use of biological assays in which the effect assessed is dependent on Fc-Fc receptor binding.

Therefore, the assay to characterize the product, to monitor lot-to-lot consistency and to assure stability of the product is of clinical importance and should be sufficiently sensitive to detect differences which may impact mechanism of action and function of the product. As shown in Example 2, the use of the cyclic peptide of the present disclosure provided for an improved ADCP assay. Surprisingly, when using the cyclic peptide as antigen instead of a TTR aggregate, the assay showed a remarkable improvement of sensitivity and reliability. Furthermore, the initially developed highly sensitive ADCP assay as described in Example 2 has been confirmed and validated; see Example 5. In particular, the assay as outlined in Example 5 has been shown to have the capacity to detect potency changes related to Fc domain alterations in the range of 40% to 180%, /.< ., up to 60% potency loss and 80% potency increase.

Thus, the present disclosure also relates to the use of the cyclic peptide compound of the present disclosure as target antigen in a method for determining the phagocytosis-related potency of an antigen binding molecule comprising an Fc domain, such as an antibody, wherein in a preferred embodiment, the antigen binding molecule is an antigen binding molecule specific for amyloidogenic proteins, preferably in an aggregated, misfolded, and non-physiological form.

In one embodiment, such potency assay comprises the steps of

(a) contacting the cyclic peptide compound of the present disclosure as target antigen with the binding molecule under conditions allowing the formation of a binding moleculeantigen complex, wherein in a preferred embodiment, the cyclic peptide compound is bound on a solid support, for example a microtiter plate;

(b) contacting the binding molecule-antigen complex with a population of effector cells, which are preferably Jurkat cells, that are engineered to express an Fc receptor, preferably a human Fc receptor FcyRI (CD64), and harbor a reporter gene, preferably encoding a bioluminescent protein, preferably a luciferase, under the control of a response element that is responsive to activation by the Fc receptor, preferably wherein the response element is an NF AT (Nuclear Factor of Activated T cells) response element, under conditions allowing for binding of the Fc domain to the Fc receptor, wherein binding of the Fc domain to the Fc receptor results in intracellular signaling and mediates a quantifiable reporter gene activity; and

(c) detecting the reporter gene activity, wherein at least one mechanism of action of the Fc domain of the binding molecule is mediated through the binding of the Fc domain to a Fc receptor, and the reporter gene activity is indicative for the potency of the binding molecule, preferably wherein the mechanism of action of the Fc domain is to induce antibody-dependent cell-mediated phagocytosis (ADCP). The detailed embodiments to the potency assay are described and claimed in WO 2023/099788 Al, which are herein incorporated by reference.

Accordingly, the cyclic peptide compound of the present disclosure which comprises an epitope of the amyloidogenic protein can be used as target antigen instead of the amyloidogenic protein itself which - as mentioned above - leads to highly sensitive and reliable potency assays. The cyclic peptide of the present disclosure is particularly suitable in an assay for determining the potency of antibodies and Fc domain containing binding molecules which bind amyloidogenic TTR or other amyloidogenic proteins that are involved in systemic amyloidosis.

The superiority of the cyclic peptide might be explained, without being bound by theory, by the extreme flexibility of linear peptides that therefore can adopt a virtually infinite number of conformations in contrast to a cyclic peptide which is constrained by having the two extremities connected together and has therefore much less flexibility and adopts more stable conformations. In different terms, a cyclic peptide has a lower entropy than the same amino acid sequence in a linear form.

Accordingly, the provision of the cyclic peptide compound in accordance with the present disclosure represents an important contribution to the art given its outstanding utility as a suitable target to study the binding between a target antigen and a corresponding target antigen binding molecule, for example in assays which require a high sensitivity. Nevertheless, the present disclosure also relates to the linear form of the cyclic peptide compound and cyclic peptide, respectively, for example for use a precursor for preparing the cyclic peptide compound or as a control in the experiments.

The assay as described above, z.e., potency assay using the cyclic peptide compound of the present disclosure, can be applied in a method of producing a pharmaceutical composition comprising a target antigen binding molecule, wherein first after production, the potency of said binding molecule is analyzed. Based on the result, it is assessed whether the binding molecule may be used in a pharmaceutical composition or not. In particular, only binding molecules that are regarded as potent according to the assay are selected for further use and formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier. The potency assay can also be used in a method for analyzing and selecting a batch of a pharmaceutical composition of a target antigen binding molecule, wherein a sample of the batch to be analyzed and a control sample are subjected to said potency assay and the reporter gene activity of the sample is compared to that of the control. The batch for which the sample shows greater, equal or not substantively less reporter gene activity compared to the control is finally chosen for further use. Thus, the assay can be used for verifying lot-to-lot consistency.

The present disclosure further relates to a kit which is preferably designed to carry out the potency assay as disclosed herein, in particular to assay the potency of a binding molecule comprising an Fc domain to induce ADCP, wherein the kit comprises at least the cyclic peptide compound of the present disclosure or the corresponding linear precursor, which could also serve a control similar as shown for the TTR peptide in the Examples. The kit may further comprise

(i) a population of effector cells engineered to express an Fc receptor, preferably a human Fc receptor FcyR, and harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor, preferably wherein the population of effector cells is a population of Jurkat cells and the reporter gene encodes a luminescence protein, preferably a luciferase under control of NF AT response element;

(ii) a corresponding substrate for the reporter; preferably wherein the kit further comprises one or more of the following:

(iii) a solid support, preferably a microtiter plate, preferably a 96-well plate including a lid;

(iv) washing, blocking and assay/sample dilution buffer; and/or

(v) a monomer control of the target antigen and/or a positive control anti-target antigen antibody.

The immunological and biological assays as described herein have been illustrated with the cyclic peptides of the present disclosure comprising a TTR epitope as the target antigen and an anti-TTR antibody, such as, e.g., NI-301.37F1, which is disclosed in international application WO 2015/092077 Al, and which has been described to be capable of activating the immune system for the elimination of TTR fibrils in an animal model; see international application WO 2020/094883 Al. TTR in its physiological form is a tetramer protein that develops amyloidogenic properties when it dissociates into monomers and forms transthyretin amyloidosis (ATTR), a systemic amyloidosis. Systemic amyloidosis is a protein misfolding disorder caused by extracellular deposition of amyloid leading to organ dysfunction while localized amyloidosis refers to intracellular and/or extracellular amyloid deposits that occur only in the organ or tissue of precursor protein synthesis such as intracellular Tau protein fibrils and extracellular amyloid-P fibrils and plaques in Alzheimer's disease. In principle, the cyclic peptide compound of the present disclosure can comprise any peptide or protein fragment which is capable of forming a cyclic peptide compound, in particular a peptide or protein fragment that comprises a neoepitope as mentioned above. In a particularly preferred embodiment, the (neo)epitope is hidden in the target antigen’s naturally folded conformation but accessible to antibody binding following unfolding and aggregation, e.g., like the linear epitope WEPFA of antibody NI-301.37F1, located in position 41-45 of mature TTR protein. Similarly, the method of the present disclosure is applicable to any cyclic peptide that contains and displays the epitope(s) of the target antigen binding molecule to be tested, for example an epitope which is only exposed in the misfolded variant, a conformational epitope on aggregates, fibrils and/or oligomers, an epitope on extracellular variant of an otherwise physiological protein that is located intracellularly, or an epitope specific for exogenous pathogens such as fungi, bacteria, and viruses.

Nevertheless, in accordance with the present Examples, the cyclic peptide compound of the present disclosure preferably comprises a peptide or protein fragment comprising an epitope from an amyloidogenic protein involved in systemic amyloidosis, preferably an epitope that is exposed in the misfolded and non-physiological form of the protein, such as transthyretin. Said cyclic peptide compound is particularly suited and thus preferably used in a method for detecting antibodies specific for amyloidogenic proteins involved in systemic amyloidosis, in particular antibodies binding the misfolded and non-physiological form of the protein and in a method for determining the potency of said antibodies, respectively.

The cyclic peptide compound of the present disclosure is particularly useful for measuring antibody potency to activate ADCP.

In addition, the cyclic peptide compound of the present disclosure are especially useful in methods for identifying and optionally obtaining an antibody which binds to an amyloidogenic protein involved in systemic amyloidosis, the method typically comprising the steps of:

(a) providing, optionally producing one or more potentially amyloidogenic protein binding antibodies or a source thereof; (b) subjecting the one or more of potentially amyloidogenic protein binding antibodies or source thereof to a binding assay comprising the cyclic peptide compound of the present disclosure; and

(c) identifying and optionally obtaining an antibody (subject antibody) that has been determined to bind to the cyclic peptide compound.

This method can be combined with the potency assay of the present disclosure and as described in international application WO 2023/099788 Al, respectively, and/or any other suitable method for further determining the diagnostic or preferably therapeutic utility of the subject antibody.

Hence, a further embodiment of the present disclosure consists in a method of producing a pharmaceutical composition comprising an antibody which binds to an amyloidogenic protein, the method comprising at least the steps of:

(a) providing, optionally producing one or more potentially amyloidogenic protein binding antibodies or a source thereof;

(b) subjecting said one or more potentially amyloidogenic protein binding antibodies or a source thereof to a binding assay comprising the cyclic peptide compound of the present disclosure;

(c) identifying and optionally obtaining an antibody (subject antibody) that binds to the cyclic peptide compound; and

(d) formulating the antibody identified and optionally obtained in step (c) or a derivative thereof with a pharmaceutically acceptable carrier.

The source of antibodies is not limited and comprises natural as well as synthetic antibodies obtained, for example from immunized laboratory animal such as a rodent, preferably mouse, most preferably Ig humanized mouse; human blood or a fraction thereof preferably comprising memory B cells; recombinant antibody libraries such as phage, yeast, and ribosome systems or mammalian cell systems such as CHO and HEK; see also the “Detailed description of the disclosure" for further sources of antibodies and other target binding molecules.

The binding assay used in the methods mentioned above preferably comprise ELISA such as performed in Example 1. In a preferred embodiment of the methods of the present disclosure for identification and obtaining subject antibodies and their further use in being formulated in a pharmaceutical composition and drug development, respectively, the antibody identified and optionally obtained in step (c) competes with a reference antibody for binding the amyloidogenic protein, preferably wherein the subject antibody has a lower ECso for the amyloidogenic protein than the reference antibody.

Unless defined otherwise in the present application, 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 disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure , the exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples are illustrative only and not intended to be limiting.

Further embodiments of the present disclosure will be apparent from the description, Examples and claims that follow. The person skilled in the art will appreciate that every characterization of a generic feature of a general embodiment in the following can and preferably are intended to be combined with the characterization of one or more of the other features of such general embodiment. In addition, unless specifically indicated otherwise, embodiments described herein for antibodies, including the Examples, though being preferred embodiments, are meant as illustration and hereby are extrapolated to any target binding molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: Improved sensitivity of ELISA assay. Comparison of the binding specificity of antibody NI-301.37F1 to (A) peptides TTR34-54cyc, TTR40-49, Biotin. TTR40-49, and mis-WT-TTR, and to (B) peptides TTR34-54cyc, Biotin. TTR34-54cyc, TTR40- 49, Biotin. TTR40-49, and mis-WT-TTR using ELISA assays showed specific binding of NI-301.37F1 to mis.WT-TTR, and showed that NI-301.37F1 binding to the cyclic TTR34-54cyc peptide is about 10-fold stronger than binding to mis.WT-TTR. The curve for peptide TTR40-49 is congruent with the one of the Biotin. TTR40-49.

Fig. 2: Improved ADCP assay by use of a cyclic peptide compound as the target antigen. Measuring the potency of antibody NI-301.37F1 in the ADCP assay with a cyclic TTR peptide (TTR34-54cyc) demonstrates the ability of antibody NI-301.37F1 RS to activate phagocytosis in a dose-response, i.e., in a dose-dependent manner, characterized by an ECso of 19.8 ng/ml.

Fig. 3: ADCP assay using TTR34-54cyc as target antigen for detecting changes in antibody activity by comparison of NI-301.37F1 reference sample (NI-301.37F1 RS) with samples with lower (NI-301.37F1 50% (A) and 70% (B)) concentration, and higher (NI-301.37F1 130% (C) and 150% (D)) concentration showed that the assay had the capacity to detect a 50% loss of antibody activity and a 50% increase in antibody activity, respectively.

DETAILED DESCRIPTION

The present disclosure relates to a cyclic compound comprising a peptide or protein fragment which comprises an epitope from an amyloidogenic protein involved in systemic amyloidosis. As described herein, typically the epitope in the peptide and protein fragment, respectively, in the cyclic compound of the present disclosure is an epitope of an antibody, i.e., an amyl oid/aggr egate specific antibody, and the cyclic compound is bound by the antibody. Accordingly, the present disclosure also relates to the use of such cyclic peptide compound in drug discovery and in the diagnostic field as well as in methods of determining the potency, in particular the potency to activate antibody-dependent cell-mediated phagocytosis (ADCP), of a target antigen binding molecule comprising an Fc domain, preferably wherein the target antigen binding molecule is an antibody which binds to amyloidogenic proteins. The present disclosure further relates to the use of the cyclic peptide compound in corresponding potency assays, which are particularly useful for batch release of a pharmaceutical composition comprising an antibody or like binding molecule, specifically when conducting clinical trials, applying for marketing authorization and for quality control of the approved drug.

Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2; Second edition published 2006, ISBN 0-19- 852917-1 978-0-19852917-0.

The term "protein" as used throughout the description includes fragments and peptides of the (full-length) protein, which contain and expose the epitope of the target antigen binding molecule to be tested, for example an antibody. Reference to the "cyclic peptide" herein can refer to a fully proteinaceous compound, e.g., wherein the linker is 2, 3, 4, 5, 6, 7 or 8 amino acids, or wherein no linker is present. For example, it is possible that the native protein sequence, i.e., amino acid stretch comprising the epitope of the antibody allows cyclization, for example due to the presence of two cysteines in appropriate distance, without addition of extra amino acids. It is understood that properties described for the cyclic peptide determined in the examples can be incorporated in other compounds, e.g., cyclic peptide compounds comprising non-amino acid linker molecules. "Cyclic peptide" and "cyclic compound" can be used interchangeably when the cyclic compound is composed of amino acids. Furthermore, the term "cyclic compound" can be used interchangeably with the term "cyclic peptide compound".

The term "linker" as used herein means a chemical moiety that can be covalently linked directly or indirectly to the protein fragment or peptide as defined herein. The linker ends can for example be joined to produce a cyclic peptide compound. The linker can be present at a location at the N- and C-termini. Alternatively, the linker may at an internal position at "some distance" from the termini. The linker can comprise one or more functionalizable moi eties such as one or more cysteine (C) residues. The linker can be also linked via the functionalizable moieties to other proteins or components. The cyclic peptide compound comprising the linker is of longer length than the peptide or protein fragment itself.

The term "functionalizable moiety" as used herein refers to a chemical entity with a "functional group" which as used herein refers to a group of atoms or a single atom that will react with another group of atoms or a single atom (so called "complementary functional group") to form a chemical interaction between the two groups or atoms. In the case of cysteine (C), the functional group can be -SH which can be reacted to form a disulfide bond. The reaction with another group of atoms can be covalent or a strong non-covalent bond, for example as in the case as biotin-streptavidin bonds, which can have dissociation constant (Kd) of about le-14. A strong non-covalent bond as used herein means an interaction with a Kd of at least 1 e-9, at least le-10, at least 1 e-11, at least 1 e-12, at least 1 e-13 or at least 1 e-14.

During the course of the experiments performed in accordance with the present disclosure, it was surprisingly found that a cyclic peptide comprising an epitope of TTR, in particular the epitope recognized by the anti-TTR antibody NI-301.37F1 (WEPFA (SEQ ID NO: 1), which binds selectively with high affinity to TTR aggregates of either wild-type or variant TTR as described in WO 2015/092077 Al, is an excellent target antigen in ELISA and ADCP assays. As described in Example 1 and illustrated in Figure 1, the antibody displays highly specific binding to said cyclic peptide compound, wherein the binding affinity of the antibody to the cyclic peptide is even an order of magnitude higher than to its native target antigen, z.e., misfolded TTR against which the antibody had been originally screened and identified. This remarkable effect was unexpected and advantageous since such cyclic peptide compound could not only substitute full-length amyloidogenic proteins and the preparation of aggregates/fibrils thereof, which is prone to variability and more time consuming than preparation of a cyclic peptide, but, as illustrated in Examples 1, 2 and 5, and shown in Figures 1 to 3, the cyclic peptide compound represents an excellent target antigen in binding assays like ELISA and functional assays such ADCP which require high sensitivity and reproducibility.

Originally, the cyclic TTR peptide was designed to resolve crystal structures of a Fab fragment of antibody NI-301.37F1 in complex with its TTR antigen to gain information about the three- dimensional structure of the antibody-antigen complex to understand its mechanism of action. It was a coincidence that the cyclic TTR peptide was used to replace full-length recombinant TTR protein in an ELISA assay for the determination of antibody NI-301.37F1, i.e., the IgG antibody, and surprisingly revealed that the ELISA assay became much more sensitive and reliable compared to the use of recombinant TTR protein; see Example 1 and Figure 1. Subsequent experiments even more surprisingly demonstrate that use of the cyclic TTR peptide substantially improves the sensitivity and reliability of the potency assay.

In the following, analysis of cryo electron microscopy (cryo-EM) structures revealed that the two ends of the unresolved loop at in contact with each other and suggests that on this basis the epitope and peptide sequence for the cyclic peptide and cyclic peptide compound, respectively, could have been selected as well. Accordingly, the use of cryo-EM structures, in addition or alternatively to peptide design in Fab-peptide antigen crystallographic structures may be used to choose the appropriate epitope and amino acid sequence containing the same for the design of the cyclic peptide of the present disclosure, which displays as advantageous properties as the cyclic TTTR peptide that has been experimentally proven to be so effective and a reliable tool in ELISA and ADCP assay. As mentioned hereinbefore, it is noteworthy that it has been shown by cryo-EM studies that the amyloid structures of systemic amyloidogenic proteins such as ATTR and AL amyloidosis caused by misfolding of immunoglobulin light chains (LCs) are on the one hand similar, but on the other hand substantially differ from those of local amyloidogenic proteins such as tau; see Figure 5 in Schmidt et al., Nat. Commun. 10 (2019), 5008, https://doi.org/10.1038/s41467-019-13038. Therefore, it is prudent to expect that the present results for cyclic peptides derived from TTR can also be applied to at least other systemic amyloidogenic proteins.

Methods for generating crystal structures of an antibody and its Fab fragment, respectively, to a peptide and its complex with peptide antigen are well known to the person skilled in the art; see, e.g., Amit et al., Science 233 (1986), 747-753. The same applies to cryo electron microscopy; see, e.g., Schmidt et al. (2019), supra.

This finding now opens the opportunity to generate further cyclic peptide compounds comprising epitopes of other amyloidogenic proteins. For example, once an epitope has been selected and fragment or peptide sequence of the amyloidogenic protein has been selected, the program PEP -FOLD, which can predict peptide structures from amino acid sequences and when applied to the TTR cyclic peptide seems to reasonably predict the presentation of the epitope, could be used to design further cyclic peptides, such as those described herein, that mimic for example the antibody binding epitopes of amyloidogenic proteins.

Accordingly, the present disclosure relates to a cyclic peptide compound and linear precursor thereof comprising a peptide containing an epitope of a systemic amyloidogenic protein, the epitope preferably being accessible to binding by an antibody only in the misfolded and/or aggregated form of the protein, as in the case of a neoepitope, and/or the epitope being at least not present in the physiologically active form of the protein, e.g. in the case of an epitope accessible in the monomer of the TTR protein, which is hidden in the physiologically active tetramer and is no longer accessible to antibody binding. Such a peptide is preferably an antigenic peptide and an antigenic cyclic peptide compound, respectively. As illustrated in Examples 1, 2 and 5, the cyclic peptide compound of the present disclosure is particularly useful in immunological assays, like ELISA assays and in biological assays, like the reporter gene assay used as potency assay as disclosed herein.

The cyclic peptide compound of the present disclosure can either comprise or consist of a protein fragment or peptide which consists of the epitope recognized by the target antigen binding molecule, e.g., an antibody or antigen binding molecule specific to amyloidogenic proteins, or which comprises the epitope recognized by the target antigen binding molecule, meaning that additional amino acids or other chemical entities used for example for cyclization of the peptide or protein fragment as described further below can be present in the protein fragment or peptide which forms the cyclic peptide compound.

The additional amino acids can be amino acids which are naturally located adjacent to the epitope sequence, z.e., amino acids that are flanking the epitope sequence, and which are present in the protein sequence the protein fragment or peptide is derived from, z.e., the protein fragment or peptide which forms the cyclic peptide compound comprises an epitope of the target antigen binding molecule and further amino acids that are adjacent to the epitope and that are flanking the epitope, respectively. The number of those adjacent/flanking amino acids can vary and can be, for example, between 1, 2, or 3 amino acid and 50 amino acids, preferably between 1, 2, or 3 amino acids and 40 amino acids, more preferably between 1, 2, or 3 and 30 amino acids, more preferably between 1, 2, or 3 and 20 amino acids, more preferably between 10 and 20 amino acids, wherein the amino acids are either distributed equally N-terminal and C-terminal to the epitope sequence or unequally, with for example 7 additional amino acids N-terminal and 9 amino acids C-terminal to the epitope.

In addition, or alternatively, the protein fragment or peptide comprises in one embodiment a linker, z.e., the protein fragment or peptide can either comprise the epitope recognized by the target antigen binding molecule without any adjacent amino acids, and a linker, or can comprise the epitope and the adjacent amino acids as defined above, and a linker. In a preferred embodiment, the protein fragment or peptide which forms the cyclic peptide compound of the present disclosure comprises the epitope which is recognized by the target antigen binding molecule as well as amino acids adjacent to the epitope, and a linker. Preferably, the linker is covalently coupled directly or indirectly to the N-terminus residue of the protein fragment or peptide and to the C-terminal residue of the protein fragment or peptide.

Methods for cyclization of peptides are generally known in the art. For example, cyclization can be performed by chemical crosslinking using inter alia chemical scaffolds. Crosslinking requires functional groups and just few protein chemical targets account for the vast majority of crosslinking techniques, e.g., primary amines (-NH2), wherein this group exists at the N- terminus of each polypeptide chain and in the side chain of lysine residues; carboxyls (- COOH), wherein this group exists at the C-terminus of each polypeptide chain and in the side chains of aspartic acid and glutamic acid; and sulfhydryls (-SH), wherein this group exists in the side chain of cysteine.

Scaffold-based cyclization is one of the most frequently used methods because it can be applied to chemically or biologically synthesized peptides. In general, scaffold compounds such as organohalides (most frequently organobromides) selectively react with the sulfhydryl group of cysteine. Non-sulfhydryl groups, such as the primary amine of lysine or N-terminal amino group in a peptide, can also be used for cyclization for example with N-hydroxysuccinimide (NHS)-containing chemicals. Especially designed unnatural amino acids can also be used for cyclization in peptides via a bio-orthogonal reaction. For example, if an azide-containing amino acid such as azidohomoalanine or azidophenylalanine exists in a peptide, a copper-mediated click reaction with an alkyne-bearing scaffold can lead to cyclization.

Furthermore, cysteines can be joined together between their side chains via disulfide bonds (- S— S— ) or amide cyclization can be performed without any scaffold (head-to-tail, or backbone cyclization).

For example, a peptide with "C" residues at its N- and C- termini, e.g., the cyclic TTR compound used in the Examples, GCGGGRKAADDTWEPFASGKTSESGEGGGCG (SEQ ID NO: 17), can be reacted by S-S-cyclization to produce a cyclic peptide. The cyclic peptide compound can be synthesized as a linear molecule with the linker covalently attached at or near the N-terminus or C-terminus of the peptide comprising the TTR peptide, or related epitopes mentioned herein prior to cyclization and provided as a precursor which is also subject of the present disclosure. Alternatively, part of the linker is covalently attached at or near the N- terminus and part is covalently attached at or near the C-terminus prior to cyclization. In either case, the linear compound is cyclized for example by S-S bond cyclization. Accordingly, the compounds may be cyclized by covalently bonding 1) at or near the N-terminus and the C- terminus of the peptide + linker to form a peptide bond (e.g., cyclizing the backbone), 2) at or near the N-terminus or the C-terminus with a side chain in the peptide + linker, or 3) two side chains in the peptide + linker. In this context, "near" is defined as being within 1, 2, or 3 amino acid residues of the N- or C-terminus. Preferably, the linker is coupled to the N-terminus or C- terminus. As mentioned above, peptides may be cyclized by oxidation of thiol- or mercaptan-containing residues at or near the N-terminus or C-terminus, or internal to the peptide, including for example cysteine and homocysteine. For example, two cysteine residues flanking the peptide may be oxidized to form a disulphide bond. Oxidative reagents that may employed include, for example, oxygen (air), dimethyl sulphoxide, oxidized glutathione, cystine, copper (II) chloride, potassium ferricyanide, thallium(III) trifluro acetate, or other oxidative reagents such as may be known to those of skill in the art and used with such methods as are known to those of skill in the art. Crosslinking agents are also known in the art and can be chosen for example based on the functional groups to be used for crosslinking, see for example the Crosslinker Selection Tool provided by Thermo Fisher Scientific.

Accordingly, in one embodiment, the linker comprises a functionalizable moiety, e.g., an amino acid with one of the above-mentioned functional groups such as lysine, aspartic acid, glutamic acid, or cysteine, a non-naturally occurring amino acid such as azidohomoalanine or azidophenylalanine, or equivalently functioning molecules such as polyethylene glycol (PEG).

In case the functionalizable moiety is a naturally occurring amino acid, such as lysine, aspartic acid, glutamic acid, serine, threonine, or cysteine, the functionalizable moiety does not necessarily have to be in the linker but can also be present in the epitope or within the adjacent amino acids present in the protein fragment or peptide forming the cyclic peptide. Thus, cyclization of the peptide and the protein fragment, respectively, can also be performed without a linker. Accordingly, in one embodiment, the protein fragment or peptide forms the cyclic peptide compound of the disclosure without a linker. The linkage may occur via the side chain of one or more amino acids, such as the sulfhydryl moiety of a cysteine residue, the carboxylic acid moiety of an aspartic acid or glutamic acid residue, the hydroxyl of a serine or threonine residue, or the amine of a lysine or arginine residue.

In a preferred embodiment, the at least one functionalizable moiety is present in the linker, i.e., the linker comprises one or more functionalizable moieties. The linker can comprise or consist of any amino acids including non-natural amino acids, but preferably comprises at least any one of the functionalizable moieties mentioned above, i.e., lysine, aspartic acid, glutamic acid, or cysteine, non-naturally occurring amino acids such as azidohomoalanine or azidophenylalanine, or equivalently functioning molecules such as polyethylene glycol (PEG). In a preferred embodiment, the linker comprises cysteine as functionalizable moiety. Accordingly, in a preferred embodiment, the linker of any length and sequence can be described with the following sequence X-nX-lFXl-Xn, wherein F is any functionalizable moiety, preferably C (cysteine), and X any amino acid including non-natural amino acids. In a further preferred embodiment, the linker amino acids are selected from alanine (A), or glycine (G), or serine (S), or from alanine (A) and glycine (G), or from glycine (G) and serine (S), but preferably glycine (G).

Even more preferred, the linker amino acids are selected from alanine (A), or glycine (G), or serine (S), or from alanine (A) and glycine (G), or from glycine (G) and serine (S), preferably glycine (G) and the functionalizable moiety is cysteine (C). Accordingly, preferably, the cyclization is performed with scaffold compounds such as organohalides, preferably organobromides, that selectively react with the sulfhydryl group of cysteine, or via a disulfide bridge. Most preferably, cyclization is performed via a disulfide bridge.

In a preferred embodiment, the linker comprises 1 to 40 amino acids, preferably 1 to 35 amino acids, more preferably 1 to 30 amino acids, more preferably 1 to 25 amino acids, more preferably 1 to 20 amino acids, more preferably 1 to 10 amino acids, more preferably 1 to 9 amino acids, and most preferably 1 to 8 amino, in particular 1, 2, 3, 4, 5, 6, 7, or 8 amino acids and/or equivalent functioning molecules, and/or a combination thereof, wherein, when the linker comprises only amino acids, there is preferably within the amino acids at least one amino acid having any of the above-mentioned functional groups, preferably cysteine. The other amino acids comprised in the linker can be chosen from any known amino acids including nonnatural amino acids, but are preferably alanine (A) and/or glycine (G), preferably glycine (G).

As mentioned above, the length of the linker can vary and can be for example 9 amino acids, for example GGGGCGGGG (SEQ ID NO: 148), or 8 amino acids, for example GGGCGGGG (SEQ ID NO: 149), GGCGGGGG (SEQ ID NO: 150) or GCGGGGGG (SEQ ID NO: 151), or 7 amino acids, for example GGGGCGG (SEQ ID NO: 152), GGGCGGG (SEQ ID NO: 153), GGCGGGG (SEQ ID NO: 154) or GCGGGGG (SEQ ID NO: 155), 6 amino acids, for example GGGCGG (SEQ ID NO: 156), GGCGGG (SEQ ID NO: 157) or GCGGGG (SEQ ID NO: 158), 5 amino acids, for example GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16), 4 amino acids such as GCGG (SEQ ID NO: 159) or GGCG (SEQ ID NO: 160) or 3 amino acids such as GCG. Most preferably, the linker in the cyclic peptide compound comprises or consists of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16).

As illustrated in the Examples, the cyclic peptide compound which comprises the epitope WEPFA of antibody NI-301.37F1 disclosed in international application WO 2015/092077 Al, which is a neoepitope in the sense that is located in position 41-45 of mature TTR protein, which is hidden in the TTR protein’s naturally folded conformation but accessible to antibody binding following unfolding and aggregation, has been shown to be a highly suitable target antigen for immunological and biological assays, e.g., reporter gene assay used as potency assay.

Thus, in a preferred embodiment, the cyclic peptide compound comprises a (neo)epitope, preferably from any protein which aggregation leads to a disease phenotype.

The physiological functions of proteins are highly dependent on their correct three-dimensional conformation. Disturbances in the proper folding of newly synthesized or pre-existing proteins as well as in pathways responsible for refolding (molecular chaperones) or degradation of misfolded proteins (ubiquitin-proteasome and autophagy systems) may lead to intra- and/or extracellular protein aggregation. These precipitates of misfolded proteins form either ordered (e.g., amyloid fibrils) or disordered (e.g., inclusion bodies) protein aggregates that dissociate only in the presence of high concentrations of detergents or denaturing buffers (Schroder, Acta Neuropathol 125 (2013), 1-2).

Amyloid diseases are characterized by the deposition of cross-P-sheet amyloid fibrils consisting of misfolded and/or misassembled proteins. The amyloid fibrils that are the pathological hallmark of these disorders can be either deposited systemically or localized to specific organs. The development of amyloidosis is often linked to ageing and is associated with a decreased quality of life and substantial suffering for both patients and their families. Alzheimer's disease is an example of a localized cerebral amyloidosis, and type 2 diabetes mellitus is an example of localized extracerebral amyloidosis; both diseases are associated with ageing. Systemic forms of amyloid disease, also often linked to ageing, are less common and include the TTR amyloidoses. The origin of amyloidosis is either sporadic, z.e., from the normal protein sequence, or hereditary (familial), i.e., from a protein harboring one or more point mutations. In addition, there are infectious forms of amyloidosis, such as the transmissible spongiform encephalopathies caused by the aggregation of prion protein (Ankarcrona et al., J Intern Med. 280 (2016), 177-202).

As illustrated in the appended Examples, the present disclosure provides cyclic peptide compounds comprising preferably an epitope which is usually exposed in the pathological protein aggregate of the amyloidogenic protein, which makes the cyclic compound especially useful in contributing to a reliable method for determining the potency of antibodies and antibody-based drugs in terms of their capability to activate an FC domain/receptor mediated effector function such as antibody-dependent cell-mediated phagocytosis (ADCP), wherein the antibody preferably targets an epitope on a pathological protein aggregate; see also Examples 5 to 8 in WO 2023/099788 Al.

In a preferred embodiment, the (neo)epitope is derived from an amyloidogenic protein involved in systemic amyloidosis or aggregate thereof, e.g., transthyretin (TTR), in particular wild type TTR and variant TTR, preferably wild type TTR, immunoglobulin light chain (LC), immunoglobulin heavy chain (LH), serum amyloid A (SAA), leucocyte chemotactic factor 2 (LECT2), gel solin, apolipoprotein Al (Apo Al), apolipoprotein All (Apo All), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, 02 microglobulin, in particular wild type and variant 02 microglobulin, cystatin C, ABriPP, prion protein, and lysozyme; see for example Benson et al., Amyloid 25 (2018), 215-219 and Muchtar et al., Journal of Internal Medicine 289 (2021), 268-292, and thus, the cyclic peptide compound comprises a peptide derived from any one of the listed proteins, preferably wherein the peptide comprises at least 4 amino acids from the protein.

In a preferred embodiment, the amyloidogenic protein is TTR and thus, the cyclic peptide compound comprises a protein fragment of TTR or a peptide derived from TTR.

The protein fragment or peptide in the cyclic peptide compound of the present disclosure is at least 4, preferably at least 5, more preferably at least 10, more preferably at least 15, most preferably at least 20, 21, 22, 23, 24, or 25 amino acid residues of an amyloidogenic protein. More specifically, at least the epitope of the target antigen binding molecule, which as known to the person skilled in the art can consist of as few amino acids as four should be present, which may be supplemented with appropriate number of amino acids and/or other linker moieties sufficient and necessary for cyclization. However, in principle there is no limitation as to length of the peptide as long as it can be cyclized, and it is recognized by the target binding molecule. Accordingly, the cyclic peptide compound of the present disclosure and as used herein may comprise a protein or fragment or peptide thereof containing between 4 amino acids and all amino acids of the amyloidogenic protein. Preferably, the protein fragment or peptide in the cyclic peptide compound comprises between 4 amino acids and 100 amino acids, more preferably between 4 amino acids and 90 amino acids, more preferably between 4 amino acids and 80 amino acids, more preferably between 4 amino acids and 70 amino acids, more preferably between 4 amino acids and 60 amino acids, more preferably between 4 amino acids and 50 amino acids, more preferably between 4 amino acids and 45 amino acids, more preferably between 4 amino acids and 40 amino acids, more preferably between 4 amino acids and 35 amino acids, more preferably between 4 amino acids and 30 amino acids, more preferably between 4 amino acids and 25 amino acids, or between 4 amino acids and 24 amino acids, or between 4 amino acids and 23 amino acids, or between 4 amino acids and 22 amino acids, or between 4 amino acids and 21 amino acids, or between 4 amino acids and 20 amino acids, preferably between 5 amino acids and 25 amino acids, or between 5 amino acids and 24 amino acids, or between 5 amino acids and 23 amino acids, or between 5 amino acids and 22 amino acids, or between 5 amino acids and 21 amino acids, or between 5 amino acids and 20 amino acids.

The amino acids either represent only the epitope recognized by a target antigen binding molecule or the epitope and adjacent amino acids present in the amyloidogenic protein. In a preferred embodiment, the protein fragment of peptide comprises amino acid residues of an amyloidogenic protein, wherein these amino acid residues comprise the epitope and adjacent amino acids.

The cyclic TTR peptide used in the appended Examples consists of the amino acid sequence H- GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (TTR34-54cyc; SEQ ID NO: 17) with a total of 31 amino acids and comprises 21 amino acids of the amyloidogenic protein TTR, including the epitope WEPFA (SEQ ID NO: 1), and linker sequences of 10 amino acids, five amino acids each the N- and C-termini of the 21 amino acid stretch from TTR. Thus, in a preferred embodiment, the cyclic peptide compound consists of a total of 20 to 40, more preferably 25 to 35 and most preferably of 30 ± one, two, three or four amino acids or, in case non-amino acid residues are incorporated, for example as a linker, is configured such that its structure resembles a corresponding peptide. In this embodiment, the amino acid sequence derived from the amyloidogenic protein present in the cyclic peptide compound may consist of 10 to 40, preferably of 15 to 25 and most preferably of 20 ± one, two, three or four amino acids and optionally supplemented with a linker, preferably 5 to 20 amino acids in length, more preferably 5 to 15 and most preferably of 10 ± one, two, three or four amino acids, either distributed on both ends, N- and C-terminus or only at one terminus. It is also conceivable that linker sequences or "filling" sequences are located within amino acid sequence derived from the amyloidogenic protein, e.g., if the epitope of the target binding molecule is a conformational epitope or discontinuous epitope.

As mentioned above, the cyclic peptide compound preferably comprises a peptide derived from an amyloidogenic protein involved in systemic amyloidosis or aggregate thereof, preferably wherein the peptide comprises at least 4 amino acids from the protein, most preferably wherein the amyloidogenic protein is TTR and thus, the cyclic peptide compound comprises a protein fragment of TTR or a peptide derived from TTR.

From the sequence of the cyclic TTR peptide set forth in SEQ ID NO: 17 it is further evident that the peptide comprises 7 amino acids (RKAADDT (SEQ ID NO: 162) appended to the N- terminus of the epitope WEPFA (SEQ ID NO: 1) and 9 amino acids SGKTSESGE (SEQ ID NO: 163) appended to the C-terminus of said epitope.

Thus, in one embodiment, the cyclic peptide of the present disclosure comprises at least 4 contiguous amino acid residues of the peptide sequence WEPFA (SEQ ID NO: 1), preferably all five residues of the peptide sequence WEPFA (SEQ ID NO: 1), wherein the cyclic peptide further comprises at least 7 amino acids appended to the N-terminus of said SEQ ID NO: 1 and/or at least 9 amino acids appended to the C-terminus of said SEQ ID NO: 1, or a variant thereof. In a preferred embodiment, the cyclic peptide comprises the sequence WEPFASG (SEQ ID NO: 4). Preferably, the amino acids in the N-terminus and/or C-terminus of the cyclic peptide compound of the present disclosure are appended to SEQ ID NO: 1 via a peptide bond. In a preferred embodiment, the cyclic peptide compound of the present disclosure comprises a first peptide sequence RKAADDT (SEQ ID NO: 162) appended to the epitope/peptide sequence WEPFA (SEQ ID NO: 1) at the N-terminus and/or a second peptide sequence SGKTSESGE (SEQ ID NO: 163) appended to the epitope/peptide sequence WEPFA (SEQ ID NO: 1) at the C-terminus. Preferably, the C-terminal threonine (T) amino acid of the first peptide sequence RKAADDT (SEQ ID NO: 162) is bonded to N-terminal tryptophan (W) of the peptide sequence WEPFA (SEQ ID NO: 1) and the N-terminal serine (S) of the second peptide sequence SGKTSESGE (SEQ ID NO: 163) is bonded to the C-terminal alanine (A) peptide sequence WEPFA (SEQ ID NO: 1), in each case, via a peptide bond. As mentioned above, the cyclic peptide compound of the present disclosure preferably comprises a linker which comprises or consists of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16). More particularly, in a preferred embodiment, the cyclic peptide of the present disclosure comprises a first linker (LI) comprising the sequence GCGGG (SEQ ID NO: 15), which is preferably directly or indirectly linked to the N-terminus of the peptide sequence WEPFA (SEQ ID NO: 1), and/or a second linker (L2) comprising the sequence GGGCG (SEQ ID NO: 16), which is preferably directly or indirectly linked to the C-terminus of the peptide sequence WEPFA (SEQ ID NO: 1). In one embodiment, the LI and L2 are linked to form a cyclic peptide, preferably wherein the N-terminus of LI is bonded to the C-terminus of L2 via a peptide bond.

In general, the protein fragment or peptide of TTR can be any fragment or peptide that is derived from the TTR protein. In a preferred embodiment, the TTR fragment or peptide in the cyclic peptide compound of the present disclosure comprises at least 4 amino acids from the TTR protein, wherein the 4 amino acids can for example be any one of those listed in Table 1, below.

Table 1: TTR peptides comprising 4 amino acid residues.

In a preferred embodiment, the TTR peptide comprises at least 4 amino acid residues and preferably all amino acids of an amino acid sequence which is exposed in the misfolded variant, and on aggregates, fibrils and/or oligomers, respectively, e.g., WEPFA (SEQ ID NO: 1), which is a peptide recognized by antibody NI-301.37F1 or by antibody NI-301.28B3 disclosed in WO 2015/092077 Al; EEFXEGIY (SEQ ID NO: 2), which is a peptide recognized for example by antibody NI-301.59F1 disclosed in WO 2015/092077 Al; ELXGLTXE (SEQ ID NO: 3), which is a peptide recognized for example by antibody NI-301.35G11 disclosed in WO 2015/092077 Al, wherein X can be any amino acid; WEPFASG (SEQ ID NO: 4), which is a peptide recognized for example by antibody NI-301.12D3 disclosed in WO 2015/092077 Al; TTAVVTNPKE (SEQ ID NO: 5), which is a peptide recognized for example by antibody NI- 301.18C4 disclosed in WO 2015/092077 Al; KCPLMVK and VFRK (SEQ ID NOs: 6 and 7), which represent peptides comprising a conformational epitope requiring at least the C of the first sequence and the V and F of the second sequence, and which is an epitope recognized by antibody NI-301.44E4 of WO 2015/092077 Al; EHAEVVFTA (SEQ ID NO: 8), which is a peptide recognized for example by antibody 14G8 / PRX004 disclosed inter alia in Higaki et al., Amyloid 23 (2016), 86-97; GPRRYTIAA (SEQ ID NO: 9), which is a peptide recognized for example by antibody 18C5 described in WO 2019/071205 Al; VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE (SEQ ID NO: 10), which is a peptide recognized for example by an antibody described in WO 2014/124334 A2 which binds to TTR30-66; ALLSPYSYSTTAV (SEQ ID NO: 11), which is a peptide recognized for example by an antibody described in WO 2014/124334 A2 which binds to TTR109-121; WKALGISPFHE (SEQ ID NO: 12), which is a peptide recognized for example by antibody 37 IM described in WO 2015/115332 Al; SYSTTAVVTN (SEQ ID NO: 13), which is a peptide recognized for example by antibody 313M (RT24) described in WO 2015/115331 Al; or LLSPYSYSTTAVVTNPKE (SEQ ID NO: 14), which is a peptide recognized for example by an antibody described in WO 2014/124334 A2 which binds to TTR100-127.

Most preferably, the TTR peptide of the present disclosure comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

The above-mentioned peptides are all derived from the TTR protein, wherein the peptide or fragment VFRK (SEQ ID NO: 7) is located N-terminally of the peptide WEPFA (SEQ ID NO: 1) and the peptide or fragment ELXGLTXE (SEQ ID NO: 3) is located C-terminally of the peptide WEPFA (SEQ ID NO: 1). Thus, in one embodiment, the cyclic peptide of the present disclosure comprises a part of or all of a first motif VFRK (SEQ ID NO: 7) at the N-terminus thereof and/or a part of or all of a second motif ELXGLTXE (SEQ ID NO: 3) at the C-terminus thereof, wherein X in SEQ ID NO: 3 is any natural amino acid, preferably wherein X at position 3 in SEQ ID NO: 3 is a histidine (H) and X at position 7 in SEQ ID NO: 3 is threonine (T).

In one embodiment, the cyclic peptide of the present disclosure comprises a part of the first motif VFRK (SEQ ID NO: 7) comprising at least 2 amino acids at the N-terminus thereof and a part of the second motif ELXGLTXE (SEQ ID NO: 3) comprising at least 1 amino acid at the C-terminus thereof, preferably wherein the amino acids that make up the part of the first motif comprises the dipeptide RK in SEQ ID NO: 7 and the amino acid that makes up the part of the second motif comprises the N-terminal E in SEQ ID NO: 3.

As mentioned above, the cyclic peptide compound comprises preferably a protein fragment or peptide comprising an epitope of an amyloidogenic protein, preferably a TTR epitope, and most preferably the epitope comprising the amino acid sequence WEPFA (SEQ ID NO: 1) as well as adjacent amino acids and a linker at the peptide N-terminus and C-terminus, wherein the linker can in principle comprise any of the above-described linker sequences, and preferably comprises the amino acid sequence GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16). Thus, a preferred embodiment, the cyclic peptide compound comprises, consists essentially of or consists of the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG- OH (TTR34-54cyc; SEQ ID NO: 17), which has been shown in as suitable target antigen in Examples 1, 2, and 5.

The present disclosure also relates to a variant of the cyclic peptide compound of the present disclosure, which comprises the sequence WEPFA (SEQ ID NO: 1) and further comprises a 5%-20% variation, in terms of amino acid sequence identity, wherein the variation is in the amino acids appended to the N-terminus and/or C-terminus of said SEQ ID NO: 1, e.g., a variant of SEQ ID NO: 17 comprising 80%-95% sequence identity to said SEQ ID NO: 17, wherein the variation is due to amino acid substitution, addition or deletion at the N-terminal sequence RKAADDT (SEQ ID NO: 162) and/or amino acid substitution, addition or deletion at the C- terminal sequence SGKTSESGE (SEQ ID NO: 163), particularly preferably wherein the variation is due to an amino acid substitution.

The cyclic peptide compound or the variant thereof of the present disclosure which is preferably characterized by comprising the sequence WEPFA (SEQ ID NO: 1), optionally including the above-indicated adjacent amino acids and by comprising the amino acid sequence H- GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 17), respectively, also includes cyclic peptide compounds, that comprise additional amino acids and peptides/protein fragments, respectively. Thus, in one embodiment, the cyclic peptide compound or the variant thereof of the present disclosure further includes at least one other immunogenic sequence selected from the group consisting of EEFXEGIY (SEQ ID NO: 2), wherein X in said SEQ ID NO: 2 is any amino acid, preferably wherein X in said SEQ ID NO: 2 is valine (V); TTAVVTNPKE (SEQ ID NO: 5); KCPLMVK (SEQ ID NO: 6); EHAEVVFTA (SEQ ID NO: 8); GPRRYTIAA (SEQ ID NO: 9); ALLSPYSYSTTAV (SEQ ID NO: 11); and/or WKALGISPFHE (SEQ ID NO: 12),

Alternatively, the cyclic peptide compound or the variant thereof of the present disclosure does not contain a second immunogenic sequence selected from the group consisting of EEFXEGIY (SEQ ID NO: 2), wherein X in said SEQ ID NO: 2 is any amino acid, preferably wherein X in said SEQ ID NO: 2 is valine (V); TTAVVTNPKE (SEQ ID NO: 5); KCPLMVK (SEQ ID NO: 6); EHAEVVFTA (SEQ ID NO: 8); GPRRYTIAA (SEQ ID NO: 9); ALLSPYSYSTTAV (SEQ ID NO: 11); and/or WKALGISPFHE (SEQ ID NO: 12). In addition, or alternatively, the cyclic peptide compound or the variant thereof of the present disclosure does not contain any immunogenic sequence (a) N-terminal to VFRK (SEQ ID NO: 7) in the human TTR sequence of SEQ ID NO: 164 and/or (b) C-terminal to ELHGLTTE (SEQ ID NO: 3) in the human TTR sequence of SEQ ID NO: 164.

When reference is made to the cyclic peptide compound, the above-described variant is also included.

In one embodiment, the cyclic peptide compound and precursor thereof, respectively, or the protein fragment or peptide within the cyclic peptide compound of the present disclosure is further derivatized or modified. For example, proteins and/or other agents may be coupled to the cyclic peptide compound, which may for example act as a probe in in vitro studies. For this purpose, any functionalizable moiety capable of reacting (e.g., making a covalent or non- covalent but strong bond) may be used. Those proteins and/or other agents can be for example a carrier protein such as bovine serum albumin (BSA) used for immunoblots or immunohistochemical assays. Furthermore, the other agent may be a dye.

The present disclosure further relates to a composition comprising the cyclic peptide compound as disclosed herein or a linear precursor thereof. The composition can have further excipients, such as buffers, stabilizing agents, and/or diluents. In one embodiment, the composition comprises the cyclic peptide compound, which is further derivatized as explained above, e.g., the composition comprises the cyclic peptide compound which further comprises a conjugate, such as a dye.

Provided herein are also peptide microarrays comprising the cyclic peptide compound of the present disclosure. Peptide arrays are known to the skilled person and comprise peptides displayed on a solid surface, usually a glass or plastic chip. Peptide arrays are commonly used to study binding properties and functionality and kinetics of protein-protein interactions in general. The synthesis of peptide arrays is for example described in Szymczak et al., Anal Chem. 90 (2018), 266-282 and Winkler et al., Methods Mol Biol. 570 (2009), 157-174.

As indicated in Example 1, the antigen binding molecule, here the anti-TTR antibody, showed a strong binding affinity to the cyclic peptide in ELISA assays. Accordingly, the cyclic peptide compound is a suitable target antigen in assays that are used to detect and quantify antigen binding molecules, such as antibodies.

The present disclosure thus further relates to the use of the cyclic peptide compound as disclosed herein or of the composition as disclosed herein in any kind of assay which concerns the analysis of the interaction between a target antigen binding molecule and a target antigen, for example the detection, which can also include the quantification of a target antigen binding molecule. In a preferred embodiment, such an assay is an ELISA assay.

The cyclic peptide has been further shown to be a particularly suitable target antigen in the potency assays as described herein. Thus, in a further preferred embodiment, the present disclosure relates to the use of the cyclic peptide compound as disclosed herein or of the composition as disclosed herein for determining the potency of an antigen binding molecular, such as an antibody or any other binding molecule comprising an Fc domain, preferably of an antibody as defined herein. The determination of the potency is preferably performed with the assays disclosed herein, z.e., the assay described in Examples 2 and 5 and in international application WO 2023/099788 Al, which content is herein incorporated by reference, respectively.

Potency tests are performed as part of product conformance testing, comparability studies and stability testing. These tests are used to measure product attributes associated with product quality and manufacturing controls, and are performed to assure identity, purity, strength (potency), and stability of products used during all phases of clinical study. Similarly, potency measurements are used to demonstrate that only product lots, i.e., batches that meet defined specifications or acceptance criteria are administered during all phases of clinical investigation and following market approval. Potency is defined as "the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result". Ideally, the potency assay will represent the product's mechanism of action (i.e., relevant therapeutic activity or intended biological effect); see Guidance for Industry - Potency Tests for Cellular and Gene Therapy Products, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologies Evaluation and Research, January 2011. In terms of the assay of the present invention, "potency" of a target antigen binding molecule, specifically an antibody as a drug product is thus a measure of its activity in the ADCP assay relative to the activity of a reference standard (of the drug product) for which the activity and level of activity, respectively, in the ADCP assay has been assessed or is known. Accordingly, a higher potency of the antibody/drug product in comparison to the reference means that the antibody/drug product shows a higher binding activity in the ADCP assay, z.e., a lower ECso value, and a lower potency of the antibody/drug product in comparison to the reference means that the antibody/drug product shows a lower binding activity in the ADCP assay, /.< ., a higher ECso value. For example, NI-301.37F1 150% mimics an antibody with a higher potency and showed 0.7 times higher EC50 value than the reference sample NI-301.37F1 RS (100%). In contrast antibody NI-301.37F1 50% mimics an antibody with a lower potency (loss of activity) and showed 2 times higher EC50 value than the reference sample NI-301.37F1 RS (100%); see Example 2. As shown in Example 5, even potency changes of 60% to 80% could be observed. Accordingly, a target antigen binding molecule (e.g., an antibody) that exhibits an increase in potency is one that is determined to have, for example, a lower EC50 value relative to a reference sample of at least 1%, e.g., at least 5%, such as at least 10%, or greater (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more), for example, as determined in the ADCP assay described herein. Alternatively, a target antigen binding molecule (e.g., an antibody) that exhibits a decrease in potency is one that is determined to have, for example, a higher EC50 value relative to a reference sample of at least 1%, e.g., at least 5%, such as at least 10%, or greater (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more), for example, as determined in the ADCP assay described herein. However, according to GLP and GMP, the acceptable variability (i.e., imprecision) for potency measurements is +/- 20% which are thus the preferred limits for the tested target antigen binding molecule.

As mentioned above, the cyclic peptide compound of the present disclosure and the corresponding composition, respectively, is preferably used for determining the potency of an antigen binding molecular, wherein determination of the potency is preferably performed with the assays disclosed herein, i.e., the assay described in Examples 2 and 5 and in international application WO 2023/099788 Al, which content is herein incorporated by reference, respectively.

Such a potency assay comprises preferably the following steps:

(a) contacting the cyclic compound of the present disclosure as target antigen with the binding molecule under conditions allowing the formation of a binding moleculeantigen complex; (b) contacting the binding molecule-antigen complex with a population of effector cells that are engineered to express an Fc receptor and harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor under conditions allowing for binding of the Fc domain to the Fc receptor, wherein binding of the Fc domain to the Fc receptor results in intracellular signaling and mediates a quantifiable reporter gene activity; and

(c) detecting the reporter gene activity, wherein at least one mechanism of action of the Fc domain of the binding molecule is mediated through the binding of the Fc domain to a Fc receptor and the reporter gene activity is indicative for the potency of the binding molecule.

In more detail, such assay comprises preferably at least the following steps: i) spotting the target antigen, i.e., the cyclic peptide compound of the present disclosure to the wells of a microplate, i.e., microplates (96-well plates) were coated with the target antigen over night (or 18 hours ± 2hrs) at 4°C, preferably wherein the cyclic compound was diluted to 3 pg/ml in PBS buffer pH 7.4; ii) contacting the target antigen with the target antigen binding molecule under conditions allowing the formation of a binding molecule-target antigen complex, preferably for 30 min at 37°C; iii) contacting the complex comprising the binding molecule and the target antigen with effector cells, i.e., effector cells, also called reporter cells, were added to the complex, wherein the effector cells express an Fc receptor und an reporter gene under control of a response element that is responsive to activation by the Fc receptor, preferably wherein the effector cells are engineered cells, more preferably Jurkat cells expressing the FcyRI receptor and the luciferase gene under control of the NF AT transcription factor, and wherein the complex is incubated for preferably 6 hours at 37°C; iv) adding a substrate solution, preferably a luminescence substrate solution; and v) detecting the signal, preferably a luminescence signal with a luminometer.

The detailed description of the steps of such a potency assay are described in WO 2023/099788 Al, in particular at pages 23 to 32 and 38 to 46, which content is herein incorporated by reference. The binding molecule which potency, in particular its potency to induce ADCP, is determined with the method as described herein, z.e., the potency assay using the cyclic peptide of the present disclosure can be any binding molecule which binds to the target antigen, z.e., the cyclic peptide compound of the present disclosure. Thus, in general the cyclic peptide compound of the present disclosure can be used in a method for determining the potency, preferably the potency to induce ADCP, of any binding molecule which binds to said cyclic peptide compound.

Preferably, the binding molecule is an antibody or any other binding molecule comprising an Fc domain. In a preferred embodiment, the cyclic peptide compound is used for determining the potency, in particular the potency to induce ADCP, of an anti-TTR antibody, most preferably of the anti-TTR antibody NI-301.37F1, which comprises in its variable region or binding domain the amino acid sequences of the VH and VL chain of SEQ ID NO: 19 and SEQ ID NO: 21 or SEQ ID NO: 23 and SEQ ID NO: 21.

Table 2: Amino acid sequences and nucleotide sequences of the variable heavy (VH) chain and variable light (VL) chain of antibody NI-301.37F1, wherein the CDRs in the amino acid sequences are underlined. The regions in between the CDRs represent the framework regions.

The present disclosure further relates to a method of producing a pharmaceutical composition comprising said binding molecule as defined above, i.e., preferably an antibody which binds to an amyloidogenic protein involved in systemic amyloidosis, most preferably an anti-TTR antibody.

In a first step, the binding molecule and the drug product, respectively, is provided, preferably produced. Systems and methods for the recombinant production of antibodies, corresponding binding molecules, fragments, derivatives and mimics thereof are known in the art. In particular, their recombinant production in a host cell, purification, modification, formulation in a pharmaceutical composition and therapeutic use as well as terms and feature common in the art can be relied upon by the person skilled in art when carrying out the present disclosure as claimed (see, e.g., Antibodies A Laboratory Manual 2nd edition, 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA; Frenzel et al., Front Immunol. 4 (2013), 217, doi: 10.3389/fimmu.2013.00217; Lalonde and Durocher, Journal of Biotechnology 251 (2017), 128-140, DOI: 10.1016/j.jbiotec.2017.04.028; Tripathi and Shrivastava, Front. Bioeng. Biotechnol. 7 (2019), 420, DOI: 10.3389/fbioe.2019.00420), wherein also antibody purification and storage; engineering antibodies, including use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis, immunoblotting protocols and the latest screening and labeling techniques are described and. The production of DARPins is for example explained in Stumpp et al., Drug Discovery Today 13 (2008), 695- 701 as well as in references cited therein and in Hanenberg et al., J Biol Chem 289 (2014), 27080-27089, DOI: 10.1074/jbc. Ml 14.564013. Furthermore, the production of the drug product may be performed in any manner as desired and/or suitable for the drug product in question.

In a next step, the binding molecule is subjected to the method as described herein. In particular, the binding molecule is subjected to a method for determining the potency of the binding molecule, in particular the potency to induce ADCP, thereby using the cyclic peptide compound of the present disclosure as target antigen. The information derived from the assay is used as part of an assessment of whether the binding molecule may be used as a pharmaceutical composition or not, i.e., whether the drug product comprising the binding molecules fulfills the criteria to be injected into a patient as agreed with the regulatory authorities in a country, where an injection of the drug product may take place. Furthermore, the information is used to identify the binding molecule for use in the pharmaceutical composition.

In a further preferred embodiment, the binding molecule is formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier, in particular that binding molecule which has been found useful by the method as described herein, i.e., the potency assay using the cyclic peptide compound of the present invention. A useful binding molecule is for example such a binding molecule which shows an ECso value in the (sub)-nanomolar range when assessed with the method of the present disclosure, or which shows a similar potency as a reference standard, for example at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100% in comparison to the potency of a positive control. Pharmaceutically acceptable carriers and administration routes can be taken from corresponding literature known to the person skilled in the art. The pharmaceutical compositions can be formulated according to methods well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols 2^nd Edition by Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2^nd Edition by Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected in different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.

The present disclosure also provides a process for preparing a pharmaceutical or diagnostic product comprising a target antigen binding molecule, wherein the potency of the binding molecule is at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100% in comparison to the potency of a positive control to activate ADCP. The process comprises the production of the binding molecule as explained above, wherein a batch of said binding molecule is obtained. Afterwards, the potency of the batch is analyzed, in particular the potency of the batch to activate ADCP as described herein. The process further comprises the preparation of the pharmaceutical or diagnostic product from the batch, but only if the batch is determined to have a potency which is at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100% in comparison to the potency of a positive control, in particular to activate ADCP.

The control is either a reference standard, an antibody which is known to have the potency to activate ADCP, for example an antibody which has been approved by the regulatory authorities, and/or the batch to be analyzed has been stored and/or was subjected to stress conditions and the control is the value of reporter gene activity of a sample taken from the batch or corresponding batch prior to storage and/or being subjected to said stress conditions.

The present disclosure also provides a method for determining the antibody’s potency as described above, wherein said method is part of an application for marketing authorization for selling said drug product as a pharmaceutical composition. The present disclosure also provides a method for applying for marketing authorization for a drug product comprising the binding molecule, which method comprises the method as described herein for determining the potency of the binding molecule of the drug product. As mentioned above, the method as described herein is used as a potency assay for batch release, z.e., the method is useful for analyzing different batches from for instance the production of a given target antigen binding molecule.

Any continuing production of drug products will result in the production of different batches of product to be released as pharmaceuticals. A key feature in the production is to ensure that the different batches live up to the same standard. This standard is typically set in cooperation with the regulatory bodies. Typically, each batch will be tested and examined by a number of different assays to ensure that the batch is of sufficient quality to be approved for the market. This can be performed with the potency assay as described herein, which used the cyclic peptide compound of the present disclosure.

Thus, the present disclosure also relates to a method for analyzing and selecting at least one batch of a pharmaceutical composition of a target antigen binding molecule as defined above, wherein the method comprises in a first step the assessment of the potency of a sample of the batch, in particular its potency to activate ADCP, with the method using the cyclic peptide compound as described herein. As mentioned above, the reporter gene activity is a measure for the potency of the binding molecule and thus, the reporter gene activity of the sample is compared to the reporter gene activity of a control and the batch is selected, for which the sample shows greater, equal or no substantial less reporter gene activity compared to the control. In one embodiment, the batch is selected, for which the sample shows greater, equal or no less than 80%, preferably 90%, preferably 95%, preferably 98%, preferably 99%, more preferably 100% reporter gene activity compared to the control. The selected batch can be further packed, for example into a kit, and distributed to the costumer.

Thus, the present disclosure relates to a process for validating a batch of a target binding molecule, i.e., determining the quality of a target antigen (e.g., aggregated protein) binding molecule, for distribution, wherein a sample of the batch is tested for its potency to activate ADCP with the potency assay as described herein using the cyclic peptide compound of the present disclosure and wherein the batch is validated for distribution only if the potency of the sample of the batch to activate ADCP is at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100% in comparison to the potency of a positive control to activate ADCP. In a preferred embodiment, the methods are particularly useful for analyzing and selecting a batch of a pharmaceutical composition comprising an anti-TTR antibody and for validating a batch of an anti-TTR antibody for distribution, respectively. The control can be a reference standard and/or in case the batch to be analyzed has been stored and/or was subjected to stress conditions, the control can be the value of reporter gene activity of a sample taken from the batch or corresponding batch prior to storage and/or being subjected to said stress conditions.

The binding of the binding molecule of the drug product to the Fc receptor is compared to the binding of the reference standard to the Fc receptor, and the therapeutic efficacy of the binding molecule of the drug product is assessed from its ability to bind the Fc receptor to the same or substantially the same degree as the reference standard.

As mentioned above, the potency of the sample of the batch should be preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, more preferably 100% in comparison to the potency of the reference standard. However, the particular degree to which the FcR binding profile of the binding molecule of the drug product and the FcR binding profile of the reference standard may differ may be established on a case-to-case basis and may for instance be determined in cooperation with the appropriate regulatory body.

To be able to determine FcR binding in a reliable and consistent manner, the FcR binding of the binding molecule of the drug product and the reference standard should be performed using the same assay, preferably using the assay as described herein. The determination of the binding of the reference standard will typically be performed first to establish a standard that any following batches of the binding molecule can be compared with. However, the determination of the binding of the reference standard may also be performed at the same time or after the determination of the FcR binding of the binding molecule of the drug product.

Thus, the present disclosure further relates to the use of the cyclic peptide compound of the present disclosure in the above-described methods and processes, /.< ., in the method of producing a pharmaceutical composition, in the process for preparing a pharmaceutical or diagnostic product, in the process for the application for marketing authorization for selling said drug product as a pharmaceutical composition, in the method for applying for marketing authorization for a drug product, in the method for analyzing and selecting at least one batch of a pharmaceutical composition, in the process for validating a batch of a target binding molecule, i.e., determining the quality of a target antigen binding molecule, for distribution, and in particular in the method for analyzing and selecting a batch of a pharmaceutical composition comprising an anti-TTR antibody and for validating a batch of an anti-TTR antibody for distribution, respectively.

The present disclosure further relates to a composition comprising the target antigen binding molecule of the present disclosure which has been analyzed, validated and selected according to the present disclosure, wherein the composition further comprises a pharmaceutically acceptable carrier.

In order to verify that the analyzed binding molecule indeed triggers phagocytosis leading to engulfment of the target antigen, for instance the cyclic peptide compound, phagocytosis assays can be performed, for example via in vivo phagocytosis assays as e.g., described in Prakash et al., Chem Sci. 12 (2021), 10901-10918 for monitoring phagocytic uptake of amyloid P in real time, or preferably via the in vitro phagocytosis assays as described in Examples 1 and 2 of WO 2023/099788 Al, which content is herein incorporated by reference. These assays showed that antibody NI-301.37F1 W1 indeed triggers phagocytosis of the TTR aggregate. Thus, the method of the present disclosure for assaying the potency of the binding molecule may be combined with a phagocytosis assay, in particular an in vitro phagocytosis assay. Furthermore, verifying that the analyzed binding molecule indeed triggers phagocytosis leading to engulfment of the target antigen, like aggregated TTR, can be performed with the patient- derived amyloid xenograft animal model as disclosed in WO 2020/094883 Al.

Furthermore, the binding of the analyzed binding molecule to its corresponding antigen might be verified by methods known in the art, for example via ELISA or BLI as shown in Example 1. Thus, the method of the present disclosure for assaying the potency of the binding molecule may be combined with a method for determining the binding of the binding molecule to its antigen.

As mentioned above, determining the potency of a drug is an important step in the development including evaluation of new therapeutics for the treatment of diseases. In the context of the present disclosure, such a method is used for the development, evaluation and batch release of antibody -based drugs and other target antigen binding molecule which make use of the effector function of the Fc domain for the treatment of diseases related to the target protein, especially protein aggregation disorders such as systemic and localized amyloidosis.

Thus, the cyclic peptide compound of the present invention is particularly useful as target antigen in assays for measuring the potency of antibodies that usually target a protein aggregate, i.e., an amyloidogenic protein and are thus useful in the therapy of protein aggregation disorders such as systemic and localized amyloidosis, in particular of disorders related to TTR aggregation.

Furthermore, the cyclic peptide compound of the present disclosure or a composition comprising the same can be used for the detection of autoantibodies against amyloidogenic proteins or fragments, oligomers or aggregates thereof. The cyclic peptide compound is in particular suitable for the detection of autoantibodies against TTR and identify antibodies equivalent to, for example NI-301.37F1. Likewise, the cyclic peptide can be used for screening of antibodies against amyloidogenic proteins and in particular of anti-TTR antibodies in general for example by phage display.

In addition, the cyclic peptide can be used for studying the pharmacokinetic profile, i.e., the half-life of the antibody in the plasma of in vivo non-human animal trials as well as clinical trials in humans, for example with antibody NI-307.37F11 (NI006), or NNC6019-0001 (PRX004). Furthermore, the cyclic peptide compound can be used during the course of for example antibody treatment to measure the plasma concentration of the antibody and support the dosing for keeping a sustained level of the antibody.

The cyclic peptide can also be used to identify antibodies equivalent to known antibodies and in particular, equivalent to the mentioned anti-TTR antibodies, in particular antibody NI- 307.37F11, for example by competition assays which are commonly known in the art. Thus, all the uses are also part of the present disclosure.

Furthermore, disclosed herein is a kit comprising at least the cyclic peptide compound of the present disclosure or a linear precursor thereof, optionally with reagents and instructions for use. The kit is preferably useful for detecting the interaction between a target antigen binding molecule and a target antigen, for example the detection, which can also include the quantification of a target antigen binding molecule, and most preferably for determining the potency of an antigen binding molecule which comprises an Fc domain, such as an antibody. In a preferred embodiment, determination of the potency is performed with the assay as disclosed herein, in particular as described in the appended Examples, and in WO 2023/099788 Al, respectively and thus, preferably the kit comprises means for conducting the corresponding potency assay. In a further preferred embodiment, the antigen binding molecule is an antigen binding molecule as defined hereinbefore, preferably an antigen binding molecule comprising an Fc domain, such as an antibody, and most preferably an anti-TTR antibody. Accordingly, the kit can be used for the purposes listed above.

In one embodiment, the kit comprising the cyclic peptide compound further comprises

(i) a population of effector cells engineered to express a human Fc receptor FcyR and harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor

(ii) a corresponding substrate for the reporter; and optionally

(iv) washing, blocking and assay/sample dilution buffer, and/or

(v) a monomer control of the target antigen and/or a positive control anti -target antigen antibody.

In a preferred embodiment, the population of effector cells is a population of Jurkat cells expressing FcyR, preferably FcyRI and a gene encoding a luminescence protein, preferably a luciferase under control of the NF AT transcription factor and wherein the kit comprises a luminescence substrate solution. Furthermore, the cyclic peptide compound preferably comprises the epitope of an anti-TTR antibody and an epitope of TTR, respectively, and the binding molecule is an anti-TTR antibody.

Preferably, this kit is adapted to assay the potency of a binding molecule comprising an Fc domain to induce ADCP.

In addition, the cyclic peptide compound of the present disclosure, and the linear precursor thereof, are especially useful in methods for identifying and optionally obtaining an antibody and equivalent binding molecules such as of the type described hereinbefore, which binds to an amyloidogenic protein involved in systemic amyloidosis, the method typically comprising the steps of: (a) providing, optionally producing one or more potentially amyloidogenic protein binding antibodies or a source thereof;

(b) subjecting the one or more of potentially amyloidogenic protein binding antibodies or source thereof to a binding assay comprising the cyclic peptide compound of the present disclosure; and

This method can be combined with the potency assay of the present disclosure and as described in WO 2023/099788 Al, and/or any other suitable method for further determining the diagnostic or preferably therapeutic utility of the subject antibody. As mentioned, the subject antibody may also be a different kind of antigen binding molecule.

Hence, a method of producing a pharmaceutical composition comprising an antibody which binds to a systemic amyloidogenic protein is provided, the method comprising at least the steps of:

The source of antibodies is not limited and comprises natural as well as synthetic antibodies obtained, for example from immunized laboratory animal such as a rodent, preferably mouse, most preferably Ig humanized mouse; human blood or a fraction thereof preferably comprising memory B cells; recombinant antibody libraries such as phage, yeast, and ribosome systems or mammalian cell systems such as CHO and HEK. In one embodiment, nanobodies, also known as VHHs, which originated from the serum of Camelidae may be screened with the cyclic peptide compound of the present disclosure; see, e.g., Lyu etal., Anal. Chem. 94 (2022), 7970- 7980; Muyldermans, The FEBS Journal 288 (2021) 2084-210. In this context, an IgG antibody of binding fragment thereof known to bind the amyloidogenic protein may be used a as reference antibody or source for identification and preparation of the nanobody, respectively. Likewise, synthetic alternatives to antibodies which may be designed computational modeling can be screened, for example modular peptide binders such as designed armadillo repeat proteins (dArmRPs); see, e.g., Gisdon et al., Biological Chemistry 403 (2022), 535-543.

The binding assay used in the methods mentioned above preferably comprise ELISA such as performed in Example 1.

In a preferred embodiment of the methods of the present disclosure for identification and obtaining subject antibodies and their further use in being formulated in a pharmaceutical composition and drug development, respectively, the antibody identified and optionally obtained in step (c) competes with a reference antibody for binding the amyloidogenic protein, preferably wherein the subject antibody has a lower ECso for the amyloidogenic protein than the reference antibody. Preparation and formulation of the subject antibody and like target binding molecule obtained by the method of the present invention can be performed as described for the target antigen binding molecule, supra.

The present disclosure also relates to an antigen binding molecule, in particular antibody, which has been screened for and thus obtained by using the cyclic peptide compound of the present disclosure, e.g., by using an immunological assay, like an ELISA assay as also described in the appended Examples for screening.

Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application including the background section and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to the following specific example which are provided herein for purposes of illustration only and is not intended to limit the scope of the invention. EXAMPLES

Example 1: Cyclic peptide as target antigen provides for higher sensitivity of ELISA assay for an antigen binding molecule

The ability of an antigen binding molecule to bind a cyclic peptide has exemplarily been evaluated with an ELISA assay using a cyclic peptide comprising the amino acid residues 34 to 54 of wild type TTR (TTR34-54cyc in biotinylated and non-biotinylated form) as target antigen and the anti-TTR antibody NI-301.37F1 as antigen binding molecule. Furthermore, as antigen controls, the TTR peptide TTR40-49, the biotinylated TTR peptide TTR40-49 as well as misfolded wild type TTR (mis.WT-TTR) were used.

The cyclic peptide TTR34-54cyc (1.36 mg/mL) has been manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. In particular, the peptide comprising the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 17) has been synthesized by solid phase peptide synthesis and cyclized via disulfide bridge between two cysteine residues within the poly-glycine stretch. The TTR peptide comprising the amino acid sequence H-TWEPFASGKT-OH (SEQ ID NO: 161) (TTR40-49, 1.25 mg/mL) has also been manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. The biotinylated peptides Biotin. TTR34-54cyc and Biotin. TTR40-49 each comprise an amino hexanoic acid (Ahx) spacer between their N-terminus and the biotin residue, /.< ., Biotin. TTR34-54cyc (Biotin-(Ahx)GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 17), 680 pg/mL) and Biotin. TTR40-49 (Biotin-(Ahx)TWEPFASGKT-OH, (SEQ ID NO: 161), 700 pg/mL). The misfolded wild type TTR has been prepared as follows:

Wild-type TTR protein purified from human plasma was obtained from Bio-Rad Laboratories, Inc. (California, USA; 7600-0604) and submitted to a custom purification through protein A/G chromatography followed by a lectin column to eliminate residual immunoglobulins. Plasma- purified WT-TTR was provided as a solution at a concentration of 1 mg/ml in PBS buffer. Misfolded WT-TTR aggregates (mis.WT-TTR) were prepared in vitro by diluting WT-TTR stock solutions to a concentration of 200 pg/ml in aggregation buffer (50 mM acetate-HCl, 100 mM KC1, 1 mM EDTA, pH 3.0) followed by incubation for 4 hours at 37°C with shaking at 1000 rpm. mis.WT-TTR was aliquoted and stored until use at -20°C. The quality of mis.WT- TTR was confirmed by ELISA and Biolayer interferometry (BLI).

Two ELISA assays have been performed, wherein in the first ELISA assay (ELISA- 1), antibody binding to the peptides TTR34-54cyc, TTR40-49, Biotin. TTR40-49, and mis-WT-TTR has been analyzed, and in the second ELISA assay (ELISA-2), antibody binding to the peptides TTR34-54cyc, Biotin. TTR34-54cyc, TTR40-49, Biotin. TTR40-49, and mis-WT-TTR has been analyzed.

In particular, 96-well microplates were coated for 1 hour at 37°C with TTR34-54cyc, TTR40- 49, Biotin. TTR40-49, and mis-WT-TTR (ELISA-1) and with TTR34-54cyc, Biotin.TTR34- 54cyc, TTR40-49, Biotin. TTR40-49, and mis-WT-TTR (ELISA-2), respectively, wherein each target antigen has been diluted to a concentration of 10 pg/ml in PBS buffer pH 7.4. Nonspecific binding sites were blocked for 1 hour at room temperature (RT) with a blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween-20 in PBS buffer. NI-301.37F1 antibody (Neurimmune AG, Zurich, Switzerland; NI-301.37F1) was diluted in duplicates to the indicated concentrations (dilution series from 400 nM to 4 pM and 0) in the blocking buffer and incubated overnight at 4°C. Binding was determined using an anti-human IgG antibody conjugated with horseradish peroxidase (HRP), followed by measurement of HRP activity in a standard colorimetric assay (ThermoFisher Scientific Inc., Waltham, Massachusetts, USA). Data were analyzed with the Prism software from GraphPad. ECso values were estimated using non-linear regression of individual data points using log(agonist) versus response model with variable slope. Data fitting was performed with the least square regression method.

The ELISA results confirmed binding of NI-301.37F1 to mis.WT-TTR. Furthermore, the ELISA assay showed that NI-301.37Fl binding to the cyclic TTR34-54cyc and Biotin. TTR34- 54cyc peptide is much stronger, /.< ., about 10-fold stronger, than binding to mis.WT-TTR. In particular, in ELISA-1 NI-301.37F1 binding ECso for the cyclic TTR34-54cyc peptide was 27 pM and NI-301.37F1 binding ECso for the mis.WT-TTR was 338 pM; see Fig. 1A. In ELISA- 2, the measured ECso values were higher, but the about 10-fold difference between NI-301.37F1 binding to the cyclic TTR34-54cyc peptide and binding to mis.WT-TTR was maintained. In particular, NI-301.37F1 binding ECso for the cyclic TTR34-54cyc peptide was 0.66 nM and NI-301.37F1 binding ECso for the mis.WT-TTR was 8.3 nM; see Fig. IB. No binding of NI- 301.37F1 to TTR40-49 and Biotin. TTR40-49 was observed in both ELISA assays.

Example 2: Cyclic peptide as target antigen provides for an improved ADCP assay

An ADCP assay for determining the potency of an antigen binding molecule was developed. The assay uses a reporter cell line expressing the human Fey receptor 1 (FcyRl) and has exemplary been evaluated for its capacity to measure the potency of antibody NI-301.37F1 to activate phagocytosis of a cyclic TTR peptide (TTR34-54cyc) in vitro by using a cyclic peptide as antigen.

ADCP reporter assay

To measure the potency of a NI-301.37Fl reference sample (NI-301.37F1 RS, 100%) and test samples with 50% lower concentration (NI-301.37F1 50%), 30% lower concentration (NI- 301.37F1 70%), 30% higher concentration (NI-301.37F1 130%), and 50% higher concentration (NI-301.37F1 150%) to activate phagocytosis of TTR34-54cyc, the commercially available FcyRl ADCP reporter bioassay (Promega, Madison, Wisconsin, USA, Cat.# GA1341, GA1345) has been applied. This bioluminescent cell-based assay relies on a genetically engineered Jurkat T cell line that expresses the human FcyRl together with a luciferase reporter driven by an NF AT -response element. FcyRl activation by the antibody-target complex leads to activation of NF AT pathway signaling and luciferase expression that is detected using a bioluminescent luciferase substrate. In brief, 96-well plates were coated over night at 4°C with TTR34-54cyc diluted to a concentration of 3 pg/ml in PBS buffer. Non-specific binding sites were blocked for 1 hour at room temperature (RT) with a blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween-20 in PBS buffer. A dilution plate for measurement was prepared, wherein NI-301.37F1 antibody was diluted to the indicated concentrations (500 ng/mL to 0.4 ng/mL) in ADCP buffer (96% RPMI 1640 Medium, 4% Low IgG Serum). The assay was performed by adding one unit of volume of the antibody dilutions and incubation was performed for 30 min at 37°C and 5% CO2 prior addition of one unit of volume of the FcyRl reporter cells at a density of about 1.65xl0^A5 cells/well) in ADCP buffer. The assay was incubated for 6 hours at 37°C and 5% CO2 before addition of the luminescent substrate (Bio- GloTM Luciferase Assay Reagent). The measurement of luminescence (integration time: 1000 ms, settle time: 0 ms) was performed after 15 min of incubation at room temperature.

NI-30L37F1 RS vs. NI-30L37F1 50%, NI-30L37F1 70%, NI-30L37F1 130%, and NI- 30L37F1 150%

In a first experiment it was shown that antibody NI-301.37F1 RS presented a dose-response characterized by an ECso of 19.8 ng/ml. In particular, the plates were coated with 3 pg/mL of the cyclic peptide TTR34-54cyc and antibody dilutions ranging from 500 - 0.4 ng/mL have been tested. These conditions resulted in a reasonable response curve with a stable slope, lower and upper asymptote; see Fig. 2. Further experiments have been performed in which the response of the assay was tested with 50%, 70%, 130% and 150% NI-301.37F1 concentration. As indicated in Fig. 3 A-D and in Table 3 below, NI-301.37F1 50% presented a dose-response characterized by an ECso of 39 ng/ml. The ECso increase by a factor of 1.97 was nearly in perfect agreement with the 2-time lower concentration in sample NI-301.37F 50%. NI-301.37F1 70% presented a dose-response characterized by an ECso of 30.4 ng/ml. The ECso increase by a factor of 1.43 was in very good agreement with the expected difference of 1.54 times. NI-301.37F1 130% presented a doseresponse characterized by an ECso of 13.6 ng/ml. The ECso increase by a factor of 0.77 was in perfect agreement with the expected difference of 0.77 times. NI-301.37F1 150% presented a dose-response characterized by an ECso of 13.5 ng/ml. The ECso increase by a factor of 0.67 was nearly in perfect agreement with the expected difference of 0.70 times.

Accordingly, there are nearly no assay variabilities and the results showed that the assay is responding to antibody concentration changes. In particular, it was shown that the FcyRl ADCP assay had the capacity to detect up to 50% loss of antibody activity, and up to 50% increase in antibody activity with excellent accuracy.

Table 3: Summary of the FcyRl ADCP assay performance.

Example 3: Evaluation of further cyclic peptides as target antigen for an antigen binding molecule

As shown in Example 1, the cyclic peptide TTR34-54cyc has been successfully used as target antigen for antibody NI-301.37F1 in an ELISA assay. Accordingly, the ability of an anti-TTR antibody to bind further cyclic peptides is analyzed. In particular, the ability of an anti-TTR antibody to bind the two cyclic peptides which comprise either the TTR epitope EHAEVVFTA (SEQ ID NO: 8) or the TTR epitope GPRRYTIAA (SEQ ID NO: 9), i.e., TTR89-97cyc and TTR101-109cyc as mentioned above, is evaluated with a further ELISA assay with said cyclic peptides as target antigens and the with the TTR peptide TTR40-49, the biotinylated TTR peptide TTR40-49 as well as misfolded wild type TTR (mis.WT-TTR) as antigen controls. The corresponding peptides and the mis.WT-TTR are prepared as described in Example 1, supra. For the ELISA assays, the 96-well microplates are coated with the two cyclic peptides TTR89- 97cyc and TTR101-109cy and the antigen controls, and the assay is performed as described in Example 5, supra.

Example 4: Evaluation of further cyclic peptides as target antigen in ADCP assays

As shown in Example 2, the potency of the anti-TTR antibody NI-301.37F1 to activate phagocytosis of a cyclic TTR peptide (TTR34-54cyc) in vitro has been successfully determined with an ADCP assay. Accordingly, the potency of an anti-TTR antibody to activate phagocytosis of the two cyclic peptides TTR89-97cyc and TTR101-109cyc is evaluated in a further ADCP assay.

To measure the potency of an anti-TTR antibody reference sample (anti-TTR antibody RS, 100%) and test samples with 50% lower concentration (anti-TTR antibody 50%), 30% lower concentration (anti-TTR antibody 70%), 30% higher concentration (anti-TTR antibody 130%), and 50% higher concentration (anti-TTR antibody 150%) to activate phagocytosis of said two cyclic peptides, the commercially available FcyRl ADCP reporter bioassay (Promega, Madison, Wisconsin, USA, Cat.# GA1341, GA1345) as described in Example 2 has been applied.

It is expected that the anti-TTR antibody will presented a dose-response, wherein anti-TTR antibody 50% will show an about 2-fold increased ECso value in comparison to the anti-TTR antibody RS, the anti-TTR antibody 70% will show an about 1.5-fold increased ECso value in comparison to the anti-TTR antibody RS, anti-TTR antibody 130% will show a decreased ECso value by a factor of about 0.77 in comparison to the anti-TTR antibody RS, and the anti-TTR antibody 150% will show a decreased ECso value by a factor of about 0.70 in comparison to the anti-TTR antibody RS.

Example 5: Validation of the ADCP assay

The ADCP assay for determining the potency of an antigen binding molecule with a cyclic peptide of the present invention is described in Example 2. The assay uses a reporter cell line expressing the human Fey receptor 1 (FcyRl) and has exemplary been evaluated for its capacity to ensure the potency of antibody NI-301.37F1 to activate phagocytosis of the cyclic TTR peptide (TTR34-54cyc) in vitro. The reliability and accuracy of the cyclic peptide based ADCP assay has been confirmed under validated experimental conditions to determine antibody potency in the range of 40% to 180% of theoretical relative activity.

ADCP reporter assay with luminescence readout for NI-301.37F 1

On day 1, the 96-well assay plates were coated with synthetic peptide TTR34-54cyc at 3 pg/mL in DBPS (Dulbecco’s phosphate buffered saline) by incubation for 18 ± 2h at 5°C. On day 2, the coating solution was removed and 200 pl of blocking buffer (2% BSA and 0.1% Tween 20 in DPBS) were added before the plates were incubated for 60 ± 5 min at room temperature on a plate shaker with 300 rpm.

In parallel, reference standard and test item working solutions were prepared; see Table 4. The reference standard (RS) corresponded to an antibody concentration of 4000 ng/mL in ADCP buffer (4% Low IgG Serum (v/v) in RPMI 1640 medium). The test items were generated from the reference standard stock solution at an antibody concentration of 49.9 mg/mL.

Table 4: Working solution of reference standard and test items

Also, the FyRl effector cell suspension was prepared using the instructions of the commercially available FcyRl ADCP reporter bioassay kit (Promega, USA, #GA1345) yielding to approximately 3 x 10⁶ cells/mL.

One assay plate was prepared to analyse one test item compared to the reference standard in triplicate per dose. After incubation of the assay plates with the blocking buffer, the blocking buffer was removed. The assay plates were washed with 200 pl DPBS before 55 pl antibody working solutions were transferred to the assay plates and incubated for 30 ± 5 min at 37°C and 5% CO2. After the incubation time of the assay plates, 55 pl of the effector cell suspension were transferred to the assay plates and incubated for 6h ± 15 min at 37°C and 5% CO2. The plate layout is provided in Table 5.

Table 5: Assay plate layout. R: reference standard, TS: test item, Cl : control 1 (TTR34-54cyc with RS, without effector cells), C2: control 2 (TTR34-54cyc without RS, with effector cells), C3: control 3 (RS, with effector cells, without TTR34-54cyc), BL: blank (ADCP buffer), # evaporation protection (ADCP buffer), *ng/mL: final concentration range of dilution series at 100% activity

The assay plates were removed from the incubator and incubated for 10 ± 5 min at Room Temperature before 110 pl of Bio-Gio™ luciferase assay reagent (Promega, USA, #GA1345) were added. Then, the assay plates were covered with a black lid and incubated for 15 ± 5 min at room temperature. Finally, the luminescence of the assay plates was measured using a multiplate reader with glow-type luminescence read capabilities. The results are indicated in Table 6. Table 6: Measured relative activity [%] was measured in triplicate with the mean relative activity values (and standard deviation) as indicated below:

Conclusion: These data show the utility of cyclic TTR peptides of the disclosure (e.g., TTR34- 54cyc) in the screening of anti-TTR antibody candidates that can potently activate phagocytosis of TTR amyloid plaques. The example further provides a validated ADCP reporter assay method for easily and reliably determining potency of anti-TTR antibodies as agents for the treatment and/or management of TTR amyloidosis.

Claims

CLAIMS A cyclic compound comprising a peptide comprising an epitope from an amyloidogenic protein involved in systemic amyloidosis. The cyclic compound of claim 1, which comprises a linker, preferably wherein the linker is an amino acid linker or a non-amino acid linker. The cyclic compound of claim 2, wherein the linker is covalently coupled at or near the peptide N-terminus residue and the C-terminus residue of the peptide. The cyclic compound of any one of claims 1 to 3, wherein the peptide in the cyclic compound comprises at least 5, preferably at least 10, more preferably at least 15, most preferably at least 20, 21, 22, 23, 24, or 25 amino acid residues of the amyloidogenic protein. The cyclic compound of any one of claims 1 to 4, wherein the amyloidogenic protein is selected from transthyretin (TTR), immunoglobulin light chain (LC), and serum amyloid A (SA A). The cyclic compound of claim 5, wherein the amyloidogenic protein is TTR and the peptide is a TTR peptide. The cyclic compound of claim 6, wherein the TTR peptide comprises at least 4 amino acid residues of any one of the amino acid sequences selected from: WEPFA (SEQ ID NO: 1), EEFXEGIY (SEQ ID NO: 2), ELXGLTXE (SEQ ID NO: 3), WEPFASG (SEQ ID NO: 4), TTAVVTNPKE (SEQ ID NO: 5), KCPLMVK and VFRK (SEQ ID NOs: 6 and 7), EHAEVVFTA (SEQ ID NO: 8), GPRRYTIAA (SEQ ID NO: 9), VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE (SEQ ID NO: 10), ALLSPYSYSTTAV (SEQ ID NO: 11), WKALGISPFHE (SEQ ID NO: 12), SYSTTAVVTN (SEQ ID NO: 13), and LLSPYSYSTTAVVTNPKE (SEQ ID NO: 14), wherein X can be any naturally occurring amino acid.

8. The cyclic compound of claim 6 or 7, wherein the cyclic compound comprises at least 4 contiguous amino acid residues of the peptide sequence WEPFA (SEQ ID NO: 1), wherein the cyclic compound further comprises at least 7 amino acids appended to the N-terminus of said SEQ ID NO: 1 and/or at least 9 amino acids appended to the C- terminus of said SEQ ID NO: 1, or a variant thereof.

9. The cyclic compound of any one of claims 6 to 8, wherein the TTR peptide comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

10. The cyclic compound of any one of claims 6 to 9, wherein the TTR peptide comprises the amino acid sequence WEPFASG (SEQ ID NO: 4).

11. The cyclic compound of any one of claims 8 to 10, wherein the amino acids in the N- terminus and/or C-terminus are appended to SEQ ID NO: 1 via a peptide bond.

12. The cyclic compound of any one of claims 8 to 11, comprising a first peptide sequence RKAADDT (SEQ ID NO: 162) appended to the peptide sequence WEPFA (SEQ ID NO: 1) at the N-terminus and/or a second peptide sequence SGKTSESGE (SEQ ID NO: 163) appended to the peptide sequence WEPFA (SEQ ID NO: 1) at the C-terminus.

13. The cyclic compound of claim 12, wherein the C-terminal threonine (T) amino acid of the first peptide sequence RKAADDT (SEQ ID NO: 162) is bonded to N-terminal tryptophan (W) of the peptide sequence WEPFA (SEQ ID NO: 1) and the N-terminal serine (S) of the second peptide sequence SGKTSESGE (SEQ ID NO: 163) is bonded to the C-terminal alanine (A) peptide sequence WEPFA (SEQ ID NO: 1), in each case, via a peptide bond.

14. The cyclic compound of any one of claims 2 to 13, wherein the linker comprises or consists of 1-8 amino acids and/or one or more functionalizable moi eties.

15. The cyclic compound of claim 14, wherein the linker amino acids are selected from alanine (A), glycine (G), and/or serine (S), and/or wherein the functionalizable moiety is cysteine (C), lysine (K), arginine (R), aspartic acid (D), or glutamic acid (E).

16. The cyclic compound of claim 15, wherein the compound is cyclized via a disulfide bridge.

17. The cyclic compound of any one of claims 1 to 16, wherein the compound comprises a first linker which comprises or consists of the amino acid sequence GCGGG (SEQ ID NO: 15) and/or a second linker which comprises or consists of the amino acid sequence GGGCG (SEQ ID NO: 16).

18. The cyclic compound of claim 17, wherein the LI is linked, directly or indirectly, to the N-terminus of the peptide sequence WEPFA (SEQ ID NO: 1) and the L2 is linked, directly or indirectly, to the C-terminus of the peptide sequence WEPFA (SEQ ID NO: 1).

19. The cyclic compound of claim 18, wherein the LI and L2 are linked to form a cyclic peptide, preferably wherein the N-terminus of LI is bonded to the C-terminus of L2 via a peptide bond.

20. The cyclic compound of any one of claims 1 to 19, wherein the epitope is an epitope of an antibody, and the cyclic compound is bound by the antibody.

21. The cyclic compound of claim 20, wherein the epitope is accessible to binding by the antibody only in the misfolded and/or aggregated form of the protein, preferably wherein the epitope is exposed in the pathological protein aggregate.

22. The cyclic compound of claim 20 or 21, which provides for a higher binding affinity to the antibody than the amyloidogenic protein or protein aggregate, preferably also higher than a corresponding linear peptide in an ELISA assay.

23. The cyclic compound of any one of claims 8 to 22, which comprises a part of or all of a first motif VFRK (SEQ ID NO: 7) at the N-terminus thereof and/or a part of or all of a second motif ELXGLTXE (SEQ ID NO: 3) at the C-terminus thereof, wherein X in SEQ ID NO: 3 is any natural amino acid, preferably wherein X at position 3 in SEQ ID NO: 3 is a histidine (H) and X at position 7 in SEQ ID NO: 3 is threonine (T). The cyclic compound of any one of claims 8 to 22, which comprises a part of the first motif VFRK (SEQ ID NO: 7) comprising at least 2 amino acids at the N-terminus thereof and a part of the second motif ELXGLTXE (SEQ ID NO: 3) comprising at least 1 amino acid at the C-terminus thereof, preferably wherein the amino acids that make up the part of the first motif comprises the dipeptide RK in SEQ ID NO: 7 and the amino acid that makes up the part of the second motif comprises the N-terminal E in SEQ ID NO: 3. The cyclic compound of any one of claims 1 to 24, wherein the cyclic compound comprises or consists of the amino acid sequence H- GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (TTR34-54cyc; SEQ ID NO: 17). The variant of the cyclic compound of claim 8, which comprises the sequence WEPFA (SEQ ID NO: 1) and further comprises a 5%-20% variation, in terms of amino acid sequence identity, wherein the variation is in the amino acids appended to the N- terminus and/or C-terminus of said SEQ ID NO: 1, e.g., a variant of SEQ ID NO: 17 comprising 80%-95% sequence identity to said SEQ ID NO: 17, wherein the variation is due to amino acid substitution, addition or deletion at the N-terminal sequence RKAADDT (SEQ ID NO: X) and/or amino acid substitution, addition or deletion at the C-terminal sequence SGKTSESGE (SEQ ID NO: Y), particularly preferably wherein the variation is due to an amino acid substitution. The cyclic compound of any one of claims 1 to 26, which further includes at least one other immunogenic sequence selected from the group consisting of EEFXEGIY (SEQ ID NO: 2), wherein X in said SEQ ID NO: 2 is any amino acid, preferably wherein X in said SEQ ID NO: 2 is valine (V); TTAVVTNPKE (SEQ ID NO: 5); KCPLMVK (SEQ ID NO: 6); EHAEVVFTA (SEQ ID NO: 8); GPRRYTIAA (SEQ ID NO: 9); ALLSPYSYSTTAV (SEQ ID NO: 11); and/or WKALGISPFHE (SEQ ID NO: 12). The cyclic compound of any one of claims 1 to 26, which does not contain a second immunogenic sequence selected from the group consisting of EEFXEGIY (SEQ ID NO: 2), wherein X in said SEQ ID NO: 2 is any amino acid, preferably wherein X in said SEQ ID NO: 2 is valine (V); TTAVVTNPKE (SEQ ID NO: 5); KCPLMVK (SEQ ID NO: 6); EHAEVVFTA (SEQ ID NO: 8); GPRRYTIAA (SEQ ID NO: 9); ALLSPYSYSTTAV (SEQ ID NO: 11); and/or WKALGISPFHE (SEQ ID NO: 12).

29. The cyclic compound of any one of claims 1 to 26, which does not contain any immunogenic sequence (a) N-terminal to VFRK (SEQ ID NO: 7) in the human TTR sequence of SEQ ID NO: T and/or (b) C-terminal to ELHGLTTE (SEQ ID NO: 3) in the human TTR sequence of SEQ ID NO: 164.

30. The cyclic compound of any one of claims 1 to 29, wherein the peptide is further derivatized.

31. A precursor of the cyclic compound of any one of claims 1 to 30, wherein the compound is in linear form.

32. A composition comprising the cyclic compound of any one of claims 1 to 30, and optionally one or more excipients.

33. The composition of claim 32, wherein the composition comprises the cyclic compound of any one of claims 1 to 30 and a conjugate, preferably a dye.

34. A peptide array comprising the cyclic compound of any one of claims 1 to 30.

35. A kit which comprises at least the cyclic compound of any one of claims 1 to 30 or the precursor of claim 31, optionally with reagents and/or instructions for use.

36. The kit of claim 35, further comprising

(ii) a corresponding substrate for the reporter; preferably wherein the kit further comprises one or more of the following: (iii) a solid support, preferably a microtiter plate, preferably a 96-well plate including a lid;

(iv) washing, blocking and assay/sample dilution buffer; and/or

(v) a monomer control of the target antigen and/or a positive control anti-target antigen antibody. Use of a cyclic compound of any one of claims 1 to 30, or the composition of claim 32 or 33, the array of claim 34, or the kit of claim 35 or 36 for detecting or quantifying an antigen binding molecule. The use of claim 37, wherein detecting or quantifying is performed by an immunological assay, preferably by ELISA. Use of a cyclic compound of any one of claims 1 to 30, or the composition of claim 32 or 33, the array of claim 34, or the kit of claim 35 or 36 for determining the potency of an antigen binding molecule comprising an Fc domain. The use of claim 39, wherein determining the potency of the antigen binding molecule is performed with a method comprising the steps of:

(a) contacting the cyclic compound of any one of claims 1 to 30 with the binding molecule under conditions allowing the formation of a binding molecule-antigen complex;

(b) contacting the binding molecule-antigen complex with a population of effector cells that are engineered to express an Fc receptor and harbor a reporter gene under the control of a response element that is responsive to activation by the Fc receptor under conditions allowing for binding of the Fc domain to the Fc receptor, wherein binding of the Fc domain to the Fc receptor results in intracellular signaling and mediates a quantifiable reporter gene activity; and

41. The use of claim 40, wherein a mechanism of action of the Fc domain is to induce antibody-dependent cell-mediated phagocytosis (ADCP).

42. The use of claim 40 or 41, wherein the Fc receptor is a human Fc receptor FcyRI (CD64).

43. The use of any one of claims 40 to 42, wherein the cell does not overexpress FcyRIIa (CD32a).

44. The use of any one of claims 40 to 43, wherein the cell does not overexpress FcyRIII (CD 16).

45. The use of any one of claims 40 to 44, wherein the effector cell is a Jurkat cell.

46. The use of any one of claims 40 to 45, wherein the response element is an NF AT (Nuclear Factor of Activated T cells) response element.

47. The use of any one of claims 40 to 46, wherein the reporter gene encodes a bioluminescent protein, preferably a luciferase.

48. The use of any one of claims 37 to 47, wherein the binding molecule is selected or derived from an antibody such as a monoclonal antibody or an antigen-binding fragment thereof, preferably wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

49. The use of claim 48, wherein the antibody is an IgGl antibody, such as an IgGl, X antibody or an IgGl, K antibody.

50. The use of any one of claims 37 to 49, wherein the binding molecule is an anti-TTR antibody.

51. The use of any one of claims 37 to 50, wherein the binding molecule is an anti-TTR antibody which is NI-301.37F1 and which comprises in its variable region or binding domain the amino acid sequences of the VH and VL chain of SEQ ID NO: 19 and SEQ ID NO: 21 or SEQ ID NO: 23 and SEQ ID NO: 21.

2. The use of any one of claims 37 to 51, wherein the cyclic peptide is bound on a solid support, preferably on a microtiter plate. 3. The use of claim 52, wherein at least step (b) of claim 40 is performed in a vertical plate layout.

54. A method for identifying and optionally obtaining an antibody or a corresponding antigen binding molecule thereof, which binds to an amyloidogenic protein involved in systemic amyloidosis, the method comprising the steps of:

(a) providing, optionally producing one or more potentially amyloidogenic protein binding antibodies or amyloidogenic protein binding molecules thereof, or a source thereof;

(b) subjecting the one or more of potentially amyloidogenic protein binding antibodies or amyloidogenic protein binding molecules thereof, or source thereof to a binding assay comprising the cyclic compound of any one of claims 1 to 30; and

(c) identifying and optionally obtaining an antibody (subject antibody) or an binding molecule that has been determined to bind to the cyclic compound.

55. A method of producing a pharmaceutical composition comprising an antibody or an antigen binding molecule thereof which binds to an amyloidogenic protein, the method comprising the steps of:

(b) subjecting said one or more potentially amyloidogenic protein binding antibodies or amyloidogenic protein binding molecules thereof, or a source thereof to a binding assay comprising the cyclic compound of any one of claims 1 to 30;

(c) identifying and optionally obtaining an antibody (subject antibody) or a binding molecule that binds to the cyclic compound; and

(d) formulating the antibody or binding molecule identified and optionally obtained in step (c) or a derivative thereof with a pharmaceutically acceptable carrier. The method of claim 54 or 55, wherein the source of antibodies is selected form the group consisting of: immunized laboratory animal such as a rodent, preferably mouse, most preferably Ig humanized mouse; human blood or a fraction thereof, preferably comprising memory B cells; recombinant antibody libraries such as phage, yeast, and ribosome systems or mammalian cell systems such as CHO and HEK. The method of any one of claims 54 to 56, wherein the binding assay comprises ELISA. The method of any one of claims 54 to 57, wherein the antibody identified and optionally obtained in step (c) competes with a reference antibody for binding the amyloidogenic protein, preferably wherein the subject antibody has a lower ECso for the amyloidogenic protein than the reference antibody. A method for analyzing and selecting at least one candidate target antigen binding molecule, the method comprising:

(a) subjecting at least two target antigen binding molecules to an assay for determining the potency by using the cyclic compound of any one of claims 1 to 30, preferably wherein the assay is the one as defined in any one of claims 39 to 53;

(b) comparing the reporter gene activities of the antigen binding molecules;

(c) selecting the antigen binding molecule which shows the greatest reporter gene activity. A method for analyzing and selecting at least one batch of a pharmaceutical composition of a target antigen binding molecule, the method comprising:

(a) subj ecting a sample of the batch to an assay for determining the potency by using the cyclic compound of any one of claims 1 to 30, preferably wherein the assay is the one as defined in any one of claims 39 to 53;

(b) comparing the reporter gene activity of the sample to the reporter gene activity of a control; and

(c) selecting the batch for which the sample shows greater, equal or not substantively less reporter gene activity compared to the control, preferably the batch for which the sample shows greater, equal or no less than 80% reporter gene activity compared to the control, preferably wherein the control is a reference standard and/or the batch to be analyzed has been stored and/or was subjected to stress conditions and the control is the value of reporter gene activity of a sample taken from the batch or corresponding batch prior to storage and/or being subjected to said stress conditions. The method of any one of claims 54 to 60, wherein in a further step the antigen binding molecule is assayed with a full-length amyloidogenic protein, preferably with a misfolded/aggregated amyloidogenic protein for its capability to trigger phagocytosis of the misfolded/aggregated amyloidogenic protein and/or for its capability to bind to the misfolded/aggregated amyloidogenic protein. Composition comprising an antibody or antigen binding molecule thereof obtainable by the method of claim 59, which shows greater, equal or at least 80% reporter gene activity in comparison to the reference antibody NI006.