Cyclic compounds and use of cyclic compounds in assays for detecting antibodies

Abstract

The present invention provides cyclic compounds based on peptides comprising epitopes from amyloid-producing proteins involved in systemic amyloidosis, which are useful in immunoassays and potency assays in antibody drug research and development.

Description

Technical Field

[0001] The present disclosure generally relates to cyclic compounds comprising peptides containing epitopes of systemic amyloidogenic proteins, and the use of the cyclic compounds 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 pharmaceutical compositions comprising antibodies or similar binding molecules, particularly when conducting clinical trials, applying for marketing authorization, and for quality control of approved pharmaceutical products. Background Art

[0002] Over the past three decades, monoclonal antibody drugs have matured, from research objectives to technological advancements, and from clinical research to commercialization. In recent years, the number of approved monoclonal antibody drugs has increased rapidly, with the milestone approval of the 100th monoclonal antibody product by the US Food and Drug Administration (FDA) in 2021. In 2019, nine of the 20 best-selling drugs were monoclonal antibodies (Mullard, Nature Reviews Drug Discovery 20, 491–495 (2021), DOI: 10.1038 / d41573-021-00079-7).

[0003] A promising application of therapeutic antibodies is therapeutic amyloidosis, which occurs due to the aggregation of toxic amyloid proteins. Neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and Huntington's disease represent a class of highly popular fatal local amyloidosis, in which amyloid deposits form in the nervous system, where they induce the death of specific nerve cell types. In systemic amyloidosis (such as immunoglobulin light chains, transthyretin and dialysis-related amyloidosis), because amyloidogenic proteins move from the synthesis site and are distributed in different parts of the body, several organs are affected. Antibodies and antibody fragments have been shown to be effective anti-amyloid molecules. For example, aducanumab shows a dose-dependent removal of amyloid deposits in Alzheimer's patients and has recently been approved by the FDA for the treatment of Alzheimer's disease.

[0004] Antibody drug discovery and development typically relies on, for example, immunoassays for detecting and quantifying antibodies, where the sensitivity of the assay depends on the affinity of the antibody for its target protein. Similarly, the sensitivity and reliability of efficacy assays depend, in particular, on the affinity of the antibody for its target in cell-based assays. Therefore, due to the importance of immunoassays at different stages of antibody screening and drug development, there is a constant need to improve assays such as those involving optimization of various parameters and the use of different strategies. The goal is to improve the sensitivity, specificity, accuracy, and reproducibility of assays. Key parameters and approaches that are typically subject to improvement include optimizing antigen coating, using effective blocking agents to prevent nonspecific binding, antibody dilution, incubation conditions, detection systems, and signal amplification, to name a few.

[0005] Therefore, the problem underlying the present disclosure is to provide systems and methods for immunoassays for detecting, isolating and characterizing antibodies. Summary of the Invention

[0006] The present disclosure generally relates to cyclic compounds comprising peptides or protein fragments, also referred to as cyclic peptide compounds, and to the use of such cyclic peptide compounds in methods for determining the efficacy of antibodies or binding molecules, and in the general field of drug discovery and diagnosis, wherein the cyclic compound comprises an epitope of an antibody or equivalent binding molecule. The present disclosure also relates to the use of cyclic peptide compounds in efficacy assays, for example, in determining or comparing the efficacy of binding molecules (such as anti-TTR antibodies). The assay methods disclosed herein can be used to test batch release of pharmaceutical compositions comprising antibodies or other binding molecules, for example, for evaluating drug candidates for clinical trials, applying for marketing authorization, and for quality control of approved drugs. More specifically, the cyclic peptide compounds comprise an epitope of an amyloidogenic protein and can be used in assays for detecting amyloid-specific antibodies and their corresponding binding fragments. In this context, the cyclic compounds of the present disclosure comprise an epitope of an antibody, and thus the cyclic compounds are designed to be bound by the antibody. As further explained below, preferably, the epitope of the peptide is selected from epitopes that can be bound by the antibody only in the misfolded and / or aggregated form of the protein, for example, the epitope is only exposed in pathological protein aggregates. As discussed further below and demonstrated in the accompanying Examples and in the Examples of WO 2023 / 099788 A1, the cyclic compounds of the present disclosure preferably provide higher binding affinity to antibodies than amyloidogenic proteins or protein aggregates, preferably also higher than the corresponding linear peptides in an ELISA assay.

[0007] As shown in the accompanying Examples, cyclic peptides comprising a TTR epitope (cyclic TTR peptides) have been used as target antigens in immunological and biological assays for detecting antibodies and for measuring their efficacy, respectively.

[0008] Specifically, as shown in Example 1, cyclic peptides as target antigens provide higher sensitivity to antibodies in ELISA assays than native antigens (i.e., aggregated TTR, also known as misfolded TTR). Similar results were observed in reporter gene assays as shown in Examples 2 and 5, where cyclic peptides were shown to provide improved ADCP assays. Surprisingly, the assays showed significant improvements in sensitivity and reliability when cyclic peptides were used instead of TTR aggregates as antigens. Without wishing to be bound by theory, the extraordinary performance of assays performed with cyclic peptides may be due to the very stable conformation adopted by the cyclic peptides, as they are constrained by having their two ends linked together and thus mimic the stability of protein aggregates. However, as shown in Examples 2 and 5, even taking into account such theoretical considerations, assays using cyclic peptides as target antigens are an order of magnitude more accurate and sensitive than assays using target antigens present as protein aggregates. Again, without wishing to be bound by theory, this may be due to the smaller size of peptides compared to proteins and the resulting higher epitope density, which translates into higher binding capacity and a higher avidity effect. In addition, epitopes in cyclic peptides may be more accessible than in full-length proteins. However, since the cyclic TTR peptide, including the linker amino acids with a total of 31 amino acids, is only 1 / 4 the size of the full-length TTR protein, the size of the cyclic peptide cannot a priori explain the observed effect. Another reason may be the better conformational control of the synthetic peptide compared to the recombinant protein. Specifically, more than 95% of the peptide is cyclized, and it is unknown which portion of the misfolded, aggregated TTR protein adopts the amyloid conformation. Because mis.WT-TTR is a heterogeneous mixture of conformations, a significant portion of the protein may form amorphous aggregates rather than amyloid. In summary, although the experimental results described in the accompanying examples now allow for the retrospective development of good explanatory methods and theories to envision additional cyclic peptides with suitable epitopes for the detection and identification of potent antibodies against amyloid production (particularly systemic amyloid production), this would not have been foreseeable without the knowledge of the present invention. In this context, and again without wishing to be bound by any theory, it is worth noting that recent studies using cryo-EM have shown that the amyloid structure of systemic amyloidogenic proteins (such as ATTR and AL amyloidosis) caused by misfolding of immunoglobulin light chains (LC) is similar on the one hand to the amyloid structure of localized amyloidogenic proteins (such as tau), but is significantly different on the other hand; see Figure 5 of Schmidt et al., Nat. Commun. 10 (2019), 5008, https: / / doi.org / 10.1038 / s41467-019-13038. Therefore, it is reasonable to assume that the results of the present invention with the cyclic peptides derived from TTR can also be applied to other systemic amyloidogenic proteins.

[0009] Thus, the present disclosure provides cyclic compounds comprising peptides containing epitopes of systemic amyloidogenic proteins, wherein the epitopes are preferably only bindable by antibodies in misfolded and / or aggregated forms of the protein, such as in the case of neo-epitopes, and / or the epitopes are at least not bindable by antibodies in the physiologically active form of the protein, such as in the case of epitopes accessible in monomers of the TTR protein, which are hidden in the physiologically active tetramer and are no longer accessible to antibody binding. As shown in the accompanying examples, the cyclic peptide compounds of the present disclosure are particularly useful in immunological assays, such as ELISA assays, which can be used, for example, in drug discovery or diagnostic methods, as well as in potency assays for characterizing therapeutically useful antibodies and equivalent binding molecules, where antibody Fc-mediated activity plays a key role in the mechanism of action.

[0010] Specifically, the outstanding performance of cyclic peptide compounds as target antigens was first demonstrated in the ELISA assay described in Example 1. 50 The values are comparable to the EC values for antibody binding to protein aggregates and to a linear peptide containing the same epitope as the cyclic peptide. 50 The cyclic peptide performed best because it showed the highest binding affinity to the antibody, i.e., the lowest EC 50 As mentioned above, the higher binding affinity between the antibody and the cyclic peptide compared to the protein aggregate may be due to a higher epitope density, which leads to a better apparent binding affinity due to a higher binding capacity and a higher avidity effect, due to better accessibility of the epitope in the cyclic peptide compared to the full-length protein, and / or due to better conformational control of the synthetic peptide compared to the recombinant protein. Thus, in one embodiment, the cyclic peptide compounds and cyclic peptides of the present disclosure, respectively, provide a binding affinity to the antibody that is higher than the binding affinity to the target protein from which it is derived, and higher than the corresponding linear peptide in an ELISA assay (such as described in the accompanying Example 1), preferably at least 2-fold higher, more preferably at least 3-fold, 4-fold or 5-fold higher, and most preferably at least 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher than the full-length target protein and / or linear peptide.

[0011] The therapeutic efficacy of antibodies, particularly as effective medicines for the treatment of amyloidosis, depends not only on the ability of the antibodies to bind aggregates, but also on the activity mediated by the antibody Fc, which plays a key role in the mechanism of action. The binding of antibodies to Fc receptors on the cell surface triggers many important and diverse biological responses, including the phagocytosis and destruction of antibody-coated particles (called antibody-dependent cellular phagocytosis or ADCP). When producing a pharmaceutical composition, it is not enough to formulate the drug substance into a pharmaceutical product, and it is also necessary for the resulting pharmaceutical product to be approved by the regulatory authorities of the country using the pharmaceutical composition. In the U.S., the responsible regulatory authority is the FDA (http: / / www.fda.gov / ), and in Europe, it is, for example, the European Agency for the Evaluation of Medicinal Products (EMEA) (http: / / www.emea.eu.int / ).

[0012] The approval process is strictly regulated, and drug developers are required to submit a large amount of information about drug product candidates to regulatory agencies in order to obtain approval. This may include information about the potency of the drug product candidate and the corresponding assays used to determine potency. Such potency assays are used to characterize the product, monitor lot-to-lot consistency, and ensure product stability. The potency of antibodies, for which Fc binding to Fc receptors plays a key role in the mechanism of action, has traditionally been measured using biological assays in which the effect assessed depends on Fc-Fc receptor binding.

[0013] Therefore, assays to characterize the product, monitor batch-to-batch consistency, and ensure product stability are of clinical importance and should be sensitive enough to detect differences that may affect the product's mechanism of action and function.

[0014] As shown in Example 2, the use of the cyclic peptides of the present disclosure provides an improved ADCP assay. Surprisingly, the assay showed significant improvements in sensitivity and reliability when the cyclic peptides were used instead of TTR aggregates as the antigen. Furthermore, the high-sensitivity ADCP assay initially developed as described in Example 2 has been confirmed and validated; see Example 5. Specifically, the assay outlined in Example 5 has been shown to have the ability to detect potency changes associated with Fc domain alterations ranging from 40% to 180%, i.e., up to 60% potency loss and 80% potency gain.

[0015] Therefore, the present disclosure also relates to the use of the cyclic peptide compounds of the present disclosure as target antigens in a method for determining the phagocytosis-related efficacy of an antigen binding molecule (such as an antibody) comprising an Fc domain, wherein in a preferred embodiment, the antigen binding molecule is an antigen binding molecule specific for an amyloidogenic protein (preferably in an aggregated, misfolded and non-physiological form).

[0016] In one embodiment, the potency assay comprises the following steps:

[0017] (a) contacting a cyclic peptide compound of the present disclosure as a target antigen with a binding molecule under conditions that allow formation of a binding molecule-antigen complex, wherein in a preferred embodiment, the cyclic peptide compound is bound to a solid support (e.g., a microtiter plate);

[0018] (b) contacting the binding molecule-antigen complex with a population of effector cells, preferably Jurkat cells, engineered to express an Fc receptor, preferably the human Fc receptor FcγRI (CD64), and having a reporter gene, preferably encoding a bioluminescent protein, preferably luciferase, under the control of a response element responsive to Fc receptor activation, preferably wherein the response element is a NFAT (nuclear factor of activated T cells) response element, under conditions permissive for binding of the Fc domain to an Fc receptor, wherein binding of the Fc domain to the Fc receptor results in intracellular signaling and mediates quantifiable reporter gene activity; and

[0019] (c) detecting reporter gene activity,

[0020] wherein at least one mechanism of action of the Fc domain of the binding molecule is mediated by binding of the Fc domain to an Fc receptor and the reporter gene activity is indicative of the potency of the binding molecule, preferably wherein the mechanism of action of the Fc domain is induction of antibody-dependent cell-mediated phagocytosis (ADCP).

[0021] Detailed embodiments of the efficacy assay are described and claimed in WO 2023 / 099788 A1, which is incorporated herein by reference.

[0022] Thus, the cyclic peptide compounds of the present disclosure comprising an epitope of an amyloidogenic protein can be used as a target antigen rather than the amyloidogenic protein itself, as mentioned above, resulting in highly sensitive and reliable potency assays. The cyclic peptides of the present disclosure are particularly useful in assays for determining the potency of antibodies and Fc domain-containing binding molecules that bind to amyloidogenic TTR or other amyloidogenic proteins involved in systemic amyloidosis.

[0023] Without wishing to be bound by theory, the superiority of cyclic peptides can be explained by the extreme flexibility of linear peptides, which can adopt an almost unlimited number of conformations compared to cyclic peptides that are constrained by the two ends being linked together and therefore have less flexibility and adopt more stable conformations. In other words, cyclic peptides have lower entropy than the same amino acid sequence in linear form.

[0024] Thus, providing cyclic peptide compounds according to the present disclosure represents an important contribution to the art because of their outstanding utility as suitable targets for studying the binding between a target antigen and a corresponding target antigen binding molecule, for example in assays requiring high sensitivity. However, the present disclosure also relates to linear forms of cyclic peptide compounds and linear forms of cyclic peptides, respectively, which are used, for example, as precursors for preparing cyclic peptide compounds or as controls in experiments.

[0025] The assays described above, i.e., potency assays using the disclosed cyclic peptide compounds, can be applied to methods for producing pharmaceutical compositions comprising target antigen binding molecules, wherein the potency of the binding molecules is first analyzed after production. Based on the results, a determination is made as to whether the binding molecules can be used in pharmaceutical compositions. Specifically, only binding molecules that are considered potent according to the assay are selected for further use and formulated into pharmaceutical compositions together with a pharmaceutically acceptable carrier.

[0026] The efficacy assay can also be used for analyzing and selecting a batch of target antigen binding molecules in a pharmaceutical composition method, wherein the sample and the control sample of the batch to be analyzed are subjected to the efficacy assay, and the reporter gene activity of the sample is compared with the reporter gene activity of the control. The batch of reporter gene activity that the sample shows larger, equal or does not actually reduce compared with the control is finally selected for further use. Therefore, this assay can be used for verifying batch consistency.

[0027] The present disclosure also relates to a kit, which is preferably designed for performing a potency assay as disclosed herein, in particular for determining the potency of a binding molecule comprising an Fc domain to induce ADCP, wherein the kit comprises at least a cyclic peptide compound of the present disclosure or a corresponding linear precursor, which can also be used as a control similar to that shown for TTR peptide in the Examples. The kit may also comprise:

[0028] (i) an effector cell population engineered to express an Fc receptor, preferably a human Fc receptor FcγR, and having a reporter gene under the control of a response element that responds to activation of the Fc receptor, preferably wherein the effector cell population is a Jurkat cell population and the reporter gene encodes a luminescent protein, preferably luciferase, under the control of an NFAT response element;

[0029] (ii) a corresponding substrate for a reporter gene; preferably, the kit further comprises one or more of the following:

[0030] (iii) a solid support, preferably a microtiter plate, preferably a 96-well plate including a lid;

[0031] (iv) wash, blocking, and assay / sample dilution buffers; and / or

[0032] (v) Monomer control of target protein and / or positive control anti-target antigen antibody.

[0033] Immunological and biological assays as described herein have been described using the cyclic peptides of the present disclosure, which comprise a TTR cyclic peptide, a TTR epitope as a target antigen, and an anti-TTR antibody, such as, for example, NI-301.37F1, which is disclosed in International Application WO 2015 / 092077A1 and has been described as being able to activate the immune system to eliminate TTR fibrils in animal models; see International Application WO 2020 / 094883 A1. TTR in its physiological form is a tetrameric protein that produces 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 organs or tissues where the precursor protein is synthesized, such as intracellular Tau fibrils and extracellular amyloid-β fibrils and plaques in Alzheimer's disease. In principle, the cyclic peptide compounds of the present disclosure may comprise any peptide or protein fragment capable of forming a cyclic peptide compound, in particular a peptide or protein fragment comprising a new epitope as mentioned above. In a particularly preferred embodiment, the (new) epitope is hidden in the native folded conformation of the target antigen, but can be bound by an antibody after unfolding and aggregation, for example, the linear epitope WEPFA of the antibody NI-301.37F1, which is located at position 41 to position 45 of the mature TTR protein. Similarly, the method of the present disclosure is applicable to any cyclic peptide containing and displaying an epitope of the target antigen binding molecule to be tested, such as an epitope that is exposed only in a misfolded variant, a conformational epitope on an aggregate, fibril and / or oligomer, an epitope on an extracellular variant of other physiological proteins located in the cell, or an epitope that is specific for exogenous pathogens (such as fungi, bacteria and viruses).

[0034] However, according to an embodiment of the present invention, 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 exposed in a misfolded and non-physiological form of the protein (e.g., transthyretin). The cyclic peptide compound is particularly suitable and therefore preferably used in a method for detecting an antibody specific for an amyloidogenic protein involved in systemic amyloidosis (particularly an antibody that binds to a misfolded and non-physiological form of the protein), and in a method for determining the efficacy of the antibody, respectively.

[0035] The cyclic peptide compounds of the present disclosure are particularly useful for measuring the potency of antibodies to activate ADCP.

[0036] Additionally, the cyclic peptide compounds of the present disclosure are particularly useful in a method of identifying and optionally obtaining antibodies that bind to an amyloidogenic protein involved in systemic amyloidosis, the method generally comprising the steps of:

[0037] (a) providing, optionally producing, one or more potential amyloidogenic protein binding antibodies or sources thereof;

[0038] (b) subjecting one or more potential amyloidogenic protein binding antibodies, or a source thereof, to a binding assay comprising a cyclic peptide compound of the present disclosure; and

[0039] (c) identifying and optionally obtaining an antibody determined to bind to the cyclic peptide compound (the subject antibody).

[0040] This method can be combined with the potency assays of the present disclosure and as described in International Application WO 2023 / 099788A1, respectively, and / or with any other suitable method for further determining the diagnostic or, preferably, therapeutic utility of the subject antibodies.

[0041] Therefore, another embodiment of the present disclosure is a method for producing a pharmaceutical composition comprising an antibody that binds to an amyloidogenic protein, the method comprising at least the steps of:

[0042] (a) providing, optionally producing, one or more potential amyloidogenic protein binding antibodies or sources thereof;

[0043] (b) subjecting the one or more potential amyloidogenic protein binding antibodies, or a source thereof, to a binding assay comprising a cyclic peptide compound of the disclosure;

[0044] (c) identifying and optionally obtaining an antibody that binds to the cyclic peptide compound (the subject antibody); and

[0045] (d) formulating the antibody or derivative thereof identified and optionally obtained in step (c) with a pharmaceutically acceptable carrier.

[0046] The source of antibodies is not limited and includes, for example, natural antibodies obtained from immunized laboratory animals (such as rodents, preferably mice, most preferably Ig humanized mice) as well as synthetic antibodies; human blood or fractions thereof, preferably containing memory B cells; recombinant antibody libraries, such as phage, yeast and ribosomal systems or mammalian cell systems, such as CHO and HEK; for other sources of antibodies and other target binding molecules, see also "Detailed Description of the Disclosure".

[0047] The binding assay used in the above-mentioned methods preferably comprises an ELISA, such as that performed in Example 1.

[0048] In preferred embodiments of the methods of the present disclosure for identifying and obtaining subject antibodies and their further use in formulation into pharmaceutical compositions and drug product development, respectively, the antibody identified and optionally obtained in step (c) competes with a reference antibody for binding to an amyloidogenic protein, preferably wherein the EC of the subject antibody to the amyloidogenic protein is greater than or equal to 0. 50 lower than the reference antibody.

[0049] Unless otherwise defined in this application, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Materials, methods, and examples are illustrative only and are not intended to be limiting.

[0050] Other embodiments of the present disclosure will be apparent from the following description, examples and claims. It will be understood by those skilled in the art that each characterization of the general features of the following general embodiments can and is preferably intended to be combined with the characterization of one or more other features of this general embodiment. In addition, unless specifically indicated otherwise, although the embodiments described herein for antibodies (including examples) are preferred embodiments, they are intended to be illustrated and particularly extrapolated to any target binding molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Improved sensitivity of ELISA assays. ELISA assays were used to compare the binding specificity of antibody NI-301.37F1 to (A) the peptides TTR34-54cyc, TTR40-49, Biotin.TTR40-49, and mis-WT-TTR, and (B) the peptides TTR34-54cyc, Biotin.TTR34-54cyc, TTR40-49, Biotin.TTR40-49, and mis-WT-TTR. NI-301.37F1 specifically binds to mis.WT-TTR, and binding to the cyclic TTR34-54cyc peptide is approximately 10-fold stronger than binding to mis.WT-TTR. The curve for peptide TTR40-49 is consistent with that for Biotin.TTR40-49.

[0052] Figure 2 : Improved ADCP assay using cyclic peptide compounds as target antigens. The potency of antibody NI-301.37F1 was measured in the ADCP assay with cyclic TTR peptide (TTR34-54cyc) and demonstrated that antibody NI-301.37F1 RS could inhibit the expression of cyclic TTR peptide in a dose-responsive, i.e., dose-dependent manner (with an EC of 19.8 ng / ml). 50 as a characteristic) ability to activate phagocytosis.

[0053] Figure 3 : ADCP assay using TTR34-54cyc as the target antigen for detecting changes in antibody activity. By comparing the NI-301.37F1 reference sample (NI-301.37F1 RS) with samples having lower (NI-301.37F1 50% (A) and 70% (B)) and higher (NI-301.37F1 130% (C) and 150% (D)) concentrations, the assay was shown to have the ability to detect a 50% loss and a 50% increase in antibody activity, respectively. DETAILED DESCRIPTION

[0054] The present disclosure relates to a cyclic compound comprising a peptide or protein fragment, the peptide or protein fragment comprising an epitope from an amyloidogenic protein involved in systemic amyloidosis. As described herein, typically the epitopes in the peptide and protein fragments in the cyclic compound of the present disclosure are respectively epitopes of antibodies, i.e., amyloid / aggregate-specific antibodies, and the cyclic compound is bound by the antibody. Therefore, the present disclosure also relates to the use of such cyclic peptide compounds in the field of drug discovery and diagnosis, and in determining the efficacy of target antigen binding molecules comprising an Fc domain, in particular the use in a method for activating the efficacy of antibody-dependent cell-mediated phagocytosis (ADCP), preferably wherein the target antigen binding molecule is an antibody that binds to an amyloidogenic protein. The present disclosure also relates to the use of cyclic peptide compounds in corresponding efficacy assays, which are particularly useful for batch release of pharmaceutical compositions comprising antibodies or similar binding molecules, particularly when conducting clinical trials, applying for marketing authorization, and for quality control of approved drugs.

[0055] Unless otherwise indicated, terms used herein are given the definitions given in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised in 2000, reprinted in 2003, ISBN 0 19 8506732; second edition published in 2006, ISBN 0-19-852917-1978-0-19852917-0.

[0056] The term "protein" as used throughout the specification includes fragments and peptides of the (full-length) protein, which fragments and peptides contain and expose the epitope of the target antigen binding molecule (eg antibody) to be tested.

[0057] " Cyclic peptide " mentioned herein can refer to complete protein compound, for example, wherein the linker is 2, 3, 4, 5, 6, 7 or 8 amino acids, or wherein there is no linker. For example, it is possible that the natural protein sequence, that is, the amino acid segment comprising the antibody epitope allows cyclization, for example, due to the presence of two cysteines at an appropriate distance, without adding additional amino acids. It should be understood that the properties described for the cyclic peptide determined in the embodiment can be incorporated into other compounds, for example, in cyclic peptide compounds comprising non-amino acid linker molecules. When the cyclic compound is composed of amino acids, "cyclic peptide" and "cyclic compound" can be used interchangeably. In addition, the term "cyclic compound" can be used interchangeably with the term "cyclic peptide compound".

[0058] As used herein, the term "linker" means a chemical moiety that can be covalently linked directly or indirectly to a protein fragment or peptide as defined herein. The end of the linker can, for example, be joined to produce a cyclic peptide compound. The linker can be present at the position of the N-terminus and the C-terminus. Alternatively, the linker can be at an internal position "a certain distance" from the end. The linker can comprise one or more functionalizable moieties, such as one or more cysteine (C) residues. The linker can also be connected to other proteins or components via the functionalizable moiety. The length of the cyclic peptide compound comprising the linker is longer than the peptide or protein fragment itself.

[0059] As used herein, the term "functionalizable moiety" refers to a chemical entity having 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 (a 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 react to form a disulfide bond. The reaction with the other group of atoms can be a covalent bond or a strong non-covalent bond, such as in the case of a biotin-streptavidin bond, which can have a dissociation constant (Kd) of about 1e-14. A strong non-covalent bond, as used herein, means an interaction with a Kd of at least 1e-9, at least 1e-10, at least 1e-11, at least 1e-12, at least 1e-13, or at least 1e-14.

[0060] During experiments performed according to the present disclosure, it was unexpectedly discovered 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 selectively binds to TTR aggregates of wild-type or variant TTR with high affinity, is an excellent target antigen in ELISA and ADCP assays. As described in Example 1 and Figure 1 As shown in , the antibodies exhibited highly specific binding to the cyclic peptide compounds, wherein the binding affinity of the antibodies to the cyclic peptide was even an order of magnitude higher than the binding affinity to its natural target antigen (i.e., the misfolded TTR for which the antibodies were initially screened and identified). This significant effect is unexpected and advantageous because such cyclic peptide compounds can not only replace the preparation of full-length amyloidogenic proteins and their aggregates / fibrils (the preparation of full-length amyloidogenic proteins and their aggregates / fibrils is prone to variability and is more time-consuming than the preparation of cyclic peptides), but also as shown in Examples 1, 2, and 5, and as shown in Figures 1 to 3 As shown in , cyclic peptide compounds represent excellent target antigens in binding assays (such as ELISA) and functional assays (such as ADCP) that require high sensitivity and reproducibility.

[0061] Initially, a cyclic TTR peptide was designed to solve the crystal structure of the complex of the Fab fragment of the antibody NI-301.37F1 and its TTR antigen to obtain information about the three-dimensional structure of the antibody-antigen complex and thus understand its mechanism of action. Coincidentally, in an ELISA assay for determining the antibody NI-301.37F1 (i.e., IgG antibody), the cyclic TTR peptide was used instead of the full-length recombinant TTR protein, and surprisingly, it was revealed that the ELISA assay became more sensitive and reliable than that using the recombinant TTR protein; see Examples 1 and Figure 1 Subsequent experiments demonstrated, even more surprisingly, that the use of a cyclic TTR peptide significantly improved the sensitivity and reliability of the potency assay.

[0062] In the following, analysis of the cryo-electron microscopy (cryo-EM) structure revealed that the two ends of the unresolved loop are in contact with each other, and it was shown that on this basis, the epitope and peptide sequence of the cyclic peptide and cyclic peptide compound, respectively, can also be selected. Therefore, in addition to or as an alternative to the peptide design in the Fab-peptide antigen crystal structure, the use of cryo-EM structure can be used to select appropriate epitopes and amino acid sequences containing the epitopes for use in designing the cyclic peptides of the present disclosure, which exhibit the same favorable properties as the cyclic TTTR peptides that have been experimentally proven to be such effective and reliable tools in ELISA and ADCP assays. As mentioned above, it is noteworthy that cryo-EM studies have shown that the amyloid structure of systemic amyloidogenic proteins (such as ATTR and AL amyloidosis) caused by misfolding of immunoglobulin light chains (LC) is similar to that of localized amyloidogenic proteins (e.g., tau) on the one hand, but significantly different on the other hand; see Figure 5 of Schmidt et al., Nature Communications 10 (2019), 5008, https: / / doi.org / 10.1038 / s41467-019-13038. Therefore, it is prudent to expect that the results of the present invention for cyclic peptides derived from TTR can also be applied to at least other systemic amyloidogenic proteins.

[0063] Methods for generating crystal structures of antibodies and Fab fragments thereof directed against a peptide and its complex with a peptide antigen, respectively, are well known to those skilled in the art; see, for example, Amit et al., Science 233 (1986), 747-753. The same applies to cryo-electron microscopy; see, for example, Schmidt et al. (2019), supra.

[0064] This discovery now opens up opportunities for the generation of other cyclic peptide compounds that contain epitopes of other amyloidogenic proteins. For example, once an epitope is selected and a fragment or peptide sequence of an amyloidogenic protein is chosen, the program PEP-FOLD, which can predict peptide structure from amino acid sequence and appears to reasonably predict epitope presentation when applied to TTR cyclic peptides, can be used to design other cyclic peptides that mimic, for example, antibody-binding epitopes of amyloidogenic proteins, such as those described herein.

[0065] Thus, the present disclosure relates to cyclic peptide compounds and linear precursors thereof comprising peptides containing epitopes of systemic amyloidogenic proteins, which epitopes are preferably only bindable to antibodies in misfolded and / or aggregated forms of the protein, such as in the case of neo-epitopes, and / or the epitopes are at least not present in the physiologically active form of the protein and can be bound by antibodies, such as in the case of epitopes accessible in monomers of the TTR protein, which are hidden in the physiologically active tetramer and are no longer bindable to antibodies. Such peptides are preferably antigenic peptides and antigenic cyclic peptide compounds, respectively. As shown in Examples 1, 2, and 5, the cyclic peptide compounds of the present disclosure are particularly useful in immunological assays (such as ELISA assays) and biological assays (such as the reporter gene assays disclosed herein for potency assays).

[0066] The cyclic peptide compounds of the present disclosure may comprise or consist of a protein fragment or peptide consisting of an epitope recognized by a target antigen binding molecule (e.g., an antibody or antigen binding molecule specific for an amyloidogenic protein), or comprise an epitope recognized by a target antigen binding molecule, which means that additional amino acids or other chemical entities used, for example, to cyclize the peptide or protein fragment (as further described below) may be present in the protein fragment or peptide that forms the cyclic peptide compound.

[0067] The additional amino acids may be naturally located near the epitope sequence, i.e., amino acids flanking the epitope sequence, and present in the protein sequence from which the protein fragment or peptide is derived, i.e., the protein fragment or peptide forming the cyclic peptide compound comprises the epitope of the target antigen binding molecule and additional amino acids adjacent to and flanking the epitope, respectively. The number of those adjacent / flanking amino acids may vary, and may be, for example, between 1, 2, or 3 amino acids 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 equally distributed at the N-terminus and the C-terminus of the epitope sequence, or unequally, e.g., having 7 additional amino acids at the N-terminus of the epitope and 9 amino acids at the C-terminus of the epitope.

[0068] Additionally or alternatively, in one embodiment, the protein fragment or peptide comprises a linker, that is, the protein fragment or peptide may comprise an epitope recognized by the target antigen binding molecule without any adjacent amino acids, and a linker, or may comprise an epitope and adjacent amino acids as defined above, and a linker. In a preferred embodiment, the protein fragment or peptide forming the cyclic peptide compound of the present disclosure comprises an epitope recognized by the target antigen binding molecule and amino acids adjacent to the epitope, and a linker. Preferably, the linker is covalently coupled directly or indirectly to the N-terminal residue of the protein fragment or peptide and the C-terminal residue of the protein fragment or peptide.

[0069] Methods for peptide cyclization are well known in the art. For example, cyclization can be performed by chemical cross-linking using, inter alia, chemical scaffolds. Cross-linking requires functional groups, and only a few protein chemical targets constitute the vast majority of cross-linking techniques, such as primary amines (-NH2), which are present at the N-terminus of each polypeptide chain and in the side chains of lysine residues; carboxyl groups (-COOH), which are present at the C-terminus of each polypeptide chain and in the side chains of aspartic acid and glutamic acid; and sulfhydryl groups (-SH), which are present in the side chains of cysteine.

[0070] Scaffold-based cyclization is one of the most commonly used methods because it can be applied to chemically or biologically synthesized peptides. Generally speaking, scaffold compounds (such as organic halides (the most common being organic bromides)) react selectively with the sulfhydryl groups of cysteine. Non-sulfhydryl groups (such as the primary amine of lysine or the N-terminal amino group in the peptide) can also be used for cyclization, for example, with chemicals containing N-hydroxysuccinimide (NHS). Specially designed non-natural amino acids can also be used for cyclization in peptides via bioorthogonal reactions. For example, if there is an azide-containing amino acid (such as azidohomoalanine or azidophenylalanine) in the peptide, a copper-mediated click reaction with an alkyne-bearing scaffold can result in cyclization.

[0071] Furthermore, cysteines can be joined together via disulfide bonds (-SS-) between their side chains, or amide cyclization can be performed without any scaffold (head-to-tail or backbone cyclization).

[0072] For example, a peptide having a "C" residue at the N-terminus and C-terminus (e.g., the cyclic TTR compound used in the Examples, GCGGGRKAADDTWEPFASGKTSESGEGGGCG (SEQ ID NO: 17)) can be subjected to an SS-cyclization reaction to produce a cyclic peptide. Cyclic peptide compounds can be synthesized as linear molecules in which a linker is covalently attached to or near the N-terminus or C-terminus of a peptide comprising a TTR peptide or a related epitope mentioned herein before cyclization, and provided as a precursor, which is also the subject of the present disclosure. Alternatively, prior to cyclization, a portion of the linker is covalently attached to or near the N-terminus and a portion is covalently attached to or near the C-terminus. In either case, the linear compound is cyclized, for example, by SS bond cyclization. Thus, the compound can be cyclized by 1) covalent bonding at or near the N- and C-termini of the peptide + linker to form a peptide bond (e.g., a cyclized backbone), 2) covalent bonding at or near the N- or C-termini to a side chain in the peptide + linker, or 3) covalent bonding of two side chains in the peptide + linker. In this context, "near" is defined as within 1, 2, or 3 amino acid residues of the N- or C-termini. Preferably, the linker is coupled to the N- or C-termini.

[0073] As mentioned above, peptide can be by the oxidation of the residue (comprising such as cysteine and homocysteine) containing thiol (thiol / mercaptan) at N-terminal or C-terminal or its vicinity or inside peptide and cyclization.For example, two cysteine residues of peptide flank can be oxidized to form disulfide bond.Adoptable oxidizing agent comprises such as oxygen (air), dimethyl sulfoxide, oxidized glutathione, cystine, copper chloride (II), potassium ferricyanide, trifluoroacetic acid thallium (III) or other oxidizing agent, such as oxidizing agent known to persons skilled in the art, and used together with method known to persons skilled in the art.Crosslinking agent is also known in the art, and can such as be selected based on the functional group to be used for cross-linking, referring to the crosslinking agent selection tool (CrosslinkerSelectionTool) provided by Sai Mo Fei Shi Er science and technology company (Thermo Fisher Scientific).

[0074] Thus, in one embodiment, the linker comprises a functionalizable moiety, for example, an amino acid having one of the functional groups mentioned above, such as lysine, aspartic acid, glutamic acid or cysteine, a non-naturally occurring amino acid, such as azidohomoalanine or azidophenylalanine, or an equivalent functional molecule, such as polyethylene glycol (PEG).

[0075] In the case where 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 a linker, but may also be present in an epitope or in adjacent amino acids in a protein fragment or peptide forming a cyclic peptide. Therefore, the cyclization of peptides and protein fragments may also be performed in the absence of a linker, respectively. Therefore, in one embodiment, a protein fragment or peptide forms a cyclic peptide compound of the present disclosure in the absence of a linker. Connection may occur via one or more amino acid side chains, 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.

[0076] In a preferred embodiment, at least one functionalizable moiety is present in the linker, i.e., the linker comprises one or more functionalizable moieties. The linker may comprise or consist of any amino acid (including non-natural amino acids), but preferably comprises at least any 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 equivalent functional molecules (such as polyethylene glycol (PEG)). In a preferred embodiment, the linker comprises cysteine as a functionalizable moiety.

[0077] Thus, in a preferred embodiment, linkers of any length and sequence can be described by the following sequence X-nX-1FX1-Xn, wherein F is any functionalizable moiety, preferably C (cysteine), and X is any amino acid, including unnatural amino acids. In another preferred embodiment, the linker amino acid is 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).

[0078] Even more preferably, the linker amino acid is 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). Thus, preferably, the cyclization is performed with a scaffold compound that reacts selectively with the sulfhydryl group of cysteine (such as an organic halide, preferably an organic bromide) or via a disulfide bridge. Most preferably, the cyclization is performed via a disulfide bridge.

[0079] 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 acids, in particular 1, 2, 3, 4, 5, 6, 7 or 8 amino acids and / or equivalent functional molecules, and / or combinations thereof, wherein when the linker comprises only amino acids, preferably at least one amino acid having any of the functional groups mentioned above is present among the amino acids, preferably cysteine. The other amino acids contained in the linker can be selected from any known amino acids, including unnatural amino acids, but are preferably alanine (A) and / or glycine (G), preferably glycine (G).

[0080] As mentioned above, the length of the linker can vary and can be, for example, 9 amino acids, such as GGGGCGGGG (SEQ ID NO: 148), or 8 amino acids, such as GGGCGGGG (SEQ ID NO: 149), GGCGGGGG (SEQ ID NO: 150) or GCGGGGGG (SEQ ID NO: 151), or 7 amino acids, such as GGGGCGG (SEQ ID NO: 152), GGGCGGG (SEQ ID NO: 153), GGCGGGG (SEQ ID NO: 154) or GCGGGGG (SEQ ID NO: 155), 6 amino acids, such as GGGCGG (SEQ ID NO: 156), GGCGGG (SEQ ID NO: 157) or GCGGGG (SEQ ID NO: 158), 5 amino acids, such as GCGGG (SEQ ID NO: 159) or GGGCG (SEQ ID NO: 160). 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.

[0081] Most preferably, the linker in the cyclic peptide compound comprises or consists of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16).

[0082] As shown in the Examples, cyclic peptide compounds comprising the epitope WEPFA of the antibody NI-301.37F1 disclosed in International Application WO 2015 / 092077A1 have been shown to be very suitable target antigens for immunological and biological assays (e.g., reporter gene assays used for potency assays). This epitope WEPFA is a new epitope located at positions 41 to 45 of the mature TTR protein, which is hidden in the native folded conformation of the TTR protein but can be bound by antibodies after unfolding and aggregation.

[0083] Thus, in a preferred embodiment, the cyclic peptide compound comprises a (neo)epitope, preferably from any protein whose aggregation leads to a disease phenotype.

[0084] The physiological functions of proteins are highly dependent on their correct three-dimensional conformation. Disturbances in the correct folding of newly synthesized or pre-existing proteins and the pathways responsible for refolding (molecular chaperones) or degradation of misfolded proteins (ubiquitin-proteasome and autophagy systems) may lead to protein aggregation within and / or outside the cell. These misfolded protein precipitates form ordered protein aggregates (e.g., amyloid fibrils) or disordered protein aggregates (e.g., inclusion bodies), which dissociate only in the presence of high concentrations of detergents or denaturing buffers ( Acta Neuropathol 125 (2013), 1-2).

[0085] Amyloidosis is characterized by the deposition of cross-β-sheet amyloid fibrils composed of misfolded proteins and / or misassembled proteins. The amyloid fibrils, which are the pathological hallmarks of these disorders, can be deposited systemically or localized in specific organs. The development of amyloidosis is generally associated with aging and is associated with reduced quality of life and severe pain in patients and their families. Alzheimer's disease is an example of localized brain amyloidosis, and type 2 diabetes is an example of localized extracerebral amyloidosis; both diseases are associated with aging. Systemic forms of amyloidosis, also often associated with aging, are less common and include TTR amyloidosis. The origin of amyloidosis is sporadic (i.e., from normal protein sequences) or hereditary (familial) (i.e., from proteins with one or more point mutations). In addition, there are infectious forms of amyloidosis, such as transmissible spongiform encephalopathy (Ankarcrona et al., Journal of Internal Medicine (J Intern Med.) 280 (2016), 177-202) caused by prion protein aggregation.

[0086] As shown in the accompanying examples, the present disclosure provides cyclic peptide compounds that preferably comprise epitopes that are normally exposed in pathological protein aggregates of amyloidogenic proteins, which makes the cyclic compounds particularly useful in helping to provide reliable methods for determining the efficacy of antibodies and antibody-based pharmaceuticals in terms of their ability to activate FC domain / receptor-mediated effector functions (such as antibody-dependent cell-mediated phagocytosis (ADCP)), wherein the antibodies preferably target epitopes on pathological protein aggregates; see Examples 5 to 8 in WO 2023 / 099788 A1.

[0087] In a preferred embodiment, the (neo)epitope is derived from an amyloidogenic protein involved in systemic amyloidosis or an aggregate thereof, such as transthyretin (TTR) (particularly wild-type TTR and variant TTR, preferably wild-type TTR), immunoglobulin light chain (LC), immunoglobulin heavy chain (LH), serum amyloid A (SAA), leukocyte chemoattractant factor 2 (LECT2), gelsolin, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, β2 microglobulin (particularly wild-type and variant β2 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. 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.

[0088] 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.

[0089] 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 the amyloidogenic protein. More specifically, at least the epitope of the target antigen binding molecule should be present, as known to those skilled in the art, the epitope can be composed of as few as four amino acids, and the epitope can be supplemented with an appropriate number of amino acids and / or other linker moieties sufficient and necessary for cyclization.

[0090] However, in principle, there is no limit to the length of the peptide, as long as it can be cyclized and it is recognized by the target binding molecule. Therefore, the cyclic peptide compounds disclosed and used herein may include proteins or fragments or peptides containing 4 to all amino acids of an amyloidogenic protein. Preferably, the protein fragment or peptide in the cyclic peptide compound comprises 4 amino acids to 100 amino acids, more preferably 4 amino acids to 90 amino acids, more preferably 4 amino acids to 80 amino acids, more preferably 4 amino acids to 70 amino acids, more preferably 4 amino acids to 60 amino acids, more preferably 4 amino acids to 50 amino acids, more preferably 4 amino acids to 45 amino acids, more preferably 4 amino acids to 40 amino acids, more preferably 4 amino acids to 35 amino acids, more preferably 4 amino acids to 30 amino acids, more preferably 4 amino acids to 25 amino acids, or 4 amino acids to 24 amino acids, or 4 amino acids to 23 amino acids, or 4 amino acids to 22 amino acids, or 4 amino acids to 21 amino acids, or 4 amino acids to 20 amino acids, preferably 5 amino acids to 25 amino acids, or 5 amino acids to 24 amino acids, or 5 amino acids to 23 amino acids, or 5 amino acids to 22 amino acids, or 5 amino acids to 21 amino acids, or 5 amino acids to 20 amino acids.

[0091] The amino acids represent only the epitope recognized by the target antigen binding molecule or the epitope and adjacent amino acids present in the amyloidogenic protein. In a preferred embodiment, the protein fragment of the peptide comprises amino acid residues of the amyloidogenic protein, wherein these amino acid residues comprise the epitope and adjacent amino acids.

[0092] The cyclic TTR peptide used in the accompanying examples consists of the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (TTR34-54cyc; SEQ ID NO: 17), which has a total of 31 amino acids and contains 21 amino acids of the amyloidogenic protein TTR, including the epitope WEPFA (SEQ ID NO: 1), and a 10-amino acid linker sequence, with five amino acids each from the N-terminus and C-terminus of the 21-amino acid stretch of TTR. Thus, in preferred embodiments, the cyclic peptide compound consists of a total of 20 to 40, more preferably 25 to 35, and most preferably 30 ± 1, 2, 3, or 4 amino acids, or, in the case of the incorporation of non-amino acid residues, such as as linkers, is configured so that its structure is similar to the corresponding peptide. In this embodiment, the amino acid sequence derived from the amyloidogenic protein present in the cyclic peptide compound can be composed of 10 to 40, preferably 15 to 25 and most preferably 20 ± 1, 2, 3 or 4 amino acids, and is optionally supplemented with a linker, preferably 5 to 20 amino acids in length, more preferably 5 to 15, most preferably 10 ± 1, 2, 3 or 4 amino acids, distributed at both ends (N-terminus and C-terminus) or only at one end. It is also conceivable that, for example, if the epitope of the target binding molecule is a conformational epitope or a discontinuous epitope, the linker sequence or "stuffer" sequence is located within the amino acid sequence derived from the amyloidogenic protein.

[0093] As mentioned above, the cyclic peptide compound preferably comprises a peptide derived from an amyloidogenic protein involved in systemic amyloidosis or an 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.

[0094] From the sequence of the cyclic TTR peptide shown in SEQ ID NO: 17, it is further apparent 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 the epitope.

[0095] Thus, in one embodiment, the cyclic peptide of the present disclosure comprises at least 4 consecutive 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 SEQ ID NO: 1 and / or at least 9 amino acids appended to the C-terminus of SEQ ID NO: 1, or variants thereof. In a preferred embodiment, the cyclic peptide comprises the sequence WEPFASG (SEQ ID NO: 4). Preferably, the amino acid at the N-terminus and / or C-terminus of the cyclic peptide compound of the present disclosure is 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 the 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) of the peptide sequence WEPFA (SEQ ID NO: 1), in each case via a peptide bond. As mentioned above, the cyclic peptide compounds of the present disclosure preferably comprise a linker comprising or consisting of GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16). More specifically, in a preferred embodiment, the cyclic peptide of the present disclosure comprises a first linker (L1) 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, L1 and L2 are linked to form a cyclic peptide, preferably wherein the N-terminus of L1 is bonded to the C-terminus of L2 via a peptide bond.

[0096] Generally speaking, the protein fragment or peptide of TTR can be any fragment or peptide 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 be any of the amino acids listed in Table 1 below.

[0097] Table 1: TTR peptides containing 4 amino acid residues .

[0098]

[0099]

[0100]

[0101] In preferred embodiments, the TTR peptide comprises at least 4 amino acid residues, and preferably all amino acids, of an amino acid sequence that are exposed in misfolded variants, and on aggregates, fibrils and / or oligomers, respectively, such as WEPFA (SEQ ID NO: 1), which is a peptide recognized by antibody NI-301.37F1 or antibody NI-301.28B3 disclosed in WO 2015 / 092077A1; EEFXEGIY (SEQ ID NO: 2), which is a peptide recognized, for example, by antibody NI-301.59F1 disclosed in WO 2015 / 092077A1; ELXGLTXE (SEQ ID NO: 3), which is a peptide recognized, for example, by antibody NI-301.35G11 disclosed in WO 2015 / 092077A1, wherein X can be any amino acid; WEPFASG (SEQ ID NO: 4), which is a peptide recognized, for example, by antibody NI-301.37F1 or antibody NI-301.28B3 disclosed in WO 2015 / 092077A1; NO: 4), WEPFASG is a peptide recognized, for example, by the antibody NI-301.12D3 disclosed in WO 2015 / 092077A1; TTAVVTNPKE (SEQ ID NO: 5), which is a peptide recognized, for example, by the antibody NI-301.18C4 disclosed in WO 2015 / 092077A1; KCPLMVK and VFRK (SEQ ID NO: 6 and SEQ ID NO: 7), which represent peptides comprising a conformational epitope requiring at least a first sequence of C and a second sequence of V and F, and which are recognized by the antibody NI-301.18C4 disclosed in WO 2015 / 092077A1.Epitopes recognized by 44E4: EHAEVVFTA (SEQ ID NO: 8), which is a peptide recognized, for example, by the antibody 14G8 / PRX004 disclosed in Higaki et al., Amyloid, 23 (2016), 86-97; GPRRYTIAA (SEQ ID NO: 9), which is a peptide recognized, for example, by the antibody 18C5 described in WO 2019 / 071205A1; VHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVE (SEQ ID NO: 10), which is a peptide recognized, for example, by the antibody binding to TTR30-66 described in WO 2014 / 124334 A2; ALLSPYSYSTTAV (SEQ ID NO: 11), which is a peptide recognized, for example, by the antibody 18C5 described in WO 2019 / 071205A1. 2014 / 124334 A2, which is a peptide recognized by the antibody that binds to TTR109-121; WKALGISPFHE (SEQ ID NO: 12), which is a peptide recognized, for example, by the antibody 371M described in WO 2015 / 115332 A1; SYSTTAVVTN (SEQ ID NO: 13), which is a peptide recognized, for example, by the antibody 313M (RT24) described in WO 2015 / 115331 A1; or LLSPYSYSTTAVVTNPKE (SEQ ID NO: 14), which is a peptide recognized, for example, by the antibody that binds to TTR100-127 described in WO 2014 / 124334 A2.

[0102] Most preferably, the TTR peptide of the present disclosure comprises the amino acid sequence WEPFA (SEQ ID NO: 1).

[0103] The peptides mentioned above are all derived from TTR protein, wherein the peptide or fragment VFRK (SEQ ID NO: 7) is located at the N-terminus of the peptide WEPFA (SEQ ID NO: 1), and the peptide or fragment ELXGLTXE (SEQ ID NO: 3) is located at the C-terminus of the peptide WEPFA (SEQ ID NO: 1). Therefore, in one embodiment, the cyclic peptide of the present disclosure comprises a portion or all of the first motif VFRK (SEQ ID NO: 7) at its N-terminus and / or a portion or all of the second motif ELXGLTXE (SEQ ID NO: 3) at its C-terminus, wherein X in SEQ ID NO: 3 is any natural amino acid, preferably wherein X at position 3 in SEQ ID NO: 3 is histidine (H), and X at position 7 in SEQ ID NO: 3 is threonine (T).

[0104] In one embodiment, the cyclic peptide of the present disclosure comprises at its N-terminus a portion of a first motif VFRK (SEQ ID NO: 7) comprising at least 2 amino acids and at its C-terminus a portion of a second motif ELXGLTXE (SEQ ID NO: 3) comprising at least 1 amino acid, preferably wherein the amino acids constituting the portion of the first motif comprise the dipeptide RK in SEQ ID NO: 7, and the amino acids constituting the portion of the second motif comprise the N-terminal E in SEQ ID NO: 3.

[0105] As mentioned above, the cyclic peptide compound preferably comprises a protein fragment or peptide comprising an epitope of an amyloidogenic protein, preferably a TTR epitope, and most preferably comprising the amino acid sequence WEPFA (SEQ ID NO: 1) and the epitope of adjacent amino acids, and a linker at the N-terminus and C-terminus of the peptide, wherein the linker can in principle comprise any of the linker sequences described above, and preferably comprises the amino acid sequence GCGGG (SEQ ID NO: 15) or GGGCG (SEQ ID NO: 16). Thus, in 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 to be a suitable target antigen in Examples 1, 2, and 5.

[0106] The present disclosure also relates to variants of the cyclic peptide compounds of the present disclosure, which variants comprise the sequence WEPFA (SEQ ID NO: 1) and further comprise a 5% to 20% variation in amino acid sequence identity, wherein the variation is in the amino acids appended to the N-terminus and / or C-terminus of SEQ ID NO: 1, for example, a variant of SEQ ID NO: 17 having 80% to 95% sequence identity with SEQ ID NO: 17, wherein the variation is due to an amino acid substitution, addition or deletion at the N-terminal sequence RKAADDT (SEQ ID NO: 162) and / or an 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.

[0107] Preferably, the cyclic peptide compounds of the present disclosure or variants thereof, characterized in that they each comprise the sequence WEPFA (SEQ ID NO: 1), optionally comprise the adjacent amino acids indicated above, and comprise the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 17), also include cyclic peptide compounds comprising additional amino acids and peptide / protein fragments, respectively. Thus, in one embodiment, the cyclic peptide compounds of the present disclosure or variants thereof further comprise 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),

[0108] Alternatively, the cyclic peptide compounds of the present disclosure or variants thereof do 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).

[0109] Additionally or alternatively, the cyclic peptide compounds or variants thereof of the present disclosure do not contain any immunogenic sequence (a) the N-terminus of VFRK (SEQ ID NO: 7) in the human TTR sequence of SEQ ID NO: 164 and / or (b) the C-terminus of ELHGLTTE (SEQ ID NO: 3) in the human TTR sequence of SEQ ID NO: 164.

[0110] When referring to cyclic peptide compounds, the above-mentioned variants are also included.

[0111] In one embodiment, the cyclic peptide compounds of the present disclosure and their precursors, or protein fragments or peptides within the cyclic peptide compounds are further derivatized or modified, respectively. For example, proteins and / or other reagents can be coupled to the cyclic peptide compounds, which can, for example, serve as probes in in vitro studies. For this purpose, any functionalizable moiety capable of reacting (e.g., forming a covalent or non-covalent but strong bond) can be used. These proteins and / or other reagents can be, for example, carrier proteins such as bovine serum albumin (BSA) for immunoblotting or immunohistochemical assays. In addition, other reagents can be dyes.

[0112] The present disclosure also relates to a composition comprising a cyclic peptide compound as disclosed herein or a linear precursor thereof. The composition may have additional excipients, such as a buffer, a stabilizer and / or a diluent. In one embodiment, the composition comprises a cyclic peptide compound that is further derivatized as explained above, for example, the composition comprises a cyclic peptide compound that further comprises a conjugate (such as a dye).

[0113] Also provided herein are peptide microarrays comprising cyclic peptide compounds of the present disclosure. Peptide arrays are known to those skilled in the art and include peptides displayed on a solid surface (typically a glass or plastic chip). Peptide arrays are commonly used to study the binding properties and functionality and kinetics of protein-protein interactions. The synthesis of peptide arrays is described, for example, in: Szymczak et al., Anal Chem. 90 (2018), 266-282 and Winkler et al., Methods Mol Biol. 570 (2009), 157-174.

[0114] As noted in Example 1, antigen-binding molecules (here, anti-TTR antibodies) show strong binding affinity to cyclic peptides in ELISA assays. Therefore, cyclic peptide compounds are suitable target antigens for use in assays for detecting and quantifying antigen-binding molecules (such as antibodies).

[0115] Therefore, the present disclosure also relates to the use of the cyclic peptide compounds disclosed herein or the compositions disclosed herein in any type of assay involving analysis of the interaction between a target antigen binding molecule and a target antigen, such as detection, which may also include quantification of the target antigen binding molecule. In a preferred embodiment, such an assay is an ELISA assay.

[0116] It has been further shown that cyclic peptides are particularly suitable target antigens in efficacy assays as described herein. Therefore, in another preferred embodiment, the present disclosure relates to cyclic peptide compounds as disclosed herein or compositions as disclosed herein for determining the effectiveness of antigen binding molecules (such as antibodies or any other binding molecules comprising an Fc domain, preferably antibodies as defined herein). The determination of effectiveness is preferably performed using the assays disclosed herein, i.e., Examples 2 and 5 and the assays described in International Application WO 2023 / 099788A1, the contents of which are incorporated herein by reference.

[0117] Efficacy testing is performed as part of product consistency testing, comparability studies, and stability testing. These tests are used to measure product attributes related to product quality and manufacturing control and are performed to ensure the identity, purity, strength (potency), and stability of the product used during all stages of clinical research. Similarly, efficacy measurements are used to demonstrate that only product batches (i.e., batches) that meet specified specifications or acceptance criteria can be administered during all stages of clinical research and after market approval. Efficacy is defined as "the specific ability (ability / capacity) of a product to achieve a given result, as indicated by appropriate laboratory tests or by well-controlled clinical data obtained by administering the product in the intended manner." Ideally, the potency assay will represent the product's mechanism of action (i.e., the 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, U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, January 2011. In the assay aspects of the present invention, the "potency" of a target antigen binding molecule, particularly an antibody, as a drug product is therefore a measure of its activity in an ADCP assay relative to the activity of a reference standard (of the drug product) whose activity and activity level in the ADCP assay have been assessed or are known, respectively. Thus, a higher potency of an antibody / drug product compared to a reference means that the antibody / drug product exhibits higher binding activity in the ADCP assay, i.e., a lower EC 50 values, and a lower potency of the antibody / drug product compared to the reference means that the antibody / drug product shows lower binding activity in the ADCP assay, i.e., a higher EC 50 For example, NI-301.37F1 150% mimics an antibody with higher potency and shows an EC 50 The value is 0.7 times that of the reference sample NI-301.37F1 RS (100%). In contrast, the antibody NI-301.37F1 50% mimics an antibody with lower potency (loss of activity) and shows an EC 50The value is twice that of the reference sample NI-301.37F1 RS (100%); see Example 2. As shown in Example 5, even a 60% to 80% potency change can be observed. Thus, a target antigen-binding molecule (e.g., an antibody) that exhibits increased potency is determined to have, for example, a lower EC 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 greater)) relative to a reference sample. 50 Alternatively, a target antigen binding molecule (e.g., an antibody) that exhibits reduced potency is one that is determined to have, for example, a higher EC 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 greater)) relative to a reference sample. 50 The target antigen binding molecule of the value of , for example, as determined in the ADCP assay described herein. However, according to GLP and GMP, the acceptable variability (i.e., imprecision) of potency measurement is + / - 20%, so this is the preferred limit for the target antigen binding molecule tested.

[0118] As mentioned above, the cyclic peptide compounds and corresponding compositions of the present disclosure are preferably used to determine the potency of the antigen binding molecules, respectively, wherein the determination of potency is preferably performed using the assays disclosed herein, i.e., Examples 2 and 5 and the assays described in International Application WO 2023 / 099788 A1, the contents of which are each incorporated herein by reference.

[0119] Such a potency assay preferably comprises the following steps:

[0120] (a) contacting a cyclic compound of the present disclosure, which is a target antigen, with a binding molecule under conditions that allow formation of a binding molecule-antigen complex;

[0121] (b) contacting the binding molecule-antigen complex with a population of effector cells engineered to express the Fc receptor and having a reporter gene under the control of a response element that is responsive to activation of the Fc receptor, under conditions permissive for binding of the Fc domain to an Fc receptor, wherein binding of the Fc domain to the Fc receptor results in intracellular signaling and mediates quantifiable reporter gene activity; and

[0122] (c) detecting the activity of the reporter gene,

[0123] wherein at least one mechanism of action of the Fc domain of the binding molecule is mediated through binding of the Fc domain to an Fc receptor, and reporter gene activity is indicative of the potency of the binding molecule.

[0124] In more detail, such determination preferably comprises at least the following steps:

[0125] i) spotting the target antigen (i.e., the cyclic peptide compound of the present disclosure) into the wells of a microplate, i.e., coating the microplate (96-well plate) with the target antigen at 4°C overnight (or 18 hours ± 2 hours), preferably wherein the cyclic compound is diluted to 3 μg / ml in PBS buffer pH 7.4;

[0126] ii) contacting the target antigen with the target antigen binding molecule, preferably at 37° C. for 30 minutes, under conditions that allow formation of a binding molecule-target antigen complex;

[0127] iii) contacting the complex comprising the binding molecule and the target antigen with an effector cell, i.e., adding an effector cell (also called a reporter cell) to the complex, wherein the effector cell expresses an Fc receptor and a reporter gene under the control of a response element responsive to Fc receptor activation, preferably wherein the effector cell is an engineered cell, more preferably a Jurkat cell, which expresses an FcγRI receptor and a luciferase gene under the control of a NFAT transcription factor, and wherein the complex is incubated at 37° C., preferably for 6 hours;

[0128] iv) adding a substrate solution, preferably a luminescent substrate solution; and

[0129] v) detecting a signal, preferably a luminescent signal, using a luminometer.

[0130] A detailed description of the steps of such a potency assay is described in WO 2023 / 099788 A1, in particular on pages 23 to 32 and 38 to 46, the contents of which are incorporated herein by reference.

[0131] The binding molecules whose potency, in particular their potency to induce ADCP, is determined using the methods described herein (i.e., potency assays using the cyclic peptides of the present disclosure) can be any binding molecule that binds to the target antigen (i.e., the cyclic peptide compounds of the present disclosure). Thus, in general, the cyclic peptide compounds of the present disclosure can be used in methods for determining the potency (preferably the potency to induce ADCP) of any binding molecule that binds to the cyclic peptide compound.

[0132] 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 to determine the potency, in particular the potency to induce ADCP, of an anti-TTR antibody, most preferably the anti-TTR antibody NI-301.37F1, comprising the amino acid sequences of the VH and VL chains of SEQ ID NO: 19 and SEQ ID NO: 21 or SEQ ID NO: 23 and SEQ ID NO: 21 in its variable region or binding domain.

[0133] Table 2: Variable weight (V) of antibody NI-301.37F1 H ) chain and variable light (V L ) chain amino acid sequence and nucleotide sequence The CDRs in the amino acid sequence are underlined. The regions between the CDRs represent the framework regions.

[0134]

[0135]

[0136] The present disclosure also relates to a method of producing a pharmaceutical composition comprising said binding molecule as defined above, ie preferably an antibody that binds to an amyloidogenic protein involved in systemic amyloidosis, most preferably an anti-TTR antibody.

[0137] In the first step, respectively provide, preferably produce binding molecule and pharmaceutical product.Systems and methods for recombinant production of antibodies, corresponding binding molecules, fragments thereof, derivatives and mimetics are known in the art. Specifically, when implementing the claimed disclosure, those skilled in the art can rely on their recombinant production in host cells, purification, modification, formulation in pharmaceutical compositions and therapeutic uses and terms and features common in the art (see, for example, Antibodies A Laboratory Manual 2nd edition, 2014, 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 5 (2013). 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, which also describe antibody purification and storage; engineered antibodies, including the use of degenerate oligonucleotides, 5′-RACE, phage display and mutagenesis, immunoblotting protocols, and the latest screening and labeling technologies. The production of DARPins is explained, for example, in Stumpp et al., Drug Discovery Today 13 (2008), 695-701 and references cited therein and in Hanenberg et al., J Biol Chem 289 (2014), 27080-27089, DOI: 10.1074 / jbc.M114.564013. Furthermore, the production of the drug product can be performed in any manner that is desired and / or appropriate for the drug product in question.

[0138] In the next step, the binding molecules are subjected to a method as described herein. Specifically, the binding molecules are subjected to a method for determining the potency of the binding molecules, in particular the potency to induce ADCP, thereby using the cyclic peptide compounds of the present disclosure as target antigens. The information derived from the assay is used to assess whether the binding molecules can be used as part of a pharmaceutical composition, i.e., whether the drug product containing the binding molecules meets the criteria for injection into patients agreed upon by the regulatory authorities of the country where the drug product injection may occur. In addition, this information is used to identify the binding molecules for use in pharmaceutical compositions.

[0139] In another preferred embodiment, the binding molecules are formulated into pharmaceutical compositions together with a pharmaceutically acceptable carrier, in particular binding molecules found to be useful by the methods described herein (i.e., potency assays using the cyclic peptide compounds of the invention). Useful binding molecules are, for example, binding molecules that exhibit an EC in the (sub) nanomolar range when assessed using the methods disclosed herein. 50Value, or show a potency similar to that of 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% compared to the potency of a positive control. Pharmaceutically acceptable carriers and routes of administration can be obtained from the corresponding literature known to those skilled in the art. Pharmaceutical compositions can be formulated according to methods well known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000), University of Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols, 2nd ed., Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd ed., 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, and the like. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject at a suitable dose. Administration of suitable compositions can be achieved in various ways. Examples include administering compositions comprising pharmaceutically acceptable carriers via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.

[0140] The present disclosure also provides a method for preparing a drug 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%, and more preferably 100% compared to the potency of the positive control in activating ADCP. The method comprises the production of the binding molecule as explained above, wherein a batch of the binding molecule is obtained. The potency of the batch is then analyzed, in particular the potency of the batch in activating ADCP as described herein. The method also comprises preparing a drug or diagnostic product from the batch, provided that the potency of the batch, in particular the potency of the batch in activating ADCP, is determined to be at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, and more preferably 100% compared to the potency of the positive control.

[0141] The control is a reference standard, an antibody known to have potency to activate ADCP, such as an antibody that has been approved by a regulatory agency, and / or the batch to be analyzed has been stored and / or subjected to stress conditions, and the control is the value of the reporter gene activity of a sample taken from the batch or a corresponding batch before storage and / or subjection to said stress conditions.

[0142] The present disclosure also provides a method for determining the efficacy of an antibody as described above, wherein the method is part of an application for marketing authorization for the pharmaceutical product as a pharmaceutical composition. The present disclosure also provides a method for applying for marketing authorization for a pharmaceutical product comprising a binding molecule, the method comprising a method for determining the efficacy of a binding molecule in a pharmaceutical product as described herein.

[0143] As mentioned above, the method described herein is used as a potency assay for batch release, ie the method can be used to analyze different batches from the production of, for example, a given target antigen binding molecule.

[0144] The continuous production of any pharmaceutical product will result in different batches of product being released as a drug. A key feature in production is ensuring that different batches meet the same standards. These standards are typically developed in collaboration with regulatory agencies. Typically, each batch will be tested and inspected using a variety of different assays to ensure that the batch is of sufficient quality to be approved for marketing. This can be performed using the potency assays described herein, which utilize the cyclic peptide compounds of the present disclosure.

[0145] Therefore, the present disclosure also relates to a method for analyzing and selecting at least one batch of pharmaceutical compositions of target antigen binding molecules as defined above, wherein the method includes assessing the effectiveness of the batch sample in a first step, particularly the effectiveness of its activation of ADCP, wherein the method uses a cyclic peptide compound as described herein. As mentioned above, reporter gene activity is a measure of the effectiveness of the binding molecule, and therefore, the reporter gene activity of the sample is compared with the reporter gene activity of the control, and the sample is selected to show a batch of larger, equal or no significantly smaller reporter gene activity compared to the control. In one embodiment, the sample is selected to show a batch of larger, 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 packaged, for example, loaded into a test kit, and distributed to consumers.

[0146] Thus, the present disclosure relates to a method for validating a batch of target binding molecules for distribution (i.e., determining the quality of target antigen (e.g., aggregated protein) binding molecules), wherein the potency of the batch is tested for ADCP activation using a potency assay as described herein using a cyclic peptide compound of the present disclosure, and wherein the batch is validated for distribution only if the potency 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% compared to the potency of the positive control to activate ADCP.

[0147] In preferred embodiments, the method is particularly useful for analyzing and selecting a batch of pharmaceutical compositions comprising an anti-TTR antibody, and for validating a batch of an anti-TTR antibody for distribution, respectively. The control may be a reference standard and / or, in the case where the batch to be analyzed has been stored and / or subjected to stress conditions, the control may be the value of reporter gene activity of a sample taken from the batch or a corresponding batch prior to storage and / or subjecting to the stress conditions.

[0148] Binding of the drug product's binding molecule to the Fc receptor is compared to binding of a reference standard to the Fc receptor, and therapeutic efficacy is assessed by the ability of the drug product's binding molecule to bind to the Fc receptor to the same or substantially the same extent as the reference standard.

[0149] As mentioned above, the potency of the batch should preferably be at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, and more preferably 100% compared to the potency of the reference standard. However, the specific extent 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 can be established on a case-by-case basis and can be determined, for example, in collaboration with the appropriate regulatory authorities.

[0150] In order to be able to determine FcR binding in a reliable and consistent manner, the FcR binding of the binding molecule of the pharmaceutical product and the reference standard should be performed using the same assay, preferably an assay as described herein. Typically, the determination of binding of the reference standard is performed first to establish a standard with which any subsequent batches of binding molecules can be compared. However, the determination of binding of the reference standard can also be performed simultaneously with or after the determination of FcR binding of the binding molecule of the pharmaceutical product.

[0151] Thus, the present disclosure also relates to the use of the cyclic peptide compounds of the present disclosure in the above methods and processes, i.e., in a method of producing a pharmaceutical composition, in a process for preparing a pharmaceutical or diagnostic product, in a process for applying for marketing authorization for selling the pharmaceutical product as a pharmaceutical composition, in a method for applying for marketing authorization for a pharmaceutical product, in a method for analyzing and selecting at least one batch of pharmaceutical compositions, in a process for validating a batch of target binding molecules for distribution (i.e., determining the quality of the target antigen binding molecules), and in particular in a method for analyzing and selecting a batch of pharmaceutical compositions comprising anti-TTR antibodies and validating a batch of anti-TTR antibodies for distribution, respectively.

[0152] The present disclosure also relates to a composition comprising a target antigen binding molecule of the present disclosure that has been analyzed, validated, and selected according to the present disclosure, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0153] In order to verify that the analyzed binding molecules do trigger phagocytosis, thereby resulting in the engulfment of the target antigen (e.g., cyclic peptide compound), a phagocytosis assay can be performed, for example, via an in vivo phagocytosis assay, as described in, for example, Prakash et al., Chem Sci. 12 (2021), 10901-10918 for real-time monitoring of phagocytic uptake of beta-amyloid, or preferably via an in vitro phagocytosis assay as described in Examples 1 and 2 of WO 2023 / 099788A1, the contents of which are incorporated herein by reference. These assays show that antibody NI-301.37F1 W1 does trigger phagocytosis of TTR aggregates. Therefore, the disclosed method for determining the efficacy of binding molecules can be combined with a phagocytosis assay, in particular an in vitro phagocytosis assay. In addition, a patient-derived amyloid xenograft animal model as disclosed in WO 2020 / 094883A1 can be used to verify that the analyzed binding molecules do trigger phagocytosis, thereby resulting in the engulfment of the target antigen (e.g., aggregated TTR).

[0154] Furthermore, the binding of the analyzed binding molecules to their corresponding antigens can be verified by methods known in the art, for example via ELISA or BLI as shown in Example 1. Thus, the methods of the present disclosure for analyzing the potency of binding molecules can be combined with methods for determining the binding of binding molecules to their antigens.

[0155] As mentioned above, determining the efficacy of a drug is an important step in development, including the evaluation of new therapeutic agents for the treatment of disease. In the context of the present disclosure, this method is used to develop, evaluate, and batch release antibody-based drugs and other target antigen binding molecules that utilize the effector function of the Fc domain to treat diseases associated with the target protein, particularly protein aggregation disorders (such as systemic and localized amyloidosis).

[0156] Thus, the cyclic peptide compounds of the present invention are particularly useful as target antigens in assays for measuring the efficacy of antibodies that typically target protein aggregates, i.e., amyloidogenic proteins, and are therefore useful in the treatment of protein aggregation disorders such as systemic and localized amyloidosis, particularly disorders associated with TTR aggregation.

[0157] In addition, the cyclic peptide compounds of the present disclosure or compositions comprising the cyclic peptide compounds of the present disclosure can be used to detect autoantibodies against amyloidogenic proteins or fragments, oligomers or aggregates thereof. The cyclic peptide compounds are particularly suitable for detecting autoantibodies against TTR and identifying antibodies equivalent to, for example, NI-301.37F1. Similarly, cyclic peptides can be used to screen for antibodies against amyloidogenic proteins, particularly anti-TTR antibodies, generally, for example, by phage display.

[0158] In addition, cyclic peptides can be used to study the pharmacokinetic profile, i.e., the half-life of antibodies in plasma in in vivo non-human animal studies as well as in human clinical trials (e.g., with antibodies NI-307.37F11 (NI006) or NNC6019-0001 (PRX004). In addition, cyclic peptide compounds can be used, for example, during the course of antibody therapy to measure the plasma concentration of the antibody and support dosing for maintaining sustained levels of the antibody.

[0159] The cyclic peptides can also be used to identify antibodies that are equivalent to known antibodies and in particular to the mentioned anti-TTR antibodies (in particular antibody NI-307.37F11), for example by competition assays well known in the art. Therefore, all uses are also part of the present disclosure.

[0160] In addition, a kit is disclosed herein, which comprises at least a 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 (e.g., detecting, which may also include quantitative analysis of the target antigen binding molecule), and is most preferably useful for determining the effectiveness of antigen binding molecules (such as antibodies) comprising Fc domains. In a preferred embodiment, effectiveness is determined using assays disclosed herein, particularly as described in the accompanying examples and WO 2023 / 099788A1, and therefore, preferably, the kit comprises a device for performing corresponding effectiveness assays. In another preferred embodiment, the antigen binding molecule is an antigen binding molecule as defined above, preferably comprising an antigen binding molecule of an Fc domain, such as an antibody, and most preferably an anti-TTR antibody. Therefore, the kit can be used for the purposes listed above.

[0161] In one embodiment, the kit comprising the cyclic peptide compound further comprises:

[0162] (i) an effector cell population engineered to express a human Fc receptor FcγR and having a reporter gene under the control of a response element that responds to activation of the Fc receptor,

[0163] (ii) a corresponding substrate for the reporter gene; and optionally

[0164] (iii) a solid support, preferably a microtiter plate, preferably a 96-well plate including a lid;

[0165] (iv) washing, blocking and assay / sample dilution buffers, and / or

[0166] (v) Monomer control of target protein and / or positive control anti-target antigen antibody.

[0167] In a preferred embodiment, the effector cell population is a Jurkat cell population that expresses FcγR (preferably FcγRI) and a gene encoding a luminescent protein, preferably luciferase, under the control of an NFAT transcription factor, and wherein the kit comprises a luminescent substrate solution. In addition, the cyclic peptide compound preferably comprises an epitope of an anti-TTR antibody and an epitope of TTR, respectively, and the binding molecule is an anti-TTR antibody.

[0168] Preferably, the kit is suitable for analyzing the potency of a binding molecule comprising an Fc domain to induce ADCP.

[0169] In addition, the cyclic peptide compounds of the present disclosure and their linear precursors are particularly useful in a method for identifying and optionally obtaining antibodies and equivalent binding molecules (e.g., antibodies and equivalent binding molecules of the type described above) that bind to amyloidogenic proteins involved in systemic amyloidosis, which method generally comprises the following steps:

[0170] (a) providing, optionally producing, one or more potential amyloidogenic protein binding antibodies or sources thereof;

[0171] (b) subjecting one or more potential amyloidogenic protein binding antibodies, or a source thereof, to a binding assay comprising a cyclic peptide compound of the present disclosure; and

[0172] (c) identifying and optionally obtaining an antibody determined to bind to the cyclic peptide compound (the subject antibody).

[0173] This method can be combined with the potency assays of the present disclosure and the potency assays as described in WO 2023 / 099788A1, and / or with any other suitable method for further determining the diagnostic or, preferably, therapeutic utility of the subject antibodies. As mentioned, the subject antibodies can also be different kinds of antigen binding molecules.

[0174] Thus, there is provided a method for preparing a pharmaceutical composition comprising an antibody that binds to a systemic amyloidogenic protein, the method comprising at least the steps of:

[0175] (a) providing, optionally producing, one or more potential amyloidogenic protein binding antibodies or sources thereof;

[0176] (b) subjecting the one or more potential amyloidogenic protein binding antibodies, or a source thereof, to a binding assay comprising a cyclic peptide compound of the disclosure;

[0177] (c) identifying and optionally obtaining an antibody that binds to the cyclic peptide compound (the subject antibody); and

[0178] (d) formulating the antibody or derivative thereof identified and optionally obtained in step (c) with a pharmaceutically acceptable carrier.

[0179] The source of the antibody is not limited and includes, for example, natural antibodies obtained from immunized laboratory animals (such as rodents, preferably mice, most preferably Ig humanized mice) as well as synthetic antibodies; preferably human blood or fractions thereof containing memory B cells; recombinant antibody libraries, such as phage, yeast and ribosomal systems or mammalian cell systems such as CHO and HEK. In one embodiment, the cyclic peptide compounds of the present disclosure can be used to screen for nanobodies, also known as VHHs, derived from serum of camelids; see, for example, Lyu et al., Anal. Chem. 94 (2022), 7970-7980; Muyldermans, The FEBS Journal 288 (2021) 2084-210. In this context, IgG antibodies or binding fragments thereof known to bind to amyloidogenic proteins can be used as reference antibodies or sources for identifying and preparing nanobodies, respectively. Likewise, synthetic alternatives to computationally modeled antibodies can be screened, e.g., modular peptide binders such as designed armadillo repeat proteins (dArmRPs); see, e.g., Gisdon et al., Biological Chemistry 403 (2022), 535-543.

[0180] The binding assay used in the above-mentioned methods preferably comprises an ELISA, such as that performed in Example 1.

[0181] In preferred embodiments of the methods of the present disclosure for identifying and obtaining subject antibodies and their further use in formulation into pharmaceutical compositions and drug product development, respectively, the antibody identified and optionally obtained in step (c) competes with a reference antibody for binding to an amyloidogenic protein, preferably wherein the EC of the subject antibody to the amyloidogenic protein is greater than or equal to 0. 50 Preparation and formulation of the subject antibodies and similar target binding molecules obtained by the methods of the present invention can be performed as described for the target antigen binding molecules, supra.

[0182] The present disclosure also relates to antigen-binding molecules (particularly antibodies) that have been screened and thus obtained by using the cyclic peptide compounds of the present disclosure, for example by using an immunological assay (such as the ELISA assay also described in the accompanying Examples for screening).

[0183] Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications, including background sections and manufacturer's specifications, instructions, etc., as cited throughout this application) are hereby expressly incorporated herein by reference; however, no admission is made that any cited document is prior art with respect to the present invention.

[0184] A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

[0185] Example

[0186] Example 1: Cyclic peptides as target antigens provide higher sensitivity for ELISA assays of antigen-binding molecules

[0187] The ability of the antigen-binding molecule to bind to the cyclic peptide was exemplified by ELISA assay using a cyclic peptide comprising amino acid residues 34 to 54 of wild-type TTR (TTR34-54cyc in biotinylated and non-biotinylated forms) as a target antigen and the anti-TTR antibody NI-301.37F1 as an antigen-binding molecule. In addition, as antigen controls, the TTR peptide TTR40-49, the biotinylated TTR peptide TTR40-49, and misfolded wild-type TTR (mis.WT-TTR) were used.

[0188] The cyclic peptide TTR34-54cyc (1.36 mg / mL) was manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. Specifically, a peptide comprising the amino acid sequence H-GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 17) was synthesized by solid-phase peptide synthesis and cyclized via a disulfide bridge between two cysteine residues within a polyglycine stretch. A TTR peptide (TTR40-49, 1.25 mg / mL) comprising the amino acid sequence H-TWEPFASGKT-OH (SEQ ID NO: 161) was also manufactured by Schafer-N (Copenhagen, Denmark) and stored at -20°C. The biotinylated peptides Biotin.TTR34-54cyc and Biotin.TTR40-49 each contained an aminohexanoic acid (Ahx) spacer between their N-termini and biotin, i.e., Biotin.TTR34-54cyc (biotin-(Ahx)GCGGGRKAADDTWEPFASGKTSESGEGGGCG-OH (SEQ ID NO: 17), 680 μg / mL) and Biotin.TTR40-49 (biotin-(Ahx)TWEPFASGKT-OH, (SEQ ID NO: 161), 700 μg / mL). Misfolded wild-type TTR was prepared as follows:

[0189] Wild-type TTR protein purified from human plasma was obtained from Bio-Rad Laboratories, Inc. (Bio-Rad Laboratories, Inc., CA 7600-0604, USA) and custom purified by protein A / G chromatography followed by elimination of residual immunoglobulins by lectin column. Plasma-purified WT-TTR was provided at a concentration of 1 mg / ml in PBS buffer. Misfolded WT-TTR aggregates (mis.WT-TTR) were prepared in vitro by diluting the WT-TTR stock solution to a concentration of 200 μg / ml in aggregation buffer (50 mM acetate-HCl, 100 mM KCl, 1 mM EDTA, pH 3.0) and then incubating at 37°C with shaking at 1000 rpm for 4 hours. The mis.WT-TTR was aliquoted and stored at -20°C until use. The quality of the mis.WT-TTR was confirmed by ELISA and biolayer interferometry (BLI).

[0190] Two ELISA assays have been performed, wherein in the first ELISA assay (ELISA-1), the binding of the antibodies 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), the binding of the antibodies to the peptides TTR34-54cyc, Biotin.TTR34-54cyc, TTR40-49, Biotin.TTR40-49 and mis-WT-TTR has been analyzed.

[0191] Specifically, 96-well microtiter plates were coated 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) for 1 hour at 37° C., wherein each target antigen had been diluted to a concentration of 10 μg / ml in PBS buffer, pH 7.4. Nonspecific binding sites were blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween-20 in PBS buffer for 1 hour at room temperature (RT). NI-301.37F1 antibody (Neurimmune AG, Zurich, Switzerland; NI-301.37F1) was diluted in duplicate in blocking buffer to the indicated concentrations (dilution series from 400 nM to 4 pM and 0) and incubated overnight at 4°C. Binding was determined using an anti-human IgG antibody conjugated to horseradish peroxidase (HRP), and HRP activity was measured in a standard colorimetric assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Data were analyzed with Prism software from GraphPad. EC values were estimated using nonlinear regression of individual data points using a log(agonist) versus response model with a variable slope. 50 The data were fitted using the least squares regression method.

[0192] ELISA results confirmed the binding of NI-301.37F1 to mis.WT-TTR. Furthermore, ELISA assays showed that NI-301.37F1 bound to cyclic TTR34-54cyc and Biotin.TTR34-54cyc peptides much more strongly than to mis.WT-TTR, i.e., approximately 10-fold more strongly. Specifically, in ELISA-1, the EC binding of NI-301.37F1 to the cyclic TTR34-54cyc peptide was 50 is 27 pM, and the binding EC of NI-301.37F1 to mis.WT-TTR 50 is 338 pM; see Figure 1 A. In ELISA-2, the measured EC 50 The values were higher, but the approximately 10-fold difference between NI-301.37F1 binding to the cyclic TTR34-54cyc peptide and mis.WT-TTR was maintained. Specifically, the EC value of NI-301.37F1 binding to the cyclic TTR34-54cyc peptide was 50is 0.66 nM, and the binding EC of NI-301.37F1 to mis.WT-TTR is 50 is 8.3 nM; see Figure 1 B. No binding of NI-301.37F1 to TTR40-49 and Biotin.TTR40-49 was observed in both ELISA assays.

[0193] Example 2: Cyclic peptides as target antigens provide improved ADCP assays

[0194] An ADCP assay was developed to determine the potency of antigen-binding molecules. The assay uses a reporter cell line expressing human Fcγ receptor 1 (FcγR1) and has been evaluated for its ability to measure the potency of the antibody NI-301.37F1 to activate phagocytosis of cyclic TTR peptide (TTR34-54cyc) in vitro using the cyclic peptide as an antigen.

[0195] ADCP reporter gene assay

[0196] To measure the potency of the NI-301.37F1 reference sample (NI-301.37F1 RS, 100%) and test samples having a low 50% concentration (NI-301.37F1 50%), a low 30% concentration (NI-301.37F1 70%), a high 30% concentration (NI-301.37F1 130%), and a high 50% concentration (NI-301.37F1 150%) to activate phagocytosis of TTR34-54cyc, a commercially available FcγR1 ADCP reporter gene bioassay (Promega, Madison, Wisconsin, USA, Catalog Nos. GA1341, GA1345) was applied. This bioluminescent cell-based assay relies on a genetically engineered Jurkat T cell line that expresses human FcγR1 and a luciferase reporter gene driven by an NFAT response element. Activation of FcγR1 by the antibody-target complex leads to activation of NFAT pathway signaling and luciferase expression, which is detected using a bioluminescent luciferase substrate. Briefly, a 96-well plate was coated overnight at 4°C with TTR34-54cyc diluted to a concentration of 3 μg / ml in PBS buffer. 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. Dilution plates were prepared for measurement in which the NI-301.37F1 antibody was diluted to a specified concentration (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 volume of antibody dilution and incubating at 37°C and 5% CO2 for 30 minutes. One unit volume of FcγR1 reporter cells in ADCP buffer was then added at a density of approximately 1.65 × 10^5 cells / well. The assay was incubated at 37°C and 5% CO2 for 6 hours before adding the luminescent substrate (Bio-Glo™ Luciferase Assay Reagent). Luminescence was measured after incubation at room temperature for 15 minutes (integration time: 1000 ms, settling time: 0 ms).

[0197] NI-301.37F1 RS relative to NI-301.37F1 50%, NI-301.37F1 70%, NI-301.37F1 130% and NI-301.37F1 150%

[0198] In the first experiment, the antibody NI-301.37F1 RS was shown to exhibit an EC of 19.8 ng / ml. 50Specifically, the plates were coated with 3 μg / mL of the cyclic peptide TTR34-54cyc, and antibody dilutions ranging from 500 ng / mL to 0.4 ng / mL were tested. These conditions resulted in a reasonable response curve with a stable slope, lower asymptote, and upper asymptote; see Figure 2 .

[0199] Further experiments were performed in which the response of the assay was tested with 50%, 70%, 130% and 150% concentrations of NI-301.37F1. Figure 3 A to Figure 3 As shown in Table 3, NI-301.37F1 50% exhibited an EC of 39 ng / ml. 50 is characterized by a dose-response. EC 50 The increase was 1.97 times, which is almost exactly the same as the lower half concentration of sample NI-301.37F 50%. NI-301.37F1 70% showed an EC of 30.4 ng / ml. 50 is characterized by a dose-response. EC 50 The difference was 1.43-fold, which is in good agreement with the expected difference of 1.54-fold. NI-301.37F1 130% showed an EC of 13.6 ng / ml. 50 is characterized by a dose-response. EC 50 The difference was 0.77-fold, which is consistent with the expected difference of 0.77-fold. NI-301.37F1 150% showed an EC of 13.5 ng / ml. 50 is characterized by a dose-response. EC 50 This increased to 0.67 times, which is almost exactly the same as the expected difference of 0.70 times.

[0200] Thus, there is little assay variability, and the results show that the assay is responsive to changes in antibody concentration. Specifically, the FcγR1 ADCP assay was shown to have the ability to detect up to 50% loss and up to 50% gain in antibody activity with excellent accuracy.

[0201] Table 3: Summary of FcγR1 ADCP assay performance .

[0202]

[0203] Example 3: Evaluation of other cyclic peptides as target antigens for antigen-binding molecules

[0204] As shown in Example 1, the cyclic peptide TTR34-54cyc has been successfully used as the target antigen for the antibody NI-301.37F1 in an ELISA assay. Therefore, the ability of anti-TTR antibodies to bind to additional cyclic peptides was analyzed. Specifically, the ability of anti-TTR antibodies to bind to two cyclic peptides containing 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) was evaluated using additional ELISA assays, using the cyclic peptides as target antigens and the TTR peptide TTR40-49, biotinylated TTR peptide TTR40-49, and misfolded wild-type TTR (mis.WT-TTR) as antigen controls. The corresponding peptides and mis.WT-TTR were prepared as described in Example 1 (described above). For the ELISA assay, 96-well microplates were coated with the two cyclic peptides TTR89-97cyc and TTR101-109cy and an antigen control, and the assay was performed as described in Example 5 (described above).

[0205] Example 4: Evaluation of other cyclic peptides as target antigens in ADCP assays

[0206] The potency of the anti-TTR antibody NI-301.37F1 to activate phagocytosis of a cyclic TTR peptide (TTR34-54cyc) in vitro was successfully determined using an ADCP assay as shown in Example 2. Therefore, the potency of the anti-TTR antibody to activate phagocytosis of two cyclic peptides, TTR89-97cyc and TTR101-109cyc, was evaluated in a further ADCP assay.

[0207] To measure the efficacy of an anti-TTR antibody reference sample (anti-TTR antibody RS, 100%) and test samples having a 50% lower concentration (anti-TTR antibody 50%), a 30% lower concentration (anti-TTR antibody 70%), a 30% higher concentration (anti-TTR antibody 130%), and a 50% higher concentration (anti-TTR antibody 150%) in activating phagocytosis of the two cyclic peptides, a commercially available FcγR1 ADCP reporter gene bioassay (Promega, Madison, WI, USA, Catalog Nos. GA1341, GA1345) as described in Example 2 was applied.

[0208] It is expected that the anti-TTR antibodies will exhibit a dose-response, with anti-TTR antibody 50% showing an approximately 2-fold increase in EC compared to anti-TTR antibody RS. 50 The anti-TTR antibody 70% will show an EC value increased to about 1.5 times compared with the anti-TTR antibody RS. 50 The EC values of anti-TTR antibody 130% were reduced to about 0.77 times compared with those of anti-TTR antibody RS. 50values, and anti-TTR antibody 150% would show an EC value reduced to approximately 0.70-fold compared to anti-TTR antibody RS. 50 value.

[0209] Example 5: Validation of ADCP Assay

[0210] The ADCP assay used to determine the potency of antigen-binding molecules with cyclic peptides of the present invention is described in Example 2. This assay uses a reporter cell line expressing human Fcγ receptor 1 (FcγR1) and has been exemplarily evaluated for its ability to ensure the potency of the antibody NI-301.37F1 in activating phagocytosis of cyclic TTR peptide (TTR34-54cyc) in vitro.

[0211] The reliability and accuracy of the cyclic peptide-based ADCP assay have been confirmed under validated experimental conditions to determine antibody potency within the range of 40% to 180% of theoretical relative activity.

[0212] have NI-301.37F1 ADCP reporter gene assay with luminescent readout

[0213] On day 1, 96-well assay plates were coated with 3 μg / mL of the synthetic peptide TTR34-54cyc in DBPS (Dulbecco's phosphate-buffered saline) by incubation for 18 hours ± 2 hours at 5° C. On day 2, the coating solution was removed, and 200 μl of blocking buffer (2% BSA and 0.1% Tween 20 in DPBS) was added, and the plates were incubated at room temperature on a plate shaker at 300 rpm for 60 minutes ± 5 minutes.

[0214] In parallel, reference standard and test item working solutions were prepared; see Table 4. The reference standard (RS) corresponds to an antibody concentration of 4000 ng / mL in ADCP buffer (4% low IgG serum (v / v) in RPMI 1640 medium). Test items were generated from a reference standard stock solution with an antibody concentration of 49.9 mg / mL.

[0215] Table 4: Working solutions of reference standards and test items

[0216] project Working solution RS 4000 ng / mL antibody in ADCP buffer TS 40% 1600 ng / mL antibody in ADCP buffer TS 71% 2840 ng / mL antibody in ADCP buffer TS 100% 4000 ng / mL antibody in ADCP buffer TS 131% 5240 ng / mL antibody in ADCP buffer TS 168% 6720 ng / mL antibody in ADCP buffer TS 180% 7200 ng / mL antibody in ADCP buffer

[0217] In addition, a commercially available FcγR1 ADCP reporter gene bioassay kit (Promega, USA, No. GA1345) was used to prepare a FγR1 effector cell suspension to generate approximately 3×10 6 cells / mL.

[0218] Prepare one assay plate to analyze one test item, comparing it to a reference standard, in triplicate for each dose. After incubating the assay plate with blocking buffer, remove the blocking buffer. Wash the assay plate with 200 μl of DPBS, then transfer 55 μl of the antibody working solution to the assay plate and incubate at 37°C and 5% CO₂ for 30 min ± 5 min. Following the assay plate incubation time, transfer 55 μl of the effector cell suspension to the assay plate and incubate at 37°C and 5% CO₂ for 6 h ± 15 min. The plate layout is provided in Table 5.

[0219] Table 5: Assay plate layout. R: Reference standard, TS: Test item, C1: Control 1 (TTR34-54cyc, with RS, invalid 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: dilution series at 100% activity Final concentration range

[0220]

[0221]

[0222] Remove the assay plate from the incubator and incubate at room temperature for 10 min ± 5 min before adding 110 μl of Bio-Glo TM Luciferase assay reagent (Promega, USA, No. GA1345). The assay plate was then covered with a black lid and incubated at room temperature for 15 minutes ± 5 minutes. Finally, the luminescence of the assay plate was measured using a multi-plate reader with a glow-type luminescence reading capability. The results are shown in Table 6.

[0223] Table 6: Measured relative activity [%] The measurements were performed in triplicate and the average relative activity values (and standard deviations) are as follows shown :

[0224]

[0225] Conclusion: These data demonstrate the utility of the disclosed cyclic TTR peptides (e.g., TTR34-54cyc) in screening for anti-TTR antibody candidates that can effectively activate phagocytosis of TTR amyloid plaques. This example further provides a validated ADCP reporter gene assay for easily and reliably determining the efficacy of anti-TTR antibodies as agents for treating and / or managing TTR amyloidosis.

Claims

1. A cyclic compound comprising a peptide comprising an epitope from an amyloidogenic protein involved in systemic amyloidosis.

2. The cyclic compound according to claim 1, comprising a linker, preferably wherein the linker is an amino acid linker or a non-amino acid linker. 3 . The cyclic compound according to claim 2 , wherein the linker is covalently coupled at or near the N-terminal residue of the peptide and the C-terminal residue of the peptide.

4. The cyclic compound according to 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. 5 . The cyclic compound according to claim 1 , wherein the amyloidogenic protein is selected from the group consisting of transthyretin (TTR), immunoglobulin light chain (LC), and serum amyloid A (SAA).

6. The cyclic compound according to claim 5, wherein the amyloidogenic protein is TTR, and the peptide is a TTR peptide.

7. The cyclic compound of claim 6, wherein the TTR peptide comprises at least 4 amino acid residues selected from any one of the following amino acid sequences: 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 NO: 6 and SEQ ID NO: 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 according to claim 6 or 7, wherein the cyclic compound comprises at least 4 consecutive 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 SEQ ID NO: 1 and / or at least 9 amino acids appended to the C-terminus of SEQ ID NO: 1, or a variant thereof.

9. The cyclic compound according to 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 according to 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 according to claim 8 , wherein the amino acid in the N-terminus and / or the C-terminus is attached to SEQ ID NO: 1 via a peptide bond.

12. The cyclic compound according to 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 according to claim 12, wherein the C-terminal threonine (T) of the first peptide sequence RKAADDT (SEQ ID NO: 162) is bonded to the 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) of the peptide sequence WEPFA (SEQ ID NO: 1), in each case via a peptide bond.

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

15. The cyclic compound of claim 14, wherein the linker amino acid is 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 according to claim 15, wherein the compound is cyclized via a disulfide bridge.

17. The compound according to any one of claims 1 to 16, wherein the compound comprises a first linker comprising or consisting of the amino acid sequence GCGGG (SEQ ID NO: 15) and / or a second linker comprising or consisting of the amino acid sequence GGGCG (SEQ ID NO: 16). The cyclic compound according to claim 17 , wherein the L1 is directly or indirectly linked to the N-terminus of the peptide sequence WEPFA (SEQ ID NO: 1), and the L2 is directly or indirectly linked to the C-terminus of the peptide sequence WEPFA (SEQ ID NO: 1). 19 . The cyclic compound according to claim 18 , wherein the L1 and the L2 are linked to form a cyclic peptide, preferably wherein the N-terminus of the L1 is bonded to the C-terminus of the L2 via a peptide bond. 20 . The cyclic compound according to claim 1 , wherein the epitope is an epitope of an antibody, and the cyclic compound is bound by the antibody.

21. The cyclic compound according to claim 20, wherein the epitope can be bound by the antibody only in a misfolded and / or aggregated form of the protein, preferably wherein the epitope is exposed in pathological protein aggregates.

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

23. The cyclic compound according to any one of claims 8 to 22, comprising a part or all of the first motif VFRK (SEQ ID NO: 7) at its N-terminus and / or a part or all of the second motif ELXGLTXE (SEQ ID NO: 3) at its C-terminus, wherein X in SEQ ID NO: 3 is any natural amino acid, preferably wherein X at position 3 in SEQ ID NO: 3 is histidine (H), and X at position 7 in SEQ ID NO: 3 is threonine (T).

24. The cyclic compound according to any one of claims 8 to 22, comprising at its N-terminus a portion of a first motif VFRK (SEQ ID NO: 7) comprising at least 2 amino acids and at its C-terminus a portion of a second motif ELXGLTXE (SEQ ID NO: 3) comprising at least 1 amino acid, preferably wherein the amino acids constituting the portion of the first motif comprise the dipeptide RK in SEQ ID NO: 7, and the amino acids constituting the portion of the second motif comprise the N-terminal E in SEQ ID NO:

3.

25. The cyclic compound according to 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).

26. A variant of the cyclic compound according to claim 8, comprising the sequence WEPFA (SEQ ID NO: 1) and further comprising a 5% to 20% variation in amino acid sequence identity, wherein the variation is in the amino acids appended to the N-terminus and / or the C-terminus of SEQ ID NO: 1, for example, a variant of SEQ ID NO: 17 comprising 80% to 95% sequence identity with SEQ ID NO: 17, wherein the variation is due to an amino acid substitution, addition or deletion at the N-terminal sequence RKAADDT (SEQ ID NO: X) and / or an 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.

27. The cyclic compound according to any one of claims 1 to 26, further comprising at least one other immunogenic sequence selected from the group consisting of: EEFXEGIY (SEQ ID NO: 2), wherein X in SEQ ID NO: 2 is any amino acid, preferably wherein X in 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).

28. The cyclic compound according to 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 SEQ ID NO: 2 is any amino acid, preferably wherein X in 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 according to any one of claims 1 to 26, which does not contain any immunogenic sequence: (a) the N-terminus of VFRK (SEQ ID NO: 7) in the human TTR sequence of SEQ ID NO: T and / or (b) the C-terminus of ELHGLTTE (SEQ ID NO: 3) in the human TTR sequence of SEQ ID NO:

164.

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

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

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

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

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

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

36. The kit according to claim 35, further comprising: (i) an effector cell population engineered to express an Fc receptor, preferably a human Fc receptor FcγR, and having a reporter gene under the control of a response element, said response element is responsive to activation of said Fc receptor, preferably wherein said effector cell population is a Jurkat cell population, and said reporter gene encodes a photoprotein, preferably luciferase, under the control of a NFAT response element; (ii) a corresponding substrate for a reporter gene; preferably, 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) wash, blocking, and assay / sample dilution buffers; and / or (v) a monomer control of the target protein and / or a positive control anti-target antigen antibody.

37. Use of the cyclic compound according to any one of claims 1 to 30, or the composition according to claim 32 or 33, the array according to claim 34, or the kit according to claim 35 or 36 for detecting or quantifying an antigen binding molecule.

38. Use according to claim 37, wherein the detection or quantification is performed by an immunological assay, preferably by ELISA.

39. Use of the cyclic compound according to any one of claims 1 to 30, or the composition according to claim 32 or 33, the array according to claim 34, or the kit according to claim 35 or 36 for determining the potency of an antigen binding molecule comprising an Fc domain.

40. The use according to claim 39, wherein determining the potency of the antigen binding molecule is performed using a method comprising the steps of: (a) contacting the cyclic compound according to any one of claims 1 to 30 with the binding molecule under conditions that allow formation of a binding molecule-antigen complex; (b) contacting the binding molecule-antigen complex with a population of effector cells engineered to express an Fc receptor and having a reporter gene under the control of a response element that is responsive to activation of the Fc receptor, under conditions permissive 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 quantifiable reporter gene activity; and (c) detecting the activity of the reporter gene, wherein at least one mechanism of action of said Fc domain of said binding molecule is mediated through said binding of said Fc domain to an Fc receptor, and said reporter gene activity is indicative of said potency of said binding molecule.

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

42. The use according to claim 40 or 41, wherein the Fc receptor is the human Fc receptor FcγRI (CD64).

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

44. The use according to any one of claims 40 to 43, wherein the cells do not overexpress FcγRIII (CD16).

45. The use according to any one of claims 40 to 44, wherein the effector cells are Jurkat cells.

46. Use according to any one of claims 40 to 45, wherein the response element is a NFAT (nuclear factor of activated T cells) response element.

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

48. Use according to any one of claims 37 to 47, wherein the binding molecule is selected from 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 according to claim 48, wherein the antibody is an IgG1 antibody, such as an IgG1, lambda antibody or an IgG1, kappa antibody.

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

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

52. Use according to any one of claims 37 to 51, wherein the cyclic peptide is bound to a solid support, preferably to a microtiter plate.

53. The use according to claim 52, wherein at least step (b) of claim 40 is performed in a vertical plate configuration.

54. A method for identifying and optionally obtaining an antibody or its corresponding antigen binding molecule that binds to an amyloidogenic protein involved in systemic amyloidosis, the method comprising the steps of: (a) providing, optionally producing, one or more potential amyloidogenic protein-binding antibodies or amyloidogenic protein-binding molecules thereof or sources thereof; (b) subjecting the one or more potential amyloidogenic protein binding antibodies or amyloidogenic protein binding molecules thereof, or a source thereof, to a binding assay comprising a cyclic compound according to any one of claims 1 to 30; and (c) identifying and optionally obtaining an antibody (subject antibody) or binding molecule that has been determined to bind to the cyclic compound.

55. A method for producing a pharmaceutical composition comprising an antibody or antigen-binding molecule thereof that binds to an amyloidogenic protein, the method comprising the steps of: (a) providing, optionally producing, one or more potential amyloidogenic protein-binding antibodies or amyloidogenic protein-binding molecules thereof or sources thereof; (b) subjecting the one or more potential amyloidogenic protein binding antibodies or amyloidogenic protein binding molecules thereof, or a source thereof, to a binding assay comprising a cyclic compound according to any one of claims 1 to 30; (c) identifying and optionally obtaining an antibody (subject antibody) or binding molecule that binds to the cyclic compound; as well as (d) formulating the antibody or binding molecule or derivative thereof identified and optionally obtained in step (c) with a pharmaceutically acceptable carrier.

56. The method of claim 54 or 55, wherein the antibody source is selected from the group consisting of: immunized laboratory animals, such as rodents, preferably mice, most preferably Ig humanized mice; human blood or fractions thereof, preferably containing memory B cells; recombinant antibody libraries, such as phage, yeast and ribosomal systems or mammalian cell systems, such as CHO and HEK.

57. The method of any one of claims 54 to 56, wherein the binding assay comprises an ELISA.

58. 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 to the amyloidogenic protein, preferably wherein the EC of the subject antibody to the amyloidogenic protein is 50 lower than the reference antibody.

59. 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 said potency by using the cyclic compound according to any one of claims 1 to 30, preferably wherein said assay is an assay as defined in any one of claims 39 to 53; (b) comparing the reporter gene activity of the antigen-binding molecules; (c) selecting the antigen-binding molecule that exhibits the greatest reporter gene activity.

60. A method for analyzing and selecting a pharmaceutical composition of at least one batch of target antigen binding molecules, the method comprising: (a) subjecting a sample of said batch to an assay for determining said potency by using a cyclic compound according to any one of claims 1 to 30, preferably wherein said assay is an assay as defined in any one of claims 39 to 53; (b) comparing the reporter gene activity of the sample with the reporter gene activity of a control; as well as (c) selecting a batch in which the sample shows a greater, equal or no substantially reduced reporter gene activity compared to the control, preferably a batch in which the sample shows a greater, equal or not 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 subjected to stress conditions, and the control is the value of the reporter gene activity of a sample taken from the batch or a corresponding batch before storage and / or subjection to the stress conditions.

61. according to the method described in any one of claim 54 to 60, wherein in another step, the ability of described antigen binding molecule to trigger phagocytosis of described misfolded / aggregated amyloidogenic protein and / or its ability to bind to described misfolded / aggregated amyloidogenic protein is analyzed with full-length amyloidogenic protein, preferably with misfolded / aggregated amyloidogenic protein.

62. A composition comprising an antibody or antigen-binding molecule thereof obtainable by the method according to claim 59, wherein the antibody or antigen-binding molecule thereof exhibits a reporter gene activity that is greater, equal to, or at least 80% greater than that of the reference antibody NI006.

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